2019 |
LeDuc, R D; Fellers, R T; Early, B P; Greer, J B; Shams, D P; Thomas, P; Kelleher, N L Accurate Estimation of Context-Dependent False Discovery Rates in Top-Down Proteomics Journal Article Mol Cell Proteomics., 18 (4), pp. 796-805, 2019. Abstract | Links | BibTeX | Tags: @article{LeDuc2019, title = {Accurate Estimation of Context-Dependent False Discovery Rates in Top-Down Proteomics}, author = {LeDuc, R.D and Fellers, R.T and Early, B.P and Greer, J.B and Shams, D.P and Thomas, P and Kelleher, N.L. }, url = {https://www.mcponline.org/content/18/4/796.long}, doi = {10.1074/mcp.RA118.000993}, year = {2019}, date = {2019-04-01}, journal = {Mol Cell Proteomics.}, volume = {18}, number = {4}, pages = {796-805}, abstract = {Within the last several years, top-down proteomics has emerged as a high throughput technique for protein and proteoform identification. This technique has the potential to identify and characterize thousands of proteoforms within a single study, but the absence of accurate false discovery rate (FDR) estimation could hinder the adoption and consistency of top-down proteomics in the future. In automated identification and characterization of proteoforms, FDR calculation strongly depends on the context of the search. The context includes MS data quality, the database being interrogated, the search engine, and the parameters of the search. Particular to top-down proteomics-there are four molecular levels of study: proteoform spectral match (PrSM), protein, isoform, and proteoform. Here, a context-dependent framework for calculating an accurate FDR at each level was designed, implemented, and validated against a manually curated training set with 546 confirmed proteoforms. We examined several search contexts and found that an FDR calculated at the PrSM level under-reported the true FDR at the protein level by an average of 24-fold. We present a new open-source tool, the TDCD_FDR_Calculator, which provides a scalable, context-dependent FDR calculation that can be applied post-search to enhance the quality of results in top-down proteomics from any search engine.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Within the last several years, top-down proteomics has emerged as a high throughput technique for protein and proteoform identification. This technique has the potential to identify and characterize thousands of proteoforms within a single study, but the absence of accurate false discovery rate (FDR) estimation could hinder the adoption and consistency of top-down proteomics in the future. In automated identification and characterization of proteoforms, FDR calculation strongly depends on the context of the search. The context includes MS data quality, the database being interrogated, the search engine, and the parameters of the search. Particular to top-down proteomics-there are four molecular levels of study: proteoform spectral match (PrSM), protein, isoform, and proteoform. Here, a context-dependent framework for calculating an accurate FDR at each level was designed, implemented, and validated against a manually curated training set with 546 confirmed proteoforms. We examined several search contexts and found that an FDR calculated at the PrSM level under-reported the true FDR at the protein level by an average of 24-fold. We present a new open-source tool, the TDCD_FDR_Calculator, which provides a scalable, context-dependent FDR calculation that can be applied post-search to enhance the quality of results in top-down proteomics from any search engine. |
Cline, E N; Das, A; Bicca, M A; Mohammad, S N; Schachner, L F; Kamel, J M; DiNunno, N; Weng, A; Paschall, J D; Bu, R L; Khan, F M; Rollins, M G; Ives, A N; Shekhawat, G; Nunes-Tavares, N; de Mello, F G; Compton, P D; Kelleher, N L; Klein, W L A novel crosslinking protocol stabilizes amyloid β oligomers capable of inducing Alzheimer's-associated pathologies Journal Article J Neurochem., 148 (6), pp. 822-836, 2019. Abstract | Links | BibTeX | Tags: @article{Cline2018, title = {A novel crosslinking protocol stabilizes amyloid β oligomers capable of inducing Alzheimer's-associated pathologies}, author = {Cline, E.N and Das, A and Bicca, M.A and Mohammad, S.N and Schachner, L.F and Kamel, J.M and DiNunno, N and Weng, A and Paschall, J.D and Bu, R.L and Khan, F.M and Rollins, M.G and Ives, A.N and Shekhawat, G and Nunes-Tavares, N and de Mello, F.G and Compton, P.D and Kelleher, N.L and Klein, W.L. }, doi = {10.1111/jnc.14647}, year = {2019}, date = {2019-03-01}, journal = {J Neurochem.}, volume = {148}, number = {6}, pages = {822-836}, abstract = {Amyloid β oligomers (AβOs) accumulate early in Alzheimer's disease (AD) and experimentally cause memory dysfunction and the major pathologies associated with AD, for example, tau abnormalities, synapse loss, oxidative damage, and cognitive dysfunction. In order to develop the most effective AβO-targeting diagnostics and therapeutics, the AβO structures contributing to AD-associated toxicity must be elucidated. Here, we investigate the structural properties and pathogenic relevance of AβOs stabilized by the bifunctional crosslinker 1,5-difluoro-2,4-dinitrobenzene (DFDNB). We find that DFDNB stabilizes synthetic Aβ in a soluble oligomeric conformation. With DFDNB, solutions of Aβ that would otherwise convert to large aggregates instead yield solutions of stable AβOs, predominantly in the 50-300 kDa range, that are maintained for at least 12 days at 37°C. Structures were determined by biochemical and native top-down mass spectrometry analyses. Assayed in neuronal cultures and i.c.v.-injected mice, the DFDNB-stabilized AβOs were found to induce tau hyperphosphorylation, inhibit choline acetyltransferase, and provoke neuroinflammation. Most interestingly, DFDNB crosslinking was found to stabilize an AβO conformation particularly potent in inducing memory dysfunction in mice. Taken together, these data support the utility of DFDNB crosslinking as a tool for stabilizing pathogenic AβOs in structure-function studies.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Amyloid β oligomers (AβOs) accumulate early in Alzheimer's disease (AD) and experimentally cause memory dysfunction and the major pathologies associated with AD, for example, tau abnormalities, synapse loss, oxidative damage, and cognitive dysfunction. In order to develop the most effective AβO-targeting diagnostics and therapeutics, the AβO structures contributing to AD-associated toxicity must be elucidated. Here, we investigate the structural properties and pathogenic relevance of AβOs stabilized by the bifunctional crosslinker 1,5-difluoro-2,4-dinitrobenzene (DFDNB). We find that DFDNB stabilizes synthetic Aβ in a soluble oligomeric conformation. With DFDNB, solutions of Aβ that would otherwise convert to large aggregates instead yield solutions of stable AβOs, predominantly in the 50-300 kDa range, that are maintained for at least 12 days at 37°C. Structures were determined by biochemical and native top-down mass spectrometry analyses. Assayed in neuronal cultures and i.c.v.-injected mice, the DFDNB-stabilized AβOs were found to induce tau hyperphosphorylation, inhibit choline acetyltransferase, and provoke neuroinflammation. Most interestingly, DFDNB crosslinking was found to stabilize an AβO conformation particularly potent in inducing memory dysfunction in mice. Taken together, these data support the utility of DFDNB crosslinking as a tool for stabilizing pathogenic AβOs in structure-function studies. |
Kafader, J O; Melani, R D; Senko, M W; Makarov, A A; Kelleher, N L; Compton, P D Measurement of Individual Ions Sharply Increases the Resolution of Orbitrap Mass Spectra of Proteins Journal Article Anal Chem., 91 (4), pp. 2776-2783, 2019. Abstract | Links | BibTeX | Tags: @article{Kafader2019, title = {Measurement of Individual Ions Sharply Increases the Resolution of Orbitrap Mass Spectra of Proteins}, author = {Kafader, J.O and Melani, R.D and Senko, M.W and Makarov, A.A and Kelleher, N.L and Compton, P.D. }, url = {https://pubs.acs.org/doi/10.1021/acs.analchem.8b04519}, doi = {10.1021/acs.analchem.8b04519}, year = {2019}, date = {2019-02-19}, journal = {Anal Chem.}, volume = {91}, number = {4}, pages = {2776-2783}, abstract = {It is well-known that with Orbitrap-based Fourier-transform-mass-spectrometry (FT-MS) analysis, longer-time-domain signals are needed to better resolve species of interest. Unfortunately, increasing the signal-acquisition period comes at the expense of increasing ion decay, which lowers signal-to-noise ratios and ultimately limits resolution. This is especially problematic for intact proteins, including antibodies, which demonstrate rapid decay because of their larger collisional cross-sections, and result in more frequent collisions with background gas molecules. Provided here is a method that utilizes numerous low-ion-count spectra and single-ion processing to reconstruct a conventional m/ z spectrum. This technique has been applied to proteins varying in molecular weight from 8 to 150 kDa, with a resolving power of 677 000 achieved for transients of carbonic anhydrase (29 kDa) with a duration of only ∼250 ms. A resolution improvement ranging from 10- to 20-fold was observed for all proteins, providing isotopic resolution where none was previously present.}, keywords = {}, pubstate = {published}, tppubtype = {article} } It is well-known that with Orbitrap-based Fourier-transform-mass-spectrometry (FT-MS) analysis, longer-time-domain signals are needed to better resolve species of interest. Unfortunately, increasing the signal-acquisition period comes at the expense of increasing ion decay, which lowers signal-to-noise ratios and ultimately limits resolution. This is especially problematic for intact proteins, including antibodies, which demonstrate rapid decay because of their larger collisional cross-sections, and result in more frequent collisions with background gas molecules. Provided here is a method that utilizes numerous low-ion-count spectra and single-ion processing to reconstruct a conventional m/ z spectrum. This technique has been applied to proteins varying in molecular weight from 8 to 150 kDa, with a resolving power of 677 000 achieved for transients of carbonic anhydrase (29 kDa) with a duration of only ∼250 ms. A resolution improvement ranging from 10- to 20-fold was observed for all proteins, providing isotopic resolution where none was previously present. |
Swaroop, A; Oyer, J A; Will, C M; Huang, X; Yu, W; Troche, C; Bulic, M; Durham, B H; Wen, Q J; Crispino, J D; MacKerell, Jr A D; Bennett, R L; Kelleher, N L; Licht, J D An activating mutation of the NSD2 histone methyltransferase drives oncogenic reprogramming in acute lymphocytic leukemia Journal Article Oncogene., 38 (5), pp. 671-686, 2019. Abstract | Links | BibTeX | Tags: @article{Swaroop2019, title = {An activating mutation of the NSD2 histone methyltransferase drives oncogenic reprogramming in acute lymphocytic leukemia}, author = {Swaroop, A. and Oyer, J.A and Will, C.M and Huang, X and Yu, W and Troche, C and Bulic, M and Durham, B.H and Wen, Q.J and Crispino, J.D and MacKerell, A.D. Jr and Bennett, R.L and Kelleher, N.L and Licht, J.D. }, url = {https://www.nature.com/articles/s41388-018-0474-y}, doi = {10.1038/s41388-018-0474-y}, year = {2019}, date = {2019-01-01}, journal = {Oncogene.}, volume = {38}, number = {5}, pages = {671-686}, abstract = {NSD2, a histone methyltransferase specific for methylation of histone 3 lysine 36 (H3K36), exhibits a glutamic acid to lysine mutation at residue 1099 (E1099K) in childhood acute lymphocytic leukemia (ALL), and cells harboring this mutation can become the predominant clone in relapsing disease. We studied the effects of this mutant enzyme in silico, in vitro, and in vivo using gene edited cell lines. The E1099K mutation altered enzyme/substrate binding and enhanced the rate of H3K36 methylation. As a result, cell lines harboring E1099K exhibit increased H3K36 dimethylation and reduced H3K27 trimethylation, particularly on nucleosomes containing histone H3.1. Mutant NSD2 cells exhibit reduced apoptosis and enhanced proliferation, clonogenicity, adhesion, and migration. In mouse xenografts, mutant NSD2 cells are more lethal and brain invasive than wildtype cells. Transcriptional profiling demonstrates that mutant NSD2 aberrantly activates factors commonly associated with neural and stromal lineages in addition to signaling and adhesion genes. Identification of these pathways provides new avenues for therapeutic interventions in NSD2 dysregulated malignancies.}, keywords = {}, pubstate = {published}, tppubtype = {article} } NSD2, a histone methyltransferase specific for methylation of histone 3 lysine 36 (H3K36), exhibits a glutamic acid to lysine mutation at residue 1099 (E1099K) in childhood acute lymphocytic leukemia (ALL), and cells harboring this mutation can become the predominant clone in relapsing disease. We studied the effects of this mutant enzyme in silico, in vitro, and in vivo using gene edited cell lines. The E1099K mutation altered enzyme/substrate binding and enhanced the rate of H3K36 methylation. As a result, cell lines harboring E1099K exhibit increased H3K36 dimethylation and reduced H3K27 trimethylation, particularly on nucleosomes containing histone H3.1. Mutant NSD2 cells exhibit reduced apoptosis and enhanced proliferation, clonogenicity, adhesion, and migration. In mouse xenografts, mutant NSD2 cells are more lethal and brain invasive than wildtype cells. Transcriptional profiling demonstrates that mutant NSD2 aberrantly activates factors commonly associated with neural and stromal lineages in addition to signaling and adhesion genes. Identification of these pathways provides new avenues for therapeutic interventions in NSD2 dysregulated malignancies. |
Jin, Q; Martinez, C A; Arcipowski, K M; Zhu, Y; Gutierrez-Diaz, B T; Wang, K K; Johnson, M R; Volk, A G; Wang, F; Wu, J; Grove, C; Wang, H; Sokirniy, I; Thomas, P M; Goo, Y A; Abshiru, N A; Hijiya, N; Peirs, S; Vandamme, N; Berx, G; Goosens, S; Marshall, S A; Rendleman, E J; Takahashi, Y H; Wang, L; Rawat, R; Bartom, E T; Collings, C K; Van Vlierberghe, P; Strikoudis, A; Kelly, S; Ueberheide, B; Mantis, C; Kandela, I; Bourquin, J P; Bornhauser, B; Serafin, V; Bresolin, S; Paganin, M; Accordi, B; Basso, G; Kelleher, N L; Weinstock, J; Kumar, S; Crispino, J D; Shilatifard, A; Ntziachristos, P USP7 Cooperates with NOTCH1 to Drive the Oncogenic Transcriptional Program in T-Cell Leukemia Journal Article Clin Cancer Res., 25 (1), pp. 222-239, 2019. Abstract | Links | BibTeX | Tags: @article{Jin2019, title = {USP7 Cooperates with NOTCH1 to Drive the Oncogenic Transcriptional Program in T-Cell Leukemia}, author = {Jin, Q and Martinez, C.A and Arcipowski, K.M and Zhu, Y and Gutierrez-Diaz, B.T and Wang, K.K and Johnson, M.R and Volk, A.G and Wang, F and Wu, J and Grove, C and Wang, H and Sokirniy, I and Thomas, P.M and Goo, Y.A and Abshiru, N.A and Hijiya, N and Peirs, S and Vandamme, N and Berx, G and Goosens, S and Marshall, S.A and Rendleman, E.J and Takahashi, Y.H and Wang, L and Rawat, R and Bartom, E.T and Collings, C.K and Van Vlierberghe, P and Strikoudis, A and Kelly, S and Ueberheide, B and Mantis, C and Kandela, I and Bourquin, J.P and Bornhauser, B and Serafin, V and Bresolin, S and Paganin, M and Accordi, B and Basso, G and Kelleher, N.L and Weinstock, J and Kumar, S and Crispino, J.D and Shilatifard, A and Ntziachristos, P. }, url = {http://clincancerres.aacrjournals.org/content/25/1/222.long}, doi = {10.1158/1078-0432.CCR-18-1740}, year = {2019}, date = {2019-01-01}, journal = {Clin Cancer Res.}, volume = {25}, number = {1}, pages = {222-239}, abstract = {T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease, affecting children and adults. Chemotherapy treatments show high response rates but have debilitating effects and carry risk of relapse. Previous work implicated NOTCH1 and other oncogenes. However, direct inhibition of these pathways affects healthy tissues and cancer alike. Our goal in this work has been to identify enzymes active in T-ALL whose activity could be targeted for therapeutic purposes. EXPERIMENTAL DESIGN: To identify and characterize new NOTCH1 druggable partners in T-ALL, we coupled studies of the NOTCH1 interactome to expression analysis and a series of functional analyses in cell lines, patient samples, and xenograft models. RESULTS: We demonstrate that ubiquitin-specific protease 7 (USP7) interacts with NOTCH1 and controls leukemia growth by stabilizing the levels of NOTCH1 and JMJD3 histone demethylase. USP7 is highly expressed in T-ALL and is transcriptionally regulated by NOTCH1. In turn, USP7 controls NOTCH1 levels through deubiquitination. USP7 binds oncogenic targets and controls gene expression through stabilization of NOTCH1 and JMJD3 and ultimately H3K27me3 changes. We also show that USP7 and NOTCH1 bind T-ALL superenhancers, and inhibition of USP7 leads to a decrease of the transcriptional levels of NOTCH1 targets and significantly blocks T-ALL cell growth in vitro and in vivo. CONCLUSIONS: These results provide a new model for USP7 deubiquitinase activity through recruitment to oncogenic chromatin loci and regulation of both oncogenic transcription factors and chromatin marks to promote leukemia. Our studies also show that targeting USP7 inhibition could be a therapeutic strategy in aggressive leukemia.}, keywords = {}, pubstate = {published}, tppubtype = {article} } T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease, affecting children and adults. Chemotherapy treatments show high response rates but have debilitating effects and carry risk of relapse. Previous work implicated NOTCH1 and other oncogenes. However, direct inhibition of these pathways affects healthy tissues and cancer alike. Our goal in this work has been to identify enzymes active in T-ALL whose activity could be targeted for therapeutic purposes. EXPERIMENTAL DESIGN: To identify and characterize new NOTCH1 druggable partners in T-ALL, we coupled studies of the NOTCH1 interactome to expression analysis and a series of functional analyses in cell lines, patient samples, and xenograft models. RESULTS: We demonstrate that ubiquitin-specific protease 7 (USP7) interacts with NOTCH1 and controls leukemia growth by stabilizing the levels of NOTCH1 and JMJD3 histone demethylase. USP7 is highly expressed in T-ALL and is transcriptionally regulated by NOTCH1. In turn, USP7 controls NOTCH1 levels through deubiquitination. USP7 binds oncogenic targets and controls gene expression through stabilization of NOTCH1 and JMJD3 and ultimately H3K27me3 changes. We also show that USP7 and NOTCH1 bind T-ALL superenhancers, and inhibition of USP7 leads to a decrease of the transcriptional levels of NOTCH1 targets and significantly blocks T-ALL cell growth in vitro and in vivo. CONCLUSIONS: These results provide a new model for USP7 deubiquitinase activity through recruitment to oncogenic chromatin loci and regulation of both oncogenic transcription factors and chromatin marks to promote leukemia. Our studies also show that targeting USP7 inhibition could be a therapeutic strategy in aggressive leukemia. |
