Biochemistry Biochemistry最新文献

{"title":"CRISPR Screen Identifies BAP1 as a Deubiquitinase Regulating SPIN4 Stability.","authors":"Alondra Sanchez, Chen Zhou, Rima Tulaiha, Francisco Ramirez, Lu Wang, Xiaoyu Zhang","doi":"10.1021/acs.biochem.5c00493","DOIUrl":"https://doi.org/10.1021/acs.biochem.5c00493","url":null,"abstract":"<p><p>Protein homeostasis is tightly controlled by the coordinated actions of E3 ubiquitin ligases and deubiquitinases (DUBs). We previously identified Spindlin-4 (SPIN4), a histone H3K4me3 reader, as a degradation substrate of DCAF16. In this study, we confirmed this degradation pathway using an E3 ligase-focused CRISPR-Cas9 knockout screen. Furthermore, through a DUB-focused CRISPR-Cas9 knockout screen and biochemical analyses, we demonstrated that the deubiquitinase BAP1 interacts with and stabilizes SPIN4 via its deubiquitination activity. Inhibition or loss of BAP1 reduces SPIN4 levels, highlighting its critical role in maintaining SPIN4 homeostasis. Proteomics and interactome analyses further support this regulatory axis. These findings reveal a dynamic balance controlling SPIN4 stability, with potential implications for epigenetic regulation and disease processes.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":""},"PeriodicalIF":3.0,"publicationDate":"2025-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145190446","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Structure and Dynamics of the Magainin 2 Antimicrobial Peptide in Biomimetic Lipid Bilayers by Solid-State NMR.","authors":"Ahmad Saad, Jesus Raya, Burkhard Bechinger","doi":"10.1021/acs.biochem.5c00467","DOIUrl":"https://doi.org/10.1021/acs.biochem.5c00467","url":null,"abstract":"<p><p>In this study, we present an atomic-level structural investigation of the magainin 2 antimicrobial peptide reconstituted in extended lipid bilayers that closely mimic the composition of bacterial membranes. Using state-of-the-art solid-state NMR spectroscopy, we show that within liquid-crystalline membranes the peptide exhibits site-specific motional regimes, which correlate with its amphipathic character. Peptide-lipid interactions are identified at the polar headgroup region consistent with an in-plane topology also observed by oriented ¹⁵N solid-state NMR spectroscopy. While ¹³C chemical shift analysis reveals α-helical conformations, the NMR line shapes indicate pronounced conformational heterogeneity, which can be explained by the existence of higher order arrangements along the membrane surface. A reduced degree of helicity is observed when the membrane is in the gel phase suggesting more superficial interactions of magainin 2. Notably, our NMR data show that membrane-associated magainin 2 can evolve into amyloid-like β-sheet structures, forming large peptide-lipid aggregates. This behavior occurs only in bacterial and not in mammalian membrane models, paving the way for a new understanding of the role of these supramolecular assemblies in host defense mechanisms, and highlighting a potential relationship between antimicrobial peptides and functional amyloid structures.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":""},"PeriodicalIF":3.0,"publicationDate":"2025-09-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145190474","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Molecular Mechanisms Underlying the Construction of Ciliary Doublet Microtubules.","authors":"Zhe Chen, Guangshuo Ou","doi":"10.1021/acs.biochem.5c00468","DOIUrl":"https://doi.org/10.1021/acs.biochem.5c00468","url":null,"abstract":"<p><p>Motile cilia and flagella are complex microtubule-based organelles essential for cell motility, fluid flow, and sensory functions. At the core of these organelles lie doublet microtubules (DMTs), specialized structures consisting of an A-tubule and a B-tubule that serve as the scaffold for axonemal complexes. Recent advances have uncovered the intricate architecture of DMTs, highlighting inner and outer junction proteins, luminal scaffolds, and interdoublet linkers critical for structural stability and function. Studies in model organisms such as <i>Chlamydomonas</i>, <i>Tetrahymena</i>, and <i>Caenorhabditis elegans</i> have identified conserved regulators orchestrating DMT assembly. Parallel insights from human genetics reveal that mutations in DMT-associated proteins underlie a subset of cases of primary ciliary dyskinesia and other ciliopathies. This review synthesizes current understanding of DMT biogenesis from molecular, structural, and disease perspectives, illuminating how coordinated assembly ensures ciliary function and how its disruption leads to human disease.