bioRxiv - Biochemistry最新文献

{"title":"Molecular mechanisms of uric acid transport by the native human URAT1 and its inhibition by anti-gout drugs","authors":"Canrong Wu, Chao Zhang, Sanshan Jin, James Jiqi Wang, Antao Dai, Jiuyin Xu, Heng Zhang, Xuemei Yang, Xinheng He, Qingning Yuan, Wen Hu, Youwei Xu, Ming-Wei Wang, Yi Jiang, Dehua Yang, H. Eric Xu","doi":"10.1101/2024.09.11.612394","DOIUrl":"https://doi.org/10.1101/2024.09.11.612394","url":null,"abstract":"Gout, a common and painful disease, stems from hyperuricemia, where elevated blood uric acid levels lead to urate crystal formation in joints and kidneys. The human urate transporter 1 (hURAT1) plays a critical role in urate homeostasis by facilitating urate reabsorption in the renal proximal tubule, making it a key target for gout therapy. Pharmacological inhibition of hURAT1 with drugs such as dotinurad, benzbromarone, lesinurad, and verinurad promotes uric acid excretion and alleviates gout symptoms. Here we present cryo-electron microscopy structures of native hURAT1 bound with these anti-gout drugs in the inward-open state, and with uric acid in inward-open, outward-open, and occluded states. Complemented by mutagenesis and cell-based assays, these structures reveal the mechanisms of uric acid reabsorption and hURAT1 inhibition. Our findings elucidate the molecular basis of uric acid transport and anti-gout medication action, and provide a structural framework for the rational design of next-generation therapies for hyperuricemia and gout.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"7 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142175902","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}

{"title":"Molecular recording of cellular protein kinase activity with chemical labeling","authors":"De-en Sun, Siu Wang Ng, Yu Zheng, Shu Xie, Niklas Schwan, Paula Breuer, Dirk C. Hoffmann, Julius Michel, Daniel D. Azorin, Kim E. Boonekamp, Frank Winkler, Wolfgang Wick, Michael Boutros, Yulong Li, Kai Johnsson","doi":"10.1101/2024.09.11.611894","DOIUrl":"https://doi.org/10.1101/2024.09.11.611894","url":null,"abstract":"Protein kinases control most cellular processes and aberrant kinase activity is involved in numerous diseases. To investigate the link between specific kinase activities and cellular phenotypes in heterogeneous cell populations and in vivo, we introduce molecular recorders of kinase activities for later analysis. Based on split-HaloTag and a phosphorylation-dependent molecular switch, our recorders become rapidly labeled in the presence of a specific kinase activity and a fluorescent HaloTag substrate. The kinase activity in a given cell controls the degree of fluorescent labeling whereas the recording window is set by the presence of the fluorescent substrate. We have designed specific recorders for four protein kinases, including protein kinase A. We apply our protein kinase A recorder for the sorting of heterogeneous cell populations and subsequent transcriptome analysis, in genome-wide CRISPR screens to discover regulators of PKA activity and for the tracking of neuromodulation in freely moving mice.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"106 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142175903","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}

{"title":"ATP-dependent remodeling of chromatin condensates uncovers distinct mesoscale effects of two remodelers","authors":"Camille Marie Moore, Upneet Kaur, Emily Wong, Un Seng Marie Chio, Ziling Zhou, Megan Ostrowski, Ke Wu, Iryna Irkliyenko, Sean Wang, Vijay Ramani, Geeta Narlikar","doi":"10.1101/2024.09.10.611504","DOIUrl":"https://doi.org/10.1101/2024.09.10.611504","url":null,"abstract":"ATP-dependent chromatin remodeling enzymes mobilize nucleosomes, but how such mobilization affects chromatin condensation is unclear. Here, we investigate effects of two major remodelers, ACF and RSC using chromatin condensates and single-molecule footprinting. We find that both remodelers inhibit the formation of condensed chromatin. However, the remodelers have distinct effects on pre-formed chromatin condensates. ACF spaces nucleosomes without de-condensing the chromatin, explaining how ACF maintains nucleosome organization in transcriptionally repressed genomic regions. In contrast, RSC catalyzes ATP-dependent de-condensation of chromatin. Surprisingly, RSC also drives micron-scale movements of entire condensates. These newly uncovered activities of RSC explain its central role in transcriptional activation. The biological importance of remodelers may thus reflect both their effects on nucleosome mobilization and the corresponding consequences on chromatin dynamics at the mesoscale.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"40 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142175907","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}

