C/EBPalpha is required for proteolytic cleavage of cyclin A by calpain 3 in myeloid precursor cells

In this report, we present novel findings that implicate CCAAT/enhancer-binding protein (C/EBPalpha) in regulating the expression and activity of calpain 3 in vivo and data showing a new physiological substrate for calpain 3, cyclin A. Our results demonstrate that cleavage of cyclin A by calpain 3 occurs in mouse and human myeloid precursor cells. Calpain 3 cleaves cyclin A in vitro and in vivo, resulting in the production of a truncated product that lacks the N-terminal destruction box required for its degradation at the end of mitosis. The cleaved form of cyclin A retains the cyclin-dependent kinase (cdk) binding domain and forms active complexes with cdk2. Calpain 3-mediated cleavage of cyclin A is lacking in C/EBPalpha-/- mice, which are not able to produce mature granulocytes. Our data support a model in which calpain 3-mediated cleavage of cyclin A in dividing myeloid progenitor cells is important for the onset of differentiation. Deficits in this pathway in C/EBPalpha-/- mice might contribute to the failure of these mice to produce mature granulocytes. These data reveal a new pathway involving tightly controlled post-translational processing of cyclin A during differentiation of granulocytes.     

Talin contains a C-terminal calpain2 cleavage site important in focal adhesion dynamics

Talin is a large (～2540 residues) dimeric adaptor protein that associates with the integrin family of cell adhesion molecules in cell-extracellular matrix junctions (focal adhesions; FAs), where it both activates integrins and couples them to the actin cytoskeleton. Calpain2-mediated cleavage of talin between the head and rod domains has previously been shown to be important in FA turnover. Here we identify an additional calpain2-cleavage site that removes the dimerisation domain from the C-terminus of the talin rod, and show that an E2492G mutation inhibits calpain cleavage at this site in vitro, and increases the steady state levels of talin1 in vivo. Expression of a GFP-tagged talin1 E2492G mutant in CHO.K1 cells inhibited FA turnover and the persistence of cell protrusion just as effectively as a L432G mutation that inhibits calpain cleavage between the talin head and rod domains. Moreover, incorporation of both mutations into a single talin molecule had an additive effect clearly demonstrating that calpain cleavage at both the N- and C-terminal regions of talin contribute to the regulation of FA dynamics. However, the N-terminal site was more sensitive to calpain cleavage suggesting that lower levels of calpain are required to liberate the talin head and rod fragments than are needed to clip off the C-terminal dimerisation domain. The talin head and rod liberated by calpain2 cleavage have recently been shown to play roles in an integrin activation cycle important in FA turnover and in FAK-dependent cell cycle progression respectively. The half-life of the talin head is tightly regulated by ubiquitination and we suggest that removal of the C-terminal dimerisation domain from the talin rod may provide a mechanism both for terminating the signalling function of the talin rod and indeed for inactivating full-length talin thereby promoting FA turnover at the rear of the cell.     

Perturbation with intrabodies reveals that calpain cleavage is required for degradation of huntingtin exon 1

Background

Proteolytic processing of mutant huntingtin (mHtt), the protein that causes Huntington''s disease (HD), is critical for mHtt toxicity and disease progression. mHtt contains several caspase and calpain cleavage sites that generate N-terminal fragments that are more toxic than full-length mHtt. Further processing is then required for the degradation of these fragments, which in turn, reduces toxicity. This unknown, secondary degradative process represents a promising therapeutic target for HD.

