Calcium-dependent Cysteine Protease Research Articles

Detecting Colorectal Neoplasia Using Specific Fecal Fluorogenic Protease-Sensitive Substrates: A Pilot Study.

identification and removal of advanced adenomas (AA) reduce colorectal cancer (CRC) incidence and potentially mortality. CRC screening often uses fecal immunochemical testing to select high-risk individuals for colonoscopy, despite its low sensitivity for AA and relatively high false-positivity rate. Previous studies have linked proteases to CRC development through their ability to facilitate angiogenesis and immunoregulation. This study aims to identify colorectal neoplasia-associated proteases and their substrates as a potential noninvasive screening test, introducing an innovative application of fecal protease profiling, which has previously been limited to tissue samples. eighteen fluorogenic substrates were designed based on literature. Proteolytic degradation of these substrates was measured in fecal samples of patients with CRC (n = 12), AA (n = 9), nonadvanced adenomas (n = 10), and controls (n = 14). Substrate degradation was correlated to a matched human proteome data set, and underlying proteases were identified based on their recognition patterns. Experiments with protease inhibitors and ZnCl2 were performed to further characterize the involved proteases. in total, 7 of the 18 substrates tested showed a significantly decreased proteolytic degradation in feces from patients with any colorectal neoplasia compared to the control group. The l-aspartic acid-l-glutamic acid substrate (ED) showed significantly decreased degradation in AA and CRC patients. ED degradation significantly decreased with the addition of ZnCl2 and the cysteine protease inhibitor NEM. we successfully developed colorectal neoplasia-specific fluorogenic substrates, highlighting the ED substrate as a potential substrate for the detection of AA and CRC. Although the responsible proteases require further identification, our results suggest an association with calcium-dependent cysteine proteases.

Abstract A016: Targeting calpain-1 and calpain-2 for prevention of breast cancer metastasis: In vivo insights and drug discovery approaches

Abstract Calpain-1 and calpain-2 are heterodimeric calcium-dependent cysteine proteases composed of the catalytic subunits CAPN1 or CAPN2, respectively, and the common regulatory subunit CAPNS1. They are associated with cancer progression, metastasis, and treatment resistance in breast cancer. Here, we present novel insights into the therapeutic potential of calpain inhibition by combining genetic disruption and preclinical mouse tumor models and biosensor-based detection of calpain heterodimerization with ongoing efforts to discover small-molecule and peptide inhibitors. CRISPR-Cas9-mediated knockout of CAPN1, CAPN2, or CAPNS1 in MDA-MB-231 human triple-negative breast cancer (TNBC) cells revealed that disruption of both calpains, through CAPNS1 knockout, significantly reduced cell migration and inhibited spontaneous metastasis in a mouse orthotopic engraftment model by over 80%. Individual knockouts of CAPN1 or CAPN2 also reduced metastasis but to a lesser extent, highlighting the necessity of dual inhibition for optimal therapeutic effect. In parallel, we have been actively seeking small molecules and peptide inhibitors to target calpain through two approaches: inhibiting the PEF-PEF interaction that mediates heterodimerization using small molecules; and targeting the active site using a calpastatin (CAST)-based peptide. To identify small molecules capable of disrupting the PEF-PEF interaction, we conducted in silico screens of over 3.6 million compounds from several libraries. These compounds were evaluated based on their calculated binding affinity and potential to sterically hinder the conformational changes required for calpain activity. The CAST-based peptide inhibitor was designed based on the active site binding B-domain and tested on a purified calpain-2. These efforts lay the groundwork for novel therapeutic approaches targeting calpain-1 and calpain-2, which have emerged as promising targets for preventing metastasis in TNBC. We will present our progress in identifying and validating these small molecules and peptides for further development. Citation Format: Ivan Shapovalov, Pitambar Poudel, Shailesh K. Panday, Danielle Harper, Jung Yeon Min, Yan Gao, Kazem Nouri, Emil Alexov, Peter A. Greer. Targeting calpain-1 and calpain-2 for prevention of breast cancer metastasis: In vivo insights and drug discovery approaches. [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Optimizing Therapeutic Efficacy and Tolerability through Cancer Chemistry; 2024 Dec 9-11; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(12_Suppl):Abstract nr A016

Calpain 8 as a potential biomarker regulates the progression of pancreatic cancer via EMT and AKT/ERK pathway

