Rapid proteolysis of brain MAP-1 related cytoskeleton-associated 350kd protein by purified calpain

Microtubule associated protein-1 of brain and its intracellular 350kd analogues were highly sensitive to purified Ca2+-dependent cysteine proteinase (calpain). After 15 second digestion, we detected intermediate degradation products of MAP-1 by immunoblotting using anti-MAP-1 antibody as 290, 260, 220, 170, 140, 112, 80, 68, and 32kd polypeptides. These values corresponded to the molecular weights of the immunoreactive polypeptides of microtubule-enriched cytoskeletons isolated from HeLa and SV-3Y1 cells, suggesting the action of endogenous calpain on intracellular MAP-1 analogues in vivo or during the course of preparation.     

Tripeptide analogues of MG132 as protease inhibitors

The 26S proteasome and calpain are linked to a number of important human diseases. Here, we report a series of analogues of the prototypical tripeptide aldehyde inhibitor MG132 that show a unique combination of high activity and selectivity for calpains over proteasome. Tripeptide aldehydes (1–3) with an aromatic P3 substituent show enhanced activity and selectivity against ovine calpain 2 relative to chymotrypsin-like activity of proteasome. Docking studies reveal the key contacts between inhibitors and calpain to confirm the importance of the S3 pocket with respect to selectivity between calpains 1 and 2 and the proteasome.     

Effects of L-1-methyl-histidine and the muscle dipeptides carnosine and anserine on the activities of muscle calpains

1. Carnosine, anserine and L-1-methyl-histidine activated muscle calpain II assayed at 2.5 mM Ca2+. 2. At 5 microM Ca2+, none of these compounds activated calpain II sufficiently to bring its activity up to the level measured at 2.5 mM Ca2+. 3. Carnosine increased, whereas both anserine and L-1-methyl-histidine decreased the inhibitory effect of calpastatin on calpain II. 4. These results suggest that although the compounds are not potent activators of calpain II, the ratio of the dipeptides in muscle may have an effect on calpain II-calpastatin interaction.     

Differential effects of aluminum ion on smooth muscle calpain I and calpain II activities.

1. In millimolar Ca2+, smooth muscle calpains I and II were inhibited by aluminum ion. 2. At sub-millimolar Ca2+, calpain II, but not calpain I, was activated by low millimolar aluminum ion. 3. Calpastatin inhibited aluminum ion-activated calpain II. 4. Aluminum ion-activated and Ca(2+)-activated calpain II gave almost identical patterns of desmin cleavage. 5. Aluminum-activated calpain II, unlike the Ca(2+)-activated enzyme, did not autolyze and retained its proteolytic activity over extended periods of time.     

The synthesis of ketomethylene pseudopeptide analogues of dipeptide aldehyde inhibitors of calpain

The ketomethylene phenylalanal and alanal analogues of Cbz-Val-Phe-H and Cbz-Val-Ala-H have been prepared and the Ki values versus chicken gizzard smooth muscle calpain were determined. The ketomethylene isosteres were significantly less potent than the corresponding dipeptide aldehydes, and this loss in activity is attributed to the absence of a critical interaction between the P1-P2 amide bond and the peptide binding region of calpain.     

Characteristics of various synthetic peptide calpain inhibitors and their application for the analysis of platelet reaction

Calpeptin (a cell permeable synthetic peptide calpain inhibitor) inhibited the generation of thromboxane B2 (TxB2) by the direct inhibition on Tx synthetase in platelets at the concentrations more than 30 microM. Calpeptin, its analogues and E-64d (EST) were further examined with regard to cell permiability and inhibitory spectra. Among all compounds, only calpeptin inhibited the degradation of substrate proteins of calpain with negligible effect on TxB2 generation in intact platelets at the concentrations less than 30 microM. These concentrations of calpeptin did not inhibit the platelet aggregation, the elevation of [Ca2+], nor the formation of inositol 1,4,5-trisphosphate (IP3) in thrombin or collagen activated platelets. These results indicate that calpain dose not participate in the process of platelet activation induced by thrombin or collagen.     

