Pub Date : 2024-09-04DOI: 10.1101/2024.09.04.611269
Pooja Anantha, Xiangdong Wu, Salaheldeen Elsaid, Piyush Raj, Ishan Barman, Sui Seng Tee
The thermogenic capacity of brown adipose tissue (BAT) has garnered much attention for its potential to regulate systemic energy balance. BAT depot size and function need to be tightly to prevent loss of metabolic homeostasis due to energy dissipation via non-shivering thermogenesis. While adipocyte-intrinsic mechanisms controlling thermogenesis are critical, an increasing appreciation for the role of the BAT microenvironment is emerging. For example, changes in circulating hexoses due to dietary intake have shown to impact BAT function. Here, we show that murine BAT preadipocytes metabolism is impacted when fructose is used as the sole carbon source. Similarly, differentiation medium containing only fructose yield mature adipocytes with fewer lipid droplets, with a concomitant decrease in adipogenic genes. These deficiencies are also observed in human BAT preadipocytes, where cutting-edge optical imaging modalities show a decrease in total cell mass and lipid mass in fructose only medium. Taken together, the metabolic microenvironment significantly impacts BAT growth and function, with implications for the role of diets potentially mitigating the efficacy of BAT-targeted therapies.
棕色脂肪组织（BAT）的产热能力因其调节全身能量平衡的潜力而备受关注。需要严格控制棕色脂肪组织的大小和功能，以防止由于非颤抖性产热导致能量耗散而失去代谢平衡。虽然控制产热的脂肪细胞内在机制至关重要，但人们对 BAT 微环境作用的认识也在不断提高。例如，膳食摄入导致的循环己糖变化已被证明会影响脂肪细胞的功能。在这里，我们发现当果糖被用作唯一碳源时，小鼠 BAT 前脂肪细胞的新陈代谢会受到影响。同样，仅含果糖的分化培养基产生的成熟脂肪细胞脂滴较少，同时脂肪生成基因也会减少。在人类 BAT 前脂肪细胞中也观察到了这些缺陷，最先进的光学成像模式显示，在仅含果糖的培养基中，细胞总质量和脂质质量都有所下降。总之，代谢微环境对 BAT 的生长和功能有重大影响，对饮食的作用有潜在影响，可能会减轻 BAT 靶向疗法的疗效。
{"title":"Sweet science: Exploring the impact of fructose and glucose on brown adipocyte differentiation using optical diffraction tomography","authors":"Pooja Anantha, Xiangdong Wu, Salaheldeen Elsaid, Piyush Raj, Ishan Barman, Sui Seng Tee","doi":"10.1101/2024.09.04.611269","DOIUrl":"https://doi.org/10.1101/2024.09.04.611269","url":null,"abstract":"The thermogenic capacity of brown adipose tissue (BAT) has garnered much attention for its potential to regulate systemic energy balance. BAT depot size and function need to be tightly to prevent loss of metabolic homeostasis due to energy dissipation via non-shivering thermogenesis. While adipocyte-intrinsic mechanisms controlling thermogenesis are critical, an increasing appreciation for the role of the BAT microenvironment is emerging. For example, changes in circulating hexoses due to dietary intake have shown to impact BAT function. Here, we show that murine BAT preadipocytes metabolism is impacted when fructose is used as the sole carbon source. Similarly, differentiation medium containing only fructose yield mature adipocytes with fewer lipid droplets, with a concomitant decrease in adipogenic genes. These deficiencies are also observed in human BAT preadipocytes, where cutting-edge optical imaging modalities show a decrease in total cell mass and lipid mass in fructose only medium. Taken together, the metabolic microenvironment significantly impacts BAT growth and function, with implications for the role of diets potentially mitigating the efficacy of BAT-targeted therapies.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"18 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142175860","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 : 2024-08-11DOI: 10.1101/2024.08.11.607481
Alexander Helmut Rotsch, Delong Li, Maud Dupont, Tim Krischuns, Christiane Oberthuer, Alice Stelfox, Maria Lukarska, Isaac Fianu, Michael Lidschreiber, Nadia Naffakh, Christian Dienemann, Stephen Cusack, Patrick Cramer
Influenza virus mRNA is stable and competent for nuclear export and translation because it re-ceives a 5′ cap(1) structure in a process called cap-snatching1. During cap-snatching, the viral RNA-dependent RNA polymerase (FluPol) binds to host RNA polymerase II (Pol II) and the emerging transcript2,3. The FluPol endonuclease then cleaves a capped RNA fragment that sub-sequently acts as a primer for the transcription of viral genes4,5. Here, we present the cryo-EM structure of FluPol bound to a transcribing Pol II in complex with the elongation factor DSIF in the pre-cleavage state. The structure shows that FluPol directly interacts with both Pol II and DSIF, which position the FluPol endonuclease domain near the RNA exit channel of Pol II. These interactions are important for the endonuclease activity of FluPol and FluPol activity in cells. A second structure trapped after cap-snatching shows that cleavage rearranges the capped RNA primer within the FluPol, directing the capped RNA 3′-end towards the FluPol polymer-ase active site for viral transcription initiation. Altogether, our results provide the molecular mechanisms of co-transcriptional cap-snatching by FluPol.
