Protein Science最新文献

{"title":"Conkazal-M1 from the MKAVA family of conotoxins: A dual-function protease inhibitor and neuroactive peptide.","authors":"Celeste M Hackney, Thomas Lund Koch, Nicklas Lund Ryding, Aymeric Rogalski, Kevin Chase, Matías Leonel Giglio, Samuel S Espino, Zildjian G Acyatan, Maren Watkins, Baldomero M Olivera, Helena Safavi-Hemami, Kaare Teilum, Lars Ellgaard","doi":"10.1002/pro.70580","DOIUrl":"https://doi.org/10.1002/pro.70580","url":null,"abstract":"<p><p>Marine cone snails produce a diverse array of bioactive peptides, known as conotoxins, in their venom. Given their high target potency and specificity, conotoxins are attractive compounds for the development of precision research tools and pharmacological agents. Here, we provide the first experimental characterization of a conotoxin from the MKAVA superfamily, conkazal-M1, from Conus magus. Using NMR spectroscopy, we show that conkazal-M1 adopts a fold characteristic of the Kazal-type protease inhibitor family, featuring a Glu residue at the inhibitory P1 position. Recombinantly expressed conkazal-M1 inhibits the proteolytic activity of Subtilisin A with an apparent Ki of 1.1 μM. In addition, conkazal-M1 partially inhibits calcium transients in mouse sensory neurons, suggesting a potential role in modulating ion-channel activity, as seen for many other toxins. The dual function of conkazal-M1 in protease inhibition and neuroactivity is analogous to the dual function of several toxins harboring a Kunitz-type fold. The well-conserved sequence of the MKAVAs indicates an evolutionary trajectory in which these proteins face an adaptive conflict, where mutations that enhance one activity compromise the other. Collectively, this work provides new structural and functional insights into a previously uncharacterized toxin superfamily in cone snails, illustrates how structural scaffolds can be repurposed for functions that diverge from the original while retaining their overall structure, and expands our understanding of the toxin arsenal available to venomous animals.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"35 5","pages":"e70580"},"PeriodicalIF":5.2,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13101452/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147779495","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"ATP modulates holdase activity of fungal and human 110-kilodalton heat shock proteins to promote protein folding.","authors":"Justin M Kidd, Cancan Sun, Alissa Garay, Mitchell Keplinger, Colin Richter, Aaron E May, Qinglian Liu","doi":"10.1002/pro.70575","DOIUrl":"https://doi.org/10.1002/pro.70575","url":null,"abstract":"<p><p>Heat shock proteins of the 70-kDa family (Hsp70s) are highly abundant and conserved molecular chaperones that help preserve proteostasis primarily by facilitating proper protein folding. Heat shock protein of the 110-kDa family (Hsp110s), a specialized branch of the Hsp70/Hsp110 superfamily, function both as nucleotide exchange factor (NEF) cochaperones for Hsp70s and as independent \"holdase\" chaperones that stabilize non-native polypeptides to prevent aggregation and facilitate downstream refolding by Hsp70s. While Hsp110 NEF activity is well characterized, the consequences of adenosine 5'-triphosphate (ATP) binding for Hsp110 holdase behavior have remained largely unexplored. Although holdase activity is generally considered nucleotide-independent, reports of ATP-dependent effects have raised questions about the underlying mechanism. Here, we examined the biochemical properties of Multicopy Suppressor of ira1 3 (Msi3), the sole Hsp110 in Candida albicans, to dissect the role of ATP in holdase function. We first identified an inhibitory effect of elevated Mg²⁺ concentrations on Msi3 holdase activity. This inhibitory effect is counteracted by the intrinsically disordered C-terminal segment, revealing a distinct stabilization role for this region, previously of unknown function. In addition, ATP alleviates inhibition by elevated Mg²⁺, providing an explanation for an apparent ATP-dependence observed previously. Interestingly, although dispensable for aggregation suppression, ATP modulates Msi3 holdase activity for refolding competence by broadening the concentration range over which it remains productive. Increasing Msi3 concentration improved overall downstream refolding recovery but slowed refolding kinetics, and ATP alleviated this kinetic constraint. Analyses of Hsp105, the major human Hsp110, suggest that these biochemical properties are largely conserved. Together, these findings suggest that ATP modulates Hsp110 holdase activity by tuning the balance between substrate sequestration and engagement dynamics, revealing an ATP-dependent regulatory dimension of Hsp110 holdase function that is mechanistically distinct from its NEF activity.