Cyclin-dependent kinase 5 (CDK5) inhibitors in Parkinson disease.

Cyclin-dependent kinase 5 (Cdk5) is a protein expressed in postmitotic neurons in the central nervous system (CNS). Cdk5 is activated by p35 and p39 which are neuron regulatory subunits. Cdk5/p35 complex is activated by calpain protease to form Cdk5/p35 which has a neuroprotective effect by regulating the synaptic plasticity and memory functions. However, exaggerated Cdk5 is implicated in different types of neurodegenerative diseases including Parkinson disease (PD). Therefore, modulation of Cdk5 signalling may mitigate PD neuropathology. Therefore, the aim of the present review was to discuss the critical role of Cdk5 in the pathogenesis of PD, and how Cdk5 inhibitors are effectual in the management of PD. In conclusion, overactivated Cdk5 is involved the development of neurodegeneration, and Cdk5/calpain inhibitors such as statins, metformin, fenofibrates and rosiglitazone can attenuate the progression of PD neuropathology.

Proteasome activation is critical for cell death induced by inhibitors of polo-like kinase 1 (PLK1) in multiple cancers.

Inhibitors of polo-like kinase (PLK) are currently being evaluated as anticancer drugs. However, the molecular mechanism of PLK inhibitor-induced cell death is not fully understood. In this study, we found that GW843682X and BI2536, two inhibitors of PLK1, significantly induced cell death in multiple type cells. The induction of cell death was related to the preferring expression of PLK1. However, in human umbilical vascular endothelial cells (HUVEC) and human colorectal carcinoma cells, which expressed higher levels of both PLK1 and PLK2, PLK1 inhibitors induced very low levels of cell death. Clinical analysis reveals PLK1 presence in 26 of 30 NPC tumor tissues. In in vivo NPC lung metastasis nude mouse models, PLK1 inhibitors decreased NPC progress. Mechanistically, the PLK1 inhibitor did not activate p53, and the cell death was not reversed by p53 inhibition. Moreover, PLK1 inhibitor-induced cell death was PARP- and caspase-independent. Although PLK1 inhibitors induced down-regulation of calpain inhibitor calpastatin and calpain was activated by PLK1 inhibition, calpain blocking did not reverse cell death induced by PLK1 inhibitors, suggesting the non-involvement of calpain. Surprisingly, we found that PLK1 inhibitors induced the activation of proteasome, and the treatment of cells with PLK1 inhibitors reduced the levels of ubiquitinated proteins. And proteasome inhibitors reversed cell death induced by PLK1 inhibitors in various cell types in which PLK1 was preferentially expressed. Moreover, PLK1 inhibition reversed the degradation of proteins including p53, caspase 8, PARP and calpastatin. These results suggest that the activation of proteasome is critical for cell death induced by PLK1 inhibition.

The effect of calpain inhibitor-I on copper oxide nanoparticle-induced damage and cerebral ischemia-reperfusion in a rat model.

This study aimed to investigate the effects of the calpain inhibitor N-Acetyl-Leu-Leu-norleucinal (ALLN) on neuroapoptotic cell damage caused by Copper Oxide Nanoparticles (CuO-NP) and exacerbation of damage through brain ischemia/reperfusion (I/R) in a rat model. Male Wistar Albino rats (n=80) were divided into eight groups: Control, I/R, CuO-NP, CuO-NP+I/R, I/R+ALLN, CuO-NP+ALLN, CuO-NP+I/R+ALLN, and DMSO. Biochemical markers (MBP, S100B, NEFL, NSE, BCL-2, Cyt-C, Calpain, TNF-α, Caspase-3, MDA, and CAT) were measured in serum and brain tissue samples. Histological examinations (H&E staining), DNA fragmentation analysis (TUNEL) were performed, along with Caspase-3 assessment. The ALLN-treated groups exhibited significant improvements in biochemical markers and a remarkable reduction in apoptosis compared to the damaged groups (CuO-NP and I/R). H&E and Caspase-3 staining revealed damage-related morphological changes and reduced apoptosis in the ALLN-treated group. However, no differences were observed among the groups with TUNEL staining. The findings suggest that ALLN, as a calpain inhibitor, has potential implications for anti-apoptotic treatment, specifically in mitigating neuroapoptotic cell damage caused by CuO-NP and I/R.

