Calcium-activated Protease Calpain Research Articles

Disturbances in Calcium Homeostasis Promotes Skeletal Muscle Atrophy: Lessons From Ventilator-Induced Diaphragm Wasting.

Mechanical ventilation (MV) is often a life-saving intervention for patients in respiratory failure. Unfortunately, a common and undesired consequence of prolonged MV is the development of diaphragmatic atrophy and contractile dysfunction. This MV-induced diaphragmatic weakness is commonly labeled “ventilator-induced diaphragm dysfunction” (VIDD). VIDD is an important clinical problem because diaphragmatic weakness is a major risk factor for the failure to wean patients from MV; this inability to remove patients from ventilator support results in prolonged hospitalization and increased morbidity and mortality. Although several processes contribute to the development of VIDD, it is clear that oxidative stress leading to the rapid activation of proteases is a primary contributor. While all major proteolytic systems likely contribute to VIDD, emerging evidence reveals that activation of the calcium-activated protease calpain plays a required role. This review highlights the signaling pathways leading to VIDD with a focus on the cellular events that promote increased cytosolic calcium levels and the subsequent activation of calpain within diaphragm muscle fibers. In particular, we discuss the emerging evidence that increased mitochondrial production of reactive oxygen species promotes oxidation of the ryanodine receptor/calcium release channel, resulting in calcium release from the sarcoplasmic reticulum, accelerated proteolysis, and VIDD. We conclude with a discussion of important and unanswered questions associated with disturbances in calcium homeostasis in diaphragm muscle fibers during prolonged MV.

The calcium-activated protease calpain regulates netrin-1 receptor deleted in colorectal cancer-induced axon outgrowth in cortical neurons.

During development, neurons extend axons toward their appropriate synaptic targets to establish functional neuronal connections. The growth cone, a highly motile structure at the tip of the axon, is capable of recognizing extracellular guidance cues and translating them into directed axon outgrowth through modulation of the actin cytoskeleton. Netrin-1 mediates its attractive function through the receptor deleted in colorectal cancer (DCC) to promote axon outgrowth and guidance. The calcium-activated protease calpain is involved in the cleavage of cytoskeletal proteins, which plays an important role during adhesion turnover and cell migration. However, its function during neuronal development is less understood. Here we demonstrate that netrin-1 activated calpain in embryonic rat cortical neurons in an extracellular-regulated kinase 1/2-dependent manner. In addition, we found that netrin-1 stimulation led to an increase in calpain-1 localization in the axon, whereas its endogenous inhibitor calpastatin was decreased in the growth cones of cortical neurons by indirect immunofluorescence. Interestingly, calpain-1 was able to cleave DCC in vitro. Furthermore, netrin-1 induced the cleavage of the cytoskeletal proteins spectrin and focal adhesion kinase concomitantly with the intracellular domain of DCC in a calpain-dependent manner in embryonic rat cortical neurons. Cortical neurons over-expressing calpastatin or calpain-depleted neurons displayed increased basal axon length and were unresponsive to netrin-1 stimulation. Altogether, we propose a novel model whereby netrin-1/DCC-mediated axon outgrowth is modulated by calpain-mediated proteolysis of DCC and cytoskeletal targets in embryonic cortical neurons. Open Science: This manuscript was awarded with the Open Materials Badge For more information see: https://cos.io/our-services/open-science-badges/.

