Effect of outer membrane vesicles of Lactobacillus pentosus on Tau phosphorylation and CDK5-Calpain pathway in mice.

Alzheimer's disease (AD) stands as a neurodegenerative disorder causing cognitive decline, posing a significant health concern for the elderly population in China. This study explored the effects of outer membrane vesicles (OMVs) from the gut microbiota of AD patients on learning and memory abilities and Tau protein phosphorylation in mice. In contrast to the OMVs from healthy controls and the PBS treatment group, mice treated with AD-OMVs exhibited notable declines in learning and memory capabilities, as evidenced by results from the Morris water maze, Y-maze, and novel object recognition tests. Immunohistochemistry and Western blot assessments unveiled elevated levels of hyperphosphorylated Tau in the cortex and hippocampus of mice treated with AD-OMVs. However, there were no alterations observed in the total Tau levels. In addition, AD-OMVs treated mice showed increased neuroinflammation indicated by elevated astrocytes and microglia. Molecular mechanism studies demonstrated that AD-OMVs could activate GSK3ß, CDK5-Calpain and NF-κB pathways in mice hippocampus. These studies suggest AD patient gut microbiota derived OMVs can promote host Tau phosphorylation and improved neuroinflammation.

17ß-Estradiol promotes metastasis in triple-negative breast cancer through the Calpain/YAP/ß-catenin signaling axis.

ß-catenin is an important regulator of malignant progression. 17ß-Estradiol (E2), an important sex hormone in women, promotes the growth and metastasis of triple-negative breast cancer (TNBC). However, whether ß-catenin is involved in E2-induced metastasis of TNBC remains unknown. In this study, we show that E2 induces the proliferation, migration, invasion, and metastasis of TNBC cells. E2 induces ß-catenin protein expression and nuclear translocation, thereby regulating the expression of target genes such as Cyclin D1 and MMP-9. The inhibition of ß-catenin reversed the E2-induced cell malignant behaviors. Additionally, E2 activated Calpain by increasing intracellular Ca2+ levels and reducing calpastatin levels. When Calpain was inhibited, E2 did not induce the proliferation, migration, invasion, or metastasis of TNBC cells. In addition, E2 promoted translocation of YAP into the nucleus by inhibiting its phosphorylation. Calpain inhibition reversed the E2-induced YAP dephosphorylation. Inhibition of YAP transcriptional activity reversed the effects of E2 on the proliferation, migration, invasion, and ß-catenin of TNBC cells. In conclusion, we demonstrated that E2 induced metastasis-related behaviors in TNBC cells and this effect was mediated through the Calpain/YAP/ß-catenin signaling pathway.

Experimental and computational evidence that Calpain-10 binds to the carboxy terminus of NaV1.2 and NaV1.6.

Voltage-gated sodium channels (NaV) are pivotal proteins responsible for initiating and transmitting action potentials. Emerging evidence suggests that proteolytic cleavage of sodium channels by calpains is pivotal in diverse physiological scenarios, including ischemia, brain injury, and neuropathic pain associated with diabetes. Despite this significance, the precise mechanism by which calpains recognize sodium channels, especially given the multiple calpain isoforms expressed in neurons, remains elusive. In this work, we show the interaction of Calpain-10 with NaV's C-terminus through a yeast 2-hybrid assay screening of a mouse brain cDNA library and in vitro by GST-pulldown. Later, we also obtained a structural and dynamic hypothesis of this interaction by modeling, docking, and molecular dynamics simulation. These results indicate that Calpain-10 interacts differentially with the C-terminus of NaV1.2 and NaV1.6. Calpain-10 interacts with NaV1.2 through domains III and T in a stable manner. In contrast, its interaction with NaV1.6 involves domains II and III, which could promote proteolysis through the Cys-catalytic site and C2 motifs.

A novel combination approach to effectively reduce inflammation and neurodegeneration in multiple sclerosis models.

