Pub Date : 2024-10-30DOI: 10.1016/j.jmb.2024.168836
Jack Boylan , Rebecca A Shrem , Isabel C. Vallecillo-Viejo , Craig L. Duvall , Brian E. Wadzinski , Benjamin W. Spiller
Cas9s and fusions of Cas9s have emerged as powerful tools for genetic manipulations. Fusions of Cas9 with other DNA editing enzymes have led to variants capable of single base editing and catalytically dead Cas9s have emerged as tools to specifically target desired regions of a genome. Here we describe the generation of a panel of nanobodies directed against three unique epitopes on Streptococcus pyogenes Cas9. The nanobodies were identified from a nanobody library derived from an alpaca that had been immunized with Cas9. The most potent binders recognize Cas9 and RNA bound Cas9 equally well and do not inhibit Cas9 cleavage of target DNA. These nanobodies bind non-overlapping epitopes as determined by ELISA based epitope binning experiments and mass photometry. We present the sequences of these clones and supporting biochemical data so the broader scientific community can access these reagents.
{"title":"A Nanobody Toolbox for Recognizing Distinct Epitopes on Cas9","authors":"Jack Boylan , Rebecca A Shrem , Isabel C. Vallecillo-Viejo , Craig L. Duvall , Brian E. Wadzinski , Benjamin W. Spiller","doi":"10.1016/j.jmb.2024.168836","DOIUrl":"10.1016/j.jmb.2024.168836","url":null,"abstract":"<div><div>Cas9s and fusions of Cas9s have emerged as powerful tools for genetic manipulations. Fusions of Cas9 with other DNA editing enzymes have led to variants capable of single base editing and catalytically dead Cas9s have emerged as tools to specifically target desired regions of a genome. Here we describe the generation of a panel of nanobodies directed against three unique epitopes on <em>Streptococcus pyogenes</em> Cas9. The nanobodies were identified from a nanobody library derived from an alpaca that had been immunized with Cas9. The most potent binders recognize Cas9 and RNA bound Cas9 equally well and do not inhibit Cas9 cleavage of target DNA. These nanobodies bind non-overlapping epitopes as determined by ELISA based epitope binning experiments and mass photometry. We present the sequences of these clones and supporting biochemical data so the broader scientific community can access these reagents.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"436 23","pages":"Article 168836"},"PeriodicalIF":4.7,"publicationDate":"2024-10-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142556804","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-30DOI: 10.1016/j.jmb.2024.168850
Dmitri E. Andreev , Jack A.S. Tierney , Pavel V. Baranov
Upstream open reading frames (uORFs) are a class of translated regions (translons) in mRNA 5′ leaders. uORFs are believed to be pervasive regulators of the translation of mammalian mRNAs. Some uORFs are highly repressive but others have little or no impact on downstream mRNA translation either due to inefficient recognition of their start codon(s) or/and due to efficient reinitiation after uORF translation. While experiments with uORF reporter constructs proved to be instrumental in the investigation of uORF-mediated mechanisms of translation control, they can have serious limitations as manipulations with uORF sequences can yield various artefacts. Here we propose a general approach for using translation complex profiling (TCP-seq) data for exploring uORF regulatory characteristics. Using several examples, we show how TCP-seq could be used to estimate both repressiveness and modes of action of individual uORFs. We demonstrate how this approach could be used to assess the mechanisms of uORF-mediated translation control in the mRNA of several human genes, including EIF5, IFRD1, MDM2, MIEF1, PPP1R15B, TAF7, and UCP2.