Identification and neuroprotective properties of NA-184, a calpain-2 inhibitor.

Our laboratory has shown that calpain-2 activation in the brain following acute injury is directly related to neuronal damage and the long-term functional consequences of the injury, while calpain-1 activation is generally neuroprotective and calpain-1 deletion exacerbates neuronal injury. We have also shown that a relatively selective calpain-2 inhibitor, referred to as C2I, enhanced long-term potentiation and learning and memory, and provided neuroprotection in the controlled cortical impact (CCI) model of traumatic brain injury (TBI) in mice. Using molecular dynamic simulation and Site Identification by Ligand Competitive Saturation (SILCS) software, we generated about 130 analogs of C2I and tested them in a number of in vitro and in vivo assays. These led to the identification of two interesting compounds, NA-112 and NA-184. Further analyses indicated that NA-184, (S)-2-(3-benzylureido)-N-((R,S)-1-((3-chloro-2-methoxybenzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide, selectively and dose-dependent inhibited calpain-2 activity without evident inhibition of calpain-1 at the tested concentrations in mouse brain tissues and human cell lines. Like NA-112, NA-184 inhibited TBI-induced calpain-2 activation and cell death in mice and rats, both male and females. Pharmacokinetic and pharmacodynamic analyses indicated that NA-184 exhibited properties, including stability in plasma and liver and blood-brain barrier permeability, that make it a good clinical candidate for the treatment of TBI.

Targeting histone deacetylase-3 blocked epithelial-mesenchymal plasticity and metastatic dissemination in gastric cancer.

BACKGROUND AND PURPOSE: Histone deacetylase (HDAC) inhibitors (HDIs) can modulate the epithelial-mesenchymal transition (EMT) progression and inhibit the migration and invasion of cancer cells. Emerging as a novel class of anti-cancer drugs, HDIs are attracted much attention in the field of drug discovery. This study aimed to discern the underlying mechanisms of Honokiol in preventing the metastatic dissemination of gastric cancer cells by inhibiting HDAC3 activity/expression. EXPERIMENTAL APPROACH: Clinical pathological analysis was performed to determine the relationship between HDAC3 and tumor progression. The effects of Honokiol on pharmacological characterization, functional, transcriptional activities, organelle structure changes, and molecular signaling were analyzed using binding assays, differential scanning calorimetry, luciferase reporter assay, HDAC3 activity, ER stress response element activity, transmission electron microscopy, immune-blotting, and Wnt/ß-catenin activity assays. The in vivo effects of Honokiol on peritoneal dissemination were determined by a mouse model and detected by PET/CT tomography. KEY RESULTS: HDAC3 over-expression was correlated with poor prognosis. Honokiol significantly abolished HDAC3 activity (Y298) via inhibition of NFκBp65/CEBPß signaling, which could be reversed by the over-expression of plasmids of NFκBp65/CEBPß. Treatments with 4-phenylbutyric acid (a chemical chaperone) and calpain-2 gene silencing inhibited Honokiol-inhibited NFκBp65/CEBPß activation. Honokiol increased ER stress markers and inhibited EMT-associated epithelial markers, but decreased Wnt/ß-catenin activity. Suppression of HDAC3 by both Honokiol and HDAC3 gene silencing decreased cell migration and invasion in vitro and metastasis in vivo. CONCLUSIONS AND IMPLICATIONS: Honokiol acts by suppressing HDAC3-mediated EMT and metastatic signaling. By prohibiting HDAC3, metastatic dissemination of gastric cancer may be blocked. Conceptual model showing the working hypothesis on the interaction among Honokiol, HDAC3, and ER stress in the peritoneal dissemination of gastric cancer. Honokiol targeting HDAC3 by ER stress cascade and mitigating the peritoneal spread of gastric cancer. Honokiol-induced ER stress-activated calpain activity targeted HDAC3 and blocked Tyr298 phosphorylation, subsequently blocked cooperating with EMT transcription factors and cancer progression. The present study provides evidence to demonstrate that HDAC3 is a positive regulator of EMT and metastatic growth of gastric cancer cells. The findings here imply that overexpressed HDAC3 is a potential therapeutic target for honokiol to reverse EMT and prevent gastric cancer migration, invasion, and metastatic dissemination. • Honokiol significantly abolished HDAC3 activity on catalytic tyrosine 298 residue site. In addition, Honokiol-induced ER stress markedly inhibited HDAC3 expression via inhibition of NFκBp65/CEBPß signaling. • HDAC3, which is a positive regulator of metastatic gastric cancer cell growth, can be significantly inhibited by Honokiol. • Opportunities for HDAC3 inhibition may be a potential therapeutic target for preventing gastric cancer metastatic dissemination.

