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1.
Sci Transl Med ; 13(581)2021 02 17.
Article in English | MEDLINE | ID: mdl-33597260

ABSTRACT

Myocyte death occurs in many inherited and acquired cardiomyopathies, including arrhythmogenic cardiomyopathy (ACM), a genetic heart disease plagued by the prevalence of sudden cardiac death. Individuals with ACM and harboring pathogenic desmosomal variants, such as desmoglein-2 (DSG2), often show myocyte necrosis with progression to exercise-associated heart failure. Here, we showed that homozygous Dsg2 mutant mice (Dsg2 mut/mut), a model of ACM, die prematurely during swimming and display myocardial dysfunction and necrosis. We detected calcium (Ca2+) overload in Dsg2 mut/mut hearts, which induced calpain-1 (CAPN1) activation, association of CAPN1 with mitochondria, and CAPN1-induced cleavage of mitochondrial-bound apoptosis-inducing factor (AIF). Cleaved AIF translocated to the myocyte nucleus triggering large-scale DNA fragmentation and cell death, an effect potentiated by mitochondrial-driven AIF oxidation. Posttranslational oxidation of AIF cysteine residues was due, in part, to a depleted mitochondrial thioredoxin-2 redox system. Hearts from exercised Dsg2 mut/mut mice were depleted of calpastatin (CAST), an endogenous CAPN1 inhibitor, and overexpressing CAST in myocytes protected against Ca2+ overload-induced necrosis. When cardiomyocytes differentiated from Dsg2 mut/mut embryonic stem cells (ES-CMs) were challenged with ß-adrenergic stimulation, CAPN1 inhibition attenuated CAPN1-induced AIF truncation. In addition, pretreatment of Dsg2 mut/mut ES-CMs with an AIF-mimetic peptide, mirroring the cyclophilin-A (PPIA) binding site of AIF, blocked PPIA-mediated AIF-nuclear translocation, and reduced both apoptosis and necrosis. Thus, preventing CAPN1-induced AIF-truncation or barring binding of AIF to the nuclear chaperone, PPIA, may avert myocyte death and, ultimately, disease progression to heart failure in ACM and likely other forms of cardiomyopathies.


Subject(s)
Apoptosis Inducing Factor , Calpain , Cardiomyopathies , Myocytes, Cardiac/pathology , Physical Conditioning, Animal , Animals , Apoptosis Inducing Factor/metabolism , Calpain/metabolism , Cardiomyopathies/metabolism , Cell Death , Mice , Mitochondria/metabolism , Myocytes, Cardiac/metabolism
2.
Nat Prod Res ; 33(10): 1449-1455, 2019 May.
Article in English | MEDLINE | ID: mdl-29298502

ABSTRACT

We are reporting in the present study that molecules extracted from olive pomace prevent cell death induced by Ca2+-overloading in different cell types. Exposure of cells to these molecules counteracts the Ca2+-induced cell damages by reducing the activation of the Ca2+-dependent protease calpain, acting possibly through the modification of the permeability to Ca2+ of the plasma membrane. The purification step by RP-HPLC suggests that effective compound(s), differing from the main biophenols known to be present in the olive pomace extract, could be responsible for this effect. Our observations suggest that bioactive molecules present in the olive pomace could be potential candidates for therapeutic applications in pathologies characterised by alterations of intracellular Ca2+ homeostasis.


Subject(s)
Calcium/metabolism , Cytoprotection/drug effects , Olive Oil/analysis , Plant Extracts/pharmacology , Cell Line, Tumor , Cell Survival/drug effects , Cells, Cultured , Chromatography, High Pressure Liquid , Chromatography, Reverse-Phase , Homeostasis , Humans , Ion Transport
3.
Biosci Rep ; 38(2)2018 04 26.
Article in English | MEDLINE | ID: mdl-29572388

ABSTRACT

Autoproteolysis of human erythrocyte calpain-1 proceeds in vitro at high [Ca2+], through the conversion of the 80-kDa catalytic subunit into a 75-kDa activated enzyme that requires lower [Ca2+] for catalysis. Importantly, here we detect a similar 75 kDa calpain-1 form also in vivo, in human meningiomas. Although calpastatin is so far considered the specific inhibitor of calpains, we have previously identified in rat brain a calpastatin transcript truncated at the end of the L-domain (cast110, L-DOM), coding for a protein lacking the inhibitory units. Aim of the present study was to characterize the possible biochemical role of the L-DOM during calpain-1 autoproteolysis in vitro, at high (100 µM) and low (5 µM) [Ca2+]. Here we demonstrate that the L-DOM binds the 80 kDa proenzyme in the absence of Ca2+ Consequently, we have explored the ability of the 75 kDa activated protease to catalyze at 5 µM Ca2+ the intermolecular activation of native calpain-1 associated with the L-DOM. Notably, this [Ca2+] is too low to promote the autoproteolytic activation of calpain-1 but enough to support the catalysis of the 75 kDa calpain. We show for the first time that the L-DOM preserves native calpain-1 from the degradation mediated by the 75 kDa form. Taken together, our data suggest that the free L-domain of calpastatin is a novel member of the calpain/calpastatin system endowed with a function alternative to calpain inhibition. For this reason, it will be crucial to define the intracellular relevance of the L-domain in controlling calpain activation/activity in physiopathological conditions having altered Ca2+ homeostasis.


