Trpv6 channel targeting using monoclonal antibody induces prostate cancer cell apoptosis and tumor regression.

TRPV6 calcium channel is a prospective target in prostate cancer (PCa) since it is not expressed in healthy prostate while its expression increases during cancer progression. Despite the role of TRPV6 in PCa cell survival and apoptotic resistance has been already established, no reliable tool to target TRPV6 channel in vivo and thus to reduce tumor burden is known to date. Here we report the generation of mouse monoclonal antibody mAb82 raised against extracellular epitope of the pore region of the channel. mAb82 inhibited TRPV6 currents by 90% at 24 µg/ml in a dose-dependent manner while decreasing store-operated calcium entry to 56% at only 2.4 µg/ml. mAb82 decreased PCa survival rate in vitro by 71% at 12 µg/ml via inducing cell death through the apoptosis cascade via activation of the protease calpain, following bax activation, mitochondria enlargement, and loss of cristae, Cyt C release, pro-caspase 9 cleavage with the subsequent activation of caspases 3/7. In vivo, mice bearing either PC3Mtrpv6+/+ or PC3Mtrpv6-/-+pTRPV6 tumors were successfully treated with mAb82 at the dose as low as 100 µg/kg resulting in a significant reduction tumor growth by 31% and 90%, respectively. The survival rate was markedly improved by 3.5 times in mice treated with mAb82 in PC3Mtrpv6+/+ tumor group and completely restored in PC3Mtrpv6-/-+pTRPV6 tumor group. mAb82 showed a TRPV6-expression dependent organ distribution and virtually no toxicity in the same way as mAbAU1, a control antibody of the same Ig2a isotype. Overall, our data demonstrate for the first time the use of an anti-TRPV6 monoclonal antibody in vitro and in vivo in the treatment of the TRPV6-expressing PCa tumors.

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.

CAPN2 promotes apalutamide resistance in metastatic hormone-sensitive prostate cancer by activating protective autophagy.

Apalutamide, a novel endocrine therapy agent, has been shown to significantly improve the prognosis of patients with metastatic hormone-sensitive prostate cancer (mHSPC). However, resistance to apalutamide has also been reported, and the underlying mechanism for this response has yet to be clearly elucidated. First, this study established apalutamide-resistant prostate cancer (PCa) cells, and confirmed that apalutamide activated the release of calcium ions (Ca2+) and endoplasmic reticulum stress (ERS) to enhance autophagy. Second, RNA sequencing, western blotting, and immunohistochemistry revealed significantly decreased Calpain 2 (CAPN2) expression in the apalutamide-resistant PCa cells and tissues. Furthermore, immunofluorescence and transmission electron microscopy (TEM) showed that CAPN2 promoted apalutamide resistance by activating protective autophagy. CAPN2 promoted autophagy by reducing Forkhead Box O1 (FOXO1) degradation while increasing nuclear translocation via nucleoplasmic protein isolation and immunofluorescence. In addition, FOXO1 promoted protective autophagy through the transcriptional regulation of autophagy-related gene 5 (ATG5). Furthermore, a dual-fluorescence assay confirmed that transcription factor 3 (ATF3) stimulation promoted CAPN2-mediated autophagy activation via transcriptional regulation. In summary, CAPN2 activated protective autophagy by inhibiting FOXO1 degradation and promoting its nuclear translocation via transcriptional ATG5 regulation. ATF3 activation and transcriptional CAPN2 regulation jointly promoted this bioeffect. Thus, our findings have not only revealed the mechanism underlying apalutamide resistance, but also provided a promising new target for the treatment of metastatic PCa.

Genetic Linkage between CAPN5 and TYR Variants in the Context of Albinism and Autosomal Dominant Neovascular Inflammatory Vitreoretinopathy Absence: A Case Report.