Multiple sclerosis (MS) is an autoimmune disease characterized by immune-mediated attacks on the central nervous system (CNS), resulting in demyelination and recurring T-cell responses. Unfortunately, there is no cure for it. Current therapies that target immunomodulation and/or immunosuppression show only modest beneficial effects, have many side effects, and do not block neurodegeneration or progression of the disease. Since neurodegeneration and in particular axonal degeneration is implicated in disability in progressive MS, development of novel therapeutic strategies to attenuate the neurodegenerative processes is imperative. This study aims to develop new safe and efficacious treatments that address both the inflammatory and neurodegenerative aspects of MS using its animal model, experimental allergic encephalomyelitis (EAE). In EAE, the cysteine protease calpain is upregulated in CNS tissue, and its activity correlates with neurodegeneration. Our immunologic studies on MS have indicated that increased calpain activity promotes pro-inflammatory T helper (Th)1 cells and the severity of the disease in EAE, suggesting that calpain inhibition could be a novel target to combat neurodegeneration in MS/EAE. While calpain inhibition by SNJ1945 reduced disease severity, treatment of EAE animals with a novel protease-resistant altered small peptide ligand (3aza-APL) that mimic myelin basic protein (MBP), also decreased the incidence of EAE, disease severity, infiltration of inflammatory cells, and protected myelin. A reduction in inflammatory T-cells with an increase in Tregs and myeloid suppressor cells is also found in EAE mice treated with SNJ1945 and 3aza-APL. Thus, a novel combination strategy was tested in chronic EAE mouse model in B10 mice which showed multiple pathological mechanisms could be addressed by simultaneous treatment with calpain inhibitor SNJ1945 and protease-resistant 3aza-APL to achieve a stronger therapeutic effect.

T-type voltage-gated channels, Na+/Ca2+-exchanger, and calpain-2 promote photoreceptor cell death in inherited retinal degeneration.

Inherited retinal degenerations (IRDs) are a group of untreatable and commonly blinding diseases characterized by progressive photoreceptor loss. IRD pathology has been linked to an excessive activation of cyclic nucleotide-gated channels (CNGC) leading to Na+- and Ca2+-influx, subsequent activation of voltage-gated Ca2+-channels (VGCC), and further Ca2+ influx. However, a connection between excessive Ca2+ influx and photoreceptor loss has yet to be proven.Here, we used whole-retina and single-cell RNA-sequencing to compare gene expression between the rd1 mouse model for IRD and wild-type (wt) mice. Differentially expressed genes indicated links to several Ca2+-signalling related pathways. To explore these, rd1 and wt organotypic retinal explant cultures were treated with the intracellular Ca2+-chelator BAPTA-AM or inhibitors of different Ca2+-permeable channels, including CNGC, L-type VGCC, T-type VGCC, Ca2+-release-activated channel (CRAC), and Na+/Ca2+ exchanger (NCX). Moreover, we employed the novel compound NA-184 to selectively inhibit the Ca2+-dependent protease calpain-2. Effects on the retinal activity of poly(ADP-ribose) polymerase (PARP), sirtuin-type histone-deacetylase, calpains, as well as on activation of calpain-1, and - 2 were monitored, cell death was assessed via the TUNEL assay.While rd1 photoreceptor cell death was reduced by BAPTA-AM, Ca2+-channel blockers had divergent effects: While inhibition of T-type VGCC and NCX promoted survival, blocking CNGCs and CRACs did not. The treatment-related activity patterns of calpains and PARPs corresponded to the extent of cell death. Remarkably, sirtuin activity and calpain-1 activation were linked to photoreceptor protection, while calpain-2 activity was related to degeneration. In support of this finding, the calpain-2 inhibitor NA-184 protected rd1 photoreceptors.These results suggest that Ca2+ overload in rd1 photoreceptors may be triggered by T-type VGCCs and NCX. High Ca2+-levels likely suppress protective activity of calpain-1 and promote retinal degeneration via activation of calpain-2. Overall, our study details the complexity of Ca2+-signalling in photoreceptors and emphasizes the importance of targeting degenerative processes specifically to achieve a therapeutic benefit for IRDs. Video Abstract.

The neuronal transcription factor MEIS2 is a calpain-2 protease target.

