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Analysis of mitochondrial protein aggregation and disaggregation. 线粒体蛋白质聚集和分解分析。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-08-20 DOI: 10.1016/bs.mie.2024.07.048
Wolfgang Voos, Anne Wilkening, Robin Ostermann, Michael Bruderek, Witold Jaworek, Laura Ruland
{"title":"Analysis of mitochondrial protein aggregation and disaggregation.","authors":"Wolfgang Voos, Anne Wilkening, Robin Ostermann, Michael Bruderek, Witold Jaworek, Laura Ruland","doi":"10.1016/bs.mie.2024.07.048","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.07.048","url":null,"abstract":"<p><p>Deficits of mitochondrial functions have been identified in many human pathologies, in particular in age-related human neurodegenerative diseases. Hence, the molecular causes for mitochondrial dysfunction and potential protection mechanisms have become a major topic in modern cell biology. Apart from defects in their structural integrity, problems in mitochondrial protein biogenesis, including polypeptide transport, folding and assembly to active enzymes, all may result in some degree of functional defects of the organelle. An accumulation of misfolded polypeptides inside mitochondria, confounded by the dual source of mitochondrial polypeptides, will result in the formation of protein aggregates. Such aggregate accumulation bears a cell-toxic potential, resulting in mitochondrial and correlated cellular damages, summarized in the term \"aggregate proteotoxicity\". Here, we discuss methods to analyze protein aggregation in the mitochondrial matrix compartment. We also address techniques to characterize the biochemical mechanisms that reduce aggregate proteotoxicity, the disaggregation or resolubilization of aggregated polypeptides and the sequestration and neutralization of mitochondrial aggregates at specific sites inside a cell.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"707 ","pages":"475-498"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142564294","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}
引用次数: 0
Analysis of mitochondrial protein translocation by disulfide bond formation and cysteine specific crosslinking. 通过二硫键形成和半胱氨酸特异性交联分析线粒体蛋白质的转运。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-08-31 DOI: 10.1016/bs.mie.2024.07.057
Laura F Fielden, Jakob D Busch, Caroline Lindau, Jian Qiu, Nils Wiedemann
{"title":"Analysis of mitochondrial protein translocation by disulfide bond formation and cysteine specific crosslinking.","authors":"Laura F Fielden, Jakob D Busch, Caroline Lindau, Jian Qiu, Nils Wiedemann","doi":"10.1016/bs.mie.2024.07.057","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.07.057","url":null,"abstract":"<p><p>Protein translocation is a highly dynamic process and, in addition, mitochondrial protein import is especially complicated as the majority of nuclear encoded precursor proteins must engage with multiple translocases before they are assembled in the correct mitochondrial subcompartment. In this chapter, we describe assays for engineered disulfide bond formation and cysteine specific crosslinking to analyze the rearrangement of translocase subunits or to probe protein-protein interactions between precursor proteins and translocase subunits. Such assays were used to characterize the translocase of the outer membrane, the presequence translocase of the inner membrane and the sorting and assembly machinery for the biogenesis of β-Barrel proteins. Moreover, these approaches were also employed to determine the translocation path of precursor proteins (identification of import receptors and specific domains required for translocation) as well as the analysis, location and translocase subunit dependence for the formation of β-Barrel proteins. Here we describe how engineered disulfide bond formation and cysteine specific crosslinking assays are planned and performed and discuss important aspects for its application to study mitochondrial protein translocation.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"707 ","pages":"257-298"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142564306","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}
引用次数: 0
Strand-specific PCR-competitive replication and adduct bypass assay for assessing how DNA adducts perturb DNA replication in mammalian cells. 链特异性 PCR 竞争性复制和加合物旁路测定法，用于评估 DNA 加合物如何干扰哺乳动物细胞中的 DNA 复制。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-08-10 DOI: 10.1016/bs.mie.2024.07.013
Jun Yuan, Chen Wang, Xiaomei He, Yinsheng Wang
