Methods in enzymology最新文献

{"title":"Regulation of phospholipid scramblases by cholesterol.","authors":"Sabita Chourasia, Haodong Wang, Anant K Menon","doi":"10.1016/bs.mie.2026.01.020","DOIUrl":"10.1016/bs.mie.2026.01.020","url":null,"abstract":"<p><p>The activity of membrane proteins is often regulated by their lipid environment, either via physical properties of the membrane or by direct interaction with specific lipids. Testing these effects with purified proteins reconstituted into lipid vesicles is challenging because of the compositional heterogeneity of the vesicle sample. Cholesterol influences membrane properties and binds to a variety of membrane proteins, thereby regulating their activity. To study the effect of cholesterol on the phospholipid scramblase activity of G protein-coupled receptors (GPCRs), we developed a protocol to introduce cholesterol into vesicles after reconstituting the protein. This approach sidesteps the problem of having to account for the possible effect of cholesterol on the reconstitution process itself, enabling direct evaluation of the effect of cholesterol on activity. Here we describe the cholesterol loading protocol and how to quantify the amount loaded by colorimetric assays and membrane fluidity measurements. We provide sample data on the effect of cholesterol on GPCRs. Our protocol is broadly applicable and can be used in any study of the effect of cholesterol on a reconstituted membrane protein.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"727 ","pages":"1-16"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147326721","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Detecting ZDHHC acyltransferase inhibition using acylation-coupled lipophilic induction of polarization (Acyl-cLIP) or LC-MS.","authors":"Wenzhe Chen, Jiaqi Zhao, Zhenquan Sun, Kewen Peng, Wendy K Greentree, Maurine E Linder, Hening Lin","doi":"10.1016/bs.mie.2026.01.015","DOIUrl":"10.1016/bs.mie.2026.01.015","url":null,"abstract":"<p><p>Acylation of protein substrates by ZDHHC (zinc finger Asp-His-His-Cys) acyltransferases is a key regulator of cell signaling. Potent and selective small molecule inhibitors for ZDHHCs have been long desired for the field but so far still do not exist. Robust activity assay methods are needed to discover selective ZDHHC inhibitors. Here we present two complementary assays for in vitro ZDHHC activity assay and inhibitor profiling. The Acylation-Coupled Lipophilic Induction of Polarization (Acyl-cLIP) assay is a homogeneous fluorescence polarization (FP) assay that uses a fluorophore-tagged peptide substrate; upon palmitoylation by ZDHHC enzymes, the substrate partitions into detergent micelles, increasing FP signal. This assay enables real-time kinetic measurements in 384-well plates suitable for high-throughput screening. As an orthogonal validation method, we use liquid chromatography-mass spectrometry (LC-MS) to directly detect palmitoylation through a +238 Da mass shift on model peptide substrates, allowing more reliable quantification and confirmation. Together, these methods offer a platform for identifying and validating ZDHHC inhibitors.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"728 ","pages":"129-141"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147530474","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Reconstitution strategies for exploring interactions of Ebola virus matrix protein with host membrane mimetics.","authors":"Sreetama Pal, Shalini T Low-Nam, Robert V Stahelin","doi":"10.1016/bs.mie.2026.01.006","DOIUrl":"10.1016/bs.mie.2026.01.006","url":null,"abstract":"<p><p>Ebola virus (EBOV), an enveloped virus of the Filoviridae family, is an excellent biophysical model for studying enveloped virus assembly, despite its status as a BSL-4 pathogen. The expression of the EBOV matrix protein VP40 (viral protein 40 kDa) is necessary and sufficient for virus assembly/budding in human cells. VP40, in its dimeric form, interacts with the host plasma membrane (PM) inner leaflet and forms higher-order hexamers critical for EBOV assembly. The dependence of viral maturation on VP40 dynamics provides the opportunity to quantify biophysical requirements for the EBOV life cycle using a reductionist approach to measure VP40-membrane interactions. There are significant gaps in our understanding of the regulation underlying VP40 recruitment at the host cell PM, and how it culminates in the assembly/budding of mature, infectious virions. Here, we describe (i) reconstitution strategies for mimicking the VP40-host cell membrane interface in a controlled, biologically relevant manner and (ii) total internal reflection fluorescence microscopy-based detection of endogenous VP40 association with membranes under regulation of anionic lipid content. These methods are applicable for analyzing peripheral proteins and their interactions with lipid membranes.