Tight control over transcription factor activity is necessary for a sensible balance between cellular proliferation and differentiation in the embryo and during tissue homeostasis by adult stem cells, but mechanistic details have remained incomplete. The homeodomain transcription factor MEIS2 is an important regulator of neurogenesis in the ventricular-subventricular zone (V-SVZ) adult stem cell niche in mice. We here identify MEIS2 as direct target of the intracellular protease calpain-2 (composed of the catalytic subunit CAPN2 and the regulatory subunit CAPNS1). Phosphorylation at conserved serine and/or threonine residues, or dimerization with PBX1, reduced the sensitivity of MEIS2 towards cleavage by calpain-2. In the adult V-SVZ, calpain-2 activity is high in stem and progenitor cells, but rapidly declines during neuronal differentiation, which is accompanied by increased stability of MEIS2 full-length protein. In accordance with this, blocking calpain-2 activity in stem and progenitor cells, or overexpression of a cleavage-insensitive form of MEIS2, increased the production of neurons, whereas overexpression of a catalytically active CAPN2 reduced it. Collectively, our results support a key role for calpain-2 in controlling the output of adult V-SVZ neural stem and progenitor cells through cleavage of the neuronal fate determinant MEIS2.

Calpain-2 mediates SARS-CoV-2 entry via regulating ACE2 levels.

Since the beginning of the coronavirus disease 2019 (COVID-19) pandemic, much effort has been dedicated to identifying effective antivirals against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). A number of calpain inhibitors show excellent antiviral activities against SARS-CoV-2 by targeting the viral main protease (Mpro), which plays an essential role in processing viral polyproteins. In this study, we found that calpain inhibitors potently inhibited the infection of a chimeric vesicular stomatitis virus (VSV) encoding the SARS-CoV-2 spike protein but not Mpro. In contrast, calpain inhibitors did not exhibit antiviral activities toward the wild-type VSV with its native glycoprotein. Genetic knockout of calpain-2 by CRISPR/Cas9 conferred resistance of the host cells to the chimeric VSV-SARS-CoV-2 virus and a clinical isolate of wild-type SARS-CoV-2. Mechanistically, calpain-2 facilitates SARS-CoV-2 spike protein-mediated cell attachment by positively regulating the cell surface levels of ACE2. These results highlight an Mpro-independent pathway targeted by calpain inhibitors for efficient viral inhibition. We also identify calpain-2 as a novel host factor and a potential therapeutic target responsible for SARS-CoV-2 infection at the entry step. IMPORTANCE: Many efforts in small-molecule screens have been made to counter SARS-CoV-2 infection by targeting the viral main protease, the major element that processes viral proteins after translation. Here, we discovered that calpain inhibitors further block SARS-CoV-2 infection in a main protease-independent manner. We identified the host cysteine protease calpain-2 as an important positive regulator of the cell surface levels of SARS-CoV-2 cellular receptor ACE2 and, thus, a facilitator of viral infection. By either pharmacological inhibition or genetic knockout of calpain-2, the SARS-CoV-2 binding to host cells is blocked and viral infection is decreased. Our findings highlight a novel mechanism of ACE2 regulation, which presents a potential new therapeutic target. Since calpain inhibitors also potently interfere with the viral main protease, our data also provide a mechanistic understanding of the potential use of calpain inhibitors as dual inhibitors (entry and replication) in the clinical setting of COVID-19 diseases. Our findings bring mechanistic insights into the cellular process of SARS-CoV-2 entry and offer a novel explanation to the mechanism of activities of calpain inhibitors.

New perspective for Calpain-Mediated regulation of meat Quality: Unveiling the impact on mitochondrial pathway apoptosis in post-mortem.

While calpain's role in myofibrillar protein degradation is well-established, its impact on post-mortem apoptosis remains fully elucidated. This study aimed to examine how calpain influences the mitochondrial apoptotic pathway in post-mortem muscle cells and assess its potential impact on chicken tenderness. The findings indicate that the calpain inhibitor treatment could decelerate the rate of lysosome destruction in post-mortem chicken, which is a crucial factor in delaying the mitochondrial apoptotic pathway. Subsequently, this inhibition enhanced the mitochondrial membrane's stability and suppressed the apoptosis-inducing factor Cyt c release into the sarcoplasm. The Western blot results in a greater myofibrillar protein degradation degree in the caspase inhibitor samples compared to the calpain inhibitor samples. Interestingly, the two groups had no significant difference in shear force. Based on these reasons, a novel perspective was introduced in this paper: Calpain could affect the change in meat tenderness by regulating mitochondrial apoptosis in the post-mortem period.