Calpain regulates CVB3 induced viral myocarditis by promoting autophagic flux upon infection

Calpains are calcium-activated neutral cysteine proteases. The dysregulation of calpain activity has been found to be related to cardiovascular diseases, for which calpain inhibition is used as a treatment. Viral myocarditis (VMC) is primarily caused by Coxsackievirus group B3 virus infection (CVB3). CVB3 virus infection induces autophagy and hijacks this process to facilitate its replication. In this study, we found that calpain was significantly activated in hearts affected by VMC. However, pharmacologically inhibiting calpain aggravated VMC symptoms in mice due to myocardial inflammation and cardiac dysfunction. The inhibition of calpain activity in vitro led to the accumulation of LC3-II and increased levels of p62/SQSTM1 protein expression, suggesting that autophagic flux was impaired by calpain inhibition. These effects of calpain inhibition were also observed in capn4-specific myocardial knockout mice in vivo. Furthermore, our results provided evidence that calpain inhibition in VMC, unlike other cardiovascular diseases, exacerbated the disease symptom by impairing CVB3-induced autophagic flux, which may subsequently reduce virus autolysosome degradation. Our findings indicated that calpain inhibition may not be a good treatment for VMC disease in a clinical setting.     

Comparison of calpain I and calpain II from carp muscle

1. The content of calpain II is 3.4 times more than that of calpain I when estimated by the elution profiles from a column of DEAE-cellulose. 2. Calpain I required 1 mM Ca2+ and calpain II required 5 mM Ca2+ to show the full activities. These data demonstrated that Ca2+-sensitivities of both calpains were lower than those of mammalian calpains, respectively. 3. The optimum caseinolytic activity was pH 7.2 for calpain I and pH 7.5 for calpain II. 4. The molecular weight of calpain I was estimated to be 110 k and that of calpain II to be 120 k by gel filtration. 5. Calpain I was much more heat-stable than calpain II around 50-60 degrees C. 6. Both calpains were sensitive to calpastatin, an endogenous inhibitor for calpain.     

Inhibition of chicken calpain II by proteins of the cystatin superfamily and alpha 2-macroglobulin.

Inhibition of chicken calpain II by proteins of the cystatin superfamily and alpha 2-macroglobulin was investigated. Human liver cystatins A and B, human cystatin C, chicken cystatin and rat T-kininogen were found not to be inhibitory. Inhibition was, however, observed for bovine and rat kininogens, with Ki (inhibition constant) values of 0.8 nM and 30 nM respectively. alpha 2-Macroglobulin inhibits calpain with an initial rate constant of the order of 3 X 10(4) M-1.S-1. Calpain complexed with alpha 2-macroglobulin showed only limited reactivity towards azocasein, but reacted readily with the peptide substrate Suc-Leu-Tyr-4-methyl-7-coumarylamide and with L-3-carboxy-trans-2,3-epoxypropionyl-leucylamido-(4-guanidin o)butane (E-64). The calpain in the complexes was at least partially protected from loss of activity due to autolysis. The calpain-alpha 2-macroglobulin complexes contained both the calpain subunits.     

Calpain‐truncated CRMP‐3 and ‐4 contribute to potassium deprivation‐induced apoptosis of cerebellar granule neurons

