Identification of putative in vivo substrates of calpain 3 by comparative proteomics of overexpressing transgenic and nontransgenic mice
Identification of putative in vivo substrates of calpain 3 by comparative proteomics of overexpressing transgenic and nontransgenic mice
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DOI：
10.1002/pmic.200600199
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发表时间：
2006-11-01
期刊：
影响因子：
3.4
通讯作者：
Spencer, Melissa J.
中科院分区：
文献类型：
--
作者：
Cohen, Niaz;Kudryashova, Elena;Spencer, Melissa J.
钙蛋白酶3（CAPN 3）是一种钙依赖性蛋白酶，其突变导致肢带型肌营养不良症2A型。为了探索CAPN 3的生理功能，我们比较了过表达CAPN 3的转基因小鼠（CAPN 3 Tg）和非转基因小鼠（non-Tg）的蛋白质组。我们首先检查了已知的肌营养不良症相关蛋白，以确定CAPN 3的过度表达是否会导致其分布或浓度的变化。该分析没有鉴定出任何已知的肌营养不良蛋白作为CAPN 3的底物。接下来，我们使用蛋白质组学方法来比较和鉴定CAPN 3 Tg和非Tg小鼠的2-DE中差异表达的蛋白质。LC-MS/MS分析鉴定出CAPN 3的十种可能底物，分为两大功能类别：代谢和肌原纤维。肌球蛋白轻链1（MLC 1）是重点，因为我们以前的研究表明CAPN 3在肌节重塑中的作用。在这项研究中，CAPN 3显示出在体外蛋白水解MLC 1。这些研究是第一个在体内系统中鉴定CAPN 3的可能底物，并支持CAPN 3通过切割肌原纤维蛋白如MLC 1在肌节重塑中的作用。此外，这些数据还表明CAPN 3在线粒体蛋白质周转中的作用。
Calpain 3 (CAPN3) is a calcium-dependent protease, mutations in which cause limb girdle muscular dystrophy type 2A. To explore the physiological function of CAPN3, we compared the proteomes of transgenic mice that overexpress CAPN3 (CAPN3 Tg) and their nontransgenic (non-Tg) counterparts. We first examined known muscular dystrophy-related proteins to determine if overexpression of CAPN3 results in a change in their distribution or concentration. This analysis did not identify any known muscular dystrophy proteins as substrates of CAPN3. Next, we used a proteomic approach to compare and identify differentially represented proteins in 2-DE of CAPN3 Tg and non-Tg mice. LC-MS/MS analysis led to the identification of ten possible substrates for CAPN3, classified into two major functional categories: metabolic and myofibrillar. Myosin light chain 1 (MLC1) was focused upon because our previous studies suggested a role for CAPN3 in sarcomere remodeling. In this study, CAPN3 was shown to proteolyze MLC1 in vitro. These studies are the first to identify possible substrates for CAPN3 in an in vivo system and support a role for CAPN3 in sarcomere remodeling by cleavage of myofibrillar proteins such as MLC1. In addition, these data also suggest a role for CAPN3 in mitochondrial protein turnover.