Astragaloside IV mitigates hypoxia-induced cardiac hypertrophy through calpain-1-mediated mTOR activation.

BACKGROUND: Astragaloside IV (AsIV), a key functioning element of Astragalus membranaceus, has been recognized for its potential cardiovascular protective properties. However, there is a need to elucidate the impacts of AsIV on myocardial hypertrophy under hypoxia conditions and its root mechanisms. PURPOSE: This study scrutinized the influence of AsIV on cardiac injury under hypoxia, with particular emphasis on the role of calpain-1 (CAPN1) in mediating mTOR pathways. METHODS: Hypoxia-triggered cardiac hypertrophy was examined in vivo with CAPN1 knockout and wild-type C57BL/6 mice and in vitro with H9C2 cells. The impacts of AsIV, 3-methyladenine, and CAPN1 inhibition on hypertrophy, autophagy, apoptosis, [Ca2+]i, and CAPN1 and mTOR levels in cardiac tissues and H9C2 cells were investigated. RESULTS: Both AsIV treatment and CAPN1 knockout mitigated hypoxia-induced cardiac hypertrophy, autophagy, and apoptosis in mice and H9C2 cells. Moreover, AsIV, 3-methyladenine, and CAPN1 inhibition augmented p-mTOR level but reduced [Ca2+]i and CAPN1 level. Additionally, lentivirus-mediated CAPN1 overexpression in H9C2 cells exacerbated myocardial hypertrophy, apoptosis, and p-mTOR inhibition under hypoxia. Specifically, AsIV treatment reversed the impacts of increased CAPN1 expression on cardiac injury and the inhibition of p-mTOR. CONCLUSION: These findings suggest that AsIV may alleviate cardiac hypertrophy under hypoxia by attenuating apoptosis and autophagy through CAPN1-mediated mTOR activation.

SARM1 is responsible for calpain-dependent dendrite degeneration in mouse hippocampal neurons.

Sterile alpha and toll/interleukin receptor motif-containing 1 (SARM1) is a critical regulator of axon degeneration that acts through hydrolysis of NAD+ following injury. Recent work has defined the mechanisms underlying SARM1's catalytic activity and advanced our understanding of SARM1 function in axons, yet the role of SARM1 signaling in other compartments of neurons is still not well understood. Here, we show in cultured hippocampal neurons that endogenous SARM1 is present in axons, dendrites, and cell bodies and that direct activation of SARM1 by the neurotoxin Vacor causes not just axon degeneration, but degeneration of all neuronal compartments. In contrast to the axon degeneration pathway defined in dorsal root ganglia, SARM1-dependent hippocampal axon degeneration in vitro is not sensitive to inhibition of calpain proteases. Dendrite degeneration downstream of SARM1 in hippocampal neurons is dependent on calpain 2, a calpain protease isotype enriched in dendrites in this cell type. In summary, these data indicate SARM1 plays a critical role in neurodegeneration outside of axons and elucidates divergent pathways leading to degeneration in hippocampal axons and dendrites.

NADH improves AIF dimerization and inhibits apoptosis in iPSCs-derived neurons from patients with auditory neuropathy spectrum disorder.