Subject(s)
Calcium-Binding Proteins/chemistry , Calcium/metabolism , Calpain/chemistry , Erythrocytes/chemistry , Proteolysis , Calcium/chemistry , Calcium-Binding Proteins/metabolism , Calpain/metabolism , Erythrocytes/metabolism , Humans , Protein Domains
4.
Arch Biochem Biophys ; 604: 103-12, 2016 08 15.
Article in English | MEDLINE | ID: mdl-27349634

ABSTRACT

Matrix metalloproteinase 9 (MMP9) is physiologically involved in remodeling the extracellular matrix components but its abnormal release has been observed in several human pathologies. We here report that peripheral blood mononuclear cells (PBMCs), isolated from cystic fibrosis (CF) patients homozygous for F508del-cystic fibrosis transmembrane conductance regulator (CFTR), express constitutively and release at high rate MMP9 due to the alteration in their intracellular Ca(2+) homeostasis. This spontaneous and sustained MMP9 secretion may contribute to the accumulation of this protease in fluids of CF patients. Conversely, in PBMCs isolated from healthy donors, expression and secretion of MMP9 are undetectable but can be evoked, after 12 h of culture, by paracrine stimulation which also promotes an increase in [Ca(2+)]i. We also demonstrate that in both CF and control PBMCs the Ca(2+)-dependent MMP9 secretion is mediated by the concomitant activation of calpain and protein kinase Cα (PKCα), and that MMP9 expression involves extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) phosphorylation. Our results are supported by the fact that either the inhibition of Ca(2+) entry or chelation of [Ca(2+)]i as well as the inhibition of single components of the signaling pathway or the restoration of CFTR activity all promote the reduction of MMP9 secretion.


Subject(s)
Calpain/blood , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis/blood , Leukocytes, Mononuclear/metabolism , Matrix Metalloproteinase 9/blood , Protein Kinase C-alpha/blood , Adolescent , Adult , Aged , Calcium/metabolism , Enzyme Activation , Epithelial Cells/cytology , Extracellular Signal-Regulated MAP Kinases/metabolism , Gene Deletion , Gene Expression Regulation, Enzymologic , Homeostasis , Homozygote , Humans , Middle Aged , Phosphorylation , Predictive Value of Tests , Young Adult
5.
PLoS One ; 10(10): e0139750, 2015.
Article in English | MEDLINE | ID: mdl-26431040

ABSTRACT

We have recently demonstrated that in resting conditions calpain 1, but not calpain 2, is specifically associated to the N-Methyl-D-Aspartate receptor (NMDAR) multiprotein complex. We are here reporting that in SKNBE neuroblastoma cells or in freshly isolated nerve terminals from adult rat hippocampus, the proteolytic activity of calpain 1 resident at the NMDAR is very low under basal conditions and greatly increases following NMDAR stimulation. Since the protease resides at the NMDAR in saturating amounts, variations in Ca2+ influx promote an increase in calpain 1 activity without affecting the amount of the protease originally associated to NMDAR. In all the conditions examined, resident calpain 1 specifically cleaves NR2B at the C-terminal region, leading to its internalization together with NR1 subunit. While in basal conditions intracellular membranes include small amounts of NMDAR containing the calpain-digested NR2B, upon NMDAR stimulation nearly all the receptor molecules are internalized. We here propose that resident calpain 1 is involved in NMDAR turnover, and following an increase in Ca2+ influx, the activated protease, by promoting the removal of NMDAR from the plasma membranes, can decrease Ca2+ entrance through this channel. Due to the absence of calpastatin in such cluster, the activity of resident calpain 1 may be under the control of HSP90, whose levels are directly related to the activation of this protease. Observations of different HSP90/calpain 1 ratios in different ultrasynaptic compartments support this conclusion.