Toby, T K; Fornelli, L; Srzentić, K; DeHart, C J; Levitsky, J; Friedewald, J; Kelleher, N L A comprehensive pipeline for translational top-down proteomics from a single blood draw Journal Article Nat Protoc., 14 (1), pp. 119-152, 2019. Abstract | Links | BibTeX | Tags: @article{Toby2019, title = {A comprehensive pipeline for translational top-down proteomics from a single blood draw}, author = {Toby, T.K and Fornelli, L and Srzentić, K and DeHart, C.J and Levitsky, J and Friedewald, J and Kelleher, N.L.}, url = {https://www.nature.com/articles/s41596-018-0085-7}, doi = {10.1038/s41596-018-0085-7}, year = {2019}, date = {2019-01-01}, journal = {Nat Protoc.}, volume = {14}, number = {1}, pages = {119-152}, abstract = {Top-down proteomics (TDP) by mass spectrometry (MS) is a technique by which intact proteins are analyzed. It has become increasingly popDesalting and concentrating GELFrEEular in translational research because of the value of characterizing distinct proteoforms of intact proteins. Compared to bottom-up proteomics (BUP) strategies, which measure digested peptide mixtures, TDP provides highly specific molecular information that avoids the bioinformatic challenge of protein inference. However, the technique has been difficult to implement widely because of inherent limitations of existing sample preparation methods and instrumentation. Recent improvements in proteoform pre-fractionation and the availability of high-resolution benchtop mass spectrometers have made it possible to use high-throughput TDP for the analysis of complex clinical samples. Here, we provide a comprehensive protocol for analysis of a common sample type in translational research: human peripheral blood mononuclear cells (PBMCs). The pipeline comprises multiple workflows that can be treated as modular by the reader and used for various applications. First, sample collection and cell preservation are described for two clinical biorepository storage schemes. Cell lysis and proteoform pre-fractionation by gel-eluted liquid fractionation entrapment electrophoresis are then described. Importantly, instrument setup and liquid chromatography-tandem MS are described for TDP analyses, which rely on high-resolution Fourier-transform MS. Finally, data processing and analysis are described using two different, application-dependent software tools: ProSight Lite for targeted analyses of one or a few proteoforms and TDPortal for high-throughput TDP in discovery mode. For a single sample, the minimum completion time of the entire experiment is 72 h.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Top-down proteomics (TDP) by mass spectrometry (MS) is a technique by which intact proteins are analyzed. It has become increasingly popDesalting and concentrating GELFrEEular in translational research because of the value of characterizing distinct proteoforms of intact proteins. Compared to bottom-up proteomics (BUP) strategies, which measure digested peptide mixtures, TDP provides highly specific molecular information that avoids the bioinformatic challenge of protein inference. However, the technique has been difficult to implement widely because of inherent limitations of existing sample preparation methods and instrumentation. Recent improvements in proteoform pre-fractionation and the availability of high-resolution benchtop mass spectrometers have made it possible to use high-throughput TDP for the analysis of complex clinical samples. Here, we provide a comprehensive protocol for analysis of a common sample type in translational research: human peripheral blood mononuclear cells (PBMCs). The pipeline comprises multiple workflows that can be treated as modular by the reader and used for various applications. First, sample collection and cell preservation are described for two clinical biorepository storage schemes. Cell lysis and proteoform pre-fractionation by gel-eluted liquid fractionation entrapment electrophoresis are then described. Importantly, instrument setup and liquid chromatography-tandem MS are described for TDP analyses, which rely on high-resolution Fourier-transform MS. Finally, data processing and analysis are described using two different, application-dependent software tools: ProSight Lite for targeted analyses of one or a few proteoforms and TDPortal for high-throughput TDP in discovery mode. For a single sample, the minimum completion time of the entire experiment is 72 h. |
2018 |
Alvares, K; DeHart, C J; Thomas, P M; Kelleher, N L; Veis, A The unique biomineralization transcriptome and proteome of Lytechinus variegatus teeth Journal Article Connect Tissue Res., 59 (sup1), pp. 20-29, 2018. Abstract | Links | BibTeX | Tags: @article{Alvares2018, title = {The unique biomineralization transcriptome and proteome of Lytechinus variegatus teeth}, author = {Alvares, K and DeHart, C.J and Thomas, P.M and Kelleher, N.L and Veis, A. }, url = {https://www.tandfonline.com/doi/full/10.1080/03008207.2017.1408605}, doi = {10.1080/03008207.2017.1408605}, year = {2018}, date = {2018-12-01}, journal = {Connect Tissue Res.}, volume = {59}, number = {sup1}, pages = {20-29}, abstract = {BACKGROUND: Matrix-regulated biomineralization involves the specific nucleation and growth of mineral phases within or upon preformed structured organic matrices. We hypothesized that there might be a general mechanism whereby anionic, phosphorylated mineral ion-binding proteins assist in specifically locating the mineral ions with respect to the mineralizing structural organic matrix. Here we extended these studies to invertebrate mineralization in Lytechinus variegatus (Lv) teeth. MATERIALS AND METHODS: The tooth proteins were extracted and the phosphoproteins occluded in the mineral were enriched by passage through a ProQ Diamond phosphoprotein enrichment column, and subjected to MS/MS analysis. A Lv RNA-seq derived transcriptome database was generated. The MS/MS data found 25 proteins previously classified as "Predicted uncharacterized proteins" and many of the spicule matrix proteins. As these 25 proteins were also identified with the transcriptome analysis, and were thus no longer "hypothetical" but real proteins in the Lv tooth. Each protein was analyzed for the presence of a signal peptide, an acidic pI≤4, and the ability to be phosphorylated. RESULTS: Four new Lv tooth specific Pro-Ala-rich proteins were found, representing a new class of proteins. CONCLUSION: The tooth is different from the spicules and other urchin skeletal elements in that only the tooth contains both "high" and "very high" magnesium calcite, [Ca(1-X) Mg(X) CO3], where X is the mole fraction of Mg. We speculate that our newly discovered proline-alanine rich proteins, also containing sequences of acidic amino acids, may be involved in the formation of high magnesium and very high magnesium calcite.}, keywords = {}, pubstate = {published}, tppubtype = {article} } BACKGROUND: Matrix-regulated biomineralization involves the specific nucleation and growth of mineral phases within or upon preformed structured organic matrices. We hypothesized that there might be a general mechanism whereby anionic, phosphorylated mineral ion-binding proteins assist in specifically locating the mineral ions with respect to the mineralizing structural organic matrix. Here we extended these studies to invertebrate mineralization in Lytechinus variegatus (Lv) teeth. MATERIALS AND METHODS: The tooth proteins were extracted and the phosphoproteins occluded in the mineral were enriched by passage through a ProQ Diamond phosphoprotein enrichment column, and subjected to MS/MS analysis. A Lv RNA-seq derived transcriptome database was generated. The MS/MS data found 25 proteins previously classified as "Predicted uncharacterized proteins" and many of the spicule matrix proteins. As these 25 proteins were also identified with the transcriptome analysis, and were thus no longer "hypothetical" but real proteins in the Lv tooth. Each protein was analyzed for the presence of a signal peptide, an acidic pI≤4, and the ability to be phosphorylated. RESULTS: Four new Lv tooth specific Pro-Ala-rich proteins were found, representing a new class of proteins. CONCLUSION: The tooth is different from the spicules and other urchin skeletal elements in that only the tooth contains both "high" and "very high" magnesium calcite, [Ca(1-X) Mg(X) CO3], where X is the mole fraction of Mg. We speculate that our newly discovered proline-alanine rich proteins, also containing sequences of acidic amino acids, may be involved in the formation of high magnesium and very high magnesium calcite. |
Fisher, O S; Kenney, G E; Ross, M O; Ro, S Y; Lemma, B E; Batelu, S; Thomas, P M; Sosnowski, V C; DeHart, C J; Kelleher, N L; Stemmler, T L; Hoffman, B M; Rosenzweig, A C Characterization of a long overlooked copper protein from methane- and ammonia-oxidizing bacteria Journal Article Nat Commun., 9 (1), pp. 4276, 2018. Abstract | Links | BibTeX | Tags: @article{Fisher2018, title = {Characterization of a long overlooked copper protein from methane- and ammonia-oxidizing bacteria}, author = {Fisher, O.S and Kenney, G.E and Ross, M.O and Ro, S.Y and Lemma, B.E and Batelu, S and Thomas, P.M and Sosnowski, V.C and DeHart, C.J and Kelleher, N.L and Stemmler, T.L and Hoffman, B.M and Rosenzweig, A.C. }, url = {https://www.nature.com/articles/s41467-018-06681-5}, doi = {10.1038/s41467-018-06681-5}, year = {2018}, date = {2018-10-15}, journal = {Nat Commun.}, volume = {9}, number = {1}, pages = {4276}, abstract = {Methane-oxidizing microbes catalyze the oxidation of the greenhouse gas methane using the copper-dependent enzyme particulate methane monooxygenase (pMMO). Isolated pMMO exhibits lower activity than whole cells, however, suggesting that additional components may be required. A pMMO homolog, ammonia monooxygenase (AMO), converts ammonia to hydroxylamine in ammonia-oxidizing bacteria (AOB) which produce another potent greenhouse gas, nitrous oxide. Here we show that PmoD, a protein encoded within many pmo operons that is homologous to the AmoD proteins encoded within AOB amo operons, forms a copper center that exhibits the features of a well-defined CuA site using a previously unobserved ligand set derived from a cupredoxin homodimer. PmoD is critical for copper-dependent growth on methane, and genetic analyses strongly support a role directly related to pMMO and AMO. These findings identify a copper-binding protein that may represent a missing link in the function of enzymes critical to the global carbon and nitrogen cycles.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Methane-oxidizing microbes catalyze the oxidation of the greenhouse gas methane using the copper-dependent enzyme particulate methane monooxygenase (pMMO). Isolated pMMO exhibits lower activity than whole cells, however, suggesting that additional components may be required. A pMMO homolog, ammonia monooxygenase (AMO), converts ammonia to hydroxylamine in ammonia-oxidizing bacteria (AOB) which produce another potent greenhouse gas, nitrous oxide. Here we show that PmoD, a protein encoded within many pmo operons that is homologous to the AmoD proteins encoded within AOB amo operons, forms a copper center that exhibits the features of a well-defined CuA site using a previously unobserved ligand set derived from a cupredoxin homodimer. PmoD is critical for copper-dependent growth on methane, and genetic analyses strongly support a role directly related to pMMO and AMO. These findings identify a copper-binding protein that may represent a missing link in the function of enzymes critical to the global carbon and nitrogen cycles. |
Mullowney, M W; McClure, R A; Robey, M T; Kelleher, N L; Thomson, R J Natural products from thioester reductase containing biosynthetic pathways Journal Article Nat Prod Rep., 35 (9), pp. 847-878, 2018. Abstract | Links | BibTeX | Tags: @article{Mulowney2018, title = {Natural products from thioester reductase containing biosynthetic pathways}, author = {Mullowney, M.W and McClure, R.A and Robey, M.T and Kelleher, N.L and Thomson, R.J. }, url = {https://pubs.rsc.org/en/content/articlelanding/2018/NP/C8NP00013A#!divAbstract}, doi = {10.1039/c8np00013a}, year = {2018}, date = {2018-09-19}, journal = {Nat Prod Rep.}, volume = {35}, number = {9}, pages = {847-878}, abstract = {Covering: up to 2018 Thioester reductase domains catalyze two- and four-electron reductions to release natural products following assembly on nonribosomal peptide synthetases, polyketide synthases, and their hybrid biosynthetic complexes. This reductive off-loading of a natural product yields an aldehyde or alcohol, can initiate the formation of a macrocyclic imine, and contributes to important intermediates in a variety of biosyntheses, including those for polyketide alkaloids and pyrrolobenzodiazepines. Compounds that arise from reductase-terminated biosynthetic gene clusters are often reactive and exhibit biological activity. Biomedically important examples include the cancer therapeutic Yondelis (ecteinascidin 743), peptide aldehydes that inspired the first therapeutic proteasome inhibitor bortezomib, and numerous synthetic derivatives and antibody drug conjugates of the pyrrolobenzodiazepines. Recent advances in microbial genomics, metabolomics, bioinformatics, and reactivity-based labeling have facilitated the detection of these compounds for targeted isolation. Herein, we summarize known natural products arising from this important category, highlighting their occurrence in Nature, biosyntheses, biological activities, and the technologies used for their detection and identification. Additionally, we review publicly available genomic data to highlight the remaining potential for novel reductively tailored compounds and drug leads from microorganisms. This thorough retrospective highlights various molecular families with especially privileged bioactivity while illuminating challenges and prospects toward accelerating the discovery of new, high value natural products.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Covering: up to 2018 Thioester reductase domains catalyze two- and four-electron reductions to release natural products following assembly on nonribosomal peptide synthetases, polyketide synthases, and their hybrid biosynthetic complexes. This reductive off-loading of a natural product yields an aldehyde or alcohol, can initiate the formation of a macrocyclic imine, and contributes to important intermediates in a variety of biosyntheses, including those for polyketide alkaloids and pyrrolobenzodiazepines. Compounds that arise from reductase-terminated biosynthetic gene clusters are often reactive and exhibit biological activity. Biomedically important examples include the cancer therapeutic Yondelis (ecteinascidin 743), peptide aldehydes that inspired the first therapeutic proteasome inhibitor bortezomib, and numerous synthetic derivatives and antibody drug conjugates of the pyrrolobenzodiazepines. Recent advances in microbial genomics, metabolomics, bioinformatics, and reactivity-based labeling have facilitated the detection of these compounds for targeted isolation. Herein, we summarize known natural products arising from this important category, highlighting their occurrence in Nature, biosyntheses, biological activities, and the technologies used for their detection and identification. Additionally, we review publicly available genomic data to highlight the remaining potential for novel reductively tailored compounds and drug leads from microorganisms. This thorough retrospective highlights various molecular families with especially privileged bioactivity while illuminating challenges and prospects toward accelerating the discovery of new, high value natural products. |
Gruppuso, P A; Boylan, J M; Zabala, V; Neretti, N; Abshiru, N A; Sikora, J W; Doud, E H; Camarillo, J M; Thomas, P M; Kelleher, N L; Sanders, J A Stability of histone post-translational modifications in samples derived from liver tissue and primary hepatic cells Journal Article PLoS One., 13 (9), 2018. Abstract | Links | BibTeX | Tags: @article{Gruppuso2018, title = {Stability of histone post-translational modifications in samples derived from liver tissue and primary hepatic cells}, author = {Gruppuso, P.A and Boylan, J.M and Zabala, V and Neretti, N and Abshiru, N.A and Sikora, J.W and Doud, E.H. and Camarillo, J.M and Thomas, P.M and Kelleher, N.L and Sanders, J.A.. }, url = {https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0203351}, doi = {10.1371/journal.pone.0203351}, year = {2018}, date = {2018-09-07}, journal = {PLoS One.}, volume = {13}, number = {9}, abstract = {Chromatin structure, a key contributor to the regulation of gene expression, is modulated by a broad array of histone post-translational modifications (PTMs). Taken together, these "histone marks" comprise what is often referred to as the "histone code". The quantitative analysis of histone PTMs by mass spectrometry (MS) offers the ability to examine the response of the histone code to physiological signals. However, few studies have examined the stability of histone PTMs through the process of isolating and culturing primary cells. To address this, we used bottom-up, MS-based analysis of histone PTMs in liver, freshly isolated hepatocytes, and cultured hepatocytes from adult male Fisher F344 rats. Correlations between liver, freshly isolated cells, and primary cultures were generally high, with R2 values exceeding 0.9. However, a number of acetylation marks, including those on H2A K9, H2A1 K13, H3 K4, H3 K14, H4 K8, H4 K12 and H4 K16 differed significantly among the three sources. Inducing proliferation of primary adult hepatocytes in culture affected several marks on histones H3.1/3.2 and H4. We conclude that hepatocyte isolation, culturing and cell cycle status all contribute to steady-state changes in the levels of a number of histone PTMs, indicating changes in histone marks that are rapidly induced in response to alterations in the cellular milieu. This has implications for studies aimed at assigning biological significance to histone modifications in tumors versus cancer cells, the developmental behavior of stem cells, and the attribution of changes in histone PTMs to altered cell metabolism.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Chromatin structure, a key contributor to the regulation of gene expression, is modulated by a broad array of histone post-translational modifications (PTMs). Taken together, these "histone marks" comprise what is often referred to as the "histone code". The quantitative analysis of histone PTMs by mass spectrometry (MS) offers the ability to examine the response of the histone code to physiological signals. However, few studies have examined the stability of histone PTMs through the process of isolating and culturing primary cells. To address this, we used bottom-up, MS-based analysis of histone PTMs in liver, freshly isolated hepatocytes, and cultured hepatocytes from adult male Fisher F344 rats. Correlations between liver, freshly isolated cells, and primary cultures were generally high, with R2 values exceeding 0.9. However, a number of acetylation marks, including those on H2A K9, H2A1 K13, H3 K4, H3 K14, H4 K8, H4 K12 and H4 K16 differed significantly among the three sources. Inducing proliferation of primary adult hepatocytes in culture affected several marks on histones H3.1/3.2 and H4. We conclude that hepatocyte isolation, culturing and cell cycle status all contribute to steady-state changes in the levels of a number of histone PTMs, indicating changes in histone marks that are rapidly induced in response to alterations in the cellular milieu. This has implications for studies aimed at assigning biological significance to histone modifications in tumors versus cancer cells, the developmental behavior of stem cells, and the attribution of changes in histone PTMs to altered cell metabolism. |