We present a case involving a patient whose clinical phenotype aligns with oculocutaneous albinism (OCA), yet exhibits a complex genotype primarily characterized by variants of unknown significance (VUS). An 11-year-old boy manifested iris hypopigmentation and translucency, pronounced photophobia, diminished visual acuity and stereopsis, nystagmus, reduced pigmentation of the retina, and foveal hypoplasia. Genetic testing was performed. A heterozygous missense VUS CAPN5 c.230A>G, p.(Gln77Arg), a heterozygous missense VUS TYR c.1307G>C, p.(Gly436Ala), and a heterozygous missense variant TYR c.1205G>A, p.(Arg402Gln) which was classified as a risk factor, were identified. We hypothesized that the TYR c.1307G>C, p.(Gly436Ala) variant is in genetic disequilibrium with the TYR c.1205G>A, p.(Arg402Gln) variant leading to deficient expression of melanogenic enzymes in retinal cells, resulting in the manifestation of mild OCA. Additionally, this study represents the case where we did not detect chiasmal misrouting in visual evoked potentials, nor did we observe a shift in the distribution of ganglion cell thickness from a temporal to a central position. Moreover, our patient's case supports the probable benign nature of the CAPN5 c.230A>G, p.(Gln77Arg) variant.

Otud6b induces pulmonary arterial hypertension by mediating the Calpain-1/HIF-1α signaling pathway.

Pulmonary hypertension (PAH) is a cardiopulmonary disease in which pulmonary artery pressure continues to rise, leading to right heart failure and death. Otud6b is a member of the ubiquitin family and is involved in cell proliferation, apoptosis and inflammation. The aim of this study was to understand the role and mechanism of Otud6b in PAH. C57BL/6 and Calpain-1 knockout (KO) mice were exposed to a PAH model induced by 10% oxygen. Human pulmonary artery endothelial cells (HPACEs) and human pulmonary artery smooth muscle cells (HPASMCs) were exposed to 3% oxygen to establish an in vitro model. Proteomics was used to determine the role of Otud6b and its relationship to Calpain-1/HIF-1α signaling. The increased expression of Otud6b is associated with the progression of PAH. ROtud6b activates Otud6b, induces HIF-1α activation, increases the production of ET-1 and VEGF, and further aggravates endothelial injury. Reducing Otud6b expression by tracheal infusion of siOtud6b has the opposite effect, improving hemodynamic and cardiac response to PAH, reducing the release of Calpain-1 and HIF-1α, and eliminating the pro-inflammatory and apoptotic effects of Otud6b. At the same time, we also found that blocking Calpain-1 reduced the effect of Otud6b on HIF-1α, and inhibiting HIF-1α reduced the expression of Calpain-1 and Otud6b. Our study shows that increased Otud6b expression during hypoxia promotes the development of PAH models through a positive feedback loop between HIF-1α and Calpain-1. Therefore, we use Otud6b as a biomarker of PAH severity, and regulating Otud6b expression may be an effective target for the treatment of PAH.

Formation of the calpain-1/calpastatin complex promotes activation of calpain-1 under oxidizing conditions.

Calpains are cysteine proteinases responsible for many biological roles in muscle, including protein degradation, muscle growth, and myoblast fusion. Calpains are inhibited by calpastatin, an endogenous inhibitor. Other factors, such as variations in pH, ionic strength, and oxidation influence calpain activity. This study aimed to determine the extent to which oxidation influences calpastatin inhibition of calpain-1. A series of order of addition assays were used to determine calpain-1 calcium activation and autolysis after exposure to an oxidizing agent (n-ethylmaleimide [NEM] or hydrogen peroxide [H2O2]. In the first series, purified calpastatin was added to the assay before or after oxidizing exposure at 165 mM NaCl, pH 6.5. In the second series, incubation buffer ionic strength (165 mM or 295 mM NaCl) was evaluated. The inhibitory activities of purified porcine calpastatin, purified human calpastatin domain I, or a subdomain B inhibitor peptide were evaluated in the third series. In the fourth series, a maleimide-polyethylene glycol molecule (MAL-PEG; MW = 5,000 Dalton) was used to evaluate the accessibility of free sulfhydryl groups and tagging of calpain-1 under each condition through a molecular weight shift assay. Results from this study indicate that autolysis of calpain-1, when used as an indicator of activation, occurred when the calpain-1/calpastatin complex was exposed to an oxidant or cysteine modifier such as NEM. However, when calpain-1 was exposed to the cysteine modifier before calpastatin, autolysis of calpain-1 did not occur or was significantly decreased (P < 0.05). Irreversible modification of cysteine residues by NEM prevented activation of calpain-1 in the absence of calpastatin, but if the cysteine modification is potentially reversible (H2O2), calpain-1 activity can be recovered. Results from this study indicate that when calpastatin is bound to calpain-1, calpain-1 activation can occur even after being exposed to a cysteine modifier (NEM) or hydrogen peroxide (H2O2). Calpain-1 is not tagged with maleimide-polyethylene glycol (MAL-PEG) in the presence of calpastatin, indicating that calpastatin blocks or covers free cysteines on calpain-1 from modification. Moreover, exposure to calpain-1/calpastatin complex with a cysteine modifier allows activation of calpain-1, indicating that the inhibitory action of calpastatin is compromised. These results indicate a regulatory role for calpastatin that is not inhibitory but protective for calpain-1.