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":""},"PeriodicalIF":3.0,"publicationDate":"2025-09-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145190430","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Potential Activation Mechanism of Calcium Indicator WHaloCaMP1a Revealed by the Crystal Structure and Molecular Dynamics Simulations.","authors":"Kecheng Zhang, Nanhao Chen, Junwei Zhang, Zhixing Chen","doi":"10.1021/acs.biochem.5c00352","DOIUrl":"https://doi.org/10.1021/acs.biochem.5c00352","url":null,"abstract":"<p><p>Chemigenetic indicators combined with advanced organic fluorophores have become increasingly popular in biosensor development due to their integration of protein biocompatibility and the superior optical properties of dyes. However, despite the development of various biosensors based on these systems, the underlying activation mechanisms remain obscure. Here, we report the first crystal structure of the recently developed calcium indicator WHaloCaMP1a in complex with the rhodamine-based fluorophore BD566_HTL. Integrating structural analysis with molecular dynamics simulations, we identify a potential activation mechanism of WHaloCaMP1a and reveal novel interactions between the sensor and BD566. These findings provide new insights for the rational design of biosensors and the study of protein-dye coevolution.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":""},"PeriodicalIF":3.0,"publicationDate":"2025-09-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145172114","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Structural Insights into the Role of the Stereochemistry of the Cyclopropyl Ring in the Inhibitory Activity of Xeruborbactam against SME-1 Class A Carbapenemase.","authors":"Kunal Dhankhar, Adwaita S R Nair, Mousumi Hazra, Alaa Eddin Alhmeidi Alkhatib, Subhecchha Baidya, Narayan C Mishra, Saugata Hazra","doi":"10.1021/acs.biochem.5c00336","DOIUrl":"https://doi.org/10.1021/acs.biochem.5c00336","url":null,"abstract":"<p><p>Xeruborbactam is a boronic acid-based transition-state analogue that has exhibited great potential as a clinically relevant inhibitor of carbapenemase enzymes, including class A carbapenemases. In this work, we have investigated the mechanism of inhibition of xeruborbactam against SME-1 carbapenemase using kinetic, structural, and thermodynamic approaches. With a <i>K</i>_i(app) of 4 nM, xeruborbactam shows more potent inhibitory activity than any other beta-lactamase inhibitor available until now. Structural data from crystal complexes revealed that xeruborbactam covalently engages Ser70 at the active site and forms stabilizing interactions; in particular, the cyclopropyl group forms hydrophobic interactions with His105, further stabilizing the adduct, which correlates with a high rate of borylation and minimal deborylation. We investigated xeruborbactam with its 2<i>R</i>,3<i>S</i>-cyclopropyl isomer to grasp the influence of the stereochemistry of the cyclopropyl ring. Although both inhibitors bind covalently to Ser70 in SME-1, the 2<i>R</i>,3<i>S</i>-isomer adopts a different conformation of the cyclopropyl ring, which makes the C3 carbon much farther from His105, Asn132, and Lys73, thereby decreasing the binding affinity and <i>K</i>_i(app) of the isomer. Furthermore, the fluorine-12 atom takes different conformations in the two structures, changing the terrain of interaction with the protein. Consistent with its lowered inhibition efficiency, the 2<i>R</i>,3<i>S</i>-isomer shows a lower borylation rate and weaker enzyme-inhibitor binding. In the molecular dynamics, xeruborbactam stabilized SME-1 more than its isomer, which is consistent with our experimental findings. These results together show the strong inhibitory profile of xeruborbactam and highlight the importance of stereochemistry in the design of next-generation β-lactamase inhibitors and diagnostics for AMR.