Specific Proteolysis of Neuronal Protein GAP-43 by Calpain: Characterization,Regulation, and Physiological Role

The mechanism of specific proteolysis of the neuronal protein GAP-43 in axonal terminals has been investigated. In synaptic terminals in vivo and in synaptosomes in vitro GAP-43 is cleaved only at the single peptide bond formed by Ser41; this is within the main effector domain of GAP-43. Proteolysis at this site involves the cysteine calcium-dependent neutral protease calpain. The following experimental evidences support this conclusion: 1) calcium-dependent proteolysis of GAP-43 in synaptosomes is insensitive to selective inhibitor of micro-calpain (PD151746), but it is completely blocked by micro- and m-calpain inhibitor PD150606; 2) GAP-43 proteolysis in the calcium ionophore A23187-treated synaptosomes is activated by millimolar concentration of calcium ions; 3) the pattern of fragmentation of purified GAP-43 by m-calpain (but not by micro-calpain) is identical to that observed in synaptic terminals in vivo. GAP-43 phosphorylated at Ser41 by protein kinase C (PKC) is resistant to the cleavage by calpain. In addition, calmodulin binding to GAP-43 decreases the rate of calpain-mediated GAP-43 proteolysis. Our results indicate that m-calpain-mediated GAP-43 proteolysis regulated by PKC and calmodulin is of physiological relevance, particularly in axonal growth cone guidance. We suggest that the function of the N-terminal fragment of GAP-43 (residues 1-40) formed during cleavage by m-calpain consists in activation of neuronal heterotrimeric GTP-binding protein G(o); this results in growth cone turning in response to repulsive signals.     

Short window of opportunity for calpain induced growth cone formation after axotomy of Aplysia neurons

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.     

Aminoguanidine-treatment results in the inhibition of lens opacification and calpain-mediated proteolysis in Shumiya cataract rats (SCR)

The Shumiya cataract rat (SCR) is a hereditary cataract model in which lens opacity appears spontaneously in the nuclear and perinuclear portions at 11-12 weeks of age. We found incidentally that the oral administration of aminoguanidine (AG), an inhibitor of inducible nitric oxide synthase (iNOS), strongly inhibits the development of lens opacification in SCR. Since our previous results strongly suggested that calpain-mediated proteolysis contributes to lens opacification during cataract formation in SCR, we examined the calpain-mediated proteolysis in AG-treated SCR lenses in detail. The results show that the calpain-mediated limited proteolysis of crystallins is also inhibited by AG-treatment. However, the administration of AG has no effect on the substrate susceptibility to calpain. On the other hand, the autolytic activation of calpain in AG-treated lenses is strongly inhibited, although AG itself does not inhibit calpain activity in vitro. Then, we analyzed the effect of AG-treatment on calcium concentrations in lens, and found that the elevation in calcium concentration that should occur prior to cataractogenesis in lenses is strongly suppressed by AG-treatment. These results strengthen our previous conclusion that calpain-mediated proteolysis plays a critical role in the development of lens opacification in SCR. Moreover, our results indicate that the inhibition of calpain-mediated proteolysis by AG-treatment is due to the suppression of calcium ion influx into the lens cells.     

Identification of a novel calpain inhibitor using phage display

Calpains are calcium- and thiol-dependent proteases that cleave a variety of intracellular substrates. Overactivation of the calpains has been implicated in a number of diseases and conditions such as ischemic stroke indicating a need for the development of calpain inhibitors. A major problem with current calpain inhibitors has been specific targeting to calpain. To identify highly specific calpain interacting peptides, we developed a peptide-phage library screening method based on the calcium-dependent conformation change associated with calpain activation. A phage-peptide library representing greater than 2 billion expressed 12-mers was incubated with calpain I in the presence of calcium. The calcium-dependent bound phage was then eluted by addition of EGTA. After four rounds of selection we found a conserved 5-mer sequence represented by LSEAL. Synthetic LSEAL inhibited tau-calpain interaction and in vitro proteolysis of tau- and alpha-synuclein by calpains. Deletion of the portion of the tau protein containing a homologous sequence to LSEAL resulted in decreased calpain-mediated tau degradation. These data suggest that these peptides may represent novel calpastatin mimetics.     

