Pub Date : 2024-08-09DOI: 10.1101/2024.08.09.606993
Thierry D. Marti, Diana Schweizer, Yaochun Yu, Milo R. Schaerer, Silke I. Probst, Serina L Robinson
Organic micropollutants - including pharmaceuticals, personal care products, pesticides and food additives - are prevalent in the environment and have unknown and potentially toxic effects. Humans are a direct source of micropollutants as the majority of pharmaceuticals are primarily excreted through urine. Urine contains its own microbiota with the potential to catalyze micropollutant biotransformations. Amidase signature (AS) enzymes are known for their promiscuous activity in micropollutant biotransformations, but the potential for AS enzymes from the urinary microbiota to transform micropollutants is not known. Moreover, characterization of AS enzymes to identify key chemical and enzymatic features predictive of biotransformation profiles is critical for developing benign-by-design chemicals and micropollutant removal strategies. In this study, we biochemically characterized a new AS enzyme with arylamidase activity from a urine isolate, Lacticaseibacillus rhamnosus< and demonstrated its capability to hydrolyze pharmaceuticals and other micropollutants. To uncover the signatures of AS enzyme-substrate specificity, we then designed a targeted enzyme library consisting of 40 arylamidase homologs from diverse urine isolates and tested it against 17 structurally diverse compounds. We found that 16 out of the 40 enzymes showed activity on at least one substrate and exhibited diverse substrate specificities, with the most promiscuous enzymes active on nine different substrates. Using an interpretable gradient boosting machine learning model, we identified chemical and amino acid features predictive of arylamidase biotransformations. Key chemical features from our substrates included the molecular weight of the amide carbonyl substituent and the number of charges in the molecule. Important amino acid features were found to be located on the protein surface and four predictive residues were located in close proximity of the substrate tunnel entrance. Overall, this work highlights the understudied role of urine-derived microbial arylamidases and contributes to enzyme sequence-structure-substrate-based predictions of micropollutant biotransformations.
{"title":"Machine learning reveals signatures of promiscuous microbial amidases for micropollutant biotransformations","authors":"Thierry D. Marti, Diana Schweizer, Yaochun Yu, Milo R. Schaerer, Silke I. Probst, Serina L Robinson","doi":"10.1101/2024.08.09.606993","DOIUrl":"https://doi.org/10.1101/2024.08.09.606993","url":null,"abstract":"Organic micropollutants - including pharmaceuticals, personal care products, pesticides and food additives - are prevalent in the environment and have unknown and potentially toxic effects. Humans are a direct source of micropollutants as the majority of pharmaceuticals are primarily excreted through urine. Urine contains its own microbiota with the potential to catalyze micropollutant biotransformations. Amidase signature (AS) enzymes are known for their promiscuous activity in micropollutant biotransformations, but the potential for AS enzymes from the urinary microbiota to transform micropollutants is not known. Moreover, characterization of AS enzymes to identify key chemical and enzymatic features predictive of biotransformation profiles is critical for developing benign-by-design chemicals and micropollutant removal strategies. In this study, we biochemically characterized a new AS enzyme with arylamidase activity from a urine isolate, Lacticaseibacillus rhamnosus< and demonstrated its capability to hydrolyze pharmaceuticals and other micropollutants. To uncover the signatures of AS enzyme-substrate specificity, we then designed a targeted enzyme library consisting of 40 arylamidase homologs from diverse urine isolates and tested it against 17 structurally diverse compounds. We found that 16 out of the 40 enzymes showed activity on at least one substrate and exhibited diverse substrate specificities, with the most promiscuous enzymes active on nine different substrates. Using an interpretable gradient boosting machine learning model, we identified chemical and amino acid features predictive of arylamidase biotransformations. Key chemical features from our substrates included the molecular weight of the amide carbonyl substituent and the number of charges in the molecule. Important amino acid features were found to be located on the protein surface and four predictive residues were located in close proximity of the substrate tunnel entrance.\u0000Overall, this work highlights the understudied role of urine-derived microbial arylamidases and contributes to enzyme sequence-structure-substrate-based predictions of micropollutant biotransformations.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"43 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141944462","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-09DOI: 10.1101/2024.08.09.607156
Arisa Suto, Takashi Matsui, Yoshio Kodera
Alkylation of the thiol group in cysteine (Cys) residues using halide reagents is a significant step in proteomics. However, non-specific modifications to the N-terminus and other amino acids are known. Thus promiscuous offsite alkylation in the peptide further complicated the MS spectra and thus made the difficulty of identification and quantification of all peptides. 2-mercaptoethanol (2-ME) is not only a regent for the reduction of the disulfide bond but also is bound to the Cys residue. Furthermore, it is known that dimethyl sulfoxide (DMSO) enhances the disulfide bond formation. Thus, based on these facts, we developed a method for specifical modification of Cys residues using 2-ME and DMSO. The specific modification of Cys residue by 2-ME were promoted by the concentration-dependent manner of DMSO with quite less offsite modification reaction compared with recent procedures. This Cys-specific modification technique may not only improve the quantification of peptides containing cysteine but also enhance the quantification accuracy of all peptides.