Increasing evidence shows that calpain‐mediated proteolytic processing of a selective number of proteins plays an important role in neuronal apoptosis. Study of calpain‐mediated cleavage events and related functions may contribute to a better understanding of neuronal apoptosis and neurodegenerative diseases. We, therefore, investigated the role of calpain substrates in potassium deprivation‐induced apoptosis of cerebellar granule neurons (CGNs). Twelve previously known and seven novel candidates of calpain substrates were identified by 2‐D DIGE and MALDI‐TOF/TOF MS analysis. Further, the identified novel calpain substrates were validated by Western blot analysis. Moreover, we focused on the collapsin response mediator proteins (CRMP‐1, ‐2, ‐3 and ‐4 isoforms) and found that CRMPs were proteolytically processed by calpain but not by caspase, both in vivo and in vitro. To clarify the properties of the calpain‐mediated proteolysis of CRMPs, we constructed the deletion mutants of CRMPs for additional biochemical studies. In vitro cleavage assays revealed that CRMP‐1, ‐2 and ‐4 were truncated by calpain at the C‐terminus, whereas CRMP‐3 was cleaved at the N‐terminus. Finally, we assessed the role of CRMPs in the process of potassium deprivation‐triggered neuronal apoptosis by overexpressing the truncated CRMPs in CGNs. Our data clearly showed that the truncated CRMP‐3 and ‐4, but not CRMP‐1 and ‐2, significantly induced neuronal apoptosis. These findings demonstrated that calpain‐truncated CRMP‐3 and ‐4 act as pro‐apoptotic players when CGNs undergo apoptosis.     

High dose of lipoxin A4 induces apoptosis in rat renal interstitial fibroblasts

Studies have implicated that lipoxinA4 (LXA4) inhibited nuclear factor-kappaB (NF-kappaB), Akt/PKB and PI 3-kinase activity and proliferation of glomerular mesangial cells. It is speculated that LXA4 might serve as pro-apoptotic factor. Rat renal interstitial fibroblasts (NRK-49F cells) were exposed to LXA4 in 5% FCS for 24 h. LXA4 at 0.1 and 1 microM induced 9.83% and 33.82% apoptosis of the cells, respectively, upregulated the expression of calpain 10 and Smac, the levels of [Ca2+]i and activity of caspase-3, and downregulated the activity of STAT3 and threonine phosphorylated Akt1. Transfection of calpain 10 or Smac antisense oligodeoxynucleotide into the cells inhibited the LXA4-induced apoptosis, activity of caspase-3. Pretreatment of the cells with calcium inhibitor SK&F96365 inhibited the LXA4-induced apoptosis, levels of [Ca2+]i, expression of calpain 10 and Smac. In conclusion, LXA4 at high concentrations induced apoptosis of renal interstitial fibroblasts via [Ca2+]i-dependent upregulation of calpain 10 and Smac expression.     

Calpain activation is not required for AIF translocation in PARP-1-dependent cell death (parthanatos)

Apoptosis-inducing factor (AIF) is critical for poly(ADP-ribose) polymerase-1 (PARP-1)-dependent cell death (parthanatos). The molecular mechanism of mitochondrial AIF release to the nucleus remains obscure, although a possible role of calpain I has been suggested. Here we show that calpain is not required for mitochondrial AIF release in parthanatos. Although calpain I cleaved recombinant AIF in a cell-free system in intact cells under conditions where endogenous calpain was activated by either NMDA or N -methyl- N '-nitro- N -nitrosoguanidine (MNNG) administration, AIF was not cleaved, and it was released from mitochondria to the nucleus in its 62-kDa uncleaved form. Moreover, NMDA administration under conditions that failed to activate calpain still robustly induced AIF nuclear translocation. Inhibition of calpain with calpastatin or genetic knockout of the regulatory subunit of calpain failed to prevent NMDA- or MNNG-induced AIF nuclear translocation and subsequent cell death, respectively, which was markedly prevented by the PARP-1 inhibitor, 3,4-dihydro-5-[4-(1-piperidinyl)butoxyl]-1(2H)-iso-quinolinone. Our study clearly shows that calpain activation is not required for AIF release during parthanatos, suggesting that other mechanisms rather than calpain are involved in mitochondrial AIF release in parthanatos.     

