sq-23377 and calpeptin

Calpains are a family of calcium-dependent cysteine proteases that are ubiquitously expressed in mammals and play critical roles in neuronal death by catalyzing substrate proteolysis. Here, we developed two-dimensional gel electrophoresis-based protease proteomics to identify putative calpain substrates. To accomplish this, cellular lysates from neuronal cells were first separated by pI, and the immobilized sample on a gel strip was incubated with a recombinant calpain and separated by molecular weight. Among 25 altered protein spots that were differentially expressed by at least 2-fold, we confirmed that arsenical pump-driving ATPase, optineurin, and peripherin were cleaved by calpain using in vitro and in vivo cleavage assays. Furthermore, we found that all of these substrates were cleaved in MN9D cells treated with either ionomycin or 1-methyl-4-phenylpyridinium, both of which cause a calcium-mediated calpain activation. Their cleavage was blocked by calcium chelator or calpain inhibitors. In addition, calpain-mediated cleavage of these substrates and its inhibition by calpeptin were confirmed in a middle cerebral artery occlusion model of cerebral ischemia, as well as a stereotaxic brain injection model of Parkinson disease. Transient overexpression of each protein was shown to attenuate 1-methyl-4-phenylpyridinium-induced cell death, indicating that these substrates may confer protection of varying magnitudes against dopaminergic injury. Taken together, the data indicate that our protease proteomic method has the potential to be applicable for identifying proteolytic substrates affected by diverse proteases. Moreover, the results described here will help us decipher the molecular mechanisms underlying the progression of neurodegenerative disorders where protease activation is critically involved.

Topics: 1-Methyl-4-phenylpyridinium; Animals; Arsenite Transporting ATPases; Calpain; Cell Death; Cell Line; Dipeptides; Dopaminergic Neurons; Electrophoresis, Gel, Two-Dimensional; Glycine; Infarction, Middle Cerebral Artery; Ionomycin; Peripherins; Proteome; Proteomics; Rats; Rats, Sprague-Dawley

Activation of calpains by calcium flux leading to talin cleavage is thought to be an important process of LFA-1 activation by inside-out signalling. Here, we tested the effects of the calcium ionophore ionomycin and calpain inhibitor calpeptin on LFA-1-mediated adhesion of a T cell hybridoma line, cytotoxic T cells and primary resting T cells. Ionomycin activated LFA-1-mediated adhesion of all three types of T cells, and calpeptin inhibited the effects of ionomycin. However, calpeptin also inhibited activation of LFA-1 by PMA, which did not induce calcium flux. Cleavage of talin was undetectable in ionomycin-treated T cells. Furthermore, treatment with ionomycin and calpeptin induced apoptosis of T cells. Inhibitors of phosphatidyl Inositol-3 kinase inhibited activation of LFA-1 by ionomycin, but not by PMA, whereas the protein kinase C inhibitor inhibited the effects of PMA, but not ionomycin. Thus, activation of LFA-1 by ionomycin is independent of calpain-mediated talin cleavage.

Topics: Acrylates; Androstadienes; Animals; Apoptosis; Calpain; Cell Adhesion; Cell Line, Tumor; Chromones; Cysteine Proteinase Inhibitors; Dipeptides; Dose-Response Relationship, Drug; Enzyme Inhibitors; Humans; Hybridomas; Ionomycin; Ionophores; Lymphocyte Function-Associated Antigen-1; Mice; Mice, Inbred C57BL; Mice, Transgenic; Morpholines; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase C; Receptors, Antigen, T-Cell; T-Lymphocytes; Talin; Wortmannin

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

Prostate carcinoma is the most commonly diagnosed cancer in men and the second leading cause of death due to cancer in Western civilization. Androgen ablation therapy is effective in treating androgen-dependent tumors, but eventually, androgen-independent tumors recur and are refractory to conventional chemotherapeutics. Hence, the emergence of androgen independence is the most challenging problem in managing prostate tumors. We report a novel mechanism of androgen independence: calpain cleaves the androgen receptor (AR) into an androgen-independent isoform. In vitro and in vivo analyses show that calpain removes the COOH-terminal ligand binding domain generating a constitutively active molecule. Analysis of human prostate tumors indicates that several tumors express higher levels of this truncated AR than noncancerous prostate tissue. In transient transfection studies, the truncated AR is three to five times more potent than the full-length receptor in transactivating transcription. The androgen-independent Rv1 cells express high levels of the truncated AR, and treatment of these cells with a calpain inhibitor reduces truncated AR expression. In the absence of androgen, inhibition of calpain activity induces apoptosis. The HIV protease inhibitor amprenavir inhibits calpain activity and is also effective in inducing apoptosis in the Rv1 cell line. The cell culture studies were reproduced in a mouse xenograft model, where, in the absence of androgens, amprenavir significantly reduces tumor growth. Together, these studies indicate that calpain-dependent proteolysis of the AR may be a mechanism of androgen independence. The calpain inhibition studies suggest that inhibiting this activity may be a potential treatment for some androgen-independent prostate tumors.

