Calpains: physiological and pathophysiological significance (original) (raw)
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Journal of Biological Chemistry, 2006
It is generally accepted that the Ca 2؉ -dependent interaction of calpain with calpastatin is the most relevant mechanism involved in the regulation of Ca 2؉ -induced proteolysis. We now report that a calpain-calpastatin association can occur also in the absence of Ca 2؉ or at very low Ca 2؉ concentrations, reflecting the physiological conditions under which calpain retains its inactive conformational state. The calpastatin binding region is localized in the non-inhibitory L-domain containing the amino acid sequences encoded by exons 4 -7. This calpastatin region recognizes a calpain sequence located near the end of the DIIdomain. Interaction of calpain with calpastatins lacking these sequences becomes strictly Ca 2؉ -dependent because, under these conditions, the transition to an active state of the protease is an obligatory requirement. The occurrence of the molecular association between Ca 2؉ -free calpain and various recombinant calpastatin forms has been demonstrated by the following experimental results. Addition of calpastatin protected calpain from trypsin digestion. Calpain was coprecipitated when calpastatin was immunoprecipitated. The calpastatin molecular size increased following exposure to calpain. The two proteins comigrated in zymogram analysis. Furthermore, calpain-calpastatin interaction was perturbed by protein kinase C phosphorylation occurring at sites located at the exons involved in the association. At a functional level, calpain-calpastatin interaction at a physiological concentration of Ca 2؉ represents a novel mechanism for the control of the amount of the active form of the protease potentially generated in response to an intracellular Ca 2؉ influx.
Role of Calpain in Pathogenesis of Human Disease Processes
Journal of nature and science, 2016
Calpains are a 15-member class of calcium activated nonlysosomal neutral proteases which are involved in a broad range of cellular function. Calpains are usually localized to the cytosol and within mitochondria. Calpastatin is an endogenous protein that specifically binds to and inhibits calpain. Overactivation of calpain has been implicated in a number of disease processes of the brain, eyes, heart, lungs, pancreas, kidneys, vascular system and skeletal muscle. Therefore, calpain may serve as a potential therapeutic target for a wide variety of disease processes. This review briefly outlines the current literature regarding the involvement of calpain overactivation in the pathogenesis of almost every organ in the body.
Implications of calpains in health and diseases
Indian journal of biochemistry & biophysics, 2012
The number of mammalian calpain protease family members has grown as many as 15 till recent count. Although initially described as a cytosolic protease, calpains have now been found in almost all subcellular locations i.e., from mitochondria to endoplasmic reticulum and from caveolae to Golgi bodies. Importantly, some calpains do not possess the 28 kDa regulatory subunit and have only the 80 kDa catalytic subunit. In some instances, the 80 kDa subunit by itself confers the calpain proteolytic activity. Calpains have been shown to be involved in a number of physiological processes such as cell cycle progression, remodeling of cytoskeletal-cell membrane attachments, signal transduction, gene expression and apoptosis. Recent studies have linked calpain deficiencies or it's over production with a variety of diseases, such as muscular dystrophies, gastropathy, diabetes, Alzheimer's and Parkinson's diseases, atherosclerosis and pulmonary hypertension. Herein, we present a brie...
Journal of Biological Chemistry, 1996
Calpains are intracellular Ca 2؉-dependent proteases that are thought to participate in Ca 2؉-associated signal transduction pathways. It has been proposed that calpains are activated by an autoproteolytic mechanism. If this is true one would expect a relatively short half-life for calpain protein in cells. To test this hypothesis, WI-38 human diploid fibroblasts were pulse-labeled with [ 35 S]methionine, and calpain was immunoprecipitated at various times after chasing with nonradioactive methionine to determine residual radioactivity. The results demonstrated that the two major calpain isozymes, m-calpain and-calpain, had metabolic half-lives of approximately 5 days. Calpains were long-lived proteins in several human cell lines, A-431, HeLa, VA-13, C-33A, and TE2 cells. In addition, calpastatin, the calpain-specific inhibitor protein, also had a long metabolic half-life. These observations suggest that the model for calpain activation by autoproteolysis requires re-investigation. Based on a knowledge of calpain metabolic stability, a protocol was devised for chronic exposure of WI-38 cells and HeLa cells to a calpain small subunit antisense oligodeoxyribonucleotide. Depletion of calpain small subunit after 5 or more days of treatment led to inhibition of cell proliferation that could be reversed by removal of antisense oligodeoxyribonucleotide from the culture medium. Together with previous studies, these results indicate a requirement for calpains in mammalian cell proliferation.
Folia histochemica et cytobiologica / Polish Academy of Sciences, Polish Histochemical and Cytochemical Society, 2007
Limited proteolysis of multiple intracellular proteins by endogenous Ca-dependent cysteine proteases--calpains--is an important regulatory mechanism for cell proliferation, apoptosis etc. Its importance for cellular functions is stressed by existence of endogenous calpain inhibitors--calpastatins. The calpain-calpastatin system within living cells is in a fragile balance, which depends on both partners. The interdependence of calpain--a protease--and calpastatin--an endogenous inhibitor and at the same time a substrate for this enzyme makes any assessment of actual activity of this enzyme in the cells very difficult. In this work we made an attempt to estimate and compare the activity of calpain in human peripheral blood lymphocytes by assessing the levels of limited proteolysis of calpastatin in these cells by western blot, while at the same time the levels of calpain protein inside these cells was measured by flow cytometry. Our results indicate that it is possible to compare (sem...