In vivo targeted deletion of calpain small subunit, Capn4, in cells of the osteoblast lineage impairs cell proliferation, differentiation, and bone formation

Calpains are intracellular cysteine proteases, which include widely expressed mu- and m-calpains (1). Both mu-calpains and m-calpains are heterodimers consisting of a large catalytic subunit and a small regulatory subunit. The calpain small subunit encoded by the gene Capn4 directly binds to the intracellular C-terminal tail (C-tail) of the receptor for parathyroid hormone and parathyroid hormone-related peptide and modulates its cellular functions in osteoblasts in vitro (2). To investigate a potential role of the calpain small subunit in osteoblasts in vivo, we generated osteoblast-specific Capn4 knock-out mice using the Cre-LoxP system (3). Mutant mice had smaller bodies with shorter limbs, reduced trabecular bone with thinner cortices, and decreased osteoblast number. In vitro analysis confirmed that deletion of Capn4 in osteoblasts severely affected multiple osteoblast functions including proliferation, differentiation, and matrix mineralization. Collectively, our findings provide the first in vivo demonstration that the calpain small subunit is essential for proper osteoblast activity and bone remodeling.     

Calpain protects the heart from hemodynamic stress

Calpains make up a family of Ca(2+)-dependent intracellular cysteine proteases that include ubiquitously expressed μ- and m-calpains. Both are heterodimers consisting of a distinct large catalytic subunit (calpain 1 for μ-calpain and calpain 2 for m-calpain) and a common regulatory subunit (calpain 4). The physiological roles of calpain remain unclear in the organs, including the heart, but it has been suggested that calpain is activated by Ca(2+) overload in diseased hearts, resulting in cardiac dysfunction. In this study, cardiac-specific calpain 4-deficient mice were generated to elucidate the role of calpain in the heart in response to hemodynamic stress. Cardiac-specific deletion of calpain 4 resulted in decreased protein levels of calpains 1 and 2 and showed no cardiac phenotypes under base-line conditions but caused left ventricle dilatation, contractile dysfunction, and heart failure with interstitial fibrosis 1 week after pressure overload. Pressure-overloaded calpain 4-deficient hearts took up a membrane-impermeant dye, Evans blue, indicating plasma membrane disruption. Membrane repair assays using a two-photon laser-scanning microscope revealed that calpain 4-deficient cardiomyocytes failed to reseal a plasma membrane that had been disrupted by laser irradiation. Thus, the data indicate that calpain protects the heart from hemodynamic stresses, such as pressure overload.     

Four genes for the calpain family locate on four distinct human chromosomes

Calcium dependent proteases (calpains, CAPNs, E.C.3.4.22.17) constitute a family of proteins which share a homologous cysteine-protease domain (large subunits, L1, L2, and L3) and an E-F hand Ca2(+)-binding domain (L1, L2, L3, and small subunit, S). We have mapped the genes for four calpain proteins (L1, L2, L3, and S) on four distinct human chromosomes by a combination of spot-blot hybridization to flow-sorted chromosomes and Southern hybridization of DNAs from a human x mouse hybrid cell panel. The genes for calpain L1 (CAPN1, large subunit of calpain I), L2 (CAPN2, large subunit of calpain II), L3 (CAPN3, a protein related to the large subunits), and S (CAPN4, a small subunit common to calpains I and II) were assigned to human chromosomes 11, 1, 15, and 19, respectively.     

Targeted suppression of μ‐calpain and caspase 9 expression and its effect on caspase 3 and caspase 7 in satellite cells of Korean Hanwoo cattle