Abstract 824: Thromboxane/Prostanoid Receptor Activation Increases Calpain-Mediated Proteolysis and Alters Calcium Handling and Fibrosis Following Right Ventricular Pressure Overload

In pulmonary arterial hypertension (PAH), the right ventricle undergoes remodeling and fibrosis as it struggles to adapt to the increased pressure overload. RV dysfunction and failure is the primary cause of death in PAH patients. The G protein-coupled thromboxane/prostanoid receptor (TPr) is expressed in vascular smooth muscle, myofibroblasts, and immune cells, and is upregulated in cardiomyocytes following PAH. Activation of the cardiomyocyte TPr increases intracellular calcium via G αq /IP 3 ; activation of the receptor in other cells leads to fibrosis and vasoconstriction. The TPr is activated by isoprostane as well as thromboxane, which suggests that the receptor could contribute to deleterious remodeling during cardiac stress. Our previous studies demonstrate that TPr antagonism prevents RV fibrosis and dilatation in murine models of PAH, without affecting pressures. Because the TPr can signal through Gq, we hypothesized that its activation in PAH causes changes in cardiomyocyte calcium-handling proteins which contribute to remodeling and failure. In this study, we used pulmonary arterial banding (PAB) to induce fixed pressure overload of the RV. Mice were treated for 4 weeks past PAB with normal drinking water or water containing 25 mg/kg/day of the TPr antagonist ifetroban, and either underwent pressure-volume catheterization and whole RV evaluation, or cardiomyocytes were isolated for calcium handling and protein. PAB caused an increase in cardiomyocyte resting (end-diastolic) intracellular calcium, which was ameliorated in mice given TPr antagonist. The increased intracellular calcium following PAB was associated with increased activity of the calcium-activated protease calpain, also blocked with TPr antagonism. There was no decrease in caffeine-mediated release of calcium from the sarcoplasmic reticulum (SR) at 4 weeks past PAB, and phosphorylation of phospholamban was increased, suggesting compensation to drive calcium into the SR. Our findings suggest that TPr activation produces alterations in RV calcium handling, signaling, and calpain activity that contribute to deleterious remodeling and early failure in pressure overload. Therapeutic TPr antagonism may help preserve RV function in patients with PAH.

Methylglyoxal Disrupts Paranodal Axoglial Junctions via Calpain Activation.

Nodes of Ranvier and associated paranodal and juxtaparanodal domains along myelinated axons are essential for normal function of the peripheral and central nervous systems. Disruption of these domains as well as increases in the reactive carbonyl species methylglyoxal are implicated as a pathophysiology common to a wide variety of neurological diseases. Here, using an ex vivo nerve exposure model, we show that increasing methylglyoxal produces paranodal disruption, evidenced by disorganized immunostaining of axoglial cell-adhesion proteins, in both sciatic and optic nerves from wild-type mice. Consistent with previous studies showing that increase of methylglyoxal can alter intracellular calcium homeostasis, we found upregulated activity of the calcium-activated protease calpain in sciatic nerves after methylglyoxal exposure. Methylglyoxal exposure altered clusters of proteins that are known as calpain substrates: ezrin in Schwann cell microvilli at the perinodal area and zonula occludens 1 in Schwann cell autotypic junctions at paranodes. Finally, treatment with the calpain inhibitor calpeptin ameliorated methylglyoxal-evoked ezrin loss and paranodal disruption in both sciatic and optic nerves. Our findings strongly suggest that elevated methylglyoxal levels and subsequent calpain activation contribute to the disruption of specialized axoglial domains along myelinated nerve fibers in neurological diseases.

Activity-Dependent Cleavage of the K-Cl Cotransporter KCC2 Mediated by Calcium-Activated Protease Calpain