Methodology/Principal Findings

We have used intrabodies, intracellularly expressed antibody fragments, to gain insight into the mechanism of mutant huntingtin exon 1 (mHDx-1) clearance. Happ1, an intrabody recognizing the proline-rich region of mHDx-1, reduces the level of soluble mHDx-1 by increasing clearance. While proteasome and macroautophagy inhibitors reduce turnover of mHDx-1, Happ1 is still able to reduce mHDx-1 under these conditions, indicating Happ1-accelerated mHDx-1 clearance does not rely on these processes. In contrast, a calpain inhibitor or an inhibitor of lysosomal pH block Happ1-mediated acceleration of mHDx-1 clearance. These results suggest that mHDx-1 is cleaved by calpain, likely followed by lysosomal degradation and this process regulates the turnover rate of mHDx-1. Sequence analysis identifies amino acid (AA) 15 as a potential calpain cleavage site. Calpain cleavage of recombinant mHDx-1 in vitro yields fragments of sizes corresponding to this prediction. Moreover, when the site is blocked by binding of another intrabody, V_L12.3, turnover of soluble mHDx-1 in living cells is blocked.

Conclusions/Significance

These results indicate that calpain-mediated removal of the 15 N-terminal AAs is required for the degradation of mHDx-1, a finding that may have therapeutic implications.     

Developmental and cell-selective variations in N-methyl-D-aspartate receptor degradation by calpain

NMDA receptors play critical roles in synaptic modulation and neurological disorders. In this study, we investigated the developmental changes in NR2 cleavage by NMDA receptor-activated calpain in cultured cortical and hippocampal neurons. Calpain activity increased with development, associated with increased expression of NMDA receptors but not of calpain I. The activation of calpain in immature and mature cortical cultures was inhibited by antagonists of NR1/2B and NR1/2A/2B receptors, whereas the inhibition of NR1/2B receptors did not alter calpain activation in mature hippocampal cultures. The degradation of NR2 subunits by calpain differed with developmental age. NR2A was not a substrate of calpain in mature hippocampal cultures, but was cleaved in immature cortical and hippocampal cultures. NR2B degradation by calpain in cortical cultures decreased with development, but the level of degradation of NR2B in hippocampal cultures did not change. The kinetics of NMDA receptor-gated whole cell currents were also modulated by calpain activation in a manner that varied with developmental stage in vitro. In early (but not later) developmental stages, calpain activation altered the NMDA-evoked current rise time and time constants for both desensitization and deactivation. Our data suggest that the susceptibility of the NMDA receptor to cleavage by calpain varies with neuronal maturity in a manner that may alter its electrophysiological properties.     

In vivo administration of calpeptin attenuates calpain activation and cardiomyocyte loss in pressure-overloaded feline myocardium

Calpain activation is linked to the cleavage of several cytoskeletal proteins and could be an important contributor to the loss of cardiomyocytes and contractile dysfunction during cardiac pressure overload (PO). Using a feline right ventricular (RV) PO model, we analyzed calpain activation during the early compensatory period of cardiac hypertrophy. Calpain enrichment and its increased activity with a reduced calpastatin level were observed in 24- to 48-h-PO myocardium, and these changes returned to basal level by 1 wk of PO. Histochemical studies in 24-h-PO myocardium revealed the presence of TdT-mediated dUTP nick-end label (TUNEL)-positive cardiomyocytes, which exhibited enrichment of calpain and gelsolin. Biochemical studies showed an increase in histone H2B phosphorylation and cytoskeletal binding and cleavage of gelsolin, which indicate programmed cardiomyocyte cell death. To test whether calpain inhibition could prevent these changes, we administered calpeptin (0.6 mg/kg iv) by bolus injections twice, 15 min before and 6 h after induction of 24-h PO. Calpeptin blocked the following PO-induced changes: calpain enrichment and activation, decreased calpastatin level, caspase-3 activation, enrichment and cleavage of gelsolin, TUNEL staining, and histone H2B phosphorylation. Although similar administration of a caspase inhibitor, N-benzoylcarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VD-fmk), blocked caspase-3 activation, it did not alleviate other aforementioned changes. These results indicate that biochemical markers of cardiomyocyte cell death, such as sarcomeric disarray, gelsolin cleavage, and TUNEL-positive nuclei, are mediated, at least in part, by calpain and that calpeptin may serve as a potential therapeutic agent to prevent cardiomyocyte loss and preserve myocardial structure and function during cardiac hypertrophy.     