Subject(s)
Calpain/physiology , Receptors, N-Methyl-D-Aspartate/physiology , Animals , HSP90 Heat-Shock Proteins/physiology , Male , Rats , Rats, Sprague-Dawley
6.
PLoS One ; 10(1): e0116738, 2015.
Article in English | MEDLINE | ID: mdl-25575026

ABSTRACT

Here we demonstrate that heat shock protein 90 (HSP90) interacts with calpain-1, but not with calpain-2, and forms a discrete complex in which the protease maintains its catalytic activity, although with a lower affinity for Ca2+. Equilibrium gel distribution experiments show that this complex is composed by an equal number of molecules of each protein partner. Moreover, in resting cells, cytosolic calpain-1 is completely associated with HSP90. Since calpain-1, in association with HSP90, retains its proteolytic activity, and the chaperone is displaced by calpastatin also in the absence of Ca2+, the catalytic cleft of the protease is not involved in this association. Thus, calpain-1 can form two distinct complexes depending on the availability of calpastatin in the cytosol. The occurrence of a complex between HSP90 and calpain-1, in which the protease is still activable, can prevent the complete inhibition of the protease even in the presence of high calpastatin levels. We also demonstrate that in basal cell conditions HSP90 and calpain-1, but not calpain-2, are inserted in the multi-protein N-Methyl-D-Aspartate receptor (NMDAR) complex. The amount of calpain-1 at the NMDAR cluster is not modified in conditions of increased [Ca2+]i, and this resident protease is involved in the processing of NMDAR components. Finally, the amount of calpain-1 associated with NMDAR cluster is independent from Ca2+-mediated translocation. Our findings show that HSP90 plays an important role in maintaining a given and proper amount of calpain-1 at the functional sites.


Subject(s)
Calpain/metabolism , HSP90 Heat-Shock Proteins/metabolism , Animals , Calcium/chemistry , Calcium/metabolism , Calcium-Binding Proteins/chemistry , Calcium-Binding Proteins/genetics , Calcium-Binding Proteins/metabolism , Calpain/chemistry , Cell Line , HSP90 Heat-Shock Proteins/chemistry , HSP90 Heat-Shock Proteins/genetics , Humans , Immunoprecipitation , Ions/chemistry , Male , Mice , Microscopy, Confocal , Peptide Hydrolases/metabolism , Protein Binding , Rats , Receptors, N-Methyl-D-Aspartate/chemistry , Receptors, N-Methyl-D-Aspartate/metabolism , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics
7.
Biochim Biophys Acta ; 1843(11): 2583-91, 2014 Nov.
Article in English | MEDLINE | ID: mdl-25026177

ABSTRACT

Here we demonstrate that the presence of the L-domain in calpastatins induces biphasic interaction with calpain. Competition experiments revealed that the L-domain is involved in positioning the first inhibitory unit in close and correct proximity to the calpain active site cleft, both in the closed and in the open conformation. At high concentrations of calpastatin, the multiple EF-hand structures in domains IV and VI of calpain can bind calpastatin, maintaining the active site accessible to substrate. Based on these observations, we hypothesize that two distinct calpain-calpastatin complexes may occur in which calpain can be either fully inhibited (I) or fully active (II). In complex II the accessible calpain active site can be occupied by an additional calpastatin molecule, now a cleavable substrate. The consequent proteolysis promotes the accumulation of calpastatin free inhibitory units which are able of improving the capacity of the cell to inhibit calpain. This process operates under conditions of prolonged [Ca(2+)] alteration, as seen for instance in Familial Amyotrophic Lateral Sclerosis (FALS) in which calpastatin levels are increased. Our findings show that the L-domain of calpastatin plays a crucial role in determining the formation of complexes with calpain in which calpain can be either inhibited or still active. Moreover, the presence of multiple inhibitory domains in native full-length calpastatin molecules provides a reservoir of potential inhibitory units to be used to counteract aberrant calpain activity.

8.
PLoS One ; 8(6): e66089, 2013.
Article in English | MEDLINE | ID: mdl-23785472

ABSTRACT

A basal calpain activity promotes the limited proteolysis of wild type (WT) cystic fibrosis conductance regulator (CFTR), inducing the internalization of the split channel. This process contributes to the regulation in the level of the active CFTR at the plasma membranes. In peripheral blood mononuclear cells (PBMC) from 16 healthy donors, the inhibition of calpain activity induces a 3-fold increase in the amount of active WT CFTR at the plasma membranes. Instead, in PBMC from cystic fibrosis (CF) patients, calpain activity is expressed at aberrant levels causing the massive removal of F(508)del-CFTR from the cell surface. In these patients, the inhibition of such abnormal proteolysis rescues physiological amounts of active mutated CFTR in 90% of the patients (25 over 28). The recovery of functional F(508)del-CFTR at the physiological location, in cells treated with a synthetic calpain inhibitor, indicates that F(508)del-CFTR folding, maturation, and trafficking operate in CF-PBMC at significant rate. Thus, an increase in the basal calpain activity seems primarily involved in the CFTR defect observed in various CF cells. Furthermore, in CF-PBMC the recovery of the scaffolding protein Na(+)/H(+) exchanger regulatory factor 1 (NHERF-1), occurring following inhibition of the aberrant calpain activity, can contribute to rescue CFTR-functional clusters.


