Calpain 10 is required for cell viability and is decreased in the aging kidney.

Aging is associated with abnormalities in kidney function, but the exact mechanisms are unknown. We examined calpains 1, 2, and 10 protein levels in kidneys from rats, mice, and humans of various ages and determined whether calpain 10 is required for cell viability. Calpain 10 protein expression decreased in the kidney, but not in the liver, of aging Fischer 344 rats, and this decrease was attenuated with caloric restriction. There was no change in calpains 1 or 2 levels in the kidney or liver in control and caloric-restricted aging rats. Aging mice also exhibited decreased calpain 10 protein levels. Calpain 10 protein and mRNA levels decreased linearly in human kidney samples with age in the absence of changes in calpains 1 or 2. Our laboratory previously found calpain 10 to be expressed in both the cytosol and mitochondria of rabbit renal proximal tubular cells (RPTC). Adenoviral-delivered shRNA to rabbit RPTC decreased mitochondrial calpain 10 expression below detectable levels by 3 days while cytosolic calpain 10 levels remained unchanged at 3 days and decreased to approximately 20% of control by 5 days. Knockdown of mitochondrial calpain 10 resulted in nuclear condensation and cleaved procaspase 3, markers of apoptosis. In summary, mitochondrial calpain 10 is required for cell viability and calpain 10 levels specifically decrease in aging rat, mice, and human kidney tissues when renal function decreases, suggesting that calpain 10 is required for renal function and is a biomarker of the aging kidney.

Identification and optimization of a novel inhibitor of mitochondrial calpain 10.

Calpain 10 has been localized to the mitochondria and is a key mediator of Ca(2+) induced mitochondrial dysfunction. A peptide screen followed by a series of modifications identified the homodisulfide form of CYGAK (CYGAK)(2) as an inhibitor of calpain 10 while showing no inhibitory activity against calpain 1. Methylation or truncation of the N-terminal cysteine significantly reduced the inhibitory activity of (CYGAK)(2) and inhibition was reversed by reducing agents, suggesting that CYGAK forms a disulfide with a cysteine near the active site. Data suggests CYGAK may be a P' calpain inhibitor and may achieve its specificity through this mechanism. CYGAK inhibited calpain activity in intact mitochondria, renal cells, and hepatocytes, prevented Ca(2+) induced cleavage of NDUFV2, and blocked Ca(2+) induced state III dysfunction. (CYGAK)(2) is the first P' specific calpain inhibitor and will be a valuable tool to prevent Ca(2+) induced mitochondrial dysfunction and explore the function of calpain 10.

Calpain 10: a mitochondrial calpain and its role in calcium-induced mitochondrial dysfunction.

Calpains, Ca(2+)-activated cysteine proteases, are cytosolic enzymes implicated in numerous cellular functions and pathologies. We identified a mitochondrial Ca(2+)-inducible protease that hydrolyzed a calpain substrate (SLLVY-AMC) and was inhibited by active site-directed calpain inhibitors as calpain 10, an atypical calpain lacking domain IV. Immunoblot analysis and activity assays revealed calpain 10 in the mitochondrial outer membrane, intermembrane space, inner membrane, and matrix fractions. Mitochondrial staining was observed when COOH-terminal green fluorescent protein-tagged calpain 10 was overexpressed in NIH-3T3 cells and the mitochondrial targeting sequence was localized to the NH(2)-terminal 15 amino acids. Overexpression of mitochondrial calpain 10 resulted in mitochondrial swelling and autophagy that was blocked by the mitochondrial permeability transition (MPT) inhibitor cyclosporine A. With the use of isolated mitochondria, Ca(2+)-induced MPT was partially decreased by calpain inhibitors. More importantly, Ca(2+)-induced inhibition of Complex I of the electron transport chain was blocked by calpain inhibitors and two Complex I proteins were identified as targets of mitochondrial calpain 10, NDUFV2, and ND6. In conclusion, calpain 10 is the first reported mitochondrially targeted calpain and is a mediator of mitochondrial dysfunction through the cleavage of Complex I subunits and activation of MPT.