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"35 5","pages":"e70575"},"PeriodicalIF":5.2,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13114805/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147779513","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Correction to \"Detergent-mediated cryoprotection: Optimizing freezing and storage conditions for G protein-coupled receptors\".","authors":"","doi":"10.1002/pro.70597","DOIUrl":"10.1002/pro.70597","url":null,"abstract":"","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"35 5","pages":"e70597"},"PeriodicalIF":5.2,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13114779/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147779458","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Proteolytic control of mitochondrial protein translocases.","authors":"Lara Kroczek, Thomas Langer","doi":"10.1002/pro.70553","DOIUrl":"10.1002/pro.70553","url":null,"abstract":"<p><p>Mitochondria are essential organelles that drive numerous cellular processes, including energy metabolism, ion homeostasis, and programmed cell death. This functional versatility relies on a highly dynamic proteome whose composition is continuously remodeled to meet changing cellular and environmental demands. Central to this remodeling are mitochondrial proteases (termed mitoproteases), which maintain protein quality and regulate mitochondrial function through selective processing and degradation events. Their activity ensures rapid degradation of regulatory proteins and dynamically adjusts components of multiprotein complexes. Among their most critical targets are elements of the mitochondrial protein import machinery. By modulating translocase stability and by processing preproteins during translocation, mitoproteases enable precise control over the organelle's proteome, aligning mitochondrial function with the cell's metabolic state. This review discusses how mitoproteases maintain translocase integrity and dynamically regulate mitochondrial protein import and the mitochondrial proteome.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"35 5","pages":"e70553"},"PeriodicalIF":5.2,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13092810/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147723651","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Quality control of protein import into mammalian mitochondria.","authors":"Madeleine Goldstein, Laurie Lee-Glover, Hilla Weidberg","doi":"10.1002/pro.70585","DOIUrl":"10.1002/pro.70585","url":null,"abstract":"<p><p>Mitochondrial function depends on the continuous import of hundreds of nuclear-encoded proteins. Targeting and translocation of mitochondrial proteins is a multistep process that is inherently vulnerable to defects in cytosolic quality control systems as well as perturbations in mitochondrial protein import machinery and organelle function. Failure of mitochondrial protein import has dual consequences: it compromises mitochondrial biogenesis and activity, and it poses a cytosolic proteotoxic threat due to the accumulation of unimported precursor proteins. Accordingly, mitochondrial protein import defects are detrimental to cellular homeostasis and are associated with a wide range of disorders, including metabolic and neurodegenerative diseases. Cells therefore rely on layered quality control systems that monitor mitochondrial protein biogenesis and mitigate stress arising from mislocalized mitochondrial proteins. In this review, we summarize recent progress in understanding pathways that modulate mitochondrial protein import and the fate of unimported proteins in mammals. We highlight cytosolic and mitochondrial protein quality control mechanisms and discuss how import defects are translated into cellular stress responses and mitochondrial protective programs to restore cellular and mitochondrial homeostasis.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"35 5","pages":"e70585"},"PeriodicalIF":5.2,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13125379/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147779036","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Family-level specialization in protein domain insertion architectures.","authors":"R Dustin Schaeffer, Rui Guo, Jing Zhang, Qian Cong, Nick V Grishin","doi":"10.1002/pro.70586","DOIUrl":"https://doi.org/10.1002/pro.70586","url":null,"abstract":"<p><p>Domain insertion creates architectures where one domain interrupts another's sequence. Analysis across 2.7 million classified domains reveals that insertions occur in 20% of multidomain proteins, with 