Calpain Cleavage Research Articles

Imiquimod‐induced apoptosis of melanoma cells is mediated by ER stress‐dependent Noxa induction and enhanced by NF‐κB inhibition

Melanoma is characterized by dysregulated intracellular signalling pathways including an impairment of the cell death machinery, ultimately resulting in melanoma resistance, survival and progression. This explains the tumour's extraordinary resistance to the standard treatment. Imiquimod is a topical immune response modifier (imidazoquinoline) with both antiviral and antitumour activities. The mechanism by which imiquimod triggers the apoptosis of melanoma cells has now been carefully elucidated. Imiquimod-induced apoptosis is associated with the activation of apoptosis signalling regulating kinase1/c-Jun-N-terminal kinase/p38 pathways and the induction of endoplasmic stress characterized by the activation of the protein kinase RNA-like endoplasmic reticulum kinase signalling pathway, increase in intracellular Ca(2+) release, degradation of calpain and subsequent cleavage of caspase-4. Moreover, imiquimod triggers the activation of NF-κB and the expression of the inhibitor of apoptosis proteins (IAPs) such as, X-linked IAP (XIAP) together with the accumulation of reactive oxygen species (ROS). Also, imiquimod triggers mitochondrial dysregulation characterized by the loss of mitochondrial membrane potential (Δψm), the increase in cytochrome c release, and cleavage of caspase-9, caspase-3 and poly(ADP-ribose) polymerase (PARP). Inhibitors of specific pathways, permit the elucidation of possible mechanisms of imiquimod-induced apoptosis. They demonstrate that inhibition of NF-kB by the inhibitor of nuclear factor kappa-B kinase (IKK) inhibitor Bay 11-782 or knockdown of XIAP induces melanoma apoptosis in cells exposed to imiquimod. These findings support the use of either IKK inhibitors or IAP antagonists as adjuvant therapies to improve the effectiveness topical imiquimod in the treatment of melanoma.

STIM1 is cleaved by calpain

Store-operated calcium entry (SOCE) is a pathway that moves Ca2+ across the plasma membrane and is mediated by two major proteins, STIM1 and Orai1. Here, we discovered that the cytoplasmic domain of STIM1 is a target for calpains, a family of Ca2+-activated proteases. We found that calpain cleavage of STIM1 serves to control its cellular abundance and was noticeably increased under conditions of cellular stress and apoptosis. Dysregulation of STIM1 levels has been reported to have human disease consequences and our results suggest a mechanism for controlling STIM1 abundance.

Abstract IA06: Recycling Myc to survive stress

Abstract Missexpression of Myc family members has been linked to numerous abnormalities ranging from developmental defects to cancer. Indeed there is considerable evidence that Myc expression is controlled at every level of gene regulation, including protein degradation. Recently we found that Myc is targeted by calcium-dependent cytosolic proteases from the calpain family. Unlike proteasomal degradation resulting in complete protein destruction, calpain mediated proteolysis leads to partial cleavage of substrates. While regulated calpain activity is essential for embryonic development and terminal differentiation, aberrant calpain levels and activity have been correlated with tumor development and metastasis. We found that upon calpain cleavage, the C-terminus of Myc is degraded, while an N-terminal segment of about 300 aminoacids is preserved and gives rise to Myc-nick. The removal of the C-terminus of Myc, which is essential for nuclear translocation and to binding to DNA and to Max, renders Myc-nick transcriptionally inactive and cytoplasmically localized. The conversion of Myc into Myc-nick is significantly enhanced in cancer cells exposed to stress conditions such as hypoxia and nutrient deprivation. Importantly, we found that Myc-nick expression increases the survival of colon cancer cells following nutrient deprivation, hypoxia, and treatment chemotherapeutic agents such as etoposide, cisplatin and imatinib. Myc-nick expressing cells display an increase in autophagy and treatment with inhibitors of autophagy prevents cell survival. Therefore, we propose that activation of autophagy is the major mechanism by which Myc-nick promotes cell survival under adverse conditions and drug treatment. The ability of Myc-nick to augment autophagy and cell survival requires its binding to acetyltransferases which correlates with acetylation of cytoplasmic proteins. We find that Myc-nick augments acetylation of ATG3 and alpha-tubulin, which are required in the acetylated form for the migration of autophagosomes and their subsequent fusion with lysosomes. Overexpression of Myc-nick not only promotes the survival of colon cancer cells, but also increases their motility. Importantly, Myc-nick induces an increase in the formation of filopodia and the expression of fascin, which is a robust driver of invasiveness in solid tumors. Myc-nick expressing cells, while highly motile, do not appear to undergo epithelial to mesenchymal transition (EMT). These cells migrate while maintaining their epithelial shape by making use of very distinct filopodia. This is likely to be related to the recently described phenomenon called “collective invasion”, where cancer cells become invasive in the absence of EMT. Our results indicate that Myc-nick-induced survival and motility play important roles in promoting the progression of Myc-dependant tumors. Citation Format: Maralice Conacci-Sorrell, Celine Ngouenet, Sarah Anderson, Robert Eisenman. Recycling Myc to survive stress. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr IA06.