Calpain inhibition delays neutrophil apoptosis via cyclic AMP-independent activation of protein kinase A and protein kinase A-mediated stabilization of Mcl-1 and X-linked inhibitor of apoptosis (XIAP)

Human neutrophils underwent spontaneous apoptosis, which was accompanied with proteasome-mediated degradation of Mcl-1 and X-linked inhibitor of apoptosis (XIAP). Calpain inhibitors (PD150606 and N-acetyl-Leu-Leu-Nle-CHO) prevented spontaneous neutrophil apoptosis and degradation of Mcl-1 and XIAP, and the effects of calpain inhibitors on neutrophils were resistant to cycloheximide. Calpain inhibitors induced protein kinase A (PKA) activation, which was unaccompanied with an increase in intracellular cyclic AMP. Calpain inhibition-mediated delayed neutrophil apoptosis, stabilization of Mcl-1 and XIAP, and phosphorylation of PKA substrates were suppressed by H-89 (specific PKA inhibitor). These findings suggest that calpain inhibition delays neutrophil apoptosis via cyclic AMP-independent activation of PKA and PKA-mediated stabilization of Mcl-1 and XIAP.     

Calpain-dependent calpastatin cleavage regulates caspase-3 activation during apoptosis of Jurkat T cells induced by Entamoeba histolytica

In this study, we investigated whether there is a signalling interaction between calpain and caspase-3 during apoptosis in Jurkat T cells by Entamoeba histolytica. When Jurkat cells were co-incubated with E. histolytica, phosphatidylserine externalisation and DNA fragmentation markedly increased compared with results for cells incubated with medium alone. In addition, E. histolytica strongly induced cleavage of caspases-3, -6, -7 and poly(ADP-ribose) polymerase. A rise in intracellular calcium levels and activation of calpain were seen in Jurkat cells after exposure to E. histolytica. Pretreatment of Jurkat cells with calpain inhibitor calpeptin effectively blocked E. histolytica-triggered cleavage of caspase-3 as well as calpain. In contrast, pan-caspase inhibitor did not affect E. histolytica-induced calpain activation. In addition, incubation with E. histolytica resulted in multiple fragmented bands of calpastatin, which is an endogenous inhibitor of calpain, in Jurkat T cells. Moreover, Entamoeba-induced calpastatin degradation was dramatically suppressed by pretreatment with calpeptin, but not by z-VAD-fmk. Entamoeba-induced DNA fragmentation was strongly retarded by z-VAD-fmk, but not calpeptin. Our results suggest that calpain-mediated calpastatin degradation plays a crucial role in regulation of caspase-3 activation during apoptosis of Jurkat T cells by E. histolytica.     

Pak regulates calpain-dependent degradation of E3b1

E3b1, a binding partner of Eps8, plays a critical role in receptor tyrosine kinase (RTK)-mediated Rac activation by facilitating the interaction of Eps8 with Sos-1 and the consequent activation of the Rac-specific guanine nucleotide exchange factor activity of Sos-1. Here we present evidence that E3b1 levels are regulated by the Ca(2+)-activated protease calpain, and also by Pak, a downstream target of Rac signaling. Serum starvation of Rat2 or COS7 cells resulted in rapid loss of E3b1 that was reversed by calpain inhibitors. Loss was also prevented by expressing the constitutively active Pak1 mutant, Pak1(H83,86L). Activation of endogenous Pak by platelet-derived growth factor or the constitutively active Rac1 mutant, Rac1(G12V), also inhibited degradation. In contrast, inhibition of endogenous Pak activity by expressing the Pak auto-inhibitory domain caused degradation of over-expressed E3b1 even in the presence of serum. Taken together, these findings indicate that E3b1 is down-regulated by calpain activation and stabilized by Pak activation. They also suggest that RTK-mediated Rac activation can be modulated by changes in the level of E3b1 in response to signals that affect the activity of calpain or Pak.     