{"title":"Mechanism of Co-Transcriptional Cap-Snatching by Influenza Polymerase","authors":"Alexander Helmut Rotsch, Delong Li, Maud Dupont, Tim Krischuns, Christiane Oberthuer, Alice Stelfox, Maria Lukarska, Isaac Fianu, Michael Lidschreiber, Nadia Naffakh, Christian Dienemann, Stephen Cusack, Patrick Cramer","doi":"10.1101/2024.08.11.607481","DOIUrl":"https://doi.org/10.1101/2024.08.11.607481","url":null,"abstract":"Influenza virus mRNA is stable and competent for nuclear export and translation because it re-ceives a 5′ cap(1) structure in a process called cap-snatching1. During cap-snatching, the viral RNA-dependent RNA polymerase (FluPol) binds to host RNA polymerase II (Pol II) and the emerging transcript2,3. The FluPol endonuclease then cleaves a capped RNA fragment that sub-sequently acts as a primer for the transcription of viral genes4,5. Here, we present the cryo-EM structure of FluPol bound to a transcribing Pol II in complex with the elongation factor DSIF in the pre-cleavage state. The structure shows that FluPol directly interacts with both Pol II and DSIF, which position the FluPol endonuclease domain near the RNA exit channel of Pol II. These interactions are important for the endonuclease activity of FluPol and FluPol activity in cells. A second structure trapped after cap-snatching shows that cleavage rearranges the capped RNA primer within the FluPol, directing the capped RNA 3′-end towards the FluPol polymer-ase active site for viral transcription initiation. Altogether, our results provide the molecular mechanisms of co-transcriptional cap-snatching by FluPol.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"198 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141944456","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 : 2024-08-11DOI: 10.1101/2024.08.11.607463
Gabriel Arini, Tiago Cabral Borelli, Elthon Gois Ferreira, Rafael de Felicio, Paula Rezende-Teixeira, Matheus Pedrino Goncalves, Franciene Rabico Oliveira, Guilherme Viana de Siqueira, Luiz Gabriel Mencucini, Henrique Tsuji, Lucas Sousa Neves Andrade, Leandro Garrido, Gabriel Padilla, Alberto Gil-de-la-Fuentes, Mingxun Wang, Norberto Peporine Lopes, Daniela BB Trivella, Leticia V Costa-Lotufo, Maria-Eugenia Guazzaroni, Ricardo Roberto da Silva
Exploiting microbial natural products is a key pursuit of the bioactive compound discovery field. Recent advances in modern analytical techniques have increased the volume of microbial genomes and their encoded biosynthetic products measured by mass spectrometry-based metabolomics. However, connecting multi-omics data to uncover metabolic processes of interest is still challenging. This results in a large portion of genes and metabolites remaining unannotated. Further exacerbating the annotation challenge, databases and tools for annotation and omics integration are scattered, requiring complex computations to annotate and integrate omics datasets. Here we performed a two-way integrative analysis combining genomics and metabolomics data to describe a new approach to characterize the marine bacterial isolate BRA006 and to explore its biosynthetic gene cluster (BGC) content as well as the bioactive compounds detected by metabolomics. We described BRA006 genomic content and structure by comparing Illumina and Oxford Nanopore MinION sequencing approaches. Digital DNA:DNA hybridization (dDDH) taxonomically assigned BRA006 as a potential new species of the Micromonospora genus. Starting from LC-ESI(+)-HRMS/MS data, and mapping the annotated enzymes and metabolites belonging to the same pathways, our integrative analysis allowed us to correlate the compound Brevianamide F to a new BGC, previously assigned to other function.