Compton, P D; Kelleher, N L; Gunawardena, J Estimating the Distribution of Protein Post-Translational Modification States by Mass Spectrometry Journal Article J Proteome Res., 17 (8), pp. 2727-2734, 2018. Abstract | Links | BibTeX | Tags: @article{Compton2018, title = {Estimating the Distribution of Protein Post-Translational Modification States by Mass Spectrometry}, author = {Compton, P.D and Kelleher, N.L and Gunawardena, J. }, url = {https://pubs.acs.org/doi/10.1021/acs.jproteome.8b00150}, doi = {10.1021/acs.jproteome.8b00150}, year = {2018}, date = {2018-08-03}, journal = {J Proteome Res.}, volume = {17}, number = {8}, pages = {2727-2734}, abstract = {Post-translational modifications (PTMs) of proteins play a central role in cellular information encoding, but the complexity of PTM state has been challenging to unravel. A single molecule can exhibit a "modform" or combinatorial pattern of co-occurring PTMs across multiple sites, and a molecular population can exhibit a distribution of amounts of different modforms. How can this "modform distribution" be estimated by mass spectrometry (MS)? Bottom-up MS, based on cleavage into peptides, destroys correlations between PTMs on different peptides, but it is conceivable that multiple proteases with appropriate patterns of cleavage could reconstruct the modform distribution. We introduce a mathematical language for describing MS measurements and show, on the contrary, that no matter how many distinct proteases are available, the shortfall in information required for reconstruction worsens exponentially with increasing numbers of sites. Whereas top-down MS on intact proteins can do better, current technology cannot prevent the exponential worsening. However, our analysis also shows that all forms of MS yield linear equations for modform amounts. This permits different MS protocols to be integrated and the modform distribution to be constrained within a high-dimensional "modform region", which may offer a feasible proxy for analyzing information encoding.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Post-translational modifications (PTMs) of proteins play a central role in cellular information encoding, but the complexity of PTM state has been challenging to unravel. A single molecule can exhibit a "modform" or combinatorial pattern of co-occurring PTMs across multiple sites, and a molecular population can exhibit a distribution of amounts of different modforms. How can this "modform distribution" be estimated by mass spectrometry (MS)? Bottom-up MS, based on cleavage into peptides, destroys correlations between PTMs on different peptides, but it is conceivable that multiple proteases with appropriate patterns of cleavage could reconstruct the modform distribution. We introduce a mathematical language for describing MS measurements and show, on the contrary, that no matter how many distinct proteases are available, the shortfall in information required for reconstruction worsens exponentially with increasing numbers of sites. Whereas top-down MS on intact proteins can do better, current technology cannot prevent the exponential worsening. However, our analysis also shows that all forms of MS yield linear equations for modform amounts. This permits different MS protocols to be integrated and the modform distribution to be constrained within a high-dimensional "modform region", which may offer a feasible proxy for analyzing information encoding. |
Fornelli, L; Srzentić, K; Huguet, R; Mullen, C; Sharma, S; Zabrouskov, V; Fellers, R T; Durbin, K R; Compton, P D; Kelleher, N L Anal Chem., 90 (14), pp. 8421-8429, 2018. Abstract | Links | BibTeX | Tags: @article{Fornelli2018, title = {Accurate Sequence Analysis of a Monoclonal Antibody by Top-Down and Middle-Down Orbitrap Mass Spectrometry Applying Multiple Ion Activation Techniques}, author = {Fornelli, L and Srzentić, K and Huguet, R and Mullen, C and Sharma, S and Zabrouskov, V and Fellers, R.T and Durbin, K.R and Compton, P.D and Kelleher, N.L. }, url = {https://pubs.acs.org/doi/10.1021/acs.analchem.8b00984}, doi = {10.1021/acs.analchem.8b00984}, year = {2018}, date = {2018-07-17}, journal = {Anal Chem.}, volume = {90}, number = {14}, pages = {8421-8429}, abstract = {Targeted top-down (TD) and middle-down (MD) mass spectrometry (MS) offer reduced sample manipulation during protein analysis, limiting the risk of introducing artifactual modifications to better capture sequence information on the proteoforms present. This provides some advantages when characterizing biotherapeutic molecules such as monoclonal antibodies, particularly for the class of biosimilars. Here, we describe the results obtained analyzing a monoclonal IgG1, either in its ∼150 kDa intact form or after highly specific digestions yielding ∼25 and ∼50 kDa subunits, using an Orbitrap mass spectrometer on a liquid chromatography (LC) time scale with fragmentation from ion-photon, ion-ion, and ion-neutral interactions. Ultraviolet photodissociation (UVPD) used a new 213 nm solid-state laser. Alternatively, we applied high-capacity electron-transfer dissociation (ETD HD), alone or in combination with higher energy collisional dissociation (EThcD). Notably, we verify the degree of complementarity of these ion activation methods, with the combination of 213 nm UVPD and ETD HD producing a new record sequence coverage of ∼40% for TD MS experiments. The addition of EThcD for the >25 kDa products from MD strategies generated up to 90% of complete sequence information in six LC runs. Importantly, we determined an optimal signal-to-noise threshold for fragment ion deconvolution to suppress false positives yet maximize sequence coverage and implemented a systematic validation of this process using the new software TDValidator. This rigorous data analysis should elevate confidence for assignment of dense MS2 spectra and represents a purposeful step toward the application of TD and MD MS for deep sequencing of monoclonal antibodies.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Targeted top-down (TD) and middle-down (MD) mass spectrometry (MS) offer reduced sample manipulation during protein analysis, limiting the risk of introducing artifactual modifications to better capture sequence information on the proteoforms present. This provides some advantages when characterizing biotherapeutic molecules such as monoclonal antibodies, particularly for the class of biosimilars. Here, we describe the results obtained analyzing a monoclonal IgG1, either in its ∼150 kDa intact form or after highly specific digestions yielding ∼25 and ∼50 kDa subunits, using an Orbitrap mass spectrometer on a liquid chromatography (LC) time scale with fragmentation from ion-photon, ion-ion, and ion-neutral interactions. Ultraviolet photodissociation (UVPD) used a new 213 nm solid-state laser. Alternatively, we applied high-capacity electron-transfer dissociation (ETD HD), alone or in combination with higher energy collisional dissociation (EThcD). Notably, we verify the degree of complementarity of these ion activation methods, with the combination of 213 nm UVPD and ETD HD producing a new record sequence coverage of ∼40% for TD MS experiments. The addition of EThcD for the >25 kDa products from MD strategies generated up to 90% of complete sequence information in six LC runs. Importantly, we determined an optimal signal-to-noise threshold for fragment ion deconvolution to suppress false positives yet maximize sequence coverage and implemented a systematic validation of this process using the new software TDValidator. This rigorous data analysis should elevate confidence for assignment of dense MS2 spectra and represents a purposeful step toward the application of TD and MD MS for deep sequencing of monoclonal antibodies. |
Riley, N M; Sikora, J W; Seckler, H S; Greer, J B; Fellers, R T; LeDuc, R D; Westphall, M S; Thomas, P M; Kelleher, N L; Coon, J J The Value of Activated Ion Electron Transfer Dissociation for High-Throughput Top-Down Characterization of Intact Proteins Journal Article Anal Chem., 90 (14), pp. 8553-8560, 2018. Abstract | Links | BibTeX | Tags: @article{Riley2018, title = {The Value of Activated Ion Electron Transfer Dissociation for High-Throughput Top-Down Characterization of Intact Proteins}, author = {Riley, N.M and Sikora, J.W and Seckler, H.S and Greer, J.B and Fellers, R.T and LeDuc, R.D and Westphall, M.S and Thomas, P.M and Kelleher, N.L and Coon, J.J. }, url = {https://pubs.acs.org/doi/10.1021/acs.analchem.8b01638}, doi = {10.1021/acs.analchem.8b01638}, year = {2018}, date = {2018-07-17}, journal = {Anal Chem.}, volume = {90}, number = {14}, pages = {8553-8560}, abstract = {High-throughput top-down proteomic experiments directly identify proteoforms in complex mixtures, making high quality tandem mass spectra necessary to deeply characterize proteins with many sources of variation. Collision-based dissociation methods offer expedient data acquisition but often fail to extensively fragment proteoforms for thorough analysis. Electron-driven dissociation methods are a popular alternative approach, especially for precursor ions with high charge density. Combining infrared photoactivation concurrent with electron transfer dissociation (ETD) reactions, i.e., activated ion ETD (AI-ETD), can significantly improve ETD characterization of intact proteins, but benefits of AI-ETD have yet to be quantified in high-throughput top-down proteomics. Here, we report the first application of AI-ETD to LC-MS/MS characterization of intact proteins (<20 kDa), highlighting improved proteoform identification the method offers over higher energy-collisional dissociation (HCD), standard ETD, and ETD followed by supplemental HCD activation (EThcD). We identified 935 proteoforms from 295 proteins from human colorectal cancer cell line HCT116 using AI-ETD compared to 1014 proteoforms, 915 proteoforms, and 871 proteoforms with HCD, ETD, and EThcD, respectively. Importantly, AI-ETD outperformed each of the three other methods in MS/MS success rates and spectral quality metrics (e.g., sequence coverage achieved and proteoform characterization scores). In all, this four-method analysis offers the most extensive comparisons to date and demonstrates that AI-ETD both increases identifications over other ETD methods and improves proteoform characterization via higher sequence coverage, positioning it as a premier method for high-throughput top-down proteomics.}, keywords = {}, pubstate = {published}, tppubtype = {article} } High-throughput top-down proteomic experiments directly identify proteoforms in complex mixtures, making high quality tandem mass spectra necessary to deeply characterize proteins with many sources of variation. Collision-based dissociation methods offer expedient data acquisition but often fail to extensively fragment proteoforms for thorough analysis. Electron-driven dissociation methods are a popular alternative approach, especially for precursor ions with high charge density. Combining infrared photoactivation concurrent with electron transfer dissociation (ETD) reactions, i.e., activated ion ETD (AI-ETD), can significantly improve ETD characterization of intact proteins, but benefits of AI-ETD have yet to be quantified in high-throughput top-down proteomics. Here, we report the first application of AI-ETD to LC-MS/MS characterization of intact proteins (<20 kDa), highlighting improved proteoform identification the method offers over higher energy-collisional dissociation (HCD), standard ETD, and ETD followed by supplemental HCD activation (EThcD). We identified 935 proteoforms from 295 proteins from human colorectal cancer cell line HCT116 using AI-ETD compared to 1014 proteoforms, 915 proteoforms, and 871 proteoforms with HCD, ETD, and EThcD, respectively. Importantly, AI-ETD outperformed each of the three other methods in MS/MS success rates and spectral quality metrics (e.g., sequence coverage achieved and proteoform characterization scores). In all, this four-method analysis offers the most extensive comparisons to date and demonstrates that AI-ETD both increases identifications over other ETD methods and improves proteoform characterization via higher sequence coverage, positioning it as a premier method for high-throughput top-down proteomics. |
Park, Y J; Kenney, G E; Schachner, L F; Kelleher, N L; Rosenzweig, A C Repurposed HisC Aminotransferases Complete the Biosynthesis of Some Methanobactins Journal Article Biochemistry, 57 (25), pp. 3515-3523, 2018. Abstract | Links | BibTeX | Tags: @article{Park2018, title = {Repurposed HisC Aminotransferases Complete the Biosynthesis of Some Methanobactins}, author = {Park, Y.J and Kenney, G.E and Schachner, L.F and Kelleher, N.L and Rosenzweig, A.C. }, url = {https://pubs.acs.org/doi/10.1021/acs.biochem.8b00296}, doi = {10.1021/acs.biochem.8b00296}, year = {2018}, date = {2018-06-26}, journal = {Biochemistry}, volume = {57}, number = {25}, pages = {3515-3523}, abstract = {Methanobactins (Mbns) are ribosomally produced, post-translationally modified bacterial natural products with a high affinity for copper. MbnN, a pyridoxal 5'-phosphate-dependent aminotransferase, performs a transamination reaction that is the last step in the biosynthesis of Mbns produced by several Methylosinus species. Our bioinformatic analyses indicate that MbnNs likely derive from histidinol-phosphate aminotransferases (HisCs), which play a key role in histidine biosynthesis. A comparison of the HisC active site with the predicted MbnN structure suggests that MbnN's active site is altered to accommodate the larger and more hydrophobic substrates necessary for Mbn biosynthesis. Moreover, we have confirmed that MbnN is capable of catalyzing the final transamination step in Mbn biosynthesis in vitro and in vivo. We also demonstrate that without this final modification, Mbn exhibits significantly decreased stability under physiological conditions. An examination of other Mbns and Mbn operons suggests that N-terminal protection of this family of natural products is of critical importance and that several different means of N-terminal stabilization have evolved independently in Mbn subfamilies.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Methanobactins (Mbns) are ribosomally produced, post-translationally modified bacterial natural products with a high affinity for copper. MbnN, a pyridoxal 5'-phosphate-dependent aminotransferase, performs a transamination reaction that is the last step in the biosynthesis of Mbns produced by several Methylosinus species. Our bioinformatic analyses indicate that MbnNs likely derive from histidinol-phosphate aminotransferases (HisCs), which play a key role in histidine biosynthesis. A comparison of the HisC active site with the predicted MbnN structure suggests that MbnN's active site is altered to accommodate the larger and more hydrophobic substrates necessary for Mbn biosynthesis. Moreover, we have confirmed that MbnN is capable of catalyzing the final transamination step in Mbn biosynthesis in vitro and in vivo. We also demonstrate that without this final modification, Mbn exhibits significantly decreased stability under physiological conditions. An examination of other Mbns and Mbn operons suggests that N-terminal protection of this family of natural products is of critical importance and that several different means of N-terminal stabilization have evolved independently in Mbn subfamilies. |
Clevenger, K D; Ye, R; Bok, J W; Thomas, P M; Islam, M N; Miley, G P; Robey, M T; Chen, C; Yang, K; Swyers, M; Wu, E; Gao, P; Wu, C C; Keller, N P; Kelleher, N L Biochemistry, 57 (23), pp. 3237-3243, 2018. Abstract | Links | BibTeX | Tags: @article{Clevenger2018, title = {Interrogation of Benzomalvin Biosynthesis Using Fungal Artificial Chromosomes with Metabolomic Scoring (FAC-MS): Discovery of a Benzodiazepine Synthase Activity.}, author = {Clevenger, K.D. and Ye, R and Bok, J.W. and Thomas, P.M. and Islam, M.N. and Miley, G.P. and Robey, M.T. and Chen, C and Yang, K and Swyers, M and Wu, E and Gao, P and Wu, C.C. and Keller, N.P. and Kelleher, N.L.}, url = {https://pubs.acs.org/doi/10.1021/acs.biochem.8b00076}, doi = {10.1021/acs.biochem.8b00076}, year = {2018}, date = {2018-06-12}, journal = {Biochemistry}, volume = {57}, number = {23}, pages = {3237-3243}, abstract = {The benzodiazepine benzomalvin A/D is a fungally derived specialized metabolite and inhibitor of the substance P receptor NK1, biosynthesized by a three-gene nonribosomal peptide synthetase cluster. Here, we utilize fungal artificial chromosomes with metabolomic scoring (FAC-MS) to perform molecular genetic pathway dissection and targeted metabolomics analysis to assign the in vivo role of each domain in the benzomalvin biosynthetic pathway. The use of FAC-MS identified the terminal cyclizing condensation domain as BenY-CT and the internal C-domains as BenZ-C1 and BenZ-C2. Unexpectedly, we also uncovered evidence suggesting BenY-CT or a yet to be identified protein mediates benzodiazepine formation, representing the first reported benzodiazepine synthase enzymatic activity. This work informs understanding of what defines a fungal CT domain and shows how the FAC-MS platform can be used as a tool for in vivo analyses of specialized metabolite biosynthesis and for the discovery and dissection of new enzyme activities.}, keywords = {}, pubstate = {published}, tppubtype = {article} } The benzodiazepine benzomalvin A/D is a fungally derived specialized metabolite and inhibitor of the substance P receptor NK1, biosynthesized by a three-gene nonribosomal peptide synthetase cluster. Here, we utilize fungal artificial chromosomes with metabolomic scoring (FAC-MS) to perform molecular genetic pathway dissection and targeted metabolomics analysis to assign the in vivo role of each domain in the benzomalvin biosynthetic pathway. The use of FAC-MS identified the terminal cyclizing condensation domain as BenY-CT and the internal C-domains as BenZ-C1 and BenZ-C2. Unexpectedly, we also uncovered evidence suggesting BenY-CT or a yet to be identified protein mediates benzodiazepine formation, representing the first reported benzodiazepine synthase enzymatic activity. This work informs understanding of what defines a fungal CT domain and shows how the FAC-MS platform can be used as a tool for in vivo analyses of specialized metabolite biosynthesis and for the discovery and dissection of new enzyme activities. |