Molecular Determinants of Calpain-dependent Cleavage of Junctophilin-2 Protein in Cardiomyocytes

Junctophilin-2 (JP2), a membrane-binding protein that provides a structural bridge between the plasmalemma and sarcoplasmic reticulum, is essential for precise Ca(2+)-induced Ca(2+) release during excitation-contraction coupling in cardiomyocytes. In animal and human failing hearts, expression of JP2 is decreased markedly, but the molecular mechanisms underlying JP2 down-regulation remain incompletely defined. In mouse hearts, ischemia/reperfusion injury resulted in acute JP2 down-regulation, which was attenuated by pretreatment with the calpain inhibitor MDL-28170 or by transgenic overexpression of calpastatin, an endogenous calpain inhibitor. Using a combination of computational analysis to predict calpain cleavage sites and in vitro calpain proteolysis reactions, we identified four putative calpain cleavage sites within JP2 with three N-terminal and one C-terminal cleavage sites. Mutagenesis defined the C-terminal region of JP2 as the predominant calpain cleavage site. Exogenous expression of putative JP2 cleavage fragments was not sufficient to rescue Ca(2+) handling in JP2-deficient cardiomyocytes, indicating that cleaved JP2 is non-functional for normal Ca(2+)-induced Ca(2+) release. These data provide new molecular insights into the posttranslational regulatory mechanisms of JP2 in cardiac diseases.

191 Interleukin-1 Alpha is Directly Activated During Haemostasis by Thrombin Cleavage

<h3></h3> The clinical consequences of atherosclerosis are closely associated with thrombus formation following plaque rupture. Thrombin primarily cleaves fibrinogen into fibrin which forms a thrombus at sites of injury, but can also induce inflammation indirectly via PAR receptors. Chronic inflammation is a key feature of atherosclerosis that promotes both plaque growth and instability, and links between inflammation and haemostasis are widely proposed. We now present data showing that the powerful proatherogenic cytokine IL-1α is directly activated by thrombin. We find that thrombin treatment of pro-IL-1α produces a truncated form, indicating cleavage of the protein in a similar manner to its canonical calpain cleavage. Mutation of the thrombin consensus in IL-1α prevented cleavage, confirming the site. Thrombin cleavage of pro-IL-1α induced similar cytokine activity as calpain, while recombinant proteins equivalent to the thrombin- and calpain-cleaved forms had identical activity. Importantly, cleavage and activation of pro-IL-1α also occurred during coagulation of platelet-rich plasma and whole blood, suggesting physiological relevance. Again, thrombin-site mutant pro-IL-1α was uncleaved and lacked activity, proving the specific action of thrombin. Murine pro-IL-1α also requires processing for cytokine activity and again thrombin cleavage induced full activity, supporting an evolutionary conserved mechanism. Importantly, specific mutation of the site in mouse pro-IL-1α prevents thrombin cleavage, which has enabled us to generate a novel knock-in mouse to study the <i>in vivo</i> relevance of these findings. We show that thrombin cleaves and activates pro-IL-1α at a novel site that is conserved across species. This suggests that thrombin-mediated activation of IL-1α during thrombosis may directly promote inflammation. We propose that this represents an unappreciated fundamental link between the key enzyme of thrombosis and the principal cytokine within immunity. These findings have broad implications for normal physiology and the pathogenesis of diseases such as atherothrombosis.

Discovery of novel isoforms of huntingtin reveals a new hominid-specific exon.