{"title":"Characterization of a marine bacteria through a novel metabologenomics approach","authors":"Gabriel Arini, Tiago Cabral Borelli, Elthon Gois Ferreira, Rafael de Felicio, Paula Rezende-Teixeira, Matheus Pedrino Goncalves, Franciene Rabico Oliveira, Guilherme Viana de Siqueira, Luiz Gabriel Mencucini, Henrique Tsuji, Lucas Sousa Neves Andrade, Leandro Garrido, Gabriel Padilla, Alberto Gil-de-la-Fuentes, Mingxun Wang, Norberto Peporine Lopes, Daniela BB Trivella, Leticia V Costa-Lotufo, Maria-Eugenia Guazzaroni, Ricardo Roberto da Silva","doi":"10.1101/2024.08.11.607463","DOIUrl":"https://doi.org/10.1101/2024.08.11.607463","url":null,"abstract":"Exploiting microbial natural products is a key pursuit of the bioactive compound discovery field. Recent advances in modern analytical techniques have increased the volume of microbial genomes and their encoded biosynthetic products measured by mass spectrometry-based metabolomics. However, connecting multi-omics data to uncover metabolic processes of interest is still challenging. This results in a large portion of genes and metabolites remaining unannotated. Further exacerbating the annotation challenge, databases and tools for annotation and omics integration are scattered, requiring complex computations to annotate and integrate omics datasets. Here we performed a two-way integrative analysis combining genomics and metabolomics data to describe a new approach to characterize the marine bacterial isolate BRA006 and to explore its biosynthetic gene cluster (BGC) content as well as the bioactive compounds detected by metabolomics. We described BRA006 genomic content and structure by comparing Illumina and Oxford Nanopore MinION sequencing approaches. Digital DNA:DNA hybridization (dDDH) taxonomically assigned BRA006 as a potential new species of the Micromonospora genus. Starting from LC-ESI(+)-HRMS/MS data, and mapping the annotated enzymes and metabolites belonging to the same pathways, our integrative analysis allowed us to correlate the compound Brevianamide F to a new BGC, previously assigned to other function.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"220 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141944461","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 : 2024-08-10DOI: 10.1101/2024.08.09.606997
Wenqing Xu, Zhenguang Zhao, Matthew Su, Atul Jain, Hannah C. Lloyd, Ethan Yang Feng, Nick Cox, Christina M. Woo
C-Terminal cyclic imides are post-translational modifications (PTMs) that can arise from spontaneous intramolecular cleavage of asparagine or glutamine residues resulting in a form of irreversible protein damage. These protein damage events are recognized and removed by the E3 ligase substrate adapter cereblon (CRBN), indicating that these aging-related modifications may require cellular quality control mechanisms to prevent deleterious effects. However, the factors that determine protein or peptide susceptibility to C-terminal cyclic imide formation or their effect on protein stability have not been explored in detail. Here, we characterize the primary and secondary structures of peptides and proteins that promote intrinsic formation of C-terminal cyclic imides in comparison to deamidation, a related form of protein damage. Extrinsic effects from solution properties and stressors on the cellular proteome additionally promote C-terminal cyclic imide formation on proteins like glutathione synthetase (GSS) that are susceptible to aggregation if the protein damage products are not removed by CRBN. This systematic investigation provides insight to the regions of the proteome that are prone to these unexpectedly frequent modifications, the effects of this form of protein damage on protein stability, and the biological role of CRBN.