Seckler, H D S; Fornelli, L; Mutharasan, R K; Thaxton, C S; Fellers, R; Daviglus, M; Sniderman, A; Rader, D; Kelleher, N L; Lloyd-Jones, D M; Compton, P D; Wilkins, J T J Proteome Res., 17 (6), pp. 2156-2164, 2018. Abstract | Links | BibTeX | Tags: @article{Seckler2018, title = {A Targeted, Differential Top-Down Proteomic Methodology for Comparison of ApoA-I Proteoforms in Individuals with High and Low HDL Efflux Capacity.}, author = {Seckler, H.D.S. and Fornelli, L and Mutharasan, R.K. and Thaxton, C.S. and Fellers, R and Daviglus, M and Sniderman, A and Rader, D and Kelleher, N.L. and Lloyd-Jones, D.M. and Compton, P.D. and Wilkins, J.T.}, url = {https://pubs.acs.org/doi/10.1021/acs.jproteome.8b00100}, doi = {10.1021/acs.jproteome.8b00100}, year = {2018}, date = {2018-06-01}, journal = {J Proteome Res.}, volume = {17}, number = {6}, pages = {2156-2164}, abstract = {Top-down proteomics (TDP) allows precise determination/characterization of the different proteoforms derived from the expression of a single gene. In this study, we targeted apolipoprotein A-I (ApoA-I), a mediator of high-density-lipoprotein cholesterol efflux (HDL-E), which is inversely associated with coronary heart disease risk. Absolute ApoA-I concentration and allelic variation only partially explain interindividual HDL-E variation. Therefore, we hypothesize that differences in HDL-E are associated with the abundances of different ApoA-I proteoforms. Here, we present a targeted TDP methodology to characterize ApoA-I proteoforms in serum samples and compare their abundances between individuals. We characterized 18 ApoA-I proteoforms using selected-ion monitoring coupled to electron-transfer dissociation mass spectrometry. We then compared the abundances of these proteoforms between two groups of four participants, representing the individuals with highest and lowest HDL-E values within the Chicago Healthy Aging Study ( n = 420). Six proteoforms showed significantly ( p < 0.0005) higher intensity in high HDL-E individuals: canonical ApoA-I [fold difference (fd) = 1.17], carboxymethylated ApoA-I (fd = 1.24) and, with highest difference, four fatty acylated forms: palmitoylated (fd = 2.16), oleoylated (fd = 2.08), arachidonoylated (fd = 2.31) and one bearing two modifications: palmitoylation and truncation (fd = 2.13). These results demonstrate translational potential for targeted TDP in revealing, with high sensitivity, associations between interindividual proteoform variation and physiological differences underlying disease risk.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Top-down proteomics (TDP) allows precise determination/characterization of the different proteoforms derived from the expression of a single gene. In this study, we targeted apolipoprotein A-I (ApoA-I), a mediator of high-density-lipoprotein cholesterol efflux (HDL-E), which is inversely associated with coronary heart disease risk. Absolute ApoA-I concentration and allelic variation only partially explain interindividual HDL-E variation. Therefore, we hypothesize that differences in HDL-E are associated with the abundances of different ApoA-I proteoforms. Here, we present a targeted TDP methodology to characterize ApoA-I proteoforms in serum samples and compare their abundances between individuals. We characterized 18 ApoA-I proteoforms using selected-ion monitoring coupled to electron-transfer dissociation mass spectrometry. We then compared the abundances of these proteoforms between two groups of four participants, representing the individuals with highest and lowest HDL-E values within the Chicago Healthy Aging Study ( n = 420). Six proteoforms showed significantly ( p < 0.0005) higher intensity in high HDL-E individuals: canonical ApoA-I [fold difference (fd) = 1.17], carboxymethylated ApoA-I (fd = 1.24) and, with highest difference, four fatty acylated forms: palmitoylated (fd = 2.16), oleoylated (fd = 2.08), arachidonoylated (fd = 2.31) and one bearing two modifications: palmitoylation and truncation (fd = 2.13). These results demonstrate translational potential for targeted TDP in revealing, with high sensitivity, associations between interindividual proteoform variation and physiological differences underlying disease risk. |
Robey, M T; Ye, R; Bok, J W; Clevenger, K D; Islam, M N; Chen, C; Gupta, R; Swyers, M; Wu, E; Gao, P; Thomas, P M; Wu, C C; Keller, N P; Kelleher, N L Identification of the First Diketomorpholine Biosynthetic Pathway Using FAC-MS Technology. Journal Article ACS Chem Biol., 13 (5), pp. 1142-1147, 2018. Abstract | Links | BibTeX | Tags: @article{Robey2018, title = {Identification of the First Diketomorpholine Biosynthetic Pathway Using FAC-MS Technology.}, author = {Robey, M.T. and Ye, R and Bok, J.W. and Clevenger, K.D. and Islam, M.N. and Chen, C and Gupta, R and Swyers, M and Wu, E and Gao, P and Thomas, P.M. and Wu, C.C. and Keller, N.P. and Kelleher, N.L.}, url = {https://pubs.acs.org/doi/10.1021/acschembio.8b00024}, doi = {10.1021/acschembio.8b00024}, year = {2018}, date = {2018-05-18}, journal = {ACS Chem Biol.}, volume = {13}, number = {5}, pages = {1142-1147}, abstract = {Filamentous fungi are prolific producers of secondary metabolites with drug-like properties, and their genome sequences have revealed an untapped wealth of potential therapeutic leads. To better access these secondary metabolites and characterize their biosynthetic gene clusters, we applied a new platform for screening and heterologous expression of intact gene clusters that uses fungal artificial chromosomes and metabolomic scoring (FAC-MS). We leverage FAC-MS technology to identify the biosynthetic machinery responsible for production of acu-dioxomorpholine, a metabolite produced by the fungus, Aspergilllus aculeatus. The acu-dioxomorpholine nonribosomal peptide synthetase features a new type of condensation domain (designated CR) proposed to use a noncanonical arginine active site for ester bond formation. Using stable isotope labeling and MS, we determine that a phenyllactate monomer deriving from phenylalanine is incorporated into the diketomorpholine scaffold. Acu-dioxomorpholine is highly related to orphan inhibitors of P-glycoprotein targets in multidrug-resistant cancers, and identification of the biosynthetic pathway for this compound class enables genome mining for additional derivatives.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Filamentous fungi are prolific producers of secondary metabolites with drug-like properties, and their genome sequences have revealed an untapped wealth of potential therapeutic leads. To better access these secondary metabolites and characterize their biosynthetic gene clusters, we applied a new platform for screening and heterologous expression of intact gene clusters that uses fungal artificial chromosomes and metabolomic scoring (FAC-MS). We leverage FAC-MS technology to identify the biosynthetic machinery responsible for production of acu-dioxomorpholine, a metabolite produced by the fungus, Aspergilllus aculeatus. The acu-dioxomorpholine nonribosomal peptide synthetase features a new type of condensation domain (designated CR) proposed to use a noncanonical arginine active site for ester bond formation. Using stable isotope labeling and MS, we determine that a phenyllactate monomer deriving from phenylalanine is incorporated into the diketomorpholine scaffold. Acu-dioxomorpholine is highly related to orphan inhibitors of P-glycoprotein targets in multidrug-resistant cancers, and identification of the biosynthetic pathway for this compound class enables genome mining for additional derivatives. |
Zheng, J; L, Chen.; Skinner, O S; Ysselstein, D; Remis, J; Lansbury, P; Skerlj, R; Mrosek, M; Heunisch, U; Krapp, S; Charrow, J; Schwake, M; Kelleher, N L; Silverman, R B; Krainc, D β-Glucocerebrosidase Modulators Promote Dimerization of β-Glucocerebrosidase and Reveal an Allosteric Binding Site Journal Article J Am Chem Soc., 140 (18), pp. 5914-5924, 2018. Abstract | Links | BibTeX | Tags: @article{Zheng2018, title = {β-Glucocerebrosidase Modulators Promote Dimerization of β-Glucocerebrosidase and Reveal an Allosteric Binding Site}, author = {Zheng, J and Chen. L and Skinner, O.S and Ysselstein, D and Remis, J and Lansbury, P and Skerlj, R and Mrosek, M and Heunisch, U and Krapp, S and Charrow, J and Schwake, M and Kelleher, N.L and Silverman, R.B and Krainc, D.}, url = {https://pubs.acs.org/doi/10.1021/jacs.7b13003}, doi = {10.1021/jacs.7b13003}, year = {2018}, date = {2018-05-09}, journal = {J Am Chem Soc.}, volume = {140}, number = {18}, pages = {5914-5924}, abstract = {β-Glucocerebrosidase (GCase) mutations cause Gaucher's disease and are a high risk factor in Parkinson's disease. The implementation of a small molecule modulator is a strategy to restore proper folding and lysosome delivery of degradation-prone mutant GCase. Here, we present a potent quinazoline modulator, JZ-4109, which stabilizes wild-type and N370S mutant GCase and increases GCase abundance in patient-derived fibroblast cells. We then developed a covalent modification strategy using a lysine targeted inactivator (JZ-5029) for in vitro mechanistic studies. By using native top-down mass spectrometry, we located two potentially covalently modified lysines. We obtained the first crystal structure, at 2.2 Å resolution, of a GCase with a noniminosugar modulator covalently bound, and were able to identify the exact lysine residue modified (Lys346) and reveal an allosteric binding site. GCase dimerization was induced by our modulator binding, which was observed by native mass spectrometry, its crystal structure, and size exclusion chromatography with a multiangle light scattering detector. Finally, the dimer form was confirmed by negative staining transmission electron microscopy studies. Our newly discovered allosteric site and observed GCase dimerization provide a new mechanistic insight into GCase and its noniminosugar modulators and facilitate the rational design of novel GCase modulators for Gaucher's disease and Parkinson's disease.}, keywords = {}, pubstate = {published}, tppubtype = {article} } β-Glucocerebrosidase (GCase) mutations cause Gaucher's disease and are a high risk factor in Parkinson's disease. The implementation of a small molecule modulator is a strategy to restore proper folding and lysosome delivery of degradation-prone mutant GCase. Here, we present a potent quinazoline modulator, JZ-4109, which stabilizes wild-type and N370S mutant GCase and increases GCase abundance in patient-derived fibroblast cells. We then developed a covalent modification strategy using a lysine targeted inactivator (JZ-5029) for in vitro mechanistic studies. By using native top-down mass spectrometry, we located two potentially covalently modified lysines. We obtained the first crystal structure, at 2.2 Å resolution, of a GCase with a noniminosugar modulator covalently bound, and were able to identify the exact lysine residue modified (Lys346) and reveal an allosteric binding site. GCase dimerization was induced by our modulator binding, which was observed by native mass spectrometry, its crystal structure, and size exclusion chromatography with a multiangle light scattering detector. Finally, the dimer form was confirmed by negative staining transmission electron microscopy studies. Our newly discovered allosteric site and observed GCase dimerization provide a new mechanistic insight into GCase and its noniminosugar modulators and facilitate the rational design of novel GCase modulators for Gaucher's disease and Parkinson's disease. |
Parkinson, E I; Tryon, J H; Goering, A W; Ju, K S; McClure, R A; Kemball, J D; Zhukovsky, S; Labeda, D P; Thomson, R J; Kelleher, N L; Metcalf, W W Discovery of the Tyrobetaine Natural Products and Their Biosynthetic Gene Cluster via Metabologenomics. Journal Article ACS Chem Biol., 13 (4), pp. 1029-1037, 2018. Abstract | Links | BibTeX | Tags: @article{Parkinson2018, title = {Discovery of the Tyrobetaine Natural Products and Their Biosynthetic Gene Cluster via Metabologenomics.}, author = {Parkinson, E.I. and Tryon, J.H. and Goering, A.W. and Ju, K.S. and McClure, R.A. and Kemball, J.D. and Zhukovsky, S and Labeda, D.P. and Thomson, R.J. and Kelleher, N.L. and Metcalf, W.W.}, url = {https://pubs.acs.org/doi/10.1021/acschembio.7b01089}, doi = {10.1021/acschembio.7b01089}, year = {2018}, date = {2018-04-20}, journal = {ACS Chem Biol.}, volume = {13}, number = {4}, pages = {1029-1037}, abstract = {Natural products (NPs) are a rich source of medicines, but traditional discovery methods are often unsuccessful due to high rates of rediscovery. Genetic approaches for NP discovery are promising, but progress has been slow due to the difficulty of identifying unique biosynthetic gene clusters (BGCs) and poor gene expression. We previously developed the metabologenomics method, which combines genomic and metabolomic data to discover new NPs and their BGCs. Here, we utilize metabologenomics in combination with molecular networking to discover a novel class of NPs, the tyrobetaines: nonribosomal peptides with an unusual trimethylammonium tyrosine residue. The BGC for this unusual class of compounds was identified using metabologenomics and computational structure prediction data. Heterologous expression confirmed the BGC and suggests an unusual mechanism for trimethylammonium formation. Overall, the discovery of the tyrobetaines shows the great potential of metabologenomics combined with molecular networking and computational structure prediction for identifying interesting biosynthetic reactions and novel NPs.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Natural products (NPs) are a rich source of medicines, but traditional discovery methods are often unsuccessful due to high rates of rediscovery. Genetic approaches for NP discovery are promising, but progress has been slow due to the difficulty of identifying unique biosynthetic gene clusters (BGCs) and poor gene expression. We previously developed the metabologenomics method, which combines genomic and metabolomic data to discover new NPs and their BGCs. Here, we utilize metabologenomics in combination with molecular networking to discover a novel class of NPs, the tyrobetaines: nonribosomal peptides with an unusual trimethylammonium tyrosine residue. The BGC for this unusual class of compounds was identified using metabologenomics and computational structure prediction data. Heterologous expression confirmed the BGC and suggests an unusual mechanism for trimethylammonium formation. Overall, the discovery of the tyrobetaines shows the great potential of metabologenomics combined with molecular networking and computational structure prediction for identifying interesting biosynthetic reactions and novel NPs. |
Miley, G P; Rote, J C; Silverman, R B; Kelleher, N L; Thomson, R J Total Synthesis of Tambromycin Enabled by Indole C-H Functionalization. Journal Article Org Lett., 20 (8), pp. 2369-2373, 2018. Abstract | Links | BibTeX | Tags: @article{Miley2018, title = {Total Synthesis of Tambromycin Enabled by Indole C-H Functionalization.}, author = {Miley, G.P. and Rote, J.C. and Silverman, R.B. and Kelleher, N.L. and Thomson, R.J.}, url = {https://pubs.acs.org/doi/10.1021/acs.orglett.8b00700}, doi = {10.1021/acs.orglett.8b00700}, year = {2018}, date = {2018-04-20}, journal = {Org Lett.}, volume = {20}, number = {8}, pages = {2369-2373}, abstract = {The total synthesis of tambromycin (1), a recently isolated tetrapeptide, is reported. This unusual natural product possesses a highly modified tryptophan-derived indole fragment fused to an α-methylserine-derived oxazoline ring, and a unique noncanonical amino acid residue named tambroline (11). A convergent synthesis of tambromycin was achieved by a 13-step route that leveraged recent developments in the field of C-H functionalization to prepare the complex indole fragment, as well as an efficient synthesis of tambroline that featured a diastereoselective amination of homoproline.}, keywords = {}, pubstate = {published}, tppubtype = {article} } The total synthesis of tambromycin (1), a recently isolated tetrapeptide, is reported. This unusual natural product possesses a highly modified tryptophan-derived indole fragment fused to an α-methylserine-derived oxazoline ring, and a unique noncanonical amino acid residue named tambroline (11). A convergent synthesis of tambromycin was achieved by a 13-step route that leveraged recent developments in the field of C-H functionalization to prepare the complex indole fragment, as well as an efficient synthesis of tambroline that featured a diastereoselective amination of homoproline. |
