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.

中文翻译：

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心脏组织中细胞内 MMP-2 底物的 N 末端鉴定

蛋白酶是通过水解蛋白质中的酰胺键诱导不可逆翻译后修饰的酶。其中一种蛋白酶是基质金属蛋白酶-2 (MMP-2)，它已被证明可以在心肌损伤期间调节细胞外基质重塑和细胞内蛋白水解。然而，心脏组织中 MMP-2 的底物有限，而且切割位点鲜为人知。在这里，我们使用降解组学来研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的、组成型活性 MMP-2 融合蛋白 (MMP-2-Fc)，并从哺乳动物细胞中表达和纯化。使用这种蛋白酶，我们对心室提取物进行蛋白水解，并使用枯草连接酶介导的 N 末端标记，利用质谱法鉴定了 95 个假定的 MMP-2-Fc 蛋白水解切割位点。在心脏组织提取物中鉴定出的细胞内 MMP-2 裂解位点富含主要参与代谢以及脂肪酸和氨基酸分解的蛋白质。我们根据基因本体分析进一步表征了其中三种 MMP-2-Fc 底物​​的裂解。我们首先描述了肌浆/内质网钙 ATP 酶 (SERCA2a) 的裂解，SERCA2a 是心肌损伤中已知的 MMP-2 底物。然后，我们对代表新心脏组织底物的苹果酸脱氢酶 (MDHM) 和磷酸甘油酸激酶 1 (PGK1) 的裂解进行了表征。我们的研究结果提供了对心肌细胞中 MMP-2 胞内底物的深入了解，表明 MMP-2 激活在心脏代谢中发挥作用。