{"title":"Translation Complex Profile Sequencing Allows Discrimination of Leaky Scanning and Reinitiation in Upstream Open Reading Frame-controlled Translation","authors":"Dmitri E. Andreev , Jack A.S. Tierney , Pavel V. Baranov","doi":"10.1016/j.jmb.2024.168850","DOIUrl":"10.1016/j.jmb.2024.168850","url":null,"abstract":"<div><div>Upstream open reading frames (uORFs) are a class of translated regions (translons) in mRNA 5′ leaders. uORFs are believed to be pervasive regulators of the translation of mammalian mRNAs. Some uORFs are highly repressive but others have little or no impact on downstream mRNA translation either due to inefficient recognition of their start codon(s) or/and due to efficient reinitiation after uORF translation. While experiments with uORF reporter constructs proved to be instrumental in the investigation of uORF-mediated mechanisms of translation control, they can have serious limitations as manipulations with uORF sequences can yield various artefacts. Here we propose a general approach for using translation complex profiling (TCP-seq) data for exploring uORF regulatory characteristics. Using several examples, we show how TCP-seq could be used to estimate both repressiveness and modes of action of individual uORFs. We demonstrate how this approach could be used to assess the mechanisms of uORF-mediated translation control in the mRNA of several human genes, including <em>EIF5</em>, <em>IFRD1</em>, <em>MDM2</em>, <em>MIEF1</em>, <em>PPP1R15B</em>, <em>TAF7,</em> and <em>UCP2</em>.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"436 23","pages":"Article 168850"},"PeriodicalIF":4.7,"publicationDate":"2024-10-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142563952","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-29DOI: 10.1016/j.jmb.2024.168845
Lucas Farnung
In eukaryotic cells, transcription by RNA polymerase II occurs in the context of chromatin, requiring the transcription machinery to navigate through nucleosomes as it traverses gene bodies. Recent advances in structural biology have provided unprecedented insights into the mechanisms underlying transcription elongation. This review presents a structural perspective on transcription through chromatin, focusing on the latest findings from high-resolution structures of transcribing RNA polymerase II-nucleosome complexes. I discuss how RNA polymerase II, in concert with elongation factors such as SPT4/5, SPT6, ELOF1, and the PAF1 complex, engages with and transcribes through nucleosomes. The review examines the stepwise unwrapping of nucleosomal DNA as polymerase advances, the roles of elongation factors in facilitating this process, and the mechanisms of nucleosome retention and transfer during transcription. This structural perspective provides a foundation for understanding the intricate interplay between the transcription machinery and chromatin, offering insights into how cells balance the need for genetic accessibility with the maintenance of genome stability and epigenetic regulation.
在真核细胞中，RNA聚合酶II的转录是在染色质背景下进行的，转录机器在穿越基因体时需要穿过核小体。结构生物学的最新进展让人们对转录伸长的内在机制有了前所未有的深入了解。这篇综述从结构的角度阐述了通过染色质进行转录的问题，重点是转录 RNA 聚合酶 II-核小体复合物高分辨率结构的最新发现。我将讨论 RNA 聚合酶 II 如何与 SPT4/5、SPT6、ELOF1 和 PAF1 复合物等延伸因子协同作用，并通过核小体进行转录。这篇综述探讨了随着聚合酶的推进，核糖体 DNA 逐步解开的过程、延伸因子在促进这一过程中的作用，以及转录过程中核糖体保留和转移的机制。这一结构性视角为理解转录机制与染色质之间错综复杂的相互作用奠定了基础，有助于深入了解细胞如何在遗传可及性需求与维持基因组稳定性和表观遗传调控之间取得平衡。
{"title":"Chromatin Transcription Elongation - A Structural Perspective.","authors":"Lucas Farnung","doi":"10.1016/j.jmb.2024.168845","DOIUrl":"10.1016/j.jmb.2024.168845","url":null,"abstract":"<p><p>In eukaryotic cells, transcription by RNA polymerase II occurs in the context of chromatin, requiring the transcription machinery to navigate through nucleosomes as it traverses gene bodies. Recent advances in structural biology have provided unprecedented insights into the mechanisms underlying transcription elongation. This review presents a structural perspective on transcription through chromatin, focusing on the latest findings from high-resolution structures of transcribing RNA polymerase II-nucleosome complexes. I discuss how RNA polymerase II, in concert with elongation factors such as SPT4/5, SPT6, ELOF1, and the PAF1 complex, engages with and transcribes through nucleosomes. The review examines the stepwise unwrapping of nucleosomal DNA as polymerase advances, the roles of elongation factors in facilitating this process, and the mechanisms of nucleosome retention and transfer during transcription. This structural perspective provides a foundation for understanding the intricate interplay between the transcription machinery and chromatin, offering insights into how cells balance the need for genetic accessibility with the maintenance of genome stability and epigenetic regulation.