Calpains: Structure, Regulation, And Cell Survival

The calpain system

Calpains are a well-conserved family of intracellular cysteine proteases that were first described in 1964. Fourteen different isoforms of calpain have been identified by the presence of protease domains. These include micro-calpains (µ-calpains) and milli calpains (m-calpains). µ-calpains are the most extensively studied isoforms of calpains. However, the precise physiological protease activity of these isoforms still need to be elucidated, studies have demonstrated their significant role in several cellular activities such as- cell motility and apoptosis.

Calpain structure:

A few members of the calpain family rely on calcium ions and a neutral pH for their proteolytic activity. The µ-calpains and the m-calpains were named based on their calcium ion concentration required for activation. Both µ-calpains and m-calpains are heterodimers consisting of a catalytic subunit (80 kDa) and a regulatory subunit (120 kDa). The catalytic subunits differ between the two isoforms as they are formed by calpain 1 (encoded by CAPN1) and calpain 2 (encoded by CAPN2) respectively. The regulatory subunit is common to both the isoforms and is encoded by CAPNS1. The catalytic subunit has four domains- D1 to DIV and the regulatory subunit has two DV to DVI. D1 is autolysed when calpains are activated by calcium. D2 is a conserved protease domain which is divided in to IIa and IIb that on binding with calcium forms a signal domain (II) that has the catalytic cleft. The DIII domain is involved in structural changes and has the characteristic C2 domains. The DIV and DVI are carboxy terminal domains and have five EF hands. The fifth EF hand aids in dimerization of the subunits. Despite the DII protease subunit being conserved, there exists a divergence in these subunits within the isoforms as a result, not all calpains are activated by calcium or require the regulatory subunit.

Calpain regulation by calpastatin:

Calpastatin (CAST) is the endogenous inhibitor of µ-calpain and m-calpain and is expressed ubiquitously. It consists of an L domain that contains an N terminal XL region, and four inhibitory domains (I-IV) and can reversibly inhibit up to four calpain heterodimers. Structural changes in calpain allow inhibition by each inhibitory domain of the calpastatin. These are liberated from intracellular storage aggregates into the cytosol as a result of calcium influx for calpain interaction. CAST is a single gene with several promoters that generates distinct calpastatin isoforms with a variation at the N terminal. Type I and Type II calpastatins have the L domain but differ in N terminal sequences. Type III calpastatin is a product of a promoter that is associated with the untranslated region 1u. Type IV calpastatin is a testis specific isoform which is a product of promoter between exons 14 and 15. Additionally, to the alternative promoters, transcripts that are alternatively spliced at the L-domain, results in the generation of several polypeptides arising from a single gene. A small region at the calpastatin L domain has been shown to partially reprime L-type calcium channels after voltage gated activation. The inhibitory specificity, efficacy and cellular location of calpastatins is effected by the post-translational phosphorylation by PKA.

Calpain activity in cell survival and death:

Pro-survival: Calpain functions promotes pro-survival activities of the cell via p53 and nuclear factor-kB (NF-κB). It has been shown that calpains cleave the wild type p53 which affects the protein stability and inhibits p53 mediated apoptosis. The growth arrest specific protein 2 (GAS2) has been shown to associate with calpain and prevent the cleavage of p53 leading to enhanced p53 stability. Additionally, calpains promote survival through the activation of NF-κB by the proteolysis of its inhibitor IκBα. Activation of epidermal growth factor receptor (EGFR) and tumour necrosis factor (TNF) in breast cancer cells regulates the cleavage of IκBα. Proteolysis of

IκBα is mediated by µ-calpain, m-calpain, and calpain-3 and can be disrupted by overexpression of calpastatins. Calpains are also responsible for proteolysis of several other substrates that promote apoptosis,- such as the transcription factor MYC. MYC has shown to reduce calpastatin expression thus increasing calpain activity.

Pro apoptotic: Calpains have been shown to associate with caspase cysteine protease machinery such as- caspase 7, caspase 9, caspase 10, and caspase 12 in the induction of apoptosis. Caspase 7 and caspase 10 are activated by calpain cleavage while caspase 9 is inactivated by calpain cleavage. Calpain can activate caspase 12 during endoplasmic reticulum stress causing apoptosis. Caspase 8 has shown to disrupt calpastatin mediated inhibition of m-calpain by interfering with their interaction and resulting in tumour cell migration. Calpains cleave members of the apoptosis regulating BCL-2 family, thus promoting apoptosis. These include BAX and BID- mediated cytochrome c release, and cleavage of BCL-2. In some cellular models calpains can cleave their inhibitor calpastatin.

The prosurvival or proapototic activity is very context, and cell type, dependent. Abnormal calpain activity can cause or accelerate disease progression. Some of these include: neurodegenerative disease, cataract formation, myocardial infarction and limb-girdle muscular dystrophy type 2A (LGMD2A).