Protein degradation in skeletal muscle is a key component of protein turnover and maintenance of muscle function. Protein degradation in postmortem muscle is commonly observed and is associated with the accumulation of degradation products and improved meat tenderness. Because there is significant evidence that calpain-1 is involved with proteolysis of muscle proteins in both situations, defining the factors that regulate calpain activity will position scientists to improve calpain-1 activity in both contexts. Calpain-1 is a neutral calcium-dependent proteinase that is inhibited by calpastatin, oxidation, and slightly acidic pH environments. Because oxidation of the calpain/calpastatin complex with hydrogen peroxide appeared to activate calpain-1, we hypothesize that calpastatin binding to calpain may protect the active site cysteine. In the current study, we tested this hypothesis and investigated how n-ethyl maleimide (NEM), an alkylating agent, affects the regulation of calpain in the presence and absence of calpastatin molecules. The results suggest that calpastatin can protect calpain-1 from reacting with maleimide-polyethylene glycol but that exposure of calpain-1/calpastatin complex to NEM or hydrogen peroxide resulted in autolysis and activation of calpain. Under some circumstances, calpastatin appears to protect calpain-1 from inhibition by modification of active site cysteine. These novel observations show a different role for calpastatin and give reason to interpret calpastatin abundance and activity data in a different light.

A retrospective study on the clinical and molecular outcomes of calpainopathy in a Turkish patient cohort.

Background and aim: Calpainopathy, also known as limb-girdle muscular dystrophy recessive type 1, is a progressive muscle disorder that impacts the muscles around the hips and shoulders. The disease is caused by defects in the CAPN3 gene and can be inherited in both recessive and dominant forms. In this retrospective study, we aimed to evaluate the clinical and molecular results of our patients with calpainopathy and to examine the CAPN3 variants in Turkish and global populations. Materials and methods: Molecular analyses were performed using the next-generation sequencing (NGS) method. CAPN3 variants were identified through the examination of various databases. Results: In this retrospective study, the cohort consisted of seven patients exhibiting the CAPN3 (NM_000070.3) mutation and a phenotype compatible with calpainopathy at a single center in Türkiye. All patients displayed high CK levels and muscle weakness. We report a novel missense c.2437G>A variant that causes the autosomal dominant form of calpainopathy. Interestingly, the muscle biopsy report for the patient with the novel mutation indicated sarcoglycan deficiency. Molecular findings for the remaining individuals in the cohort included a compound heterozygous variant (frameshift and missense), one homozygous nonsense, one homozygous intronic deletion, and three homozygous missense variants. The most common variant in the Turkish population was c.550del. In both populations, pathogenic variants were most frequently located in exon 21, according to exon length. Variants were stochastically distributed based on consequences in CAPN3 domains. Conclusion: Therefore, the NGS method proves highly effective in diagnosing rare diseases characterized by clinical heterogeneity. Assessing variants based on ethnicity holds significance in the development of precise therapies.