Calpain is a non-lysozyme, calcium-dependent intracellular cysteine protease that has been shown to play a role in tumor proliferation, survival, migration, invasion, and apoptosis. Dysregulation of calpain expression is closely related to tumorigenesis. However, the role of calpain-8 (CAPN8), as a member of the calpain family, in pancreatic cancer (PC) is remains unclear. In elucidating the mechanism of CAPN8 in PC, a comprehensive bioinformatics analysis and in vitro experiments were conducted. The TCGA database was used to explore the expression level of CAPN8, and the results in PC tissues and cell lines were verified. Then, the correlation between CAPN8 and clinicopathological features was analyzed. Additionaly, promoter methylation, immune infiltration, and GO/KEGG enrichment analyses were performed. Lastly, the molecular mechanism of CAPN8 in PC was investigated by using cell counting kit (CCK) 8, transwell, wound healing, Western blot assays, and so on. Results indicate that CAPN8 was highly expressed in PC and correlated with poor prognosis and advanced TNM stage. In addition, a low level of immune infiltration was closely associated with the high expression level of CAPN8. Based on these findings, we hypothesized that CAPN8 is a potential biomarker that regulates progression of PC via EMT and the AKT/ERK pathway. SignificanceThrough comprehensive biological information and in vitro experiments, CAPN8 has been confirmed to play an important role in regulating pancreatic cancer (PC) proliferation, migration and invasion. CAPN8 is found to be closely related to the diagnosis, survival and prognosis of PC. Above all, CAPN8 may be a potential biomarker for the diagnosis and prognosis of PC.

Abstract 16819: Activation of Mitochondrial Calpain 1 Contributes to Complex I Damage During ER Stress

Introduction: ER (Endoplasmic reticulum) stress is increased in failing hearts. The increased ER stress impairs complex I in cardiac mitochondria (MITO). Calpain 1 (CPN1) and calpain 2 (CPN2) are calcium-dependent cysteine proteases located in cytosol and MITO (mCPN1 and mCPN2). Activation of mCPN1 contributes to complex I damage during ischemia-reperfusion (IR) and aging. Activation of the mCPN2 causes complex I damage following IR. However. the role of mCPN1 and mCPN2 activation in ER stress-mediated complex I damage remains unclear. Hypothesis: ER stress leads to complex I damage by activating mCPN1 and mCPN2. Methods: Wild type (WT) and conditional cardiomyocyte specific calpain 4 (CPN4) knockout (KO) mice were used. CPN4 is a small regulatory subunit of CPN1 and 2, and KO of CPN4 eliminates CPN1 and CPN2 activities. Tunicamycin (TUNI, 0.4 mg/kg) was used to induce ER stress in both WT and KO mice through one time IP injection. DMSO was used as vehicle treatment. Mice were sacrificed for cardiac mitochondrial isolation after 72 hours of vehicle or TUNI treatment. Results: ER stress was increased in both WT and KO mice, as shown by increased BIP in TUNI-treated mice (Figure, Panel A). Compared to vehicle, the rate of oxidative phosphorylation (OXPHOS) was decreased in TUNI-treated WT when glutamate + malate was used as complex I substrate (Panel B). In contrast, TUNI treatment did not alter OXPHOS with complex I substrate in KO (Panel B). Direct measurement of complex I activity showed that TUNI treatment only decreased complex I in MITO from WT but not from KO (Panel C). Proteomic study showed that the contents of complex I subunits including NDUFV2 and ND5 were decreased in WT but not in KO mice (Panel D); confirmed by western blotting (data not shown). These results indicate that activation of mCPN1 and mCPN2 contributes to the degradation of complex I subunits during ER stress. Conclusion: Induction of acute ER stress using TUNI damages complex I by activating mCPN1 and mCPN2.

Myeloid-specific deletion of Capns1 attenuates myocardial infarction injury via restoring mitochondrial function and inhibiting inflammasome activation.