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":""},"PeriodicalIF":3.0,"publicationDate":"2025-09-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145147129","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Single Particle Dynamics of Protein Aggregation and Disaggregation in the Presence of the sHsp Proteins IbpAB.","authors":"Andrew Roth, YuChen Yang, Jason Puchalla, Hays S Rye","doi":"10.1021/acs.biochem.5c00312","DOIUrl":"10.1021/acs.biochem.5c00312","url":null,"abstract":"<p><p>The small heat shock proteins (sHsps) are a key class of molecular chaperones that can inhibit protein aggregation and enhance protein recovery from aggregates. However, the mechanisms sHsps employ to carry out these roles are not well understood, in part because the highly heterogeneous and dynamic particles they form with aggregating proteins are difficult to study with traditional biophysical tools. Here we have applied a novel single particle fluorescence technique known as Burst Analysis Spectroscopy (BAS) to the study of the <i>Escherichia coli</i> sHsps IbpA and IbpB (IbpAB). We show that in the presence of IbpAB, two different model proteins converge toward similar, limited aggregate particle size distributions. Additionally, while IbpAB dramatically accelerates the disassembly of protein aggregates by the bacterial KJEB bichaperone disaggregase, this enhancement does not appear to be strongly influenced by aggregate particle size. Rather, it is the ability of IbpAB to alter aggregate structure during particle formation that appears to be essential for stimulated disassembly. These observations support a model of aggregate recognition by IbpAB that is not only highly adaptable but capable of shaping aggregate particles into a specialized range of physical properties that are necessary for efficient protein disaggregation.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":""},"PeriodicalIF":3.0,"publicationDate":"2025-09-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145147073","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Small Ankyrin 1 Interacts with Phospholamban and Forms a Three-Way Complex with SERCA1.","authors":"Amanda Labuza, Yi Li, Megan A Rizzo, Robert J Bloch","doi":"10.1021/acs.biochem.5c00143","DOIUrl":"https://doi.org/10.1021/acs.biochem.5c00143","url":null,"abstract":"<p><p>The activity of the sarco(endo)plasmic reticulum Ca²⁺-ATPase 1 (SERCA1) of muscle is inhibited by small ankyrin1 (sAnk1), a ∼17 kDa transmembrane protein that stabilizes the network compartment of the sarcoplasmic reticulum (SR). sAnk1 binds to sarcolipin (SLN), a 31 amino acid peptide inhibitor of SERCA1, to ablate its inhibitory activity. SERCA1 is also inhibited by phospholamban (PLN), which shares homology with both sAnk1 and SLN. Here we use cotransfection of COS7 cells, coimmunoprecipitation and bimolecular fluorescent complementation (BiFC) to show that sAnk1 associates with PLN and forms a 3-way complex with PLN and SERCA1. Anisotropy-based FRET (AFRET) studies of Cerulean-SERCA1 with Venus-tagged sAnk1 and PLN confirmed the presence of a 3-way complex. ATPase assays showed that, unlike its effects on SLN, sAnk1 does not ablate PLN's inhibition of SERCA1 activity. Our results are consistent with a model in which, in forming a three-way complex, PLN binds to SERCA1 first, followed by binding to sAnk1. This modeling suggests that the interactions of PLN, SLN and sAnk1 with SERCA1, either alone or in pairs, are distinct and have different effects on SERCA1's enzymatic activity.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":""},"PeriodicalIF":3.0,"publicationDate":"2025-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145147054","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Mapping of Residues in Leishmanial Glyceraldehyde-3-phosphate Dehydrogenase Crucial for Binding to 3'-UTR of TNF-α mRNA.","authors":"Puja Panja, Sumit Das, Yuthika Dholey, Gaurab Chowdhury, Subrata Adak","doi":"10.1021/acs.biochem.5c00252","DOIUrl":"https://doi.org/10.1021/acs.biochem.5c00252","url":null,"abstract":"<p><p>Recently, we described that glyceraldehyde-3-phosphate dehydrogenase from <i>Leishmania major</i> (LmGAPDH) is present in extracellular vesicles and inhibits host TNF-α expression during infection via post-transcriptional repression. LmGAPDH binding to AU-rich elements in the 3'-untranslated region of TNF-α mRNA (TNF-α ARE) was sufficient for limiting cytokine production, but the TNF-α ARE binding residues in LmGAPDH remain unexplored. RNA electrophoretic mobility shift assay (REMSA) and catalytic activity measurements revealed that the inhibition by the TNF-α ARE was competitive with respect to the cofactor NAD⁺ in LmGAPDH. To identify the TNF-α ARE binding residues of LmGAPDH, we performed a systematic mutational analysis of its NAD⁺ binding domain. Catalytic activity measurements indicated that both R13 and N336 amino acids in the NAD⁺ binding site are absolutely required for activity, whereas other mutants, including I14A, R16A, D39A, and T112A, showed higher <i>K</i>_m (lower affinity) values for NAD⁺ binding and lower catalytic activity. REMSA studies revealed that the replacement of Arg13 with Ala/Lys or Asn336 with Ala resulted in a complete loss of binding to the TNF-α ARE. I14A, R16A, D39A, and T112A residues at or near the NAD⁺ binding site showed lower binding to the TNF-α ARE compared to the wild-type protein. Protein-induced fluorescence enhancement (PIFE) studies and <i>in vitro</i> protein translation assays further confirmed the REMSA results. Based on our findings, the NAD⁺ binding residues in LmGAPDH are important for TNF-α ARE binding.