331 families exhibiting consistent architectural roles: 162 function exclusively as hosts, while 169 exclusively serve as inserted modules, such as zinc-binding dehydrogenases appearing as insertions across 450 events. The remaining 1116 families with sufficient insertion activity demonstrate versatile behavior, adopting different roles depending on partnership context. Size analysis shows inserted domains are consistently smaller than their hosts (median 115 vs. 199 residues), with role-consistent families exhibiting 1.7-fold size differences. Insertions frequently involve domains from different structural superfamilies: 31,925 events (65.8% of total) occur between families from different H-groups, such as P-loop hydrolases with tRNA modification domains. While most insertions are simple single-level architectures, insertion mechanisms can create complex organizations, including six-level nested structures in cyanobacterial RNA polymerase. This work provides a comprehensive dataset of 48,551 insertion events across 5701 families, with quantitative characterization of size relationships and partnership patterns that can inform structure prediction and protein design efforts.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"35 5","pages":"e70586"},"PeriodicalIF":5.2,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13114784/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147779527","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"AlphaFold-driven discovery of oxysterol-binding protein-related protein-phosphoinositide 3-, 4-, and 5-phosphatase interactions using new generation confidence scores.","authors":"Filippo Dall'Armellina, Sylvie Urbé, Daniel J Rigden","doi":"10.1002/pro.70572","DOIUrl":"10.1002/pro.70572","url":null,"abstract":"<p><p>Non-vesicular lipid transport contributes to the regulation of membrane composition and organelle function at membrane contact sites. OSBP-related proteins (ORPs) are central to this process, yet their interaction networks remain incompletely defined. Here, we systematically screened potential interactions between ORPs and phosphoinositide 3-, 4-, and 5-phosphatases using AlphaPulldown2, AlphaFold2-Multimer, and AlphaFold3. We established a protocol for model generation by combining AlphaFold2-Multimer predictions (including five-replicates) with an AlphaPulldown2 interaction screen across around 200 protein pairs, and with AlphaFold3 predictions including lipid-bound and multimeric assemblies. Interface confidence was assessed for consistency using the weighted ipTM + pTM metric, actifpTM, new generation ipSAE scoring, and FoldSeek-Multimer clustering. We further evaluated the protein pairs' biological plausibility based on subcellular localization data, in silico membrane insertion, evolutionary conservation via ConSurf, and protein binding interface analysis using the deep learning tool PeSTo. This integrative protocol uncovered functionally conserved binding modes in the SAC1 lipid phosphatase with the ORP family, particularly with ORP11, and predicted functionally relevant protein-lipid interfaces.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"35 5","pages":"e70572"},"PeriodicalIF":5.2,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13081691/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147691848","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Biophysical characterization of Cyclophilin B reveals membrane localization as its primary functional determinant as a prolyl isomerase.","authors":"Sarah C DeVoe, Thomas C Yost, Ashley J Newton, Gavin A Grever, Melissa Fernandez Ayala, Wendell P Griffith, Robert D Latvala, Philipp A M Schmidpeter","doi":"10.1002/pro.70579","DOIUrl":"10.1002/pro.70579","url":null,"abstract":"<p><p>The endoplasmic reticulum (ER) provides a specialized environment for the folding of secreted and membrane proteins, a process supported by many different chaperones. Among these chaperones, peptidyl-prolyl cis/trans isomerases (PPIases) catalyze a rate-limiting conformational step in protein folding, yet the principles governing isoform-specific function of PPIases remain poorly defined. Cyclophilin B (CypB), an ER-resident PPIase, has been implicated in early folding events, but whether its activity reflects biochemical adaptation to the ER environment is unclear. Here, we report the biophysical characterization of human CypB and compare it with the cytosolic isoform Cyclophilin A. Spectroscopic and enzymatic analyses show that CypB adopts the canonical cyclophilin fold and displays catalytic activity toward multiple substrates under both cytosolic- and ER-mimicking conditions, indicating that its enzymatic properties are not uniquely tuned to the ER milieu. Confocal imaging confirms that full-length CypB is enriched in the ER, and that removal of its N-terminal segment disrupts this localization. Together, these results indicate that subcellular localization, mediated by an N-terminal membrane anchor, rather than catalytic specialization, may define the physiological role of CypB. Our findings underscore compartmentalization as a central organizing principle of proteostasis in the secretory pathway.