C 端环状酰亚胺是一种翻译后修饰（PTM），可产生于天冬酰胺或谷氨酰胺残基的自发分子内裂解，导致一种不可逆的蛋白质损伤。E3 连接酶底物适配器脑龙（CRBN）可识别并清除这些蛋白质损伤事件，这表明这些与衰老相关的修饰可能需要细胞质量控制机制来防止有害影响。然而，决定蛋白质或肽易受 C 端环状亚胺形成影响的因素及其对蛋白质稳定性的影响尚未得到详细探讨。在这里，我们描述了肽和蛋白质的一级和二级结构与脱酰胺（一种相关的蛋白质损伤形式）相比促进 C 端环状亚胺内在形成的特性。溶液特性和应激因素对细胞蛋白质组的外在影响也促进了谷胱甘肽合成酶（GSS）等蛋白质上 C 端环状亚胺的形成，如果蛋白质损伤产物没有被 CRBN 清除，这些蛋白质就很容易发生聚集。这项系统性研究深入探讨了蛋白质组中容易发生这些意外频繁修饰的区域、这种形式的蛋白质损伤对蛋白质稳定性的影响以及 CRBN 的生物学作用。
{"title":"Genesis and regulation of C-terminal cyclic imides from protein damage","authors":"Wenqing Xu, Zhenguang Zhao, Matthew Su, Atul Jain, Hannah C. Lloyd, Ethan Yang Feng, Nick Cox, Christina M. Woo","doi":"10.1101/2024.08.09.606997","DOIUrl":"https://doi.org/10.1101/2024.08.09.606997","url":null,"abstract":"C-Terminal cyclic imides are post-translational modifications (PTMs) that can arise from spontaneous intramolecular cleavage of asparagine or glutamine residues resulting in a form of irreversible protein damage. These protein damage events are recognized and removed by the E3 ligase substrate adapter cereblon (CRBN), indicating that these aging-related modifications may require cellular quality control mechanisms to prevent deleterious effects. However, the factors that determine protein or peptide susceptibility to C-terminal cyclic imide formation or their effect on protein stability have not been explored in detail. Here, we characterize the primary and secondary structures of peptides and proteins that promote intrinsic formation of C-terminal cyclic imides in comparison to deamidation, a related form of protein damage. Extrinsic effects from solution properties and stressors on the cellular proteome additionally promote C-terminal cyclic imide formation on proteins like glutathione synthetase (GSS) that are susceptible to aggregation if the protein damage products are not removed by CRBN. This systematic investigation provides insight to the regions of the proteome that are prone to these unexpectedly frequent modifications, the effects of this form of protein damage on protein stability, and the biological role of CRBN.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"30 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141944459","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 : 2024-08-10DOI: 10.1101/2024.08.09.604662
Alireza Ghanbarpour, Bertina Telusma, Barrett M. Powell, Jia Jia Zhang, Isabella Bolstad, Carolyn Vargas, Sandro Keller, Tania A. Baker, Robert T. Sauer, Joseph H. Davis
FtsH, a AAA protease, associates with HflK/C subunits to form a megadalton complex that spans the inner membrane and extends into the periplasm of E. coli. How this complex and homologous assemblies in eukaryotic organelles recruit, extract, and degrade membrane-embedded substrates is unclear. Following overproduction of protein components, recent cryo-EM structures reveal symmetric HflK/C cages surrounding FtsH in a manner proposed to inhibit degradation of membrane-embedded substrates. Here, we present structures of native complexes in which HflK/C instead forms an asymmetric nautilus-like assembly with an entryway for membrane-embedded substrates to reach and be engaged by FtsH. Consistent with this nautilus-like structure, proteomic assays suggest that HflK/C enhances FtsH degradation of certain membrane-embedded substrates. Membrane curvature in our FtsH*HflK/C complexes is opposite that of surrounding membrane regions, a property that correlates with lipid-scramblase activity and possibly with FtsH's function in the degradation of membrane-embedded proteins.