Calpain activates caspase-8 in neuron-like differentiated PC12 cells via the amyloid-β-peptide and CD95 pathways

The neurotoxic amyloid-β-peptide (Aβ) is important in the pathogenesis of Alzheimer's disease (AD). Calpain (Ca²⁺-dependent protease) and caspase-8 (the initiating caspase for the extrinsic, receptor-mediated apoptosis pathway) have been implicated in AD/Aβ toxicity. We previously found that Aβ promoted degradation of calpastatin (the specific endogenous calpain inhibitor); calpastatin degradation was prevented by inhibitors of either calpain or caspase-8. The results implied a cross-talk between the two proteases and suggested that one protease was responsible for the activity of the other one. We now report on the previously unrecognized caspase-8 activation by calpain. In neuron-like differentiated PC12 cells, calpain promotes active caspase-8 formation from procaspase-8 via the Aβ and CD95 pathways, along with degradation of the procaspase-8 processing inhibitor caspase-8 (FLICE)-like inhibitory protein, short isoform (FLIP_S). Inhibition of calpain (by pharmacological inhibitors and by overexpression of calpastatin) prevents the cleavage of procaspase-8 to mature, active caspase-8, and inhibits FLIP_S degradation in the Aβ-treated and CD95-triggered cells. Increased cellular Ca²⁺ per se results in calpain activation but does not lead to caspase-8 activation or FLIP_S degradation. The results suggest that procaspase-8 and FLIP_S association with cell membrane receptor complexes is required for calpain-induced caspase-8 activation. The results presented here add to the understanding of the roles of calpain, caspase-8, and CD95 pathway in AD/Aβ toxicity. Calpain-promoted activation of caspase-8 may have implications for other types of CD95-induced cell damage, and for nonapoptotic functions of caspase-8. Inhibition of calpain may be useful for modulating certain caspase-8-dependent processes.     

Group B Streptococcus induces macrophage apoptosis by calpain activation

Group B Streptococcus (GBS) has developed several strategies to evade immune defenses. We show that GBS induces macrophage (Mphi) membrane permeability defects and apoptosis, prevented by inhibition of calcium influx but not caspases. We analyze the molecular mechanisms of GBS-induced murine Mphi apoptosis. GBS causes a massive intracellular calcium increase, strictly correlated to membrane permeability defects and apoptosis onset. Calcium increase was associated with activation of calcium-dependent protease calpain, demonstrated by casein zymography, alpha-spectrin cleavage to a calpain-specific fragment, fluorogenic calpain-substrate cleavage, and inhibition of these proteolyses by calpain inhibitors targeting the calcium-binding, 3-(4-Iodophenyl)-2-mercapto-(Z)-2-propenoic acid, or active site (four different inhibitors), by calpain small-interfering-RNA (siRNA) and EGTA. GBS-induced Mphi apoptosis was inhibited by all micro- and m-calpain inhibitors used and m-calpain siRNA, but not 3-(5-Fluoro-3-indolyl)-2-mercapto-(Z)-2-propenoic acid (micro-calpain inhibitor) and micro-calpain siRNA indicating that m-calpain plays a central role in apoptosis. Calpain activation is followed by Bax and Bid cleavage, cytochrome c, apoptosis-inducing factor, and endonuclease G release from mitochondria. In GBS-induced apoptosis, cytochrome c did not induce caspase-3 and -7 activation because they and APAF-1 were degraded by calpains. Therefore, apoptosis-inducing factor and endonuclease G seem the main mediators of the calpain-dependent but caspase-independent pathway of GBS-induced apoptosis. Proapoptotic mediator degradations do not occur with nonhemolytic GBS, not inducing Mphi apoptosis. Apoptosis was reduced by Bax siRNA and Bid siRNA suggesting Bax and Bid degradation is apoptosis correlated. This signaling pathway, different from that of most pathogens, could represent a GBS strategy to evade immune defenses.     