The K-Cl cotransporter KCC2 plays a crucial role in neuronal chloride regulation. In mature central neurons, KCC2 is responsible for the low intracellular Cl(-) concentration ([Cl(-)](i)) that forms the basis for hyperpolarizing GABA(A) receptor-mediated responses. Fast changes in KCC2 function and expression have been observed under various physiological and pathophysiological conditions. Here, we show that the application of protein synthesis inhibitors cycloheximide and emetine to acute rat hippocampal slices have no effect on total KCC2 protein level and K-Cl cotransporter function. Furthermore, blocking constitutive lysosomal degradation with leupeptin did not induce significant changes in KCC2 protein levels. These findings indicate a low basal turnover rate of the total KCC2 protein pool. In the presence of the glutamate receptor agonist NMDA, the total KCC2 protein level decreased to about 30% within 4 h, and this effect was blocked by calpeptin and MDL-28170, inhibitors of the calcium-activated protease calpain. Interictal-like activity induced by incubation of hippocampal slices in an Mg(2+)-free solution led to a fast reduction in KCC2-mediated Cl(-) transport efficacy in CA1 pyramidal neurons, which was paralleled by a decrease in both total and plasmalemmal KCC2 protein. These effects were blocked by the calpain inhibitor MDL-28170. Taken together, these findings show that calpain activation leads to cleavage of KCC2, thereby modulating GABAergic signaling.

Fibroblast growth factor‐2 exerts protective effects on cardiac mitochondria

Fibroblast growth factor‐2 (FGF‐2) is a potent cardioprotective agent, by a mechanism linked to connexin43 (Cx43) phosphorylation at protein kinase C (PKC) target serines such as S262. Here we have examined the effects of FGF‐2 on mitochondrial resistance to calcium‐ induced opening of the permeability transition pore (swelling), in relation to cardiac mitochondrial Cx43 levels and phosphorylation.Subsarcolemmal mitochondria isolated from FGF‐2‐treated hearts: (a) showed 1.5–2 fold increase in total Cx43, PKCε, TOM20 translocase, and a over 30‐fold increase in phospho‐S262‐Cx43, compared to control mitochondria obtained from untreated hearts; (b) were more resistant to calcium‐induced swelling and cytochrome C release; (c) were resistant to calcium‐induced mitochondrial Cx43 degradation. Calpeptin, a potent inhibitor of the calcium activated protease calpain, prevented Cx43 degradation in control mitochondria. Stimulation of control mitochondria with the phorbol‐ester PMA, or FGF‐2, increased both mitochondrial Cx43 phosphorylation at S262 and resistance to calcium‐induced swelling / cytochrome c release.Our data imply that FGF‐2 cardioprotection is mediated by increasing mitochondrial resistance to calcium damage, via Cx43 translocation to mitochondria and mitochondrial Cx43 phosphorylation.This work is supported by Heart and Stroke Foundation of Canada and St. Boniface Hospital Research Foundation.

Calpain 2 is required for the invasion of glioblastoma cells in the zebrafish brain microenvironment

Glioblastoma is an aggressive primary brain tumor with a 5-year survival rate of less than 5%. The ability of glioblastoma cells to invade surrounding brain tissue presents the primary challenge for the success of focal therapeutic approaches. We previously reported that the calcium-activated protease calpain 2 is critical for glioblastoma cell invasion in vitro. Here, we show that expression of calpain 2 is required for the dispersal of glioblastoma cells in a living brain microenvironment. Knockdown of calpain 2 resulted in a 2.9-fold decrease in the invasion of human glioblastoma cells in zebrafish brain. Control cells diffusely migrated up to 450 μm from the site of injection, whereas knockdown cells remained confined in clusters. The invasion study was repeated in organotypic mouse brain tissues, and calpain 2 knockdown cells demonstrated a 2.3-fold lower area of dispersal compared with control cells. In zebrafish brain, glioblastoma cells appeared to migrate in part along the blood vessels of the host. Furthermore, angiogenesis was detected in 27% of zebrafish injected with control cells, whereas only 12.5% of fish receiving knockdown cells showed the formation of new vessels, suggesting a role for calpain 2 in tumor cell angiogenesis. Consistent with the progression of glioblastoma in humans, transplanted tumor cells were not observed to metastasize outside the brain of zebrafish. This study demonstrates that calpain 2 expression is required for the dispersal of glioblastoma cells within the dynamic microenvironment of the brain, identifying zebrafish as a valuable orthotopic system for studying glioblastoma cell invasion.

