Calpastatin participates in the regulation of cell migration in BAP1-deficient uveal melanoma cells.
ABSTRACT: AIM:To detect how BRCA-associated protein 1 (BAP1) regulates cell migration in uveal melanoma (UM) cells. METHODS:Wound healing and transwell assays were performed to detect UM cell migration abilities. Protein chip, immunoprecipitations and surface plasmon resonance analyses were applied to identify BAP1 protein partners. Western blot and calpain activity assays were used to test the expression and function of calpastatin (CAST). RESULTS:CAST protein was confirmed as a new BAP1 protein partner, and loss of BAP1 reduced the expression and function of CAST in UM cells. The overexpression of CAST rescued the cell migration phenotype caused by BAP1 loss. CONCLUSION:BAP1 interacts with CAST in UM cells, and CAST and its subsequent calpain pathway may mediate BAP1-related cell migration regulation.
Project description:Increased activity of calpains is implicated in synaptic dysfunction and neurodegeneration in Alzheimer's disease (AD). The molecular mechanisms responsible for increased calpain activity in AD are not known. Here, we demonstrate that disease progression is propelled by a marked depletion of the endogenous calpain inhibitor, calpastatin (CAST), from AD neurons, which is mediated by caspase-1, caspase-3, and calpains. Initial CAST depletion focally along dendrites coincides topographically with calpain II and ERK 1/2 activation, tau cleavage by caspase-3, and tau and neurofilament hyperphosphorylation. These same changes, together with cytoskeletal proteolysis and neuronal cell death, accompany CAST depletion after intrahippocampal kainic acid administration to mice, and are substantially reduced in mice overexpressing human CAST. Moreover, CAST reduction by shRNA in neuronal cells causes calpain-mediated death at levels of calcium-induced injury that are sublethal to cells normally expressing CAST. Our results strongly support a novel hypothesis that CAST depletion by multiple abnormally activated proteases accelerates calpain dysregulation in AD leading to cytoskeleton disruption and neurodegeneration. CAST mimetics may, therefore, be neuroprotective in AD.
Project description:The calpain gene family and its inhibitors have diverse effects, many related to protein turnover, which appear to affect a range of phenotypes such as diabetes, exercise-induced muscle injury, and pathological events associated with degenerative neural diseases in humans, fertility, longevity, and postmortem effects on meat tenderness in livestock species. The calpains are inhibited by calpastatin, which binds directly to calpain. Here we report the direct measurement of epistatic interactions of causative mutations for quantitative trait loci (QTL) at calpain 1 (CAPN1), located on chromosome 29, with causative mutations for QTL variation at calpastatin (CAST), located on chromosome 7, in cattle. First we identified potential causative mutations at CAST and then genotyped these along with putative causative mutations at CAPN1 in >1500 cattle of seven breeds. The maximum allele substitution effect on the phenotype of the CAPN1:c.947G>C single nucleotide polymorphism (SNP) was 0.14 sigma(p) (P = 0.0003) and of the CAST:c.155C>T SNP was also 0.14 sigma(p) (P = 0.0011) when measured across breeds. We found significant epistasis between SNPs at CAPN1 and CAST in both taurine and zebu derived breeds. There were more additive x dominance components of epistasis than additive x additive and dominance x dominance components combined. A minority of breed comparisons did not show epistasis, suggesting that genetic variation at other genes may influence the degree of epistasis found in this system.
Project description:We report that neuronal overexpression of the endogenous inhibitor of calpains, calpastatin (CAST), in a mouse model of human Alzheimer's disease (AD) ?-amyloidosis, the APP23 mouse, reduces ?-amyloid (A?) pathology and A? levels when comparing aged, double transgenic (tg) APP23/CAST with APP23 mice. Concurrent with A? plaque deposition, aged APP23/CAST mice show a decrease in the steady-state brain levels of the amyloid precursor protein (APP) and APP C-terminal fragments (CTFs) when compared with APP23 mice. This CAST-dependent decrease in APP metabolite levels was not observed in single tg CAST mice expressing endogenous APP or in younger, A? plaque predepositing APP23/CAST mice. We also determined that the CAST-mediated inhibition of calpain activity in the brain is greater in the CAST mice with A? pathology than in non-APP tg mice, as demonstrated by a decrease in calpain-mediated cytoskeleton protein cleavage. Moreover, aged APP23/CAST mice have reduced extracellular signal-regulated kinase 1/2 (ERK1/2) activity and tau phosphorylation when compared with APP23 mice. In summary, in vivo calpain inhibition mediated by CAST transgene expression reduces A? pathology in APP23 mice, with our findings further suggesting that APP metabolism is modified by CAST overexpression as the mice develop A? pathology. Our results indicate that the calpain system in neurons is more responsive to CAST inhibition under conditions of A? pathology, suggesting that in the disease state neurons may be more sensitive to the therapeutic use of calpain inhibitors.