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. SIGNIFICANCE: Through 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.

Secondary Lipid Oxidation Products as Modulators of Calpain-2 Functionality In Vitro.

The objective was to understand the impacts of secondary lipid oxidation products on calpain-2 activity and autolysis and, subsequently, to determine the quantity and localization of modification sites. 2-Hexenal and 4-hydroxynonenal incubation significantly decreased calpain-2 activity and slowed the progression of autolysis, while malondialdehyde had minimal impact on calpain-2 activity and autolysis. Specific modification sites were determined with LC-MS/MS, including distinct malondialdehyde modification sites on the calpain-2 catalytic and regulatory subunits. 2-Hexenal modification sites were observed on the calpain-2 catalytic subunit. Intact protein mass analysis with MALDI-MS revealed that a significant number of modifications on the calpain-2 catalytic and regulatory subunits are likely to exist. These observations confirm that specific lipid oxidation products modify calpain-2 and may affect the calpain-2 functionality. The results of these novel experiments have implications for healthy tissue metabolism, skeletal muscle growth, and post-mortem meat tenderness development.

Complement Cascade 8 - Alpha and Calpain-2 in Extracellular Vesicles of Human Vitreous as Biomarkers of Infectious Endophthalmitis.

Purpose: Extracellular vesicles (EVs) are messenger pigeons of the cells that communicate about cellular microenvironment. In this study, we evaluated the expression of C8α and calpain-2 in EVs from vitreous of patients with bacterial endophthalmitis to assess its utility as a diagnostic marker. Methods: EVs were isolated from vitreous of patients with bacterial endophthalmitis (culture positive and culture negative) and noninfectious control by exosome isolation reagent and characterized, and the levels of C8α and calpain-2 was assessed by enzyme-linked immunosorbent assay in isolated EVs and direct vitreous. The receiver operating characteristic curve was generated to assess the diagnostic performance. Results: Scanning electron microscopy (SEM) and dynamic light scattering (DLS) confirmed the presence of EVs having a diameter (nm) of 275.2 ± 93, 92 ± 22, and 77.28 ± 12 in culture-positive (CP), culture-negative (CN), and control respectively. The expression level (ng/mL) of C8α in the EVs obtained from CP was 144 ± 22 and CN was 31.2 ± 9.8, which was significantly higher (P < 0.01) than control 3.7 ± 2.4. Interestingly, C8α is not expressed directly in the vitreous of CN and controls. Calpain-2 was significantly downregulated (P ≤ 0.0001) in CP (0.94 ± 0.16) and CN (0.70 ± 0.14) than control. The sensitivity and specificity of 1 for C8α and calpain-2 in the EVs implied that its diagnostic accuracy was significant. Conclusions: This study showed that the EV proteins C8α and calpain-2 could be suitable diagnostic markers for endophthalmitis. However, the presence of C8α in the EVs of CN samples but not in direct vitreous promises EVs as the future of diagnostics. Translational Relevance: Expression levels of EV-calpain-2 and EV-C8α could diagnose CN bacterial endophthalmitis.

LC-MS-based metabolomics reveals metabolite dynamic changes of beef after superchilling early post-mortem.

To explore the underlying mechanisms by which superchilling (SC, -3 °C within 5 h of slaughter) improves beef tenderness, an untargeted metabolomics strategy was employed. M. Longissimus lumborum (LL) muscles from twelve beef carcasses were assigned to either SC or very fast chilling (VFC, 0 °C within 5 h of slaughter) treatments, with conventional chilling (CC, 0 âˆ¼ 4 °C until 24 h post-mortem) serving as the control (6 per group). Biochemical properties and metabolites were investigated during the early post-mortem period. The results showed that the degradation of µ-calpain and caspase 3 occurred earlier in SC treated sample, which might be attributed to the accelerated accumulation of free Ca2+. The metabolomic profiles of samples from the SC and CC treatments were clearly distinguished based on partial least squares-discriminant analysis (PLS-DA) at each time point. It is noteworthy that more IMP and 4-hydroxyproline were found in the comparison between SC and CC treatments. According to the results of metabolic pathways analysis and the correlation analysis between traits related to tenderness and metabolites with significant differences (SC vs. CC), it can be suggested that the tenderization effect of the SC treatment may be related to the alteration of arginine and proline metabolism, and purine metabolism in the early post-mortem phase.