[Effect of extracellular signal-regulated kinase inhibitor on calpain in rat cerebral cortex after cardiopulmonary resuscitation].

OBJECTIVE: To explore the effect of extracellular signal-regulated kinase (ERK) inhibitor PD98059 on calpain-related proteins in the brain, and to understand the pathophysiological changes of calpain in cerebral ischemia/reperfusion injury (CIRI). METHODS: Forty-two rats were divided into sham operation (Sham) group (n = 6), model group (n = 12), dimethyl sulfoxide (DMSO) control group (n = 12), and PD98059 group (n = 12) by random number table. The rat model of CIRI induced by cardiac arrest-cardiopulmonary resuscitation (CA-CPR) was reproduced by transesophageal electrical stimulation to induce ventricular fibrillation. In the Sham group, only the basic operations such as anesthesia, tracheal intubation, and arteriovenous catheterization were performed without CA-CPR. The rats in the DMSO control group and PD98059 group were injected with DMSO or PD98059 0.30 mg/kg via femoral vein, respectively, 30 minutes after the restoration of spontaneous circulation (ROSC), and rats in the Sham group and model group were given the same amount of normal saline. The duration of CPR, 24-hour survival rate and neurological deficit score (NDS) after ROSC were recorded. Hematoxylin-eosin (HE) staining and Nissl staining were used to observe the pathological changes of the cerebral cortex. The expressions of phosphorylated ERK (p-ERK), ERK, calpastatin, calpain-1, and calpain-2 were detected by Western blotting. The co-expression of p-ERK and calpain-2 was detected by double immunofluorescence. RESULTS: There were no significant differences in the duration of CPR and 24-hour survival rate among all groups. In the model group, the nuclei of the cerebral cortex were obviously deformed and pyknotic, cells vacuoles and tissues were arranged disorderly, Nissl corpuscles were significantly reduced, NDS scores were also significantly reduced, level of ERK phosphorylation was increased, and calpain-2 protein was significantly up-regulated compared with the Sham group. There was no significant difference in the above parameters between the DMSO control group and the model group. After intervention with PD98059, the pathological injury of brain tissue was significantly improved, Nissl corpuscles were significantly increased, the NDS score was significantly higher than that in the model group [75.0 (72.0, 78.0) vs. 70.0 (65.0, 72.0), P < 0.05], the level of ERK phosphorylation and calpain-2 protein expression were significantly lower than those in the model group [p-ERK (p-ERK/ERK): 0.65±0.12 vs. 0.92±0.05, calpain-2 protein (calpain-2/GAPDH): 0.73±0.10 vs. 1.07±0.14, both P < 0.05], while there was no significant difference in the expressions of calpastatin and calpain-1 in the cerebral cortex among all the groups. Double immunofluorescence staining showed that p-ERK and calpain-2 were co-expressed in cytosol and nucleus, and the co-expression rate of p-ERK and calpain-2 in the model group was significantly higher than that in the Sham group [(38.6±4.3)% vs. (9.2±3.5)%, P < 0.05], while it was significantly lowered in the PD98059 group compared with the model group [(18.2±7.0)% vs. (38.6±4.3)%, P < 0.05]. CONCLUSIONS: ERK together with calpain-2 participated in CIRI induced by CA-CPR. PD98059 inhibited the expression of calpain-2 and ERK phosphorylation. Therefore, ERK/calpain-2 may be a novel therapeutic target for CIRI.