Positive regulation of mu-calpain action by polyphosphoinositides.

Whether calcium is the only major intracellular activator of calpain has not yet been established. Here we demonstrate that polyphosphoinositides may play critical roles in the activation process of mu-calpain. Experiments with purified enzyme, substrate (fodrin), and phospholipids show that only polyphosphoinositides but not other lipids significantly promote calpain action in the physiological intracellular calcium range of 10(-7) to 10(-6) M. The effect of polyphosphoinositide is exerted through both a reduction in the calcium concentration required for calpain autolysis and an increase in the Vmax of the proteolytic reaction. Neomycin, a polyphosphoinositide-binding antibiotic, inhibits both polyphosphoinositide-assisted proteolysis in test tubes and calcium-induced calpain activation coupled with substrate proteolysis in intact cells. This implies that the presence of polyphosphoinositides may actually be a prerequisite for calpain activation inside cells.     

Calcium Influx into Human Neuroblastoma Cells Induces ALZ-50 Immunoreactivity: Involvement of Calpain-Mediated Hydrolysis of Protein Kinase C

Abstract: Calcium influx into SH-SY5Y human neuroblastoma cells after ionophore treatment or transient permeabilization in calcium-containing medium increased ALZ-50 immunoreactivity markedly. This increase was prevented by inhibitors active against calpain or against protein kinase C (PKC), suggesting that both of these enzymes were required to mediate the effect of calcium influx on ALZ-50 immunoreactivity. Treatment with PKC activator TPA increased ALZ-50 immunoreactivity in the absence of calcium influx or after intracellular delivery of the specific calpain inhibitor calpastatin, indicating that the influence of PKC was downstream from that of calpain. Calcium influx also resulted in μ-calpain autolysis (one index of calpain activation) and the transient appearance of PKM (i.e., free PKC catalytic subunits, generated by calpain-mediated cleavage of the regulatory and catalytic PKC domains). Inhibition of calpain within intact cells resulted in a dramatic increase in steady-state levels of total τ (migrating at 46–52 kDa) but resulted in a relatively minor increase in 68-kDa ALZ-50-immunoreactive τ isoforms. Although calcium influx into intact cells resulted in accumulation of ALZ-50 immunoreactivity, total τ levels were, by contrast, rapidly depleted. Incubation of isolated fractions with calpain in the presence of calcium indicated that ALZ-50-immunoreactive τ isoforms were more resistant to calpain-mediated proteolysis than were non-ALZ-50 reactive τ isoforms. These data therefore indicate that calpain may regulate τ levels directly via proteolysis and indirectly through PKC activation. A consequence of the latter action is altered τ phosphorylation, perhaps involving one or more kinase cascades, and the preferential accumulation of ALZ-50-immunoreactive τ isoforms due to their relative resistance to degradation. These findings provide a basis for the possibility that disregulation of calcium homeostasis may contribute to the pathological levels of conversion of τ to A68 by hyperactivation of the calpain/PKC system.     

Protection of neuronal calcium sensor 1 protein in cells treated with paclitaxel

Paclitaxel (Taxol) is one of the most effective treatment options for patients suffering from a variety of cancers. A major side effect seen in a high percentage of patients treated with paclitaxel is irreversible peripheral neuropathy. We previously reported that prolonged treatment with paclitaxel activates a calcium-dependent enzyme, calpain, which degrades neuronal calcium sensor 1 (NCS-1) and subsequent loss of intracellular calcium signaling. Because it appears that activation of calpain is an early step in this destructive cascade, we proposed that inhibition of calpain will protect against the unwanted side effects of paclitaxel treatment. First, NCS-1 levels and intracellular calcium signaling were found to be protected by the presence of lactacystin, a protesome inhibitor. To reinforce the role of calpain in this process, we showed that increased concentrations of calpastatin, a naturally occurring calpain inhibitor, were protective. Next, we tested two mutated versions of NCS-1 developed with point mutations at the P2 position of the calpain cleavage site of NCS-1 to decrease the likelihood of NCS-1 degradation. One mutant was cleaved more favorably by calpain compared with NCS-1 WT, whereas the other mutant was less favorably cleaved. Expression of either mutated version of NCS-1 in neuroblastoma cells protected intracellular calcium signals from paclitaxel-induced changes. These results support our hypothesis that it is possible to protect cells from paclitaxel-induced degradation of NCS-1 by inhibiting calpain activity.     

