Protein Science最新文献

{"title":"Protein stability models fail to capture epistatic interactions of double point mutations.","authors":"Henry Dieckhaus, Brian Kuhlman","doi":"10.1002/pro.70003","DOIUrl":"10.1002/pro.70003","url":null,"abstract":"<p><p>There is strong interest in accurate methods for predicting changes in protein stability resulting from amino acid mutations to the protein sequence. Recombinant proteins must often be stabilized to be used as therapeutics or reagents, and destabilizing mutations are implicated in a variety of diseases. Due to increased data availability and improved modeling techniques, recent studies have shown advancements in predicting changes in protein stability when a single-point mutation is made. Less focus has been directed toward predicting changes in protein stability when there are two or more mutations. Here, we analyze the largest available dataset of double point mutation stability and benchmark several widely used protein stability models on this and other datasets. We find that additive models of protein stability perform surprisingly well on this task, achieving similar performance to comparable non-additive predictors according to most metrics. Accordingly, we find that neither artificial intelligence-based nor physics-based protein stability models consistently capture epistatic interactions between single mutations. We observe one notable deviation from this trend, which is that epistasis-aware models provide marginally better predictions than additive models on stabilizing double point mutations. We develop an extension of the ThermoMPNN framework for double mutant modeling, as well as a novel data augmentation scheme, which mitigates some of the limitations in currently available datasets. Collectively, our findings indicate that current protein stability models fail to capture the nuanced epistatic interactions between concurrent mutations due to several factors, including training dataset limitations and insufficient model sensitivity.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 1","pages":"e70003"},"PeriodicalIF":4.5,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11659742/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142865268","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":"Proline variants in the BRCA1 coiled-coil domain disrupt folding and binding to PALB2.","authors":"Chrissy N S Baker, Precious Grace C Pajela, Davis E Martin, Sergei V Dzyuba, Mikaela D Stewart","doi":"10.1002/pro.5240","DOIUrl":"10.1002/pro.5240","url":null,"abstract":"<p><p>Inherited mutations in the genes coding for the tumor suppressor proteins BRCA1 and PALB2 can lead to increased risk of breast and ovarian cancer. Upon DNA damage, these two proteins form a complex to promote double-stranded break repair via homologous recombination. Missense mutations in either BRCA1 or PALB2 that disrupt this important interaction result in loss of effective DNA damage repair and are associated with breast tumorigenesis. However, the overwhelming majority of missense mutations found in the binding domains of these two genes remain classified as variants of unknown significance. Here we report an in vitro assay for assessing the effect of variants of unknown significance on the heterodimerization of PALB2 and BRCA1 that recapitulates the effect of the known deleterious mutations. We apply the assay to several variants of unknown significance in BRCA1 which reveals other mutations in this region that also disrupt binding, including a mutation of a residue not predicted to directly interact with PALB2. Structural analysis indicates that all BRCA1 mutations to proline tested disrupt α-helix formation and therefore are not well tolerated even when located at positions outside of the PALB2-binding interface. This assay and the structural hypothesis described will be helpful for assessing risk for variants identified in the future in the BRCA1/PALB2 interaction domains.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 1","pages":"e5240"},"PeriodicalIF":4.5,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11645666/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142823938","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":"Ordered ATP hydrolysis in the Hsp90 chaperone is regulated by Aha1 and a conserved post-translational modification.","authors":"Desmond Prah Amoah, Solomon K Hussein, Jill L Johnson, Paul LaPointe","doi":"10.1002/pro.5255","DOIUrl":"10.1002/pro.5255","url":null,"abstract":"<p><p>Hsp90 is a dimeric molecular chaperone that is important for the folding, stabilization, activation, and maturation of hundreds of protein substrates called \"clients\" in cells. Dozens of co-chaperones and hundreds of post-translational modifications (PTMs) regulate the ATP-dependent client activation cycle. The Aha1 co-chaperone is the most potent stimulator of the ATPase cycle of Hsp90 and phosphorylation of threonine 22 in Hsp90 can regulate the recruitment of Aha1 in cells. We report here that phosphorylation of threonine 22 regulates specific aspects of Aha1 function after recruitment occurs. The phosphomimetic substitution, T22E, neutralizes the action of the Aha1 NxNNWHW motif. Moreover, this substitution can exert this effect from only one protomer of the Hsp90 dimer. This work sheds light on how asymmetric modifications in the Hsp90 dimer can functionalize individual protomers and fine-tune the Hsp90 cycle.