Calpain Inhibitors
Calpains belong to a family of calcium-dependent thiol-proteases that proteolyze a wide variety of cytoskeletal, membraneassociated, and regulatory proteins. Fifteen gene products of the calpain family are reported in mammals, which are classified as nine typical and six atypical calpains. Typical calpains are characterized by a C-terminal Ca2+- binding domain that includes EF-hand motifs, while atypical calpains lack this region, but often contain additional domains. Calpains do not generally function as destructive proteases, but act as calcium-dependent modulators that remove limited portions of protein substrates. Calpains respond to Ca2+ signals by cleaving specific proteins, frequently components of signaling cascades, thereby irreversibly modifying their function. Two major isoforms of calpain are reported in mammals, calpain-1 (µ-form) and calpain-2 (m-form). They are constitutively expressed in all tissues and differ in their calcium requirement for activation (~50 µM for calpain-1 and ~500 µM for calpain-2) and contain several calciumbinding sites, which allosterically affect the enzyme activity.
Calpain-1 and -2 exhibit about 55 to 65% sequence homology and are composed of an 80 kDa and a 30 kDa subunit. The 80 kDa subunit has the catalytic site and is unique to each isozyme, whereas the 30 kDa unit is the regulatory subunit and is common to both calpains 1 and 2. Several putative substrates of calpain-1 and -2 are known that are cleaved by both isoforms, but with different efficiencies. Recently, determinants for calpain-1 and -2 have been analyzed and it is shown that amino acid preferences extend over 11 residues around the scissile bond. Calpains prefer Leu, Thr, Val in the P2 position, Lys, Tyr, Arg in the P1 position, and proline in the region flanking the P2 - P’1 segment.
Calpain-3, another typical calpain was first described as a skeletal muscle-specific calpain isoform. However, subsequent studies have shown its presence in several other tissues. It is a 94 kDa enzyme that is structurally similar to calpain-1 and -2, however, it has an additional N-terminal sequence of 20 -30 amino acids.
More recently, attention has been focused on the pathological significance of calcium accumulation in the central nervous system following cerebral ischemia and traumatic brain injury. Over-activation of NMDA, kainate and AMPA receptors in the brain leads to sustained influx of Ca2+ through the voltage-gated calcium channels. Disturbances in calcium homeostasis result in the activation of several calcium-dependent enzymes including calpains. Over-expression of calpains has been positively linked to both acute and chronic neurodegenerative processes including ischemia, trauma, and Alzheimer’s disease. In Alzheimer’s disease the ratio of active (76 kDa) to inactive (80 kDa) calpain-1 is reported to be much higher. Calpain proteolysis is usually the late-stage common pathway towards cell death induced by excitotoxic compounds; hence, a selective inhibition of calpains to limit neuronal damage appears to be a viable therapeutic measure. However, most of the inhibitors reported are active site targeted peptides and their limited cell permeability poses problems.
References:
Bartoli, M., and Richard, I. 2005. Int. J. Biochem. Cell Biol. 37, 2115.
Tompa, P. et al, 2004. J. Biol. Chem. 279, 20775.
Goll, D.E. et al. 2003. Physiol. Rev. 83, 731.
Kinbara, K. et al. 1998. Biochem. Pharmacol. 56, 415.