BackgroundMitochondrial dysfunction of macrophage-mediated inflammatory response plays a key pathophysiological process in myocardial infarction (MI). Calpains are a well-known family of calcium-dependent cysteine proteases that regulate a variety of processes, including cell adhesion, proliferation, and migration, as well as mitochondrial function and inflammation. CAPNS1, the common regulatory subunit of calpain-1 and 2, is essential for the stabilization and activity of the catalytic subunit. Emerging studies suggest that calpains may serve as key mediators in mitochondria and NLRP3 inflammasome. This study investigated the role of myeloid cell calpains in MI. MethodsMI models were constructed using myeloid-specific Capns1 knockout mice. Cardiac function, cardiac fibrosis, and inflammatory infiltration were investigated. In vitro, bone marrow-derived macrophages (BMDMs) were isolated from mice. Mitochondrial function and NLRP3 activation were assessed in BMDMs under LPS stimulation. ATP5A1 knockdown and Capns1 knock-out mice were subjected to MI to investigate their roles in MI injury. ResultsAblation of calpain activities by Capns1 deletion improved the cardiac function, reduced infarct size, and alleviated cardiac fibrosis in mice subjected to MI. Mechanistically, Capns1 knockout reduced the cleavage of ATP5A1 and restored the mitochondria function thus inhibiting the inflammasome activation. ATP5A1 knockdown antagonized the protective effect of Capns1 mKO and aggravated MI injury. ConclusionThis study demonstrated that Capns1 depletion in macrophages mitigates MI injury via maintaining mitochondrial homeostasis and inactivating the NLRP3 inflammasome signaling pathway. This study may offer novel insights into MI injury treatment.

Calpain signaling: from biology to therapeutic opportunities in neurodegenerative disorders.

Neurodegenerative disorders represent a major and growing healthcare challenge globally. Among the numerous molecular pathways implicated in their pathogenesis, calpain signaling has emerged as a crucial player in neuronal dysfunction and cell death. Calpain is a family of calcium-dependent cysteine proteases that is involved in many biological processes, such as signal transduction, cytoskeleton remodeling, and protein turnover. Dysregulation of calpain activation and activity has been associated with several neurodegenerative diseases, including Alzheimer's, Parkinson's, and Huntington's diseases. Understanding the intricate structure of calpains is crucial for unraveling their roles in cellular physiology and their implications in pathology. In addition, the identification of diverse abnormalities in both humans and other animal models with deficiencies in calpain highlights the significant progress made in understanding calpain biology. In this comprehensive review, we delve into the recent roles attributed to calpains and provide an overview of the mechanisms that govern their activity during the progression of neurodegenerative diseases. The possibility of utilizing calpain inhibition as a potential therapeutic approach for treating neuronal dysfunctions in neurodegenerative disorders would be an area of interest in future calpain research.

Prevention of noise-induced hearing loss by calpain inhibitor MDL-28170 is associated with upregulation of PI3K/Akt survival signaling pathway.

Noise-induced calcium overload in sensory hair cells has been well documented as an early step in the pathogenesis of noise-induced hearing loss (NIHL). Alterations in cellular calcium homeostasis mediate a series of cellular events, including activation of calcium-dependent protein kinases and phosphatases. Using cell-membrane- and blood-brain-barrier-permeable calpain-1 (μ-calpain) and calpain-2 (m-calpain) inhibitor MDL-28170, we tested the involvement of calpains, a family of calcium-dependent cysteine proteases, and the potential of MDL-28170 in preventing NIHL. CBA/J mice at the age of 12 weeks were exposed to broadband noise with a frequency spectrum from 2-20 kHz for 2 h at 101 dB sound pressure level to induce permanent hearing loss as measured by auditory brainstem response and distortion product otoacoustic emissions. Morphological damage was assessed by quantification of remaining sensory hair cells and inner hair cell synapses 2 weeks after the exposure. MDL-28170 treatment by intraperitoneal injection significantly attenuated noise-induced functional deficits and cochlear pathologies. MDL-28170 treatment also prevented noise-induced cleavage of alpha-fodrin, a substrate for calpain-1. Furthermore, MDL-28170 treatment prevented reduction of PI3K/Akt signaling after exposure to noise and upregulated p85α and p-Akt (S473) in outer hair cells. These results indicate that noise-induced calpain activation negatively regulates PI3K/Akt downstream signaling, and that prevention of NIHL by treatment with MDL-28170 is associated with upregulation of PI3K/Akt survival signaling pathways.

Calpain-mediated Mechanoptosis in Breast Adenocarcinoma.