Subject(s)
Calpain/antagonists & inhibitors , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Cystic Fibrosis/genetics , Cystic Fibrosis/metabolism , Glycoproteins/pharmacology , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/metabolism , Adolescent , Adult , Aged , Aged, 80 and over , Carrier Proteins/metabolism , Cell Membrane/metabolism , Child , Female , Humans , Male , Middle Aged , Oligopeptides/pharmacology , Phosphoproteins/metabolism , Protein Binding , Sodium-Hydrogen Exchangers/metabolism , Young Adult
9.
PLoS One ; 7(8): e44518, 2012.
Article in English | MEDLINE | ID: mdl-22952988

ABSTRACT

BACKGROUND: Extracellular high mobility group box 1 (HMGB1) protein can operate in a synergistic fashion with different signal molecules promoting an increase of cell Ca(2+) influx. However, the mechanisms responsible for this effect of HMGB1 are still unknown. PRINCIPAL FINDINGS: Here we demonstrate that, at concentrations of agonist per se ineffective, HMGB1 potentiates the activation of the ionotropic glutamate N-methyl-D-aspartate receptor (NMDAR) in isolated hippocampal nerve terminals and in a neuroblastoma cell line. This effect was abolished by the NMDA channel blocker MK-801. The HMGB1-facilitated NMDAR opening was followed by activation of the Ca(2+)-dependent enzymes calpain and nitric oxide synthase in neuroblastoma cells, resulting in an increased production of NO, a consequent enhanced cell motility, and onset of morphological differentiation. We have also identified NMDAR as the mediator of HMGB1-stimulated murine erythroleukemia cell differentiation, induced by hexamethylenebisacetamide. The potentiation of NMDAR activation involved a peptide of HMGB1 located in the B box at the amino acids 130-139. This HMGB1 fragment did not overlap with binding sites for other cell surface receptors of HMGB1, such as the advanced glycation end products or the Toll-like receptor 4. Moreover, in a competition assay, the HMGB1((130-139)) peptide displaced the NMDAR/HMGB1 interaction, suggesting that it comprised the molecular and functional site of HMGB1 regulating the NMDA receptor complex. CONCLUSION: We propose that the multifunctional cytokine-like molecule HMGB1 released by activated, stressed, and damaged or necrotic cells can facilitate NMDAR-mediated cell responses, both in the central nervous system and in peripheral tissues, independently of other known cell surface receptors for HMGB1.


Subject(s)
Extracellular Space/metabolism , HMGB1 Protein/pharmacology , Receptors, N-Methyl-D-Aspartate/metabolism , Acetamides/pharmacology , Animals , Aspartic Acid/metabolism , Calcium/metabolism , Cell Death/drug effects , Cell Differentiation/drug effects , Cell Line, Tumor , Cell Movement/drug effects , Enzyme Activation/drug effects , Extracellular Space/drug effects , Humans , Male , Mice , N-Methylaspartate/pharmacology , Neurites/drug effects , Neurites/metabolism , Nitric Oxide/biosynthesis , Nitric Oxide Synthase Type I/metabolism , Presynaptic Terminals/drug effects , Presynaptic Terminals/metabolism , Protein Binding/drug effects , Rats , Rats, Sprague-Dawley , Synaptosomes/drug effects , Synaptosomes/metabolism , Tritium
10.
PLoS One ; 7(5): e37133, 2012.
Article in English | MEDLINE | ID: mdl-22615919

ABSTRACT

Earlier observations in the literature suggest that proteolytic degradation of excess unmatched α-globin chains reduces their accumulation and precipitation in ß-thalassaemia erythroid precursor cells and have linked this proteolytic degradation to the activity of calpain protease. The aim of this study was to correlate the activity of calpain and its inhibitor, calpastatin, with different degrees of disease severity in ß-thalassaemia. CD34(+) cells were enriched from peripheral blood of healthy individuals (control group) and patients with mild and severe clinical presentations of ß(0)-thalassaemia/Hb E disease. By ex vivo cultivation promoting erythroid cell differentiation for 7 days, proerythroblasts, were employed for the functional characterization of the calpain-calpastatin proteolytic system. In comparison to the control group, enzymatic activity and protein amounts of µ-calpain were found to be more than 3-fold increased in proerythroblasts from patients with mild clinical symptoms, whereas no significant difference was observed in patients with severe clinical symptoms. Furthermore, a 1.6-fold decrease of calpastatin activity and 3.2-fold accumulation of a 34 kDa calpain-mediated degradation product of calpastatin were observed in patients with mild clinical symptoms. The increased activity of calpain may be involved in the removal of excess α-globin chains contributing to a lower degree of disease severity in patients with mild clinical symptoms.


