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Global Profiling of Proteolysis from the Mitochondrial Amino Terminome during Early Intrinsic Apoptosis Prior to Caspase-3 Activation
Journal of Proteome Research ( IF 4.4 ) Pub Date : 2018-10-29 , DOI: 10.1021/acs.jproteome.8b00675
Natalie C. Marshall 1 , Theo Klein , Maichael Thejoe 1 , Niklas von Krosigk 1 , Jayachandran Kizhakkedathu 2 , B. Brett Finlay 1 , Christopher M. Overall
Affiliation  

The human genome encodes ∼20 mitochondrial proteases, yet we know little of how they sculpt the mitochondrial proteome, particularly during important mitochondrial events such as the initiation of apoptosis. To characterize global mitochondrial proteolysis we refined our technique, terminal amine isotopic labeling of substrates, for mitochondrial SILAC (MS-TAILS) to identify proteolysis across mitochondria and parent cells in parallel. Our MS-TAILS analyses identified 45% of the mitochondrial proteome and identified protein amino (N)-termini from 26% of mitochondrial proteins, the highest reported coverage of the human mitochondrial N-terminome. MS-TAILS revealed 97 previously unknown proteolytic sites. MS-TAILS also identified mitochondrial targeting sequence (MTS) removal by proteolysis during protein import, confirming 101 MTS sites and identifying 135 new MTS sites, revealing a wobbly requirement for the MTS cleavage motif. To examine the relatively unknown initial cleavage events occurring before the well-studied activation of caspase-3 in intrinsic apoptosis, we quantitatively compared N-terminomes of mitochondria and their parent cells before and after initiation of apoptosis at very early time points. By identifying altered levels of >400 N-termini, MS-TAILS analyses implicated specific mitochondrial pathways including protein import, fission, and iron homeostasis in apoptosis initiation. Notably, both staurosporine and Bax activator molecule-7 triggered in common 7 mitochondrial and 85 cellular cleavage events that are potentially part of an essential core of apoptosis-initiating events. All mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the dataset identifier PXD009054.

中文翻译:

Caspase-3激活之前的早期内在凋亡过程中从线粒体氨基末端蛋白水解的全球剖析。

人类基因组编码约20种线粒体蛋白酶,但我们对它们如何雕刻线粒体蛋白质组的了解甚少,尤其是在重要的线粒体事件(例如细胞凋亡的启动过程)中。为了表征整体线粒体蛋白水解,我们改进了技术,即底物的末端胺同位素标记,用于线粒体SILAC(MS-TAILS),以跨线粒体和亲代细胞并行鉴定蛋白水解。我们的MS-TAILS分析确定了45%的线粒体蛋白质组,并从26%的线粒体蛋白质中鉴定了蛋白质氨基(N)末端,这是人类线粒体N末端组报道的最高报道。MS-TAILS揭示了97个以前未知的蛋白水解位点。MS-TAILS还确定了蛋白质导入过程中通过蛋白水解作用去除的线粒体靶向序列(MTS),确定了101个MTS站点并确定了135个新的MTS站点,这揭示了对MTS裂解基序的不稳定要求。为了检查在充分研究caspase-3激活内在凋亡之前发生的相对未知的初始切割事件,我们定量比较了在非常早的时间点开始凋亡之前和之后的线粒体及其亲代细胞的N末端。通过鉴定> 400 N-末端的变化水平,MS-TAILS分析了凋亡起始中涉及的特定线粒体途径,包括蛋白质导入,裂变和铁稳态。值得注意的是,星形孢菌素和Bax活化剂分子7均在常见的7个线粒体和85个细胞裂解事件中触发,这可能是细胞凋亡起始事件核心的一部分。
更新日期:2018-10-30
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