Monitoring mitochondrial precursor processing and presequence peptide degradation.

4区 生物学 Q3 Biochemistry, Genetics and Molecular Biology
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
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引用次数: 0

Abstract

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.

监测线粒体前体加工和前序肽降解。
线粒体前序前体蛋白输入细胞器后的成熟是一个复杂的过程,需要多种线粒体蛋白酶的相互作用。线粒体前序蛋白酶对前体的处理直接与线粒体前序肽酶对裂解的靶向信号的蛋白水解周转相关联。这些酶的功能障碍与多种人类疾病有关,包括神经系统疾病、心肌病和肾脏疾病。在本章中,我们将介绍研究主要线粒体前序蛋白酶(MPP)和前序肽酶活性的实验方法。通过体外测定和可溶性线粒体提取物,可以使用免疫印迹法、荧光测量法和自显影法作为读数,对肽酶和蛋白酶的活性进行评估和实验操作。特别是,这些检测方法可以在体内、器官或可溶性提取物/体外等多个层面进行操作。通过纯化酵母异源二聚体 MPP,可以使用放射性标记的前体作为底物,在体外重组最初的前序处理步骤。应用可溶性线粒体提取物可以直接评估 MPP 的加工过程和前序肽的周转情况,这很容易操作,而且与蛋白质在线粒体膜上的转运无关。本章介绍的技术可对前体加工和前序肽周转进行深入分析,并直接评估患者基因突变对前序肽加工机制活性的影响。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Methods in enzymology
Methods in enzymology 生物-生化研究方法
CiteScore
2.90
自引率
0.00%
发文量
308
审稿时长
3-6 weeks
期刊介绍: The critically acclaimed laboratory standard for almost 50 years, Methods in Enzymology is one of the most highly respected publications in the field of biochemistry. Each volume is eagerly awaited, frequently consulted, and praised by researchers and reviewers alike. Now with over 500 volumes the series contains much material still relevant today and is truly an essential publication for researchers in all fields of life sciences, including microbiology, biochemistry, cancer research and genetics-just to name a few. Five of the 2013 Nobel Laureates have edited or contributed to volumes of MIE.
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