</p>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":" ","pages":"168845"},"PeriodicalIF":4.7,"publicationDate":"2024-10-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142542652","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Channelrhodopsins are light-gated ion channels consisting of seven transmembrane helices and a retinal chromophore, which are used as popular optogenetic tools for modulating neuronal activity. Cation channelrhodopsins (CCRs), first recognized as the photoreceptors in the chlorophyte Chlamydomonas reinhardtii, have since been identified in diverse species of green algae, as well in other unicellular eukaryotes. The CCRs from non-chlorophyte species are commonly referred to as bacteriorhodopsin-like cation channelrhodopsins, or BCCRs, as most of them feature the three characteristic amino acid residues of the “DTD motif” in the third transmembrane helix (TM3 or helix C) matching the canonical DTD motif of the well-studied archaeal light-driven proton pump bacteriorhodopsin. Here, we report characterization of HulaCCR1, a novel BCCR identified through metatranscriptomic analysis of a unicellular eukaryotic community in Lake Hula, Israel. Interestingly, HulaCCR1 has an ETD motif in which the first residue of the canonical motif is substituted for glutamate. Electrophysiological measurements of the wild-type and a mutant with a DTD motif of HulaCCR1 suggest the critical role of the first glutamate in spectral tuning and channel gating. Additionally, HulaCCR1 exhibits long extensions at the N- and C-termini. Photocurrents recorded from a truncated variant without the signal peptide predicted at the N-terminus were diminished, and membrane localization of the truncated variant significantly decreased, indicating that the signal peptide is important for membrane trafficking of HulaCCR1. These characteristics of HulaCCR1 would be related to a new biological significance in the original unidentified species, distinct from those known for other BCCRs.
{"title":"HulaCCR1, a pump-like cation channelrhodopsin discovered in a lake microbiome","authors":"Shunki Takaramoto , Shai Fainsod , Takashi Nagata , Andrey Rozenberg , Oded Béjà , Keiichi Inoue","doi":"10.1016/j.jmb.2024.168844","DOIUrl":"10.1016/j.jmb.2024.168844","url":null,"abstract":"<div><div>Channelrhodopsins are light-gated ion channels consisting of seven transmembrane helices and a retinal chromophore, which are used as popular optogenetic tools for modulating neuronal activity. Cation channelrhodopsins (CCRs), first recognized as the photoreceptors in the chlorophyte <em>Chlamydomonas reinhardtii</em>, have since been identified in diverse species of green algae, as well in other unicellular eukaryotes. The CCRs from non-chlorophyte species are commonly referred to as bacteriorhodopsin-like cation channelrhodopsins, or BCCRs, as most of them feature the three characteristic amino acid residues of the “DTD motif” in the third transmembrane helix (TM3 or helix C) matching the canonical DTD motif of the well-studied archaeal light-driven proton pump bacteriorhodopsin. Here, we report characterization of HulaCCR1, a novel BCCR identified through metatranscriptomic analysis of a unicellular eukaryotic community in Lake Hula, Israel. Interestingly, HulaCCR1 has an ETD motif in which the first residue of the canonical motif is substituted for glutamate. Electrophysiological measurements of the wild-type and a mutant with a DTD motif of HulaCCR1 suggest the critical role of the first glutamate in spectral tuning and channel gating. Additionally, HulaCCR1 exhibits long extensions at the N- and C-termini. Photocurrents recorded from a truncated variant without the signal peptide predicted at the N-terminus were diminished, and membrane localization of the truncated variant significantly decreased, indicating that the signal peptide is important for membrane trafficking of HulaCCR1. These characteristics of HulaCCR1 would be related to a new biological significance in the original unidentified species, distinct from those known for other BCCRs.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"436 23","pages":"Article 168844"},"PeriodicalIF":4.7,"publicationDate":"2024-10-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142542654","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-29DOI: 10.1016/j.jmb.2024.168846
José Aguilar-Rodríguez , Christopher M. Jakobson , Daniel F. Jarosz