Huntington’s disease (HD) is a devastating neurological disorder that is caused by an expansion of the poly-Q tract in exon 1 of the Huntingtin gene (HTT). HTT is an evolutionarily conserved and ubiquitously expressed protein that has been linked to a variety of functions including transcriptional regulation, mitochondrial function, and vesicle transport. This large protein has numerous caspase and calpain cleavage sites and can be decorated with several post-translational modifications such as phosphorylations, acetylations, sumoylations, and palmitoylations. However, the exact function of HTT and the role played by its modifications in the cell are still not well understood. Scrutiny of HTT function has been focused on a single, full length mRNA. In this study, we report the discovery of 5 novel HTT mRNA splice isoforms that are expressed in normal and HTT-expanded human embryonic stem cell (hESC) lines as well as in cortical neurons differentiated from hESCs. Interestingly, none of the novel isoforms generates a truncated protein. Instead, 4 of the 5 new isoforms specifically eliminate domains and modifications to generate smaller HTT proteins. The fifth novel isoform incorporates a previously unreported additional exon, dubbed 41b, which is hominid-specific and introduces a potential phosphorylation site in the protein. The discovery of this hominid-specific isoform may shed light on human-specific pathogenic mechanisms of HTT, which could not be investigated with current mouse models of the disease.

Variant Specific Cleavage of the Na+‐Ca2+ Exchanger NCX3 During Excitotoxicity

Recent studies highlighted the crucial role of the Na+‐Ca2+ exchanger isoform 3, NCX3, during stress exposure, allowing for a significant Ca2+ entry in neuronal cells. Consequently, the hyperfunctionality of NCX3 triggered via its cleavage by the calpain protease, is considered to play a neuroprotective effect in numerous pathologies such as Alzheimer's disease and ischemia.NCX3 family contains three variants hypothesized to carry diverse capacity of exchange.We aimed in dissecting the molecular mechanisms responsible for NCX3 cleavage during excitotoxicity.The capacity of exchange of each variant expressed in HEK293T cells was measured by recording single cell [Ca2+]i in response to Na+ removal. Additionally, the influence of calpain on NCX3 was investigated using overexpression of the endogeneous inhibitor, Calpastatin, and silencing of the calpain family.We observed that the variants containing the exon B, NCX3‐B and NCX3‐BC, are predominantly expressed in the brain. Measurements of NCX3 revealed a significant decrease in the capacity of exchange of NCX3‐AC and NCX3‐BC during overexpression of Calpastatin. A similar effect was observed by silencing calpain‐1. Site‐directed mutagenesis was performed on a hypothesized cleavage site for calpain located in the exon C of NCX3. The mutation of L600 and L601, at the cleavage site, caused a loss of hyperfunctionality, as observed during the silencing of calpain‐1.Calpain activity triggers an increase in the capacity of exchange of NCX3. However only the variants containing the exon C, NCX3‐AC and NCX3‐BC, are sensitive to calpain cleavage. The predominant expression of NCX3‐BC observed in the brain together with the identification of the cleavage site in NCX3 allows for potential drug design targeting these specific residues.

A Truncated Fragment of Src Protein Kinase Generated by Calpain‐mediated Cleavage is a Mediator of Neuronal Death in Excitotoxicity.

Excitotoxicity resulting from overstimulation of glutamate receptors is a major cause of neuronal death in cerebral ischemic stroke. The overstimulated ionotropic glutamate receptors exert their neurotoxic effects in part by overactivation of calpains, which induce neuronal death by catalyzing limited proteolysis of specific cellular proteins. Here, we report that in cultured cortical neurons and in vivo in a rat model of focal ischemic stroke, the tyrosine kinase Src is cleaved by calpains at a site in the N‐terminal unique domain. This generates a truncated Src fragment of ~52 kDa, which we localized predominantly to the cytosol. A cell membrane‐permeable fusion peptide derived from the unique domain of Src prevents calpain from cleaving Src in neurons and protects against excitotoxic neuronal death. To explore the role of the truncated Src fragment in neuronal death, we expressed a recombinant truncated Src fragment in cultured neurons and examined how it affects neuronal survival. Expression of this fragment, which lacks the myristoylation motif and unique domain, was sufficient to induce neuronal death. Furthermore, inactivation of the prosurvival kinase Akt is a key step in its neurotoxic signaling pathway. Because Src maintains neuronal survival, our results implicate calpain cleavage as a molecular switch converting Src from a promoter of cell survival to a mediator of neuronal death in excitotoxicity. Besides unveiling a new pathological action of Src, our discovery of the neurotoxic action of the truncated Src fragment suggests new therapeutic strategies with the potential to minimize brain damage in ischemic stroke

Calpains mediate the proteolytic modification of human cytomegalovirus UL112-113 proteins.