{"title":"Reducing Offsite Modification using 2-mercaptoethanol for Proteome Analysis.","authors":"Arisa Suto, Takashi Matsui, Yoshio Kodera","doi":"10.1101/2024.08.09.607156","DOIUrl":"https://doi.org/10.1101/2024.08.09.607156","url":null,"abstract":"Alkylation of the thiol group in cysteine (Cys) residues using halide reagents is a significant step in proteomics. However, non-specific modifications to the N-terminus and other amino acids are known. Thus promiscuous offsite alkylation in the peptide further complicated the MS spectra and thus made the difficulty of identification and quantification of all peptides. 2-mercaptoethanol (2-ME) is not only a regent for the reduction of the disulfide bond but also is bound to the Cys residue. Furthermore, it is known that dimethyl sulfoxide (DMSO) enhances the disulfide bond formation. Thus, based on these facts, we developed a method for specifical modification of Cys residues using 2-ME and DMSO. The specific modification of Cys residue by 2-ME were promoted by the concentration-dependent manner of DMSO with quite less offsite modification reaction compared with recent procedures. This Cys-specific modification technique may not only improve the quantification of peptides containing cysteine but also enhance the quantification accuracy of all peptides.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"24 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141944464","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-08DOI: 10.1101/2024.08.08.607204
Samantha F. Sedor, Sichen Shao
Codanin-1 (CDAN1) is an essential and ubiquitous protein named after congenital dyserythropoietic anemia type I (CDA-I), an autosomal recessive disease that manifests from mutations in the CDAN1 or CDIN1 (CDAN1 interacting nuclease 1) gene. CDAN1 interacts with CDIN1 and the paralogous histone H3-H4 chaperones ASF1A (Anti-Silencing Function 1A) and ASF1B, but its function remains unclear. Here, we biochemically and structurally analyze CDAN1 complexes. We find that CDAN1 dimerizes and assembles into cytosolic complexes with CDIN1 and multiple copies of ASF1A/B. Single-particle cryogenic electron microscopy (cryo-EM) structures of CDAN1 complexes identify interactions with ASF1 mediated by two CDAN1 B-domains commonly found in ASF1 binding partners and two helices that mimic histone H3 binding. We additionally observe that one CDAN1 can recruit two ASF1 molecules and that ASF1A and ASF1B have different requirements for CDAN1 engagement. Our findings explain how CDAN1 sequesters and inhibits the chaperone function of ASF1A/B and provide new molecular-level insights into this enigmatic complex.