Dapagliflozin Ameliorates STZ-Induced Cardiac Hypertrophy in Type 2 Diabetic Rats by Inhibiting the Calpain-1 Expression and Nuclear Transfer of NF-κB.

OBJECTIVE: To investigate the effect of dapagliflozin (DAPA) on cardiac hypertrophy induced by type 2 diabetes mellitus (T2DM) and its mechanism. METHODS: SD rats with T2DM were divided into a T2DM group (n = 6) and DAPA group (n = 6). They were, respectively, fed with the same amount of normal saline and 1 mg/kg DAPA. The control group (n = 6) was also fed with normal saline. The hearts were tested by the application of echocardiography and hemodynamics. Subsequently, fasting blood glucose (FBG), serum total cholesterol (TC), and triglyceride (TG) as well as interleukin- (IL-) 10, IL-6, and tumor necrosis factor (TNF)-α in serum were tested. H&E and Masson staining was performed to observe the degree of cardiac tissue lesions, and expression of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), calpain-1, p-IκBα, and p65 in myocardial tissue was tested by qRT-PCR and Western blot. RESULTS: Compared with the control group, rats in the T2DM group exhibited significant diabetic symptoms: FBG was significantly elevated, and the levels of TC, TG, IL-6, and TNF-α were significantly increased, while the levels of IL-10 and the calpain activity were evidently decreased. However, DAPA treatment could improve the above changes. At the same time, the damage and fibrosis of the heart tissue in the DAPA group were markedly improved. Additionally, the mRNA expression of ANP and BNP in myocardial tissue of the DAPA group was markedly increased. And DAPA could inhibit the expression of p-IκBα/IκBα in the cytoplasm and p65 in the nucleus as well as the expression of calpain-1 in myocardial tissue. CONCLUSION: DAPA treatment ameliorates the cardiac hypertrophy caused by T2DM by decreasing body blood glucose, while reducing the expression of calpain-1 in cardiomyocytes and inhibiting the nuclear translocation of NF-κB.

Reversing mitochondrial defects in aged hearts: role of mitochondrial calpain activation.

Aging chronically increases endoplasmic reticulum (ER) stress that contributes to mitochondrial dysfunction. Activation of calpain 1 (CPN1) impairs mitochondrial function during acute ER stress. We proposed that aging-induced ER stress led to mitochondrial dysfunction by activating CPN1. We posit that attenuation of the ER stress or direct inhibition of CPN1 in aged hearts can decrease cardiac injury during ischemia-reperfusion by improving mitochondrial function. Male young (3 mo) and aged mice (24 mo) were used in the present study, and 4-phenylbutyrate (4-PBA) was used to decrease the ER stress in aged mice. Subsarcolemmal (SSM) and interfibrillar mitochondria (IFM) were isolated. Chronic 4-PBA treatment for 2 wk decreased CPN1 activation as shown by the decreased cleavage of spectrin in cytosol and apoptosis inducing factor (AIF) and the α1 subunit of pyruvate dehydrogenase (PDH) in mitochondria. Treatment improved oxidative phosphorylation in 24-mo-old SSM and IFM at baseline compared with vehicle. When 4-PBA-treated 24-mo-old hearts were subjected to ischemia-reperfusion, infarct size was decreased. These results support that attenuation of the ER stress decreased cardiac injury in aged hearts by improving mitochondrial function before ischemia. To challenge the role of CPN1 as an effector of the ER stress, aged mice were treated with MDL-28170 (MDL, an inhibitor of calpain 1). MDL treatment improved mitochondrial function in aged SSM and IFM. MDL-treated 24-mo-old hearts sustained less cardiac injury following ischemia-reperfusion. These results support that age-induced ER stress augments cardiac injury during ischemia-reperfusion by impairing mitochondrial function through activation of CPN1.

