Journal of Molecular Biology最新文献

{"title":"Drug Discovery for Diseases with High Unmet Need Through Perturbation of Biomolecular Condensates","authors":"Bahareh Eftekharzadeh,&nbsp;Aislinn Mayfield,&nbsp;Michael G. Kauffman,&nbsp;John F. Reilly","doi":"10.1016/j.jmb.2024.168855","DOIUrl":"10.1016/j.jmb.2024.168855","url":null,"abstract":"<div><div>Biomolecular condensates (BMCs), play significant roles in organizing cellular functions in the absence of membranes through phase separation events involving RNA, proteins, and RNA-protein complexes. These membrane-less organelles form dynamic multivalent weak interactions, often involving intrinsically disordered proteins or regions (IDPs/IDRs). However, the nature of these crucial interactions, how most of these organelles are organized and are functional, remains unknown. Aberrant condensates have been implicated in neurodegenerative diseases and various cancers, presenting novel therapeutic opportunities for small molecule condensate modulators. Recent advancements in optogenetic technologies, particularly Corelet, enable precise manipulation of BMC dynamics within living cells, facilitating high-throughput screening for small molecules that target these complex structures. By elucidating the molecular mechanisms governing BMC formation and function, this innovative approach holds promise to unlock therapeutic strategies against previously “undruggable” protein targets, paving the way for effective interventions in disease.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"436 23","pages":"Article 168855"},"PeriodicalIF":4.7,"publicationDate":"2024-11-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142602659","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}

{"title":"Determinants in the HTLV-1 Capsid Major Homology Region that are Critical for Virus Particle Assembly","authors":"Huixin Yang ,&nbsp;William G. Arndt ,&nbsp;Wei Zhang ,&nbsp;Louis M. Mansky","doi":"10.1016/j.jmb.2024.168851","DOIUrl":"10.1016/j.jmb.2024.168851","url":null,"abstract":"<div><div>The Gag protein of retroviruses is the primary driver of virus particle assembly. Particle morphologies among retroviral genera are distinct, with intriguing differences observed relative to human immunodeficiency virus type 1 (HIV-1), particularly that of human T-cell leukemia virus type 1 (HTLV-1). In contrast to HIV-1 and other retroviruses where the capsid (CA) carboxy-terminal domain (CTD) possesses the key amino acid determinants involved in driving Gag-Gag interactions, we have previously demonstrated that the amino-terminal domain (NTD) encodes the key residues crucial for Gag multimerization and immature particle production. Here in this study, we sought to thoroughly interrogate the conserved HTLV-1 major homology region (MHR) of the CA_CTD to determine whether this region harbors residues important for particle assembly. In particular, site-directed mutagenesis of the HTLV-1 MHR was conducted, and mutants were analyzed for their ability to impact Gag subcellular distribution, particle production and morphology, as well as the CA-CA assembly kinetics. Several key residues (<em>i.e.</em>, Q138, E142, Y144, F147 and R150), were found to significantly impact Gag multimerization and particle assembly. Taken together, these observations imply that while the HTLV-1 CA_NTD acts as the major region involved in CA-CA interactions, residues in the MHR can impact Gag multimerization, particle assembly and morphology, and likely play an important role in the conformation the CA_CTD that is required for CA-CA interactions.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"436 24","pages":"Article 168851"},"PeriodicalIF":4.7,"publicationDate":"2024-11-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142589675","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}