Ntai, I; Fornelli, L; DeHart, C J; Hutton, J E; Doubleday, P F; LeDuc, R D; van Nispen, A J; Fellers, R T; Whiteley, G; Boja, E S; Rodriguez, H; Kelleher, N L Precise characterization of KRAS4b proteoforms in human colorectal cells and tumors reveals mutation/modification cross-talk. Journal Article Proc Natl Acad Sci USA, 115 (16), pp. 4140-4145, 2018. Abstract | Links | BibTeX | Tags: @article{Ntai2018, title = {Precise characterization of KRAS4b proteoforms in human colorectal cells and tumors reveals mutation/modification cross-talk.}, author = {Ntai, I and Fornelli, L and DeHart, C.J. and Hutton, J.E. and Doubleday, P.F. and LeDuc, R.D. and van Nispen, A.J. and Fellers, R.T. and Whiteley, G and Boja, E.S. and Rodriguez, H and Kelleher, N.L.}, url = {https://www.pnas.org/content/115/16/4140.long}, doi = {10.1073/pnas.1716122115}, year = {2018}, date = {2018-04-17}, journal = {Proc Natl Acad Sci USA}, volume = {115}, number = {16}, pages = {4140-4145}, abstract = {Mutations of the KRAS gene are found in human cancers with high frequency and result in the constitutive activation of its protein products. This leads to aberrant regulation of downstream pathways, promoting cell survival, proliferation, and tumorigenesis that drive cancer progression and negatively affect treatment outcomes. Here, we describe a workflow that can detect and quantify mutation-specific consequences of KRAS biochemistry, namely linked changes in posttranslational modifications (PTMs). We combined immunoaffinity enrichment with detection by top-down mass spectrometry to discover and quantify proteoforms with or without the Gly13Asp mutation (G13D) specifically in the KRAS4b isoform. The workflow was applied first to isogenic KRAS colorectal cancer (CRC) cell lines and then to patient CRC tumors with matching KRAS genotypes. In two cellular models, a direct link between the knockout of the mutant G13D allele and the complete nitrosylation of cysteine 118 of the remaining WT KRAS4b was observed. Analysis of tumor samples quantified the percentage of mutant KRAS4b actually present in cancer tissue and identified major differences in the levels of C-terminal carboxymethylation, a modification critical for membrane association. These data from CRC cells and human tumors suggest mechanisms of posttranslational regulation that are highly context-dependent and which lead to preferential production of specific KRAS4b proteoforms.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Mutations of the KRAS gene are found in human cancers with high frequency and result in the constitutive activation of its protein products. This leads to aberrant regulation of downstream pathways, promoting cell survival, proliferation, and tumorigenesis that drive cancer progression and negatively affect treatment outcomes. Here, we describe a workflow that can detect and quantify mutation-specific consequences of KRAS biochemistry, namely linked changes in posttranslational modifications (PTMs). We combined immunoaffinity enrichment with detection by top-down mass spectrometry to discover and quantify proteoforms with or without the Gly13Asp mutation (G13D) specifically in the KRAS4b isoform. The workflow was applied first to isogenic KRAS colorectal cancer (CRC) cell lines and then to patient CRC tumors with matching KRAS genotypes. In two cellular models, a direct link between the knockout of the mutant G13D allele and the complete nitrosylation of cysteine 118 of the remaining WT KRAS4b was observed. Analysis of tumor samples quantified the percentage of mutant KRAS4b actually present in cancer tissue and identified major differences in the levels of C-terminal carboxymethylation, a modification critical for membrane association. These data from CRC cells and human tumors suggest mechanisms of posttranslational regulation that are highly context-dependent and which lead to preferential production of specific KRAS4b proteoforms. |
Kenney, G E; Dassama, L M K; Pandelia, M E; Gizzi, A S; Martinie, R J; Gao, P; DeHart, C J; Schachner, L F; Skinner, O S; Ro, S Y; Zhu, X; Sadek, M; Thomas, P M; Almo, S C; Bollinger, J M; Jr Krebs, C; Kelleher, N L; Rosenzweig, AC The biosynthesis of methanobactin. Journal Article Science, 359 (6382), pp. 1411-1416, 2018. Abstract | Links | BibTeX | Tags: @article{Kenney2018, title = {The biosynthesis of methanobactin.}, author = {Kenney, G.E. and Dassama, L.M.K. and Pandelia, M.E. and Gizzi, A.S. and Martinie, R.J. and Gao, P and DeHart, C.J. and Schachner, L.F. and Skinner, O.S. and Ro, S.Y. and Zhu, X and Sadek, M and Thomas, P.M. and Almo, S.C. and Bollinger, J.M. and Jr, Krebs, C and Kelleher, N.L. and Rosenzweig, AC}, url = {https://science.sciencemag.org/content/359/6382/1411.long}, doi = {10.1126/science.aap9437}, year = {2018}, date = {2018-03-23}, journal = {Science}, volume = {359}, number = {6382}, pages = {1411-1416}, abstract = {Metal homeostasis poses a major challenge to microbes, which must acquire scarce elements for core metabolic processes. Methanobactin, an extensively modified copper-chelating peptide, was one of the earliest natural products shown to enable microbial acquisition of a metal other than iron. We describe the core biosynthetic machinery responsible for the characteristic posttranslational modifications that grant methanobactin its specificity and affinity for copper. A heterodimer comprising MbnB, a DUF692 family iron enzyme, and MbnC, a protein from a previously unknown family, performs a dioxygen-dependent four-electron oxidation of the precursor peptide (MbnA) to install an oxazolone and an adjacent thioamide, the characteristic methanobactin bidentate copper ligands. MbnB and MbnC homologs are encoded together and separately in many bacterial genomes, suggesting functions beyond their roles in methanobactin biosynthesis.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Metal homeostasis poses a major challenge to microbes, which must acquire scarce elements for core metabolic processes. Methanobactin, an extensively modified copper-chelating peptide, was one of the earliest natural products shown to enable microbial acquisition of a metal other than iron. We describe the core biosynthetic machinery responsible for the characteristic posttranslational modifications that grant methanobactin its specificity and affinity for copper. A heterodimer comprising MbnB, a DUF692 family iron enzyme, and MbnC, a protein from a previously unknown family, performs a dioxygen-dependent four-electron oxidation of the precursor peptide (MbnA) to install an oxazolone and an adjacent thioamide, the characteristic methanobactin bidentate copper ligands. MbnB and MbnC homologs are encoded together and separately in many bacterial genomes, suggesting functions beyond their roles in methanobactin biosynthesis. |
Martin, R W; Des Soye, B J; Kwon, Y C; Kay, J; Davis, R G; Thomas, P M; Majewska, N I; Chen, C X; Marcum, R D; Weiss, M G; Stoddart, A E; Amiram, M; Ranji Charna, A K; Patel, J R; Isaacs, F J; Kelleher, N L; Hong, S H; Jewett, MC Cell-free protein synthesis from genomically recoded bacteria enables multisite incorporation of noncanonical amino acids. Journal Article Nat. Commun., 9 (1), pp. 1203, 2018. Abstract | Links | BibTeX | Tags: @article{Martin2018, title = {Cell-free protein synthesis from genomically recoded bacteria enables multisite incorporation of noncanonical amino acids.}, author = {Martin, R.W. and Des Soye, B.J. and Kwon, Y.C. and Kay, J and Davis, R.G. and Thomas, P.M. and Majewska, N.I. and Chen, C.X. and Marcum, R.D. and Weiss, M.G. and Stoddart, A.E. and Amiram, M and Ranji Charna, A.K. and Patel, J.R. and Isaacs, F.J. and Kelleher, N.L. and Hong, S.H. and Jewett, MC}, url = {https://www.nature.com/articles/s41467-018-03469-5}, doi = {10.1038/s41467-018-03469-5}, year = {2018}, date = {2018-03-23}, journal = {Nat. Commun.}, volume = {9}, number = {1}, pages = {1203}, abstract = {Cell-free protein synthesis has emerged as a powerful approach for expanding the range of genetically encoded chemistry into proteins. Unfortunately, efforts to site-specifically incorporate multiple non-canonical amino acids into proteins using crude extract-based cell-free systems have been limited by release factor 1 competition. Here we address this limitation by establishing a bacterial cell-free protein synthesis platform based on genomically recoded Escherichia coli lacking release factor 1. This platform was developed by exploiting multiplex genome engineering to enhance extract performance by functionally inactivating negative effectors. Our most productive cell extracts enabled synthesis of 1,780 ± 30 mg/L superfolder green fluorescent protein. Using an optimized platform, we demonstrated the ability to introduce 40 identical p-acetyl-L-phenylalanine residues site specifically into an elastin-like polypeptide with high accuracy of incorporation ( ≥ 98%) and yield (96 ± 3 mg/L). We expect this cell-free platform to facilitate fundamental understanding and enable manufacturing paradigms for proteins with new and diverse chemistries.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Cell-free protein synthesis has emerged as a powerful approach for expanding the range of genetically encoded chemistry into proteins. Unfortunately, efforts to site-specifically incorporate multiple non-canonical amino acids into proteins using crude extract-based cell-free systems have been limited by release factor 1 competition. Here we address this limitation by establishing a bacterial cell-free protein synthesis platform based on genomically recoded Escherichia coli lacking release factor 1. This platform was developed by exploiting multiplex genome engineering to enhance extract performance by functionally inactivating negative effectors. Our most productive cell extracts enabled synthesis of 1,780 ± 30 mg/L superfolder green fluorescent protein. Using an optimized platform, we demonstrated the ability to introduce 40 identical p-acetyl-L-phenylalanine residues site specifically into an elastin-like polypeptide with high accuracy of incorporation ( ≥ 98%) and yield (96 ± 3 mg/L). We expect this cell-free platform to facilitate fundamental understanding and enable manufacturing paradigms for proteins with new and diverse chemistries. |
Davis, R G; Park, H M; Kim, K; Greer, J B; Fellers, R T; LeDuc, R D; Romanova, E V; Rubakhin, S S; Zombeck, J A; Wu, C; Yau, P M; Gao, P; van Nispen, A J; Patrie, S M; Thomas, P M; Sweedler, J V; Rhodes, J S; Kelleher, N L Top-Down Proteomics Enables Comparative Analysis of Brain Proteoforms Between Mouse Strains. Journal Article Anal Chem, 90 (6), pp. 3802-3810, 2018. Abstract | Links | BibTeX | Tags: @article{Davis2018, title = {Top-Down Proteomics Enables Comparative Analysis of Brain Proteoforms Between Mouse Strains.}, author = {Davis, R.G. and Park, H.M. and Kim, K and Greer, J.B. and Fellers, R.T. and LeDuc, R.D. and Romanova, E.V. and Rubakhin, S.S. and Zombeck, J.A. and Wu, C and Yau, P.M. and Gao, P and van Nispen, A.J. and Patrie, S.M. and Thomas, P.M. and Sweedler, J.V. and Rhodes, J.S. and Kelleher, N.L.}, url = {https://pubs.acs.org/doi/10.1021/acs.analchem.7b04108}, doi = {10.1021/acs.analchem.7b04108}, year = {2018}, date = {2018-03-20}, journal = {Anal Chem}, volume = {90}, number = {6}, pages = {3802-3810}, abstract = {Over the past decade, advances in mass spectrometry-based proteomics have accelerated brain proteome research aimed at studying the expression, dynamic modification, interaction and function of proteins in the nervous system that are associated with physiological and behavioral processes. With the latest hardware and software improvements in top-down mass spectrometry, the technology has expanded from mere protein profiling to high-throughput identification and quantification of intact proteoforms. Murine systems are broadly used as models to study human diseases. Neuroscientists specifically study the mouse brain from inbred strains to help understand how strain-specific genotype and phenotype affect development, functioning, and disease progression. This work describes the first application of label-free quantitative top-down proteomics to the analysis of the mouse brain proteome. Operating in discovery mode, we determined physiochemical differences in brain tissue from four healthy inbred strains, C57BL/6J, DBA/2J, FVB/NJ, and BALB/cByJ, after probing their intact proteome in the 3.5-30 kDa mass range. We also disseminate these findings using a new tool for top-down proteomics, TDViewer and cataloged them in a newly established Mouse Brain Proteoform Atlas. The analysis of brain tissues from the four strains identified 131 gene products leading to the full characterization of 343 of the 593 proteoforms identified. Within the results, singly and doubly phosphorylated ARPP-21 proteoforms, known to inhibit calmodulin, were differentially expressed across the four strains. Gene ontology (GO) analysis for detected differentially expressed proteoforms also helps to illuminate the similarities and dissimilarities in phenotypes among these inbred strains.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Over the past decade, advances in mass spectrometry-based proteomics have accelerated brain proteome research aimed at studying the expression, dynamic modification, interaction and function of proteins in the nervous system that are associated with physiological and behavioral processes. With the latest hardware and software improvements in top-down mass spectrometry, the technology has expanded from mere protein profiling to high-throughput identification and quantification of intact proteoforms. Murine systems are broadly used as models to study human diseases. Neuroscientists specifically study the mouse brain from inbred strains to help understand how strain-specific genotype and phenotype affect development, functioning, and disease progression. This work describes the first application of label-free quantitative top-down proteomics to the analysis of the mouse brain proteome. Operating in discovery mode, we determined physiochemical differences in brain tissue from four healthy inbred strains, C57BL/6J, DBA/2J, FVB/NJ, and BALB/cByJ, after probing their intact proteome in the 3.5-30 kDa mass range. We also disseminate these findings using a new tool for top-down proteomics, TDViewer and cataloged them in a newly established Mouse Brain Proteoform Atlas. The analysis of brain tissues from the four strains identified 131 gene products leading to the full characterization of 343 of the 593 proteoforms identified. Within the results, singly and doubly phosphorylated ARPP-21 proteoforms, known to inhibit calmodulin, were differentially expressed across the four strains. Gene ontology (GO) analysis for detected differentially expressed proteoforms also helps to illuminate the similarities and dissimilarities in phenotypes among these inbred strains. |
Smith, L M; Kelleher, N L Proteoforms as the next proteomics currency. Journal Article Science, 359 (6380), pp. 1106-1107, 2018. Abstract | Links | BibTeX | Tags: @article{Smith2018, title = {Proteoforms as the next proteomics currency.}, author = {Smith, L.M. and Kelleher, N.L.}, url = {https://science.sciencemag.org/content/359/6380/1106.long}, doi = {10.1126/science.aat1884}, year = {2018}, date = {2018-03-09}, journal = {Science}, volume = {359}, number = {6380}, pages = {1106-1107}, abstract = {Proteoforms—the different forms of proteins produced from the genome with a variety of sequence variations, splice isoforms, and myriad posttranslational modifications (1)—are critical elements in all biological systems (see the figure, left). Yang et al. (2) recently showed that the functions of proteins produced from splice variants from a given gene—different proteoforms—can be as different as those for proteins encoded by entirely different genes. Li et al. (3) showed that splice variants play a central role in modulating complex traits. However, the standard paradigm of proteomic analysis, the “bottom-up” strategy pioneered by Eng and Yates some 20 years ago (4), does not directly identify proteoforms. We argue that proteomic analysis needs to provide the identities and abundances of the proteoforms themselves, rather than just their peptide surrogates. Developing new proteome-wide strategies to accomplish this goal presents a formidable but not insurmountable technological challenge that will benefit the biomedical community.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Proteoforms—the different forms of proteins produced from the genome with a variety of sequence variations, splice isoforms, and myriad posttranslational modifications (1)—are critical elements in all biological systems (see the figure, left). Yang et al. (2) recently showed that the functions of proteins produced from splice variants from a given gene—different proteoforms—can be as different as those for proteins encoded by entirely different genes. Li et al. (3) showed that splice variants play a central role in modulating complex traits. However, the standard paradigm of proteomic analysis, the “bottom-up” strategy pioneered by Eng and Yates some 20 years ago (4), does not directly identify proteoforms. We argue that proteomic analysis needs to provide the identities and abundances of the proteoforms themselves, rather than just their peptide surrogates. Developing new proteome-wide strategies to accomplish this goal presents a formidable but not insurmountable technological challenge that will benefit the biomedical community. |
LeDuc, R; Schwammle, V; Shortreed, MR ; Cesnick, AJ ; Solntsev, SK ; Shaw, JB ; Martin, MJ ; Vizcaino, JA ; Alpi, E; Danis, P; Kelleher, NL ; Smith, LM ; Ge, Y; Agar, JN ; Chamot-Rooke, J; Loo, JA ; Pasa-Tolic, L; Tsybin, YO ProForma: A Standard Proteoform Notation. Journal Article J Proteome Res., 17 (3), pp. 1321-1325, 2018. Abstract | Links | BibTeX | Tags: @article{LeDuc2018, title = {ProForma: A Standard Proteoform Notation.}, author = {LeDuc, R and Schwammle, V and Shortreed, MR and Cesnick, AJ and Solntsev, SK and Shaw, JB and Martin, MJ and Vizcaino, JA and Alpi,E and Danis, P and Kelleher, NL and Smith, LM and Ge, Y and Agar, JN and Chamot-Rooke, J and Loo, JA and Pasa-Tolic, L and Tsybin, YO}, url = {https://pubs.acs.org/doi/10.1021/acs.jproteome.7b00851}, doi = {10.1021/acs.jproteome.7b00851}, year = {2018}, date = {2018-03-02}, journal = {J Proteome Res.}, volume = {17}, number = {3}, pages = {1321-1325}, abstract = {The Consortium for Top-Down Proteomics (CTDP) proposes a standardized notation, ProForma, for writing the sequence of fully characterized proteoforms. ProForma provides a means to communicate any proteoform by writing the amino acid sequence using standard one-letter notation and specifying modifications or unidentified mass shifts within brackets following certain amino acids. The notation is unambiguous, human-readable, and can easily be parsed and written by bioinformatic tools. This system uses seven rules and supports a wide range of possible use cases, ensuring compatibility and reproducibility of proteoform annotations. Standardizing proteoform sequences will simplify storage, comparison, and reanalysis of proteomic studies, and the Consortium welcomes input and contributions from the research community on the continued design and maintenance of this standard.}, keywords = {}, pubstate = {published}, tppubtype = {article} } The Consortium for Top-Down Proteomics (CTDP) proposes a standardized notation, ProForma, for writing the sequence of fully characterized proteoforms. ProForma provides a means to communicate any proteoform by writing the amino acid sequence using standard one-letter notation and specifying modifications or unidentified mass shifts within brackets following certain amino acids. The notation is unambiguous, human-readable, and can easily be parsed and written by bioinformatic tools. This system uses seven rules and supports a wide range of possible use cases, ensuring compatibility and reproducibility of proteoform annotations. Standardizing proteoform sequences will simplify storage, comparison, and reanalysis of proteomic studies, and the Consortium welcomes input and contributions from the research community on the continued design and maintenance of this standard. |
