Calcium-dependent Protease Research Articles

Molecular basis of the development of Parkinson's disease.

Parkinson's disease is one of the most prevalent neurodegenerative motor disorders worldwide with postural instability, bradykinesia, resting tremor and rigidity being the most common symptoms of the disease. Despite the fact that the molecular mechanisms of Parkinson's disease pathogenesis have already been well described, there is still no coherent picture of the etiopathogenesis of this disease. According to modern concepts, neurodegeneration is induced mainly by oxidative stress, neuroinflammation, dysregulation of cerebral proteostasis, apoptotic dysregulation, and impaired autophagy. This review describes how various factors contribute to neurodegeneration in Parkinson's disease. Understanding the factors affecting fundamental cellular processes and responsible for disease progression may help develop therapeutic strategies to improve the quality of life of patients suffering from the disease. The review also discusses the role of calpains in the development of Parkinson's disease. It is known that α-synuclein is a substrate of calcium-dependent proteases of the calpain family. Truncated forms of α-synuclein are not only involved in the process of formation of the aggregates, but also increase their toxicity.

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

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

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

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

Comprehensive bioinformatics analysis of the prognostic value and immune infiltration of CAPN2 in pancreatic adenocarcinoma

BackgroundPancreatic adenocarcinoma (PAAD) is a highly aggressive cancer with a poor prognosis, highlighting an urgent requirement for effective biomarkers for its early diagnosis and prognosis prediction. CAPN2, a calcium-dependent protease, has been implicated in various cancers, but its role in PAAD remains unclear. MethodsIn this study, we utilized multiple bioinformatics methods, including differential expression, survival, correlation, and enrichment analyses, to investigate the prognostic value of CAPN2 in PAAD using data from the TCGA and GEO databases. Additionally, the correlation between CAPN2 expression and the tumor microenvironment (TME), immunotherapy potential, and drug sensitivity was also explored. ResultsCAPN2 was upregulated in PAAD tissues and was correlated with higher tumor grade. And high expression of CAPN2 was significantly associated with reduced overall survival, establishing it as an independent prognostic biomarker for PAAD. Enrichment analysis implicated that CAPN2 was involved in multiple biological processes and pathways associated with tumor immunity. Furthermore, CAPN2 expression had a negative correlation with immune cell infiltration and a positive association with tumor mutational burden, which may have potential implications for immunotherapy strategies. ConclusionsCAPN2 is a promising biomarker for PAAD prognosis and a potential therapeutic target. Its association with the TME and immunotherapy response highlights its importance in PAAD progression and patient outcomes, warranting further investigation into its role and potential clinical applications.

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

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

Revisiting the calpain hypothesis of learning and memory 40 years later.

In 1984, Gary Lynch and Michel Baudry published in Science a novel biochemical hypothesis for learning and memory, in which they postulated that the calcium-dependent protease, calpain, played a critical role in regulating synaptic properties and the distribution of glutamate receptors, thereby participating in memory formation in hippocampus. Over the following 40 years, much work has been done to refine this hypothesis and to provide convincing arguments supporting what was viewed at the time as a simplistic view of synaptic biochemistry. We have now demonstrated that the two major calpain isoforms in the brain, calpain-1 and calpain-2, execute opposite functions in both synaptic plasticity/learning and memory and in neuroprotection/neurodegeneration. Thus, calpain-1 activation is required for triggering long-term potentiation (LTP) of synaptic transmission and learning of episodic memory, while calpain-2 activation limits the magnitude of LTP and the extent of learning. On the other hand, calpain-1 is neuroprotective while calpain-2 is neurodegenerative, and its prolonged activation following various types of brain insults leads to neurodegeneration. The signaling pathways responsible for these functions have been identified and involve local protein synthesis, cytoskeletal regulation, and regulation of glutamate receptors. Human families with mutations in calpain-1 have been reported to have impairment in motor and cognitive functions. Selective calpain-2 inhibitors have been synthesized and clinical studies to test their potential use to treat disorders associated with acute neuronal damage, such as traumatic brain injury, are being planned. This review will illustrate the long and difficult journey to validate a bold hypothesis.

Characterization of MdpS: an in-depth analysis of a MUC5B-degrading protease from Streptococcus oralis.