Auditory neuropathy spectrum disorder (ANSD) is a hearing impairment involving disruptions to inner hair cells (IHCs), ribbon synapses, spiral ganglion neurons (SGNs), and/or the auditory nerve itself. The outcomes of cochlear implants (CI) for ANSD are variable and dependent on the location of lesion sites. Discovering a potential therapeutic agent for ANSD remains an urgent requirement. Here, 293T stable transfection cell lines and patient induced pluripotent stem cells (iPSCs)-derived auditory neurons carrying the apoptosis inducing factor (AIF) p.R422Q variant were used to pursue a therapeutic regent for ANSD. Nicotinamide adenine dinucleotide (NADH) is a main electron donor in the electron transport chain (ETC). In 293T stable transfection cells with the p.R422Q variant, NADH treatment improved AIF dimerization, rescued mitochondrial dysfunctions, and decreased cell apoptosis. The effects of NADH were further confirmed in patient iPSCs-derived neurons. The relative level of AIF dimers was increased to 150.7 % (P = 0.026) from 59.2 % in patient-neurons upon NADH treatment. Such increased AIF dimerization promoted the mitochondrial import of coiled-coil-helix-coiled-coil-helix domain-containing protein 4 (CHCHD4), which further restored mitochondrial functions. Similarly, the content of mitochondrial calcium (mCa2+) was downregulated from 136.7 % to 102.3 % (P = 0.0024) in patient-neurons upon NADH treatment. Such decreased mCa2+ levels inhibited calpain activity, ultimately reducing the percentage of apoptotic cells from 30.5 % to 21.1 % (P = 0.021). We also compared the therapeutic effects of gene correction and NADH treatment on hereditary ANSD. NADH treatment had comparable restorative effects on functions of ANSD patient-specific cells to that of gene correction. Our findings offer evidence of the molecular mechanisms of ANSD and introduce NADH as a potential therapeutic agent for ANSD therapy.

Development of a monoclonal antibody specific for a calpain-generated ∆48 kDa calcineurin fragment, a marker of distressed astrocytes.

BACKGROUND: Calcineurin (CN) is a Ca2+/calmodulin-dependent protein phosphatase. In healthy tissue, CN exists mainly as a full-length (∼60 kDa) highly-regulated protein phosphatase involved in essential cellular functions. However, in diseased or injured tissue, CN is proteolytically converted to a constitutively active fragment that has been causatively-linked to numerous pathophysiologic processes. These calpain-cleaved CN fragments (∆CN) appear at high levels in human brain at early stages of cognitive decline associated with Alzheimer's disease (AD). NEW METHOD: We developed a monoclonal antibody to ∆CN, using an immunizing peptide corresponding to the C-terminal end of the ∆CN fragment. RESULTS: We obtained a mouse monoclonal antibody, designated 26A6, that selectively detects ∆CN in Western analysis of calpain-cleaved recombinant human CN. Using this antibody, we screened both pathological and normal human brain sections provided by the University of Kentucky's Alzheimer's Disease Research Center. 26A6 showed low reactivity towards normal brain tissue, but detected astrocytes both surrounding AD amyloid plaques and throughout AD brain tissue. In brain tissue with infarcts, there was considerable concentration of 26A6-positive astrocytes within/around infarcts, suggesting a link with anoxic/ischemia pathways. COMPARISON WITH EXISTING METHOD: The results obtained with the new monoclonal are similar to those obtained with a polyclonal we had previously developed. However, the monoclonal is an abundant tool available to the dementia research community. CONCLUSIONS: The new monoclonal 26A6 antibody is highly selective for the ∆CN proteolytic fragment and labels a subset of astrocytes, and could be a useful tool for marking insidious brain pathology and identifying novel astrocyte phenotypes.

Calpain as a Therapeutic Target for Hypoxic-Ischemic Encephalopathy.

Hypoxic-ischemic encephalopathy (HIE) is a complex pathophysiological process with multiple links and factors. It involves the interaction of inflammation, oxidative stress, and glucose metabolism, and results in acute and even long-term brain damage and impairment of brain function. Calpain is a family of Ca2+-dependent cysteine proteases that regulate cellular function. Calpain activation is involved in cerebral ischemic injury, and this involvement is achieved by the interaction among Ca2+, substrates, organelles, and multiple proteases in the neuronal necrosis and apoptosis pathways after cerebral ischemia. Many calpain inhibitors have been developed and tested in the biochemical and biomedical fields. This study reviewed the potential role of calpain in the treatment of HIE and related mechanism, providing new insights for future research on HIE.

Enriched environment ameliorates fear memory impairments induced by sleep deprivation via inhibiting PIEZO1/calpain/autophagy signaling pathway in the basal forebrain.