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.

Advancing Nonsmall Cell Lung Cancer Diagnosis Accuracy via Dual Detection Fluorescent Nanoprobes.

Among the primary threats to human health worldwide, nonsmall cell lung cancer (NSCLC) remains a significant factor and is a leading cause of cancer-related deaths. Due to subtle early symptoms, NSCLC patients are diagnosed at advanced stages, resulting in low survival rates. Herein, novel Au-Se bond nanoprobes (NPs) designed for the specific detection of Calpain-2 (CAPN2) and Human Neutrophil Elastase (HNE), pivotal biomarkers in NSCLC, were developed. The NPs demonstrated exceptional specificity and sensitivity toward CAPN2 and HNE, enabling dual-color fluorescence imaging to distinguish between NSCLC cells and normal lung cells effectively. The NPs' performance was consistent across a wide pH range (6.2 to 8.0), and it exhibited remarkable resistance to biological thiol interference, indicating its robustness in complex physiological environments. These findings suggest the nanoprobe is a promising tool for early NSCLC diagnosis, offering a novel approach for enhancing the accuracy of cancer detection.

CAPN2-responsive mesoporous silica nanoparticles: A promising nanocarrier for targeted therapy of pancreatic cancer.

Pancreatic adenocarcinoma (PDAC) is highly resistant to conventional chemotherapeutic interventions, resulting in exceptionally low survival rates. The limited efficacy can in part be attributed to dose limitations and treatment cessation urged by toxicity of currently used chemotherapy. The advent of targeted delivery strategies has kindled hope for circumventing off-target toxicity. We have previously reported a PDAC-specific mesoporous silica nanoparticle (MSN) containing a protease linker responsive to ADAM9, a PDAC-enriched extracellularly deposited protease. Upon loading with paclitaxel these ADAM9-MSNs reduced side effects both in vitro and in vivo, however, disappointing antitumor efficacy was observed in vivo. Here, we propose that an efficient uptake of MSNs by tumor cells might underlie the lack of antitumor efficacy of MSNs functionalized with linker responsive to extracellular proteases. Harnessing this premise to improve antitumor efficacy, we performed an in silico analysis to identify PDAC-enriched intracellular proteases. We report the identification of BACE2, CAPN2 and DPP3 as PDAC enriched intracellular proteases, and report the synthesis of BACE2-, CAPN2- and DPP3-responsive MSNs. Extensive preclinical assessments revealed that paclitaxel-loaded CAPN2- and DPP3-MSNs exhibit high PDAC specificity in vitro as opposed to free paclitaxel. The administration of paclitaxel-loaded CAPN2- and DPP3-MSNs in vivo confirmed the reduction of leukopenia and induced no organ damage. Promisingly, in two mouse models CAPN2-MSNs reduced tumor growth at least as efficiently as free paclitaxel. Taken together, our results pose CAPN2-MSNs as a promising nanocarrier for the targeted delivery of chemotherapeutics in PDAC.

Identification of novel pathogenic variants of Calpain-3 gene in limb girdle muscular dystrophy R1.