The human cytomegalovirus (HCMV) UL112-113 gene is implicated in lytic viral replication. The UL112-113 proteins p34, p43, p50 and p84 are expressed via alternative splicing. However, the mechanism for the generation of three additional virus-associated proteins (p20, p26 and p28), which share the UL112 reading frame, remains unknown. Bioinformatic analyses indicated that p34, p43, p50 and p84 contain potential PEST-like degradation motifs. In this study, inhibitors of calpains, lysosomes and proteasomes reduced p20, p26 and p28 levels in virus-infected cells, suggesting the involvement of proteolytic modification. Moreover, maitotoxin, which increases intracellular calcium levels and activates calpain activity, induced the intracellular proteolysis of p34 into p20, p26 and p28 and the cleavage of p43, p50 and p84 into p38 and a novel protein, p34c. Proteolytic assays further indicated that p34, p43, p50 and p84 were substrates of calpain-1 and calpain-2 and that they generated proteolytic products that corresponded to those detected during the HCMV infectious period. Furthermore, substitution mutations in the putative calpain cleavage sites of p34 reduced accumulation of proteolytic products. The knockdown of endogenous calpain-1 and calpain-2 by RNA interference reduced accumulation of p20, p26 and p28 and concurrently increased levels of nascent p43, p50 and p84 during the infectious cycle. Intriguingly, calpain depletion enhanced viral genome synthesis. Moreover, HCMV-permissive cells that stably expressed p20, p26 or p28 exhibited reduced viral genome synthesis and mature virus production. Our findings suggest that cognate UL112-113 proteins derived from calpain-catalysed proteolysis are involved in the HCMV replication process.

Complete sequencing and characterization of equine aggrecan.

To fully sequence and characterize equine aggrecan and confirm conservation of major aggrecanase, calpain and matrix metalloproteinase (MMP) cleavage sites. Reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends were used to generate clones that encompassed the complete equine aggrecan sequence. Clones were sequenced and compared with the equine genome database to determine intron-exon boundaries. The aggrecan gene spans over 61 kb on chromosome 1 and is encoded by 17 exons. Two major variants of aggrecan were cloned; one containing 8187 bp (2728 amino acids) and a second sequence of 8061 nucleotides (2686 amino acids). The variation was due to a CS1 domain polymorphism. Both sequences are substantially larger than predicted by the genomic database; 11 CS1 repeat elements are absent in the database sequence. The equine amino acid sequence was compared with human, bovine and murine sequences. Globular domains 1, 2 and 3 are highly conserved (overall identity over 80%). Equine CS1 is considerably larger than in other species and, therefore, is the least conserved domain (an overall amino acid identity of 22%). Previously defined aggrecanase, calpain and MMP cleavage sites were identified. Western blotting of chondrocyte culture samples showed complex post-secretion processing. The complete equine aggrecan sequence will support more in-depth research on aggrecan processing and degradation in equine articular cartilage and other musculoskeletal tissues.

Molecular Basis of Calpain Cleavage and Inactivation of the Sodium-Calcium Exchanger 1 in Heart Failure

Cardiac sodium (Na(+))-calcium (Ca(2+)) exchanger 1 (NCX1) is central to the maintenance of normal Ca(2+) homeostasis and contraction. Studies indicate that the Ca(2+)-activated protease calpain cleaves NCX1. We hypothesized that calpain is an important regulator of NCX1 in response to pressure overload and aimed to identify molecular mechanisms and functional consequences of calpain binding and cleavage of NCX1 in the heart. NCX1 full-length protein and a 75-kDa NCX1 fragment along with calpain were up-regulated in aortic stenosis patients and rats with heart failure. Patients with coronary artery disease and sham-operated rats were used as controls. Calpain co-localized, co-fractionated, and co-immunoprecipitated with NCX1 in rat cardiomyocytes and left ventricle lysate. Immunoprecipitations, pull-down experiments, and extensive use of peptide arrays indicated that calpain domain III anchored to the first Ca(2+) binding domain in NCX1, whereas the calpain catalytic region bound to the catenin-like domain in NCX1. The use of bioinformatics, mutational analyses, a substrate competitor peptide, and a specific NCX1-Met(369) antibody identified a novel calpain cleavage site at Met(369). Engineering NCX1-Met(369) into a tobacco etch virus protease cleavage site revealed that specific cleavage at Met(369) inhibited NCX1 activity (both forward and reverse mode). Finally, a short peptide fragment containing the NCX1-Met(369) cleavage site was modeled into the narrow active cleft of human calpain. Inhibition of NCX1 activity, such as we have observed here following calpain-induced NCX1 cleavage, might be beneficial in pathophysiological conditions where increased NCX1 activity contributes to cardiac dysfunction.