Topics: Androgens; Animals; Apoptosis; Calpain; Carbamates; Cell Line, Tumor; Dipeptides; Enzyme Activation; Furans; Humans; Ionomycin; Male; Mice; Mice, Nude; Neoplasms, Hormone-Dependent; Prostatic Neoplasms; Receptors, Androgen; Sulfonamides; Transcriptional Activation; Transfection; Transplantation, Heterologous

Apoptosis of retinal ganglion cells (RGCs) impairs vision in glaucoma patients. RGCs are also degenerated in multiple sclerosis (MS), resulting in loss of visual perception in MS patients. We examined the involvement of calpain and caspase cascades in apoptosis of the rat retinal ganglion cell line RGC-5 following 24 h of exposure to 250 nM ionomycin (IMN) or 300 units/ml interferon-gamma (IFN-gamma) and then evaluated functional neuroprotection with 2 microM calpeptin (CP, a calpain-specific inhibitor). Morphological and biochemical features of apoptosis were detected in RGC-5 cells following exposure to IMN or IFN-gamma. Fura-2 assay determined significant increases in intracellular free [Ca2+] following exposure to IMN or IFN-gamma. Pretreatment with CP for 1 h prevented Ca2+ influx, proteolytic activities, and apoptosis in RGC-5 cells. Western blot analyses showed an increase in activities of calpain and caspase-12, upregulation of Bax:Bcl-2 ratio, release of cytochrome c from mitochondria, and increase in caspase-9 and caspase-3 activities during apoptosis. Increased caspase-3 activity was also confirmed by a colorimetric assay. Activation of caspase-8 and cleavage of Bid to tBid in RGC-5 cells following exposure to IFN-gamma indicated co-operation between extrinsic and intrinsic pathways of apoptosis. Patch-clamp recordings showed that pretreatment with CP attenuated apoptosis and maintained normal whole-cell membrane potential, indicating functional neuroprotection. Taken together, our results demonstrated that Ca2+ overload could be responsible for activation of calpain and caspase cascades leading to apoptotic death of RGC-5 cells and CP provided functional neuroprotection.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Calcium; Caspases; Cell Line; Cytochromes c; Dipeptides; Drug Interactions; Enzyme Activation; Interferon-gamma; Ionomycin; Ionophores; Membrane Potentials; Mitochondria; Models, Biological; Neuroprotective Agents; Patch-Clamp Techniques; Proto-Oncogene Proteins c-bcl-2; Rats; Retinal Ganglion Cells

Glutamate toxicity has been implicated in cell death in neurodegenerative diseases and injuries. Glutamate-induced Ca2+ influx may mediate activation of calpain, a Ca2+-dependent cysteine protease, which in turn may degrade key cytoskeletal proteins. We investigated glutamate-mediated apoptosis of VSC4.1 motoneurons and functional neuroprotection by calpain inhibition. Exposure of VSC4.1 cells to 10 microM glutamate for 24 hr caused significant increases in intracellular free [Ca2+], as determined by fura-2 assay. Pretreatment of cells with 10 or 25 microM calpeptin (a cell-permeable calpain-specific inhibitor) for 1 hr prevented glutamate-induced Ca2+ influx. Western blot analyses showed an increase in Bax:Bcl-2 ratio, release of cytochrome c from mitochondria, and calpain and caspase-3 activities during apoptosis. Cell morphology, as evaluated by Wright staining, indicated predominantly apoptotic features following glutamate exposure. ApopTag assay further substantiated apoptotic features morphologically as well as biochemically. Our data showed that calpeptin mainly prevented calpain-mediated proteolysis and apoptosis and maintained whole-cell membrane potential, indicating functional neuroprotection. The results imply that calpeptin may serve as a therapeutic agent for preventing motoneuron degeneration, which occurs in amyotrophic lateral sclerosis and spinal cord injury. In this investigation, we also examined glutamate receptor subtypes involved in the initiation of apoptosis in VSC4.1 cells following exposure to glutamate. Our results indicated that the N-methyl-D-aspartate (NMDA) receptors contributed more than alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) receptors to glutamate-mediated Ca2+ influx and cell death mechanism. Inhibition of the activities of both NMDA and AMPA receptors protected VSC4.1 cells from glutamate toxicity and preserved whole-cell membrane potential.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Calcium; Calpain; Caspase 3; Caspases; Cell Fusion; Cysteine Proteinase Inhibitors; Cytochromes c; Dipeptides; Glutamic Acid; Humans; Ionomycin; Ionophores; Mice; Mitochondria; Motor Neurons; Neuroblastoma; Neuroprotective Agents; Patch-Clamp Techniques; Proto-Oncogene Proteins c-bcl-2; Rats; Spectrin; Spinal Cord