{"title":"An asymmetric nautilus-like HflK/C assembly controls FtsH proteolysis of membrane proteins","authors":"Alireza Ghanbarpour, Bertina Telusma, Barrett M. Powell, Jia Jia Zhang, Isabella Bolstad, Carolyn Vargas, Sandro Keller, Tania A. Baker, Robert T. Sauer, Joseph H. Davis","doi":"10.1101/2024.08.09.604662","DOIUrl":"https://doi.org/10.1101/2024.08.09.604662","url":null,"abstract":"FtsH, a AAA protease, associates with HflK/C subunits to form a megadalton complex that spans the inner membrane and extends into the periplasm of E. coli. How this complex and homologous assemblies in eukaryotic organelles recruit, extract, and degrade membrane-embedded substrates is unclear. Following overproduction of protein components, recent cryo-EM structures reveal symmetric HflK/C cages surrounding FtsH in a manner proposed to inhibit degradation of membrane-embedded substrates. Here, we present structures of native complexes in which HflK/C instead forms an asymmetric nautilus-like assembly with an entryway for membrane-embedded substrates to reach and be engaged by FtsH. Consistent with this nautilus-like structure, proteomic assays suggest that HflK/C enhances FtsH degradation of certain membrane-embedded substrates. Membrane curvature in our FtsH*HflK/C complexes is opposite that of surrounding membrane regions, a property that correlates with lipid-scramblase activity and possibly with FtsH's function in the degradation of membrane-embedded proteins.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"4 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141944460","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 : 2024-08-10DOI: 10.1101/2024.08.08.606885
Jimin Lee, James Brett Case, Rashmi Ravichandran, Daniel Asarnow, M. Alejandra Tortorici, Jack T Brown, Shilpa Sanapala, Lauren Carter, David Baker, Michael S Diamond, David Veesler
The continued evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has compromised neutralizing antibody responses elicited by prior infection or vaccination and abolished the utility of most monoclonal antibody therapeutics. We previously described a computationally-designed, homotrimeric miniprotein inhibitor, designated TRI2-2, that protects mice against pre-Omicron SARS-CoV-2 variants. Here, we show that TRI2-2 exhibits pan neutralization of variants that evolved during the 4.5 years since the emergence of SARS-CoV-2 and protects mice against BQ.1.1, XBB.1.5 and BA.2.86 challenge when administered post-exposure by an intranasal route. The resistance of TRI2-2 to viral escape and its direct delivery to the upper airways rationalize a path toward clinical advancement.
{"title":"A pan-variant miniprotein inhibitor protects against SARS-CoV-2 variants","authors":"Jimin Lee, James Brett Case, Rashmi Ravichandran, Daniel Asarnow, M. Alejandra Tortorici, Jack T Brown, Shilpa Sanapala, Lauren Carter, David Baker, Michael S Diamond, David Veesler","doi":"10.1101/2024.08.08.606885","DOIUrl":"https://doi.org/10.1101/2024.08.08.606885","url":null,"abstract":"The continued evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has compromised neutralizing antibody responses elicited by prior infection or vaccination and abolished the utility of most monoclonal antibody therapeutics. We previously described a computationally-designed, homotrimeric miniprotein inhibitor, designated TRI2-2, that protects mice against pre-Omicron SARS-CoV-2 variants. Here, we show that TRI2-2 exhibits pan neutralization of variants that evolved during the 4.5 years since the emergence of SARS-CoV-2 and protects mice against BQ.1.1, XBB.1.5 and BA.2.86 challenge when administered post-exposure by an intranasal route. The resistance of TRI2-2 to viral escape and its direct delivery to the upper airways rationalize a path toward clinical advancement.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"13 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141944458","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 : 2024-08-10DOI: 10.1101/2024.06.17.599268
Amrita Rai, Petra Janning, Ingrid R. Vetter, Roger S. Goody
Mical1 regulates F-actin dynamics through the reversible oxidation of actin, a process controlled by its interactions with various proteins. Upon binding to Rab8 family members, Mical1 links endosomes to the cytoskeleton, promoting F-actin disassembly. In the absence of Rab, Mical1 exists in an auto-inhibited state, but its biochemical characterization remains incomplete. Our study reveals that the N-terminal MO-CH-LIM domains of Mical1 form an intramolecular complex with its C-terminal bMERB domain. Mutational analysis, guided by the AlphaFold2 model, identifies critical residues at the binding interface. Additionally, we demonstrate that full-length Mical1 binds to Rab8 in a 1:2 stoichiometry, thereby releasing auto-inhibition. Through structure-based mutational studies, we uncover allostery between the N and C-terminal Rab binding sites. Notably, Rab binding at the high-affinity C-terminal site precedes binding at the N-terminal site, suggesting a sequential binding mode. These findings elucidate how Rab8 binding releases the MO-CH-LIM domains from the Mical1 bMERB domain, facilitating interactions with other proteins and the actin cytoskeleton, thereby modulating actin dynamics.