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 1","pages":"e5255"},"PeriodicalIF":4.5,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11635476/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142813923","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 \"The transmission of mutation effects in a multiprotein machine: A comprehensive metadynamics study of the cardiac thin filament\".","authors":"","doi":"10.1002/pro.70002","DOIUrl":"10.1002/pro.70002","url":null,"abstract":"","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 1","pages":"e70002"},"PeriodicalIF":4.5,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11670301/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142897240","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":"Exploring the druggability of the UEV domain of human TSG101 in search for broad-spectrum antivirals.","authors":"Fernando Montero, Marisa Parra-López, Alejandro Rodríguez-Martínez, Javier Murciano-Calles, Pedro Buzon, Ziying Han, L-Y Lin, Maria C Ramos, Javier Ruiz-Sanz, Jose C Martinez, Marco Radi, Christiane Moog, Sandra Diederich, Ronald N Harty, Horacio Pérez-Sánchez, Francisca Vicente, Francisco Castillo, Irene Luque","doi":"10.1002/pro.70005","DOIUrl":"10.1002/pro.70005","url":null,"abstract":"<p><p>The ubiquitin E2 variant domain of TSG101 (TSG101-UEV) plays a pivotal role in protein sorting and virus budding by recognizing PTAP motifs within ubiquitinated proteins. Disruption of TSG101-UEV/PTAP interactions has emerged as a promising strategy for the development of host-oriented broad-spectrum antivirals with low susceptibility to resistance. TSG101 is a challenging target characterized by an extended and flat binding interface, low affinity for PTAP ligands, and complex binding energetics. Here, we assess the druggability of the TSG101-UEV/PTAP binding interface by searching for drug-like inhibitors and evaluating their ability to block PTAP recognition, impair budding, and inhibit viral proliferation. A discovery workflow was established by combining in vitro miniaturized HTS assays and a set of cell-based activity assays including high-content bimolecular complementation, virus-like particle release measurement, and antiviral testing in live virus infection. This approach has allowed us to identify a set of chemically diverse molecules that block TSG101-UEV/PTAP binding with IC50s in the low μM range and are able to disrupt the interaction between full-length TSG101 and viral proteins in human cells and inhibit viral replication. State-of-the-art molecular docking studies reveal that the active compounds exploit binding hotspots at the PTAP binding site, unlocking the full binding potential of the TSG101-UEV binding pockets. These inhibitors represent promising hits for the development of novel broad-spectrum antivirals through targeted optimization and are also valuable tools for investigating the involvement of ESCRT in the proliferation of different virus families and study the secondary effects induced by the disruption of ESCRT/virus interactions.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 1","pages":"e70005"},"PeriodicalIF":4.5,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11670305/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142897242","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":"First-principles simulations of the fluorescence modulation of a COX-2-specific fluorogenic probe upon protein dimerization for cancer discrimination.","authors":"Álex Pérez-Sánchez, Carles Curutchet, Àngels González-Lafont, José M Lluch","doi":"10.1002/pro.70001","DOIUrl":"10.1002/pro.70001","url":null,"abstract":"<p><p>Cyclooxygenase-2 (COX-2) plays a crucial role in inflammation and has been implicated in cancer development. Understanding the behavior of COX-2 in different cellular contexts is essential for developing targeted therapeutic strategies. In this study, we investigate the fluorescence spectrum of a fluorogenic probe, NANQ-IMC6, when bound to the active site of human COX-2 in both its monomeric and homodimeric forms. We employ a multiscale first-principles simulation protocol that combines ground state MM-MD simulations with multiple excited state adiabatic QM/MM Born-Oppenheimer MD simulations based on linear response TD-DFT, which allows to