Project description:The Ca(2+)-dependent cysteine proteases, calpains, regulate cell migration, cell death, insulin secretion, synaptic function and muscle homeostasis. Their endogenous inhibitor, calpastatin, consists of four inhibitory repeats, each of which neutralizes an activated calpain with exquisite specificity and potency. Despite the physiological importance of this interaction, the structural basis of calpain inhibition by calpastatin is unknown. Here we report the 3.0 A structure of Ca(2+)-bound m-calpain in complex with the first calpastatin repeat, both from rat, revealing the mechanism of exclusive specificity. The structure highlights the complexity of calpain activation by Ca(2+), illustrating key residues in a peripheral domain that serve to stabilize the protease core on Ca(2+) binding. Fully activated calpain binds ten Ca(2+) atoms, resulting in several conformational changes allowing recognition by calpastatin. Calpain inhibition is mediated by the intimate contact with three critical regions of calpastatin. Two regions target the penta-EF-hand domains of calpain and the third occupies the substrate-binding cleft, projecting a loop around the active site thiol to evade proteolysis.
Project description:Calpain is activated following myocardial infarction and ablation of calpastatin (CAST), an endogenous inhibitor of calpains, promotes left ventricular remodeling after myocardial infarction (MI). The present study aimed to investigate the effect of transgenic over-expression of CAST on the post-infarction myocardial remodeling process.We established transgenic mice (TG) ubiquitously over-expressing human CAST protein and produced MI in TG mice and C57BL/6J wild-type (WT) littermates.The CAST protein expression was profoundly upregulated in the myocardial tissue of TG mice compared with WT littermates (P < 0.01). Overexpression of CAST significantly reduced the infarct size (P < 0.01) and blunted MI-induced interventricular hypertrophy, global myocardial fibrosis and collagen I and collagen III deposition, hypotension and hemodynamic disturbances at 21 days after MI. Moreover, the MI-induced up-regulation and activation of calpains were obviously attenuated in CAST TG mice. MI-induced down-regulation of CAST was partially reversed in TG mice. Additionally, the MI-caused imbalance of matrix metalloproteinases and their inhibitors was improved in TG mice.Transgenic over-expression of CAST inhibits calpain activation and attenuates post-infarction myocardial remodeling.
Project description:In the adult mammalian brain, new neurons continue to be produced throughout life in two main regions in the brain, the subgranular zone (SGZ) in the hippocampus and the subventricular zone in the walls of the lateral ventricles. Neural stem cells (NSCs) proliferate in these niches, and migrate as neuroblasts, to further differentiate in locations where new neurons are needed, either in normal or pathological conditions. However, the endogenous attempt of brain repair is not very efficient. Calpains are proteases known to be involved in neuronal damage and in cell proliferation, migration and differentiation of several cell types, though their effects on neurogenesis are not well known. Previous work by our group has shown that the absence of calpastatin (CAST), the endogenous inhibitor of calpains, impairs early stages of neurogenesis. Since the hippocampus is highly associated with learning and memory, we aimed to evaluate whether calpain inhibition would help improve cognitive recovery after lesion and efficiency of post-injury neurogenesis in this region. For that purpose, we used the kainic acid (KA) model of seizure-induced hippocampal lesion and mice overexpressing CAST. Selected cognitive tests were performed on the 3rd and 8th week after KA-induced lesion, and cell proliferation, migration and differentiation in the dentate gyrus (DG) of the hippocampus of adult mice were analyzed using specific markers. Cognitive recovery was evaluated by testing the animals for recognition, spatial and associative learning and memory. Cognitive function was preserved by CAST overexpression following seizures, while modulation of post-injury neurogenesis was similar to wild type (WT) mice. Calpain inhibition could still be potentially able to prevent the impairment in the formation of new neurons, given that the levels of calpain activity could be reduced under a certain threshold and other harmful effects from the pathological environment could also be controlled.
Project description:Syk is a 72kDa non-receptor tyrosine kinase that is best characterized in hematopoietic cells. While Syk is pro-tumorigenic in some cancer cell types, it also has been reported as a negative regulator of metastatic cell growth in others. An examination of the RelA (p65) subunit of NF-?B expressed in MCF7 breast cancer cells indicated that either treatment with pervanadate or stable expression of Syk protected RelA from calpain-mediated proteolysis. Similar results were observed with the tyrosine phosphatase, PTP1B, another sensitive calpain substrate. The activity of calpain in MCF7 cell lysates was inhibited by both treatment with hydrogen peroxide and expression of Syk, the former due to oxidative inactivation of calpain and the latter to enhanced expression of calpastatin (CAST), the endogenous calpain inhibitor. The level of CAST was elevated in the cytosolic fraction of Syk-positive breast cancer cells resulting in more CAST present in complex with calpain in cell lysates. The high levels of CAST coincided with elevated basal levels of calcium-and of intracellular calpain activity-in Syk-expressing cells resulting from decreased levels of Bcl-2, an inhibitor of IP3-receptor-mediated calcium release. The inhibition of cellular calpain stimulated the Syk-mediated enhancement of NF-?B induced by TNF-?, enhanced tyrosine phosphorylation resulting from integrin crosslinking, and increased the localization of Syk to the plasma membrane.