{"title":"Strand-specific PCR-competitive replication and adduct bypass assay for assessing how DNA adducts perturb DNA replication in mammalian cells.","authors":"Jun Yuan, Chen Wang, Xiaomei He, Yinsheng Wang","doi":"10.1016/bs.mie.2024.07.013","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.07.013","url":null,"abstract":"<p><p>Human genomes are susceptible to damage by a variety of endogenous and exogenous agents. If not repaired, the resulting DNA lesions can potentially lead to mutations, genome instability, and cell death. While existing in vitro experiments allow for characterizing replication outcomes from the use of purified translesion synthesis (TLS) DNA polymerases, such studies often lack the sophistication and dynamic nature of cellular contexts. Here, we present a strand-specific PCR-based Competitive Replication and Adduct Bypass (ssPCR-CRAB) assay designed to investigate quantitatively the impact of DNA lesions on replication efficiency and fidelity in mammalian cells. Combined with genetic manipulation, this approach facilitates the revelation of diverse functions of TLS polymerases in replication across DNA lesions.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"705 ","pages":"251-270"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142400662","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}
引用次数: 0
Monitoring mitochondrial precursor processing and presequence peptide degradation. 监测线粒体前体加工和前序肽降解。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-09-10 DOI: 10.1016/bs.mie.2024.07.018
Cansu Kücükköse, F-Nora Vögtle, Annette Flotho
{"title":"Monitoring mitochondrial precursor processing and presequence peptide degradation.","authors":"Cansu Kücükköse, F-Nora Vögtle, Annette Flotho","doi":"10.1016/bs.mie.2024.07.018","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.07.018","url":null,"abstract":"<p><p>The maturation of mitochondrial presequence precursor proteins after their import into the organelle is a complex process that requires the interaction of several mitochondrial proteases. Precursor processing by the mitochondrial presequence proteases is directly coupled to the proteolytic turnover of the cleaved targeting signal by mitochondrial presequence peptidases. Dysfunction of these enzymes is associated with a variety of human diseases, including neurological disorders, cardiomyopathies and renal diseases. In this chapter, we describe experimental approaches to study the activity of the major mitochondrial presequence protease (MPP) and of the presequence peptidases. In vitro assays and soluble mitochondrial extracts allow the assessment and experimental manipulation of peptidase and protease activity using immunoblotting, fluorescence measurements and autoradiography as readouts. In particular, the assays allow manipulation at multiple levels including in vivo, in organello or in soluble extracts/in vitro. Purification of the yeast heterodimeric MPP allows in vitro reconstitution of the initial presequence processing step using radiolabeled precursors as substrates. Application of soluble mitochondrial extracts enables direct assessment of MPP processing and presequence peptide turnover which can be easily manipulated and is uncoupled from protein translocation across the mitochondrial membranes. The techniques presented in this chapter allow in-depth analysis of precursor processing and presequence turnover as well as direct assessment of the impact of patient mutations on the activity of the presequence processing machinery.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"706 ","pages":"193-213"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142504054","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}
引用次数: 0
Analysis of mitochondrial translation using click chemistry. 利用点击化学分析线粒体翻译。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-08-23 DOI: 10.1016/bs.mie.2024.07.044
Roya Yousefi, Sven Dennerlein
{"title":"Analysis of mitochondrial translation using click chemistry.","authors":"Roya Yousefi, Sven Dennerlein","doi":"10.1016/bs.mie.2024.07.044","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.07.044","url":null,"abstract":"<p><p>Mitochondria contain their own gene expression machinery, which synthesizes core subunits of the oxidative phosphorylation system. Monitoring mitochondrial translation within spatial compartments of cells is difficult. Here we describe a method to visualize mitochondrial translation within defined parts of cells, using a click chemistry approach. This method can be applied to different cell types such as neurons and allows detection of newly synthesized mitochondrial proteins in spatial resolution using microscopy techniques. Furthermore, using click chemistry, mitochondrial translation can also be monitored by standard SDS-PAGE. The described method avenues the analysis of newly synthesized mitochondrial encoded proteins in the cellular context, by avoiding the usage of radioactive components.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"706 ","pages":"533-547"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142504041","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}