Synaptic transmission is conducted by neurotransmitters released from presynaptic nerve terminals by means of Ca2+-dependent exocytosis of synaptic vesicles. Formation of a complex of soluble N-ethylmaleimide-sensitive fusion protein receptor (SNARE) proteins, including vesicle-associated membrane protein-2 (VAMP-2) in the synaptic vesicle membrane, and syntaxin 1 and synaptosomal-associated protein of 25 kDa (SNAP-25) in the plasma membrane, is essential for exocytosis. Ionomycin treatment of cultured rat cerebellar granule cells led to cleavage of SNAP-25, but not syntaxin 1 and VAMP-2, that was dependent on extracellular Ca2+. Cleavage was also induced by N-methyl-D-aspartate (NMDA) treatment, but not by depolarization. The use of various site-specific antibodies to SNAP-25, suggested that the cleavage site was in the N-terminal domain of SNAP-25. Calpain inhibitors abolished the Ca2+-dependent cleavage of SNAP-25 and markedly facilitated Ca2+-dependent glutamate (Glu) release from cerebellar granule cells. These results suggest that calpain may play an important role in the long-lasting regulation of synaptic transmission by suppressing neurotransmitter release, possibly through the proteolytic cleavage of SNAP-25.

Topics: Age Factors; Animals; Animals, Newborn; Antibodies; Blotting, Western; Calcium; Calpain; Cells, Cultured; Cerebellum; Chromatography, High Pressure Liquid; Dipeptides; Dose-Response Relationship, Drug; Down-Regulation; Drug Interactions; Excitatory Amino Acid Agonists; Glutamic Acid; Glycoproteins; Ionomycin; Ionophores; Membrane Proteins; N-Methylaspartate; Nerve Tissue Proteins; Neurons; Potassium; Rats; Rats, Wistar; Synaptosomal-Associated Protein 25; Time Factors

At fertilization in mammals, the spermatozoon triggers a unique signal transduction mechanism within the egg, leading to its activation. It is well accepted that the earliest event observed in all activated eggs is an abrupt rise in intracellular calcium concentrations. However, little is known regarding the downstream proteins that are activated by this rise in calcium. Calpains constitute a family of intracellular calcium-dependent cysteine proteases whose members are expressed widely in a variety of cells. We investigated the expression and possible role of the calpain isoforms mu and m throughout egg activation. Both calpains were expressed in the rat egg and localized at the egg cortex as well as in the meiotic spindle. m Calpain translocated to the membrane and to the spindle area during parthenogenetic egg activation and during in vivo fertilization, upon sperm binding to the egg. The cytoskeletal protein alpha-spectrin (fodrin) was proteolysed by calpain during the egg-activation process, as demonstrated by specific calpain-breakdown products. Following parthenogenetic activation by ionomycin or puromycin, the calpain-selective permeable inhibitor, calpeptin, inhibited the resumption of meiosis and cortical reaction in a dose-dependent manner. Calpeptin was also effective in inhibiting in vitro fertilization. These results may imply a correlation between calpain activation and mammalian egg activation at fertilization and a possible role for calpain in the cascade of cellular events leading to resumption of meiosis.

Topics: Animals; Calcium Signaling; Calpain; Dipeptides; Female; Fertilization in Vitro; Fluorescent Antibody Technique; Ionomycin; Ionophores; Male; Meiosis; Microscopy, Confocal; Oligopeptides; Ovum; Parthenogenesis; Puromycin; Rats; Rats, Wistar; Sperm-Ovum Interactions

Our laboratory has established that local activation of calpain by a transient elevation of the free intracellular calcium concentration is crucial for the induction of growth cone (GC) formation in cultured Aplysia neurons. The mechanisms and stages in which calpain is involved in the formation of a GC are not known. We began to study these questions by determining the nature of calpain's action and the stages in which calpain activity affects the cascade of events that leads to the formation of the GC and its extension. We report that the calpain-dependent transformation of an axonal segment into a GC occurs within a narrow window of opportunity that lasts approximately 5 min. If calpain is inhibited during this window of opportunity, GC formation does not occur. Inhibition of calpain after the window of opportunity slows down the rate of lamellipodial extension but doesn't arrest it. The proteolysis of spectrin, a calpain substrate and a major component of the membrane skeleton, occurs within this window of opportunity, in agreement with the hypothesis that spectrin proteolysis is an early step in the formation of the GC. If the onset of proteolysis is deferred, spectrin remains unchanged and GC formation is compromised. We suggest that calpain participates in two different processes: it is critical for the triggering of GC formation and plays a modulatory role during the extension of the GC's lamellipodia.

Topics: Animals; Aplysia; Axotomy; Calpain; Cells, Cultured; Cysteine Proteinase Inhibitors; Dipeptides; Growth Cones; Immunohistochemistry; Ionomycin; Ionophores; Nerve Regeneration; Neurons; Signal Transduction; Spectrin; Time Factors