Calpains belong to a family of important calcium-dependent cysteine proteases. They are involved in intracellular processes including cytoskeleton disorganization and substrate proteolysis. They also enhance apoptosis and cell to cell adhesion. Calpains demonstrate also a mechanosensory function in neoplastic and malignant cells due to their implication in mechanoptosis. This is a specific type of apoptotic death induced by strong external mechanical stimuli. Anti-cytoskeleton rigidity inhibition strategies based on calpain induction lead to increased apoptosis of tumor transformed cells. Elevated intracellular calcium concentration mediated by specific receptors and channels activates calpains. In the current molecular review, we explored the role of calpains in calcium-dependent signa transduction pathways in breast adenocarcinoma in conjunction with novel agents that activate their important anti-tumor functions.

Missense mutation of c.635 T > C in CAPN3 impairs muscle injury repair in a Limb-Girdel Muscular Dystropy Model.

Limb-girdle muscular dystrophy recessive 1 (LGMDR1), previously known as LGMD2A, is a specific LGMD caused by a gene mutation encoding the calcium-dependent neutral cysteine protease calpain-3 (CAPN3). In our study, the compound heterozygosity with two missense variants c.635 T > C (p.Leu212Pro) and c.2120A > G (p.Asp707Gly) was identified in patients with LGMDR1. However, the pathogenicity of c.635 T > C has not been investigated. To evaluate the effects of this novel likely pathogenic variant to the motor system, the mouse model with c.635 T > C variant was prepared by CRISPR/Cas9 gene editing technique. The pathological results revealed that a limited number of inflammatory cells infiltrated the endomyocytes of certain c.635 T > C homozygous mice at 10 months of age. Compared with wild-type mice, motor function was not significantly impaired in Capn3 c. 635 T > C homozygous mice. Western blot and immunofluorescence assays further indicated that the expression levels of the Capn3 protein in muscle tissues of homozygous mice were similar to those of wild-type mice. However, the arrangement and ultrastructural alterations of the mitochondria in the muscular tissues of homozygous mice were confirmed by electron microscopy. Subsequently, muscle regeneration of LGMDR1 was simulated using cardiotoxin (CTX) to induce muscle necrosis and regeneration to trigger the injury modification process. The repair of the homozygous mice was significantly worse than that of the control mice at day 15 and day 21 following treatment, the c.635 T > C variant of Capn3 exhibited a significant effect on muscle regeneration of homozygous mice and induced mitochondrial damage. RNA-sequencing results demonstrated that the expression levels of the mitochondrial-related functional genes were significantly downregulated in the mutant mice. Taken together, the results of the present study strongly suggested that the LGMDR1 mouse model with a novel c.635 T > C variant in the Capn3 gene was significantly dysfunctional in muscle injury repair via impairment of the mitochondrial function.

Osteocyte-Derived CaMKK2 Regulates Osteoclasts and Bone Mass in a Sex-Dependent Manner through Secreted Calpastatin.

Calcium/calmodulin (CaM)-dependent protein kinase kinase 2 (CaMKK2) regulates bone remodeling through its effects on osteoblasts and osteoclasts. However, its role in osteocytes, the most abundant bone cell type and the master regulator of bone remodeling, remains unknown. Here we report that the conditional deletion of CaMKK2 from osteocytes using Dentine matrix protein 1 (Dmp1)-8kb-Cre mice led to enhanced bone mass only in female mice owing to a suppression of osteoclasts. Conditioned media isolated from female CaMKK2-deficient osteocytes inhibited osteoclast formation and function in in vitro assays, indicating a role for osteocyte-secreted factors. Proteomics analysis revealed significantly higher levels of extracellular calpastatin, a specific inhibitor of calcium-dependent cysteine proteases calpains, in female CaMKK2 null osteocyte conditioned media, compared to media from female control osteocytes. Further, exogenously added non-cell permeable recombinant calpastatin domain I elicited a marked, dose-dependent inhibition of female wild-type osteoclasts and depletion of calpastatin from female CaMKK2-deficient osteocyte conditioned media reversed the inhibition of matrix resorption by osteoclasts. Our findings reveal a novel role for extracellular calpastatin in regulating female osteoclast function and unravel a novel CaMKK2-mediated paracrine mechanism of osteoclast regulation by female osteocytes.

Distinct Changes in Calpain and Calpastatin during PNS Myelination and Demyelination in Rodent Models.