account for protein heterogeneity effects on excited-state properties. Emission is then estimated from polarizable embedding TD-DFT QM/MMPol calculations. Our findings indicate that the emission shift arises from dimerization of the highly overexpressed COX-2 in cancer tissues, in contrast to the monomer structure present in inflammatory lesions and in normal cells with constitutive COX-2. This spectral shift is linked to changes in specific protein-probe interactions upon dimerization due to changes in the environment, whereas steric effects related to modulation of the NANQ geometry by the protein scaffold are found to be minor. This research paves the way for detailed investigations on the impact of environment structural transitions on the spectral properties of fluorogenic probes. Moreover, the fact that COX-2 exists as homodimer just in cancer tissues, but as monomer elsewhere, gives novel hints for therapeutical avenues to fight cancer and contributes to the development of drugs targeted to COX-2 dimer in cancer, but without affecting constitutive COX-2, thus minimizing off-target effects.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 1","pages":"e70001"},"PeriodicalIF":4.5,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11669116/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142886234","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":"Screening microorganisms with robust and stable protein expression and secretion capacity.","authors":"Li-Hua Liu, Yu Guo, Min Yang, Yang Zhang, Yi-Rui Wu, Ao Jiang, Zhiqian Zhang","doi":"10.1002/pro.70007","DOIUrl":"10.1002/pro.70007","url":null,"abstract":"<p><p>Robust and stable protein secretion is crucial for efficient recombinant protein production. Here, a novel and powerful platform using split GFP activated droplet sorting (SGADS) has been developed to significantly boost the yields of the protein of interest (POI). The SGADS platform leverages solubilizing peptide P17 and secretory expression in Bacillus subtilis to optimize two split GFP sensors: the P17-GFP1-9/GFP10-POI-GFP11 sensor for assessing protease activity and the P17-GFP1-10/GFP11-POI sensor for measuring secretion capacity. This innovative platform has demonstrated its effectiveness by successfully screening high-performance mutant strains capable of producing collagen, amylase, and protein glutaminase across a range of host organisms, including Escherichia coli, Bacillus subtilis, and Pichia pastoris. The substantial increases in production achieved with the SGADS platform highlight its broad applicability and potential in enhancing recombinant protein production.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 1","pages":"e70007"},"PeriodicalIF":4.5,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11651217/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142839014","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":"PairK: Pairwise k-mer alignment for quantifying protein motif conservation in disordered regions.","authors":"Jackson C Halpin, Amy E Keating","doi":"10.1002/pro.70004","DOIUrl":"10.1002/pro.70004","url":null,"abstract":"<p><p>Protein-protein interactions are often mediated by a modular peptide recognition domain binding to a short linear motif (SLiM) in the disordered region of another protein. To understand the features of SLiMs that are important for binding and to identify motif instances that are important for biological function, it is useful to examine the evolutionary conservation of motifs across homologous proteins. However, the intrinsically disordered regions (IDRs) in which SLiMs reside evolve rapidly. Consequently, multiple sequence alignment (MSA) of IDRs often misaligns SLiMs and underestimates their conservation. We present PairK (pairwise k-mer alignment), an MSA-free method to align and quantify the relative local conservation of subsequences within an IDR. Lacking a ground truth for conservation, we tested PairK on the task of distinguishing biologically important motif instances from background motifs, under the assumption that biologically important motifs are more conserved. The method outperforms both standard MSA-based conservation scores and a modern LLM-based conservation score predictor. PairK can quantify conservation over wider phylogenetic distances than MSAs, indicating that some SLiMs are more conserved than MSA-based metrics imply. PairK is available as an open-source python package at https://github.com/jacksonh1/pairk. It is designed to be easily adapted for use with other SLiM tools and for diverse applications.