Kindlin-3 Undergoes Endoproteolysis in Stored Platelets and After Platelet Stimulation

Abstract 1255Kindlins are a family of FERM domain proteins that are essential for inside-out integrin activation. In particular, kindlin-3 is required for the conversion of the major platelet integrin αIIbβ3 from its resting conformation to its active ligand binding conformation. Moreover, naturally-occurring kindlin-3 mutations result in the inherited disorder leukocyte adhesion deficiency III, one component of which is defective platelet function that mimics Glanzmann thrombasthenia. Despite the importance of kindlin-3 in initiating αIIbβ3 function in platelets, little is known about its regulation in resting platelets or its fate in activated platelets. To address these questions, we purified full-length kindlin-3 from outdated human platelets where it is present in substantial amounts and also developed a procedure to synthesize substantial amounts of recombinant kindlin-3 in SF9 cells. We found that in stored human platelets, kindlin-3 is cleaved into two fragments as a function of the time of storage. We also found that kindlin-3 is cleaved into identical fragments when fresh human platelets are stimulated with the thrombin receptor activating peptide (TRAP) for 5 minutes. To identify the site of kindlin-3 cleavage, as well as the responsible protease, we used a proteomics method in which an engineered peptide ligase, subtiligase, was used to selectively biotinylate the unblocked α amines of proteins obtained from platelet lysates (Mahrus et al, Cell 134:866–76, 2008). Biotinylated proteins were digested with trypsin and the resulting biotinylated peptide fragments were then captured using avidin agarose and identified using tandem mass spectrometry. Using this method, we identified the kindlin-3 peptide (G)SAPTDVLDSLTTIPELKDHL in lysates of stored platelets, thereby mapping the cleavage site to residues 335–336. We obtained identical results using proteins isolated from TRAP-stimulated platelets. Further, we were able to recapitulate these results in vitro using purified kindlin-3 and the calcium-activated protease calpain, implying that calpain is the responsible protease in vivo. Kindlin-3 is thought to initiate αIIbβ3 function by binding to the distal NITY motif in the β3 cytosolic tail in an interaction that also involves S752. Previously, we reported a model for αIIbβ3 regulation based on an NMR structure of the β3 cytosolic tail (Metcalf et al, PNAS 107:24775–83, 2010). In this structure, the NITY motif is located in a distal dynamic amphiphilic helix where the motif is transiently masked by interacting with the membrane. To validate this model, we have studied the interaction of both purified and recombinant kindlin-3 with the β3 tail using surface plasmon resonance (SPR). A peptide corresponding to β3 residues 719–762, encompassing the complete β3 tail, was immobilized on a CM5 chip and kindlin-3 was flowed over the chip surface. The resulting sensorgrams could then be fit to two binding events with dissociation constants of 2.2 nM and 2.8 μM. The biphasic behavior could have resulted from heterogeneity of the β3 tail on the chip surface or heterogeneity of the interaction between kindlin-3 and the carboxymethylated dextran. Similar SPR experiments measuring binding of the talin-1 FERM domain to the β3 tail also could be fit to two binding events with dissociation constants of 155 nM and 3.5 μM. Thus, under these experimental conditions, kindlin-3 binds approximately 70-fold more tightly to the β3 tail than does the talin-1 FERM domain. In summary, these studies demonstrate that kindlin-3 undergoes calpain-mediated endoproteolysis during platelet storage, an event that may contribute to the development of the platelet storage lesion. Kindlin-3 also undergoes an identical cleavage following platelet stimulation by agonists such as thrombin. Since high affinity binding of kindlin-3 to the β3 cytosolic tail is required for physiologic αIIbβ3 activation, it is possible that kindlin-3 cleavage attenuates αIIbβ3 activity. Disclosures:No relevant conflicts of interest to declare.

