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

Area of Science:

• Biochemistry
• Molecular Biology
• Cardiology

Background:

• Proteases, like matrix metalloproteinase-2 (MMP-2), modify proteins, impacting cellular processes.
• MMP-2 is implicated in myocardial injury, but its intracellular substrates and cleavage sites in heart tissue are not well-defined.

Purpose of the Study:

• To investigate the intracellular substrates and cleavage sites of MMP-2 in rat ventricular extracts using a novel degradomics approach.
• To identify novel cardiac tissue substrates of MMP-2 and understand its role in cardiac metabolism.

Main Methods:

• Developed and purified a constitutively active MMP-2 fusion protein (MMP-2-Fc) for proteolysis.
• Utilized subtiligase-mediated N-terminomic labeling and mass spectrometry to identify MMP-2 cleavage sites.
• Characterized novel MMP-2 substrates (malate dehydrogenase and phosphoglycerate kinase 1) and a known substrate (SERCA2a) via gene ontology analysis.

Main Results:

• Identified 95 putative MMP-2-Fc proteolytic cleavage sites in rat ventricular extracts.
• Proteins cleaved by MMP-2 were enriched in pathways related to metabolism, fatty acid, and amino acid breakdown.
• Confirmed cleavage of SERCA2a and identified malate dehydrogenase (MDHM) and phosphoglycerate kinase 1 (PGK1) as new cardiac MMP-2 substrates.

Conclusions:

• This study reveals novel intracellular substrates of MMP-2 in cardiac cells, expanding the understanding of its function beyond extracellular matrix remodeling.
• MMP-2 activation appears to play a significant role in regulating cardiac metabolism through the proteolysis of key metabolic enzymes.