sq-23377 and calpastatin

Malignant gliomas (MG), including grades III and IV astrocytomas, are the most common adult brain tumors. These tumors are highly aggressive with a median survival of less than 2 years. Nuclear factor I (NFI) is a family of transcription factors that regulates the expression of glial genes in the developing brain. We have previously shown that regulation of the brain fatty acid-binding protein (B-FABP; FABP7) and glial fibrillary acidic protein (GFAP) genes in MG cells is dependent on the phosphorylation state of NFI, with hypophosphorylation of NFI correlating with GFAP and B-FABP expression. Importantly, NFI phosphorylation is dependent on phosphatase activity that is enriched in GFAP/B-FABP+ve cells. Using chromatin immunoprecipitation, we show that NFI occupies the GFAP and B-FABP promoters in NFI-hypophosphorylated GFAP/B-FABP+ve MG cells. NFI occupancy, NFI-dependent transcriptional activity, and NFI phosphorylation are all modulated by the serine/threonine phosphatase calcineurin. Importantly, a cleaved form of calcineurin, associated with increased phosphatase activity, is specifically expressed in NFI-hypophosphorylated GFAP/B-FABP+ve MG cells. Calcineurin in GFAP/B-FABP+ve MG cells localizes to the nucleus. In contrast, calcineurin is primarily found in the cytoplasm of GFAP/B-FABP-ve cells, suggesting a dual mechanism for calcineurin activation in MG. Finally, our results demonstrate that calcineurin expression is up-regulated in areas of high infiltration/migration in grade IV astrocytoma tumor tissue. Our data suggest a critical role for calcineurin in NFI transcriptional regulation and in the determination of MG infiltrative properties.

Topics: Adult; Astrocytoma; Calcineurin; Calcium-Binding Proteins; Cell Line, Tumor; Chromatin Immunoprecipitation; Cyclosporine; Glioma; Humans; Immunohistochemistry; Immunoprecipitation; Ionomycin; NFI Transcription Factors; Phosphorylation; Promoter Regions, Genetic; Protein Binding; Protein Transport

Calpains are involved in calcium-induced neuronal cell toxicity, which is associated with the pathophysiology of Alzheimer's disease (AD). The activity of calpains is regulated by the inhibitor calpastatin, and increased activity of calpains and decreased calpastastin are often found in AD. Histone deacetylase (HDAC) inhibitors are implicated in AD treatment through the improvement of learning and memory but the underlying mechanism is yet to be understood. Here, using SH-SY5Y neuroblastoma cells and a calcium ionophore ionomycin, we examined whether and how HDAC inhibitor trichostatin A (TSA) inhibits calcium-induced neuronal cell death. TSA increased both the mRNA and protein levels of calpastatin, with no alterations in those of calpain 1 and calpain 2. Furthermore, TSA-stimulated increase of calpastatin was accompanied by a significant attenuation of ionomycin-induced autolysis of calpain 1, but not of calpain 2, and calpain-dependent 150 kDa αII spectrin cleavage. Under these conditions, however, caspase activity was unaltered. Moreover, ectopic expression of small interfering RNA of calpastatin reversed the inhibitory effect of TSA on ionomycin-induced calpain 1 autolysis and αII spectrin cleavage. Chromatin immunoprecipitation assay revealed the increased levels of acetylation at lysine 5 of histone H4 (H4K5-Ac), H3K9-Ac and H3K14-Ac within the calpastatin promoter region in TSA-treated cells relative to control cells. Finally, TSA significantly decreased ionomycin-induced cell toxicity. This study demonstrates that TSA attenuates calcium-induced neuronal cell death by the inhibition of calpain activity which is mediated in part by increased calpastatin expression via histone hyperacetylation within the calpastatin promoter region. Our study provides a novel mechanism for the neuroprotective effect of HDAC inhibitors on AD.