Oral biofilms, comprising hundreds of bacteria and other microorganisms on oral mucosal and dental surfaces, play a central role in oral health and disease dynamics. Streptococcus oralis, a key constituent of these biofilms, contributes significantly to the formation of which, serving as an early colonizer and microcolony scaffold. The interaction between S. oralis and the orally predominant mucin, MUC5B, is pivotal in biofilm development, yet the mechanism underlying MUC5B degradation remains poorly understood. This study introduces MdpS (Mucin Degrading Protease from Streptococcus oralis), a protease that extensively hydrolyses MUC5B and offers an insight into its evolutionary conservation, physicochemical properties, and substrate- and amino acid specificity. MdpS exhibits high sequence conservation within the species and also explicitly among early biofilm colonizing streptococci. It is a calcium or magnesium dependent serine protease with strict physicochemical preferences, including narrow pH and temperature tolerance, and high sensitivity to increasing concentrations of sodium chloride and reducing agents. Furthermore, MdpS primarily hydrolyzes proteins with O-glycans, but also shows activity toward immunoglobulins IgA1/2 and IgM, suggesting potential immunomodulatory effects. Significantly, MdpS extensively degrades MUC5B in the N- and C-terminal domains, emphasizing its role in mucin degradation, with implications for carbon and nitrogen sequestration for S. oralis or oral biofilm cross-feeding. Moreover, depending on substrate glycosylation, the amino acids serine, threonine or cysteine triggers the enzymatic action. Understanding the interplay between S. oralis and MUC5B, facilitated by MdpS, has significant implications for the management of a healthy eubiotic oral microenvironment, offering potential targets for interventions aimed at modulating oral biofilm composition and succession. Additionally, since MdpS does not rely on O-glycan removal prior to extensive peptide backbone hydrolysis, the MdpS data challenges the current model of MUC5B degradation. These findings emphasize the necessity for further research in this field.

Aberrant mitochondrial aggregation of TDP-43 activated mitochondrial unfolded protein response and contributed to recovery of acetaminophen induced acute liver injury.

Mitochondrial dysfunction is a key pathological event in the acute liver injury following the overdose of acetaminophen (APAP). Calpain is the calcium-dependent protease, recent studies demonstrate that it is involved in the impairment of mitochondrial dynamics. The mitochondrial unfolded protein response (UPRmt) is commonly activated in the context of mitochondrial damage following pathological insults and contributes to the maintenance of the mitochondrial quality control through regulating a wide range of gene expression. More importantly, it is reported that abnormal aggregation of TDP-43 in mitochondria induced the activation of UPRmt. However, whether it is involved in APAP induced-hepatotoxicity remains unclear. In the present study, C57/BL6 mice were given 300mg/kg APAP to establish a time-course model of acute liver injury. Furthermore, Calpeptin, the specific inhibiter of calpains, was used to conduct the intervention experiment. Our results showed, APAP exposure produced severe liver injury. Moreover, TDP-43 was obviously accumulated within mitochondria whereas mitochondrial protease LonP1 was significantly decreased. However, these changes exhibited significant recovery at 48h. By contrast, the mitochondrial protease ClpP and chaperone mtHSP70 and HSP60 were consistently increased, which supported the UPRmt was activated to promote protein homeostasis. Further investigation revealed that calpain-mediated cleavage of TDP-43 could promote the accumulation of TDP-43 in mitochondria compartment, thereby facilitating the activation of UPRmt. Additionally, Calpeptin pretreatment not only protected against APAP-induced liver injury, but also suppressed the formation of TDP-43 aggregates and the activation of UPRmt. Taken together, our findings indicated that in APAP-induced acute liver injury, calpain-mediated cleavage of TDP43 caused its aberrant aggregation on the mitochondria. As a stress-protective response, the induction of UPRmt contributed to the recovery of mitochondrial function.

A Rhodnius prolixus catalytically inactive Calpain protease patterns the insect embryonic dorsal-ventral axis

The calcium dependent Calpain proteases are modulatory enzymes with important roles in cell cycle control, development and immunity. In the fly model Drosophila melanogaster Calpain A cleaves Cactus/IkappaB and consequently modifies Toll signals during embryonic dorsal-ventral (DV) patterning. Here we explore the role of Calpains in the hemiptera Rhodnius prolixus, an intermediate germband insect where the Bone Morphogenetic Protein (BMP) instead of the Toll pathway plays a major role in DV patterning. Phylogenetic analysis of Calpains in species ranging from Isoptera to Diptera indicates an increase of Calpain sequences in the R. prolixus genome and other hemimetabolous species. One locus encoding each of the CalpC, CalpD and Calp7 families, and seven Calpain A/B loci are present in the R. prolixus genome. Several predicted R. prolixus Calpains display a unique architecture, such as loss of Calcium-binding EF-hand domains and loss of catalytic residues in the active site CysPc domain, yielding catalytically dead Calpains A/B. Knockdown for one of these inactive Calpains results in embryonic DV patterning defects, with expansion of ventral and lateral gene expression domains and consequent failure of germ band elongation. In conclusion, our results reveal that Calpains may exert a conserved function in insect DV patterning, despite the changing role of the Toll and BMP pathways in defining gene expression territories along the insect DV axis.