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"728 ","pages":"289-303"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147530707","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Assembling Retromer-coated membrane tubules for biochemical and structural studies.","authors":"Kai-En Chen, Vikas A Tillu, Nicholas Ariotti, Brett M Collins","doi":"10.1016/bs.mie.2026.01.012","DOIUrl":"10.1016/bs.mie.2026.01.012","url":null,"abstract":"<p><p>The evolutionarily conserved Retromer complex, composed of Vps29, Vps26 and Vps35, is an essential regulator of endosomal retrieval of transmembrane cargo proteins. For cargo sorting and trafficking to take place, Retromer assembles into coated tubulovesicular carriers together with various sorting nexin (SNX) adaptor proteins including SNX3 and SNX27 in metazoans, and Snx3 or the dimeric Vps5-Vps17 SNX-BAR proteins in yeast. Although Retromer-coated tubulovesicular carriers are vital for its function, the in vitro reconstitution of these membrane assemblies for structural and functional studies can be technically challenging. Approaches include the use of giant unilamellar vesicles and supported membrane tubules for fluorescence imaging, or smaller multilamellar vesicles (MLVs) to generate uniform tubules for imaging by cryoelectron tomography (CryoET). This chapter describes protocols for producing MLVs for membrane binding studies of Retromer and assembling the yeast Retromer-Vps5-Vps17 heteropentameric complex for reconstituting membrane tubulation for CryoET studies. We also discuss our observations of both poorly ordered and well-ordered Retromer coats observed in this experimental setup.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"728 ","pages":"101-116"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147530286","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Real-time measurement of the ATG8 lipidation reaction by fluorescence spectroscopy.","authors":"Wenxin Zhang, Sharon A Tooze, Taki Nishimura","doi":"10.1016/bs.mie.2026.01.011","DOIUrl":"10.1016/bs.mie.2026.01.011","url":null,"abstract":"<p><p>Autophagy is a highly conserved intracellular degradation pathway, in which damaged organelles and/or dysfunctional cytosolic components are enveloped via double-membraned autophagosomes and subsequently delivered to lysosomes for degradation. The ubiquitin-like ATG8 family proteins (LC3s and GABARAPs) are covalently conjugated to phosphatidylethanolamine (PE) on autophagic membranes via ubiquitin-like conjugation systems, a process known as ATG8 lipidation. Lipidated ATG8 is the most widely used membrane marker for autophagosomes, and its flux is commonly used as a readout for autophagy activity. In vitro reconstitution of the ATG8 lipidation reaction is well-established, and the end-point reaction is typically resolved by SDS-PAGE. This endpoint readout is not suitable to monitor the kinetics of this reaction, and tools to study this process have been lacking. Here, we describe a real-time assay to measure the ATG8 lipidation reaction. This approach not only reveals the subsequential formation of covalently bound intermediates of ATG8 with the E1 (ATG7) and E2 (ATG3) enzymes, as well as ATG8-PE itself, but also provides insights into the interaction interface of ATG8 with proteins and membranes during the conjugation reaction.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"728 ","pages":"327-345"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147530745","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Genetic code expansion for single noncanonical amino acid incorporation into SARS-CoV-2 Omicron spike on amber-free lentiviral particles.","authors":"Wang Xu, Narendra Kumar Gonepudi, Yang Han, Maolin Lu","doi":"10.1016/bs.mie.2026.05.022","DOIUrl":"10.1016/bs.mie.2026.05.022","url":null,"abstract":"<p><p>Fluorescent labeling or tagging of viral proteins is crucial for understanding molecular virology and guiding antiviral interventions. However, traditional methods using large tags are often invasive, impeding visualization of viral proteins in their native states. We detail a methodology combining an amber-free lentiviral packaging system with amber (TAG stop codon) suppression - genetic code expansion and site-specific bioorthogonal click labeling. Amber suppression enables site-specific incorporation of a non-canonical amino acid (ncAA) bearing strained alkynes or alkenes into a target protein. The ncAA-containing viral protein can then be labeled with a fluorophore conjugated with a click-chemistry-reactive functional group via copper-free click chemistry, allowing single-residue fluorescent labeling with minimal impact on protein structure and function. However, amber