Aebersold, R; Agar, JN ; Amster, IJ ; Baker, MS ; Bertozzi, CR ; Boja, ES ; Costello, CE ; Cravatt, BF ; Fenselau, C; Garcia, BA ; Ge, Y; Gunawardena, J; Hendrickson, RC ; Hergenrother, PJ ; Huber, CG ; Ivanov, AR ; Jensen, ON ; Jewett, MC ; Kelleher, NL ; Kiessling, LL ; Krogan, NJ ; Larsen, MR ; Loo, JA ; Ogorzalek, Loo RR ; Lundberg, E; MacCoss, MJ ; Mallick, P; Mootha, VK ; Mrksich, M; Muir, TW ; Patrie, SM ; Pesavento, JJ ; Pitteri, SJ ; Rodriguez, H; Saghatelian, A; Sandoval, W; Schlüter, H; Sechi, S; Slavoff, SA ; Smith, LM ; Snyder, MP ; Thomas, PM ; Uhlén, M; Van Eyk, JE ; Vidal, M; Walt, DR ; White, FM ; Williams, ER ; Wohlschlager, T; Wysocki, VH ; Yates, NA ; Young, NL ; Zhang, B How many human proteoforms are there? Journal Article Nat Chem Biol., 14 (3), pp. 206-214, 2018. Abstract | Links | BibTeX | Tags: @article{Aebersold2018, title = {How many human proteoforms are there?}, author = {Aebersold, R and Agar, JN and Amster, IJ and Baker, MS and Bertozzi, CR and Boja, ES and Costello, CE and Cravatt, BF and Fenselau, C and Garcia, BA and Ge, Y and Gunawardena, J and Hendrickson, RC and Hergenrother, PJ and Huber, CG and Ivanov, AR and Jensen, ON and Jewett, MC and Kelleher, NL and Kiessling, LL and Krogan, NJ and Larsen, MR and Loo, JA and Ogorzalek, Loo RR and Lundberg, E and MacCoss, MJ and Mallick, P and Mootha, VK and Mrksich, M and Muir, TW and Patrie, SM and Pesavento, JJ and Pitteri, SJ and Rodriguez, H and Saghatelian, A and Sandoval, W and Schlüter, H and Sechi, S and Slavoff, SA and Smith, LM and Snyder, MP and Thomas, PM and Uhlén, M and Van Eyk, JE and Vidal, M and Walt, DR and White, FM and Williams, ER and Wohlschlager, T and Wysocki, VH and Yates, NA and Young, NL and Zhang, B}, url = {https://www.nature.com/articles/nchembio.2576}, doi = {10.1038/nchembio.2576}, year = {2018}, date = {2018-02-14}, journal = {Nat Chem Biol.}, volume = {14}, number = {3}, pages = {206-214}, abstract = {Despite decades of accumulated knowledge about proteins and their post-translational modifications (PTMs), numerous questions remain regarding their molecular composition and biological function. One of the most fundamental queries is the extent to which the combinations of DNA-, RNA- and PTM-level variations explode the complexity of the human proteome. Here, we outline what we know from current databases and measurement strategies including mass spectrometry-based proteomics. In doing so, we examine prevailing notions about the number of modifications displayed on human proteins and how they combine to generate the protein diversity underlying health and disease. We frame central issues regarding determination of protein-level variation and PTMs, including some paradoxes present in the field today. We use this framework to assess existing data and to ask the question, "How many distinct primary structures of proteins (proteoforms) are created from the 20,300 human genes?" We also explore prospects for improving measurements to better regularize protein-level biology and efficiently associate PTMs to function and phenotype.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Despite decades of accumulated knowledge about proteins and their post-translational modifications (PTMs), numerous questions remain regarding their molecular composition and biological function. One of the most fundamental queries is the extent to which the combinations of DNA-, RNA- and PTM-level variations explode the complexity of the human proteome. Here, we outline what we know from current databases and measurement strategies including mass spectrometry-based proteomics. In doing so, we examine prevailing notions about the number of modifications displayed on human proteins and how they combine to generate the protein diversity underlying health and disease. We frame central issues regarding determination of protein-level variation and PTMs, including some paradoxes present in the field today. We use this framework to assess existing data and to ask the question, "How many distinct primary structures of proteins (proteoforms) are created from the 20,300 human genes?" We also explore prospects for improving measurements to better regularize protein-level biology and efficiently associate PTMs to function and phenotype. |
2017 |
Harris, D; Lukoyanov, D; Shaw, S; Compton, P D; Tokmina-Lukaszewska, M; Bothner, B; Kelleher, N L; Dean, D R; Hoffman, B M; Seefeldt, L C The Mechanism of N2 Reduction Catalyzed by Fe-Nitrogenase Involves Reductive Elimination of H2 Journal Article Biochemistry, 57 (5), pp. 701-710, 2017. Abstract | Links | BibTeX | Tags: @article{Harris2017, title = {The Mechanism of N2 Reduction Catalyzed by Fe-Nitrogenase Involves Reductive Elimination of H2}, author = {D. Harris and D. Lukoyanov and S. Shaw and P.D. Compton and M. Tokmina-Lukaszewska and B. Bothner and N.L. Kelleher and D.R. Dean and B.M. Hoffman and L.C. Seefeldt}, url = {http://pubs.acs.org/doi/10.1021/acs.biochem.7b01142}, doi = {10.1021/acs.biochem.7b01142}, year = {2017}, date = {2017-12-28}, journal = {Biochemistry}, volume = {57}, number = {5}, pages = {701-710}, abstract = {Of the three forms of nitrogenase (Mo-nitrogenase, V-nitrogenase, and Fe-nitrogenase), the Fe-nitrogenase has the poorest ratio of N2 reduction relative to H2 evolution. Recent work on the Mo-nitrogenase has revealed that reductive elimination of two bridging Fe-H-Fe hydrides on the active site FeMo-cofactor to yield H2 is a key feature in the N2 reduction mechanism. The N2 reduction mechanism for the Fe-nitrogenase active site FeFe-cofactor was unknown. Here, we have purified both component proteins of the Fe-nitrogenase system, the electron-delivery Fe protein (AnfH) plus the catalytic FeFe protein (AnfDGK), and established its mechanism of N2 reduction. Inductively coupled plasma emission spectroscopy and protein mass spectrometry show that the FeFe protein component does not contain significant amounts of Mo or V, thus ruling out a requirement of these metals for N2 reduction. The fully functioning Fe-nitrogenase system was found to have specific activities for N2 reduction (1 atm) of 181 ± 5 nmol NH3 min-1 mg-1 FeFe protein, for proton reduction (in the absence of N2) of 1085 ± 41 nmol H2 min-1 mg-1 FeFe protein, and for acetylene reduction (0.3 atm) of 306 ± 3 nmol C2H4 min-1 mg-1 FeFe protein. Under turnover conditions N2 reduction is inhibited by H2 and the enzyme catalyzes the formation of HD when presented with N2 and D2. These observations are explained by the accumulation of four reducing equivalents as two metal-bound hydrides and two protons at the FeFe-cofactor, with activation for N2 reduction occurring by reductive elimination of H2.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Of the three forms of nitrogenase (Mo-nitrogenase, V-nitrogenase, and Fe-nitrogenase), the Fe-nitrogenase has the poorest ratio of N2 reduction relative to H2 evolution. Recent work on the Mo-nitrogenase has revealed that reductive elimination of two bridging Fe-H-Fe hydrides on the active site FeMo-cofactor to yield H2 is a key feature in the N2 reduction mechanism. The N2 reduction mechanism for the Fe-nitrogenase active site FeFe-cofactor was unknown. Here, we have purified both component proteins of the Fe-nitrogenase system, the electron-delivery Fe protein (AnfH) plus the catalytic FeFe protein (AnfDGK), and established its mechanism of N2 reduction. Inductively coupled plasma emission spectroscopy and protein mass spectrometry show that the FeFe protein component does not contain significant amounts of Mo or V, thus ruling out a requirement of these metals for N2 reduction. The fully functioning Fe-nitrogenase system was found to have specific activities for N2 reduction (1 atm) of 181 ± 5 nmol NH3 min-1 mg-1 FeFe protein, for proton reduction (in the absence of N2) of 1085 ± 41 nmol H2 min-1 mg-1 FeFe protein, and for acetylene reduction (0.3 atm) of 306 ± 3 nmol C2H4 min-1 mg-1 FeFe protein. Under turnover conditions N2 reduction is inhibited by H2 and the enzyme catalyzes the formation of HD when presented with N2 and D2. These observations are explained by the accumulation of four reducing equivalents as two metal-bound hydrides and two protons at the FeFe-cofactor, with activation for N2 reduction occurring by reductive elimination of H2. |
Rickels, R; Herz, H M; Sze, C C; Cao, K; Morgan, M A; Collings, C K; Gause, M; Takahashi, Y H; Wang, L; Rendleman, E J; Marshall, S A; Krueger, A; Bartom, E T; Piunti, A; Smith, E R; Abshiru, N A; Kelleher, N L; Dorsett, D; Shilatifard, A Histone H3K4 monomethylation catalyzed by Trr and mammalian COMPASS-like proteins at enhancers is dispensable for development and viability. Journal Article Nat. Genet., 49 (11), pp. 1647-1653, 2017. Abstract | Links | BibTeX | Tags: @article{Rickels2017, title = {Histone H3K4 monomethylation catalyzed by Trr and mammalian COMPASS-like proteins at enhancers is dispensable for development and viability.}, author = {Rickels, R and Herz, H.M. and Sze, C.C. and Cao, K and Morgan, M.A. and Collings, C.K. and Gause, M and Takahashi, Y.H. and Wang, L and Rendleman, E.J. and Marshall, S.A. and Krueger, A and Bartom, E.T. and Piunti, A and Smith, E.R. and Abshiru, N.A. and Kelleher, N.L. and Dorsett, D and Shilatifard, A}, url = {https://www.nature.com/articles/ng.3965}, doi = {10.1038/ng.3965}, year = {2017}, date = {2017-11-30}, journal = {Nat. Genet.}, volume = {49}, number = {11}, pages = {1647-1653}, abstract = {Histone H3 lysine 4 monomethylation (H3K4me1) is an evolutionarily conserved feature of enhancer chromatin catalyzed by the COMPASS-like methyltransferase family, which includes Trr in Drosophila melanogaster and MLL3 (encoded by KMT2C) and MLL4 (encoded by KMT2D) in mammals. Here we demonstrate that Drosophila embryos expressing catalytically deficient Trr eclose and develop to productive adulthood. Parallel experiments with a trr allele that augments enzyme product specificity show that conversion of H3K4me1 at enhancers to H3K4me2 and H3K4me3 is also compatible with life and results in minimal changes in gene expression. Similarly, loss of the catalytic SET domains of MLL3 and MLL4 in mouse embryonic stem cells (mESCs) does not disrupt self-renewal. Drosophila embryos with trr alleles encoding catalytic mutants manifest subtle developmental abnormalities when subjected to temperature stress or altered cohesin levels. Collectively, our findings suggest that animal development can occur in the context of Trr or mammalian COMPASS-like proteins deficient in H3K4 monomethylation activity and point to a possible role for H3K4me1 on cis-regulatory elements in specific settings to fine-tune transcriptional regulation in response to environmental stress.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Histone H3 lysine 4 monomethylation (H3K4me1) is an evolutionarily conserved feature of enhancer chromatin catalyzed by the COMPASS-like methyltransferase family, which includes Trr in Drosophila melanogaster and MLL3 (encoded by KMT2C) and MLL4 (encoded by KMT2D) in mammals. Here we demonstrate that Drosophila embryos expressing catalytically deficient Trr eclose and develop to productive adulthood. Parallel experiments with a trr allele that augments enzyme product specificity show that conversion of H3K4me1 at enhancers to H3K4me2 and H3K4me3 is also compatible with life and results in minimal changes in gene expression. Similarly, loss of the catalytic SET domains of MLL3 and MLL4 in mouse embryonic stem cells (mESCs) does not disrupt self-renewal. Drosophila embryos with trr alleles encoding catalytic mutants manifest subtle developmental abnormalities when subjected to temperature stress or altered cohesin levels. Collectively, our findings suggest that animal development can occur in the context of Trr or mammalian COMPASS-like proteins deficient in H3K4 monomethylation activity and point to a possible role for H3K4me1 on cis-regulatory elements in specific settings to fine-tune transcriptional regulation in response to environmental stress. |
Turcan, S; Makarov, V; Taranda, J; Wang, Y; Fabius, A W M; Wu, W; Zheng, Y; El-Amine, N; Haddock, S; Nanjangud, G; LeKaye, H C; Brennan, C; Cross, J; Huse, J T; Kelleher, N L; Osten, P; Thompson, C B; Chan, T A Mutant-IDH1-dependent chromatin state reprogramming, reversibility, and persistence. Journal Article Nat. Genet., 50 (1), pp. 62-72, 2017. Abstract | Links | BibTeX | Tags: @article{S.Turcan2017, title = {Mutant-IDH1-dependent chromatin state reprogramming, reversibility, and persistence.}, author = {Turcan, S and Makarov, V and Taranda, J and Wang, Y and Fabius, A.W.M. and Wu, W and Zheng, Y and El-Amine, N and Haddock, S and Nanjangud, G and LeKaye, H.C. and Brennan, C and Cross, J and Huse, J.T. and Kelleher, N.L. and Osten, P and Thompson, C.B. and Chan, T.A.}, url = {https://www.nature.com/articles/s41588-017-0001-z}, doi = {10.1038/s41588-017-0001-z}, year = {2017}, date = {2017-11-27}, journal = {Nat. Genet.}, volume = {50}, number = {1}, pages = {62-72}, abstract = {Mutations in IDH1 and IDH2 (encoding isocitrate dehydrogenase 1 and 2) drive the development of gliomas and other human malignancies. Mutant IDH1 induces epigenetic changes that promote tumorigenesis, but the scale and reversibility of these changes are unknown. Here, using human astrocyte and glioma tumorsphere systems, we generate a large-scale atlas of mutant-IDH1-induced epigenomic reprogramming. We characterize the reversibility of the alterations in DNA methylation, the histone landscape, and transcriptional reprogramming that occur following IDH1 mutation. We discover genome-wide coordinate changes in the localization and intensity of multiple histone marks and chromatin states. Mutant IDH1 establishes a CD24+ population with a proliferative advantage and stem-like transcriptional features. Strikingly, prolonged exposure to mutant IDH1 results in irreversible genomic and epigenetic alterations. Together, these observations provide unprecedented high-resolution molecular portraits of mutant-IDH1-dependent epigenomic reprogramming. These findings have substantial implications for understanding of mutant IDH function and for optimizing therapeutic approaches to targeting IDH-mutant tumors.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Mutations in IDH1 and IDH2 (encoding isocitrate dehydrogenase 1 and 2) drive the development of gliomas and other human malignancies. Mutant IDH1 induces epigenetic changes that promote tumorigenesis, but the scale and reversibility of these changes are unknown. Here, using human astrocyte and glioma tumorsphere systems, we generate a large-scale atlas of mutant-IDH1-induced epigenomic reprogramming. We characterize the reversibility of the alterations in DNA methylation, the histone landscape, and transcriptional reprogramming that occur following IDH1 mutation. We discover genome-wide coordinate changes in the localization and intensity of multiple histone marks and chromatin states. Mutant IDH1 establishes a CD24+ population with a proliferative advantage and stem-like transcriptional features. Strikingly, prolonged exposure to mutant IDH1 results in irreversible genomic and epigenetic alterations. Together, these observations provide unprecedented high-resolution molecular portraits of mutant-IDH1-dependent epigenomic reprogramming. These findings have substantial implications for understanding of mutant IDH function and for optimizing therapeutic approaches to targeting IDH-mutant tumors. |
Skinner, O S; Haverland, N A; Fornelli, L; Melani, R D; Vale, Do L H F; Seckler, H S; Doubleday, P F; Schachner, L F; Srzentić, K; Kelleher, N L; Compton, P D Top-down characterization of endogenous protein complexes with native proteomics. Journal Article Nat. Chem. Biol., 14 (1), pp. 36-41, 2017. Abstract | Links | BibTeX | Tags: @article{Skinner2017, title = {Top-down characterization of endogenous protein complexes with native proteomics.}, author = {O.S. Skinner and N.A. Haverland and L. Fornelli and R.D. Melani and L.H.F. Do Vale and H.S. Seckler and P.F. Doubleday and L.F. Schachner and K. Srzentić and N.L. Kelleher and P.D. Compton}, url = {https://www.nature.com/articles/nchembio.2515}, doi = {10.1038/nchembio.2515}, year = {2017}, date = {2017-11-13}, journal = {Nat. Chem. Biol.}, volume = {14}, number = {1}, pages = {36-41}, abstract = {Protein complexes exhibit great diversity in protein membership, post-translational modifications and noncovalent cofactors, enabling them to function as the actuators of many important biological processes. The exposition of these molecular features using current methods lacks either throughput or molecular specificity, ultimately limiting the use of protein complexes as direct analytical targets in a wide range of applications. Here, we apply native proteomics, enabled by a multistage tandem MS approach, to characterize 125 intact endogenous complexes and 217 distinct proteoforms derived from mouse heart and human cancer cell lines in discovery mode. The native conditions preserved soluble protein-protein interactions, high-stoichiometry noncovalent cofactors, covalent modifications to cysteines, and, remarkably, superoxide ligands bound to the metal cofactor of superoxide dismutase 2. These data enable precise compositional analysis of protein complexes as they exist in the cell and demonstrate a new approach that uses MS as a bridge to structural biology.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Protein complexes exhibit great diversity in protein membership, post-translational modifications and noncovalent cofactors, enabling them to function as the actuators of many important biological processes. The exposition of these molecular features using current methods lacks either throughput or molecular specificity, ultimately limiting the use of protein complexes as direct analytical targets in a wide range of applications. Here, we apply native proteomics, enabled by a multistage tandem MS approach, to characterize 125 intact endogenous complexes and 217 distinct proteoforms derived from mouse heart and human cancer cell lines in discovery mode. The native conditions preserved soluble protein-protein interactions, high-stoichiometry noncovalent cofactors, covalent modifications to cysteines, and, remarkably, superoxide ligands bound to the metal cofactor of superoxide dismutase 2. These data enable precise compositional analysis of protein complexes as they exist in the cell and demonstrate a new approach that uses MS as a bridge to structural biology. |
