Calcium-dependent Cysteine Proteinases Research Articles

Evidence for calpains in cancer metastasis

Metastatic dissemination represents the final stage of tumor progression as well as the principal cause of cancer-associated deaths. Calpains are a conserved family of calcium-dependent cysteine proteinases with ubiquitous or tissue-specific expression. Accumulating evidence indicates a central role for calpains in tumor migration and invasion via participating in several key processes, including focal adhesiondynamics, cytoskeletal remodeling, epithelial-to-mesenchymal transition, and apoptosis. Activated after the increased intracellular calcium concentration ( ) induced by membrane channels and extracellular or intracellular stimuli, calpains induce the limited cleavage or functional modulation of various substrates that serve as metastatic mediators. This review covers established literature to summarize the mechanisms and underlying signaling pathways of calpains in cancer metastasis, making calpains attractive targets for aggressive tumor therapies.

Impact of PRRS Challenge on Calpain and Calpastatin Activity in Skeletal Muscle of Young Pigs

ObjectivesPorcine Reproductive and Respiratory Syndrome (PRRS) virus challenges in growing pigs are known to negatively affect growth performance. One hypothesis to explain this observation is that an increase in calpain system initiated protein degradation in muscle contributes to the reduction of muscle protein accretion. Calpain-1 and –2 are calcium dependent cysteine proteinases that are regulated by an endogenous inhibitor, calpastatin. The objective of this experiment was to define the extent to which PRRS challenge influences skeletal muscle calpain and calpastatin activity at 2 stages of infection (viremia and seroconversion).Materials and MethodsThirty-three pigs (11.3 ± 1.54 kg body weight (BW), approximately 6 wk of age) were assigned to a contemporary group (3 pigs per group) based on age and weight. Pigs were housed in individual pens in separate rooms depending on treatment. One pig per group was assigned to 1 of 3 treatments, 1)PRRS [PRRS challenged with ORF5 RFLP1–3-4 isolate (n = 11)], 2)Pair-fed [Non-challenged, daily feed intake matched to challenged pigs (n = 11)], and 3)Ad-lib [Non-challenged, Ad libitum fed (n = 11)]. Weekly BW, feed disappearance, and feed efficiency were assessed over each period. At days post inoculation (dpi) 9–10 (7 groups; viremia period) and 16 to 17 (4 groups; seroconversion period), pigs were euthanized and a 50-g sample of longissimus muscle was collected and homogenized in pre-rigor extraction buffer (100 mM Tris-HCl pH 8.3, 10 mM EDTA, 100 mg/L trypsin inhibitor, 2 μM E-64, and 0.1% 2-mercaptoethanol). Calpains and calpastatins were separated using anion exchange chromatography and quantified using casein as a substrate. Data from each time period were analyzed as a separate experiment. Data were analyzed using the mixed procedure in JMP Pro version 13.1 with fixed effects of treatment and a random effect of contemporary group. Means separations were conducted via Student t test with P < 0.05 considered significantly different.ResultsAt both time points, PRRS pigs had reduced ADG and G:F compared with Ad-lib and Pair-fed pigs (P < 0.01). Additionally, ADFI was reduced in PRRS and Pair-fed pigs compared with Ad-lib pigs (P < 0.001). At dpi 9–10, PRRS challenge resulted in greater calpastatin-2 (1.04) and total calpastatin (1.70) activity compared with the Pair-fed pigs (0.58, 1.06 respectively; P < 0.05). By contrast, at dpi 16 to 17, PRRS challenge resulted in greater calpastatin-1 (1.08) and total calpastatin activity (1.98) compared with Pair-fed controls (0.54, 0.89, respectively; P < 0.05). Calpain-1 and –2 were not significantly altered by treatment at either time point. The ratio of calpain-2 to total calpastatin activity was significantly (P < 0.05) less in muscle from PRRS challenged pigs (1.16) compared with muscle from the Pair-fed (2.47), but not the Ad-lib pigs (1.58).ConclusionThe results demonstrate that a decline in voluntary feed intake was not singularly responsible for the observed response to PRRS challenge. These results are counter to the hypothesis that PRRS challenge would decrease calpastatin activity. Moreover, the results of this study indicate a difference in calpastatin response to PRRS challenge, notably that PRRS challenge increased calpastatin-2 activity at dpi 9 to 10 and calpastatin-1 activity at dpi 16 to 17. The switch indicates a temporal response in skeletal muscle proteolysis due to PRRS virus challenge.