Phosphoinositide binding to the substrate regulates susceptibility to proteolysis by calpain

Calpain-mediated proteolysis regulates cytoskeletal dynamics and is altered during aging and the progression of numerous diseases or pathological conditions. Although several cytoskeletal proteins have been identified as substrates, how localized calpain activity is regulated and the mechanisms controlling substrate recognition are not clear. In this study, we report that phosphoinositide binding regulates the susceptibility of the cytoskeletal adhesion protein alpha-actinin to proteolysis by calpains 1 and 2. At first, alpha-actinin did not appear to be a substrate for calpain 2; however, phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) binding to alpha-actinin resulted in nearly complete proteolysis of the full-length protein, producing stable breakdown products. Calpain 1 was able to cleave alpha-actinin in the absence of phosphoinositide binding; however, PtdIns(3,4,5)P(3) binding increased the rate of proteolysis, and phosphatidylinositol 4,5-diphosphate (PtdIns(4,5)P(2)) binding significantly inhibited cleavage. Phosphoinositide binding appeared to regulate calpain proteolysis of alpha-actinin by modulating the exposure of a highly sensitive cleavage site within the calponin homology 2 domain. In U87MG glioblastoma cells, which contain elevated levels of PtdIns(3,4,5)P(3), alpha-actinin colocalized with calpain within dynamic actin cytoskeletal structures. Furthermore, proteolysis of alpha-actinin producing stable breakdown products was observed in U87MG cells treated with calcium ionophore to activate the calcium-dependent calpains. Additional evidence of PtdIns(3,4,5)P(3)-mediated calpain proteolysis of alpha-actinin was observed in rat embryonic fibroblasts. These results suggest that PtdIns(3,4,5)P(3) binding is a critical determinant for alpha-actinin proteolysis by calpain. In conclusion, phosphoinositide binding to the substrate is a potential mechanism for regulating susceptibility to proteolysis by calpain.     

Oxidized lipoproteins inhibit surfactant phosphatidylcholine synthesis via calpain-mediated cleavage of CTP:phosphocholine cytidylyltransferase

We investigated effects of pro-atherogenic oxidized lipoproteins on phosphatidylcholine (PtdCho) biosynthesis in murine lung epithelial cells (MLE-12). Cells surface-bound, internalized, and degraded oxidized low density lipoproteins (Ox-LDL). Ox-LDL significantly reduced [3H]choline incorporation into PtdCho in cells by selectively inhibiting the activity of the rate-regulatory enzyme, CTP:phosphocholine cytdylyltransferase (CCT). Ox-LDL coordinately increased the cellular turnover of CCTalpha protein as determined by [35S]methionine pulse-chase studies by inducing the calcium-activated proteinase, calpain. Forced expression of calpain or exposure of cells to the calcium ionophore, A23187, increased CCTalpha degradation, whereas overexpression of the endogenous calpain inhibitor, calpastatin, attenuated Ox-LDL-induced CCTalpha degradation. The effects of Ox-LDL on CCTalpha breakdown were attenuated in calpain-deficient cells. In vitro calpain digestion of CCTalpha isolated from cells transfected with truncated or internal deletion mutants indicated multiple cleavage sites within the CCTalpha primary structure, leading to the generation of a 26-kDa (p26) fragment. Calpain hydrolysis of purified CCTalpha generated p26, which upon NH2-terminal sequencing localized a calpain attack site within the CCTalpha amino terminus. Expression of a CCTalpha mutant where the amino-terminal cleavage site and a putative carboxyl-terminal hydrolysis region were modified resulted in an enzyme that was significantly less sensitive to proteolytic cleavage and restored the ability of cells to synthesize surfactant PtdCho after Ox-LDL treatment. Thus, these results provide a critical link between proatherogenic lipoproteins and their metabolic target, CCTalpha, resulting in impaired surfactant metabolism.     