S-Nitrosylation activates Cdk5 and contributes to synaptic spine loss induced by β-amyloid peptide

The activity of Cdk5 and its regulatory subunit p35 is thought to be important in both normal brain function and neurodegenerative disease pathogenesis. Increased Cdk5 activity, via proteolytic cleavage of p35 to a p25 fragment by the calcium-activated protease calpain or by phosphorylation at Cdk5(Tyr15), can contribute to neurotoxicity. Nonetheless, our knowledge of regulation of Cdk5 activity in disease states is still emerging. Here we demonstrate that Cdk5 is activated by S-nitrosylation or reaction of nitric oxide (NO)-related species with the thiol groups of cysteine residues 83 and 157, to form SNO-Cdk5. We then show that S-nitrosylation of Cdk5 contributes to amyloid-β (Aβ) peptide-induced dendritic spine loss. Furthermore, we observed significant levels of SNO-Cdk5 in postmortem Alzheimer's disease (AD) but not in normal human brains. These findings suggest that S-nitrosylation of Cdk5 is an aberrant regulatory mechanism of enzyme activity that may contribute to the pathogenesis of AD.

Calpain 2 is required for glioblastoma cell invasion: regulation of matrix metalloproteinase 2.

Invasion of glioblastoma cells significantly reduces the effectiveness of current treatments, highlighting the importance of understanding dispersal mechanisms and characteristics of the invasive population. Induction of calcium fluxes into glioblastoma cells by autocrine glutamate is critical for invasion. However, the target(s) by which calcium acts to stimulate the dispersal of glioblastoma cells is not clear. In this study, we tested the hypothesis that the calcium-activated protease calpain 2 is required for glioblastoma cell invasion. Knockdown of calpain 2 expression using shRNA or chemical inhibition of calpain activity reduced glioblastoma cell invasion by 90%. Interestingly, decreased expression of calpain 2 did not influence morphology or migration, suggesting regulation of invasion specific mechanisms. Consistent with this idea, 39% less extracellular MMP2 was measured from knockdown cells identifying one mechanism by which calpain 2 mediates glioblastoma cell invasion. This is the first report demonstrating that calpain 2 is required for glioblastoma cell invasion.

Integrin activation by Fam38A uses a novel mechanism of R-Ras targeting to the endoplasmic reticulum

The integrin family of heterodimeric cell-surface receptors are fundamental in cell-cell and cell-matrix adhesion. Changes to either integrin-ligand affinity or integrin gene expression are central to a variety of disease processes, including inflammation, cardiovascular disease and cancer. In screening for novel activators of integrin-ligand affinity we identified the previously uncharacterised multi-transmembrane domain protein Fam38A, located at the endoplasmic reticulum (ER). siRNA knockdown of Fam38A in epithelial cells inactivates endogenous beta1 integrin, reducing cell adhesion. Fam38A mediates integrin activation by recruiting the small GTPase R-Ras to the ER, which activates the calcium-activated protease calpain by increasing Ca(2+) release from cytoplasmic stores. Fam38A-induced integrin activation is blocked by inhibition of either R-Ras or calpain activity, or by siRNA knockdown of talin, a well-described calpain substrate. This highlights a novel mechanism for integrin activation by Fam38A, utilising calpain and R-Ras signalling from the ER. These data represent the first description of a novel spatial regulator of R-Ras, of an alternative integrin activation-suppression pathway based on direct relocalisation of R-Ras to the ER, and of a mechanism linking R-Ras and calpain signalling from the ER with modulation of integrin-ligand affinity.