{"title":"Mechanism of ASF1 Inhibition by CDAN1","authors":"Samantha F. Sedor, Sichen Shao","doi":"10.1101/2024.08.08.607204","DOIUrl":"https://doi.org/10.1101/2024.08.08.607204","url":null,"abstract":"Codanin-1 (CDAN1) is an essential and ubiquitous protein named after congenital dyserythropoietic anemia type I (CDA-I), an autosomal recessive disease that manifests from mutations in the CDAN1 or CDIN1 (CDAN1 interacting nuclease 1) gene. CDAN1 interacts with CDIN1 and the paralogous histone H3-H4 chaperones ASF1A (Anti-Silencing Function 1A) and ASF1B, but its function remains unclear. Here, we biochemically and structurally analyze CDAN1 complexes. We find that CDAN1 dimerizes and assembles into cytosolic complexes with CDIN1 and multiple copies of ASF1A/B. Single-particle cryogenic electron microscopy (cryo-EM) structures of CDAN1 complexes identify interactions with ASF1 mediated by two CDAN1 B-domains commonly found in ASF1 binding partners and two helices that mimic histone H3 binding. We additionally observe that one CDAN1 can recruit two ASF1 molecules and that ASF1A and ASF1B have different requirements for CDAN1 engagement. Our findings explain how CDAN1 sequesters and inhibits the chaperone function of ASF1A/B and provide new molecular-level insights into this enigmatic complex.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"13 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141944378","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
New proteasomes are produced to accommodate increases in cellular catabolic demand and prevent the accumulation of cytotoxic proteins. Formation of the proteasomal 20S core complex relies on the function of the five chaperones PAC1-4 and POMP. To understand how these chaperones facilitate proteasome assembly, we tagged the endogenous chaperones using CRISPR/Cas gene editing and examined the chaperone-bound complexes by cryo-EM. We observed an early α-ring intermediate subcomplex that is stabilized by PAC1-4, which transitions to β-ring assembly upon dissociation of PAC3/PAC4 and rearrangement of the PAC1 N-terminal tail. Completion of the β-ring and dimerization of half-proteasomes repositions critical lysine K33 to trigger cleavage of the β pro-peptides, leading to the concerted dissociation of POMP and PAC1/PAC2 to yield mature 20S proteasomes. This study reveals structural insights into critical points along the assembly pathway of the human proteasome and provides a molecular blueprint for 20S biogenesis.
{"title":"Structural basis of human 20S proteasome biogenesis","authors":"Hanxiao Zhang, Chenyu Zhou, Zarith Sofiya Mohammad, Jianhua Zhao","doi":"10.1101/2024.08.08.607236","DOIUrl":"https://doi.org/10.1101/2024.08.08.607236","url":null,"abstract":"New proteasomes are produced to accommodate increases in cellular catabolic demand and prevent the accumulation of cytotoxic proteins. Formation of the proteasomal 20S core complex relies on the function of the five chaperones PAC1-4 and POMP. To understand how these chaperones facilitate proteasome assembly, we tagged the endogenous chaperones using CRISPR/Cas gene editing and examined the chaperone-bound complexes by cryo-EM. We observed an early α-ring intermediate subcomplex that is stabilized by PAC1-4, which transitions to β-ring assembly upon dissociation of PAC3/PAC4 and rearrangement of the PAC1 N-terminal tail. Completion of the β-ring and dimerization of half-proteasomes repositions critical lysine K33 to trigger cleavage of the β pro-peptides, leading to the concerted dissociation of POMP and PAC1/PAC2 to yield mature 20S proteasomes. This study reveals structural insights into critical points along the assembly pathway of the human proteasome and provides a molecular blueprint for 20S biogenesis.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"113 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141944377","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-08DOI: 10.1101/2024.08.08.606642
Xingyang Fu, Aaron A. Hoskins
Spliceosome assembly and catalytic site formation (called activation) involve dozens of protein and snRNA binding and unbinding events. The B-complex specific proteins Prp38, Snu23, and Spp381 have critical roles in stabilizing the spliceosome during conformational changes essential for activation. While these proteins are conserved, different mechanisms have been proposed for their recruitment to spliceosomes. To visualize recruitment directly, we used Colocalization Single Molecule Spectroscopy (CoSMoS) to study the dynamics of Prp38, Snu23, and Spp381 during splicing in real time. These proteins bind to and release from spliceosomes simultaneously and are likely associated with one another. We designate the complex of Prp38, Snu23, and Spp381 as the B Complex Protein (BCP) subcomplex. Under splicing conditions, the BCP associates with pre-mRNA after tri-snRNP binding. BCP release predominantly occurs after U4 snRNP dissociation and after NineTeen Complex (NTC) association. Under low concentrations of ATP, the BCP pre-associates with the tri-snRNP resulting in their simultaneous binding to pre-mRNA. Together, our results reveal that the BCP recruitment pathway to the spliceosome is conserved between S. cerevisiae and humans. Binding of the BCP to the tri-snRNP when ATP is limiting may result in formation of unproductive complexes that could be used to regulate splicing.