Calpain-mediated Bid cleavage and calpain-independent Bak modulation: two separate pathways in cisplatin-induced apoptosis

Calpain is a ubiquitous protease with potential involvement in apoptosis. We report that in human melanoma cells, cisplatin-induced calpain activation occurs early in apoptosis. Calpain activation and subsequent apoptosis were inhibited by calpeptin and PD150606, two calpain inhibitors with different modes of action. Furthermore, cisplatin induced cleavage of the BH3-only protein Bid, yielding a 14-kDa fragment similar to proapoptotic, caspase-cleaved Bid. However, Bid cleavage was inhibited by inhibitors of calpain, but not by inhibitors of caspases or of cathepsin L. Recombinant Bid was cleaved in vitro by both recombinant calpain and by lysates of cisplatin-treated cells. Cleavage was calpeptin sensitive, and the cleavage site was mapped between Gly70 and Arg71. Calpain-cleaved Bid induced cytochrome c release from isolated mitochondria. While calpeptin did not affect cisplatin-induced modulation of Bak to its proapoptotic conformation, a dominant-negative mutant of MEKK1 (dnMEKK) inhibited Bak modulation. dnMEKK did not, however, block Bid cleavage. The combination of dnMEKK and calpeptin had an additive inhibitory effect on apoptosis. In summary, calpain-mediated Bid cleavage is important in drug-induced apoptosis, and cisplatin induces at least two separate apoptotic signaling pathways resulting in Bid cleavage and Bak modulation, respectively.     

Investigation of the structural basis of the interaction of calpain II with phospholipid and with carbohydrate.

Two forms of pig kidney calpain II were isolated, both of which appeared to contain an intact 80 kDa large subunit, but which showed specific proteolytic degradation at the N-terminal end of the 30 kDa small subunit. The structure of each of these molecules was investigated by amino acid sequence analysis. The forms corresponded to molecules with small subunits starting at residue 38 (degraded calpain A) and at residue 62 (degraded calpain B) of the complete sequence. These molecules were tested for their ability to interact with phosphatidylinositol and with carbohydrate (agarose gel-filtration media). Calpain and degraded calpain A, but not degraded calpain B, would interact with phosphatidylinositol. Thus the sequence (G)17TAMRILG (residues 38-61) is essential for the interaction. Neither calpain nor the degraded forms of the enzyme showed specific interaction with carbohydrate.     

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

Cardiac sodium (Na⁺)-calcium (Ca²⁺) exchanger 1 (NCX1) is central to the maintenance of normal Ca²⁺ homeostasis and contraction. Studies indicate that the Ca²⁺-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 Ca²⁺ 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-Met³⁶⁹ antibody identified a novel calpain cleavage site at Met³⁶⁹. Engineering NCX1-Met³⁶⁹ into a tobacco etch virus protease cleavage site revealed that specific cleavage at Met³⁶⁹ inhibited NCX1 activity (both forward and reverse mode). Finally, a short peptide fragment containing the NCX1-Met³⁶⁹ 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.     

Caspase-dependent activation of calpain during drug-induced apoptosis

We have previously demonstrated that calpain is responsible for the cleavage of Bax, a proapoptotic protein, during drug-induced apoptosis of HL-60 cells (Wood, D. E., Thomas, A., Devi, L. A., Berman, Y., Beavis, R. C., Reed, J. C., and Newcomb, E. W. (1998) Oncogene 17, 1069-1078). Here we show the sequential activation of caspases and calpain during drug-induced apoptosis of HL-60 cells. Time course experiments using the topoisomerase I inhibitor 9-amino-20(S)-camptothecin revealed that cleavage of caspase-3 substrates poly(ADP-ribose) polymerase (PARP) and the retinoblastoma protein as well as DNA fragmentation occurred several hours before calpain activation and Bax cleavage. Pretreatment with the calpain inhibitor calpeptin blocked calpain activation and Bax cleavage but did not inhibit PARP cleavage, DNA fragmentation, or 9-amino-20(S)-camptothecin-induced morphological changes and cell death. Pretreatment with the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk) inhibited PARP cleavage, DNA fragmentation, calpain activation, and Bax cleavage and increased cell survival by 40%. Interestingly, Z-VAD-fmk-treated cells died in a caspase- and calpain-independent manner that appeared morphologically distinct from apoptosis. Our results suggest that excessive or uncontrolled calpain activity may play a role downstream of and distinct from caspases in the degradation phase of apoptosis.     