Impact of PRRS Challenge on Calpain and Calpastatin Activity in Skeletal Muscle of Young Pigs

ObjectivesPorcine Reproductive and Respiratory Syndrome (PRRS) virus challenges in growing pigs are known to negatively affect growth performance. One hypothesis to explain this observation is that an increase in calpain system initiated protein degradation in muscle contributes to the reduction of muscle protein accretion. Calpain-1 and –2 are calcium dependent cysteine proteinases that are regulated by an endogenous inhibitor, calpastatin. The objective of this experiment was to define the extent to which PRRS challenge influences skeletal muscle calpain and calpastatin activity at 2 stages of infection (viremia and seroconversion).Materials and MethodsThirty-three pigs (11.3 ± 1.54 kg body weight (BW), approximately 6 wk of age) were assigned to a contemporary group (3 pigs per group) based on age and weight. Pigs were housed in individual pens in separate rooms depending on treatment. One pig per group was assigned to 1 of 3 treatments, 1)PRRS [PRRS challenged with ORF5 RFLP1–3-4 isolate (n = 11)], 2)Pair-fed [Non-challenged, daily feed intake matched to challenged pigs (n = 11)], and 3)Ad-lib [Non-challenged, Ad libitum fed (n = 11)]. Weekly BW, feed disappearance, and feed efficiency were assessed over each period. At days post inoculation (dpi) 9–10 (7 groups; viremia period) and 16 to 17 (4 groups; seroconversion period), pigs were euthanized and a 50-g sample of longissimus muscle was collected and homogenized in pre-rigor extraction buffer (100 mM Tris-HCl pH 8.3, 10 mM EDTA, 100 mg/L trypsin inhibitor, 2 μM E-64, and 0.1% 2-mercaptoethanol). Calpains and calpastatins were separated using anion exchange chromatography and quantified using casein as a substrate. Data from each time period were analyzed as a separate experiment. Data were analyzed using the mixed procedure in JMP Pro version 13.1 with fixed effects of treatment and a random effect of contemporary group. Means separations were conducted via Student t test with P < 0.05 considered significantly different.ResultsAt both time points, PRRS pigs had reduced ADG and G:F compared with Ad-lib and Pair-fed pigs (P < 0.01). Additionally, ADFI was reduced in PRRS and Pair-fed pigs compared with Ad-lib pigs (P < 0.001). At dpi 9–10, PRRS challenge resulted in greater calpastatin-2 (1.04) and total calpastatin (1.70) activity compared with the Pair-fed pigs (0.58, 1.06 respectively; P < 0.05). By contrast, at dpi 16 to 17, PRRS challenge resulted in greater calpastatin-1 (1.08) and total calpastatin activity (1.98) compared with Pair-fed controls (0.54, 0.89, respectively; P < 0.05). Calpain-1 and –2 were not significantly altered by treatment at either time point. The ratio of calpain-2 to total calpastatin activity was significantly (P < 0.05) less in muscle from PRRS challenged pigs (1.16) compared with muscle from the Pair-fed (2.47), but not the Ad-lib pigs (1.58).ConclusionThe results demonstrate that a decline in voluntary feed intake was not singularly responsible for the observed response to PRRS challenge. These results are counter to the hypothesis that PRRS challenge would decrease calpastatin activity. Moreover, the results of this study indicate a difference in calpastatin response to PRRS challenge, notably that PRRS challenge increased calpastatin-2 activity at dpi 9 to 10 and calpastatin-1 activity at dpi 16 to 17. The switch indicates a temporal response in skeletal muscle proteolysis due to PRRS virus challenge.