Subject(s)
Calcium-Binding Proteins/metabolism , Calpain/antagonists & inhibitors , Calpain/metabolism , beta-Thalassemia/enzymology , beta-Thalassemia/pathology , Animals , Antigens, CD34/metabolism , Calcium-Binding Proteins/pharmacology , Case-Control Studies , Cell Differentiation/drug effects , Cells, Cultured , Erythroid Precursor Cells/drug effects , Erythroid Precursor Cells/metabolism , Humans , Male , Proteolysis/drug effects , Rats , Recombinant Proteins/pharmacology , Severity of Illness Index , alpha-Globins/metabolism
11.
Biochim Biophys Acta ; 1812(12): 1649-57, 2011 Dec.
Article in English | MEDLINE | ID: mdl-21983488

ABSTRACT

We are here reporting that in peripheral blood mononuclear cells (PBMC) of patients homozygous for F508del-CFTR the calpain-calpastatin system undergoes a profound alteration. In fact, calpain basal activity, almost undetectable in control PBMC, becomes measurable at a significant extent in cells from cystic fibrosis (CF) patients, also due to a 40-60% decrease in both calpastatin protein and inhibitory activity. Constitutive protease activation in CF patients' cells induces a large accumulation of the mutated cystic fibrosis transmembrane conductance regulator (CFTR) in the 100kD+70kD split forms as well as a degradation of proteins associated to the CFTR complex. Specifically, the scaffolding protein Na(+)/H(+) exchanger 3 regulatory factor-1 (NHERF-1) is converted in two distinct fragments showing masses of 35kD and 20kD, being however the latter form the most represented one, thereby indicating that in CF-PBMC the CFTR complex undergoes a large disorganization. In conclusion, our observations are providing new information on the role of calpain in the regulation of plasma membrane ion conductance and provide additional evidence on the transition of this protease activity from a physiological to a pathological function.


Subject(s)
Calcium-Binding Proteins/metabolism , Calpain/metabolism , Cystic Fibrosis/metabolism , Leukocytes, Mononuclear/metabolism , Phosphoproteins/metabolism , Sodium-Hydrogen Exchangers/metabolism , Adolescent , Adult , Calcium-Binding Proteins/chemistry , Calpain/antagonists & inhibitors , Case-Control Studies , Child , Cystic Fibrosis/blood , Cystic Fibrosis/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Cytoskeletal Proteins/metabolism , Enzyme Activation , Enzyme Assays , Female , Humans , Leukocytes, Mononuclear/enzymology , Male , Middle Aged , Mutation, Missense , Protein Isoforms/metabolism , Protein Transport , Proteolysis , Young Adult
12.
Biochim Biophys Acta ; 1813(1): 50-9, 2011 Jan.
Article in English | MEDLINE | ID: mdl-21111762

ABSTRACT

We are here showing that peripheral mononuclear blood cells (PBMC) from cystic fibrosis (CF) patients contain almost undetectable amounts of mature 170 kDa CF-transmembrane conductance regulator (CFTR) and a highly represented 100 kDa form. This CFTR protein, resembling the form produced by calpain digestion and present, although in lower amounts, also in normal PBMC, is localized in cytoplasmic internal vesicles. These observations are thus revealing that the calpain-mediated proteolysis is largely increased in cells from CF patients. To characterize the process leading to the accumulation of such split CFTR, FRT cells expressing the F508del-CFTR mutated channel protein and human leukaemic T cell line (JA3), expressing wild type CFTR were used. In in vitro experiments, the sensitivity of the mutated channel to the protease is identical to that of the wild type, whereas in Ca(2+)-loaded cells F508del-CFTR is more susceptible to digestion. Inhibition of intracellular calpain activity prevents CFTR degradation and leads to a 10-fold increase in the level of F508del-CFTR at the plasma membrane, further indicating the involvement of calpain activity in the maintenance of very low levels of mature channel form. The higher sensitivity to calpain of the mutated 170 kDa CFTR results from a reduced affinity for HSP90 causing a lower degree of protection from calpain digestion. The recovery of HSP90 binding capacity in F508del-CFTR, following digestion, explains the large accumulation of the 100 kDa CFTR form in circulating PBMC from CF patients.