Myelin forming around axons provides electrical insulation and ensures rapid and efficient transmission of electrical impulses. Disruptions to myelinated nerves often result in nerve conduction failure along with neurological symptoms and long-term disability. In the central nervous system, calpains, a family of calcium dependent cysteine proteases, have been shown to have a role in developmental myelination and in demyelinating diseases. The roles of calpains in myelination and demyelination in the peripheral nervous system remain unclear. Here, we show a transient increase of activated CAPN1, a major calpain isoform, in postnatal rat sciatic nerves when myelin is actively formed. Expression of the endogenous calpain inhibitor, calpastatin, showed a steady decrease throughout the period of peripheral nerve development. In the sciatic nerves of Trembler-J mice characterized by dysmyelination, expression levels of CAPN1 and calpastatin and calpain activity were significantly increased. In lysolecithin-induced acute demyelination in adult rat sciatic nerves, we show an increase of CAPN1 and decrease of calpastatin expression. These changes in the calpain-calpastatin system are distinct from those during central nervous system development or in acute axonal degeneration in peripheral nerves. Our results suggest that the calpain-calpastatin system has putative roles in myelination and demyelinating diseases of peripheral nerves.

Exercise mitigates calpain induced Purkinje cell loss in diabetes

Calpain-1 is a ubiquitous calcium dependent cysteine protease and found in cytoplasm as well as mitochondria. We have earlier reported that active calpain-1 is translocated from cytosol to mitochondria and activates MMP9. Calpain-1 activation is detrimental to the heart in several different ways, but there is little evidence that it can degrade Purkinje cell protein (PCP-4) and impair contractility in diabetes. Our hypothesis is that in diabetes, PCP-4 is degraded by calpain-1, causing contractile dysfunction that can be mitigated by exercise. To test this hypothesis, we recruited four groups of mice, 1) db/+ control, 2) db/+ with exercise, 3) db/db, 4) db/db with exercise. The mice were exercised on treadmill for 8 weeks as per American Veterinary Research Guidelines. Adding calcium to isolated cardiomyocytes caused them to lose shape and die. Compared with live myocytes, we observed high calpain-1 levels as well as significantly lower levels of PCP-4 and increased levels of calmodulin and calmodulin kinase II (CaMKII) in dead myocytes. We used the CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) plasmid to knock down calpain-1 in HL-1 myocytes which restored the levels of PCP-4 along with calmodulin and CaMKII. In vivo, we found upregulated levels of calpain-1 in db/db mice (diabetic) as compared to db/+ which were mitigated in the exercised mice. Conclusively our data strongly suggests that in diabetes there is high induction of calpain-1 with degrades PCP-4, a protein important for contractility and exercise can mitigate this.

Calpain-mediated protein targets in cardiac mitochondria following ischemia\u2013reperfusion

Calpain 1 and 2 (CPN1/2) are calcium-dependent cysteine proteases that exist in cytosol and mitochondria. Pharmacologic inhibition of CPN1/2 decreases cardiac injury during ischemia (ISC)–reperfusion (REP) by improving mitochondrial function. However, the protein targets of CPN1/2 activation during ISC–REP are unclear. CPN1/2 include a large subunit and a small regulatory subunit 1 (CPNS1). Genetic deletion of CPNS1 eliminates the activities of both CPN1 and CPN2. Conditional cardiomyocyte specific CPNS1 deletion mice were used in the present study to clarify the role of CPN1/2 activation in mitochondrial damage during ISC–REP with an emphasis on identifying the potential protein targets of CPN1/2. Isolated hearts from wild type (WT) or CPNS1 deletion mice underwent 25 min in vitro global ISC and 30 min REP. Deletion of CPNS1 led to decreased cytosolic and mitochondrial calpain 1 activation compared to WT. Cardiac injury was decreased in CPNS1 deletion mice following ISC–REP as shown by the decreased infarct size compared to WT. Compared to WT, mitochondrial function was improved in CPNS1 deletion mice following ischemia–reperfusion as shown by the improved oxidative phosphorylation and decreased susceptibility to mitochondrial permeability transition pore opening. H2O2 generation was also decreased in mitochondria from deletion mice following ISC–REP compared to WT. Deletion of CPNS1 also resulted in less cytochrome c and truncated apoptosis inducing factor (tAIF) release from mitochondria. Proteomic analysis of the isolated mitochondria showed that deletion of CPNS1 increased the content of proteins functioning in regulation of mitochondrial calcium homeostasis (paraplegin and sarcalumenin) and complex III activity. These results suggest that activation of CPN1 increases cardiac injury during ischemia–reperfusion by impairing mitochondrial function and triggering cytochrome c and tAIF release from mitochondria into cytosol.