The calpains play an important role in cell death and cell signalling. Caspases catalyse wholesale destruction of cellular proteins which is a major cause of cellular death. The current study looks at the function of μ‐calpain and caspase 9, using RNAi (RNA interference)‐mediated silencing, and to observe the mRNA expression level of caspase genes during satellite cell growth. The satellite cells were treated with siRNA (small interfering RNA) of μ‐calpain and caspase 9 separately. There was reduction of 16 and 24% in CAPN1 (calpain1)‐siRNA2 and CAPN1‐siRNA3 transfected cells respectively, whereas it was 60 and 56% in CAPN1‐siRNA1 and CAPN1‐siRNA4 transfected cells respectively. CAPN1‐siRNA4 and CAPN1‐siRNA1 treated cells showed more reduction in caspase 3 and 7 gene expression. CARD9 (caspase recruitment domain 9)‐siRNA1 and CARD9‐siRNA2‐treated cells showed reduction of 40 and 49% respectively. CARD9‐siRNA1 and CARD9‐siRNA2 showed an increase in caspase 3 gene expression, whereas CARD9‐siRNA2 showed reduction in caspase 7 gene expression. These results suggest a strong cross‐talk between μ‐calpain and the caspase enzyme systems. Suppression of target genes, such as μ‐calpain and caspase 9, might have genuine potential in the treatment of skeletal muscle atrophy.     

The Calpain–Calpastatin System and Cellular Proliferation and Differentiation in Rodent Osteoblastic Cells

The calpain–calpastatin system, which consists of calpains I and II (two ubiquitously distributedcium-activated pa-like cysteine proteases), as well as calpastatin (the endogenous calpain inhibitor), plays an important role in cell proliferation and differentiation in many tissues. However, its contribution to the regulation of osteoprogenitor or pluripotent stem cell proliferation and differentiation into osteoblasts remains poorly defined. In these studies, rat pluripotent mesodermal cells (ROB-C26) and mouse MC3T3-E1 preosteoblasts were induced to differentiate into osteoblasts by long-term culture or in response to bone morphogenetic protein (BMP). The occurrence and distribution of calpain–calpastatin system proteins were determined by immunofluorescent microscopy, measurement of calcium-dependent proteolytic activity, and Western blotting. Treatment of intact MC3T3-E1 cells with an irreversible, membrane-permeable cysteine protease inhibitor attenuated proliferation and alkaline phosphatase upregulation under differentiation-enhancing conditions. Calpain II activity increased during differentiation of MC3T3-E1 cells in postconfluent culture. When ROB-C26 cells were maintained in long-term culture, neutral protease, calpain I, and calpain II activities increased 2- to 3-fold in the absence of BMP. In the presence of partially purified native BMP, neutral protease and calpain I activities also increased similarly, but calpain II activity increased by 10-fold in 3 days. The maximal increase in alkaline phosphatase occurred 4 to 11 days after the calpain II activity had peaked. Induction of differentiation in long-term MC3T3-E1 cultures was associated with higher calpain II and 70- and 110-kDa calpastatin protein levels and lower 17-kDa calpastatin degradation product levels. In conclusion, cysteine protease activity is essential for preosteoblastic proliferation and differentiation. The calpain–calpastatin system is regulated during osteoprogenitor proliferation and differentiation, as it is in other cells, and bone morphogenetic protein is a specific regulator of calpain II.     

Human calpain 7/PalBH associates with a subset of ESCRT-III-related proteins in its N-terminal region and partly localizes to endocytic membrane compartments

Calpain 7 (also known as PalBH) is a mammalian homologue of the Aspergillus, atypical calpain PalB. Knowledge of the biochemical properties of calpain 7 is limited and its function is not yet known. In this study, we investigated the interactions of calpain 7 with all 11 ESCRT-III-related proteins, named charged multivesicular body proteins (CHMPs), and the subcellular localization of calpain 7. Pulldown assays using stable HEK293T transfectants of Strep-tagged calpain 7 revealed interactions of calpain 7 with a subset of FLAG-tagged CHMPs, among which CHMP1B was selected for further analyses. The N-terminal region containing a tandem repeat of MIT domains of calpain 7 was found to be necessary and sufficient for interaction with CHMP1B. Direct interaction was confirmed by a pulldown assay using recombinant proteins. Fluorescence microscopic analysis using HeLa cells revealed that overexpression of GFP-fused CHMPs or a dominant-negative construct of SKD1/Vps4B caused accumulation of epitope-tagged calpain 7 in a punctate pattern in the perinuclear area. Subcellular fractionation revealed that the most of endogenous calpain 7 is present in the cytosol but a small portion is present in particulate fractions. Punctate fluorescence signals of monomeric GFP-fused calpain 7 partly merged with those of endocytosed tetramethylrhodamine-labelled EGF. These results suggest that calpain 7 plays roles in the endosomal pathway by interacting with a subset of ESCRT-III-related proteins.     