Calpains are required for invasive and metastatic potentials of human HCC cells

Calpains are a conserved family of calcium-dependent cysteine proteinases involved in various cellular functions. Two ubiquitous isoforms, µ- and m-calpain, are key members of the calpain family that play essential roles in regulating cell migration and invasion. However, it remains unclear whether they are involved in the progression of hepatocellular carcinoma (HCC). Here, we investigated the functions of µ- and m-calpain in the invasive and metastatic processes of human hepatoma cells. Our results indicated that the expression levels of calpains were elevated in HCC cells compared with those in normal hepatic cells. Our results indicated that small interfering RNA (siRNA)-mediated silencing of µ- and m-calpain expressions significantly suppressed the adhesive, migrative and invasive potentials of human hepatoma cells. The matrix metalloproteinases (MMPs) are key regulators of malignant tumour invasion and metastasis. siRNA-mediated down-regulation of µ- and m-calpain expressions also significantly attenuated MMP-2 and MMP-9 secretion. Thus µ- and m-calpain may play important roles in the invasion and metastasis of human hepatoma cells, and calpains may be drug targets for preventing HCC metastasis.

Calcium-bound structure of calpain and its mechanism of inhibition by calpastatin

Calpains are non-lysosomal calcium-dependent cysteine proteinases that selectively cleave proteins in response to calcium signals and thereby control cellular functions such as cytoskeletal remodelling, cell cycle progression, gene expression and apoptotic cell death. In mammals, the two best-characterized members of the calpain family, calpain 1 and calpain 2 (micro-calpain and m-calpain, respectively), are ubiquitously expressed. The activity of calpains is tightly controlled by the endogenous inhibitor calpastatin, which is an intrinsically unstructured protein capable of reversibly binding and inhibiting four molecules of calpain, but only in the presence of calcium. To date, the mechanism of inhibition by calpastatin and the basis for its absolute specificity have remained speculative. It was not clear how this unstructured protein inhibits calpains without being cleaved itself, nor was it known how calcium induced changes that facilitated the binding of calpastatin to calpain. Here we report the 2.4-A-resolution crystal structure of the calcium-bound calpain 2 heterodimer bound by one of the four inhibitory domains of calpastatin. Calpastatin is seen to inhibit calpain by occupying both sides of the active site cleft. Although the inhibitor passes through the active site cleft it escapes cleavage in a novel manner by looping out and around the active site cysteine. The inhibitory domain of calpastatin recognizes multiple lower affinity sites present only in the calcium-bound form of the enzyme, resulting in an interaction that is tight, specific and calcium dependent. This crystal structure, and that of a related complex, also reveal the conformational changes that calpain undergoes on binding calcium, which include opening of the active site cleft and movement of the domains relative to each other to produce a more compact enzyme.

Trypanosoma cruzi: a stage-specific calpain-like protein is induced after various kinds of stress

Calpains are calcium-dependent cysteine proteinases found in all living organisms and are involved in diverse cellular processes. Calpain-like proteins have been reported after in silico analysis of the Tritryps genome and are believed to play important roles in cell functions of trypanosomatids. We describe the characterization of a member of this family, which is differentially expressed during the life-cycle of Trypanosoma cruzi.

Amelioration of experimental arthritis by a calpain-inhibitory compound: regulation of cytokine production by E-64-d in vivo and in vitro

Calpain, a calcium-dependent cysteine proteinase, has been reported to participate in the pathophysiology of rheumatoid arthritis (RA). The aim of this study is to investigate the therapeutic efficacy of calpain-inhibitory compounds in an animal model of RA and to clarify the underlying mechanisms in vivo and in vitro. Arthritis was induced in BALB/c mice with anti-type II collagen mAbs and LPS, and the mice were treated intra-peritoneally with a high dose (9 mg kg(-1) per day) or low dose (3 mg kg(-1) per day) of E-64-d (a membrane-permeable cysteine proteinase inhibitor) or control diluent. As a result, a high dose of E-64-d significantly alleviated the clinical arthritis and the histopathological findings, compared with the control diluent, although a low dose of E-64-d did not have a significant effect. Next, we evaluated the effects of E-64-d on cytokine mRNA expression at the inflamed joints by quantitative reverse transcription-PCR. High dose of E-64-d significantly decreased IL-6 and IL-1beta mRNA levels at the inflamed joints. The regulatory effects of E-64-d on cytokine production were also confirmed in vitro, using a synovial cell line (E11) and crude synoviocytes derived from RA patients. These results suggest the key roles of calpain in the pathophysiology of arthritis and that calpain-inhibitory compounds might be applicable to the treatment of arthritic diseases such as RA.