{"title":"Dynamics and Evolutionary Conservation of B Complex Protein Recruitment During Spliceosome Activation","authors":"Xingyang Fu, Aaron A. Hoskins","doi":"10.1101/2024.08.08.606642","DOIUrl":"https://doi.org/10.1101/2024.08.08.606642","url":null,"abstract":"Spliceosome assembly and catalytic site formation (called activation) involve dozens of protein and snRNA binding and unbinding events. The B-complex specific proteins Prp38, Snu23, and Spp381 have critical roles in stabilizing the spliceosome during conformational changes essential for activation. While these proteins are conserved, different mechanisms have been proposed for their recruitment to spliceosomes. To visualize recruitment directly, we used Colocalization Single Molecule Spectroscopy (CoSMoS) to study the dynamics of Prp38, Snu23, and Spp381 during splicing in real time. These proteins bind to and release from spliceosomes simultaneously and are likely associated with one another. We designate the complex of Prp38, Snu23, and Spp381 as the B Complex Protein (BCP) subcomplex. Under splicing conditions, the BCP associates with pre-mRNA after tri-snRNP binding. BCP release predominantly occurs after U4 snRNP dissociation and after NineTeen Complex (NTC) association. Under low concentrations of ATP, the BCP pre-associates with the tri-snRNP resulting in their simultaneous binding to pre-mRNA. Together, our results reveal that the BCP recruitment pathway to the spliceosome is conserved between <em>S. cerevisiae</em> and humans. Binding of the BCP to the tri-snRNP when ATP is limiting may result in formation of unproductive complexes that could be used to regulate splicing.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"3 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141944379","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-07DOI: 10.1101/2024.08.07.607052
Christopher E. Berndsen, Amanda R. Storm, Angelina M. Sardelli, Sheikh R. Hossain, Kristen R. Clermont, Luke M. McFather, Mafe A. Connor, Jonathan D. Monroe
Starch accumulation in plant tissues provides an important carbon source at night and for regrowth after periods of dormancy and in times of stress. Both ɑ- and β-amylases (AMYs and BAMs, respectively) catalyze starch hydrolysis, but their functional roles are unclear. Moreover, the presence of catalytically inactive amylases that show starch excess phenotypes when deleted presents an interesting series of questions on how starch degradation is regulated. Plants lacking one of these catalytically inactive β-amylases, BAM9, were shown to have enhanced starch accumulation when combined with mutations in BAM1 and BAM3, the primary starch degrading BAMs in response to stress and at night, respectively. Importantly, BAM9 has been reported to be transcriptionally induced by stress through activation of SnRK1. Using yeast two-hybrid experiments, we identified the plastid-localized AMY3 as a potential interaction partner for BAM9. We found that BAM9 interacted with AMY3 in vitro and that BAM9 enhances AMY3 activity 3-fold. Modeling of the AMY3-BAM9 complex revealed a previously undescribed N-terminal structural feature in AMY3 that we call the alpha-alpha hairpin that could serve as a potential interaction site. Additionally, AMY3 lacking the alpha-alpha hairpin is unaffected by BAM9. Structural analysis of AMY3 showed that it can form a homodimer in solution and that BAM9 appears to replace one of the AMY3 monomers to form a heterodimer. Collectively these data suggest that BAM9 is a pseudoamylase that activates AMY3 in response to cellular stress, possibly facilitating starch degradation to provide an additional energy source for stress recovery.
