Pub Date : 2024-09-11DOI: 10.1101/2024.09.11.612394
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
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.
{"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}
Pub Date : 2024-09-11DOI: 10.1101/2024.09.11.611894
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
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.
{"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}
Pub Date : 2024-09-10DOI: 10.1101/2024.09.10.611504
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
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.
依赖于 ATP 的染色质重塑酶能调动核小体，但这种调动如何影响染色质凝聚尚不清楚。在这里，我们利用染色质凝集物和单分子足迹分析研究了两种主要重塑酶 ACF 和 RSC 的影响。我们发现这两种重塑因子都会抑制染色质凝聚的形成。不过，这两种重塑因子对已形成的染色质凝聚体有不同的影响。ACF 可使核小体空间化，而不会使染色质凝结，这就解释了 ACF 如何在转录抑制的基因组区域维持核小体组织。与此相反，RSC 可催化依赖 ATP 的染色质去凝结。令人惊讶的是，RSC 还能驱动整个凝集物的微米级运动。这些新发现的 RSC 活动解释了它在转录激活中的核心作用。因此，重塑者的生物学重要性可能既反映了它们对核小体动员的影响，也反映了它们在中观尺度上对染色质动力学的相应影响。
{"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}
Pub Date : 2024-09-10DOI: 10.1101/2024.09.10.612236
Syed Zawar Shah, Thomas N. Perry, Andrea Graziadei, Valentina Cecatiello, Thangavelu Kaliyappan, Agata D. Misiaszek, Christoph W. Muller, Ewan P. Ramsay, Alessandro Vannini
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.
RNA 聚合酶 III（Pol III）专门从事短小的基本 RNA（包括 U6 小核 RNA（snRNA））的转录。在 U6 snRNA 基因上，Pol III 被 snRNA 激活蛋白复合物（SNAPc）招募，与含 Brf2 的 TFIIIB 复合物一起形成转录能力强的前启动复合物（PIC）。此外，SNAPc 还负责在其余 snRNAs 基因（U1、2、4 和 5）上招募 Pol II，是不同 RNA 聚合酶共享多亚基转录因子的一个独特例子。SNAPc 交叉聚合酶的参与机制以及 SNAPC2 和 SNAPC5 亚基在转录中的作用仍未明确。在这里，我们以 3.2-4.2 埃的分辨率展示了装配在 U6 snRNA 启动子上的全长含 SNAPc 的 Pol III PIC 在开放和融化状态下的冷冻电镜结构。结构对比分析表明了与酵母 PIC 的意想不到的差异，并揭示了 Pol III 和 Pol II PIC 内 SNAPc 选择性啮合且结构不同的分子基础。利用交联质谱法，我们还确定了 SNAPC2 和 SNAPC5 亚基与结合的启动子 DNA 的位置，拓展了现有的 snRNA Pol III PIC 结构描述。
{"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}
Pub Date : 2024-09-10DOI: 10.1101/2024.09.09.612152
Charlene V Chabata, Haixiang Yu, Lei Ke, James Walter Frederiksen, Prakash A Patel, Bruce Sullenger, Nabil K Thalji
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.
背景：安得生α（andexanet alfa）是美国食品及药物管理局批准的唯一一种直接 Xa 因子（FXa）抑制剂的解毒剂，但有报道称它会导致对非分数肝素（UFH）产生耐药性。这延误了需要心肺旁路（CPB）手术的抗凝治疗。目前尚不清楚安乃近和 UFH 剂量依赖性的机制以及安乃近相关肝素抗性的血栓风险。研究方法使用活化凝血时间（ACT）和血栓弹性成像（TEG）确定安乃近在体外的作用。使用体外 CPB 循环来确定安乃近是否会损害体外循环的抗凝作用。在安乃近存在的情况下，测量了抗凝血酶（AT）抑制 FXa 和凝血酶的动力学。利用平衡建模和凝血酶生成测定（TGA）验证来预测安乃近、AT 和 UFH 浓度在安乃近相关肝素抗性中的作用。结果显示安乃近能防止 UFH 介导的 ACT 和 TEG 时间延长。在安乃近浓度较低时，肝素抵抗可通过超药物剂量的 UFH 来克服，但在安乃近浓度较高时则无法克服。安乃近使标准剂量的 UFH 不足以防止回路血栓形成，而超药物剂量的 UFH 只能部分克服这一问题。扫描电子显微镜显示了回路中的凝血活化。Andexanet 阻止了 UFH 对 AT 介导的 FXa 和凝血酶抑制作用的增强。平衡模型和 TGA 验证表明，安乃近与 UFH 和 AT 形成了三相平衡：最初 UFH 无反应，安乃近耗尽时 UFH 反应正常，最后 AT 耗尽。只有在安乃近治疗剂量较低和 AT 水平正常的情况下，CPB 才能充分发挥肝素化作用。较高的安乃近剂量或 AT 缺乏可能需要同时补充 AT 和超高剂量的 UFH。结论：安乃近会因 UFH 结合的 AT 的重新分布而导致肝素抵抗。如果在肝素化之前不能避免使用安乃近，又不希望使用直接凝血酶抑制剂，那么我们的体外研究表明，在补充 AT 之前，应考虑将过量 UFH 作为一种可能的策略。
{"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}
Pub Date : 2024-09-10DOI: 10.1101/2024.08.25.609586
Julia Aragones Pedrola, Francoise A. Dekker, Katrin Guttmann, Litske M. van Leeuwen, Shalini Singh, Guy Mayer, Tommaso Garfagnini, Assaf Friedler, Stefan G.D. Rudiger
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.