stop codons that serve as native termination signals in viral genomes can be inadvertently suppressed during genetic code expansion, leading to protein readthrough and unpredictable experimental outcomes. Here, we provide a step-by-step method for constructing a complete amber-free lentivirus, which overcomes this limitation by providing an amber-free viral background. We then describe a stepwise workflow for single ncAA incorporation into the Omicron spike decorated on amber-free lentiviruses, followed by the attachment of organic fluorophores via click chemistry. The methods described here are, in principle, adaptable to other viral surface glycoproteins, non-surface lentiviral proteins, and pseudotyped viral systems, with appropriate optimization and validation as needed.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"731 ","pages":"141-165"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13305207/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148330993","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Epigenetic clues: Predicting maternal depression through DNA methylation.","authors":"Varghese Merin Jerusha, Chinnasamy Thirumoorthy, Kuppan Gokulakrishnan","doi":"10.1016/bs.mie.2026.06.020","DOIUrl":"https://doi.org/10.1016/bs.mie.2026.06.020","url":null,"abstract":"<p><p>Perinatal depression (PND) is a prevalent and multifactorial mood disorder affecting approximately 10-20 % of women globally, with higher burdens reported in low- and middle-income countries. Despite the availability of screening tools such as the Edinburgh Postnatal Depression Scale, these approaches primarily identify risk without elucidating underlying biological mechanisms. Emerging evidence highlights the role of epigenetic regulation particularly DNA methylation as a key mediator linking genetic susceptibility and environmental exposures during the perinatal period. This review synthesizes current knowledge on DNA methylation dynamics in maternal depression, emphasizing both candidate gene and epigenome-wide association study (EWAS) approaches. Candidate gene studies have identified differential methylation in stress-related pathways, including HPA axis genes (NR3C1, FKBP5), serotonergic signalling (SLC6A4), and oxytocin pathways (OXTR), though findings remain limited by poor reproducibility and small sample sizes. In contrast, EWAS provides a hypothesis-free framework, identifying novel differentially methylated positions and regions associated with PND, including predictive CpG panels with potential diagnostic utility. The review also highlights the importance of tissue specificity, temporal epigenetic remodeling across pregnancy, and the interplay between maternal and fetal epigenomes. Furthermore, methodological challenges such as heterogeneity in study design, lack of replication, and analytical inconsistencies remain barriers to clinical translation. Integrating genetic, epigenetic, and environmental data through multi-omics approaches may enhance predictive accuracy and improve early intervention strategies. Overall, DNA methylation represents a promising avenue for understanding the biological underpinnings of PND and developing robust biomarkers for risk prediction and personalized care.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"734 ","pages":"235-268"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148888047","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Lysine methylation is an endogenous post-translational modifications of tau protein in human brain and a modulator of aggregation propensity.","authors":"Marisel Sanchez, Jeff Kuret","doi":"10.1016/bs.mie.2026.05.044","DOIUrl":"10.1016/bs.mie.2026.05.044","url":null,"abstract":"<p><p>Tau protein undergoes a broad range of post-translational modifications in the brain, influencing its structure, solubility, and propensity to aggregate. This chapter presents an integrated methodological framework for characterizing tau methylation and evaluating its impact on tau biology. We describe procedures for isolating soluble and filamentous tau from post-mortem human brain tissue while preserving modifications for proteomic analysis. These approaches support precise mapping of methylation sites alongside other co-occurring modifications. To model methylation under controlled conditions, we outline protocols for recombinant tau expression, purification, and chemical reductive methylation, including radiolabeled assays for determining modification stoichiometry. We then detail biophysical assays used to assess how methylation alters tau conformation and aggregation propensity. This methodological framework supports experimentation seeking insight into mechanisms relevant to Alzheimer's disease and related tauopathies.