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Changes in intracellular localization of calpastatin during calpain activation.

ABSTRACT: Localization of the two main components of the Ca(2+)-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 granule-like 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(2+), which results in calpastatin progressively becoming a soluble protein. However, calpain is distributed in the soluble cell fraction and, in activating conditions, partially accumulates on the plasma membrane. Similar behaviour has been observed in calpastatin localization in LAN-5 cells induced with retinoic acid, suggesting that the proteolytic system is activated during the differentiation process of these cells. The involvement of calpastatin in controlling calpain activity, rather than its activation process, and the utilization of changes in calpastatin localization as a marker of activation of the system is discussed.

SUBMITTER: De Tullio R

PROVIDER: S-EPMC1220576 | biostudies-other | 1999 Oct

REPOSITORIES: biostudies-other

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Changes in intracellular calpastatin localization are mediated by reversible phosphorylation.

Project description:We have previously reported that, in neuroblastoma LAN-5 cells, calpastatin is in an aggregated state, close to the cell nucleus [de Tullio, Passalacqua, Averna, Salamino, Melloni and Pontremoli (1999) Biochem. J. 343, 467-472]. In the present paper, we demonstrate that aggregated calpastatin is predominantly in a phosphorylated state. An increase in intracellular free [Ca2+] induces both dephosphorylation of calpastatin, through the action of a phosphoprotein phosphatase, and its redistribution as a soluble inhibitor species. cAMP, but not PMA-induced phosphorylation, reverses calpastatin distribution favouring its aggregation. This intracellular reversible mechanism, regulating the level of cytosolic calpastatin, could be considered a strategy through which calpain can escape calpastatin inhibition, especially during earlier steps of its activation process.

| S-EPMC1221624 | biostudies-other

Unexpected role of the L-domain of calpastatin during the autoproteolytic activation of human erythrocyte calpain.

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| S-EPMC5968184 | biostudies-literature

Roles of calpain-calpastatin system (CCS) in human T cell activation.

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| S-EPMC5363525 | biostudies-literature

Over-expression of calpastatin inhibits calpain activation and attenuates post-infarction myocardial remodeling.

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Calpastatin phosphorylation regulates radiation-induced calpain activity in glioblastoma.

Project description:Glioblastoma (GBM) is an aggressive, malignant brain tumor that inevitably develops resistance to conventional chemotherapy and radiation treatments. In order to identify signaling pathways involved in the development of radiation resistance, we performed mass spectrometry-based phospho-proteomic profiling of GBM cell lines and normal human astrocytes before and after radiation treatment. We found radiation induced phosphorylation of a number of proteins including calpastatin, specifically in GBM stem cells (GSCs). Herein, we focused on calpastatin, an endogenous inhibitor of calpain proteases. Radiation-induced phosphorylation of calpastatin at Ser-633 within the inhibitory domain was validated with a phospho-specific antibody. In order to test the functional significance of phosphorylated calpastatin, we utilized site-directed mutagenesis to generate phospho-inactive (Ser633Ala) and phospho-mimetic (Ser633Glu) mutant calpastatin. GBM cell lines stably expressing the mutant calpastatin showed that phosphorylation was necessary for radiation-induced calpain activation. We also showed that casein kinase 2, a pro-survival kinase overexpressed in many cancer types, phosphorylated calpastatin at Ser-633. Our results indicate that calpastatin phosphorylation promotes radiation resistance in GBM cells by increasing the activity of calpain proteases, which are known to promote survival and invasion in cancer.

| S-EPMC5865692 | biostudies-literature

Detecting the active conformation of calpain with calpastatin-based reagents.

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| S-EPMC1805107 | biostudies-literature

Epistasis between calpain 1 and its inhibitor calpastatin within breeds of cattle.

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.

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Elevated calpain activity in acute myelogenous leukemia correlates with decreased calpastatin expression.

Project description:Calpains are intracellular cysteine proteases that have crucial roles in many physiological and pathological processes. Elevated calpain activity has been associated with many pathological states. Calpain inhibition can be protective or lethal depending on the context. Previous work has shown that c-myc transformation regulates calpain activity by suppressing calpastatin, the endogenous negative regulator of calpain. Here, we have investigated calpain activity in primary acute myelogenous leukemia (AML) blast cells. Calpain activity was heterogeneous and greatly elevated over a wide range in AML blast cells, with no correlation to FAB classification. Activity was particularly elevated in the CD34+CD38- enriched fraction compared with the CD34+CD38+ fraction. Treatment of the cells with the specific calpain inhibitor, PD150606, induced significant apoptosis in AML blast cells but not in normal equivalent cells. Sensitivity to calpain inhibition correlated with calpain activity and preferentially targeted CD34+CD38- cells. There was no correlation between calpain activity and p-ERK levels, suggesting the ras pathway may not be a major contributor to calpain activity in AML. A significant negative correlation existed between calpain activity and calpastatin, suggesting calpastatin is the major regulator of activity in these cells. Analysis of previously published microarray data from a variety of AML patients demonstrated a significant negative correlation between calpastatin and c-myc expression. Patients who achieved a complete remission had significantly lower calpain activity than those who had no response to treatment. Taken together, these results demonstrate elevated calpain activity in AML, anti-leukemic activity of calpain inhibition and prognostic potential of calpain activity measurement.

| S-EPMC3270254 | biostudies-literature

Affinity purification of human m-calpain through an intrinsically disordered inhibitor, calpastatin.

Project description:Calpains are calcium-activated proteases that have biomedical and biotechnological potential. Their activity is tightly regulated by their endogenous inhibitor, calpastatin that binds to the enzyme only in the presence of calcium. Conventional approaches to purify calpain comprise multiple chromatographic steps, and are labor-intensive, leading to low yields. Here we report a new purification procedure for the human m-calpain based on its reversible calcium-mediated interaction with the intrinsically disordered calpastatin. We exploit the specific binding properties of human calpastatin domain 1 (hCSD1) to physically capture human m-calpain from a complex biological mixture. The dissociation of the complex is mediated by chelating calcium, upon which heterodimeric calpain elutes while hCSD1 remains immobilized onto the stationary phase. This novel affinity-based purification was compared to the conventional multistep purification strategy and we find that it is robust, it yields a homogeneous preparation, it can be scaled up easily and it rests on a non-disruptive step that maintains close to physiological conditions that allow further biophysical and functional studies.

| S-EPMC5358782 | biostudies-literature

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