Project description:To gain knowledge about the behaviour of calpastatin (the specific inhibitor of the Ca(2+)-dependent thiol protease calpain) in the intact cell, we analysed the inhibitor by specific antibodies and determined its activity in erythrocytes from individuals 20-34 years old (young) and 70-93 years old (old). Differences between old and young in the behaviour of erythrocyte calpastatin were observed. Erythrocytes of old individuals had lower amounts of calpastatin and less calpastatin activity than those of young ones. A difference between old and young was also found in the molecular-mass distribution of calpastatin subunits. Increasing the erythrocyte Ca2+ induced changes in calpastatin in young individuals, rendering it similar to calpastatin in cells of old individuals. When calpastatin (isolated from erythrocytes of a young individual) was added to erythrocyte membranes, the initial binding and subsequent association of calpastatin with the membrane were lower in old than in young individuals. We had previously found that calpain binding and activation were enhanced in erythrocyte membranes from old individuals, along with enhanced degradation of band 3 (a major erythrocyte transmembrane anion-transport protein). The overall results indicate an interaction of calpain with calpastatin in the intact cell. Enhanced activation of erythrocyte calpain and degradation of calpastatin occur under conditions of increased cellular Ca2+ and in cells of the aged.
Project description:We reported that cigarette smoke extract (CSE) causes decreases in the activity and expression of endothelial nitric oxide synthase (eNOS) and calpain activity in pulmonary artery endothelial cells (PAECs). Calpains are a family of calcium-dependent endopeptidases, and their specific endogenous inhibitor is calpastatin. In this study, we evaluated the role of calpain-calpastatin in CSE-induced decrease in eNOS gene expression. PAEC were incubated with 5-10% CSE for 2-24 h. eNOS gene transcription rate, eNOS messenger ribonucleic acid (mRNA) half-life, and the activity and protein contents of calpain and calpastatin were measured. Incubation of PAEC with CSE caused significant decreases in eNOS gene transcription and calpain activity and an increase in calpastatin protein content. eNOS mRNA half-life was not significantly altered by CSE. To investigate whether CSE-induced inhibition of eNOS gene expression is caused by decreased calpain activity due to an increase in calpastatin protein content, we cloned calpastatin gene from PAEC and constructed adenovirus vectors containing calpastatin. Overexpression of calpastatin mimics the inhibitory effects of CSE on calpain activity and on the activity, protein, and mRNA of eNOS. The cell-permeable calpain inhibitor, calpastatin peptide, inhibits acetylcholine-induced endothelium-dependent relaxation of the pulmonary artery. Incubation of PAEC with an antisense oligodeoxyribonucleotide of calpastatin prevented CSE-induced increases in calpastatin protein and CSE-induced decreases in calpain activity, eNOS gene transcription, activity and protein content of eNOS, and NO release. These results indicate that CSE-induced inhibition of eNOS expression in PAEC is caused by calpain inhibition due to an increase in calpastatin protein content.
Project description:Calpain enzymes proteolytically modulate cellular function and have been implicated in inflammatory diseases. In this study, we found that calpain levels did not differ between intestinal tissues from inflammatory bowel disease (IBD) patients and healthy controls, but IBD tissues showed increased levels of the endogenous calpain inhibitor, calpastatin (CAST). To investigate the role of CAST in the immune system during IBD, mice were x-ray irradiated, reconstituted with either CAST-knockout (KO) or wild-type (WT) bone marrow, and subjected to dextran sulfate sodium-induced colitis. CAST-KO recipients with induced colitis exhibited more severe weight loss, bloody diarrhea, and anemia compared with WT controls. Histological evaluation of colons from KO recipients with colitis revealed increased inflammatory pathology. Macrophages purified from the colons of KO recipients had higher IL-6, TNF-?, and IFN-? mRNA levels compared with WT controls. Mechanistic investigations using small interfering RNA and KO bone marrow to generate CAST-deficient macrophages showed that CAST deficiency during activation with bacterial pathogen associated molecular patterns, including heat-killed Enterococcus faecalis or CpG DNA, led to increased I?B cleavage, NF-?B nuclear localization, and IL-6 and TNF-? secretion. Thus, CAST plays a central role in regulating macrophage activation and limiting pathology during inflammatory disorders like IBD.