Abstract 14216: Mitochondrial Dysfunction in Aged Hearts: Role of Mitochondrial Calpain Activation

Introduction: Mitochondrial function is impaired in aged hearts. Calpain 1 (CPN1) is a calcium-dependent cysteine protease located in cytosol (cCPN1) and mitochondria (mCPN1). Activation of CPN1 contributes to mitochondrial dysfunction during ischemia-reperfusion. Induction of acute ER (endoplasm reticulum) stress causes CPN1 activation in adult hearts. We find that aging leads to increased ER stress that may favor CPN1 activation. Hypothesis: ER stress induced CPN1 activation leads to mitochondrial dysfunction during aging. Methods: Male adult (3 mo.) and aged (24 mo.) mice were provided by NIA. 4-PBA (1g/kg/day) was dissolved in drinking water with sucrose (0.2 g/100 ml) as sweetener. Sucrose water was used as vehicle control. Mice were treated for two weeks with 4-PBA or vehicle followed by the isolation of cardiac cytosol, SSM (subsarcolemmal mitochondria) and IFM (interfibrillar mitochondria). Results: Oxidative phosphorylation was decreased in IFM from 24 mo. compared to 3 mo. when glutamate + malate was used as complex I substrate (Fig A). The 4-PBA treatment improved oxidative phosphorylation in aged IFM (Figure A). The cCPN1 was activated in 24 mo. as shown by increased spectrin cleavage (Figure B). The α1 subunit of pyruvate dehydrogenase (PDH) is a substrate of mCPN1. The content of the PDH α1 subunit was decreased in 24 mo. compared to 3 mo., indicating that the mCPN1 was activated during aging (Figure C). The 4-PBA treatment decreased the activation of cCPN1 and mCPN1 in aged mice. SSM function is also impaired during aging (data not shown). Conclusions: Aging increased the activation of both cCPN1 and mCPN1. Attenuation of ER stress in 24 mo. mice using 4-PBA decreased cCPN1 and mCPN1 activation. Decreased CPN1 activation was accompanied by improved mitochondrial function . The results support that activation of calpain 1, most likely mCPN1, is a likely cause of increased mitochondrial dysfunction during aging.

Calpain-Mediated Mitochondrial Damage: An Emerging Mechanism Contributing to Cardiac Disease.

Calpains belong to the family of calcium-dependent cysteine proteases expressed ubiquitously in mammals and many other organisms. Activation of calpain is observed in diseased hearts and is implicated in cardiac cell death, hypertrophy, fibrosis, and inflammation. However, the underlying mechanisms remain incompletely understood. Recent studies have revealed that calpains target and impair mitochondria in cardiac disease. The objective of this review is to discuss the role of calpains in mediating mitochondrial damage and the underlying mechanisms, and to evaluate whether targeted inhibition of mitochondrial calpain is a potential strategy in treating cardiac disease. We expect to describe the wealth of new evidence surrounding calpain-mediated mitochondrial damage to facilitate future mechanistic studies and therapy development for cardiac disease.

Thioredoxin-1 regulates calcium homeostasis in MPP+ /MPTP-induced Parkinson's disease models.

Disturbance in calcium (Ca2+ ) homeostasis has been involved in a variety of neuropathological conditions including Parkinson's disease (PD). The Ca2+ channel, transient receptor potential channel 1 (TRPC1), plays a protective role in regulating entry of Ca2+ activated by store depletion of Ca2+ in endoplasmic reticulum (ER). We have showed that thioredoxin-1 (Trx-1) plays a role in suppressing ER stress in PD. However, whether Trx-1 regulates TRPC1 expression in PD is still unknown. In the present study, we demonstrated that treatment of 1-methyl-4-phenylpyridinum ion (MPP+ ) significantly reduced the expression of TRPC1 in PC12 cells, which was restored by Trx-1 overexpression, and further decreased significantly by Trx-1 siRNA. Moreover, we found that Ca2+ entered into the cells was decreased by MPP+ in PC 12 cells, which was restored by Trx-1 overexpression, and further decreased by Trx-1 siRNA. MPP+ significantly increased calcium-dependent cysteine protease calpain1 expression in PC12 cells, which was suppressed by Trx-1 overexpression. Calpain1 expression was increased by Trx-1 siRNA or SKF96365, an inhibitor of TRPC1. Moreover, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) decreased TRPC1 expression in the substantia nigra pars compacta region (SNpc), which was restored in mice overexpressing Trx-1, and further decreased in mice of knockdown Trx-1. Inversely, the expression of calpain1 was increased by MPTP, which was suppressed in mice overexpressing Trx-1, and further increased in mice of knockdown Trx-1. In conclusion, Trx-1 regulates the Ca2+ entry through regulating TRPC1 expression after treatment of MPP+ /MPTP.