{"title":"The pseudoenzyme β-amylase9 from Arabidopsis binds to and enhances the activity of α-amylase3: A possible mechanism to promote stress-induced starch degradation","authors":"Christopher E. Berndsen, Amanda R. Storm, Angelina M. Sardelli, Sheikh R. Hossain, Kristen R. Clermont, Luke M. McFather, Mafe A. Connor, Jonathan D. Monroe","doi":"10.1101/2024.08.07.607052","DOIUrl":"https://doi.org/10.1101/2024.08.07.607052","url":null,"abstract":"Starch accumulation in plant tissues provides an important carbon source at night and for regrowth after periods of dormancy and in times of stress. Both ɑ- and β-amylases (AMYs and BAMs, respectively) catalyze starch hydrolysis, but their functional roles are unclear. Moreover, the presence of catalytically inactive amylases that show starch excess phenotypes when deleted presents an interesting series of questions on how starch degradation is regulated. Plants lacking one of these catalytically inactive β-amylases, BAM9, were shown to have enhanced starch accumulation when combined with mutations in BAM1 and BAM3, the primary starch degrading BAMs in response to stress and at night, respectively. Importantly, BAM9 has been reported to be transcriptionally induced by stress through activation of SnRK1. Using yeast two-hybrid experiments, we identified the plastid-localized AMY3 as a potential interaction partner for BAM9. We found that BAM9 interacted with AMY3 <em>in vitro</em> and that BAM9 enhances AMY3 activity 3-fold. Modeling of the AMY3-BAM9 complex revealed a previously undescribed N-terminal structural feature in AMY3 that we call the alpha-alpha hairpin that could serve as a potential interaction site. Additionally, AMY3 lacking the alpha-alpha hairpin is unaffected by BAM9. Structural analysis of AMY3 showed that it can form a homodimer in solution and that BAM9 appears to replace one of the AMY3 monomers to form a heterodimer. Collectively these data suggest that BAM9 is a pseudoamylase that activates AMY3 in response to cellular stress, possibly facilitating starch degradation to provide an additional energy source for stress recovery.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"10 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141944382","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-07DOI: 10.1101/2024.08.06.606224
Christopher W. Moth, Jonathan H. Sheehan, Abdullah Al Mamun, R. Michael Sivley, Alican Gulsevin, David Rinker, John A. Capra, Jens Meiler
Effective diagnosis and treatment of rare genetic disorders requires the interpretation of a patient’s genetic variants of unknown significance (VUSs). Today, clinical decision-making is primarily guided by gene-phenotype association databases and DNA-based scoring methods. Our web-accessible variant analysis pipeline, VUStruct, supplements these established approaches by deeply analyzing the downstream molecular impact of variation in context of 3D protein structure. VUStruct’s growing impact is fueled by the co-proliferation of protein 3D structural models, gene sequencing, compute power, and artificial intelligence.