Commitment of 1-Methyl-4-phenylpyrinidinium Ion-induced Neuronal Cell Death by Proteasome-mediated Degradation of p35 Cyclin-dependent Kinase 5 Activator

The dysfunction of proteasomes and mitochondria has been implicated in the pathogenesis of Parkinson disease. However, the mechanism by which this dysfunction causes neuronal cell death is unknown. We studied the role of cyclin-dependent kinase 5 (Cdk5)-p35 in the neuronal cell death induced by 1-methyl-4-phenylpyrinidinium ion (MPP+), which has been used as an in vitro model of Parkinson disease. When cultured neurons were treated with 100 microM MPP+, p35 was degraded by proteasomes at 3 h, much earlier than the neurons underwent cell death at 12-24 h. The degradation of p35 was accompanied by the down-regulation of Cdk5 activity. We looked for the primary target of MPP+ that triggered the proteasome-mediated degradation of p35. MPP+ treatment for 3 h induced the fragmentation of the mitochondria, reduced complex I activity of the respiratory chain without affecting ATP levels, and impaired the mitochondrial import system. The dysfunction of the mitochondrial import system is suggested to up-regulate proteasome activity, leading to the ubiquitin-independent degradation of p35. The overexpression of p35 attenuated MPP+-induced neuronal cell death. In contrast, depletion of p35 with short hairpin RNA not only induced cell death but also sensitized to MPP+ treatment. These results indicate that a brief MPP+ treatment triggers the delayed neuronal cell death by the down-regulation of Cdk5 activity via mitochondrial dysfunction-induced up-regulation of proteasome activity. We propose a role for Cdk5-p35 as a survival factor in countering MPP+-induced neuronal cell death.

Calpain activation impairs neuromuscular transmission in a mouse model of the slow-channel myasthenic syndrome.

The slow-channel myasthenic syndrome (SCS) is a hereditary disorder of the acetylcholine receptor (AChR) of the neuromuscular junction (NMJ) that leads to prolonged AChR channel opening, Ca(2+) overload, and degeneration of the NMJ. We used an SCS transgenic mouse model to investigate the role of the calcium-activated protease calpain in the pathogenesis of synaptic dysfunction in SCS. Cleavage of a fluorogenic calpain substrate was increased at the NMJ of dissociated muscle fibers. Inhibition of calpain using a calpastatin (CS) transgene improved strength and neuromuscular transmission. CS caused a 2-fold increase in the frequency of miniature endplate currents (MEPCs) and an increase in NMJ size, but MEPC amplitudes remained reduced. Persistent degeneration of the NMJ was associated with localized activation of the non-calpain protease caspase-3. This study suggests that calpain may act presynaptically to impair NMJ function in SCS but further reveals a role for other cysteine proteases whose inhibition may be of additional therapeutic benefit in SCS and other excitotoxic disorders.

Increased calpain correlates with Th1 cytokine profile in PBMCs from MS patients

Multiple sclerosis (MS) is a devastating autoimmune demyelinating disease of the central nervous system (CNS). This study investigated whether expression and activity of the calcium-activated protease calpain correlated with Th1/Th2 dysregulation in MS patients during states of relapse and remission. Calpain expression and activity were significantly increased in peripheral blood mononuclear cells (PBMCs) from MS patients, compared to controls, with the highest expression and activity noted during relapse. Th1 cytokines were highest and Th2 cytokines were lowest in MS patients during relapse. Treatment with calpain inhibitor, calpeptin, decreased Th1 cytokines in PBMCs from MS patients. Calpain inhibitor also reduced degradation of myelin basic protein (MBP) by inhibiting the calpain secreted from MBP-specific T cells. Taken together, these results suggested calpain involvement in Th1/Th2 dysregulation in MS patients.

Cell signaling pathways in the mechanisms of neuroprotection afforded by bergamot essential oil against NMDA‐induced cell death in vitro