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":""},"PeriodicalIF":3.0,"publicationDate":"2025-09-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145129634","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Isoform-Selective PAD2/PAD4 Substrates with Unnatural Amino Acids Enable Cellular Peptidylarginine Deiminase Activity Profiling and Reveal Vimentin Citrullination Effects in Macrophages.","authors":"Oliwia Gorzeń, Agata Mikołajczyk-Martinez, Abdulla Al Mamun, Natalia Horbach, Olha Severynovska, Grzegorz Bereta, Ewa Bielecka, Piotr Mydel, Marcin Drąg, Tomasz Kantyka, Marcin Poręba","doi":"10.1021/acs.biochem.5c00391","DOIUrl":"https://doi.org/10.1021/acs.biochem.5c00391","url":null,"abstract":"<p><p>Peptidylarginine deiminases (PADs) catalyze the calcium-dependent conversion of arginine to citrulline, which affects diverse cellular processes. Among the human PAD isoforms, PAD2 and PAD4 are particularly relevant because of their distinct tissue distributions and substrate preferences. However, the lack of isoform-selective substrates has limited our ability to discriminate between their activities in biological systems. In this study, we developed PAD2- and PAD4-selective fluorogenic peptide substrates using the Hybrid Combinatorial Substrate Library (HyCoSuL) strategy, which incorporates both natural and over 100 unnatural amino acids. Substrate specificity profiling at P4-P2 positions revealed that PAD2 tolerates a broader range of residues, particularly at the P2 position, whereas PAD4 displays more selective preferences, favoring aspartic acid at this site. Based on these insights, we designed and validated peptide substrates with high selectivity for PAD2 or PAD4, enabling isoform-specific kinetic analysis <i>in vitro</i>. We demonstrated the utility of these substrates in profiling PAD activity in THP-1 macrophages, revealing dominant PAD2 activity in PMA (phorbol 12-myristate 13-acetate)/LPS (lipopolysaccharide)-stimulated monocytes. Furthermore, PAD4-mediated citrullination of vimentin modulates its susceptibility to caspase and calpain cleavage, potentially altering its function as a damage-associated molecular pattern (DAMP). Our findings provide a framework for the development of PAD-selective inhibitors and chemical probes, enabling the precise dissection of isozyme-specific PAD functions in health and disease.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":""},"PeriodicalIF":3.0,"publicationDate":"2025-09-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145135990","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Self-Assembly Precedes Target Membrane Recruitment of a Nuclear Dynamin-Related Protein.","authors":"Sakti Ranjan Rout, Faiyaz Alam, Swapnil Sahoo, Kaustubh Prakash, Gargi Dey, Abdur Rahaman","doi":"10.1021/acs.biochem.5c00487","DOIUrl":"https://doi.org/10.1021/acs.biochem.5c00487","url":null,"abstract":"<p><p>Dynamin family proteins typically do not depend on higher-order oligomerization; instead, dimerization and/or tetramerization is sufficient for their target membrane recruitment. Here, we demonstrate that dimerization/tetramerization alone is not enough, but self-assembly into a higher-order structure is also required for targeting a dynamin-related protein, dynamin-related protein 6 (Drp6), to the nuclear membrane. We identify residues 411-GKFR-414 as important for higher-order oligomerization of Drp6 but dispensable for its dimerization/tetramerization. Furthermore, while the mutation of GKFR residues does not affect membrane-binding ability <i>in vitro</i>, it inhibits the nuclear localization of Drp6 <i>in vivo</i>. Ultrastructure expansion microscopy and fast super-resolution live cell imaging demonstrate that the cytosolic, higher-order self-assembled structure of Drp6 is recruited to the nuclear envelope. These findings establish self-assembly into a higher-order oligomer as a prerequisite for target membrane recruitment of a dynamin-related protein.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":""},"PeriodicalIF":3.0,"publicationDate":"2025-09-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145129659","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}