Evidence of new calcium-dependent cysteine protease of Melia dubia latex with fibrinolytic potential

Evidence of new calcium-dependent cysteine protease of Melia dubia latex with fibrinolytic potential

Commentary on: Calpain-2 participates in the process of calpain-1 inactivation

Calpain belongs to the calcium-dependent non-lysosomal cysteine protease. Calpain-1 (C1) and calpain-2 (C2) expression are ubiquitous in mammals and an important mediator of the action of calcium. Specific substrate cleavage by C1 and C2 is critical for several calcium-dependent cellular pathways including neuronal function, muscle contraction, signal transduction, cell differentiation, proliferation, and apoptosis. Research suggests that C1 and C2 perform similar functions due to their structurally highly similar isoforms. Increasing evidence suggests that C1 and C2 carry out their specific function in vivo. A recent paper published by Shinkai-Ouchi et al. (Bioscience Reports (2020) 40, DOI: 10.1042/BSR20200552) elucidated the mechanism to differentiate the function of each calpain with respect to the efficiency and longevity for proteolysis after activation. Further, the study represented that C1 and C2 do not synergistically perform their work in vitro. On the other hand, the activity of C1 is reduced in presence of C2. This insight establishes the platform for future studies to examine how C2 regulates the C1 for substrate proteolysis.

Intracellular versus extracellular inhibition of calpain I causes differential effects on pain in a rat model of joint inflammation.

Calpain I is a calcium-dependent cysteine protease which has dual effects on tissue inflammation depending on its cellular location. Intracellularly, calpain I has pro-inflammatory properties but becomes anti-inflammatory when exteriorised into the extracellular space. In this study, the effect of calpain I on joint pain was investigated using the kaolin/carrageenan model of acute synovitis. Evoked pain behaviour was determined by von Frey hair algesiometry and non-evoked pain was measured using dynamic hindlimb weight bearing. Local administration of calpain I reduced secondary allodynia in the acute inflammation model and this effect was blocked by the cell impermeable calpain inhibitor E-64c. Calpain I also blocked the algesic effect of the protease activated receptor-2 (PAR-2) cleaving enzyme mast cell tryptase. The cell permeable calpain blocker E-64d also produced analgesia in arthritic joints. These data suggest that calpain I produces disparate effects on joint pain viz. analgesia when present extracellularly by disarming PAR-2, and pro-algesic when the enzyme is inside the cell.

Calpain Activation and Organ Failure in Sepsis: Molecular Insights and Therapeutic Perspectives.

Sepsis is a severe systemic response to infection; its ensuing organ failure commonly portends an unfavorable prognosis. Despite the fact that sepsis has been studied for decades, the molecular mechanisms underlying sepsis-induced organ dysfunction remain elusive and more complex than previously thought, and effective therapies are extremely limited. Calpain is a type of calcium-dependent cysteine protease that includes dozens of isoforms. Calpain, as well as its endogenous-specific inhibitor calpastatin, have been implicated in the pathogenesis of sepsis-induced organ dysfunction. Further, there is an accumulating body of evidence supporting the beneficial effect of calpain inhibition or regulation on multiple organ failure in sepsis. Better understanding of the underlying molecular mechanisms is helpful in the development of calpain/calpastatin-targeted therapeutic strategies to protect against sepsis-induced organ injury. The aim of this review is to summarize the recent literature and evidence surrounding the role of the calpain/calpastatin system in the process of organ dysfunction caused by sepsis-including regulation of cell death, modulation of inflammatory response, and disruption of critical proteins-to provide guidance for future research and therapy development.