Global modifier genes influence the mapping of genotypes onto phenotypes and fitness through their epistatic interactions with genetic variants on a massive scale. The first such factor to be identified, Hsp90, is a highly conserved molecular chaperone that plays a central role in protein homeostasis. Hsp90 is a “hub of hubs” that chaperones proteins engaged in many key cellular and developmental regulatory networks. These clients, which are enriched in kinases, transcription factors, and E3 ubiquitin ligases, drive diverse cellular functions and are themselves highly connected. By contrast to many other hub proteins, the abundance and activity of Hsp90 changes substantially in response to shifting environmental conditions. As a result, Hsp90 modifies the functional impact of many genetic variants simultaneously in a manner that depends on environmental stress. Studies in diverse organisms suggest that this coupling between Hsp90 function and challenging environments exerts a substantial impact on what parts of the genome are visible to natural selection, expanding adaptive opportunities when most needed. In this Perspective, we explore the multifaceted role of Hsp90 as global modifier of the genotype-phenotype-fitness map as well as its implications for evolution in nature and the clinic.
{"title":"The Hsp90 Molecular Chaperone as a Global Modifier of the Genotype-Phenotype-Fitness Map: An Evolutionary Perspective","authors":"José Aguilar-Rodríguez , Christopher M. Jakobson , Daniel F. Jarosz","doi":"10.1016/j.jmb.2024.168846","DOIUrl":"10.1016/j.jmb.2024.168846","url":null,"abstract":"<div><div>Global modifier genes influence the mapping of genotypes onto phenotypes and fitness through their epistatic interactions with genetic variants on a massive scale. The first such factor to be identified, Hsp90, is a highly conserved molecular chaperone that plays a central role in protein homeostasis. Hsp90 is a “hub of hubs” that chaperones proteins engaged in many key cellular and developmental regulatory networks. These clients, which are enriched in kinases, transcription factors, and E3 ubiquitin ligases, drive diverse cellular functions and are themselves highly connected. By contrast to many other hub proteins, the abundance and activity of Hsp90 changes substantially in response to shifting environmental conditions. As a result, Hsp90 modifies the functional impact of many genetic variants simultaneously in a manner that depends on environmental stress. Studies in diverse organisms suggest that this coupling between Hsp90 function and challenging environments exerts a substantial impact on what parts of the genome are visible to natural selection, expanding adaptive opportunities when most needed. In this Perspective, we explore the multifaceted role of Hsp90 as global modifier of the genotype-phenotype-fitness map as well as its implications for evolution in nature and the clinic.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"436 23","pages":"Article 168846"},"PeriodicalIF":4.7,"publicationDate":"2024-10-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142556806","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-29DOI: 10.1016/j.jmb.2024.168843
Lijun Quan, Jian Wu, Yelu Jiang, Deng Pan, Lyu Qiang
Understanding drug-protein interactions is crucial for elucidating drug mechanisms and optimizing drug development. However, existing methods have limitations in representing the three-dimensional structure of targets and capturing the complex relationships between drugs and targets. This study proposes a new method, DTA-GTOmega, for predicting drug-target binding affinity. DTA-GTOmega utilizes OmegaFold to predict protein three-dimensional structure and construct target graphs, while processing drug SMILES sequences with RDKit to generate drug graphs. By employing multi-layer graph transformer modules and co-attention modules, this method effectively integrates atomic-level features of drugs and residue-level features of targets, accurately modeling the complex interactions between drugs and targets, thereby significantly improving the accuracy of binding affinity predictions. Our method outperforms existing techniques on benchmark datasets such as KIBA, Davis, and BindingDB_Kd under cold-start setting. Moreover, DTA-GTOmega demonstrates competitive performance in real-world DTI scenarios involving DrugBank data and drug-target interactions related to cardiovascular and nervous system-related diseases, highlighting its robust generalization capabilities. Additionally, the introduced DTI evaluation metrics further validate DTA-GTOmega's potential in handling imbalanced data.