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"734 ","pages":"1-28"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13542479/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148888054","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Zinc-finger ubiquitin-binding domain of histone deacetylase 6 modulates Tau structure and function.","authors":"Abhishek Ankur Balmik, Subashchandrabose Chinnathambi","doi":"10.1016/bs.mie.2026.05.036","DOIUrl":"https://doi.org/10.1016/bs.mie.2026.05.036","url":null,"abstract":"<p><p>Histone deacetylase 6 (HDAC6), a predominantly cytoplasmic deacetylase, has emerged as a critical regulator of protein homeostasis, and plays an important role in neurodegenerative disorders characterized by Tau pathology. We investigated the role of zinc-finger ubiquitin-binding domain (ZnF UBP) of HDAC6 in modulating tau structure and function. HDAC6 is well known for its deacetylase activity and involvement in aggresome formation but the functional contribution of its ZnF UBP domain in tau biology remains not well explored. Using a combination of biochemical, biophysical, and molecular approaches, we demonstrate that the ZnF UBP domain of HDAC6 directly interacts with tau, influencing its conformational dynamics and aggregation propensity. Our findings reveal that this interaction is independent of HDAC6's catalytic deacetylase activity, highlighting a non-canonical mechanism through which HDAC6 regulates tau. Structural analyses indicate that binding of ZnF UBP induces conformational rearrangements in tau, potentially altering its microtubule-binding capacity and aggregation behavior. Furthermore, we show that the HDAC6-tau interaction modulates tau aggregation, suggesting a protective or regulatory role in preventing aberrant tau aggregation. Functional assays support the notion that ZnF UBP-mediated modulation of tau contributes to maintaining cytoskeletal integrity and cellular proteostasis. These observations suggests that HDAC6 functions not only as a deacetylase but also performs regulatory functions through ZnF UBP domain that can influence the function and properties of intrinsically disordered proteins (IDPs) like tau. Overall, this chapter provides mechanistic insights into the non-enzymatic functions of HDAC6, specifically through its ZnF UBP domain, in regulating tau structure and function. Understanding this interaction expands the current perspective on HDAC6 biology and underscores its potential as a therapeutic target in tauopathies, including Alzheimer's disease and targeting domain-specific interactions of HDAC6 may offer a novel therapeutic strategy to modulate tau pathology.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"733 ","pages":"81-110"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148795519","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Experimental techniques to evaluate IA-MSCs-mediated alleviation of vasogenic edema following ischemic stroke.","authors":"Aishika Datta, Nikita Malik, Gautam Karmarkar, Anirban Barik, Deepaneeta Sarmah, Vikas Khajekar, Akshada Dubey, Ushmita Mukherjee, Anita Kumari, Swagata Bhattacharya, Bijoyani Ghosh, Pallab Bhattacharya","doi":"10.1016/bs.mie.2026.06.021","DOIUrl":"https://doi.org/10.1016/bs.mie.2026.06.021","url":null,"abstract":"<p><p>Ischemic stroke induced cerebral edema is a devastating complication which leads to excessive swelling, increased intracranial pressure (ICP) and rapid neurological deterioration. The mortality rate of post-stroke cerebral edema is approximately 80 %. However, therapeutic options for post-stroke cerebral edema are sparce, emphasizing an urgent need to explore new therapeutic strategies for post-stroke edema. In past few decades stem cell therapy has been a promising therapeutic strategy in decreasing infarct volume and promoting functional recovery by various mechanisms following stroke. Previous studies from our lab demonstrated that IA-MSCs administration were beneficial in mitigating post-stroke edema by regulating PKCδ/ MMP9/AQP4 axis and promoting blood-brain-barrier (BBB) integrity. It has also been reported that IA-MSCs administration mitigate post-stroke edema by maintaining BBB integrity, mitochondria function and regulating AQP4 expression by modulation of the SIRT-1/PKCδ/NF-κB axis. In this chapter, we illustrated a multi-level methodological approach to assess therapeutic outcomes of IA-MSCs in alleviating post-stroke cerebral edema by utilizing behavioural analyses, histological staining, biochemical assays, mitochondrial functional assays and molecular investigations by RT-PCR, western blotting and immunofluorescence staining.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"733 ","pages":"407-431"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148795511","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}