The effects of bergamot essential oil (BEO; Citrus bergamia, Risso) on excitotoxic neuronal damage was investigated in vitro. The study was performed in human SH-SY5Y neuroblastoma cells exposed to N-methyl-D-aspartate (NMDA). Cell viability was measured by dye exclusion. Reactive oxygen species (ROS) and caspase-3 activity were measured fluorimetrically. Calpain I activity and the activation (phosphorylation) of Akt and glycogen synthase kinase-3beta (GSK-3beta) were assayed by Western blotting. NMDA induced concentration-dependent, receptor-mediated, death of SH-SY5Y cells, ranging from 11 to 25% (0.25-5 mM). Cell death induced by 1 mM NMDA (21%) was preceded by a significant accumulation of intracellular ROS and by a rapid activation of the calcium-activated protease calpain I. In addition, NMDA caused a rapid deactivation of Akt kinase and this preceded the detrimental activation of the downstream kinase, GSK-3beta. BEO (0.0005-0.01%) concentration dependently reduced death of SH-SY5Y cells caused by 1 mM NMDA. In addition to preventing ROS accumulation and activation of calpain, BEO (0.01%) counteracted the deactivation of Akt and the consequent activation of GSK-3beta, induced by NMDA. Results obtained by using specific fractions of BEO, suggested that monoterpene hydrocarbons were responsible for neuroprotection afforded by BEO against NMDA-induced cell death. Our data demonstrate that BEO reduces neuronal damage caused in vitro by excitotoxic stimuli and that this neuroprotection was associated with prevention of injury-induced engagement of critical death pathways.

Tau phosphorylation and proteolysis: Insights and perspectives

In 1992 little was known about the specific protein kinases that phosphorylate tau and the proteases that regulate tau turnover. Although we had already demonstrated that tau was a substrate of the calcium-activated protease calpain (Johnson et al. (1989), Biochem Biophys Res Commun 163, 1505-1511), our publication entitled, "Phosphorylation by cAMP-dependent protein kinase inhibits the degradation of tau by calpain" (Litersky and Johnson (1992), J Biol Chem 267, 1563-1568) was the first demonstration that phosphorylation by a specific kinase could inhibit the proteolysis of tau by calpain. At the time these findings suggested that the abnormal phosphorylation of tau in Alzheimer's disease brain could result in impaired tau turnover and thus result in an abnormal accumulation of the protein that could contribute to the formation of pathological lesions. Since this initial finding, much has been learned about the proteolysis of tau, not only by calpain, but by other proteases as well. However, much remains unknown about how phosphorylation regulates tau turnover in vivo and the specific proteases involved. In this article we give a brief history of our initial findings and then discuss subsequent studies from our laboratory, as well as others, on tau proteolysis and modulation by phosphorylation and how these findings contribute to our understanding of the posttranslational processing of tau in Alzheimer's disease.

Low activity by the calpain system in primate lenses causes resistance to calcium-induced proteolysis

The human genome contains 14 genes for 80 kDa catalytic subunit of the calcium-activated protease calpain (EC 34.22.17), yet no calpain-like cleavage sites have been detected on human lens crystallins in vivo. The purpose of the present study was to provide a comprehensive study of calpain activation in human and macaque lenses developing experimental cataract due to lens culture in ionophore A23187. Zymography was used to measure calpain activity; SDS–PAGE and immunoblotting were used to detect hydrolysis of potential lens protein substrates. Quantitative PCR was used to measure transcripts for calpains and the endogenous inhibitor calpastatin. We found that the lack of appreciable calpain-induced proteolysis in primate lenses is most likely due to relatively low levels of endogenous calpain activity compared to the high levels of endogenous calpain inhibitor, calpastatin.

Caspase-dependent and -independent pathways for cadmium-induced apoptosis in cultured kidney proximal tubule cells