Mascarenhas, R; Le, H V; Clevenger, K D; Lehrer, H J; Ringe, D; Kelleher, N L; Silverman, R B; Liu, D Biochemistry, 56 (43), pp. 5844-5845, 2017. @article{Mascarenhas2017b, title = {Correction to Selective Targeting by a Mechanism-Based Inactivator against Pyridoxal 5'-Phosphate-Dependent Enzymes: Mechanisms of Inactivation and Alternative Turnover.}, author = {R. Mascarenhas and H.V. Le and K.D. Clevenger and H.J. Lehrer and D. Ringe and N.L. Kelleher and R.B. Silverman and D. Liu}, url = {http://pubs.acs.org/doi/10.1021/acs.biochem.7b00961}, doi = {10.1021/acs.biochem.7b00961}, year = {2017}, date = {2017-10-31}, journal = {Biochemistry}, volume = {56}, number = {43}, pages = {5844-5845}, keywords = {}, pubstate = {published}, tppubtype = {article} } |
Skinner, O S; McAnally, M O; Duyne, Van R P; Schatz, G C; Breuker, K; Compton, P D; Kelleher, N L Native Electron Capture Dissociation Maps to Iron-Binding Channels in Horse Spleen Ferritin Journal Article Anal. Chem., 89 (20), pp. 10711-10716, 2017. Abstract | Links | BibTeX | Tags: @article{Skinner2017b, title = {Native Electron Capture Dissociation Maps to Iron-Binding Channels in Horse Spleen Ferritin}, author = {O.S. Skinner and M.O. McAnally and R.P. Van Duyne and G.C. Schatz and K. Breuker and P.D. Compton and N.L. Kelleher}, url = {http://pubs.acs.org/doi/10.1021/acs.analchem.7b01581}, doi = {10.1021/acs.analchem.7b01581}, year = {2017}, date = {2017-10-17}, journal = {Anal. Chem.}, volume = {89}, number = {20}, pages = {10711-10716}, abstract = {Native electron capture dissociation (NECD) is a process during which proteins undergo fragmentation similar to that from radical dissociation methods, but without the addition of exogenous electrons. However, after three initial reports of NECD from the cytochrome c dimer complex, no further evidence of the effect has been published. Here, we report NECD behavior from horse spleen ferritin, a ∼490 kDa protein complex ∼20-fold larger than the previously studied cytochrome c dimer. Application of front-end infrared excitation (FIRE) in conjunction with low- and high-m/z quadrupole isolation and collisionally activated dissociation (CAD) provides new insights into the NECD mechanism. Additionally, activation of the intact complex in either the electrospray droplet or the gas phase produced c-type fragment ions. Similar to the previously reported results on cytochrome c, these fragment ions form near residues known to interact with iron atoms in solution. By mapping the location of backbone cleavages associated with c-type ions onto the crystal structure, we are able to characterize two distinct iron binding channels that facilitate iron ion transport into the core of the complex. The resulting pathways are in good agreement with previously reported results for iron binding sites in mammalian ferritin.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Native electron capture dissociation (NECD) is a process during which proteins undergo fragmentation similar to that from radical dissociation methods, but without the addition of exogenous electrons. However, after three initial reports of NECD from the cytochrome c dimer complex, no further evidence of the effect has been published. Here, we report NECD behavior from horse spleen ferritin, a ∼490 kDa protein complex ∼20-fold larger than the previously studied cytochrome c dimer. Application of front-end infrared excitation (FIRE) in conjunction with low- and high-m/z quadrupole isolation and collisionally activated dissociation (CAD) provides new insights into the NECD mechanism. Additionally, activation of the intact complex in either the electrospray droplet or the gas phase produced c-type fragment ions. Similar to the previously reported results on cytochrome c, these fragment ions form near residues known to interact with iron atoms in solution. By mapping the location of backbone cleavages associated with c-type ions onto the crystal structure, we are able to characterize two distinct iron binding channels that facilitate iron ion transport into the core of the complex. The resulting pathways are in good agreement with previously reported results for iron binding sites in mammalian ferritin. |
Haverland, N A; Wass, M; Ntai, I; Keppel, T; Gundry, R L; Kelleher, N L Cell Surface Proteomics of N-Linked Glycoproteins for Typing of Human Lymphocytes Journal Article Proteomics, 17 (19), 2017. Abstract | Links | BibTeX | Tags: @article{Haverland2017, title = {Cell Surface Proteomics of N-Linked Glycoproteins for Typing of Human Lymphocytes}, author = {N.A. Haverland and M. Wass and I. Ntai and T. Keppel and R.L. Gundry and N.L. Kelleher}, url = {http://onlinelibrary.wiley.com/doi/10.1002/pmic.201700156/epdf}, doi = {10.1002/pmic.201700156}, year = {2017}, date = {2017-10-17}, journal = {Proteomics}, volume = {17}, number = {19}, abstract = {Lymphocytes are immune cells that are critical for the maintenance of adaptive immunity. Differentiation of lymphoid progenitors yields B-, T-, and NK-cell subtypes that individually correlate with specific forms of leukemia or lymphoma. Therefore, it is imperative a precise method of cell categorization is utilized to detect differences in distinct disease states present in patients. One viable means of classification involves evaluation of the cell surface proteome of lymphoid malignancies. Specifically, this manuscript details the use of an antibody independent approach known as Cell Surface Capture Technology, to assess the N-glycoproteome of four human lymphocyte cell lines. Altogether, 404 cell surface N-glycoproteins were identified as markers for specific cell types involved in lymphocytic malignancies, including 82 N-glycoproteins that had not been previously been described for B or T cells within the Cell Surface Protein Atlas. Comparative analysis, hierarchical clustering techniques, and label-free quantitation were used to reveal proteins most informative for each cell type. Undoubtedly, the characterization of the cell surface proteome of lymphoid malignancies is a first step toward improving personalized diagnosis and treatment of leukemia and lymphoma.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Lymphocytes are immune cells that are critical for the maintenance of adaptive immunity. Differentiation of lymphoid progenitors yields B-, T-, and NK-cell subtypes that individually correlate with specific forms of leukemia or lymphoma. Therefore, it is imperative a precise method of cell categorization is utilized to detect differences in distinct disease states present in patients. One viable means of classification involves evaluation of the cell surface proteome of lymphoid malignancies. Specifically, this manuscript details the use of an antibody independent approach known as Cell Surface Capture Technology, to assess the N-glycoproteome of four human lymphocyte cell lines. Altogether, 404 cell surface N-glycoproteins were identified as markers for specific cell types involved in lymphocytic malignancies, including 82 N-glycoproteins that had not been previously been described for B or T cells within the Cell Surface Protein Atlas. Comparative analysis, hierarchical clustering techniques, and label-free quantitation were used to reveal proteins most informative for each cell type. Undoubtedly, the characterization of the cell surface proteome of lymphoid malignancies is a first step toward improving personalized diagnosis and treatment of leukemia and lymphoma. |
Ezponda, T; Dupéré-Richer, D; Will, C M; Small, E C; Varghese, N; Patel, T; Nabet, B; Popovic, R; Oyer, J; Bulic, M; Zheng, Y; Huang, X; Shah, M Y; Maji, S; Riva, A; Occhionorelli, M; Tonon, G; Kelleher, N; Keats, J; Licht, J D UTX/KDM6A Loss Enhances the Malignant Phenotype of Multiple Myeloma and Sensitizes Cells to EZH2 inhibition Journal Article Cell. Rep., 21 (3), pp. 628-640, 2017. Abstract | Links | BibTeX | Tags: @article{Ezponda2017, title = {UTX/KDM6A Loss Enhances the Malignant Phenotype of Multiple Myeloma and Sensitizes Cells to EZH2 inhibition}, author = {T. Ezponda and D. Dupéré-Richer and C.M. Will and E.C. Small and N. Varghese and T. Patel and B. Nabet and R. Popovic and J. Oyer and M. Bulic and Y. Zheng and X. Huang and M.Y. Shah and S. Maji and A. Riva and M. Occhionorelli and G. Tonon and N. Kelleher and J. Keats and J.D. Licht }, url = {http://www.sciencedirect.com/science/article/pii/S2211124717313839?via%3Dihub}, doi = {10.1016/j.celrep.2017.09.078}, year = {2017}, date = {2017-10-17}, journal = {Cell. Rep.}, volume = {21}, number = {3}, pages = {628-640}, abstract = {Loss or inactivation of the histone H3K27 demethylase UTX occurs in several malignancies, including multiple myeloma (MM). Using an isogenic cell system, we found that loss of UTX leads to deactivation of gene expression ultimately promoting the proliferation, clonogenicity, adhesion, and tumorigenicity of MM cells. Moreover, UTX mutant cells showed increased in vitro and in vivo sensitivity to inhibition of EZH2, a histone methyltransferase that generates H3K27me3. Such sensitivity was related to a decrease in the levels of IRF4 and c-MYC and an activation of repressors of IRF4 characteristic of germinal center B cells such as BCL6 and IRF1. Rebalance of H3K27me3 levels at specific genes through EZH2 inhibitors may be a therapeutic strategy in MM cases harboring UTX mutations.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Loss or inactivation of the histone H3K27 demethylase UTX occurs in several malignancies, including multiple myeloma (MM). Using an isogenic cell system, we found that loss of UTX leads to deactivation of gene expression ultimately promoting the proliferation, clonogenicity, adhesion, and tumorigenicity of MM cells. Moreover, UTX mutant cells showed increased in vitro and in vivo sensitivity to inhibition of EZH2, a histone methyltransferase that generates H3K27me3. Such sensitivity was related to a decrease in the levels of IRF4 and c-MYC and an activation of repressors of IRF4 characteristic of germinal center B cells such as BCL6 and IRF1. Rebalance of H3K27me3 levels at specific genes through EZH2 inhibitors may be a therapeutic strategy in MM cases harboring UTX mutations. |
Zha, L; Jiang, Y; Henke, M T; Wilson, M R; Wang, J X; Kelleher, N L; Balskus, E P Colibactin assembly line enzymes use S-adenosylmethionine to build a cyclopropane ring. Journal Article Nat Chem Biol, 13 (10), pp. 1063-1065, 2017. Abstract | Links | BibTeX | Tags: @article{Zha2017, title = {Colibactin assembly line enzymes use S-adenosylmethionine to build a cyclopropane ring.}, author = {L. Zha and Y. Jiang and M.T. Henke and M.R. Wilson and J.X. Wang and N.L. Kelleher and E.P. Balskus}, url = {http://dx.doi.org/10.1038/nchembio.2448}, doi = {10.1038/nchembio.2448}, year = {2017}, date = {2017-10-01}, journal = {Nat Chem Biol}, volume = {13}, number = {10}, pages = {1063-1065}, abstract = {Despite containing an α-amino acid, the versatile cofactor S-adenosylmethionine (SAM) is not a known building block for nonribosomal peptide synthetase (NRPS) assembly lines. Here we report an unusual NRPS module from colibactin biosynthesis that uses SAM for amide bond formation and subsequent cyclopropanation. Our findings showcase a new use for SAM and reveal a novel biosynthetic route to a functional group that likely mediates colibactin's genotoxicity.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Despite containing an α-amino acid, the versatile cofactor S-adenosylmethionine (SAM) is not a known building block for nonribosomal peptide synthetase (NRPS) assembly lines. Here we report an unusual NRPS module from colibactin biosynthesis that uses SAM for amide bond formation and subsequent cyclopropanation. Our findings showcase a new use for SAM and reveal a novel biosynthetic route to a functional group that likely mediates colibactin's genotoxicity. |
Morgan, M A J; Rickels, R A; Collings, C K; He, X; Cao, K; Herz, H M; Cozzolino, K A; Abshiru, N A; Marshall, S A; Rendleman, E J; Sze, C C; Piunti, A; Kelleher, N L; Savas, J N; Shilatifard, A A cryptic Tudor domain links BRWD2/PHIP to COMPASS-mediated histone H3K4 methylation. Journal Article Genes. Dev., 19 , pp. 2003-2014, 2017. Abstract | Links | BibTeX | Tags: @article{Morgan2017, title = {A cryptic Tudor domain links BRWD2/PHIP to COMPASS-mediated histone H3K4 methylation.}, author = {M.A.J. Morgan and R.A. Rickels and C.K. Collings and X. He and K. Cao and H.M. Herz and K.A. Cozzolino and N.A. Abshiru and S.A. Marshall and E.J. Rendleman and C.C. Sze and A. Piunti and N.L. Kelleher and J.N. Savas and A. Shilatifard}, url = {http://genesdev.cshlp.org/content/31/19/2003.long}, doi = {10.1101/gad.305201.117.}, year = {2017}, date = {2017-10-01}, journal = {Genes. Dev.}, volume = {19}, pages = {2003-2014}, abstract = {Histone H3 Lys4 (H3K4) methylation is a chromatin feature enriched at gene cis-regulatory sequences such as promoters and enhancers. Here we identify an evolutionarily conserved factor, BRWD2/PHIP, which colocalizes with histone H3K4 methylation genome-wide in human cells, mouse embryonic stem cells, and Drosophila Biochemical analysis of BRWD2 demonstrated an association with the Cullin-4-RING ubiquitin E3 ligase-4 (CRL4) complex, nucleosomes, and chromatin remodelers. BRWD2/PHIP binds directly to H3K4 methylation through a previously unidentified chromatin-binding module related to Royal Family Tudor domains, which we named the CryptoTudor domain. Using CRISPR-Cas9 genetic knockouts, we demonstrate that COMPASS H3K4 methyltransferase family members differentially regulate BRWD2/PHIP chromatin occupancy. Finally, we demonstrate that depletion of the single Drosophila homolog dBRWD3 results in altered gene expression and aberrant patterns of histone H3 Lys27 acetylation at enhancers and promoters, suggesting a cross-talk between these chromatin modifications and transcription through the BRWD protein family.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Histone H3 Lys4 (H3K4) methylation is a chromatin feature enriched at gene cis-regulatory sequences such as promoters and enhancers. Here we identify an evolutionarily conserved factor, BRWD2/PHIP, which colocalizes with histone H3K4 methylation genome-wide in human cells, mouse embryonic stem cells, and Drosophila Biochemical analysis of BRWD2 demonstrated an association with the Cullin-4-RING ubiquitin E3 ligase-4 (CRL4) complex, nucleosomes, and chromatin remodelers. BRWD2/PHIP binds directly to H3K4 methylation through a previously unidentified chromatin-binding module related to Royal Family Tudor domains, which we named the CryptoTudor domain. Using CRISPR-Cas9 genetic knockouts, we demonstrate that COMPASS H3K4 methyltransferase family members differentially regulate BRWD2/PHIP chromatin occupancy. Finally, we demonstrate that depletion of the single Drosophila homolog dBRWD3 results in altered gene expression and aberrant patterns of histone H3 Lys27 acetylation at enhancers and promoters, suggesting a cross-talk between these chromatin modifications and transcription through the BRWD protein family. |
Ichikawa, Y; Connelly, C F; Appleboim, A; Miller, T C; Jacobi, H; Abshiru, N A; Chou, H J; Chen, Y; Sharma, U; Zheng, Y; Thomas, P M; Chen, H V; Bajaj, V; Müller, C W; Kelleher, N L; Friedman, N; Bolon, D N; Rando, O J; Kaufman, P D A synthetic biology approach to probing nucleosome symmetry Journal Article eLife, (6), 2017. Abstract | Links | BibTeX | Tags: @article{Ichikawa2017, title = {A synthetic biology approach to probing nucleosome symmetry}, author = {Y. Ichikawa and C.F. Connelly and A. Appleboim and T.C. Miller and H. Jacobi and N.A. Abshiru and H.J. Chou and Y. Chen and U. Sharma and Y. Zheng and P.M. Thomas and H.V. Chen and V. Bajaj and C.W. Müller and N.L. Kelleher and N. Friedman and D.N. Bolon and O.J. Rando and P.D. Kaufman}, url = {https://elifesciences.org/articles/28836}, doi = {10.7554/eLife.28836.}, year = {2017}, date = {2017-09-12}, journal = {eLife}, number = {6}, abstract = {The repeating subunit of chromatin, the nucleosome, includes two copies of each of the four core histones, and several recent studies have reported that asymmetrically-modified nucleosomes occur at regulatory elements in vivo. To probe the mechanisms by which histone modifications are read out, we designed an obligate pair of H3 heterodimers, termed H3X and H3Y, which we extensively validated genetically and biochemically. Comparing the effects of asymmetric histone tail point mutants with those of symmetric double mutants revealed that a single methylated H3K36 per nucleosome was sufficient to silence cryptic transcription in vivo. We also demonstrate the utility of this system for analysis of histone modification crosstalk, using mass spectrometry to separately identify modifications on each H3 molecule within asymmetric nucleosomes. The ability to generate asymmetric nucleosomes in vivo and in vitro provides a powerful and generalizable tool to probe the mechanisms by which H3 tails are read out by effector proteins in the cell.}, keywords = {}, pubstate = {published}, tppubtype = {article} } The repeating subunit of chromatin, the nucleosome, includes two copies of each of the four core histones, and several recent studies have reported that asymmetrically-modified nucleosomes occur at regulatory elements in vivo. To probe the mechanisms by which histone modifications are read out, we designed an obligate pair of H3 heterodimers, termed H3X and H3Y, which we extensively validated genetically and biochemically. Comparing the effects of asymmetric histone tail point mutants with those of symmetric double mutants revealed that a single methylated H3K36 per nucleosome was sufficient to silence cryptic transcription in vivo. We also demonstrate the utility of this system for analysis of histone modification crosstalk, using mass spectrometry to separately identify modifications on each H3 molecule within asymmetric nucleosomes. The ability to generate asymmetric nucleosomes in vivo and in vitro provides a powerful and generalizable tool to probe the mechanisms by which H3 tails are read out by effector proteins in the cell. |