Inhibition of Piezo1/Ca2+/calpain signaling in the rat basal forebrain reverses sleep deprivation-induced fear memory impairments.

In this study, we tested the hypothesis that the Piezo1/Ca2+/calpain pathway of the basal forebrain (BF) modulates impaired fear conditioning caused by sleep deprivation. Adult male Wistar rats were subjected to 6 h of total sleep deprivation using the gentle handling protocol. Step-down inhibitory avoidance tests revealed that sleep deprivation induced substantial short- and long-term fear memory impairment in rats, which was accompanied by increased Piezo1 protein expression (P < 0.01) and increased cleavage of full-length tropomyocin receptor kinase B (TrkB-FL) (P < 0.01) in the BF area. Microinjection of the Piezo1 activator Yoda1 into the BF mimicked these sleep deprivation-induced phenomena; TrkB-FL cleavage was increased (P < 0.01) and short- and long-term fear memory was impaired (both P < 0.01) by Yoda1. Inhibition of Piezo1 by GsMTx4 in the BF area reduced TrkB-FL degradation (P < 0.01) and partially reversed short- and long-term fear memory impairments in sleep-deprived rats (both P < 0.01). Inhibition of calpain activation, downstream of Piezo1 signaling, also improved short- and long-term fear memory impairments (P = 0.038, P = 0.011) and reduced TrkB degradation (P < 0.01) in sleep-deprived rats. Moreover, sleep deprivation induced a lower pain threshold than the rest control, which was partly reversed by microinjection of GsMTx4 or PD151746. Neither sleep deprivation nor the abovementioned drugs affected locomotion and sedation. Taken together, these results indicate that BF Piezo1/Ca2+/calpain signaling plays a role in sleep deprivation-induced TrkB signaling disruption and fear memory impairments in rats.

Calpain inhibitor prevents atherosclerosis in apolipoprotein E knockout mice by regulating mRNA expression of genes related to cholesterol uptake and efflux.

PURPOSE: We previously reported that a calpain inhibitor (CAI) prevents the development of atherosclerosis in rats. This study aimed to investigate the effects of CAI (1 mg/kg) on atherosclerosis in apolipoprotein E knockout (ApoE KO) mice that were fed a high-fat diet (HFD) and explore the underlying mechanism by analyzing the expression of genes related to the uptake and efflux of cholesterol. METHODS: Atherosclerotic plaques were evaluated. The activity of calpain in the aorta and that of superoxide dismutase (SOD) in the serum were assessed. Lipid profiles in the serum and liver were examined. Serum oxidized low-density lipoprotein (oxLDL), malondialdehyde (MDA), tumor necrosis factor (TNF-α), and interleukin-6 (IL-6) levels were measured. The mRNA expressions of CD68, TNF-α, IL-6, CD36, scavenger receptor (SR-A), peroxisome proliferator-activated receptor gamma (PPAR-γ), liver-x-receptor alpha (LXR-α), and ATP-binding cassette transporter class A1 (ABCA1) in the aorta and peritoneal macrophages were also evaluated. RESULTS: CAI reduced calpain activity in the aorta. CAI also impeded atherosclerotic lesion formation and mRNA expression of CD68 in the aorta and peritoneal macrophages of ApoE KO mice compared with those of mice receiving HFD. However, CAI had no effect on body weight and lipid levels in both the serum and liver. CAI significantly decreased MDA, oxLDL, TNF-α, and IL-6 levels and increased SOD activity in the serum. Moreover, CAI significantly inhibited the mRNA expression of TNF-α and IL-6 genes in the aorta and peritoneal macrophages. In addition, CAI significantly downregulated the mRNA expression of scavenger receptors CD36 and SR-A and upregulated the expression of genes involved in the cholesterol efflux pathway, i.e., PPAR-γ, LXR-α, and ABCA1 in the aorta and peritoneal macrophages. CONCLUSIONS: CAI inhibited the development of atherosclerotic lesions in ApoE KO mice, and this effect might be related to the reduction of oxidative stress and inflammation and the improvement of cholesterol intake and efflux pathways.