BACKGROUND: Limb Girdle Muscular Dystrophy R1 (LGMDR1) is an autosomal recessive neuromuscular disease caused by mutations in the calpain-3 (CAPN3) gene. As clinical and pathological features may overlap with other types of LGMD, therefore definite molecular diagnosis is required to understand the progression of this debilitating disease. This study aims to identify novel variants of CAPN3 gene in LGMDR1 patients. RESULTS: Thirty-four patients with clinical and histopathological features suggestive of LGMD were studied. The muscle biopsy samples were evaluated using Enzyme histochemistry, Immunohistochemistry, followed by Western Blotting and Sanger sequencing. Out of 34 LGMD cases, 13 patients were diagnosed as LGMDR1 by immunoblot analysis, demonstrating reduced or absent calpain-3 protein as compared to controls. Variants of CAPN3 gene were also found and pathogenicity was predicted using in-silico prediction tools. The CAPN3 gene variants found in this study, included, two missense variants [CAPN3: c.1189T > C, CAPN3: c.2338G > C], one insertion-deletion [c.1688delinsTC], one splice site variant [c.2051-1G > T], and one nonsense variant [c.1939G > T; p.Glu647Ter]. CONCLUSIONS: We confirmed 6 patients as LGMDR1 (with CAPN3 variants) from our cohort and calpain-3 protein expression was significantly reduced by immunoblot analysis as compared to control. Besides the previously known variants, our study found two novel variants in CAPN3 gene by Sanger sequencing-based approach indicating that genetic variants in LGMDR1 patients may help to understand the etiology of the disease and future prognostication.

CAPN2 correlates with insulin resistance states in PCOS as evidenced by multi-dataset analysis.

OBJECTIVE: IR emerges as a feature in the pathophysiology of PCOS, precipitating ovulatory anomalies and endometrial dysfunctions that contribute to the infertility challenges characteristic of this condition. Despite its clinical significance, a consensus on the precise mechanisms by which IR exacerbates PCOS is still lacking. This study aims to harness bioinformatics tools to unearth key IR-associated genes in PCOS patients, providing a platform for future therapeutic research and potential intervention strategies. METHODS: We retrieved 4 datasets detailing PCOS from the GEO, and sourced IRGs from the MSigDB. We applied WGCNA to identify gene modules linked to insulin resistance, utilizing IR scores as a phenotypic marker. Gene refinement was executed through the LASSO, SVM, and Boruta feature selection algorithms. qPCR was carried out on selected samples to confirm findings. We predicted both miRNA and lncRNA targets using the ENCORI database, which facilitated the construction of a ceRNA network. Lastly, a drug-target network was derived from the CTD. RESULTS: Thirteen genes related to insulin resistance in PCOS were identified via WGCNA analysis. LASSO, SVM, and Boruta algorithms further isolated CAPN2 as a notably upregulated gene, corroborated by biological verification. The ceRNA network involving lncRNA XIST and hsa-miR-433-3p indicated a possible regulatory link with CAPN2, supported by ENCORI database. Drug prediction analysis uncovered seven pharmacological agents, most being significant regulators of the endocrine system, as potential candidates for addressing insulin resistance in PCOS. CONCLUSIONS: This study highlights the pivotal role of CAPN2 in insulin resistance within the context of PCOS, emphasizing its importance as both a critical biomarker and a potential therapeutic target. By identifying CAPN2, our research contributes to the expanding evidence surrounding the CAPN family, particularly CAPN10, in insulin resistance studies beyond PCOS. This work enriches our understanding of the mechanisms underlying insulin resistance, offering insights that bridge gaps in the current scientific landscape.

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.

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.

Regulation of developmental gatekeeping and cell fate transition by the calpain protease DEK1 in Physcomitrium patens.

Calpains are cysteine proteases that control cell fate transitions whose loss of function causes severe, pleiotropic phenotypes in eukaryotes. Although mainly considered as modulatory proteases, human calpain targets are directed to the N-end rule degradation pathway. Several such targets are transcription factors, hinting at a gene-regulatory role. Here, we analyze the gene-regulatory networks of the moss Physcomitrium patens and characterize the regulons that are misregulated in mutants of the calpain DEFECTIVE KERNEL1 (DEK1). Predicted cleavage patterns of the regulatory hierarchies in five DEK1-controlled subnetworks are consistent with a pleiotropic and regulatory role during cell fate transitions targeting multiple functions. Network structure suggests DEK1-gated sequential transitions between cell fates in 2D-to-3D development. Our method combines comprehensive phenotyping, transcriptomics and data science to dissect phenotypic traits, and our model explains the protease function as a switch gatekeeping cell fate transitions potentially also beyond plant development.