Mascarenhas, R; Le, H V; Clevenger, K D; Lehrer, H J; Ringe, D; Kelleher, N L; Silverman, R B; Liu, D Biochemistry, 56 (37), pp. 4951-4961, 2017. Abstract | Links | BibTeX | Tags: @article{Mascarenhas2017, title = {Selective Targeting by a Mechanism-Based Inactivator against Pyridoxal 5'-Phosphate-Dependent Enzymes: Mechanisms of Inactivation and Alternative Turnover.}, author = {R. Mascarenhas and H.V. Le and K.D. Clevenger and H.J. Lehrer and D. Ringe and N.L. Kelleher and R.B. Silverman and D. Liu}, url = {https://dx.doi.org/10.1021/acs.biochem.7b00499}, doi = {10.1021/acs.biochem.7b00499}, year = {2017}, date = {2017-09-06}, journal = {Biochemistry}, volume = {56}, number = {37}, pages = {4951-4961}, abstract = {Potent mechanism-based inactivators can be rationally designed against pyridoxal 5'-phosphate (PLP)-dependent drug targets, such as ornithine aminotransferase (OAT) or γ-aminobutyric acid aminotransferase (GABA-AT). An important challenge, however, is the lack of selectivity toward other PLP-dependent, off-target enzymes, because of similarities in mechanisms of all PLP-dependent aminotransferase reactions. On the basis of complex crystal structures, we investigate the inactivation mechanism of OAT, a hepatocellular carcinoma target, by (1R,3S,4S)-3-amino-4-fluorocyclopentane-1-carboxylic acid (FCP), a known inactivator of GABA-AT. A crystal structure of OAT and FCP showed the formation of a ternary adduct. This adduct can be rationalized as occurring via an enamine mechanism of inactivation, similar to that reported for GABA-AT. However, the crystal structure of an off-target, PLP-dependent enzyme, aspartate aminotransferase (Asp-AT), in complex with FCP, along with the results of attempted inhibition assays, suggests that FCP is not an inactivator of Asp-AT, but rather an alternate substrate. Turnover of FCP by Asp-AT is also supported by high-resolution mass spectrometry. Amid existing difficulties in achieving selectivity of inactivation among a large number of PLP-dependent enzymes, the obtained results provide evidence that a desirable selectivity could be achieved, taking advantage of subtle structural and mechanistic differences between a drug-target enzyme and an off-target enzyme, despite their largely similar substrate binding sites and catalytic mechanisms.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Potent mechanism-based inactivators can be rationally designed against pyridoxal 5'-phosphate (PLP)-dependent drug targets, such as ornithine aminotransferase (OAT) or γ-aminobutyric acid aminotransferase (GABA-AT). An important challenge, however, is the lack of selectivity toward other PLP-dependent, off-target enzymes, because of similarities in mechanisms of all PLP-dependent aminotransferase reactions. On the basis of complex crystal structures, we investigate the inactivation mechanism of OAT, a hepatocellular carcinoma target, by (1R,3S,4S)-3-amino-4-fluorocyclopentane-1-carboxylic acid (FCP), a known inactivator of GABA-AT. A crystal structure of OAT and FCP showed the formation of a ternary adduct. This adduct can be rationalized as occurring via an enamine mechanism of inactivation, similar to that reported for GABA-AT. However, the crystal structure of an off-target, PLP-dependent enzyme, aspartate aminotransferase (Asp-AT), in complex with FCP, along with the results of attempted inhibition assays, suggests that FCP is not an inactivator of Asp-AT, but rather an alternate substrate. Turnover of FCP by Asp-AT is also supported by high-resolution mass spectrometry. Amid existing difficulties in achieving selectivity of inactivation among a large number of PLP-dependent enzymes, the obtained results provide evidence that a desirable selectivity could be achieved, taking advantage of subtle structural and mechanistic differences between a drug-target enzyme and an off-target enzyme, despite their largely similar substrate binding sites and catalytic mechanisms. |
Wildburger, N C; Esparza, T J; LeDuc, R D; Fellers, R T; Thomas, P M; Cairns, N J; Kelleher, N L; Bateman, R J; Brody, D L Diversity of Amyloid-beta Proteoforms in the Alzheimer’s Disease Brain Journal Article Sci. Rep., 7 (1), pp. 9520, 2017. Abstract | Links | BibTeX | Tags: @article{Wildburger2017, title = {Diversity of Amyloid-beta Proteoforms in the Alzheimer’s Disease Brain}, author = {N.C. Wildburger and T.J. Esparza and R.D. LeDuc and R.T. Fellers and P.M. Thomas and N.J. Cairns and N.L. Kelleher and R.J. Bateman and D.L. Brody}, url = {https://www.nature.com/articles/s41598-017-10422-x}, doi = {10.1038/s41598-017-10422-x}, year = {2017}, date = {2017-08-25}, journal = {Sci. Rep.}, volume = {7}, number = {1}, pages = {9520}, abstract = {Amyloid-beta (Aβ) plays a key role in the pathogenesis of Alzheimer's disease (AD), but little is known about the proteoforms present in AD brain. We used high-resolution mass spectrometry to analyze intact Aβ from soluble aggregates and insoluble material in brains of six cases with severe dementia and pathologically confirmed AD. The soluble aggregates are especially relevant because they are believed to be the most toxic form of Aβ. We found a diversity of Aβ peptides, with 26 unique proteoforms including various N- and C-terminal truncations. N- and C-terminal truncations comprised 73% and 30%, respectively, of the total Aβ proteoforms detected. The Aβ proteoforms segregated between the soluble and more insoluble aggregates with N-terminal truncations predominating in the insoluble material and C- terminal truncations segregating into the soluble aggregates. In contrast, canonical Aβ comprised the minority of the identified proteoforms (15.3%) and did not distinguish between the soluble and more insoluble aggregates. The relative abundance of many truncated Aβ proteoforms did not correlate with post-mortem interval, suggesting they are not artefacts. This heterogeneity of Aβ proteoforms deepens our understanding of AD and offers many new avenues for investigation into pathological mechanisms of the disease, with implications for therapeutic development.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Amyloid-beta (Aβ) plays a key role in the pathogenesis of Alzheimer's disease (AD), but little is known about the proteoforms present in AD brain. We used high-resolution mass spectrometry to analyze intact Aβ from soluble aggregates and insoluble material in brains of six cases with severe dementia and pathologically confirmed AD. The soluble aggregates are especially relevant because they are believed to be the most toxic form of Aβ. We found a diversity of Aβ peptides, with 26 unique proteoforms including various N- and C-terminal truncations. N- and C-terminal truncations comprised 73% and 30%, respectively, of the total Aβ proteoforms detected. The Aβ proteoforms segregated between the soluble and more insoluble aggregates with N-terminal truncations predominating in the insoluble material and C- terminal truncations segregating into the soluble aggregates. In contrast, canonical Aβ comprised the minority of the identified proteoforms (15.3%) and did not distinguish between the soluble and more insoluble aggregates. The relative abundance of many truncated Aβ proteoforms did not correlate with post-mortem interval, suggesting they are not artefacts. This heterogeneity of Aβ proteoforms deepens our understanding of AD and offers many new avenues for investigation into pathological mechanisms of the disease, with implications for therapeutic development. |
Zhou, Y; Zhang, X; Fornelli, L; Compton, P D; Kelleher, N; Wirth, M J Chromatographic efficiency and selectivity in top-down proteomics of histones. Journal Article J Chromatogr B Analyt Technol Biomed Life Sci, pp. 47-53, 2017. Abstract | Links | BibTeX | Tags: @article{Zhou2017, title = {Chromatographic efficiency and selectivity in top-down proteomics of histones.}, author = {Y. Zhou and X. Zhang and L. Fornelli and P.D. Compton and N. Kelleher and M.J. Wirth}, url = {http://www.sciencedirect.com/science/article/pii/S1570023216310972?via%3Dihub}, doi = {10.1016/j.jchromb.2016.12.027}, year = {2017}, date = {2017-02-15}, journal = {J Chromatogr B Analyt Technol Biomed Life Sci}, pages = {47-53}, abstract = {Histones are involved in epigenetic control of a wide variety of cellular processes through their multiple post-translational modifications. Their strongly cationic nature makes them challenging to separate with reversed-phase liquid chromatography coupled to mass spectrometry (RPLC-MS), where trifluoroacetic acid is avoided due to adduct formation. Columns with higher resolution are needed. In this work, RPLC-MS is performed on a histone sample using difluoroacetic acid and a 20-min gradient. Columns with C18 surfaces are compared for two different types of particle morphologies: 1) fully porous particles of 5μm in diameter, 2) superficially porous particles of 3μm in diameter with a shell of 0.2μm. The resolution for the histone separation is better for the latter column, but only when the modifier is trifluoroacetic acid, which is used with UV absorbance detection. When difluoroacetic acid is used for LCMS, the peaks broaden enough to erase the advantage in efficiency for the superficially porous particles. The fully porous and superficially porous cases show similar performance in RPLC-MS, with slightly higher resolution for the fully porous particles. The expected advantage of the shorter diffusion distances for the superficially porous particles is shown to be outweighed by the lower selectivity of its bonded phase.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Histones are involved in epigenetic control of a wide variety of cellular processes through their multiple post-translational modifications. Their strongly cationic nature makes them challenging to separate with reversed-phase liquid chromatography coupled to mass spectrometry (RPLC-MS), where trifluoroacetic acid is avoided due to adduct formation. Columns with higher resolution are needed. In this work, RPLC-MS is performed on a histone sample using difluoroacetic acid and a 20-min gradient. Columns with C18 surfaces are compared for two different types of particle morphologies: 1) fully porous particles of 5μm in diameter, 2) superficially porous particles of 3μm in diameter with a shell of 0.2μm. The resolution for the histone separation is better for the latter column, but only when the modifier is trifluoroacetic acid, which is used with UV absorbance detection. When difluoroacetic acid is used for LCMS, the peaks broaden enough to erase the advantage in efficiency for the superficially porous particles. The fully porous and superficially porous cases show similar performance in RPLC-MS, with slightly higher resolution for the fully porous particles. The expected advantage of the shorter diffusion distances for the superficially porous particles is shown to be outweighed by the lower selectivity of its bonded phase. |
Anderson, L C; DeHart, C J; Kaiser, N K; Fellers, R T; Smith, D F; Greer, J B; LeDuc, R D; Blakney, G T; Thomas, P M; Kelleher, N L; Hendrickson, C L Identification and Characterization of Human Proteoforms by Top-Down LC-21 Tesla FT-ICR Mass Spectrometry Journal Article J Proteome Res, 16 (2), pp. 1087-1096, 2017, ISSN: 1535-3907 (Electronic) 1535-3893 (Linking). @article{RN469, title = {Identification and Characterization of Human Proteoforms by Top-Down LC-21 Tesla FT-ICR Mass Spectrometry}, author = { L. C. Anderson and C. J. DeHart and N. K. Kaiser and R. T. Fellers and D. F. Smith and J. B. Greer and R. D. LeDuc and G. T. Blakney and P. M. Thomas and N. L. Kelleher and C. L. Hendrickson}, url = {http://www.ncbi.nlm.nih.gov/pubmed/27936753}, doi = {10.1021/acs.jproteome.6b00696}, issn = {1535-3907 (Electronic) 1535-3893 (Linking)}, year = {2017}, date = {2017-01-01}, journal = {J Proteome Res}, volume = {16}, number = {2}, pages = {1087-1096}, keywords = {}, pubstate = {published}, tppubtype = {article} } |
Cleland, T P; DeHart, C J; Fellers, R T; VanNispen, A J; Greer, J B; LeDuc, R D; Parker, W R; Thomas, P M; Kelleher, N L; Brodbelt, J S High-Throughput Analysis of Intact Human Proteins Using UVPD and HCD on an Orbitrap Mass Spectrometer Journal Article J Proteome Res, 16 (5), pp. 2072-2079, 2017, ISSN: 1535-3907 (Electronic) 1535-3893 (Linking). @article{RN458, title = {High-Throughput Analysis of Intact Human Proteins Using UVPD and HCD on an Orbitrap Mass Spectrometer}, author = { T. P. Cleland and C. J. DeHart and R. T. Fellers and A. J. VanNispen and J. B. Greer and R. D. LeDuc and W. R. Parker and P. M. Thomas and N. L. Kelleher and J. S. Brodbelt}, url = {http://www.ncbi.nlm.nih.gov/pubmed/28412815}, doi = {10.1021/acs.jproteome.7b00043}, issn = {1535-3907 (Electronic) 1535-3893 (Linking)}, year = {2017}, date = {2017-01-01}, journal = {J Proteome Res}, volume = {16}, number = {5}, pages = {2072-2079}, keywords = {}, pubstate = {published}, tppubtype = {article} } |
Clevenger, K D; Bok, J W; Ye, R; Miley, G P; Verdan, M H; Velk, T; Chen, C; Yang, K; Robey, M T; Gao, P; Lamprecht, M; Thomas, P M; Islam, M N; Palmer, J M; Wu, C C; Keller, N P; Kelleher, N L A scalable platform to identify fungal secondary metabolites and their gene clusters Journal Article Nat Chem Biol, 2017, ISSN: 1552-4469 (Electronic) 1552-4450 (Linking). @article{RN456, title = {A scalable platform to identify fungal secondary metabolites and their gene clusters}, author = { K. D. Clevenger and J. W. Bok and R. Ye and G. P. Miley and M. H. Verdan and T. Velk and C. Chen and K. Yang and M. T. Robey and P. Gao and M. Lamprecht and P. M. Thomas and M. N. Islam and J. M. Palmer and C. C. Wu and N. P. Keller and N. L. Kelleher}, url = {http://www.ncbi.nlm.nih.gov/pubmed/28604695}, doi = {10.1038/nchembio.2408}, issn = {1552-4469 (Electronic) 1552-4450 (Linking)}, year = {2017}, date = {2017-01-01}, journal = {Nat Chem Biol}, keywords = {}, pubstate = {published}, tppubtype = {article} } |
Clevenger, K D; Mascarenhas, R; Catlin, D; Wu, R; Kelleher, N L; Drake, E J; Gulick, A M; Liu, D; Fast, W Substrate Trapping in the Siderophore Tailoring Enzyme PvdQ Journal Article ACS Chem Biol, 12 (3), pp. 643-647, 2017, ISSN: 1554-8937 (Electronic) 1554-8929 (Linking). @article{RN464, title = {Substrate Trapping in the Siderophore Tailoring Enzyme PvdQ}, author = { K. D. Clevenger and R. Mascarenhas and D. Catlin and R. Wu and N. L. Kelleher and E. J. Drake and A. M. Gulick and D. Liu and W. Fast}, url = {http://www.ncbi.nlm.nih.gov/pubmed/28186406}, doi = {10.1021/acschembio.7b00031}, issn = {1554-8937 (Electronic) 1554-8929 (Linking)}, year = {2017}, date = {2017-01-01}, journal = {ACS Chem Biol}, volume = {12}, number = {3}, pages = {643-647}, keywords = {}, pubstate = {published}, tppubtype = {article} } |
DeHart, C J; Fellers, R T; Fornelli, L; Kelleher, N L; Thomas, P M Bioinformatics Analysis of Top-Down Mass Spectrometry Data with ProSight Lite Journal Article Methods Mol Biol, 1558 , pp. 381-394, 2017, ISSN: 1940-6029 (Electronic) 1064-3745 (Linking). @article{RN466, title = {Bioinformatics Analysis of Top-Down Mass Spectrometry Data with ProSight Lite}, author = { C. J. DeHart and R. T. Fellers and L. Fornelli and N. L. Kelleher and P. M. Thomas}, url = {http://www.ncbi.nlm.nih.gov/pubmed/28150248}, doi = {10.1007/978-1-4939-6783-4_18}, issn = {1940-6029 (Electronic) 1064-3745 (Linking)}, year = {2017}, date = {2017-01-01}, journal = {Methods Mol Biol}, volume = {1558}, pages = {381-394}, keywords = {}, pubstate = {published}, tppubtype = {article} } |
Fornelli, L; Durbin, K R; Fellers, R T; Early, B P; Greer, J B; LeDuc, R D; Compton, P D; Kelleher, N L Advancing Top-down Analysis of the Human Proteome Using a Benchtop Quadrupole-Orbitrap Mass Spectrometer Journal Article J Proteome Res, 16 (2), pp. 609-618, 2017, ISSN: 1535-3907 (Electronic) 1535-3893 (Linking). @article{RN465, title = {Advancing Top-down Analysis of the Human Proteome Using a Benchtop Quadrupole-Orbitrap Mass Spectrometer}, author = { L. Fornelli and K. R. Durbin and R. T. Fellers and B. P. Early and J. B. Greer and R. D. LeDuc and P. D. Compton and N. L. Kelleher}, url = {http://www.ncbi.nlm.nih.gov/pubmed/28152595}, doi = {10.1021/acs.jproteome.6b00698}, issn = {1535-3907 (Electronic) 1535-3893 (Linking)}, year = {2017}, date = {2017-01-01}, journal = {J Proteome Res}, volume = {16}, number = {2}, pages = {609-618}, keywords = {}, pubstate = {published}, tppubtype = {article} } |
Fornelli, L; Toby, T K; Schachner, L F; Doubleday, P F; Srzentic, K; DeHart, C J; Kelleher, N L Top-down proteomics: Where we are, where we are going? Journal Article J Proteomics, 2017, ISSN: 1876-7737 (Electronic) 1874-3919 (Linking). @article{RN463, title = {Top-down proteomics: Where we are, where we are going?}, author = { L. Fornelli and T. K. Toby and L. F. Schachner and P. F. Doubleday and K. Srzentic and C. J. DeHart and N. L. Kelleher}, url = {http://www.ncbi.nlm.nih.gov/pubmed/28188863}, doi = {10.1016/j.jprot.2017.02.002}, issn = {1876-7737 (Electronic) 1874-3919 (Linking)}, year = {2017}, date = {2017-01-01}, journal = {J Proteomics}, keywords = {}, pubstate = {published}, tppubtype = {article} } |
Gan, R; Perez, J G; Carlson, E D; Ntai, I; Isaacs, F J; Kelleher, N L; Jewett, M C Translation system engineering in Escherichia coli enhances non-canonical amino acid incorporation into proteins Journal Article Biotechnol Bioeng, 114 (5), pp. 1074-1086, 2017, ISSN: 1097-0290 (Electronic) 0006-3592 (Linking). @article{RN468, title = {Translation system engineering in Escherichia coli enhances non-canonical amino acid incorporation into proteins}, author = { R. Gan and J. G. Perez and E. D. Carlson and I. Ntai and F. J. Isaacs and N. L. Kelleher and M. C. Jewett}, url = {http://www.ncbi.nlm.nih.gov/pubmed/27987323}, doi = {10.1002/bit.26239}, issn = {1097-0290 (Electronic) 0006-3592 (Linking)}, year = {2017}, date = {2017-01-01}, journal = {Biotechnol Bioeng}, volume = {114}, number = {5}, pages = {1074-1086}, keywords = {}, pubstate = {published}, tppubtype = {article} } |
Goering, A W; Li, J; McClure, R A; Thomson, R J; Jewett, M C; Kelleher, N L In Vitro Reconstruction of Nonribosomal Peptide Biosynthesis Directly from DNA Using Cell-Free Protein Synthesis Journal Article ACS Synth Biol, 6 (1), pp. 39-44, 2017, ISSN: 2161-5063 (Electronic) 2161-5063 (Linking). @article{RN475, title = {In Vitro Reconstruction of Nonribosomal Peptide Biosynthesis Directly from DNA Using Cell-Free Protein Synthesis}, author = { A. W. Goering and J. Li and R. A. McClure and R. J. Thomson and M. C. Jewett and N. L. Kelleher}, url = {http://www.ncbi.nlm.nih.gov/pubmed/27478992}, doi = {10.1021/acssynbio.6b00160}, issn = {2161-5063 (Electronic) 2161-5063 (Linking)}, year = {2017}, date = {2017-01-01}, journal = {ACS Synth Biol}, volume = {6}, number = {1}, pages = {39-44}, keywords = {}, pubstate = {published}, tppubtype = {article} } |