A novel extracellular calcium-dependent cysteine proteinase from Crithidia deanei

An extracellular cysteine proteinase from an aposymbiotic strain of Crithidia deanei was purified 39-fold by a combination of anion-exchange and gel filtration chromatographies. The native molecular mass of this proteinase was estimated to be 225 kDa by gel filtration chromatography and it migrates in SDS–PAGE as a single band of 80 kDa. The optimal enzymatic activity on gelatin was found to occur in the presence of calcium at a neutral pH and at 28 °C. The enzyme was completely blocked by E-64 and EGTA, and partially inhibited by iodoacetamide, leupeptin, and EDTA. Compounds such as PMSF, aprotinin, and pepstatin weakly inhibited the enzyme. The protein purified in the present work shares some features with those of the family of neutral calcium-dependent cysteine proteinases named calpains, previously detected in the family Trypanosomatidae as cell-associated enzymes in Leishmania donovani and Trypanosoma brucei. The cysteine proteinase from C. deanei is distinct from the well-characterized mammalian calpains, but some degree of similarity is displayed to invertebrate calpain-related enzymes.

Mutation analysis of the calpastatin gene ( CAST) in patients with Alzheimer's disease

The calpains, a family of calcium-dependent cysteine proteinases, and calpastatin, their endogenous inhibitor protein, are involved in the proteolysis of amyloid precursor protein, which is thought to be abnormal in patients with Alzheimer's disease (AD). Specific inhibitors of calpains attenuate amyloid β peptide-induced neuronal death. We hypothesized that some AD patients have functionally deficient mutation(s) of the CAST gene encoding calpastatin, and we screened 40 Japanese patients with AD for mutations in the coding region of CAST. Nine polymorphisms, -82A/G, IVS7-96A/G, 669A/G, 1223C/G (Ser408Cys), IVS20-10C/T, IVS21-65G/A, IVS22+31T/C, IVS24+38Ins/DelA, and IVS25-32A/G, were identified. The 669A allele causes skipping of exon 11, leading to the loss of 13 residues. Comparisons between 101 patients and 90 controls revealed no significant association between CAST polymorphisms and risk for AD, indicating that genomic variations of CAST are not likely to be substantially involved in the etiology of AD.

Proteolytic Processes Underlying Molt-Induced Claw Muscle Atrophy in Decapod Crustaceans

The claw muscles of large-clawed decapod crustaceans undergo a programmed atrophy in preparation for molting, or ecdysis. This is mediated by five cytosolic proteinases organized into two proteolytic pathways: calcium-dependent and ubiquitin/proteasome-dependent. The calcium-dependent system consists of four calcium-dependent cysteine proteinases (CDPs I, IIa, IIb, and III; native masses 310, 125, 195, and 59 kDa, respectively) that completely degrade myofibrillar proteins and are activated in atrophic muscles. Immunological analysis shows that the active-site sequence in CDP IIa (60-kDa subunit mass) is similar to that in mammalian CDPs (calpains), and that CDP IIb is homologous to a calpain-like gene isolated from Drosophila cDNA libraries. Increased intracellullar Ca2+ stimulates proteolysis in situ , indicating CDPs play an important role in muscle protein catabolism. The ubiquitin/proteasome-dependent system involves the ATP-dependent conjugation of multi-ubiquitin chains to protein by ubiquitin-conjugating enzymes. This acts as a signal for substrate degradation by the 26S proteasome, a multi-subunit complex consisting of a 20S proteasome catalytic “core” and two PA700 (19S) regulatory complexes. Polyubiquitin mRNA, ubiquitin-protein conjugates, and 20S proteasome are elevated about 5-, 8-, and 2-fold, respectively, during atrophy. A heat-induced form of the 20S proteasome hydrolyzes myosin, troponin, and tropomyosin to large fragments in vitro . Biochemical studies identified the branched-chain amino acid-preferring (BrAAP) activity, one of six distinct catalytic components in the complex, as the activity that carries out these initial cleavages. These results indicate that the ubiquitin/proteasome pathway is involved, but its precise role remains to be resolved.

Chemonucleolysis with calpain I in rabbits.