Topics: Acetylation; Apoptosis; Blotting, Western; Calcium; Calcium-Binding Proteins; Calpain; Cell Proliferation; Chromatin Immunoprecipitation; Epigenomics; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Ionomycin; Luciferases; Neuroblastoma; Promoter Regions, Genetic; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Tumor Cells, Cultured

Mycoplasma hyorhinis frequently contaminates cultured cells, with effects on synthetic and metabolic pathways. We demonstrated for the first time that contamination of cells by a strain of M. hyorhinis (NDMh) results in increased levels of calpastatin (the endogenous inhibitor of the ubiquitous Ca(2+) -dependent protease calpain). We now show that the calpastatin upregulation by NDMh in neuroblastoma SH-SY5Y cells resides in the NDMh lipoprotein fraction (LPP), via the NF-κB transcription pathway. NF-κB activation requires dissociation of the cytoplasmic NF-κB/IκB complex followed by NF-κB translocation to the nucleus. NDMh-LPP induced translocation of the NF-κB RelA subunit to the nucleus and upregulated calpastatin. RelA translocation and calpastatin elevation were prevented when dissociation of the NF-κB/IκB complex was inhibited either by transfection with the non-phosphorylatable IκB mutant ΔNIκBα, or by using PS1145, an inhibitor of the IκB kinase (IKK complex). Increased calpastatin levels attenuate calpain-related amyloid-β-peptide and Ca(2+) -toxicity (these are central to the pathogenesis of Alzheimer's Disease). LPP-induced elevation of calpastatin provides an example of effects on non-inflammatory intracellular proteins, the outcome being significant alterations in host cell functions. Since calpastatin level is important in the control of calpain activity, mycoplasmal LPP may be of interest in treating some pathological processes involving excessive calpain activation.

Topics: Amyloid beta-Peptides; Bacterial Outer Membrane Proteins; Calcium Ionophores; Calcium-Binding Proteins; Calpain; Carrier Proteins; Cell Line, Tumor; Host-Pathogen Interactions; Humans; Ionomycin; Lipoproteins; Microfilament Proteins; Mycoplasma hyorhinis; Mycoplasma Infections; NF-kappa B; Signal Transduction; Up-Regulation

Important to the function of calpains is temporal and spatial regulation of their proteolytic activity. Here, we demonstrate that cytoplasm-resident calpain 2 cleaves human nuclear topoisomerase I (hTOP1) via Ca(2+)-activated proteolysis and nucleoplasmic shuttling of proteases. This proteolysis of hTOP1 was induced by either ionomycin-caused Ca(2+) influx or addition of Ca(2+) in cellular extracts. Ca(2+) failed to induce hTOP1 proteolysis in calpain 2-knockdown cells. Moreover, calpain 2 cleaved hTOP1 in vitro. Furthermore, calpain 2 entered the nucleus upon Ca(2+) influx, and calpastatin interfered with this process. Calpain 2 cleavage sites were mapped at K(158) and K(183) of hTOP1. Calpain 2-truncated hTOP1 exhibited greater relaxation activity but remained able to interact with nucleolin and to form cleavable complexes. Interestingly, calpain 2 appears to be involved in ionomycin-induced protection from camptothecin-induced cytotoxicity. Thus, our data suggest that nucleocytoplasmic shuttling may serve as a novel type of regulation for calpain 2-mediated nuclear proteolysis.

Topics: Active Transport, Cell Nucleus; Amino Acid Sequence; Calcium; Calcium-Binding Proteins; Calpain; Camptothecin; Cell Line, Tumor; Cell Nucleus; Cytoplasm; DNA Topoisomerases, Type I; Gene Knockdown Techniques; Humans; Ionomycin; Molecular Sequence Data; Nucleolin; Peptide Hydrolases; Phosphoproteins; RNA-Binding Proteins

MYOC, a gene involved in different types of glaucoma, encodes myocilin, a secreted glycoprotein of unknown function, consisting of an N-terminal leucine-zipper-like domain, a central linker region, and a C-terminal olfactomedin-like domain. Recently, we have shown that myocilin undergoes an intracellular endoproteolytic processing. We show herein that the proteolytic cleavage in the linker region splits the two terminal domains. The C-terminal domain is secreted to the culture medium, whereas the N-terminal domain mainly remains intracellularly retained. In transiently transfected 293T cells, the cleavage was prevented by calpain inhibitors, such as calpeptin, calpain inhibitor IV, and calpastatin. Since calpains are calcium-activated proteases, we analyzed how changes in either intra- or extracellular calcium affected the cleavage of myocilin. Intracellular ionomycin-induced calcium uptake enhanced myocilin cleavage, whereas chelation of extracellular calcium by EGTA inhibited the proteolytic processing. Calpains I and II cleaved myocilin in vitro. However, in cells in culture, only RNA interference knockdown of calpain II reduced myocilin processing. Subcellular fractionation and digestion of the obtained fractions with proteinase K showed that full-length myocilin resides in the lumen of the endoplasmic reticulum together with a subpopulation of calpain II. These data revealed that calpain II is responsible for the intracellular processing of myocilin in the lumen of the endoplasmic reticulum. We propose that this cleavage might regulate extracellular interactions of myocilin, contributing to the control of intraocular pressure.