Identification of Phosphorylated Calpain 3 in Rat Brain Mitochondria under mPTP Opening.

The protein phosphorylation of the membrane-bound mitochondrial proteins has become of interest from the point of view of its regulatory role of the function of the respiratory chain, opening of the mitochondrial permeability transition pore (mPTP), and initiation of apoptosis. Earlier, we noticed that upon phosphorylation of proteins in some proteins, the degree of their phosphorylation increases with the opening of mPTP. Two isoforms of myelin basic protein and cyclic nucleotide phosphodiesterase were identified in rat brain non-synaptic mitochondria and it was concluded that they are involved in mPTP regulation. In the present study, using the mass spectrometry method, the phosphorylated protein was identified as Calpain 3 in rat brain non-synaptic mitochondria. In the present study, the phosphoprotein Calpain-3 (p94) (CAPN3) was identified in the rat brain mitochondria as a phosphorylated truncated form of p60-62 kDa by two-dimensional electrophoresis and mass spectrometry. We showed that the calpain inhibitor, calpeptin, was able to suppress the Ca2+ efflux from mitochondria, preventing the opening of mPTP. It was found that phosphorylated truncated CALP3 with a molecular weight of 60-62 contains p-Tyr, which indicates the possible involvement of protein tyrosine phosphatase in this process.

De novo design of tyrosine and serine kinase-driven protein switches.

Kinases play central roles in signaling cascades, relaying information from the outside to the inside of mammalian cells. De novo designed protein switches capable of interfacing with tyrosine kinase signaling pathways would open new avenues for controlling cellular behavior, but, so far, no such systems have been described. Here we describe the de novo design of two classes of protein switch that link phosphorylation by tyrosine and serine kinases to protein-protein association. In the first class, protein-protein association is required for phosphorylation by the kinase, while in the second class, kinase activity drives protein-protein association. We design systems that couple protein binding to kinase activity on the immunoreceptor tyrosine-based activation motif central to T-cell signaling, and kinase activity to reconstitution of green fluorescent protein fluorescence from fragments and the inhibition of the protease calpain. The designed switches are reversible and function in vitro and in cells with up to 40-fold activation of switching by phosphorylation.

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.

Inhibition of Calpain Alleviates Apoptosis in Coxsackievirus B3-induced Acute Virus Myocarditis Through Suppressing Endoplasmic Reticulum Stress.

Virus myocarditis (VMC) is a common cardiovascular disease and a major cause of sudden death in young adults. However, there is still a lack of effective treatments. Our previous studies found that calpain activation was involved in VMC pathogenesis. This study aims to explore the underlying mechanisms further. Neonatal rat cardiomyocytes (NRCMs) and transgenic mice overexpressing calpastatin (Tg-CAST), the endogenous calpain inhibitor, were used to establish VMC model. Hematoxylin and eosin and Masson staining revealed inflammatory cell infiltration and fibrosis. An ELISA array detected myocardial injury. Cardiac function was measured using echocardiography. CVB3 replication was assessed by capsid protein VP1. Apoptosis was measured by TUNEL staining, flow cytometry, and western blot. The endoplasmic reticulum (ER) stress-related proteins were detected by western blot. Our data showed that CVB3 infection resulted in cardiac injury, as evidenced by increased inflammatory responses and fibrosis, which induced myocardial apoptosis. Inhibiting calpain, both by PD150606 and calpastatin overexpression, could attenuate these effects. Furthermore, ER stress was activated during CVB3 infection. However, calpain inhibition could downregulate some ER stress-associated protein levels such as GRP78, pancreatic ER kinase-like ER kinase (PERK), and inositol-requiring enzyme-1α (IRE-1α), and ER stress-related apoptotic factors, during CVB3 infection. In conclusion, calpain inhibition attenuated CVB3-induced myocarditis by suppressing ER stress, thereby inhibiting cardiomyocyte apoptosis.