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 1","pages":"e70004"},"PeriodicalIF":4.5,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11669117/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142886259","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":"Enzymatic, structural, and biophysical characterization of a single-domain antibody (VHH) selectively and tightly inhibiting neutrophil elastase and exhibiting favorable developability properties.","authors":"Paola Redeghieri, Joël Moray, Frédéric Kerff, Sophie Gohy, Teresinha Leal, Serge Muyldermans, Rita Vanbever, Francisco Javier Morales-Yánez, Mireille Dumoulin","doi":"10.1002/pro.5227","DOIUrl":"10.1002/pro.5227","url":null,"abstract":"<p><p>Human neutrophil elastase (hNE), a serine protease released by neutrophils during inflammation, plays a major role in the pathophysiology of several conditions especially in inflammatory lung diseases. Its inhibition constitutes, therefore, a promising therapeutic strategy to combat these diseases. In this work, we characterized the in vitro properties of a VHH (i.e., the antigen binding domain of camelid heavy chain-only antibodies), referred to as NbE201. This VHH is able to inhibit tightly, selectively and competitively both human and murine elastases with the inhibition constants (K_i) of 4.1 ± 0.9 nM and 36.8 ± 3.9 nM, respectively. The IC₅₀ for the inhibition of the hydrolysis of elastin is in the same range to that of alpha-1 antitrypsin (i.e., the main endogenous inhibitor of hNE also used in the clinic) and 14 times better than that of Sivelestat (i.e., the 2nd clinically approved hNE inhibitor). The X-ray crystal structure of the NbE201-hNE complex reveals that the Complementarity Determining Regions CDR1 and CDR3 of the VHH bind into the substrate binding pocket of hNE and prevent the access to small or macromolecular substrates. They do not, however, bind deep enough into the pocket to be hydrolyzed. NbE201 is highly stable towards oxidation, deamidation, and chemical or thermal denaturation. NbE201 is therefore likely to tolerate manufacturing processes during drug development. These results highlight the high potential of NbE201 as a (pre)clinical tool to diagnose and treat diseases associated with excessive hNE activity, and for fundamental research to better understand the role of hNE in these conditions.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"33 12","pages":"e5227"},"PeriodicalIF":4.5,"publicationDate":"2024-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11602439/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142740249","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":"Distinct substrate specificities of the three catalytic subunits of the Trichomonas vaginalis proteasome.","authors":"Pavla Fajtova, Brianna M Hurysz, Yukiko Miyamoto, Mateus Sá M Serafim, Zhenze Jiang, Julia M Vazquez, Diego F Trujillo, Lawrence J Liu, Urvashi Somani, Jehad Almaliti, Samuel A Myers, Conor R Caffrey, William H Gerwick, Dustin L McMinn, Christopher J Kirk, Evzen Boura, Lars Eckmann, Anthony J O'Donoghue","doi":"10.1002/pro.5225","DOIUrl":"10.1002/pro.5225","url":null,"abstract":"<p><p>The protozoan parasite Trichomonas vaginalis (Tv) causes trichomoniasis, the most common non-viral sexually transmitted infection in the world. Although Tv has been linked to significant health complications, only two closely related 5-nitroimidazole drugs are approved for its treatment. The emergence of resistance to these drugs and lack of alternative treatment options poses an increasing threat to public health, making development of novel anti-Trichomonas compounds an urgent need. The proteasome, a critical enzyme complex found in all eukaryotes has three catalytic subunits, β1, β2, and β5 and has been validated as a drug target to treat trichomoniasis. With the goal of developing tools to study the Tv proteasome, we isolated the enzyme complex and identified inhibitors that preferentially inactivate either one or two of the three catalytic subunits. Using a mass spectrometry-based peptide digestion assay, these inhibitors were used to define the substrate preferences of the β1, β2 and β5 subunits. Subsequently, three model fluorogenic substrates were designed, each specific for one of the catalytic subunits. This novel substrate profiling methodology will allow for individual subunit characterization of other proteasomes of interest. Using the new substrates, we screened a library of 284 peptide epoxyketone inhibitors against Tv and determined the subunits targeted by the most active compounds. The data show that inhibition of the Tv β5 subunit alone is toxic to the parasite. Taken together, the optimized proteasome subunit substrates will be instrumental for understanding the molecular determinants of proteasome specificity and for accelerating drug development against trichomoniasis.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"33 12","pages":"e5225"},"PeriodicalIF":4.5,"publicationDate":"2024-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11590128/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142716977","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}