The nephrotoxic metal cadmium at micromolar concentrations induces apoptosis of rat kidney proximal tubule (PT) cells within 3-6 h of exposure. The underlying cell death pathways remain poorly defined. Using Hoechst 33342/ethidium bromide nuclear staining and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) cell death assays, 10-50 microM cadmium induced apoptosis of immortalized rat kidney cells derived from the S1-segment of PT at 6 and 24 h, but necrosis at 24 h only. Cadmium (10-50 microM) also caused mitochondrial cytochrome c (cyt. c)- and apoptosis-inducing factor release at 24 h, but not at 6 h, as measured by immunofluorescence imaging and immunoblotting. Caspases-9 and -3 were activated only by 10 microM cadmium for 24 h, and accordingly apoptosis was significantly reduced by the respective inhibitors (z-LEHD-fmk, z-DEVD-fmk; 10 microg/ml) at 24 h, but not at 6 h, without affecting necrosis. At 6 h, 10 microM cadmium increased the activity of the calcium-activated protease calpain, but not at 24 h, and calpain inhibitors (ALLN, PD 150606; 10-30 microM) blocked apoptosis by 10 microM cadmium at 3-6 h. However, PD-150606 also attenuated caspase-3 activity and apoptosis at 24 h, suggesting calpain-dependent caspase activation. Thus cadmium-induced apoptosis of PT cells involves a complex and sensitive interplay of signaling cascades involving mitochondrial proapoptotic factors, calpains and caspases, whose activation is also determined by cadmium concentration and the duration of cadmium exposure.

Exercise training and calpain inhibition prevent the IR‐induced degradation of myocardial calcium handling proteins and contractile dysfunction

Increases in reactive oxygen species (ROS), cytosolic Ca2+and activity of the calcium-activated protease calpain contribute to ischemia-reperfusion (IR) injury. Additionally, there is evidence to suggest that ROS may contribute to Ca2+ overload and calpain activation by damaging critical Ca2+ handling proteins. We have previously shown that exercise protects against IR-induced oxidative stress and calpain activation; however, the mechanisms of this protection are unknown. Therefore, we tested the hypothesis that exercise training attenuates IR-induced calpain activation through the preservation of critical Ca2+handling proteins. In sedentary animals, IR (in vitro) resulted in a decrease in both sarcoplasmic/endoplasmic reticulum Ca2+ATPase (SERCA2a) and L-type Ca2+channel (LTCC) protein, as well as impaired myocardial function (systolic pressure, +dp/dt and –dp/dt). Both exercise and calpain inhibition attenuated IR-induced calpain activation, the degradation of SERCA2a and LTCC and maintained myocardial function following IR. In addition, exercise training was associated with an increase in basal levels of LTCC protein in the heart. These findings suggest that exercise may regulate calpain activation, in part, through the maintenance of Ca2+handling proteins. Further, these data support the notion that calpain may regulate itself in a feed-forward mechanism, cleaving Ca2+ handling proteins, exacerbating IR-induced Ca2+overload, leading to further calpain activation and myocardial injury. Supported by NIH HL072789 (SKP), AHA 0415135B (JPF).

Protein fucosylation regulates synapsin Ia/Ib expression and neuronal morphology in primary hippocampal neurons

Although fucose-alpha(1-2)-galactose [Fucalpha(1-2)Gal] carbohydrates have been implicated in cognitive processes such as long-term memory, the molecular mechanisms by which these sugars influence neuronal communication are not well understood. Here, we present molecular insights into the functions of Fucalpha(1-2)Gal sugars, demonstrating that they play a role in the regulation of synaptic proteins and neuronal morphology. We show that synapsins Ia and Ib, synapse-specific proteins involved in neurotransmitter release and synaptogenesis, are the major Fucalpha(1-2)Gal glycoproteins in mature cultured neurons and the adult rat hippocampus. Fucosylation has profound effects on the expression and turnover of synapsin in cells and protects synapsin from degradation by the calcium-activated protease calpain. Our studies suggest that defucosylation of synapsin has critical consequences for neuronal growth and morphology, leading to stunted neurite outgrowth and delayed synapse formation. We also demonstrate that Fucalpha(1-2)Gal carbohydrates are not limited to synapsin but are found on additional glycoproteins involved in modulating neuronal architecture. Together, our studies identify important roles for Fucalpha(1-2)Gal sugars in the regulation of neuronal proteins and morphological changes that may underlie synaptic plasticity.