{"title":"DTA-GTOmega: Enhancing Drug-Target Binding Affinity Prediction with Graph Transformers Using OmegaFold Protein Structures.","authors":"Lijun Quan, Jian Wu, Yelu Jiang, Deng Pan, Lyu Qiang","doi":"10.1016/j.jmb.2024.168843","DOIUrl":"10.1016/j.jmb.2024.168843","url":null,"abstract":"<p><p>Understanding drug-protein interactions is crucial for elucidating drug mechanisms and optimizing drug development. However, existing methods have limitations in representing the three-dimensional structure of targets and capturing the complex relationships between drugs and targets. This study proposes a new method, DTA-GTOmega, for predicting drug-target binding affinity. DTA-GTOmega utilizes OmegaFold to predict protein three-dimensional structure and construct target graphs, while processing drug SMILES sequences with RDKit to generate drug graphs. By employing multi-layer graph transformer modules and co-attention modules, this method effectively integrates atomic-level features of drugs and residue-level features of targets, accurately modeling the complex interactions between drugs and targets, thereby significantly improving the accuracy of binding affinity predictions. Our method outperforms existing techniques on benchmark datasets such as KIBA, Davis, and BindingDB_Kd under cold-start setting. Moreover, DTA-GTOmega demonstrates competitive performance in real-world DTI scenarios involving DrugBank data and drug-target interactions related to cardiovascular and nervous system-related diseases, highlighting its robust generalization capabilities. Additionally, the introduced DTI evaluation metrics further validate DTA-GTOmega's potential in handling imbalanced data.</p>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":" ","pages":"168843"},"PeriodicalIF":4.7,"publicationDate":"2024-10-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142556805","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-28DOI: 10.1016/j.jmb.2024.168837
Elena S. Klimtchuk , Tatiana Prokaeva , Brian H. Spencer , Sherry Wong , Shreya Ghosh , Angela Urdaneta , Gareth Morgan , Thomas E. Wales , Olga Gursky
Immunoglobulin light chain amyloidosis (AL) is a life-threatening disease caused by the deposition of light chain (LC) and its fragments containing variable (VL) and portions of constant (CL) domains. AL patients feature either monoclonal free LCs (FLCs) circulating as covalent and noncovalent homodimers, or monoclonal immunoglobulin (Ig) wherein the LC and heavy chain (HC) form disulfide-linked heterodimers, or both. The role of full-length Ig in AL amyloidosis is unclear as prior studies focused on FLC or VL domain. We used a mammalian cell-based expression system to generate four AL patient-derived full-length IgGs, two non-AL IgG controls, and six corresponding FLC proteins derived from an IGLV6-57 germline precursor. Comparison of proteins’ secondary structure, thermal stability, proteolytic susceptibility, and disulfide link reduction suggested the importance of local vs. global conformational stability. Analysis of IgGs vs. corresponding FLCs using hydrogen–deuterium exchange mass spectrometry revealed major differences in the local conformation/dynamics of the CL domain. In all IgGs vs. FLCs, segments containing β-strand and α-helix βAC-αACBC were more dynamic/exposed while segment βDC-βEC was less dynamic/exposed. Notably, these segments overlapped proteolysis-prone regions whose in vivo cleavage generates LC fragments found in AL deposits. Altogether, the results suggest that preferential cleavage in segments βAC-αACBC of FLC or βDC-βEC of LC in IgG helps generate amyloid protein precursors. We propose that protecting these segments using small-molecule stabilizers, which bind to the interfacial cavities CL-CL in FLC and/or CL-CH1 in IgG, is a potential therapeutic strategy to complement current approaches targeting VL-VL or VL-CL stabilization of LC dimer.