罕见遗传疾病的有效诊断和治疗需要对患者的意义不明遗传变异（VUS）进行解读。目前，临床决策主要由基因表型关联数据库和基于 DNA 的评分方法指导。我们可通过网络访问的变异分析管道--VUStruct，通过深入分析变异在三维蛋白质结构背景下的下游分子影响，对这些既有方法进行了补充。蛋白质三维结构模型、基因测序、计算能力和人工智能的共同发展推动了 VUStruct 的影响力不断扩大。
{"title":"VUStruct: a compute pipeline for high throughput and personalized structural biology","authors":"Christopher W. Moth, Jonathan H. Sheehan, Abdullah Al Mamun, R. Michael Sivley, Alican Gulsevin, David Rinker, John A. Capra, Jens Meiler","doi":"10.1101/2024.08.06.606224","DOIUrl":"https://doi.org/10.1101/2024.08.06.606224","url":null,"abstract":"Effective diagnosis and treatment of rare genetic disorders requires the interpretation of a patient’s genetic variants of unknown significance (VUSs). Today, clinical decision-making is primarily guided by gene-phenotype association databases and DNA-based scoring methods. Our web-accessible variant analysis pipeline, VUStruct, supplements these established approaches by deeply analyzing the downstream molecular impact of variation in context of 3D protein structure. VUStruct’s growing impact is fueled by the co-proliferation of protein 3D structural models, gene sequencing, compute power, and artificial intelligence.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"9 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141944383","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-07DOI: 10.1101/2024.08.06.606913
Clinton Yu, Eric Novitsky, Xiaorong Wang, Ignacia Echeverria, Scott Rychnovsky, Lan Huang
Cross-linking mass spectrometry (XL-MS) is a powerful technology for mapping protein-protein interactions (PPIs) at the systems-level. By covalently connecting pairs of proximal residues, cross-linking reagents provide distance restraints to infer protein conformations and interaction interfaces. While binary cross-links have been remarkably informative, multimeric cross-links can offer enhanced spatial resolution to facilitate the characterization of dynamic and heterogeneous protein complexes. However, the identification of multimeric cross-links remains extremely challenging due to fragmentation complexity and the vast expansion of database search space. Here, we present a novel trioxane-based MS-cleavable homotrifunctional cross-linker TSTO, which can target three proximal lysine residues simultaneously. Owing to its unique structure and MS-cleavability, TSTO enables fast and unambiguous identification of cross-linked peptides using LC-MSn analysis. Importantly, we have demonstrated that the TSTO-based XL-MS platform is effective for mapping PPIs of protein complexes and cellular networks. The trimeric interactions captured by TSTO have uncovered new structural details that cannot be easily revealed by existing reagents, allowing in-depth description of PPIs to facilitate structural modeling. This development not only advances XL-MS technologies for global PPI profiling from living cells, but also offers a new direction for creating multifunctional MS-cleavable cross-linkers to further push structural systems biology forward in the future.
交联质谱（XL-MS）是在系统水平上绘制蛋白质-蛋白质相互作用（PPIs）图谱的一项强大技术。通过共价连接成对的近端残基，交联试剂可提供距离限制，从而推断蛋白质构象和相互作用界面。虽然二元交联具有显著的信息量，但多聚交联可提供更高的空间分辨率，从而促进动态和异质蛋白质复合物的表征。然而，由于片段的复杂性和数据库搜索空间的巨大扩展，多聚交联的鉴定仍然极具挑战性。在这里，我们提出了一种新型的基于三氧杂蒽的 MS 可裂解同源三官能交联剂 TSTO，它可以同时靶向三个近端赖氨酸残基。由于其独特的结构和 MS 可裂解性，TSTO 可通过 LC-MSn 分析快速、准确地鉴定交联肽。重要的是，我们已经证明，基于 TSTO 的 XL-MS 平台可有效绘制蛋白质复合物和细胞网络的 PPIs 图谱。TSTO 捕捉到的三聚体相互作用发现了现有试剂难以揭示的新结构细节，从而可以深入描述 PPIs，促进结构建模。这项研发不仅推动了 XL-MS 技术在活细胞中进行全球 PPI 分析，而且为创造多功能 MS 可溶解交联剂提供了新的方向，从而在未来进一步推动结构系统生物学的发展。