{"title":"Pim1 is Critical in T-cell-independent B-cell Response and MAPK Activation in B Cells","authors":"Dongya Cui ,&nbsp;Yongguang Zhang ,&nbsp;Baijiao Zheng ,&nbsp;Liling Chen ,&nbsp;Jianhui Wei ,&nbsp;Danfeng Lin ,&nbsp;Miaohui Huang ,&nbsp;Hekang Du ,&nbsp;Qi Chen","doi":"10.1016/j.jmb.2024.168824","DOIUrl":"10.1016/j.jmb.2024.168824","url":null,"abstract":"<div><div>The Pim family consists of three members that encode a distinct class of highly conserved serine/threonine kinases. In this study, we generated and examined mice with hematopoiesis-specific deletion of Pim1 and bone marrow (BM) chimeric mice with B-cell-specific targeted deletion of Pim1. Pim1 was expressed at all stages of B-cell development and hematopoietic-specific deletion of Pim1 altered B-cell development in BM, spleen and peritoneal. However, Pim1 deficiency did not affect T-cell development. Studies of BM chimeric mice showed that Pim1 is required in a cell-intrinsic manner to maintain normal B-cell development. Pim1 deficiency led to significant changes in B cell antibody responses. Additionally, Pim1 deficiency resulted in reduced B cell receptor (BCR)-induced cell proliferation and cell cycle progression. Examination of the various BCR-activated signaling pathways in Pim1-deficient B cells reveals defective activation of mitogen-activated protein kinases (MAPKs), which are known to regulate genes involved in cell proliferation and survival. qRT-PCR analysis of BCR-engaged B cells from Pim1-deficient B cells revealed reduced expression of cyclin-dependent kinase (CDK) and cyclin genes, including CDK2, CCNB1 and CCNE1. In conclusion, Pim1 plays a crucial role in B-cell development and B cell activation.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"436 23","pages":"Article 168824"},"PeriodicalIF":4.7,"publicationDate":"2024-11-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142589676","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}

{"title":"A Nanobody Toolbox for Recognizing Distinct Epitopes on Cas9","authors":"Jack Boylan ,&nbsp;Rebecca A Shrem ,&nbsp;Isabel C. Vallecillo-Viejo ,&nbsp;Craig L. Duvall ,&nbsp;Brian E. Wadzinski ,&nbsp;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}

{"title":"Translation Complex Profile Sequencing Allows Discrimination of Leaky Scanning and Reinitiation in Upstream Open Reading Frame-controlled Translation","authors":"Dmitri E. Andreev ,&nbsp;Jack A.S. Tierney ,&nbsp;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}

{"title":"HulaCCR1, a pump-like cation channelrhodopsin discovered in a lake microbiome","authors":"Shunki Takaramoto ,&nbsp;Shai Fainsod ,&nbsp;Takashi Nagata ,&nbsp;Andrey Rozenberg ,&nbsp;Oded Béjà ,&nbsp;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}

{"title":"The Hsp90 Molecular Chaperone as a Global Modifier of the Genotype-Phenotype-Fitness Map: An Evolutionary Perspective","authors":"José Aguilar-Rodríguez ,&nbsp;Christopher M. Jakobson ,&nbsp;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}

{"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}

{"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 ,&nbsp;Tatiana Prokaeva ,&nbsp;Brian H. Spencer ,&nbsp;Sherry Wong ,&nbsp;Shreya Ghosh ,&nbsp;Angela Urdaneta ,&nbsp;Gareth Morgan ,&nbsp;Thomas E. Wales ,&nbsp;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_L) and portions of constant (C_L) 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_L 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_L domain. In all IgGs <em>vs.</em> FLCs, segments containing β-strand and α-helix βA_C-αA_CB_C were more dynamic/exposed while segment βD_C-βE_C 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_C-αA_CB_C of FLC or βD_C-βE_C 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_L-C_L in FLC and/or C_L-C_H1 in IgG, is a potential therapeutic strategy to complement current approaches targeting V_L-V_L or V_L-C_L 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}

{"title":"Regulated Proteolysis Induces Aberrant Phase Transition of Biomolecular Condensates into Aggregates: A Protective Role for the Chaperone Clusterin","authors":"Janine Kamps ,&nbsp;Patricia Yuste-Checa ,&nbsp;Fatemeh Mamashli ,&nbsp;Matthias Schmitz ,&nbsp;Maria Georgina Herrera ,&nbsp;Susana Margarida da Silva Correia ,&nbsp;Kalpshree Gogte ,&nbsp;Verian Bader ,&nbsp;Inga Zerr ,&nbsp;F. Ulrich Hartl ,&nbsp;Andreas Bracher ,&nbsp;Konstanze F. Winklhofer ,&nbsp;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^C) 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}