Phosphoinositide 3-kinase accelerates calpain-dependent proteolysis of fodrin during hypoxic cell death

We have shown recently that phosphoinositide 3-kinase (PI 3-kinase) accelerates the hypoxia-induced necrotic cell death of H9c2, derived from rat cardiomyocytes, by enhancing metabolic acidosis. Here we show the downstream events of acidosis that cause hypoxic cell death. Hypoxia induces the proteolysis of fodrin, a substrate of calpain. Intracellular Ca(2+) chelation by BAPTA, and the addition of SJA6017, a specific peptide inhibitor of calpain, also reduces cell death and fodrin proteolysis, indicating that Ca(2+) influx and calpain activation might be involved in these events. The overexpression of wild type PI 3-kinase accelerates fodrin proteolysis, while dominant-negative PI 3-kinase reduces it. Both (N-ethyl-N-isopropyl)amiloride (EIPA), an inhibitor of the Na(+)/H(+) exchanger, and KB-R7943, an inhibitor of the Na(+)/Ca(2+) exchanger, reduce hypoxic cell death and fodrin proteolysis. The depletion of intracellular Ca(2+ )stores by thapsigargin, an inhibitor of endoplasmic reticulum Ca(2+)-ATPase, also reduces cell death and fodrin proteolysis, indicating that Ca(2+ )release from intracellular Ca(2+ )stores might be also involved. These results indicate that PI 3-kinase might accelerate hypoxic cell death by enhancing the calpain-dependent proteolysis of fodrin.     

Advanced glycation end-products induce calpain-mediated degradation of ezrin

Advanced glycation end-products (AGEs) are important mediators of diabetic complications via incompletely understood pathways. AGEs bind to intracellular ERM proteins (ezrin, radixin and moesin) that modulate cell shape, motility, adhesion and signal transduction. AGEs bind to the N-terminal domain of ezrin but not full-length ezrin. The AGE binding site may be made accessible either by proteolysis releasing an N-terminal fragment or ezrin activation by phosphorylation. Increased intracellular calcium is a primary event in cell activation by high glucose or AGEs. Calpain activity is increased concomitantly, and ezrin is a calpain substrate. The present study assessed whether glycated proteins affect ezrin cleavage and activation in renal tubule epithelial cells. After 7?days, AGE-BSA decreased ezrin levels in MDCK renal tubular cells to 66?±?4% of control. AGE-RNAse, ribosylated fetal bovine serum and methylglyoxal-BSA all had similar effects. The AGE-BSA-induced decrease in ezrin was abolished by calpastatin peptide, a specific calpain inhibitor, and 1,2-bis-aminophenoxyethane-tetraacetic acid acetoxymethyl ester (BAPTA-AM), a calcium chelator. Ezrin breakdown products were increased in AGE-BSA-treated cells, with a main fragment of ～?43?kDa. In?vitro, calpain?1 cleaved recombinant human ezrin, generating breakdown fragments including an N-terminal fragment of ～?43?kDa. Studies with ezrin mutants showed that non-phosphorylated ezrin was more susceptible to calpain cleavage. AGE-BSA decreased phosphorylated ERM levels to 31?±?12% in MDCK cells. Thus, AGE-BSA promotes calpain-mediated proteolysis of ezrin in MDCK cells by both increasing calpain activity and reducing phosphorylation. Therapies targeting both glycated proteins and calpain may provide protection against diabetic complications. Structured digital abstract ? Calpain-1?cleaves?Ezrin?by?protease assay?(View Interaction:?1,?2).     