{"title":"Trioxane-based MS-cleavable Cross-linking Mass Spectrometry for Profiling Multimeric Interactions of Cellular Networks","authors":"Clinton Yu, Eric Novitsky, Xiaorong Wang, Ignacia Echeverria, Scott Rychnovsky, Lan Huang","doi":"10.1101/2024.08.06.606913","DOIUrl":"https://doi.org/10.1101/2024.08.06.606913","url":null,"abstract":"Cross-linking mass spectrometry (XL-MS) is a powerful technology for mapping protein-protein interactions (PPIs) at the systems-level. By covalently connecting pairs of proximal residues, cross-linking reagents provide distance restraints to infer protein conformations and interaction interfaces. While binary cross-links have been remarkably informative, multimeric cross-links can offer enhanced spatial resolution to facilitate the characterization of dynamic and heterogeneous protein complexes. However, the identification of multimeric cross-links remains extremely challenging due to fragmentation complexity and the vast expansion of database search space. Here, we present a novel trioxane-based MS-cleavable homotrifunctional cross-linker TSTO, which can target three proximal lysine residues simultaneously. Owing to its unique structure and MS-cleavability, TSTO enables fast and unambiguous identification of cross-linked peptides using LC-MS<sup>n</sup> analysis. Importantly, we have demonstrated that the TSTO-based XL-MS platform is effective for mapping PPIs of protein complexes and cellular networks. The trimeric interactions captured by TSTO have uncovered new structural details that cannot be easily revealed by existing reagents, allowing in-depth description of PPIs to facilitate structural modeling. This development not only advances XL-MS technologies for global PPI profiling from living cells, but also offers a new direction for creating multifunctional MS-cleavable cross-linkers to further push structural systems biology forward in the future.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"55 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141944381","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-07DOI: 10.1101/2024.08.06.606792
Marius Landau, Sherif Elsabbagh, Harald Gross, Adrian Fuchs, Anita C.F. Schultz, Joachim E. Schultz
The biosynthesis of cAMP by mammalian membrane-bound adenylyl cyclases (mACs) is predominantly regulated by G-protein-coupled-receptors (GPCRs). Up to now the two hexahelical transmembrane domains of mACs were considered to fix the enzyme to membranes. Here we show that the transmembrane domains serve in addition as signal receptors and transmitters of lipid signals that control Gsα-stimulated mAC activities. We identify aliphatic fatty acids and anandamide as receptor ligands of mAC isoforms 1 to 7 and 9. The ligands enhance (mAC isoforms 2, 3, 7, and 9) or attenuate (isoforms 1, 4, 5, and 6) Gsα-stimulated mAC activities in vitro and in vivo. Substitution of the stimulatory membrane receptor of mAC3 by the inhibitory receptor of mAC5 results in a ligand inhibited mAC5-mAC3 chimera. Thus, we discovered a new class of membrane receptors in which two signaling modalities are at a crossing, direct tonic lipid and indirect phasic GPCR-Gsα signaling regulating the biosynthesis of cAMP.
{"title":"A new class of receptors: Lipids regulate mammalian Gsα-stimulated adenylyl cyclase activities via their membrane anchors","authors":"Marius Landau, Sherif Elsabbagh, Harald Gross, Adrian Fuchs, Anita C.F. Schultz, Joachim E. Schultz","doi":"10.1101/2024.08.06.606792","DOIUrl":"https://doi.org/10.1101/2024.08.06.606792","url":null,"abstract":"The biosynthesis of cAMP by mammalian membrane-bound adenylyl cyclases (mACs) is predominantly regulated by G-protein-coupled-receptors (GPCRs). Up to now the two hexahelical transmembrane domains of mACs were considered to fix the enzyme to membranes. Here we show that the transmembrane domains serve in addition as signal receptors and transmitters of lipid signals that control Gsα-stimulated mAC activities. We identify aliphatic fatty acids and anandamide as receptor ligands of mAC isoforms 1 to 7 and 9. The ligands enhance (mAC isoforms 2, 3, 7, and 9) or attenuate (isoforms 1, 4, 5, and 6) Gsα-stimulated mAC activities <em>in vitro</em> and <em>in vivo</em>. Substitution of the stimulatory membrane receptor of mAC3 by the inhibitory receptor of mAC5 results in a ligand inhibited mAC5-mAC3 chimera. Thus, we discovered a new class of membrane receptors in which two signaling modalities are at a crossing, direct tonic lipid and indirect phasic GPCR-Gsα signaling regulating the biosynthesis of cAMP.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"198 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141944384","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-07DOI: 10.1101/2024.08.07.606963