Topics: Calcium-Binding Proteins; Calpain; Cell Line; Cytoskeletal Proteins; Dipeptides; Egtazic Acid; Endopeptidase K; Eye Proteins; Glaucoma; Glycoproteins; Humans; Ionomycin; Mutation, Missense; Protein Conformation; Protein Structure, Tertiary; Transfection

Calpastatin is an intrinsic intracellular inhibitor of calpain, a Ca(2+)-dependent thiol protease. The calpain-calpastatin system constitutes one functional proteolytic unit whose presence and function has already been investigated in various cell types, but not in the egg. We have previously shown that calpain is expressed in rat eggs and is activated upon egg activation. The present study was designed to investigate the calpain-calpastatin interplay throughout the process. Western blot analysis revealed two main calpastatin isoforms, the erythrocyte type (77 kDa) and the muscle tissue type (110 kDa). By immunohistochemistry and confocal laser scanning microscopy, we demonstrated that the 110 kDa calpastatin was localized at the membrane area and highly abundant at the meiotic spindle in eggs at the first and second meiotic divisions. The 77 kDa calpastatin isoform appeared to be localized as a cortical sphere of clusters. The 110 kDa calpastatin and beta-tubulin have both been localized to the spindle of metaphase II eggs, both being scattered all through the cytoplasm following spindle disruption by nocodazole treatment, implying a dynamic interaction between calpastatin and microtubule elements. Upon egg activation, membranous calpastatin translocated to the cortex whereas cortical millimolar (m)-calpain shifted towards the membrane. Spindle calpastatin and calpain remained static. We suggest that calpastatin serves as a regulator of m-calpain. The counter translocation of m-calpain and calpastatin could serve as a means of calpain escape from calpastatin inhibition and may reflect a step in the process of calpain activation, throughout egg activation, that is required for calpain to exert its proteolytic activity.

Topics: Animals; Biological Transport; Blotting, Western; Calcium-Binding Proteins; Calpain; Cell Membrane; Female; Fertilization; Ionomycin; Ionophores; Microscopy, Confocal; Molecular Weight; Ovum; Parthenogenesis; Protein Isoforms; Rats; Rats, Wistar

Previously, we reported that l-glutamic acid decarboxylase isoform 65 (GAD65) could be cleaved in vitro to release a stable truncated form which lacks amino acid 1-69 from the N-terminus, GAD65(Delta1-69). However, whether such a truncated form is also present under certain physiological conditions remains elusive. In the present study, we showed that, upon sustained neuronal stimulation, GAD65 could be cleaved into a truncated form in a rat synaptosomal preparation. This truncated form had similar electrophoretic mobility to purified recombinant human GAD65(Delta1-69). Furthermore, we demonstrated that this conversion was calcium dependent. Calcium-chelating reagents such as EDTA and 1,2-bis-(o-aminphenoxy)-ethane-N,N,N',N'-tetra-acetic acid tetra-acetoxy-methyl ester prevented the cleavage of GAD65. In addition, our data suggested that calpain, a calcium-dependent cysteine protease, is activated upon neuronal stimulation and could be responsible for the conversion of full-length GAD65 to truncated GAD65 in the brain. Moreover, calpain inhibitors such as calpain inhibitor I or calpastatin could block the cleavage. Results of our in vitro cleavage assay using purified calpain and immunopurified rat GAD65 also supported the idea that GAD65 could be directly cleaved by calpain.