Calpain 1-gamma filamin interaction in muscle cells: a possible in situ regulation by PKC-alpha

Calpains are a family of calcium-dependent cysteine-proteases involved in cytoskeleton remodelling and muscle differentiation. In a recent study, we observed the presence of calpain 1 in the muscle contractile apparatus and specifically in the N1- and N2-lines. This calpain isoform was found to be involved in the degradation of muscle fibres via proteolysis of key proteins in Z-disk and costameric junctions. The goal of this study was to determine whether gamma-filamin--a specific muscle isoform of the filamin family--is a calpain 1 substrate and to characterise this interaction. Gamma-filamin is a major muscle architectural protein located in the Z-line and under the sarcolemmal membrane. This protein is a component of the chain binding the sarcolemma to the sarcomeric structure. In this study, we found that gamma-filamin formed a stable complex in vitro and in cells with calpain 1 in the absence of calcium stimulation. We also located the binding domains in the C-terminus of gamma-filamin with a cleavage site between serine 2626 and serine 2627 in the hinge 2 region. The catalytic (80 kDa) and regulatory (28 kDa) subunits of calpain 1 are both involved in high affinity binding at gamma-filamin. Moreover, we showed that phosphorylation of the filamin C-terminus domain by PKC alpha protected gamma-filamin against proteolysis by calpain 1 in COS cells. Stimulation of PKC activity in myotubes, prevented gamma-filamin proteolysis by calpain and resulted in an increase in myotube adhesion.     

Caspases cleave the amino-terminal calpain inhibitory unit of calpastatin during apoptosis in human Jurkat T cells

We have previously reported the activation of procalpain mu (precursor for low-calcium-requiring calpain) in apoptotic cells using a cleavage-site-directed antibody specific to active calpain [Kikuchi, H. and Imajoh-Ohmi, S. (1995) Cell Death Differ. 2, 195-199]. In this study, calpastatin, the endogenous inhibitor protein for calpain, was cleaved to a 90-kDa polypeptide during apoptosis in human Jurkat T cells. The limited proteolysis of calpastatin preceded the autolytic activation of procalpain. Inhibitors for caspases rescued the cells from apoptosis and simultaneously inhibited the cleavage of calpastatin. The full-length recombinant calpastatin was also cleaved by caspase-3 or caspase-7 at Asp-233 into the same size fragment. Cys-241 was also targeted by these caspases in vitro but not in apoptotic cells. Caspase-digested calpastatin lost its amino-terminal inhibitory unit, and inhibited three moles of calpain per mole. Our findings suggest that caspases trigger the decontrol of calpain activity suppression by degrading calpastatin.     

OGG1 is degraded by calpain following oxidative stress and cisplatin exposure

Deficient repair activity for 8-hydroxy-2'-deoxyguanine (8-oxoguanine), a premutagenic oxidative DNA damage, has been observed in affected tissues in neurodegenerative diseases of aging, such as Alzheimer's disease, and in ischemia/reperfusion injury, type 2 diabetes mellitus, and cancer. These conditions have in common the accumulation of oxidative DNA damage, which is believed to play a role in disease progression, and loss of intracellular calcium regulation. These observations suggest that oxidative DNA damage repair capacity may be influenced by fluctuations in cellular calcium. We have identified human 8-oxoguanine-DNA glycosylase 1 (OGG1), the major 8-oxoguanine repair activity, as a specific target of the Ca(2+)-dependent protease Calpain I. Protein sequencing of a truncated partially calpain-digested OGG1 revealed that calpain recognizes OGG1 for degradation at a putative PEST (proline, glutamic acid, serine, threonine) sequence in the C-terminus of the enzyme. Co-immunoprecipitation experiments showed that OGG1 and Calpain I are associated in human cells. Exposure of HeLa cells to hydrogen peroxide or cisplatin resulted in the degradation of OGG1. Pretreatment of cells with the calpain inhibitor calpeptin resulted in inhibition of OGG1 proteolysis and suggests that OGG1 is a target for calpain-mediated degradation in vivo during oxidative stress- and cisplatin-induced apoptosis. Polymorphic OGG1 S326C was comparatively resistant to calpain digestion in vitro, yet was also degraded by a calpain-dependent pathway in vivo following DNA damaging agent exposure. The degradation of OGG1 by calpain may contribute to decreased 8-oxoguanine repair activity and elevated levels of 8-oxoguanine reported in tissues undergoing chronic oxidative stress, ischemia/reperfusion, and other cellular stressors known to produce perturbations of intracellular calcium homeostasis which activate calpain.     