Arni Thorlacius, Maksim Rulev, Oscar Sundberg, Anna Sundborger-Lunna
Bin/Amphiphysin/Rvs (BAR) domain containing proteins are cytosolic, peripheral membrane proteins that regulate the curvature of membranes in eukaryotic cells. BAR protein endophilin B1 plays a key role in multiple cellular processes critical for oncogenesis, including autophagy and apoptosis. Amphipathic regions in endophilin B1 drive membrane association and tubulation through membrane scaffolding. Our understanding of exactly how BAR proteins like endophilin B1 promote highly diverse intracellular membrane remodeling events in the cell is severely limited due to lack of high-resolution structural information. Here we present the highest resolution cryo-EM structure of a BAR protein to date and the first structures of a BAR protein bound to nanodiscs. Using neural networks, we can effectively sort particle species of different stoichiometries, revealing the tremendous flexibility of post-membrane binding, pre-polymer BAR dimer organization and membrane deformation. We also show that endophilin B1 efficiently permeabilizes negatively charged liposomes that contain mitochondria-specific lipid cardiolipin and propose a new model for Bax-mediated cell death.
含Bin/Amphiphysin/Rvs（BAR）结构域的蛋白是一种细胞膜外周膜蛋白，可调节真核细胞中膜的弯曲度。BAR 蛋白嗜内酯蛋白 B1 在对肿瘤发生至关重要的多个细胞过程中发挥着关键作用，包括自噬和细胞凋亡。嗜内酯蛋白 B1 中的两性区域通过膜支架驱动膜结合和管化。由于缺乏高分辨率的结构信息，我们对 BAR 蛋白（如嗜内蛋白 B1）如何促进细胞内高度多样化的膜重塑事件的了解非常有限。在这里，我们展示了迄今为止分辨率最高的 BAR 蛋白低温电子显微镜结构，并首次展示了与纳米盘结合的 BAR 蛋白结构。利用神经网络，我们可以有效地对不同化学计量的颗粒物种进行分类，揭示了膜结合后、聚合物前 BAR 二聚体组织和膜变形的巨大灵活性。我们还表明，嗜内酯蛋白 B1 能有效渗透含有线粒体特异性脂质心磷脂的带负电脂质体，并提出了 Bax 介导细胞死亡的新模型。
{"title":"Peripheral membrane protein endophilin B1 probes, perturbs and permeabilizes lipid bilayers","authors":"Arni Thorlacius, Maksim Rulev, Oscar Sundberg, Anna Sundborger-Lunna","doi":"10.1101/2024.08.07.606963","DOIUrl":"https://doi.org/10.1101/2024.08.07.606963","url":null,"abstract":"Bin/Amphiphysin/Rvs (BAR) domain containing proteins are cytosolic, peripheral membrane proteins that regulate the curvature of membranes in eukaryotic cells. BAR protein endophilin B1 plays a key role in multiple cellular processes critical for oncogenesis, including autophagy and apoptosis. Amphipathic regions in endophilin B1 drive membrane association and tubulation through membrane scaffolding. Our understanding of exactly how BAR proteins like endophilin B1 promote highly diverse intracellular membrane remodeling events in the cell is severely limited due to lack of high-resolution structural information. Here we present the highest resolution cryo-EM structure of a BAR protein to date and the first structures of a BAR protein bound to nanodiscs. Using neural networks, we can effectively sort particle species of different stoichiometries, revealing the tremendous flexibility of post-membrane binding, pre-polymer BAR dimer organization and membrane deformation. We also show that endophilin B1 efficiently permeabilizes negatively charged liposomes that contain mitochondria-specific lipid cardiolipin and propose a new model for Bax-mediated cell death.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"24 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141944380","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
京公网安备 11010802042870号
京ICP备2023020795号-1
扫码关注我们
求助内容：
应助结果提醒方式：