Modulators of calpain activity: inhibitors and activators as potential drugs
Expert Opinion on Drug Discovery, 2020
Introduction Calpains are intracellular Ca 2+-dependent cysteine proteases with 15 known members in the enzyme family. They act as regulatory enzymes, their cleavage modifies the function of their substrates. As their substrates have important roles in many physiological processes, adequate function of calpains is mandatory for normal cellular functions. Adverse operation of them is often related with diseases (e.g. neurodegenerative disorders, cancer, type 2 diabetes mellitus or limb-girdle muscular dystrophy type 2A). Areas covered Herein, the authors give an overview about calpains, their structure, physiological and pathological functions. The challenges in the drug discovery of calpain inhibitors and activators are enlightened by summarising examples that eventuated good candidates. Going through these examples new and applicable strategies are discussed. Expert opinion Calpain enzymes are attractive targets to design inhibitors or activators for drug development. This research area has high potential, although it has many challenges. The selective and targeted inhibition or activation of calpains is needed. Thus the studies focused on the improvement of these properties of drug candidates. Keywordscalpain, cysteine proteases, calpastatin, calpain inhibitor, calpain activator, structure-activity-relationship (SAR) Article highlights • Calpains are intracellular cysteine proteases with very diverse physiological roles. • Aberrant activity of them; over-or under activation; is often involved in pathological functions and thus in disease. • Specific and effective inhibitors may be potent candidate to treat these diseases. • Peptide and peptidomimetic inhibitors were developed to increase the specificity and potency using different strategies. 4 calpain 1 and 2 [4]. These proteases, called conventional calpains, are ubiquitous and wellstudied members. Their activation needs micro-and millimolar concentration of Ca 2+ ion in vitro respectively [5]. They were called μand m-calpain based on this sensitivity for Ca 2+ ion. Both enzymes form heterodimers with a small regulatory subunit (calpain 4), and expressed in all tissues, while many of the later described members have tissue specific distribution (calpain 3, 6, 8, 9 11, 12, 15 and 16) [6] (Table 1). There are 15 calpain genes in the human genome (CAPN1 to CAPN16, no CAPN4). Calpains are classified as classical or non-classical calpains based on their domain structure; on what kind of conserved protein domains are attached to the well-conserved cysteine protease domain (CysPc) (Fig. 1A) [7]. The CysPc domain in calpain is composed of protease core 1 (PC1) and PC2 domains. Amino acids that form the catalytic triad were in distinct domains; the Cys is in the PC1 while the His and Asn are in the PC2 domain. The calpain 6 is the only member that does not have a catalytic triad; there is Lys instead of Cys. In the absence of Ca 2+ ions these two domains are far from each other and thus the catalytic triad is not assembled [8]. Both domains can bind Ca 2+ ion and that results in structural changes opening of the active site cleft and assembling the catalytic triad [9] (the relationship between the structure and activity is well summarised in Ref [10]). Although the whole 3D structure of only calpain 2 is known (Fig. 2A) [9,11], using this for 3D modelling of other calpains is an accepted strategy [12,13]. 2.2 Physiological role of Calpains 2.2.1 Regulation of enzyme activity Calpains are expressed in different organisms, for example in human, invertebrates, yeast, other fungi and bacteria. These intracellular enzymes cleave their substrate proteins in welldetermined manner. This limited proteolysis activates or deactivates the substrate proteins, thus calpains have regulatory or signalling function, rather than protein digestion in cells like proteasomal or lysosomal enzymes do. Because of this, their activity should be under strong
Calpains, mitochondria, and apoptosis
Cardiovascular Research, 2012
Mitochondrial activity is critical for efficient function of the cardiovascular system. In response to cardiovascular injury, mitochondrial dysfunction occurs and can lead to apoptosis and necrosis. Calpains are a 15-member family of Ca 2+-activated cysteine proteases localized to the cytosol and mitochondria, and several have been shown to regulate apoptosis and necrosis. For example, in endothelial cells, Ca 2+ overload causes mitochondrial calpain 1 cleavage of the Na + /Ca 2+ exchanger leading to mitochondrial Ca 2+ accumulation. Also, activated calpain 1 cleaves Bid, inducing cytochrome c release and apoptosis. In renal cells, calpains 1 and 2 promote apoptosis and necrosis by cleaving cytoskeletal proteins, which increases plasma membrane permeability and cleavage of caspases. Calpain 10 cleaves electron transport chain proteins, causing decreased mitochondrial respiration and excessive activation, or inhibition of calpain 10 activity induces mitochondrial dysfunction and apoptosis. In cardiomyocytes, calpain 1 activates caspase 3 and poly-ADP ribose polymerase during tumour necrosis factor-a-induced apoptosis, and calpain 1 cleaves apoptosis-inducing factor after Ca 2+ overload. Many of these observations have been elucidated with calpain inhibitors, but most calpain inhibitors are not specific for calpains or a specific calpain family member, creating more questions. The following review will discuss how calpains affect mitochondrial function and apoptosis within the cardiovascular system.
Changes in intracellular localization of calpastatin during calpain activation
Biochemical Journal, 1999
Localization of the two main components of the Ca# +-dependent proteolytic system has been investigated in human neuroblastoma LAN-5 cells. Using a monoclonal antibody which recognizes the N-terminal calpastatin domain, it has been shown that this inhibitory protein is almost completely confined in two granulelike structures not surrounded by membranes. Similar calpastatin distribution has been found in other human and in murine cell types, indicating that aggregation of calpastatin is a general property and not an exclusive characteristic of neuronal-like cells. The existence of such calpastatin aggregates is confirmed by the kinetics of calpastatin-activity release during rat liver homogenization, which does not correspond to the rate of appearance of cytosolic proteins or to the disruption of membrane-surrounded organelles. Calpastatin distribution is affected by the intracellular increase in free Ca# + , which results in