{"title":"Structural insights into distinct mechanisms of RNA polymerase II and III recruitment to snRNA promoters","authors":"Syed Zawar Shah, Thomas N. Perry, Andrea Graziadei, Valentina Cecatiello, Thangavelu Kaliyappan, Agata D. Misiaszek, Christoph W. Muller, Ewan P. Ramsay, Alessandro Vannini","doi":"10.1101/2024.09.10.612236","DOIUrl":"https://doi.org/10.1101/2024.09.10.612236","url":null,"abstract":"RNA polymerase III (Pol III) is specialized in the transcription of short, essential RNAs, including the U6 small nuclear RNAs (snRNAs). At U6 snRNA genes, Pol III is recruited by the snRNA Activating Protein Complex (SNAPc) forming, together with a Brf2-containing TFIIIB complex, a transcriptionally competent pre-initiation complex (PIC). Additionally, SNAPc is responsible for the recruitment of Pol II at the remaining snRNAs genes (U1, 2, 4 and 5), representing a unique example of a multi subunit transcription factor shared among different RNA Polymerases. The mechanism of SNAPc cross-polymerase engagement and the role of the SNAPC2 and SNAPC5 subunits in transcription remain poorly defined. Here, we present cryo-EM structures of the full-length SNAPc-containing Pol III PIC assembled on the U6 snRNA promoter in the open and melting states at 3.2-4.2 angstrom resolution. Comparative structural analysis revealed unexpected differences with the yeast PIC and revealed the molecular basis of selective and structurally distinct SNAPc engagement within Pol III and Pol II PICs. Harnessing crosslinking mass spectrometry, we also localize the SNAPC2 and SNAPC5 subunits in proximity to the bound promoter DNA, expanding upon existing descriptions of snRNA Pol III PIC structure.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"38 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142175911","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}

{"title":"Andexanet alfa-associated heparin resistance in cardiac surgery: mechanism and in vitro perspectives","authors":"Charlene V Chabata, Haixiang Yu, Lei Ke, James Walter Frederiksen, Prakash A Patel, Bruce Sullenger, Nabil K Thalji","doi":"10.1101/2024.09.09.612152","DOIUrl":"https://doi.org/10.1101/2024.09.09.612152","url":null,"abstract":"Background: Andexanet alfa (andexanet) is the only FDA-approved antidote for direct factor Xa (FXa) inhibitors but has been reported to cause resistance to unfractionated heparin (UFH). This has delayed anticoagulation for procedures requiring cardiopulmonary bypass (CPB). The mechanism, andexanet and UFH dose dependence, and thrombotic risk of andexanet-associated heparin resistance are unknown. Methods: The effect of andexanet in vitro was determined using activated clotting times (ACT) and thromboelastography (TEG). Ex vivo CPB circuits were used to determine whether andexanet impaired anticoagulation for extracorporeal circulation. Kinetics of antithrombin (AT) inhibition of FXa and thrombin were measured in the presence of andexanet. Equilibrium modeling and thrombin generation assay (TGA) validation were used to predict the role of andexanet, AT, and UFH concentrations in andexanet-associated heparin resistance. Results: Andexanet prevented UFH-mediated prolongation of ACT and TEG times. At lower concentrations of andexanet, heparin resistance could be overcome with suprapharmacologic doses of UFH, but not at higher andexanet concentrations. Andexanet rendered standard doses of UFH inadequate to prevent circuit thrombosis, and suprapharmacologic UFH doses were only partially able to overcome this. Scanning electron microscopy demonstrated coagulation activation in circuits. Andexanet prevented UFH enhancement of AT-mediated inhibition of FXa and thrombin. Equilibrium modeling and TGA validation demonstrated that andexanet creates a triphasic equilibrium with UFH and AT: initial UFH unresponsiveness, normal UFH responsiveness when andexanet is depleted, and finally AT depletion. Sufficient CPB heparinization can only occur at low therapeutic andexanet doses and normal AT levels. Higher andexanet doses or AT deficiency may require both AT supplementation and very high UFH doses. Conclusions: Andexanet causes heparin resistance due to redistribution of UFH-bound AT. If andexanet cannot be avoided prior to heparinization and direct thrombin inhibitors are undesirable, our in vitro study suggests excess UFH should be considered as a potential strategy prior to AT supplementation.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"19 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142175905","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}