Calpastatin-Mediated Inhibition of Calpain Ameliorates Skin Scar Formation after Burn Injury.

Hypertrophic scars, the most common complication of burn injuries, are characterized by excessive deposition of fibroblast-derived extracellular matrix proteins. Calpain, a calcium-dependent protease, is involved in the fibroblast proliferation and extracellular matrix production observed in certain fibrotic diseases. However, its role in the formation of post-burn hypertrophic skin scars remains largely unknown. Here, calpain expression and activity were assessed in skin fibroblasts obtained directly from patients with third-degree burns, who consequently developed post-burn hypertrophic scars. Furthermore, the antifibrotic effect of calpastatin, an endogenous calpain inhibitor, was evaluated in human fibroblasts and a murine burn model. The activity, mRNA levels, and protein levels of calpain were markedly higher in fibroblasts from the burn wounds of patients than in normal cells. Selective calpain inhibition by calpastatin markedly reduced not only the proliferation of burn-wound fibroblasts but also the mRNA and protein expression of calpain, transforming growth factor-beta 1, α-smooth muscle actin, type I and type III collagens, fibronectin, and vimentin in burn-wound fibroblasts. The anti-scarring effects of calpastatin were validated using a murine burn model by molecular, histological, and visual analyses. This study demonstrates the pathological role of calpain and the antifibrotic effect of calpastatin via calpain inhibition in post-burn hypertrophic scar formation.

Mechanosensitive Piezo1 channel activation promotes ventilator-induced lung injury via disruption of endothelial junctions in ARDS rats.

OBJECTIVE: This study aimed to investigate the role of endothelial Piezo1 in mediating ventilator-induced lung injury secondary to acute respiratory distress syndrome (ARDS). METHODS: Rats and lung endothelial cells (ECs) were transfected with Piezo1 shRNA (shPiezo1) and Piezo1 siRNA, respectively, to knock down Piezo1. Intratracheal instillation or incubation with lipopolysaccharide (LPS) was used to establish an ARDS model, and high tidal volume (HVT) ventilation or 20% cyclic stretch (CS) was administered to simulate a two-hit injury. Lung injury, alterations in lung endothelial barrier, disruption of adherens junctions (AJs), and Ca2+ influx were assessed. RESULTS: Lung vascular hyperpermeability was further increased in ARDS rats following HVT ventilation, which was abrogated in shPiezo1-treated rats. 20% CS led to severer rupture of AJs following LPS stimulation as indicated by immunofluorescence staining. The internalization and degradation of VE-cadherin were blocked by knockdown of Piezo1. Additionally, 20% CS induced Piezo1 activation, manifesting as elevated intracellular Ca2+ concentration in LPS-treated ECs, and subsequently increased calcium-dependent calpain activity. Pharmacological inhibition of calpain or Piezo1 knockdown prevented the loss of VE-cadherin, p120-catenin, and ß-catenin in ARDS rats undergoing HVT ventilation and LPS-treated ECs exposed to 20% CS. CONCLUSION: Excessive mechanical stretch during ARDS induces the activation of Piezo1 channel and its downstream target, calpain, via Ca2+ influx. This results in the disassembly of endothelial AJs and further facilitates lung endothelial barrier breakdown and vascular hyperpermeability.

Covalent Small Molecule Immunomodulators Targeting the Protease Active Site.

