N‑Terminomic Identification of Intracellular MMP‑2 Substrates in Cardiac Tissue

Proteases are enzymes
that induce irreversible post-translational
modifications by hydrolyzing amide bonds in proteins. One of these
proteases is matrix metalloproteinase-2 (MMP-2), which has been shown
to modulate extracellular matrix remodeling and intracellular proteolysis
during myocardial injury. However, the substrates of MMP-2 in heart
tissue are limited, and lesser known are the cleavage sites. Here,
we used degradomics to investigate the substrates of intracellular
MMP-2 in rat ventricular extracts. First, we designed a novel, constitutively
active MMP-2 fusion protein (MMP-2-Fc) that we expressed and purified
from mammalian cells. Using this protease, we proteolyzed ventricular
extracts and used subtiligase-mediated N-terminomic labeling which
identified 95 putative MMP-2-Fc proteolytic cleavage sites using mass
spectrometry. The intracellular MMP-2 cleavage sites identified in
heart tissue extracts were enriched for proteins primarily involved
in metabolism, as well as the breakdown of fatty acids and amino acids.
We further characterized the cleavage of three of these MMP-2-Fc substrates
based on the gene ontology analysis. We first characterized the cleavage
of sarco/endoplasmic reticulum calcium ATPase (SERCA2a), a known MMP-2
substrate in myocardial injury. We then characterized the cleavage
of malate dehydrogenase (MDHM) and phosphoglycerate kinase 1 (PGK1),
representing new cardiac tissue substrates. Our findings provide insights
into the intracellular substrates of MMP-2 in cardiac cells, suggesting
that MMP-2 activation plays a role in cardiac metabolism.

蛋白酶是一类通过水解蛋白质中的酰胺键，引发不可逆翻译后修饰的酶。其中一类为基质金属蛋白酶-2（matrix metalloproteinase-2, MMP-2），已有研究表明其在心肌损伤过程中可调控细胞外基质重塑与细胞内蛋白水解。然而，目前心脏组织中MMP-2的底物较为有限，其剪切位点的相关认知则更为匮乏。本研究借助降解组学技术，探究大鼠心室提取物中细胞内MMP-2的底物。首先，我们设计了一种新型组成型活性MMP-2融合蛋白（MMP-2-Fc），并在哺乳动物细胞中对其进行表达与纯化。利用该蛋白酶，我们对心室提取物进行蛋白水解，并采用枯草杆菌蛋白酶介导的N端组学标记技术，通过质谱鉴定得到95个推测的MMP-2-Fc剪切位点。在心脏组织提取物中鉴定得到的细胞内MMP-2剪切位点所对应的蛋白，主要富集于代谢过程，以及脂肪酸与氨基酸的分解代谢通路。基于基因本体分析，我们进一步对其中3种MMP-2-Fc底物的剪切行为进行了表征。我们首先验证了肌浆网/内质网钙ATP酶（sarco/endoplasmic reticulum calcium ATPase, SERCA2a）的剪切——该蛋白此前已被证实为心肌损伤中的MMP-2底物；随后我们分别表征了苹果酸脱氢酶（malate dehydrogenase, MDHM）与磷酸甘油酸激酶1（phosphoglycerate kinase 1, PGK1）的剪切，二者均为新发现的心脏组织底物。本研究结果为心肌细胞内MMP-2的细胞内底物提供了新的认知，提示MMP-2激活在心脏代谢调控中发挥一定作用。