Role of calcium and calpain in complement-induced vesiculation of the platelet plasma membrane and in the exposure of the platelet factor Va receptor

The role of calcium and intracellular calpains in the expression of platelet prothrombinase activity was investigated. Incubation of gel-filtered platelets with complement proteins C5b-9 resulted in alpha-granule and dense granule secretion and exposure of membrane binding sites for coagulation factors Va and Xa. This was accompanied by the release of microparticles from the cell surface that incorporated plasma membrane glycoproteins GP Ib, IIb, and IIIa and the alpha-granule membrane protein GMP-140. Generation of these membrane microparticles was dependent on the presence of extracellular calcium and was accompanied by proteolytic degradation of the cytoskeletal proteins, actin binding protein (ABP), talin, and myosin heavy chain. Microparticle formation was also detected when unstirred platelets were activated by thrombin plus collagen, although proteolysis of ABP, talin, or myosin was not observed. Preincorporation of the calpain inhibitor leupeptin into the platelet cytosol completely blocked C5b-9-induced proteolysis of ABP, talin, and myosin. However, inhibition of this calpain-mediated proteolysis had no effect on platelet secretion, the generation of microparticles, the exposure of membrane sites for factors Va and Xa, or the expression of prothrombinase activity. Furthermore, the microparticles that formed in the presence of leupeptin contained intact ABP, talin, and myosin heavy chain. Prior depletion of ATP with metabolic inhibitors eliminated all platelet responses to thrombin plus collagen, but did not affect C5b-9-induced microparticle formation or exposure of binding sites for factor Va on the microparticles. These data indicate that the formation of microparticles and the expression of platelet prothrombinase activity in response to C5b-9 are dependent upon an influx of calcium into the platelet cytosol, but do not require metabolic energy or calpain-mediated proteolysis of cytoskeletal proteins.     

Possible mechanism for the decrease of mitochondrial aspartate aminotransferase activity in ischemic and hypoxic rat retinas.

Glutamate is believed to be an excitatory amino acid neurotransmitter in the retina. Enzymes for glutamate metabolism, such as glutamate dehydrogenase, ornithine aminotransferase, glutaminase, and aspartate aminotransferase (AAT), exist mainly in the mitochondria. The abnormal increase of intracellular calcium ions in ischemic retinal cells may cause an influx of calcium ions into the mitochondria, subsequently affecting various mitochondrial enzyme activities through the activity of mitochondrial calpain. As AAT has the highest level of activity among enzymes involved in glutamate metabolism, we investigated the change of AAT activity in ischemic and hypoxic rat retinas and the protection against such activity by calpain inhibitors. We used normal RCS (rdy+/rdy+) rats. For the in vivo studies, we clamped the optic nerve of anesthetized rats to induce ischemia. In the in vitro studies, the eye cups were incubated with Locke's solution saturated with 95% N2/5% CO2. The activity of cytosolic AAT (cAAT) was about 20% of total activity, whereas mitochondrial AAT (mAAT) was about 75% in rat retina. Ninety minutes of ischemia or hypoxia caused a 20% decrease in mAAT activity, whereas cAAT activity remained unchanged. To examine the contribution of intracellular calcium ions to the degradation of mAAT, we used Ca2+-free Locke's solution containing 1 mM EGTA, ryanodine (Ca2+ channel blocker), and thapsigargin (Ca2+-ATPase inhibitor). In the present study, thapsigargin in Ca2+-free Locke's solution, but not ryanodine in this solution, was found to prevent AAT degradation. AAT degradation was also prevented by calpain inhibitors (Ca2+-dependent protease inhibitor) such as calpeptin at 1 nM, 10 nM, 0.1 microM, 1 microM and 10 microM, and by calpain inhibitor peptide, but not by other protease inhibitors (10 microM leupeptin, pepstatin, chymostatin). Additionally, we determined the subcellular localization of calpain activity and examined the change of calpain activity in ischemic rat retinas. Our results suggest that decreased activity of mAAT in ischemic and hypoxic rat retinas might be evoked by the degradation by calpain-catalyzed proteolysis in mitochondria.     