Topics: Animals; Blotting, Western; Brain; Calcium; Calcium-Binding Proteins; Calpain; Cells, Cultured; Chelating Agents; Dose-Response Relationship, Drug; Drug Interactions; Edetic Acid; Embryo, Mammalian; Enzyme Activation; Female; Glutamate Decarboxylase; Glutamic Acid; Immunoprecipitation; Ionomycin; Ionophores; Isoenzymes; Neurons; Pregnancy; Rats; Rats, Sprague-Dawley; Synaptosomes; Time Factors

The ubiquitous calpains, mu- and m-calpain, have been implicated in essential physiological processes and various pathologies. Cell-permeable specific inhibitors are important tools to elucidate the roles of calpains in cultivated cells and animal models. The synthetic N-acetylated 27-mer peptide derived from exon B of the inhibitory domain 1 of human calpastatin (CP1B) is unique as a potent and highly selective reversible calpain inhibitor, but is poorly cell-permeant. By addition of N-terminal cysteine residues we have generated a disulfide-conjugated CP1B with the cell-penetrating 16-mer peptide penetratin derived from the third helix of the Antennapedia homeodomain protein. The inhibitory potency and selectivity of CP1B for calpain versus cathepsin B and L, caspase 3 and the proteasome was not affected by the conjugation with penetratin. The conjugate was shown to efficiently penetrate into living LCLC 103H cells, since it prevents ionomycin-induced calpain activation at 200-fold lower concentration than the non-conjugated inhibitor and is able to reduce calpain-triggered apoptosis of these cells. Penetratin-conjugated CP1B seems to be a promising alternative to the widely used cell-permeable peptide aldehydes (e.g. calpain inhibitor 1) which inhibit the lysosomal cathepsins and partially the proteasome as well or even better than the calpains.

Topics: Apoptosis; Calcium-Binding Proteins; Calpain; Carrier Proteins; Cathepsins; Cell Line; Cell Membrane Permeability; Cell-Penetrating Peptides; Cysteine Proteinase Inhibitors; Erythrocytes; Exons; Humans; Ionomycin; Peptide Fragments

The integrin beta 4 subunit often undergoes proteolytic cleavage within its long cytoplasmic tail to yield a characteristic protein pattern of 205, 165, and 125 kDa. The results in this study suggest that beta 4 cleavage often occurs during or after cell lysis, where it was readily inhibitable by calcium chelators (EDTA, EGTA) and inhibitors of cysteine proteases (E64c, leupeptin). The cleavage of beta 4 is catalyzed by a calpain-like enzyme because (i) it requires calcium, (ii) it is mimicked by purified milli-calpain, and (iii) it is inhibited by several calpain inhibitors including the calpain-specific inhibitor calpastatin. Within intact cells, cleavage of beta 4 was cell type-specific and observed only when the cells were made permeable to calcium. Substantial cell viability was retained during beta 4 cleavage induced by ionomycin plus calcium, indicating that cleavage within intact cells was not necessarily a consequence of cell death. However, manipulations of cells including suspension, synchronization, and stimulation with serum, phorbol esters, or other agents all failed to induce cleavage, suggesting that if cleavage is physiologically relevant, it is not easily duplicated in vitro. Analysis of multiple cell types showed a wide variation in beta 4 sensitivity to proteolytic cleavage, suggesting that this process might be differentially regulated depending on the cellular environment.

Topics: Antigens, Surface; Artifacts; Calcium; Calcium-Binding Proteins; Calpain; Cell Adhesion; Cell Survival; Digitonin; Egtazic Acid; Endopeptidases; Humans; Integrin alpha6beta4; Integrin beta4; Integrins; Ionomycin; Peptide Fragments; Tumor Cells, Cultured

Intracellular localization of calpain (calcium dependent cysteine proteinase) was studied in resting or activated human platelets. When stimulated with 2 U/ml thrombin, approximately 40% of total cellular calpain activity and 25% of antigen translocated mainly to the intracellular membrane fractions with autolytic activation. Translocation of calpain was completely abolished by the addition of EDTA to the sonication medium. However an endogenous calpain inhibitor (calpastatin) activity was not detected in the membrane fractions both in resting and in thrombin stimulated platelets. Translocation of calpain was also observed in the platelets stimulated with ionomycin, collagen or phorbor myristate acetate (PMA). These data suggest that cytosolic calpain reversibly translocates to the intracellular membranes during platelet activation without an interference by calpastatin.

Topics: Blood Platelets; Calcium-Binding Proteins; Calpain; Cell Membrane; Collagen; Cysteine Proteinase Inhibitors; Edetic Acid; Humans; Immunoblotting; Intracellular Membranes; Ionomycin; Tetradecanoylphorbol Acetate; Thrombin