Calpain I is a calcium-dependent cysteine proteinase that has been recently shown to degrade proteoglycan in vitro. The authors injected calpain I, which was purified from human red blood cells, into the intervertebral discs of rabbits. Roentgenograms showed disc space narrowing 1 week after the injection. Histologically, proteoglycan of the nucleus pulposus and anulus fibrosus decreased and notochordal cells in the nucleus pulposus almost disappeared. Biochemical data of the nucleus pulposus showed that the amounts of smaller proteoglycans increased 1 and 4 weeks after the injection. Eight weeks after the injection, histologic and biochemical data showed recovery compared with the data 1 week after injection. These findings show that calpain I is as potent an enzyme as chondroitinase ABC and has milder chemonucleolytic action than chymopapain. Regarding its possible clinical application, autogenous calpain I as purified from the patient's own red blood cells may have advantages over chymopapain and chondroitinase ABC in that it will prevent anaphylactic reaction.

Calcium‐dependent cysteine proteinase (calpain) in human arthritic synovial joints

To study the roles of calpains in the synovial joint in rheumatoid arthritis (RA) and osteoarthritis (OA) and to verify the hypothesis that calpains present in the synovial fluid come from the synovium. We performed immunohistochemical, biochemical, and immunoblotting analyses for calpains in synovial tissues, synovial cell cultures, and synovial fluids. Immunohistochemical staining of RA synovium demonstrated specific cytoplasmic staining of cells in the synovial lining layer, storomal fibroblasts, and endothelial cells. OA synovium showed almost the same intensity and distribution of calpain staining. DEAE-cellulose chromatography of RA and OA synovial extracts and synovial fluids showed a peak of caseinolytic activity attributable to calpain, as well as an inhibitory peak of calpastatin, a specific inhibitor protein of calpains. Immunoblotting using the anticalpain antibody from the calpain peak of RA and OA synovium and synovial fluid showed identity with the heavy subunit of calpain (80 kd). Similarly, calpain existed in the same form (80 kd) in conditioned media (supernatant) obtained from synovial cell cultures, as well as in the synoviocytes. The total specific activity of the 2 calpains in the synovial fluid of RA patients was higher than that of calpastatin. The findings suggest that the extracellular appearance of calpains could be due to the secretion of these proteins from the synovial cells and that calpains may play a role in cartilage damage of RA and OA that occurs in synovial joints.

Biochemical demonstration of calpains and calpastatin in osteoarthritic synovial fluid

Calpains (calcium-dependent cysteine proteinases; optimum pH 7.0-7.5) have been regarded as intracellular proteinases. We examined the cell-free components of synovial fluid from 14 patients with osteoarthritis and demonstrated the existence of calpains, as the caseinolytic activities of chromatographic fractions, together with calpastatin, the specific endogenous inhibitor of calpains. The presence of these calpains and calpastatin was verified by immunoblotting with their respective specific antibodies. Calpain fractions showed proteoglycan-degrading activity. The results suggest that the calpain-calpastatin system may contribute to the turnover of cartilage matrix components.

Calcium dependent cysteine proteinase is a precursor of a chemotactic factor for neutrophils

A new chemotactic factor for neutrophils is generated from calcium dependent cysteine proteinase (calpain) I by autodigestion. An active peptide was isolated from the autodigest and its structure was determined to be an acetylated nonapeptide with the sequence: N-acetyl Ser-Glu-Glu-Ile-Ile-Thr-Pro-Val-Tyr. Compared with the entire sequence of human calpain I, the peptide was identical with the N-terminal amino acid sequence of the large subunit deduced from the cDNA sequence, except that the peptide was devoid of a methionine residue and acetylated at the N-terminus. The acetyl nonapeptide was synthesized and its chemotactic activity was reconfirmed. The biological significance and possible role of this calpain derived chemotactic factor in inflammation are discussed.

An overview of cystic fibrosis

A new chemotactic factor for neutrophils is generated from calcium dependent cysteine proteinase (calpain) I by autodigestion. An active peptide was isolated from the autodigest and its structure was determined to be an acetylated nonapeptide with the sequence: N-acetyl Ser-Glu-Glu-Ile-Ile-Thr-Pro-Val-Tyr. Compared with the entire sequence of human calpain I, the peptide was identical with the N-terminal amino acid sequence of the large subunit deduced from the cDNA sequence, except that the peptide was devoid of a methionine residue and acetylated at the N-terminus. The acetyl nonapeptide was synthesized and its chemotactic activity was reconfirmed. The biological significance and possible role of this calpain derived chemotactic factor in inflammation are discussed.