引用次数: 0
In organello silencing of mitochondrial gene expression. 在器官内沉默线粒体基因的表达。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-08-21 DOI: 10.1016/bs.mie.2024.07.035
Mats Koschel, Luis Daniel Cruz-Zaragoza
{"title":"In organello silencing of mitochondrial gene expression.","authors":"Mats Koschel, Luis Daniel Cruz-Zaragoza","doi":"10.1016/bs.mie.2024.07.035","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.07.035","url":null,"abstract":"<p><p>Mitochondria contain proteins from two genetic origins. Most mitochondrial proteins are encoded in the nuclear genome, translated in the cytosol, and subsequently imported into the different mitochondrial sub-compartments. A small number is encoded in the mitochondrial DNA (mtDNA). The manipulation of the mtDNA gene expression represents a challenge. Here, we present an in vitro approach using morpholinos chemically linked to a precursor protein to silence gene expression in purified human mitochondria. The protocol is demonstrated with a Jac1-morpholino chimera specifically targeting COX1 mRNA. The chimera import and mitochondrial translation requirements are described in a step-by-step procedure, where the dose-dependent effect of reducing COX1 translation is observed. The affinity and specificity of chimera-mRNA binding also show great applicability to purify transcript-associated proteins by using the imported chimera construct as bait for immunoprecipitation. This new strategy opens up the possibility to address mechanistic questions about gene expression and physiology in mitochondria.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"706 ","pages":"501-518"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142504045","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}
引用次数: 0
Methods to analyze mitochondrial protein translocation in plant mitochondria. 分析植物线粒体蛋白质转位的方法。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-08-24 DOI: 10.1016/bs.mie.2024.07.021
Saurabh Saha, Yanqiao Zhu, James Whelan, Monika W Murcha
{"title":"Methods to analyze mitochondrial protein translocation in plant mitochondria.","authors":"Saurabh Saha, Yanqiao Zhu, James Whelan, Monika W Murcha","doi":"10.1016/bs.mie.2024.07.021","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.07.021","url":null,"abstract":"<p><p>Complex processes have evolved in plants to import proteins into mitochondria. Investigating these processes in plants provides insights into the specialised machinery and pathways that have evolved to cope with; (1) the immobile nature of plants that results in exposure to environmental stresses, and (2) the more complex cell environment due to the presence of plastids, the most prevalent being chloropalst in leaves. In this chapter, we present detailed protocols for the isolation of respiratory competent, coupled mitochondria from Arabidopsis thaliana, conducting protein import assays, and analyzing protein assembly into large multi-subunit complexes. Additionally, we present straightforward protocols for examining the localization of fluorescently tagged proteins to organelles such as mitochondria through protoplast transfections.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"706 ","pages":"475-497"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142504050","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}
引用次数: 0
Isonitrile biosynthesis by non-heme iron(II)-dependent oxidases/decarboxylases. 非血红素铁（II）依赖性氧化酶/脱羧酶的异腈生物合成。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-06-29 DOI: 10.1016/bs.mie.2024.06.002
Antonio Del Rio Flores, Rui Zhai, Wenjun Zhang
{"title":"Isonitrile biosynthesis by non-heme iron(II)-dependent oxidases/decarboxylases.","authors":"Antonio Del Rio Flores, Rui Zhai, Wenjun Zhang","doi":"10.1016/bs.mie.2024.06.002","DOIUrl":"10.1016/bs.mie.2024.06.002","url":null,"abstract":"<p><p>The isonitrile group is a compact, electron-rich moiety coveted for its commonplace as a building block and bioorthogonal functionality in synthetic chemistry and chemical biology. Hundreds of natural products containing an isonitrile group with intriguing bioactive properties have been isolated from diverse organisms. Our recent discovery of a conserved biosynthetic gene cluster in some Actinobacteria species highlighted a novel enzymatic pathway to isonitrile formation involving