{"title":"Conformational Differences in the Light Chain Constant Domain of Immunoglobulin G and Free Light Chain May Influence Proteolysis in AL Amyloidosis","authors":"Elena S. Klimtchuk , Tatiana Prokaeva , Brian H. Spencer , Sherry Wong , Shreya Ghosh , Angela Urdaneta , Gareth Morgan , Thomas E. Wales , Olga Gursky","doi":"10.1016/j.jmb.2024.168837","DOIUrl":"10.1016/j.jmb.2024.168837","url":null,"abstract":"<div><div>Immunoglobulin light chain amyloidosis (AL) is a life-threatening disease caused by the deposition of light chain (LC) and its fragments containing variable (V<sub>L</sub>) and portions of constant (C<sub>L</sub>) domains. AL patients feature either monoclonal free LCs (FLCs) circulating as covalent and noncovalent homodimers, or monoclonal immunoglobulin (Ig) wherein the LC and heavy chain (HC) form disulfide-linked heterodimers, or both. The role of full-length Ig in AL amyloidosis is unclear as prior studies focused on FLC or V<sub>L</sub> domain. We used a mammalian cell-based expression system to generate four AL patient-derived full-length IgGs, two non-AL IgG controls, and six corresponding FLC proteins derived from an <em>IGLV6-57</em> germline precursor. Comparison of proteins’ secondary structure, thermal stability, proteolytic susceptibility, and disulfide link reduction suggested the importance of local <em>vs.</em> global conformational stability. Analysis of IgGs <em>vs.</em> corresponding FLCs using hydrogen–deuterium exchange mass spectrometry revealed major differences in the local conformation/dynamics of the C<sub>L</sub> domain. In all IgGs <em>vs.</em> FLCs, segments containing β-strand and α-helix βA<sub>C</sub>-αA<sub>C</sub>B<sub>C</sub> were more dynamic/exposed while segment βD<sub>C</sub>-βE<sub>C</sub> was less dynamic/exposed. Notably, these segments overlapped proteolysis-prone regions whose <em>in vivo</em> cleavage generates LC fragments found in AL deposits. Altogether, the results suggest that preferential cleavage in segments βA<sub>C</sub>-αA<sub>C</sub>B<sub>C</sub> of FLC or βD<sub>C</sub>-βE<sub>C</sub> of LC in IgG helps generate amyloid protein precursors. We propose that protecting these segments using small-molecule stabilizers, which bind to the interfacial cavities C<sub>L</sub>-C<sub>L</sub> in FLC and/or C<sub>L</sub>-C<sub>H1</sub> in IgG, is a potential therapeutic strategy to complement current approaches targeting V<sub>L</sub>-V<sub>L</sub> or V<sub>L</sub>-C<sub>L</sub> stabilization of LC dimer.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"436 23","pages":"Article 168837"},"PeriodicalIF":4.7,"publicationDate":"2024-10-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142566743","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-28DOI: 10.1016/j.jmb.2024.168839
Janine Kamps , Patricia Yuste-Checa , Fatemeh Mamashli , Matthias Schmitz , Maria Georgina Herrera , Susana Margarida da Silva Correia , Kalpshree Gogte , Verian Bader , Inga Zerr , F. Ulrich Hartl , Andreas Bracher , Konstanze F. Winklhofer , Jörg Tatzelt
Several proteins associated with neurodegenerative diseases, such as the mammalian prion protein (PrP), undergo liquid–liquid phase separation (LLPS), which led to the hypothesis that condensates represent precursors in the formation of neurotoxic protein aggregates. However, the mechanisms that trigger aberrant phase separation are incompletely understood. In prion diseases, protease-resistant and infectious amyloid fibrils are composed of N-terminally truncated PrP, termed C2-PrP. C2-PrP is generated by regulated proteolysis (β-cleavage) of the cellular prion protein (PrPC) specifically upon prion infection, suggesting that C2-PrP is a misfolding-prone substrate for the propagation of prions. Here we developed a novel assay to investigate the role of both LLPS and β-cleavage in the formation of C2-PrP aggregates. We show that β-cleavage induces the formation of C2-PrP aggregates, but only when full-length PrP had formed biomolecular condensates via LLPS before proteolysis. In contrast, C2-PrP remains soluble after β-cleavage of non-phase-separated PrP. To investigate whether extracellular molecular chaperones modulate LLPS of PrP and/or misfolding of C2-PrP, we focused on Clusterin. Clusterin does not inhibit LLPS of full-length PrP, however, it prevents aggregation of C2-PrP after β-cleavage of phase-separated PrP. Furthermore, Clusterin interferes with the in vitro amplification of infectious human prions isolated from Creutzfeldt-Jakob disease patients. Our study revealed that regulated proteolysis triggers aberrant phase transition of biomolecular condensates into aggregates and identified Clusterin as a component of the extracellular quality control pathway to prevent the formation and propagation of pathogenic PrP conformers.