Mical1 通过肌动蛋白的可逆氧化调节 F-肌动蛋白的动态，这一过程受其与各种蛋白质的相互作用控制。与 Rab8 家族成员结合后，Mical1 将内体与细胞骨架连接起来，促进 F-肌动蛋白的解体。在没有 Rab 的情况下，Mical1 处于自动抑制状态，但其生化特征描述仍不完整。我们的研究发现，Mical1 的 N 端 MO-CH-LIM 结构域与其 C 端 bMERB 结构域形成分子内复合物。以 AlphaFold2 模型为指导的突变分析确定了结合界面上的关键残基。此外，我们还证明了全长 Mical1 与 Rab8 的结合比例为 1:2，从而释放了自动抑制作用。通过基于结构的突变研究，我们发现了 N 端和 C 端 Rab 结合位点之间的异位。值得注意的是，Rab 与高亲和力 C 端位点的结合先于与 N 端位点的结合，这表明这是一种顺序结合模式。这些发现阐明了 Rab8 结合如何从 Mical1 bMERB 结构域中释放 MO-CH-LIM 结构域，从而促进与其他蛋白质和肌动蛋白细胞骨架的相互作用，进而调节肌动蛋白的动力学。
{"title":"Biochemical and structural insights into the auto-inhibited state of Mical1 and its activation by Rab8","authors":"Amrita Rai, Petra Janning, Ingrid R. Vetter, Roger S. Goody","doi":"10.1101/2024.06.17.599268","DOIUrl":"https://doi.org/10.1101/2024.06.17.599268","url":null,"abstract":"Mical1 regulates F-actin dynamics through the reversible oxidation of actin, a process controlled by its interactions with various proteins. Upon binding to Rab8 family members, Mical1 links endosomes to the cytoskeleton, promoting F-actin disassembly. In the absence of Rab, Mical1 exists in an auto-inhibited state, but its biochemical characterization remains incomplete. Our study reveals that the N-terminal MO-CH-LIM domains of Mical1 form an intramolecular complex with its C-terminal bMERB domain. Mutational analysis, guided by the AlphaFold2 model, identifies critical residues at the binding interface. Additionally, we demonstrate that full-length Mical1 binds to Rab8 in a 1:2 stoichiometry, thereby releasing auto-inhibition. Through structure-based mutational studies, we uncover allostery between the N and C-terminal Rab binding sites. Notably, Rab binding at the high-affinity C-terminal site precedes binding at the N-terminal site, suggesting a sequential binding mode. These findings elucidate how Rab8 binding releases the MO-CH-LIM domains from the Mical1 bMERB domain, facilitating interactions with other proteins and the actin cytoskeleton, thereby modulating actin dynamics.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"40 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141944463","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 : 2024-08-10DOI: 10.1101/2024.08.09.607390
Maria J. Pires, Alen Lovric, Emanuele Fabbrizi, Dante Rotili, Mikael Altun, Nicholas C.K. Valerie
PARP inhibitors (PARPi) predominantly targeting PARP1 and PARP2 have revolutionized cancer therapy by exploiting synthetic lethality and selectively killing cancer cells with defective DNA repair. However, achieving PARP1 or PARP2-selective inhibitors is difficult due to their close structural homology. Selectivity profiling is typically done with purified proteins, but these lack the complexity of intracellular environments and could therefore be inaccurate. The cellular target engagement by accumulation of mutant (CeTEAM) method provides insights into drug binding in cellulo by means of conditionally stabilized biosensors, thus offering a dynamic view of pharmacological events in living cells. Here, we duplex PARP1 L713F-GFP and PARP2 L269A-mCherry biosensors to systematically characterize potential PARPi binding and cell cycle alterations at the single cell level. Our results reveal that most PARPi are generally equipotent for both PARPs or have slight biases only towards PARP1, not PARP2. AZD5305, a reported PARP1-selective inhibitor, was the exception and appears ~1600-fold more potent towards PARP1. Surprisingly, niraparib was >10-fold more selective for PARP1, despite reported equipotent biochemical activity. Meanwhile, the next generation PARPi, senaparib, was a potent PARP1/2 binder and DNA trapper. We also assessed the effect of the PARP1/2 active site component, HPF1, on intracellular PARPi binding and see that HPF1 depletion elicits slight deviations in apparent binding potency, while contributing additively to PARP-DNA trapping. These results highlight that multiplexing CeTEAM biosensors and layered genetic perturbations can systematically profile determinants of intracellular drug selectivity. Furthermore, the PARP1/2 CeTEAM platform should facilitate the discovery of selective PARPi for better targeted therapies.