AIMS: To verify the hypothesis that an enriched environment (EE) alleviates sleep deprivation-induced fear memory impairment by modulating the basal forebrain (BF) PIEZO1/calpain/autophagy pathway. METHODS: Eight-week-old male mice were housed in a closed, isolated environment (CE) or an EE, before 6-h total sleep deprivation. Changes in fear memory after sleep deprivation were observed using an inhibitory avoidance test. Alterations in BF PIEZO1/calpain/autophagy signaling were detected. The PIEZO1 agonist Yoda1 or inhibitor GsMTx4, the calpain inhibitor PD151746, and the autophagy inducer rapamycin or inhibitor 3-MA were injected into the bilateral BF to investigate the pathways involved in the memory-maintaining role of EE in sleep-deprived mice. RESULTS: Mice housed in EE performed better than CE mice in short- and long-term fear memory tests after sleep deprivation. Sleep deprivation resulted in increased PIEZO1 expression, full-length tropomyosin receptor kinase B (TrkB-FL) degradation, and autophagy, as reflected by increased LC3 II/I ratio, enhanced p62 degradation, increased TFEB expression and nuclear translocation, and decreased TFEB phosphorylation. These molecular changes were partially reversed by EE treatment. Microinjection of Yoda1 or rapamycin into the bilateral basal forebrain induced excessive autophagy and eliminated the cognition-protective effects of EE. Bilateral basal forebrain microinjection of GsMTx4, PD151746, or 3-MA mimicked the cognitive protective and autophagy inhibitory effects of EE in sleep-deprived mice. CONCLUSIONS: EE combats sleep deprivation-induced fear memory impairments by inhibiting the BF PIEZO1/calpain/autophagy pathway.

Role of calpain-5 in cerebral ischemia and reperfusion injury.

BACKGROUND: Ischemia and reperfusion (I/R) injury exacerbate the prognosis of ischemic diseases. The cause of this exacerbation is partly a mitochondrial cell death pathway. Mitochondrial calpain-5 is proteolyzed/autolyzed under endoplasmic reticulum stress, resulting in inflammatory caspase-4 activation. However, the role of calpain-5 in I/R injury remains unclear. We hypothesized that calpain-5 is involved in ischemic brain disease. METHODS: Mitochondria from C57BL/6J mice were extracted via centrifugation with/without proteinase K treatment. The expression and proteolysis/autolysis of calpain-5 were determined using western blotting. The mouse and human brains with I/R injury were analyzed using hematoxylin and eosin staining and immunohistochemistry. HT22 cells were treated with tunicamycin and CAPN5 siRNA. RESULTS: Calpain-5 was expressed in the mitochondria of mouse tissues. Mitochondrial calpain-5 in mouse brains was responsive to calcium earlier than cytosolic calpain-5 in vitro calcium assays and in vivo bilateral common carotid artery occlusion model mice. Immunohistochemistry revealed that neurons were positive for calpain-5 in the normal brains of mice and humans. The expression of calpain-5 was increased in reactive astrocytes at human infarction sites. The knockdown of calpain-5 suppressed of cleaved caspase-11. CONCLUSIONS: The neurons of human and mouse brains express calpain-5, which is proteolyzed/autolyzed in the mitochondria in the early stage of I/R injury and upregulated in reactive astrocytes in the end-stage. GENERAL SIGNIFICANCE: Our results provide a comprehensive understanding of the mechanisms underlying I/R injury. Targeting the expression or activity of mitochondrial calpain-5 may suppress the inflammation during I/R injuries such as cerebrovascular diseases.

Remote ischemic preconditioning reduces mitochondrial apoptosis mediated by calpain 1 activation in myocardial ischemia-reperfusion injury through calcium channel subunit Cacna2d3.

Remote Ischemic Preconditioning (RIPC) can reduce myocardial ischemia-reperfusion injury, but its mechanism is not clear. In order to explore the mechanism of RIPC in myocardial protection, we collected myocardial specimens during cardiac surgery in children with tetralogy of Fallot for sequencing. Our study found RIPC reduces the expression of the calcium channel subunit cacna2d3, thereby impacting the function of calcium channels. As a result, calcium overload during ischemia-reperfusion is reduced, and the activation of calpain 1 is inhibited. This ultimately leads to a decrease in calpain 1 cleavage of Bax, consequently inhibiting increased mitochondrial permeability-mediated apoptosis. Notably, in both murine and human models of myocardial ischemia-reperfusion injury, RIPC inhibiting the expression of the calcium channel subunit cacna2d3 and the activation of calpain 1, improving cardiac function and histological outcomes. Overall, our findings put forth a proposed mechanism that elucidates how RIPC reduces myocardial ischemia-reperfusion injury, ultimately providing a solid theoretical foundation for the widespread clinic application of RIPC.