{"title":"Regulated Proteolysis Induces Aberrant Phase Transition of Biomolecular Condensates into Aggregates: A Protective Role for the Chaperone Clusterin","authors":"Janine Kamps , Patricia Yuste-Checa , Fatemeh Mamashli , Matthias Schmitz , Maria Georgina Herrera , Susana Margarida da Silva Correia , Kalpshree Gogte , Verian Bader , Inga Zerr , F. Ulrich Hartl , Andreas Bracher , Konstanze F. Winklhofer , Jörg Tatzelt","doi":"10.1016/j.jmb.2024.168839","DOIUrl":"10.1016/j.jmb.2024.168839","url":null,"abstract":"<div><div>Several proteins associated with neurodegenerative diseases, such as the mammalian prion protein (PrP), undergo liquid–liquid phase separation (LLPS), which led to the hypothesis that condensates represent precursors in the formation of neurotoxic protein aggregates. However, the mechanisms that trigger aberrant phase separation are incompletely understood. In prion diseases, protease-resistant and infectious amyloid fibrils are composed of N-terminally truncated PrP, termed C2-PrP. C2-PrP is generated by regulated proteolysis (β-cleavage) of the cellular prion protein (PrP<sup>C</sup>) specifically upon prion infection, suggesting that C2-PrP is a misfolding-prone substrate for the propagation of prions. Here we developed a novel assay to investigate the role of both LLPS and β-cleavage in the formation of C2-PrP aggregates. We show that β-cleavage induces the formation of C2-PrP aggregates, but only when full-length PrP had formed biomolecular condensates via LLPS before proteolysis. In contrast, C2-PrP remains soluble after β-cleavage of non-phase-separated PrP. To investigate whether extracellular molecular chaperones modulate LLPS of PrP and/or misfolding of C2-PrP, we focused on Clusterin. Clusterin does not inhibit LLPS of full-length PrP, however, it prevents aggregation of C2-PrP after β-cleavage of phase-separated PrP. Furthermore, Clusterin interferes with the <em>in vitro</em> amplification of infectious human prions isolated from Creutzfeldt-Jakob disease patients. Our study revealed that regulated proteolysis triggers aberrant phase transition of biomolecular condensates into aggregates and identified Clusterin as a component of the extracellular quality control pathway to prevent the formation and propagation of pathogenic PrP conformers.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"436 23","pages":"Article 168839"},"PeriodicalIF":4.7,"publicationDate":"2024-10-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142542653","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-26DOI: 10.1016/j.jmb.2024.168842
Chen-Yu Lo , Adron R. Ung , Tirthankar Koley , Scott W. Nelson , Yang Gao
The apicoplast DNA polymerase (apPol) from Plasmodium falciparum is essential for the parasite’s survival, making it a prime target for antimalarial therapies. Here, we present cryo-electron microscopy structures of the apPol in complex with DNA and incoming nucleotide, offering insights into its molecular mechanisms. Our structural analysis reveals that apPol contains critical residues for high-fidelity DNA synthesis, but lacks certain structural elements to confer processive DNA synthesis during replication, suggesting the presence of additional accessory factors. The enzyme exhibits large-scale conformational changes upon DNA and nucleotide binding, particularly within the fingers and thumb subdomains. These movements reveal potential allosteric sites that could serve as targets for drug design. Our findings provide a foundation for advancing the understanding of apPol’s unique functional mechanisms and potentially offering new avenues for the development of novel inhibitors and therapeutic interventions against malaria.