{"title":"Probing intracellular determinants of PARP inhibitor selectivity and pharmacology with CeTEAM","authors":"Maria J. Pires, Alen Lovric, Emanuele Fabbrizi, Dante Rotili, Mikael Altun, Nicholas C.K. Valerie","doi":"10.1101/2024.08.09.607390","DOIUrl":"https://doi.org/10.1101/2024.08.09.607390","url":null,"abstract":"PARP inhibitors (PARPi) predominantly targeting PARP1 and PARP2 have revolutionized cancer therapy by exploiting synthetic lethality and selectively killing cancer cells with defective DNA repair. However, achieving PARP1 or PARP2-selective inhibitors is difficult due to their close structural homology. Selectivity profiling is typically done with purified proteins, but these lack the complexity of intracellular environments and could therefore be inaccurate. The cellular target engagement by accumulation of mutant (CeTEAM) method provides insights into drug binding in cellulo by means of conditionally stabilized biosensors, thus offering a dynamic view of pharmacological events in living cells. Here, we duplex PARP1 L713F-GFP and PARP2 L269A-mCherry biosensors to systematically characterize potential PARPi binding and cell cycle alterations at the single cell level. Our results reveal that most PARPi are generally equipotent for both PARPs or have slight biases only towards PARP1, not PARP2. AZD5305, a reported PARP1-selective inhibitor, was the exception and appears ~1600-fold more potent towards PARP1. Surprisingly, niraparib was >10-fold more selective for PARP1, despite reported equipotent biochemical activity. Meanwhile, the next generation PARPi, senaparib, was a potent PARP1/2 binder and DNA trapper. We also assessed the effect of the PARP1/2 active site component, HPF1, on intracellular PARPi binding and see that HPF1 depletion elicits slight deviations in apparent binding potency, while contributing additively to PARP-DNA trapping. These results highlight that multiplexing CeTEAM biosensors and layered genetic perturbations can systematically profile determinants of intracellular drug selectivity. Furthermore, the PARP1/2 CeTEAM platform should facilitate the discovery of selective PARPi for better targeted therapies.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"80 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141944457","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: 10.1101/2024.08.08.607276
Emily J. Furlong, Ian-Blaine P. Reininger-Chatzigiannakis, Yi C. Zeng, Simon H. J. Brown, Meghna Sobti, Alastair G. Stewart
F1Fo ATP synthase is a molecular rotary motor that can generate ATP using a transmembrane proton motive force. Isolated F1-ATPase catalytic cores can hydrolyse ATP, passing through a series of conformational states involving rotation of the central γ rotor subunit and the opening and closing of the catalytic β subunits. Cooperativity in F1-ATPase has long thought to be conferred through the γ subunit, with three key interaction sites between the γ and β subunits being identified. Single molecule studies have demonstrated that the F1 complexes lacking the γ axle still “rotate” and hydrolyse ATP, but with less efficiency. We solved the cryogenic electron microscopy structure of an axle-less Bacillus sp. PS3 F1-ATPase. The unexpected binding-dwell conformation of the structure in combination with the observed lack of interactions between the axle-less γ and the open β subunit suggests that the complete γ subunit is important for coordinating efficient ATP binding of F1-ATPase.