Mapping the substrate-binding subsite specificity of a Porphyromonas gingivalis Tpr peptidase.

Calcium-dependent peptidases of the calpain family are widespread in eukaryotes but uncommon in prokaryotes. A few bacterial calpain homologs have been discovered but none of them have been characterized in detail. Here we present an in-depth substrate specificity analysis of the bacterial calpain-like peptidase Tpr from Porphyromonas gingivalis. Using the positional scanning hybrid combinatorial substrate library method, we found that the specificity of Tpr peptidase differs substantially from the papain family of cysteine proteases, showing a strong preference for proline residues at positions P2 and P3. Such a degree of specificity indicates that this P. gingivalis cell-surface peptidase has a more sophisticated role than indiscriminate protein degradation to generate peptide nutrients, and may fulfil virulence-related functions such as immune evasion.

Cornification of keratinocytes is associated with differential changes in the catalytic activity and the immunoreactivity of transglutaminase-1.

Transglutaminase 1 (TGM1) plays an essential role in skin barrier formation by cross-linking proteins in differentiated keratinocytes. Here, we established a protocol for the antibody-dependent detection of TGM1 protein and the parallel detection of TGM activity. TGM1 immunoreactivity initially increased and co-localized with membrane-associated TGM activity during keratinocyte differentiation. TGM activity persisted upon further differentiation of keratinocytes, whereas TGM1 immunoreactivity was lost under standard assay conditions. Pretreatment of tissue sections with the proteases trypsin or proteinase K enabled immunodetection of TGM1 in cornified keratinocytes, indicating that removal of other proteins was a prerequisite for TGM1 immunolabeling after cornification. The increase of TGM activity and subsequent loss of TGM1 immunoreactivity could be replicated in HEK293T cells transfected with TGM1, suggesting that protein cross-linking mediated by TGM1 itself may lead to reduced recognition of TGM1 by antibodies. To screen for proteins potentially regulating TGM1, we performed Virotrap experiments and identified the CAPNS1 subunit of calpain as an interaction partner of TGM1. Treatment of keratinocytes and TGM1-transfected HEK293T cells with chemical inhibitors of calpain suppressed transglutamination. Our findings suggest that calpain contributes to the control of TGM1-mediated transglutamination and proteins cross-linked by transglutamination mask epitopes of TGM1.

Calpain and Cardiometabolic Diseases.

Calpain is defined as a member of the superfamily of cysteine proteases possessing the CysPC motif within the gene. Calpain-1 and -2, which are categorized as conventional isozymes, execute limited proteolysis in a calcium-dependent fashion. Accordingly, the calpain system participates in physiological and pathological phenomena, including cell migration, apoptosis, and synaptic plasticity. Recent investigations have unveiled the contributions of both conventional and unconventional calpains to the pathogenesis of cardiometabolic disorders. In the context of atherosclerosis, overactivation of conventional calpain attenuates the barrier function of vascular endothelial cells and decreases the immunosuppressive effects attributed to lymphatic endothelial cells. In addition, calpain-6 induces aberrant mRNA splicing in macrophages, conferring atheroprone properties. In terms of diabetes, polymorphisms of the calpain-10 gene can modify insulin secretion and glucose disposal. Moreover, conventional calpain reportedly participates in amino acid production from vascular endothelial cells to induce alteration of amino acid composition in the liver microenvironment, thereby facilitating steatohepatitis. Such multifaceted functionality of calpain underscores its potential as a promising candidate for pharmaceutical targets for the treatment of cardiometabolic diseases. Consequently, the present review highlights the pivotal role of calpains in the complications of cardiometabolic diseases and embarks upon a characterization of calpains as molecular targets.