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.     

The calpain 1-alpha-actinin interaction. Resting complex between the calcium-dependent protease and its target in cytoskeleton.

Calpain 1 behaviour toward cytoskeletal targets was investigated using two alpha-actinin isoforms from smooth and skeletal muscles. These two isoforms which are, respectively, sensitive and resistant to calpain cleavage, interact with the protease when using in vitro binding assays. The stability of the complexes in EGTA [Kd(-Ca2+) = 0.5 +/- 0.1 microM] was improved in the presence of 1 mm calcium ions [Kd(+Ca2+) = 0.05 +/- 0.01 microM]. Location of the binding structures shows that the C-terminal domain of alpha-actinin and each calpain subunit, 28 and 80 kDa, participates in the interaction. In particular, the autolysed calpain form (76/18) affords a similar binding compared to the 80/28 intact enzyme, with an identified binding site in the catalytic subunit, located in the C-terminal region of the chain (domain III-IV). The in vivo colocalization of calpain 1 and alpha-actinin was shown to be likely in the presence of calcium, when permeabilized muscle fibres were supplemented by exogenous calpain 1 and the presence of calpain 1 in Z-line cores was shown by gold-labelled antibodies. The demonstration of such a colocalization was brought by coimmunoprecipitation experiments of calpain 1 and alpha-actinin from C2.7 myogenic cells. We propose that calpain 1 interacts in a resting state with cytoskeletal targets, and that this binding is strengthened in pathological conditions, such as ischaemia and dystrophies, associated with high calcium concentrations.     

GPS-CCD: a novel computational program for the prediction of calpain cleavage sites

As one of the most essential post-translational modifications (PTMs) of proteins, proteolysis, especially calpain-mediated cleavage, plays an important role in many biological processes, including cell death/apoptosis, cytoskeletal remodeling, and the cell cycle. Experimental identification of calpain targets with bona fide cleavage sites is fundamental for dissecting the molecular mechanisms and biological roles of calpain cleavage. In contrast to time-consuming and labor-intensive experimental approaches, computational prediction of calpain cleavage sites might more cheaply and readily provide useful information for further experimental investigation. In this work, we constructed a novel software package of GPS-CCD (Calpain Cleavage Detector) for the prediction of calpain cleavage sites, with an accuracy of 89.98%, sensitivity of 60.87% and specificity of 90.07%. With this software, we annotated potential calpain cleavage sites for hundreds of calpain substrates, for which the exact cleavage sites had not been previously determined. In this regard, GPS-CCD 1.0 is considered to be a useful tool for experimentalists. The online service and local packages of GPS-CCD 1.0 were implemented in JAVA and are freely available at: http://ccd.biocuckoo.org/.     

Calpain activation by the Shigella flexneri effector VirA regulates key steps in the formation and life of the bacterium's epithelial niche

The enteropathogen Shigella flexneri invades epithelial cells, leading to inflammation and tissue destruction. We report that Shigella infection of epithelial cells induces an early genotoxic stress, but the resulting p53 response and cell death are impaired due to the bacterium's ability to promote p53 degradation, mainly through calpain protease activation. Calpain activation is promoted by the Shigella virulence effector VirA and dependent on calcium flux and the depletion of the endogenous calpain inhibitor calpastatin. Further, although VirA-induced calpain activity is critical for regulating cytoskeletal events driving bacterial uptake, calpain activation ultimately leads to necrotic cell death, thereby restricting Shigella intracellular growth. Therefore, calpains work at multiple steps in regulating Shigella pathogenesis by disrupting the p53-dependent DNA repair response early during infection and regulating both formation and ultimate death of the Shigella epithelial replicative niche.