Global Profiling of Proteolysis from the Mitochondrial Amino Terminome during Early Intrinsic Apoptosis Prior to Caspase‑3 Activation

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.

人类基因组编码约20种线粒体蛋白酶，但我们对它们如何塑造线粒体蛋白质组，尤其是在细胞凋亡启动等重要线粒体事件中的作用仍知之甚少。为了表征全局线粒体蛋白水解过程，我们对底物末端氨基同位素标记（terminal amine isotopic labeling of substrates, TAILS）技术进行优化，将其适配于线粒体稳定同位素标记氨基酸细胞培养（stable isotope labeling with amino acids in cell culture, SILAC）体系，开发出线粒体SILAC-MS-TAILS技术，以并行鉴定线粒体及其母细胞中的蛋白水解事件。本研究的MS-TAILS分析覆盖了45%的人类线粒体蛋白质组，并鉴定了26%线粒体蛋白质的氨基（N）末端，这是目前已报道的人类线粒体N末端组（N-terminome）最高覆盖度。MS-TAILS共鉴定出97个此前未知的蛋白水解位点。MS-TAILS还鉴定了蛋白质导入过程中通过蛋白水解作用去除线粒体靶向序列（mitochondrial targeting sequence, MTS）的过程，验证了101个MTS位点，并新发现135个MTS位点，揭示了线粒体靶向序列切割基序的作用需求并非严格固定。为了探究在被广泛研究的半胱天冬酶-3（caspase-3）激活之前的、相对未知的内源性细胞凋亡早期切割事件，我们在极早期时间点下，对细胞凋亡启动前后的线粒体及其母细胞的N末端组进行了定量比较。通过鉴定超过400个N末端的水平变化，MS-TAILS分析将特定线粒体通路与细胞凋亡启动关联起来，包括蛋白质导入、线粒体裂变以及铁稳态。值得注意的是，星形孢菌素（staurosporine）和Bax激活因子7（Bax activator molecule-7）这两种处理方式，共同触发了7个线粒体和85个细胞的切割事件，这些事件可能属于细胞凋亡启动核心必需事件的一部分。所有质谱蛋白质组学数据已提交至ProteomeXchange联盟（ProteomeXchange Consortium），数据集标识符为PXD009054。