Calpastatin Prevents NF- B-Mediated Hyperactivation of Macrophages and Attenuates Colitis (original) (raw)
Related papers
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.
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: physiological and pathophysiological significance
Pathophysiology, 1999
The system of Ca-dependent neutral proteases, commonly known as the calpain system, is represented by two main isoforms, calpain 1 and calpain 2, as well as by tissue-specific enzymes. These enzymes take part in some physiological processes and are responsible for certain pathological states. The versatility of intracellular Ca 2 + mediating the majority of cellular processes is reflected on the calpain system, i.e. it activates the functional reserves of the cell at moderate Ca 2 + concentrations and causes protein degradation at excessive Ca 2 + . Besides, the activity of the calpain system of the cell is regulated by the concentration of its specific inhibitor, calpastatin, as well as by phosphorylation-dephosphorylation of proteins. Calpains can function both inside the cell and outside it (in the extracellular matrix) and exist both in soluble and membrane-bound states, where it is the most active. Calpains induce cell degradation in some pathologies and in apoptosis. Preliminary modification of proteins or their membrane microenvironment initiates enhanced proteolysis of cytosolic and membrane-bound proteins and enzymes. The key role of calpains in the development of many pathological states testifies to the high pathophysiological significance of this proteolytic system in the organism.
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...
2008
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 (semi-quantitatively) the activities of calpain in peripheral blood CD4 + and CD19 + lymphocytes from various donors that way. Preliminary results showed that calpain activity is increased in the CD4 + T cells isolated from peripheral blood of rheumatoid arthritis patients as compared to control lymphocytes. Extremely high intrinsic activity of calpain was detected in chronic lymphocytic leukemia (CD19 +) cells. All this confirms the detection of immunoreactive products of calpastatin as a good maker of endogenous calpain activity.
Roles of calpain-calpastatin system (CCS) in human T cell activation
Oncotarget, 2016
The immune response is determined by the speed of the T cell reaction to antigens assured by a state of readiness for proliferation and cytokine secretion. Proliferation, apoptosis and motion of many cell types are controlled by cytoplasmic proteases - µ- and m-calpain - and their inhibitor calpastatin, together forming the "calpain-calpastatin system" (CCS), assumed to modify their targets only upon activation-dependent cytoplasmic Ca2+ increase. Contrastingly to this notion, using quantitative real time PCR and semiquantitative flow cytometry respectively, we show here that the CCS genes are constitutively expressed, and that both calpains are constitutively active in resting, circulating human CD4+ and CD8+ lymphocytes. Furthermore, we demonstrate that calpain inhibition in the resting T cells prevents them from proliferation in vitro and greatly reduces secretion of multiple cytokines. The mechanistic reason for these effects of calpain inhibition on T cell functions m...