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"35 5","pages":"e70579"},"PeriodicalIF":5.2,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13084196/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147691868","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Proteostasis at the mitochondrial outer membrane: Quality control of mitochondrial protein transport.","authors":"Shunsuke Matsumoto, Suzuka Ono, Toshiya Endo","doi":"10.1002/pro.70587","DOIUrl":"https://doi.org/10.1002/pro.70587","url":null,"abstract":"<p><p>Mitochondria are enclosed by a double-membrane structure composed of the outer and inner membranes, and this architectural organization underlies their diverse cellular functions. In particular, the mitochondrial outer membrane serves as an essential interface between the cytosol and the mitochondrial interior, regulating the flux of proteins, lipids, small molecules, and ions through the coordinated activities of its resident proteome. Consequently, structural and functional defects of outer membrane proteins are subject to continuous surveillance, and aberrant proteins are rapidly recognized and degraded. Defects in precursor translocation or translation can lead to the stalling of precursor proteins at the primary protein import gate, the TOM complex. Such situations are resolved by multiple quality control systems operating across both the mitochondria and the cytosol. In addition, proteins normally destined for the endoplasmic reticulum or peroxisomes may be mistargeted to mitochondria, and these mislocalized proteins are likewise managed through dedicated mechanisms that promote their degradation or re-targeting. In this review, we summarize current insights into the molecular factors and mechanisms that maintain proteostasis at the mitochondrial outer membrane.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"35 5","pages":"e70587"},"PeriodicalIF":5.2,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13106219/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147779041","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"ssRNA bacteriophage metagenomes reveal a diverse set of novel protein families.","authors":"Jānis Rūmnieks, Ieva Baltā, Mihails Šišovs, Kaspars Tārs","doi":"10.1002/pro.70582","DOIUrl":"https://doi.org/10.1002/pro.70582","url":null,"abstract":"<p><p>The bacteriophages with single-stranded RNA (ssRNA) genomes (class Leviviricetes) are among the simplest known viruses that encode only three core proteins: a receptor-binding protein, a capsid protein, and an RNA-dependent RNA polymerase. The number of isolated ssRNA phages has remained very low, but the accumulating RNA metagenome data have uncovered a large variety of these viruses in many environments. Besides the core proteins, many of these genomes putatively encode additional proteins, which up to now have remained uncharacterized. We looked for non-conserved open reading frames (ORFs) in Leviviricetes sequences from the IMG/VR virus metagenome database and used sequence- and structure-based clustering to organize them into similarity groups. Potential ORFs were found throughout the ssRNA phage genomes but almost exclusively on the positive-sense RNA strand, suggestive of their protein-coding potential. The prevalence of the non-conserved ORFs varied in various phage lineages, and their distribution among different genome positions was markedly uneven. Most of the identified ORFs encode all-α proteins, a portion of which contain transmembrane segments that resemble a group of known ssRNA phage lysis proteins, while many others represent previously uncharacterized families of globular or semi-globular α-helical proteins. We additionally uncovered a major class of globular α/β proteins and experimentally determined the structure of a representative protein of this group. These results pave the way for further functional studies of novel ssRNA phage proteins for a better understanding of this diverse virus group.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"35 5","pages":"e70582"},"PeriodicalIF":5.2,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13114773/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147778984","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}