Cells of the immune system utilize multiple proteases to regulate cell functions and orchestrate innate and adaptive immune responses. Dysregulated protease activities are implicated in many immune-related disorders; thus, protease inhibitors have been actively investigated for pharmaceutical development. Although historically considered challenging with concerns about toxicity, compounds that covalently modify the protease active site represent an important class of agents, emerging not only as chemical probes but also as approved drugs. Here, we provide an overview of technologies useful for the study of proteases with the focus on recent advances in chemoproteomic methods and screening platforms. By highlighting covalent inhibitors that have been designed to target immunomodulatory proteases, we identify opportunities for the development of small molecule immunomodulators.

Inhibition of extracellular signal-regulated kinase/calpain-2 pathway reduces neuroinflammation and necroptosis after cerebral ischemia-reperfusion injury in a rat model of cardiac arrest.

BACKGROUND: Cerebral ischemia-reperfusion injury (CIRI) is the leading cause of poor neurological prognosis after cardiopulmonary resuscitation (CPR). We previously reported that the extracellular signal-regulated kinase (ERK) activation mediates CIRI. Here, we explored the potential ERK/calpain-2 pathway role in CIRI using a rat model of cardiac arrest (CA). METHODS: Adult male Sprague-Dawley rats suffered from CA/CPR-induced CIRI, received saline, DMSO, PD98059 (ERK1/2 inhibitor, 0.3 mg/kg), or MDL28170 (calpain inhibitor, 3.0 mg/kg) after spontaneous circulation recovery. The survival rate and the neurological deficit score (NDS) were utilized to assess the brain function. Hematoxylin stain, Nissl staining, and transmission electron microscopy were used to evaluate the neuron injury. The expression levels of p-ERK, ERK, calpain-2, neuroinflammation-related markers (GFAP, Iba1, IL-1ß, TNF-α), and necroptosis proteins (TNFR1, RIPK1, RIPK3, p-MLKL, and MLKL) in the brain tissues were determined by western blotting and immunohistochemistry. Fluorescent multiplex immunohistochemistry was used to analyze the p-ERK, calpain-2, and RIPK3 co-expression in neurons, and RIPK3 expression levels in microglia or astrocytes. RESULTS: At 24 h after CA/CPR, the rats in the saline-treated and DMSO groups presented with injury tissue morphology, low NDS, ERK/calpain-2 pathway activation, and inflammatory cytokine and necroptosis protein over-expression in the brain tissue. After PD98059 and MDL28170 treatment, the brain function was improved, while inflammatory response and necroptosis were suppressed by ERK/calpain-2 pathway inhibition. CONCLUSION: Inflammation activation and necroptosis involved in CA/CPR-induced CIRI were regulated by the ERK/calpain-2 signaling pathway. Inhibition of that pathway can reduce neuroinflammation and necroptosis after CIRI in the CA model rats.

Calpain-Inhibitors Protect Frataxin-Deficient Dorsal Root Ganglia Neurons from Loss of Mitochondrial Na+/Ca2+ Exchanger, NCLX, and Apoptosis.

Calpains are calcium-dependent proteases activated in apoptotic cell death and neurodegeneration. Friedreich Ataxia is a neurodegenerative rare disease caused by frataxin deficiency, a mitochondrial protein. Dorsal root ganglion (DRG) sensory neurons are among the cellular types most affected in this disease. We have previously demonstrated that frataxin-deficient DRGs show calpain activation, alteration in calcium levels and decreased content of the Na+/Ca2+ exchanger (NCLX). This transporter is involved in mitochondrial calcium efflux. In this study, we have performed a time-course analysis of several parameters altered in a frataxin-deficient DRGs. These include decline of NCLX levels, calcium accumulation, mitochondrial depolarization, α-fodrin fragmentation and apoptotic cell death. Furthermore, we have analysed the effect of the calpain inhibitors MDL28170 and Calpeptin on these parameters. We have observed that these inhibitors increase NCLX levels, protect sensory neurons from neurite degeneration and calcium accumulation, and restore mitochondrial membrane potential. In addition, calpain 1 reduction alleviated neurodegeneration in frataxin-deficient DRG neurons. These results strengthen the hypothesis of a central role for calcium homeostasis and calpains in frataxin-deficient dorsal root ganglia neurons.