Myeloid differentiation and the leukemia-initiating cell.

Acute myeloblastic leukemia (AML) is defined by a block in the differentiation of multipotent proliferating progenitor cells into mature granulocytes. C/EBPα (CCAAT enhancer binding protein alpha) is a basic leucine zipper transcription factor and one of the central factors controlling differentiation. C/EBPα is required for adult hematopoetic stem cell (HSC) quiescence, and low levels of C/EBPα lead to enhanced HSC function. C/EBPα is a crucial regulator of granulopoiesis, and its absence, as shown in C/EBPα knockout (KO) mice, results in a block in the differentiation of common myeloid progenitor cells into granulocyte–macrophage progenitor cells accompanied by an absence of mature granulocytes. C/EBPα induces cell cycle arrest and has the capacity to inhibit proliferation by inducing p21, inhibiting cyclin-dependent kinases, mediating Calpain cleavage of cyclin A, interacting with and inhibiting E2F, and downregulating C-myc. We have shown that CEBPA is mutated in 16% of AML-M2 patients. Mutations that occur at the N-terminus result in a frameshift and loss of the full-length 42-kDa C/EBPα protein. A nonfunctional 30-kDa C/EBPα form that is initiated downstream of the frameshift is still produced. The N-terminal mutation acts as a dominant negative inhibitor of full-length CEBPA.1 To generate a mouse model that recapitulates the dominant negative function of C/EBPα, which results in AML, a combination of C-terminal mutation and N-terminal mutation was used (C/EBPα K/L).2 The central question in the molecular etiology of AML is: which cell is transformed in AML, HSC or committed progenitor? Is myeloid differentiation required for the formation of the ‘leukemic stem cell' in AML? C/EBPα KOs mimic human AML and although the mice have, by definition, human AML (blasts in bone marrow and blood), the mice do not develop a malignant disease.3, 4, 5 It is known that C/EBPα suppresses targets such as Bmi-1 and N-myc.3, 4, 5 Our efforts focused on identifying other targets that may contribute to the phenotype. A short hairpin RNA (shRNA) screen done on Lin−kit+Sca1+ (LSK) cells isolated from C/EBPα KO bone marrow identified Sox4 as a C/EBPα target-mediating serial replating (‘self-renewal') and inhibiting differentiation. Sox4 is a member of the SOX transcription factor family with a highly conserved high mobility group domain. Sox4 is a frequent target of insertional mutagenesis in retrovirally induced myeloid leukemias. Sox4 is also required for self-renewal and transformation pathways during leukemic evolution of multiple murine AML models (MOZ-TIF2, AML1-ETO and NUP98-HOXA9).6, 7, 8, 9 It is known that Sox4 expression is reciprocal to that of C/EBPα in wild-type mice, and C/EBPα binds to the Sox4 promoter directly to represses its expression (like Bmi-1 and N-myc). Therefore, we were interested in comparing the phenotypes of C/EBPα and Sox4 KOs to better understand the relationship between these two transcriptional factors in the context of myeloid differentiation. We have observed that Sox4 was upregulated in C/EBPα KO LSK and that Sox4 KO rescued myeloid progenitor expansion and increased proliferation of C/EBPα KO LSK (and rescued suppression of Bmi1 and N-myc). Importantly, in humans, enhanced SOX4 levels are observed in AML patients with dysfunctional CEBPA.10 Moreover SOX4 knockdown was confirmed to rescue myeloid differentiation in human CEBPA-mutant AML, and human C/EBPα mutants derived from patients did not repress Sox4 RNA levels in C/EBPα KO LSK and did not repress Sox4 promoter activity.11 In summary, our studies showed that Sox4 mediates serial replating (‘self-renewal') of both C/EBPα KO LSK and C/EBPα-mutant AML (K/L). In addition, Sox4 is upregulated in human primary blasts with C/EBPα mutation (modeled by K/L) or silenced (modeled by C/EBPa KO). shRNA downregulation of Sox4 induces differentiation of human primary blasts with C/EBPα dysfunction.