{"title":"Fibril Paint: a class of amyloid-targeting peptides","authors":"Julia Aragones Pedrola, Francoise A. Dekker, Katrin Guttmann, Litske M. van Leeuwen, Shalini Singh, Guy Mayer, Tommaso Garfagnini, Assaf Friedler, Stefan G.D. Rudiger","doi":"10.1101/2024.08.25.609586","DOIUrl":"https://doi.org/10.1101/2024.08.25.609586","url":null,"abstract":"Neurodegenerative diseases, such as Alzheimers Disease, Parkinsons Disease and Huntingtons Disease, are characterised by accumulation of amyloid fibrils, which remain incurable. It is of great importance to develop early-diagnosis approaches as well as disease-modifying therapies. Recently, we discovered the FibrilPaint1 peptide, a specific amyloid binder that can serve for fibril diagnosis. Here we introduce a class of FibrilPaint1 derivatives that bind to protein fibrils. The modifications include variation of the charge, termini and order of residues. As a result, we generated a class of peptides with general fibril-binding properties and with the potential for further adaptation, such as linkage to multiple dyes, optimisation for specific protein and aggregate strains, or adaptation for targeted protein degradation strategies. Thus, Fibril Paint peptides are a class of promising leads for targeting amyloid fibrils for diagnostic and therapeutic purposes.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"86 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142175910","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}

{"title":"A circular engineered sortase for interrogating histone H3 in chromatin","authors":"Samuel D Whedon, Kwangwoon Lee, Zhipeng A Wang, Emily Zahn, Congcong Lu, Maheeshi Yapa Abeywardana, Louise Fairall, Eunju Nam, Sarah Dubois-Coyne, Pablo De Ioannes, Xinlei Sheng, Adelina Andrei, Emily Lundberg, Jennifer Jiang, Karim-Jean D Armache, Yingming Zhao, John W R Schwabe, Mingxuan Wu, Benjamin Garcia, P. A. Cole","doi":"10.1101/2024.09.10.612318","DOIUrl":"https://doi.org/10.1101/2024.09.10.612318","url":null,"abstract":"Reversible modification of the histone H3 N-terminal tail is critical in regulating chromatin structure, gene expression, and cell states, while its dysregulation contributes to disease pathogenesis. Understanding the crosstalk between H3 tail modifications in nucleosomes constitutes a central challenge in epigenetics. Here we describe an engineered sortase transpeptidase, cW11, that displays highly favorable properties for introducing scarless H3 tails onto nucleosomes. This approach significantly accelerates the production of both symmetrically and asymmetrically modified nucleosomes. We demonstrate the utility of asymmetrically modified nucleosomes produced in this way in dissecting the impact of multiple modifications on eraser enzyme processing and molecular recognition by a reader protein. Moreover, we show that cW11 sortase is very effective at cutting and tagging histone H3 tails from endogenous histones, facilitating multiplex cut-and-paste middle down proteomics with tandem mass tags. This cut-and-paste proteomics approach permits the quantitative analysis of histone H3 modification crosstalk after treatment with different histone deacetylase inhibitors. We propose that these chemoenzymatic tail isolation and modification strategies made possible with cW11 sortase will broadly power epigenetics discovery and therapeutic development.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"4 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142175909","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}