Subject(s)
Calpain/metabolism , Cell Membrane/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Cystic Fibrosis/metabolism , Epithelial Cells/metabolism , HSP90 Heat-Shock Proteins/metabolism , Molecular Chaperones/metabolism , Animals , Cystic Fibrosis/pathology , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Humans , Immunoblotting , Immunoprecipitation , Mutation/genetics , Protein Transport , Rats , Rats, Inbred F344 , Sequence Deletion
13.
Biochem J ; 430(2): 255-63, 2010 Sep 01.
Article in English | MEDLINE | ID: mdl-20557290

ABSTRACT

The level of the mature native 170 kDa form of CFTR (cystic fibrosis transmembrane conductance regulator) at the plasma membrane is under the control of a selective proteolysis catalysed by calpain. The product of this limited digestion, consisting of discrete fragments still associated by strong interactions, is removed from the plasma membrane and internalized in vesicles and subject to an additional degradation. This process can be monitored by visualizing the accumulation of a 100 kDa fragment in a proliferating human leukaemic T-cell line and in human circulating lymphocytes. In reconstructed systems, and in intact cells, the conversion of native CFTR into the 100 kDa fragment linearly correlated with calpain activation and was prevented by addition of synthetic calpain inhibitors. A reduction in Ca2+ influx, by blocking the NMDA (N-methyl-D-aspartate) receptor Ca2+ channel, inhibited the conversion of the native 170 kDa fragment into the 100 kDa fragment, whereas an endosome acidification blocker promoted accumulation of the digested 100 kDa CFTR form. An important role in calpain-mediated turnover of CFTR is exerted by HSP90 (heat-shock protein 90), which, via association with the protein channel, modulates the degradative effect of calpain through a selective protection. Taken together these results indicate that CFTR turnover is initiated by calpain activation, which is induced by an increased Ca2+ influx and, following internalization of the cleaved channel protein, and completed by the lysosomal proteases. These findings provide new insights into the molecular mechanisms responsible for the defective functions of ion channels in human pathologies.


Subject(s)
Calpain/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Animals , Calpain/genetics , Cell Line , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , HSP90 Heat-Shock Proteins/genetics , HSP90 Heat-Shock Proteins/metabolism , Humans , Protein Transport , Rats
14.
J Biol Chem ; 285(1): 631-43, 2010 Jan 01.
Article in English | MEDLINE | ID: mdl-19880516

ABSTRACT

Persistent dysregulation in Ca(2+) homeostasis is a pervasive pathogenic mechanism in most neurodegenerative diseases, and accordingly, calpain activation has been implicated in neuronal cells dysfunction and death. In this study we examined the intracellular functional state of the calpain-calpastatin system in -G93A(+) SOD1 transgenic mice to establish if and how uncontrolled activation of calpain can be prevented in vivo during the course of prolonged [Ca(2+)](i) elevation. The presented data indicate that 1) calpain activation is more extensive in motor cortex, in lumbar, and sacral spinal cord segments compared with the lower or almost undetectable activation of the protease in other brain areas, 2) direct measurements of the variations of Ca(2+) levels established that the degree of the protease activation is correlated to the extent of elevation of [Ca(2+)](i), 3) intracellular activation of calpain is always associated with diffusion of calpastatin from perinuclear aggregated forms into the cytosol and the formation of a calpain-calpastatin complex, and 4) a conservative fragmentation of calpastatin is accompanied by its increased expression and inhibitory capacity in conditions of prolonged increase in [Ca(2+)](i). Thus, calpastatin diffusion and formation of the calpain-calpastatin complex together with an increased synthesis of the inhibitor protein represent a cellular defense response to conditions of prolonged dysregulation in intracellular Ca(2+) homeostasis. Altogether these findings provide a new understanding of the in vivo molecular mechanisms governing calpain activation that can be extended to many neurodegenerative diseases, potentially useful for the development of new therapeutic approaches.


Subject(s)
Brain/cytology , Brain/enzymology , Calcium-Binding Proteins/metabolism , Calcium/metabolism , Calpain/metabolism , Homeostasis , Animals , Calcium-Binding Proteins/genetics , Calpain/genetics , Enzyme Activation , Gene Expression Regulation, Enzymologic , Humans , Intracellular Space/enzymology , Isoenzymes/genetics , Isoenzymes/metabolism , Mice , Mice, Transgenic , Muscles/cytology , Muscles/enzymology , Neurons/cytology , Neurons/enzymology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Spinal Cord/cytology , Spinal Cord/enzymology , Substrate Specificity , Tissue Extracts
15.
J Neurochem ; 110(1): 412-21, 2009 Jul.
Article in English | MEDLINE | ID: mdl-19457105

ABSTRACT

In resting human neuronal cells, nitric oxide synthase (nNOS) is present in its native 160 kDa form in a quiescent state predominantly co-localized on the plasma membrane, via its PDZ (Psd-95/Discs-large/Zona Occludens) domain, with NMDA receptor (NMDA-R) and in tight association with heat shock protein 90 (HSP90). Following exposure of the cells to Ca(2+)-ionophore or to NMDA, nNOS undergoes proteolytic removal of the PDZ domain being converted into a fully active 130 kDa form. The newly generated nNO synthase form dissociates from NMDA-R and extensively diffuses into the cytosol in direct correlation with NO production. Intracellular redistribution and activation of nNOS are completely prevented in cells preloaded with calpain inhibitor-1, indicating that these processes are triggered by a concomitant activation of calpain. The role of calpain has been confirmed by immunoprecipitation experiments revealing that also mu-calpain is specifically recruited into the NMDA-R-nNOS-HSP90 complex following calcium loading. Thus, the formation of clusters containing HSP90, mu-calpain, nNOS and NMDA-R represents the limiting step for the operation of the mechanism that links an efficient synthesis of NO to a local increase in Ca(2+) influx.