E2F1 in neurons is cleaved by calpain in an NMDA receptor-dependent manner in a model of HIV-induced neurotoxicity.

The transcription factor E2F1 activates gene targets required for G1 -S phase progression and for apoptosis, and exhibits increased expression levels in neurons in several CNS diseases including HIV encephalitis, Alzheimer disease, and Parkinson's Disease. While E2F1 is known to regulate cell viability through activation of caspases, here we present evidence supporting the involvement of E2F1 in N-methyl-d-aspartate (NMDA) receptor-dependent, HIV-induced neuronal death mediated by calpains. Using an in vitro model of HIV-induced neurotoxicity that is dependent on NMDA receptor and calpain activation, we have shown that cortical neurons lacking functional E2F1 are less susceptible to neuronal death. In addition, we report that neuronal E2F1 is cleaved by calpain to a stable 55-kiloDalton fragment following NR2B-dependent NMDA receptor stimulation. This cleavage of E2F1 is protein conformation-dependent and involves at least two cleavage events, one at each terminus of the protein. Intriguingly, the stabilized E2F1 cleavage product is produced in post-mitotic neurons of all ages, but fails to be stabilized in cycling cells. Finally, we show that a matching E2F1 cleavage product is produced in human fetal neurons, suggesting that calpain cleavage of E2F1 may be produced in human cortical tissue. These results suggest neuronal E2F1 is processed in a novel manner in response to NMDA receptor-mediated toxicity, a mechanism implicated in HIV-associated neurocognitive disorders pathogenesis as well as several other diseases of the CNS. After crossing the blood-brain barrier, HIV-infected monocytes differentiate into macrophages and release excitotoxins and inflammatory factors including glutamate into the brain parenchyma (1). These factors stimulate neuronal N-Methyl-d-aspartate (NMDA) receptors (2), causing calcium influx (3) and subsequent activation of the cysteine protease calpain (4). Activated calpain cleaves multiple substrates including E2F1, producing a stabilized protein fragment with truncations at the N- and C-terminus (5). Calpain-cleaved E2F1 may contribute to calpain-mediated neuronal damage observed in NMDA receptor-mediated neurotoxicity (6).

Dual role of Src kinase in governing neuronal survival

BackgroundSrc-family kinases (SFKs) are involved in neuronal survival and their aberrant regulation contributes to neuronal death. However, how they control neuronal survival and death remains unclear. ObjectiveTo define the effect of inhibition of Src activity and expression on neuronal survival. ResultsIn agreement with our previous findings, we demonstrated that Src was cleaved by calpain to form a 52-kDa truncated fragment in neurons undergoing excitotoxic cell death, and expression of the recombinant truncated Src fragment induced neuronal death. The data confirm that the neurotoxic signaling pathways are intact in the neurons we used for our study. To define the functional role of neuronal SFKs, we treated these neurons with SFK inhibitors and discovered that the treatment induced cell death, suggesting that the catalytic activity of one or more of the neuronal SFKs is critical to neuronal survival. Using small hairpin RNAs that suppress Src expression, we demonstrated that Src is indispensable to neuronal survival. Additionally, we found that neuronal death induced by expression of the neurotoxic truncated Src mutant, treatment of SFK inhibitors or knock-down of Src expression caused inhibition of the neuroprotective protein kinases Erk1/2, or Akt. ConclusionsSrc is critical to both neuronal survival and death. Intact Src sustains neuronal survival. However, in the excitotoxic condition, calpain cleavage of Src generates a neurotoxic truncated Src fragment. Both intact Src and the neurotoxic truncated Src fragment exert their biological actions by controlling the activities of neuroprotective protein kinases.

Improved myocardial function with supplement of levosimendan to Celsior solution.

Levosimendan is a calcium-sensitizing agent shown to prevent myocardical contractile depression in various heart diseases. In this study, we investigated the effect of levosimendan on cardiac dysfunction and apoptosis in hypothermic preservation rat hearts. Isolated rat hearts were preserved in Celsior solution with or without levosimendan. The left ventricular developed pressure (LVDP) recovery rate of isolated rat heart significantly decreased, and the apoptosis index increased after 9 hours of hypothermic preservation. Supplement Celsior solution with levosimendan (10 and 10 mole/L) enhanced the LVDP recovery rate and reduced apoptosis. Levosimendan inhibited the hypothermic preservation-induced calpain activation and cleavage of Bid. Levosimendam induced increased myocardial inducible nitric oxide synthase but not endothelial nitric oxide synthase expression. A selective inducible nitric oxide synthase inhibitor 1400W, and a mitochondrial ATP-sensitive potassium (KATP) channel blocker 5-hydroxydecanoate but not a sarcolemmal KATP channel blocker HMR-1098 prevented improvement effect of levosimendam on LVDP recovery rate, abolished the inhibitory effect of levosimendan on hypothermic preservation-induced activation of calpain, cleavage of Bid, and apoptosis. These data suggested that Celsior solution supplement with levosimendan improved cardiac function recovery and reduced myocyte apoptosis in hypothermic preservation rat hearts.