Role of peroxynitrite in secondary oxidative damage after spinal cord injury

Peroxynitrite (PON, ONOO(-)), formed by nitric oxide synthase-generated nitric oxide radical ( NO) and superoxide radical (O(2) (-)), is a crucial player in post-traumatic oxidative damage. In the present study, we determined the spatial and temporal characteristics of PON-derived oxidative damage after a moderate contusion injury in rats. Our results showed that 3-nitrotyrosine (3-NT), a specific marker for PON, rapidly accumulated at early time points (1 and 3 h) and a significant increase compared with sham rats was sustained to 1 week after injury. Additionally, there was a coincident and maintained increase in the levels of protein oxidation-related protein carbonyl and lipid peroxidation-derived 4-hydroxynonenal (4-HNE). The peak increases of 3-NT and 4-HNE were observed at 24 h post-injury. In our immunohistochemical results, the co-localization of 3-NT and 4-HNE results indicates that PON is involved in lipid peroxidative as well as protein nitrative damage. One of the consequences of oxidative damage is an exacerbation of intracellular calcium overload, which activates the cysteine protease calpain leading to the degradation of several cellular targets including cytoskeletal protein (alpha-spectrin). Western blot analysis of alpha-spectrin breakdown products showed that the 145-kDa fragments of alpha-spectrin, which are specifically generated by calpain, were significantly increased as soon as 1 h following injury although the peak increase did not occur until 72 h post-injury. The later activation of calpain is most likely linked to PON-mediated secondary oxidative impairment of calcium homeostasis. Scavengers of PON, or its derived free radical species, may provide an improved antioxidant neuroprotective approach for the treatment of post-traumatic oxidative damage in the injured spinal cord.     

On the sequential determinants of calpain cleavage

The structural clues of substrate recognition by calpain are incompletely understood. In this study, 106 cleavage sites in substrate proteins compiled from the literature have been analyzed to dissect the signal for calpain cleavage and also to enable the design of an ideal calpain substrate and interfere with calpain action via site-directed mutagenesis. In general, our data underline the importance of the primary structure of the substrate around the scissile bond in the recognition process. Significant amino acid preferences were found to extend over 11 residues around the scissile bond, from P(4) to P(7)'. In compliance with earlier data, preferred residues in the P(2) position are Leu, Thr, and Val, and in P(1) Lys, Tyr, and Arg. In position P(1) ', small hydrophilic residues, Ser and to a lesser extent Thr and Ala, occur most often. Pro dominates the region flanking the P(2)-P(1)' segment, i.e. positions P(3) and P(2)'-P(4)'; most notable is its occurrence 5.59 times above chance in P(3)'. Intriguingly, the segment C-terminal to the cleavage site resembles the consensus inhibitory region of calpastatin, the specific inhibitor of the enzyme. Further, the position of the scissile bond correlates with certain sequential attributes, such as secondary structure and PEST score, which, along with the amino acid preferences, suggests that calpain cleaves within rather disordered segments of proteins. The amino acid preferences were confirmed by site-directed mutagenesis of the autolysis sites of Drosophila calpain B; when amino acids at key positions were changed to less preferred ones, autolytic cleavage shifted to other, adjacent sites. Based on these preferences, a new fluorogenic calpain substrate, DABCYLTPLKSPPPSPR-EDANS, was designed and synthesized. In the case of micro- and m-calpain, this substrate is kinetically superior to commercially available ones, and it can be used for the in vivo assessment of the activity of these ubiquitous mammalian calpains.