Subject(s)
Calcium Signaling/physiology , Calpain/metabolism , Neurons/enzymology , Nitric Oxide Synthase Type I/metabolism , Nitric Oxide/biosynthesis , Receptors, N-Methyl-D-Aspartate/metabolism , Calcium/metabolism , Calcium Signaling/drug effects , Calpain/antagonists & inhibitors , Cell Line, Tumor , Cell Membrane/drug effects , Cell Membrane/enzymology , Cytosol/metabolism , Diffusion , Enzyme Activation/drug effects , Enzyme Activation/physiology , Enzyme Inhibitors/pharmacology , HSP90 Heat-Shock Proteins/drug effects , HSP90 Heat-Shock Proteins/metabolism , Humans , Ionophores/pharmacology , Neuroblastoma , Neurons/drug effects , Nitric Oxide Synthase Type I/drug effects , Peptide Hydrolases/metabolism , Protein Structure, Tertiary/drug effects , Protein Structure, Tertiary/physiology
16.
Biochim Biophys Acta ; 1790(3): 182-7, 2009 Mar.
Article in English | MEDLINE | ID: mdl-19103264

ABSTRACT

BACKGROUND: To establish the physiological role of calpain, it is necessary to define how the protease can escape from the effect of its natural inhibitor calpastatin, since both proteins co-localize into the cell cytosol. METHODS: To answer this question, we have overexpressed four fluorescent calpastatin constructs, differing in the composition of their XL- and L-domains, and the intracellular trafficking of this protein inhibitor has been followed by single cell fluorescence imaging. RESULTS AND CONCLUSIONS: By the use of these calpastatin forms differing in the type of exon-derived sequences contained in the XL- and L-domains, we have demonstrated that the sequence coded by exon 6, containing multiple phosphorylation sites, is directly involved in determining the cell localization of calpastatin. In fact, exposure to cAMP promotes the recruitment into aggregates of those calpastatin forms containing the exon 6 sequence. These protein movements are directly related to the level of cytosolic inhibitory capacity and thereby to the extent of intracellular calpain activation. GENERAL SIGNIFICANCE: The recruitment of calpastatin into aggregates allows the translocation and activation of the protease to the membranes; on the contrary, the presence of large amounts of calpastatin in the cytosol prevents both processes, protecting the cell from undesired proteolysis.


Subject(s)
Calcium-Binding Proteins/physiology , Calpain/metabolism , Exons , Base Sequence , Cell Line , Cyclic AMP/metabolism , DNA Primers , Electrophoresis, Polyacrylamide Gel , Enzyme Activation , Humans , Microscopy, Fluorescence , Subcellular Fractions/enzymology
17.
FASEB J ; 23(4): 1246-51, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19103644

ABSTRACT

A substantial body of evidence indicates that protein kinase C (PKC) is involved in the alpha-secretory processing of the amyloid precursor protein (APP), an event that reduces the formation of the pathogenic amyloid-beta peptide. Recently, we have shown that trafficking and processing of APP are both impaired by knockdown of myosin II-B, one of the major neuronal motor proteins. Here, we provide evidence that the alpha-secretory processing of APP is mediated by PKC-dependent phosphorylation of myosin II-B. This signaling pathway provides an important link between APP and the neuronal cytoskeleton and might be crucial for the understanding of the biological and pathological roles of APP.


Subject(s)
Amyloid beta-Protein Precursor/metabolism , Nonmuscle Myosin Type IIB/metabolism , Protein Kinase C-alpha/metabolism , Amyloid beta-Protein Precursor/genetics , Animals , Cell Line , Humans , Kinetics , Mice , Neurons/metabolism , Phosphorylation , Plasmids , RNA Interference , Signal Transduction , Tetradecanoylphorbol Acetate/pharmacology , Transfection
18.
Arch Biochem Biophys ; 481(2): 202-9, 2009 Jan 15.
Article in English | MEDLINE | ID: mdl-19056334

ABSTRACT

We show that protein kinase C (PKC) theta localized at the Golgi complex is partially conjugated to monoubiquitin. Using the inactive T538A and activable T538E mutants of PKCtheta, we demonstrate that the presence of an uncharged residue at the 538 position of the activation loop favors both association with the Golgi and monoubiquitination of the kinase. Moreover, the inactive PKCtheta does not translocate from the Golgi in response to a short-term cell stimulation with a phorbol ester and is subjected to different proteolytic degradation pathways compared to the activable cytosolic kinase. These findings highlight the role of T538 as a critical determinant to address the activable and the inactive PKCtheta molecules to different intracellular compartments and to specific post-transductional modifications. The functional relevance of these observations is supported by the impaired cell division observed in phenotypes expressing high levels of the inactive PKCtheta.