{"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}
Pub Date : 2024-09-10DOI: 10.1101/2024.09.10.612318
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
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.
{"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}
Pub Date : 2024-09-10DOI: 10.1101/2024.09.10.612301
Dominik Homann, Pascal Puellmann, Martin J. Weissenborn
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.
已经发现了几种非特异性过氧酶（UPOs），它们可以进行广泛的选择性氧官能化，因此是生物催化 "工具箱 "中的重要补充。为了使这些 "氧化工具 "得到广泛应用，为它们的底物偏好、化学选择性和区域选择性提供一份详细的 "用户手册 "至关重要。因此，我们选择了 16 种不同的底物和 15 种不同的 UPOs，并绘制了它们的偏好图。事实证明，各种 UPO 都具有高度选择性--根据底物的位置或化学特性进行区分--化学和区域选择性高达 99%，而周转次数 (TON) 则从几百次到数千次不等。这一 UPO 选择性图谱将成为新的化学酶途径的起点和蛋白质工程努力的起点。
{"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}
Pub Date : 2024-09-10DOI: 10.1101/2024.09.10.612341
Kyle R Barrie, Grzegorz Rebowski, Roberto Dominguez
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.
{"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 Ca<sup>2+</sup>-dependent F-actin severing and capping proteins. A structure of Ca<sup>2+</sup>-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}
Pub Date : 2024-09-09DOI: 10.1101/2024.09.09.611948
Luca Torielli, Federica Guarra, Hao Shao, Jason E Gestwicki, Stefano Artin Serapian, Giorgio Colombo
Heat Shock Protein 60 kDa (Hsp60) is a mitochondrial chaperonin that cooperates with Hsp10 to drive the correct folding of client proteins. Monomers M of Hsp60 (featuring equatorial, intermediate, and apical domains) first assemble into 7-meric Single rings (S), then pairs of S interface equatorially to form 14-meric Double rings (D) that accommodate clients into their lumen. Recruitment of 7 Hsp10 molecules per pole turns D into a 28-meric Football-shaped complex (F). Sequential hydrolysis of ATP present in each Hsp60 unit of F finally drives client folding and F 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 M, S, D, and F for both WT and mutant Hsp60, with two catalytically relevant Hsp60 aspartates in D and F modelled in three different protontion states. Additionally, D 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 M units right up to F, 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.
热休克蛋白 60 kDa（Hsp60）是一种线粒体伴侣蛋白，它与 Hsp10 相互配合，促使客户蛋白正确折叠。Hsp60 的单体 M（具有赤道、中间和顶端结构域）首先组装成 7 个聚合单环（S），然后成对的 S 在赤道上对接，形成 14 个聚合双环（D），将客户蛋白容纳到其管腔中。每极招募 7 个 Hsp10 分子，将 D 变成 28 聚合的足球状复合物（F）。F 中每个 Hsp60 单元中存在的 ATP 顺序水解最终推动了客户的折叠和 F 的分解。赤道结构域突变 V72I 发生在 SPG13 中，这是一种遗传性痉挛性截瘫：虽然它位于活性位点的远端，但却严重损害了伴侣循环和稳定性。为了了解这种损害的分子基础，我们对 WT 和突变体 Hsp60 的 M、S、D 和 F 进行了原子分子动力学（MD）模拟，其中 D 和 F 中两个与催化相关的 Hsp60 天冬氨酸被模拟为三种不同的质子状态。此外，D 在一种质子化状态下进行了水解后建模（总生成时间：36 μs）。通过结合互补的实验和计算方法来分析功能动力学和异构机制，我们一致发现，V72I突变显著地重构了 WT Hsp60 复合物中存在的异构路线，从孤立的 M 单元一直到 F 单元，通过在 Hsp60 的赤道和顶端结构域之间引入绕过 ATP 结合位点的直接异构联系（我们在此观察到驱动反应性的机制的改变），使其僵化--正如实验所观察到的那样。我们的研究结果揭示了 Hsp60 及其致病突变体功能机制的多尺度复杂性，并为设计实验以全面了解这两种变体奠定了基础。
{"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}
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：