恶性疟原虫的 apicoplast DNA 聚合酶（apPol）对寄生虫的生存至关重要，因此成为抗疟疗法的主要靶标。在这里，我们展示了apPol与DNA和传入核苷酸复合物的冷冻电子显微镜结构，为了解其分子机制提供了线索。我们的结构分析表明，apPol含有高保真DNA合成的关键残基，但缺乏某些结构元素，无法在复制过程中进行DNA合成，这表明还存在其他辅助因素。该酶在与 DNA 和核苷酸结合时，尤其是在手指和拇指亚域内，表现出大规模的构象变化。这些变化揭示了潜在的异构位点，可作为药物设计的靶点。我们的发现为进一步了解 apPol 的独特功能机制奠定了基础，并有可能为开发新型抑制剂和疟疾治疗干预措施提供新的途径。
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The prefoldin complex is a heterohexameric, evolutionarily conserved co-chaperone that assists in folding of polypeptides downstream of the protein translation machinery. Loss of prefoldin function leads to impaired solubility of cellular proteins. The degradation of proteins by the proteasome is an integral part of protein homeostasis. Failure of regulated protein degradation can lead to the accumulation of misfolded and defective proteins. We show that prefoldin subunit 5 is required for proteasome activity by contributing to the assembly of the 26S proteasome. In particular, we found that absence of the prefoldin subunit 5 impairs formation of the Rpt ring subcomplex of the proteasome. Concomitant deletion of PFD5 and HSM3, a chaperone for assembly of the ATPase subunits comprising the Rpt ring, exacerbates this effect, suggesting a synergistic relationship between the two factors in proteasome assembly. Thus, our findings reveal a regulatory mechanism wherein prefoldin subunit 5 plays a crucial role in maintaining proteasome integrity, thereby influencing the degradation of proteins.
{"title":"Identification of a Non-canonical Function of Prefoldin Subunit 5 in Proteasome Assembly","authors":"Somayeh Shahmoradi Ghahe , Krzysztof Drabikowski , Monika Stasiak , Ulrike Topf","doi":"10.1016/j.jmb.2024.168838","DOIUrl":"10.1016/j.jmb.2024.168838","url":null,"abstract":"<div><div>The prefoldin complex is a heterohexameric, evolutionarily conserved co-chaperone that assists in folding of polypeptides downstream of the protein translation machinery. Loss of prefoldin function leads to impaired solubility of cellular proteins. The degradation of proteins by the proteasome is an integral part of protein homeostasis. Failure of regulated protein degradation can lead to the accumulation of misfolded and defective proteins. We show that prefoldin subunit 5 is required for proteasome activity by contributing to the assembly of the 26S proteasome. In particular, we found that absence of the prefoldin subunit 5 impairs formation of the Rpt ring subcomplex of the proteasome. Concomitant deletion of <em>PFD5</em> and <em>HSM3</em>, a chaperone for assembly of the ATPase subunits comprising the Rpt ring, exacerbates this effect, suggesting a synergistic relationship between the two factors in proteasome assembly. Thus, our findings reveal a regulatory mechanism wherein prefoldin subunit 5 plays a crucial role in maintaining proteasome integrity, thereby influencing the degradation of proteins.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"436 23","pages":"Article 168838"},"PeriodicalIF":4.7,"publicationDate":"2024-10-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142566753","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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