{"title":"The molecular structure of an axle-less F1-ATPase","authors":"Emily J. Furlong, Ian-Blaine P. Reininger-Chatzigiannakis, Yi C. Zeng, Simon H. J. Brown, Meghna Sobti, Alastair G. Stewart","doi":"10.1101/2024.08.08.607276","DOIUrl":"https://doi.org/10.1101/2024.08.08.607276","url":null,"abstract":"F<sub>1</sub>F<sub>o</sub> ATP synthase is a molecular rotary motor that can generate ATP using a transmembrane proton motive force. Isolated F<sub>1</sub>-ATPase catalytic cores can hydrolyse ATP, passing through a series of conformational states involving rotation of the central γ rotor subunit and the opening and closing of the catalytic β subunits. Cooperativity in F<sub>1</sub>-ATPase has long thought to be conferred through the γ subunit, with three key interaction sites between the γ and β subunits being identified. Single molecule studies have demonstrated that the F<sub>1</sub> complexes lacking the γ axle still “rotate” and hydrolyse ATP, but with less efficiency. We solved the cryogenic electron microscopy structure of an axle-less <em>Bacillus</em> sp. PS3 F<sub>1</sub>-ATPase. The unexpected binding-dwell conformation of the structure in combination with the observed lack of interactions between the axle-less γ and the open β subunit suggests that the complete γ subunit is important for coordinating efficient ATP binding of F<sub>1</sub>-ATPase.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"80 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141944465","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: 10.1101/2024.08.09.606901
Emily G. Saccuzzo, Alicia S. Robang, Yuan Gao, Bo J Chen, Raquel L Lieberman, Anant Krishna Paravastu
Myocilin-associated glaucoma is a protein-conformational disorder associated with formation of a toxic amyloid-like aggregate. Numerous destabilizing single point variants, distributed across the myocilin olfactomedin β-propeller (OLF, myocilin residues 245-504, 30 kDa) are associated with accelerated disease progression. In vitro, wild type (WT) OLF can be promoted to form thioflavin T (ThT)-positive fibrils under mildly destabilizing (37°C, pH 7.2) conditions. Consistent with the notion that only a small number of residues within a protein are responsible for amyloid formation, 3D 13C-13C solid-state NMR spectra show that OLF fibrils are likely to be composed of only about one third of the overall sequence. Here, we probe the residue composition of fibrils formed de novo from purified full-length OLF. We were able to make sequential assignments consistent with the sequence S331-G-S-L334. This sequence appears once within a previously identified amyloid-prone region (P1, G326AVVYSGSLYFQ) internal to OLF. Since nearly half of the pairs of adjacent residues (di-peptides) in OLF occur only once in the primary structure and almost all the 3-residue sequences (tri-peptides) are unique, remarkably few sequential assignments are necessary to uniquely identify specific regions of the amyloid core. This assignment approach could be applied to other systems to expand our molecular comprehension of how folded proteins undergo fibrillization.
{"title":"Evidence for S331-G-S-L within the amyloid core of myocilin olfactomedin domain fibrils based on low-resolution 3D solid-state NMR spectra","authors":"Emily G. Saccuzzo, Alicia S. Robang, Yuan Gao, Bo J Chen, Raquel L Lieberman, Anant Krishna Paravastu","doi":"10.1101/2024.08.09.606901","DOIUrl":"https://doi.org/10.1101/2024.08.09.606901","url":null,"abstract":"Myocilin-associated glaucoma is a protein-conformational disorder associated with formation of a toxic amyloid-like aggregate. Numerous destabilizing single point variants, distributed across the myocilin olfactomedin β-propeller (OLF, myocilin residues 245-504, 30 kDa) are associated with accelerated disease progression. In vitro, wild type (WT) OLF can be promoted to form thioflavin T (ThT)-positive fibrils under mildly destabilizing (37°C, pH 7.2) conditions. Consistent with the notion that only a small number of residues within a protein are responsible for amyloid formation, 3D <sup>13</sup>C-<sup>13</sup>C solid-state NMR spectra show that OLF fibrils are likely to be composed of only about one third of the overall sequence. Here, we probe the residue composition of fibrils formed de novo from purified full-length OLF. We were able to make sequential assignments consistent with the sequence S<sub>331</sub>-G-S-L<sub>334</sub>. This sequence appears once within a previously identified amyloid-prone region (P1, G<sub>326</sub>AVVYSGSLYFQ) internal to OLF. Since nearly half of the pairs of adjacent residues (di-peptides) in OLF occur only once in the primary structure and almost all the 3-residue sequences (tri-peptides) are unique, remarkably few sequential assignments are necessary to uniquely identify specific regions of the amyloid core. This assignment approach could be applied to other systems to expand our molecular comprehension of how folded proteins undergo fibrillization.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"198 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141944376","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}
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