{"title":"Mapping Selective Oxidations of Unspecific Peroxygenases","authors":"Dominik Homann, Pascal Puellmann, Martin J. Weissenborn","doi":"10.1101/2024.09.10.612301","DOIUrl":"https://doi.org/10.1101/2024.09.10.612301","url":null,"abstract":"Several unspecific peroxygenases (UPOs) have been identified that perform a broad range of selective oxyfunctionalizations and hence represent a pivotal addition to the biocatalysis 'toolbox'. To make these 'oxidation tools' broadly applicable it is crucial to provide a detailed 'user manual' for their substrate preference, chemo- and regioselectivity. We therefore selected 16 different substrates with a panel of 15 diverse UPOs and mapped their preferences. Various UPOs proved to be highly selective - discriminating based on either position or chemical properties of the substrate - with up to 99 % chemo- and regioselectivity while achieving turnover numbers (TONs) of a few hundred up to multiple thousands. This map of UPO selectivity shall serve as a starting point for new chemoenzymatic routes and starting points for protein engineering endeavors.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"116 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142175906","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}

{"title":"Mechanism of Actin Filament Severing and Capping by Gelsolin","authors":"Kyle R Barrie, Grzegorz Rebowski, Roberto Dominguez","doi":"10.1101/2024.09.10.612341","DOIUrl":"https://doi.org/10.1101/2024.09.10.612341","url":null,"abstract":"Gelsolin is the prototypical member of a family of Ca²⁺-dependent F-actin severing and capping proteins. A structure of Ca²⁺-bound full-length gelsolin at the barbed end shows domains G1G6 and the inter-domain linkers wrapping around F-actin. Another structure shows domains G1G3, a fragment produced during apoptosis, on both sides of F-actin. Conformational changes that trigger severing occur on one side of F-actin with G1G6 and on both sides with G1G3. Gelsolin remains bound after severing, blocking subunit exchange.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"75 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142175908","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}

{"title":"How a pathogenic mutation impairs Hsp60 functional dynamics from monomeric to fully assembled states","authors":"Luca Torielli, Federica Guarra, Hao Shao, Jason E Gestwicki, Stefano Artin Serapian, Giorgio Colombo","doi":"10.1101/2024.09.09.611948","DOIUrl":"https://doi.org/10.1101/2024.09.09.611948","url":null,"abstract":"Heat Shock Protein 60 kDa (Hsp60) is a mitochondrial chaperonin that cooperates with Hsp10 to drive the correct folding of client proteins. Monomers <strong>M</strong> of Hsp60 (featuring equatorial, intermediate, and apical domains) first assemble into 7-meric Single rings (<strong>S</strong>), then pairs of <strong>S</strong> interface equatorially to form 14-meric Double rings (<strong>D</strong>) that accommodate clients into their lumen. Recruitment of 7 Hsp10 molecules per pole turns <strong>D</strong> into a 28-meric Football-shaped complex (<strong>F</strong>). Sequential hydrolysis of ATP present in each Hsp60 unit of <strong>F</strong> finally drives client folding and <strong>F</strong> disassembly. Equatorial domain mutation V72I occurs in SPG13, a form of hereditary spastic paraplegia: while distal to the active site, this severely impairs the chaperone cycle and stability. To understand the molecular bases of this impairment we have run atomistic molecular dynamics (MD) simulations of <strong>M</strong>, <strong>S</strong>, <strong>D</strong>, and <strong>F</strong> for both WT and mutant Hsp60, with two catalytically relevant Hsp60 aspartates in <strong>D</strong> and <strong>F</strong> modelled in three different protontion states. Additionally, <strong>D</strong> in one protonation state was modelled post-hydrolysis (total production time: 36 μs). By combining complementary experimental and computational approaches for the analysis of functional dynamics and allosteric mechanisms, we consistently find that mutaNon V72I significantly rewires allosteric routes present in WT Hsp60 across its complexes, from isolated <strong>M</strong> units right up to <strong>F</strong>, rigidifying them—as observed experimentally—by introducing a direct allosteric link between equatorial and apical Hsp60 domains that bypasses the ATP binding site (wherein we observe the alteraNon of mechanisms driving reactivity). Our results reveal a multiscale complexity of functional mechanisms for Hsp60 and its pathogenic mutant, and may lay the foundation for the design of experiments to fully understand both variants.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"11 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142175912","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}