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（Proteases）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（post-translational modifications）的酶。其中一类为基质金属蛋白酶-2（matrix metalloproteinase-2, MMP-2），已有研究证实其可在心肌损伤（myocardial injury）进程中调控细胞外基质重塑（extracellular matrix remodeling）与细胞内蛋白水解（intracellular proteolysis）。然而，当前心脏组织中MMP-2的已知底物较为匮乏，其对应的酶切位点的相关认知亦十分有限。本研究借助降解组学（degradomics）技术，对大鼠心室提取物中的细胞内MMP-2底物开展系统性探究。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（mammalian cells）中完成其表达与纯化。利用该蛋白酶消化大鼠心室提取物，结合枯草杆菌蛋白酶（subtiligase）介导的N端组学标记（subtiligase-mediated N-terminomic labeling）技术，通过质谱（mass spectrometry）分析共鉴定得到95个潜在的MMP-2-Fc酶切位点。对心脏组织提取物中鉴定得到的细胞内MMP-2切割位点所富集的蛋白进行分析后发现，这些蛋白主要参与代谢过程，以及脂肪酸与氨基酸的分解代谢。基于基因本体分析（gene ontology analysis）结果，我们进一步对其中3种MMP-2-Fc底物的酶切特性进行了表征：首先验证了肌浆网/内质网钙ATP酶（sarco/endoplasmic reticulum calcium ATPase, SERCA2a）的切割——这是一种已被报道的心肌损伤过程中的MMP-2底物；随后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）的酶切过程，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全新的研究视角，提示MMP-2激活在心脏代谢调控中发挥了潜在作用。