Calpain cleavage within dysferlin exon 40a releases a synaptotagmin-like module for membrane repair.

Dysferlin and calpain are important mediators of the emergency response to repair plasma membrane injury. Our previous research revealed that membrane injury induces cleavage of dysferlin to release a synaptotagmin-like C-terminal module we termed mini-dysferlinC72. Here we show that injury-activated cleavage of dysferlin is mediated by the ubiquitous calpains via a cleavage motif encoded by alternately spliced exon 40a. An exon 40a-specific antibody recognizing cleaved mini-dysferlinC72 intensely labels the circumference of injury sites, supporting a key role for dysferlinExon40a isoforms in membrane repair and consistent with our evidence suggesting that the calpain-cleaved C-terminal module is the form specifically recruited to injury sites. Calpain cleavage of dysferlin is a ubiquitous response to membrane injury in multiple cell lineages and occurs independently of the membrane repair protein MG53. Our study links calpain and dysferlin in the calcium-activated vesicle fusion of membrane repair, placing calpains as upstream mediators of a membrane repair cascade that elicits cleaved dysferlin as an effector. Of importance, we reveal that myoferlin and otoferlin are also cleaved enzymatically to release similar C-terminal modules, bearing two C2 domains and a transmembrane domain. Evolutionary preservation of this feature highlights its functional importance and suggests that this highly conserved C-terminal region of ferlins represents a functionally specialized vesicle fusion module.

A protease storm cleaves a cell-cell adhesion molecule in cancer: multiple proteases converge to regulate PTPmu in glioma cells.

Cleavage of the cell-cell adhesion molecule, PTPµ, occurs in human glioblastoma multiforme brain tumor tissue and glioma cell lines. PTPµ cleavage is linked to increased cell motility and growth factor independent survival of glioma cells in vitro. Previously, PTPµ was shown to be cleaved by furin in the endoplasmic reticulum to generate membrane associated E- (extracellular) and P- (phosphatase) subunits, and by ADAMs and the gamma secretase complex at the plasma membrane. We also identified the presence of additional extracellular and intracellular PTPµ fragments in brain tumors. We set out to biochemically analyze PTPµ cleavage in cancer cells. We determined that, in addition to the furin-processed form of PTPµ, a pool of 200 kDa full-length PTPµ exists at the plasma membrane that is cleaved directly by ADAM to generate a larger shed form of the PTPµ extracellular segment. Notably, in glioma cells, full-length PTPµ is also subject to calpain cleavage, which generates novel PTPµ fragments not found in other immortalized cells. We also observed glycosylation and phosphorylation differences in the cancer cells. Our data suggest that an additional serine protease also contributes to PTPµ shedding in glioma cells. We hypothesize that a "protease storm" occurs in cancer cells whereby multiple proteases converge to reduce the presence of cell-cell adhesion molecules at the plasma membrane and to generate protein fragments with unique biological functions. As a consequence, the "protease storm" could promote the migration and invasion of tumor cells.

Calpain‐Dependent Cleavage of Junctophilin‐2 and T‐Tubule Remodeling in a Mouse Model of Reversible Heart Failure