Evidence for involvement of calpain in c-Myc proteolysis in vivo

Precise control of the level of c-Myc protein is important to normal cellular homeostasis, and this is accomplished in part by degradation through the ubiquitin-proteasome pathway. The calpains are a family of calcium-dependent proteases that play important roles in proteolysis of some proteins, and their possible participation in degradation of intracellular c-Myc was therefore investigated. Activation of calpain with the cell-permeable calcium ionophore A23187 in Rat1a-myc or ts85 cells in culture induced rapid cleavage of c-Myc. This degradation was both calpain- and calcium-dependent since it was inhibited by preincubation with either the calpain-inhibitory peptide calpeptin or the calcium-chelating agent EGTA. A23187-induced c-Myc cleavage occurred in a time-dependent manner comparable to that of FAK, a known calpain substrate, and while calpeptin was able to significantly protect c-Myc from degradation, inhibitors of the proteasome or caspase proteases could not. Exposure of Rat1a-myc or ts85 cells in culture to calpeptin, or to the thiol-protease inhibitor E64d, resulted in the accumulation of c-Myc protein without an impact on ubiquitin-protein conjugates. Using an in vitro assay, calpain-mediated degradation occurred rapidly with wild-type c-Myc as the substrate, but was significantly prolonged in some c-Myc mutants with increased transforming activity derived from lymphoma patients. Those mutants with a prolonged half-life in vitro were also more resistant to A23187-induced cleavage in intact cells. These studies support a role for calpain in the control of c-Myc levels in vivo, and suggest that mutations impacting on sensitivity to calpain may contribute to c-Myc-mediated tumorigenesis.     

Calpain 1-titin interactions concentrate calpain 1 in the Z-band edges and in the N2-line region within the skeletal myofibril

Calpain 1, a ubiquitous calcium-dependent intracellular protease, was recently found in a tight association with myofibrils in skeletal muscle tissue [Delgado EF, Geesink GH, Marchello JA, Goll DE & Koohmaraie M (2001) J Anim Sci79, 2097-2107). Our immunofluorescence and immunoelectron microscopy investigations restrain the protease location at the periphery of the Z-band and at the midpoint of the I-band. Furthermore, calpain 1 is found to localize in myofibril fractures, described as proteolysis sites, in postmortem bovine skeletal red muscles, near the calcium deposits located at the N1 and N2 level. This in situ localization of calpain 1 is substantiated by binding assays with two titin regions covering the I-band region: a native fragment of 150 kDa (identified by mass spectrometry) that includes the N-terminal Z8-I5 region and the N1-line region of titin, and an 800 kDa fragment external to the N1 line that bears the PEVK/N2 region. These two titin fragments are shown to tightly bind calpain 1 in the presence of CaCl(2) and E64, a calpain inhibitor. In the absence of E64, they are cleaved by calpain 1. We conclude that titin affords binding sites to calpain 1, which concentrates the protease in the regions restrained by the Z-band edge and the N1-line as well as at the N2-line level, two sarcomeric regions where early postmortem proteolysis is detected.     

Excitatory amino acids activate calpain I and induce structural protein breakdown in vivo

Neuronal activity regulates the catabolism of specific structural proteins in adult mammalian brain. Pharmacological stimulation of rat hippocampal neurons by systemic or intraventricular administration of the excitatory amino acids kainate or N-methyl-D-aspartate induces selective loss of brain spectrin and the microtubule-associated protein MAP2, as determined by quantitative immunoblotting, but not of actin, the high molecular weight neurofilament polypeptide, or glial fibrillary acidic protein. The spectrin decrease occurs primarily by enhanced proteolysis, as levels of the major breakdown products of the alpha-subunit increase more than 7-fold. This proteolysis may occur from activation of the calcium-dependent neutral protease calpain I. The immunopeptide maps produced by alpha-spectrin degradation, selective loss of spectrin and MAP2, and decrease in calpain I levels are all consistent with calpain I activation accompanied by autoproteolysis. We propose that calcium influx and calpain I activation provide a mechanism by which neuronal activity regulates the degradation of specific neuronal structural proteins and may thereby modify neuronal morphology.     