Subject(s)
Isoenzymes/metabolism , Protein Kinase C/metabolism , Animals , Binding Sites , Cell Line, Tumor , Genetic Vectors , Isoenzymes/chemistry , Isoenzymes/genetics , Isoenzymes/isolation & purification , Leukemia, Erythroblastic, Acute/enzymology , Leukemia, Erythroblastic, Acute/genetics , Mice , Microscopy, Confocal , Mutagenesis , Plasmids , Protein Kinase C/chemistry , Protein Kinase C/genetics , Protein Kinase C/isolation & purification , Protein Kinase C-theta , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Subcellular Fractions/enzymology , Ubiquitin/metabolism
19.
Arch Biochem Biophys ; 479(2): 145-52, 2008 Nov 15.
Article in English | MEDLINE | ID: mdl-18809371

ABSTRACT

In dividing cells calpastatin diffuses from aggregates into cytosol, indicating the requirement for a tight regulation of calpain. Accordingly, the involvement of the calpain-calpastatin system in cell proliferation and in the density-dependent growth arrest was studied in JA3 cells stably transfected with a calpastatin form permanently localized in cytosol. In calpastatin overexpressing cells, cell cycle rate is 50% reduced, and cells enter the ungrowing, still fully reversible, stage at a 3-fold higher cell density. Furthermore, in cell density growth arrest phase, down regulation of alpha- and theta-PKC isoforms, as well as FAK and talin occurs. In calpastatin overexpressing cells, degradation of these calpain substrate proteins is prevented and delayed. Thus, calpain activity plays a crucial role in inducing the cell entry into a functional quiescent phase.


Subject(s)
Calcium-Binding Proteins/metabolism , Calpain/metabolism , Cell Proliferation , Cytosol/metabolism , Animals , Calcium-Binding Proteins/genetics , Calpain/genetics , Focal Adhesion Kinase 1/genetics , Focal Adhesion Kinase 1/metabolism , Gene Expression , Humans , Isoenzymes/genetics , Isoenzymes/metabolism , Jurkat Cells , Protein Kinase C/genetics , Protein Kinase C/metabolism , Protein Kinase C-alpha/genetics , Protein Kinase C-alpha/metabolism , Protein Kinase C-theta , Rats
20.
J Biol Chem ; 283(43): 29069-76, 2008 Oct 24.
Article in English | MEDLINE | ID: mdl-18682401

ABSTRACT

Although several reports have indicated that eNOS is a highly sensitive calpain substrate, the occurrence of a concomitant Ca(2+)-dependent activation of the synthase and of the protease has never been analyzed in specific direct experiments. In this study, we have explored in vivo how eNOS can undergo Ca(2+)-dependent translocation and activation, protected against degradation by activated calpain. Here we demonstrate that following a brief exposure to Ca(2+)-loading, the cytosolic eNOS-HSP90 complex recruits calpain in a form in which the chaperone and the synthase are almost completely resistant to digestion by the protease. Furthermore, in the presence of the HSP90 inhibitor geldanamycin, a significant decrease in NO production and an extensive degradation of eNOS protein occurs, indicating that dissociation from membranes and association with the chaperone is correlated to the protection of the synthase. Experiments with isolated membrane preparations confirm the primary role of HSP90 in dissociation of eNOS from caveolae. Prolonged exposure of cells to Ca(2+)-loading resulted in an extensive degradation of both eNOS and HSP90, accompanied by a large suppression of NO production. We propose that the protective effect exerted by HSP90 on eNOS degradation mediated by calpain represents a novel and critical mechanism that assures the reversibility of the intracellular trafficking and activation of the synthase.


Subject(s)
Calpain/chemistry , HSP90 Heat-Shock Proteins/metabolism , Nitric Oxide Synthase Type III/metabolism , Nitric Oxide/chemistry , Animals , Benzoquinones/pharmacology , Calcium/chemistry , Cattle , Caveolin 1/metabolism , Cell Line , Cysteine Proteinase Inhibitors/pharmacology , Cytosol/metabolism , Lactams, Macrocyclic/pharmacology , Microscopy, Confocal , Models, Biological , Protein Folding
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