a non-heme iron(II) and α-ketoglutarate-dependent dioxygenase. Here, we focus this chapter on recent advances in understanding and probing the biosynthetic machinery for isonitrile synthesis by non-heme iron(II) and α-ketoglutarate-dependent dioxygenases. We will begin by describing how to harness isonitrile enzymatic machinery through heterologous expression, purification, synthetic strategies, and in vitro biochemical/kinetic characterization. We will then describe a generalizable strategy to probe the mechanism for isonitrile formation by combining various spectroscopic methods. The chapter will also cover strategies to study other enzyme homologs by implementing coupled assays using biosynthetic pathway enzymes. We will conclude this chapter by addressing current challenges and future directions in understanding and engineering isonitrile synthesis.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"704 ","pages":"143-172"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11424024/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142291299","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}
引用次数: 0
Expression, purification, kinetics, and crystallization of non-heme mononuclear iron enzymes: Biphenyl, Phthalate, and Terephthalate dioxygenases. 非血红素单核铁酶的表达、纯化、动力学和结晶：联苯、邻苯二甲酸盐和对苯二甲酸盐二氧酶。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-06-08 DOI: 10.1016/bs.mie.2024.05.014
Jai Krishna Mahto, Arpan Kayastha, Pravindra Kumar
{"title":"Expression, purification, kinetics, and crystallization of non-heme mononuclear iron enzymes: Biphenyl, Phthalate, and Terephthalate dioxygenases.","authors":"Jai Krishna Mahto, Arpan Kayastha, Pravindra Kumar","doi":"10.1016/bs.mie.2024.05.014","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.05.014","url":null,"abstract":"<p><p>Non-heme iron oxygenases constitute a versatile enzyme family that is crucial for incorporating molecular oxygen into diverse biomolecules. Despite their importance, only a limited number of these enzymes have been structurally and functionally characterized. Surprisingly, there remains a significant gap in understanding how these enzymes utilize a typical architecture and reaction mechanism to catalyze a wide range of reactions. Improving our understanding of these catalysts holds promise for advancing both fundamental enzymology and practical applications. This chapter aims to outline methods for heterologous expression, enzyme preparation, in vitro enzyme assays, and crystallization of biphenyl dioxygenase, phthalate dioxygenase and terephthalate dioxygenase. These enzymes catalyze the dihydroxylation of biphenyl, phthalate and terephthalate molecules, serving as a model for functional and structural analysis of other non-heme iron oxygenases.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"704 ","pages":"39-58"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142291218","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}
引用次数: 0
Expression, purification and characterization of non-heme iron-dependent mono-oxygenase OzmD in oxazinomycin biosynthesis. 草嗪霉素生物合成过程中的非血红素铁依赖性单氧化酶 OzmD 的表达、纯化和特征。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-06-08 DOI: 10.1016/bs.mie.2024.05.006
Daan Ren, Yu-Hsuan Lee, Hung-Wen Liu
{"title":"Expression, purification and characterization of non-heme iron-dependent mono-oxygenase OzmD in oxazinomycin biosynthesis.","authors":"Daan Ren, Yu-Hsuan Lee, Hung-Wen Liu","doi":"10.1016/bs.mie.2024.05.006","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.05.006","url":null,"abstract":"<p><p>Oxazinomycin is a C-nucleoside natural product characterized by a 1,3-oxazine ring linked to ribose via a C-C glycosidic bond. Construction of the 1,3-oxazine ring depends on the activity of OzmD, which is a mononuclear non-heme iron-dependent enzyme from a family of enzymes that contain a domain of unknown function (DUF) 4243. OzmD catalyzes an unusual oxidative ring rearrangement of a pyridine derivative that releases cyanide as a by-product in the final stage of oxazinomycin biosynthesis. The intrinsic sensitivity of the OzmD substrate to oxygen along with the oxygen dependency of catalysis presents significant challenges in conducting in vitro enzymatic assays. This chapter describes the detailed procedures that have been used to characterize OzmD, including protein preparation, activity assays, and reaction by-product identification.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"704 ","pages":"113-142"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142291216","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}
引用次数: 0
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