BackgroundA highly organized transverse tubule (T‐tubule) network is necessary for efficient Ca2+‐induced Ca2+ release and synchronized contraction of ventricular myocytes. Increasing evidence suggests that T‐tubule remodeling due to junctophilin‐2 (JP‐2) downregulation plays a critical role in the progression of heart failure. However, the mechanisms underlying JP‐2 dysregulation remain incompletely understood.Methods and ResultsA mouse model of reversible heart failure that is driven by conditional activation of the heterotrimeric G protein Gαq in cardiac myocytes was used in this study. Mice with activated Gαq exhibited disruption of the T‐tubule network and defects in Ca2+ handling that culminated in heart failure compared with wild‐type mice. Activation of Gαq/phospholipase Cβ signaling increased the activity of the Ca2+‐dependent protease calpain, leading to the proteolytic cleavage of JP‐2. A novel calpain cleavage fragment of JP‐2 is detected only in hearts with constitutive Gαq signaling to phospholipase Cβ. Termination of the Gαq signal was followed by normalization of the JP‐2 protein level, repair of the T‐tubule network, improvements in Ca2+ handling, and reversal of heart failure. Treatment of mice with a calpain inhibitor prevented Gαq‐dependent JP‐2 cleavage, T‐tubule disruption, and the development of heart failure.ConclusionsDisruption of the T‐tubule network in heart failure is a reversible process. Gαq‐dependent activation of calpain and subsequent proteolysis of JP‐2 appear to be the molecular mechanism that leads to T‐tubule remodeling, Ca2+ handling dysfunction, and progression to heart failure in this mouse model.

A calpain-cleaved fragment of β-catenin promotes BCRABL1 + cell survival evoked by autophagy induction in response to imatinib

Autophagy protects chronic myeloid leukemia stem cells from tyrosine kinase inhibitors hence supporting the disease persistence under therapy. However, the signals involved in autophagy regulation relative to BCR–ABL1 are still elusive. The autophagic flux proceeding from the inhibition of BCR–ABL1 tyrosine kinase represents a regulatory mechanism of β-catenin stability through events encompassing the activation of calpain, which targets β-catenin for proteasome-independent degradation. Accordingly, its inactivation may contribute to induce autophagy and autophagy induction may, in turn, promote β-catenin autolysosomal degradation to originate a regulatory loop where β-catenin plays a central role in cell decision between life and death. Here we proved that the cytoplasmic accumulation of β-catenin driven by up-regulation of its antagonist Chibby1 is a component of autophagy induction in response to imatinib in BCR–ABL1+ cells opposing the apoptotic death. It is contingent upon ER stress and elevation of free Ca2+ cytosolic concentration and results in the calpain cleavage into a 28kDa fragment implicated in β-catenin proteasome-independent degradation. More important for BCR–ABL1+ cell survival and proliferation following IM treatment, might be the calpain-mediated cleavage of β-catenin accumulated within the cytoplasmic compartment into a 75kDa fragment, still owning TCF-dependent transcriptional activity. Such a β-catenin fragment might be crucial for BCR–ABL1+ cell survival following the fusion protein TK inhibition.

Cleavage of E-Cadherin and β-Catenin by Calpain Affects Wnt Signaling and Spheroid Formation in Suspension Cultures of Human Pluripotent Stem Cells

The envisioned clinical and industrial use of human pluripotent stem cells and their derivatives has given major momentum to the establishment of suspension culture protocols that enable the mass production of cells. Understanding molecular changes accompanying the transfer from adherent to suspension culture is of utmost importance because this information can have a direct effect on the development of optimized culture conditions. In this study we assessed the gene expression of human embryonic stem cells and induced pluripotent stem cells grown in surface-adherent culture (two-dimensional) versus free-floating suspension culture spheroids (three-dimensional). We combined a quantitative proteomic approach based on stable isotope labeling by amino acids in cell culture with deep-sequencing-based transcriptomics. Cells in three-dimensional culture showed reduced expression of proteins forming structural components of cell-cell and cell-extracellular matrix junctions. However, fully unexpected, we found up-regulation of secreted inhibitors of the canonical Wnt signaling pathway and, concomitantly, a reduction in the level of active β-catenin and in the expression of Wnt target genes. In Western blot analyses the cysteine protease calpain was shown to cleave E-cadherin and β-catenin under three-dimensional culture conditions. Our data allowed the development of a model in which calpain cleavage of E-cadherin induces the disintegration of focal cell contacts and generates a 100-kDa E-cadherin fragment required for the formation of three-dimensional cell-cell contacts in spheroids. The parallel release of β-catenin and its potential activation by calpain cleavage are counterbalanced by the overexpression of soluble Wnt pathway inhibitors. According to this model, calpain has a key function in the interplay between E-cadherin and β-catenin-mediated intercellular adhesion and the canonical Wnt signaling pathway. Supporting this model, we show that pharmacological modulation of calpain activity prevents spheroid formation and causes disassembly of preexisting spheroids into single cells, thereby providing novel strategies for improving suspension culture conditions for human pluripotent stem cells in the future.