Mechanism of arsenite-mediated decreases in CYP3A23 in rat hepatocytes

In primary cultures of rat hepatocytes, exposure to arsenite causes a major decrease in dexamethasone (DEX)-mediated induction of CYP3A23 hemoprotein, with a minor decrease in CYP3A23 mRNA. Here we show that addition of heme did not prevent the arsenite-mediated decreases in CYP3A23 protein, and arsenite did not decrease intracellular glutathione levels, indicating that heme and glutathione were not limiting for formation of holoCYP3A23. We also investigated whether arsenite decreases CYP3A23 protein by increasing CYP3A23 degradation by the calpain pathway. The calpain inhibitor, calpeptin, caused greater than a 90% inhibition of calpain-mediated proteolysis, but had no effect on DEX-mediated induction of CYP3A23 protein following 24h treatments. However, calpeptin enhanced the effect of arsenite to decrease induction of CYP3A23 protein. In addition, in short-term studies, calpeptin appeared to be a suicidal inhibitor of CYP3A-catalyzed enzyme activity. Our findings suggest that CYP3A23 protein is not degraded by calpain-mediated proteolysis, even in the presence of arsenite.     

Calpain-mediated proteolytic cleavage of the neuronal glycine transporter, GlyT2

The glycine transporter 2 (GlyT2) belongs to the family of Na+/CL--dependent plasma membrane transporters and is localized on the presynaptic terminals of glycinergic neurons. GlyT2 differs from other family members by its extended N-terminal cytoplasmic region. We report that activation of a Ca2+-dependent protease, most likely calpain, in spinal cord synaptosomes or cultured spinal cord neurons, results in partial proteolysis of GlyT2. Regions sensitive to calpain cleavage in vivo are located in the N-terminal and, to a lesser extent, C-terminal regions of the transporter protein. Incubation of a GlyT2 N-terminal fusion protein with spinal cord extract in the presence of calcium followed by protein sequence analysis localized the major N-terminal cleavage site after methionine 156, with a second cleavage site being situated after glycine 164. Interestingly, the size of the N-terminally truncated GlyT2 protein (70 kDa) is similar to that of most other transporter family members, and truncated GlyT2 displayed full transport activity upon expression in HEK293 cells. Our data suggest that Ca2+-triggered proteolysis may contribute to the regulation of GlyT2 trafficking and/or function in the neuronal plasma membrane.     

The endoplasmic reticulum chaperone glycoprotein GRP94 with Ca(2+)-binding and antiapoptotic properties is a novel proteolytic target of calpain during etoposide-induced apoptosis.

GRP94 is a 94-kDa chaperone glycoprotein with Ca(2+)-binding properties. We report here that during apoptosis induced by the topoisomerase II inhibitor etoposide, a fraction of GRP94 associated with the endoplasmic reticulum membrane undergoes specific proteolytic cleavage, coinciding with the activation of the caspase CPP32 and initiation of DNA fragmentation. In vivo, inhibitors of caspases able to block etoposide-induced apoptosis can only partially protect GRP94 from proteolytic cleavage, whereas complete inhibition is observed with calpain inhibitor I but not with the proteasome inhibitor. In vitro, GRP94 is not a substrate for CPP32; rather, it can be completely cleaved by calpain, a Ca(2+)-regulated protease. The cleavage of GRP94 by calpain is Ca(2+)-dependent and generates a discrete polypeptide of 80 kDa. In contrast, calpain has no effect on other stress proteins such as GRP78 or HSP70. Further, immunohistochemical staining reveals specific co-localization of GRP94 with calpain in the perinuclear region following etoposide treatment. We further showed that reduction of GRP94 by antisense decreased cell viability in etoposide-treated Jurkat cells. Our studies provide new evidence that the cytoprotective GRP94, as in the case of the antiapoptotic protein Bcl-2, can be targets of proteolytic cleavage themselves during the apoptotic process.