Targeting the Mitochondrial Chaperone TRAP1 Alleviates Vascular Pathologies in Ischemic Retinopathy.

Activation of hypoxia-inducible factor 1α (HIF1α) contributes to blood-retinal barrier (BRB) breakdown and pathological neovascularization responsible for vision loss in ischemic retinal diseases. During disease progression, mitochondrial biology is altered to adapt to the ischemic environment created by initial vascular dysfunction, but the mitochondrial adaptive mechanisms, which ultimately contribute to the pathogenesis of ischemic retinopathy, remain incompletely understood. In the present study, it is identified that expression of mitochondrial chaperone tumor necrosis factor receptor-associated protein 1 (TRAP1) is essential for BRB breakdown and pathologic retinal neovascularization in mouse models mimicking ischemic retinopathies. Genetic Trap1 ablation or treatment with small molecule TRAP1 inhibitors, such as mitoquinone (MitoQ) and SB-U015, alleviate retinal pathologies via proteolytic HIF1α degradation, which is mediated by opening of the mitochondrial permeability transition pore and activation of calcium-dependent protease calpain-1. These findings suggest that TRAP1 can be a promising target for the development of new treatments against ischemic retinopathy, such as retinopathy of prematurity and proliferative diabetic retinopathy.

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

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

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

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

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

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

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

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

Calpain-mediated Mechanoptosis in Breast Adenocarcinoma.

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

Interplay between Furin and Sialoglycans in Modulating Adeno-Associated Viral Cell Entry.

Adeno-associated viruses (AAVs) are small, helper-dependent, single-stranded DNA viruses that exploit a broad spectrum of host factors for cell entry. During the course of infection, several AAV serotypes have been shown to transit through the trans-Golgi network within the host cell. In the current study, we investigated whether the Golgi-localized, calcium-dependent protease furin influences AAV transduction. While CRISPR/Cas9-mediated knockout (KO) of the Furin gene minimally affected the transduction efficiency of most recombinant AAV serotypes tested, we observed a striking increase in transgene expression (~2 log orders) for the African green monkey isolate AAV4. Interrogation of different steps in the infectious pathway revealed that AAV4 binding, uptake, and transcript levels are increased in furin KO cells, but postentry steps such as uncoating or nuclear entry remain unaffected. Recombinant furin does not cleave AAV4 capsid proteins nor alter cellular expression levels of essential factors such as AAVR or GPR108. Interestingly, fluorescent lectin screening revealed a marked increase in 2,3-O-linked sialoglycan staining on the surface and perinuclear space of furin KO cells. The essential nature of increased sialoglycan expression in furin KO cells in enhancing AAV4 transduction was further corroborated by (i) increased transduction by the closely related isolates AAVrh.32.33 and sea lion AAV and (ii) selective blockade or removal of cellular 2,3-O-linked sialoglycans by specific lectins or neuraminidase, respectively. Based on the overall findings, we postulate that furin likely plays a key role in regulating expression of cellular sialoglycans, which in turn can influence permissivity to AAVs and possibly other viruses. IMPORTANCE Adeno-associated viruses (AAVs) are a proven recombinant vector platform for gene therapy and have demonstrated success in the clinic. Continuing to improve our knowledge of AAV-host cell interactions is critical for improving the safety and efficacy. The current study dissects the interplay between furin, a common intracellular protease, and certain cell surface sialoglycans that serve as viral attachment factors for cell entry. Based on the findings, we postulate that differential expression of furin in host cells and tissues is likely to influence gene expression by certain recombinant AAV serotypes.

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

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

The protease calpain2a limits innate immunity by targeting TRAF6 in teleost fish

TNF receptor-associated factor 6 (TRAF6) plays a key signal transduction role in both antibacterial and antiviral signaling pathways. However, the regulatory mechanisms of TRAF6 in lower vertebrates are less reported. In this study, we identify calpain2a, is a member of the calcium-dependent proteases family with unique hydrolytic enzyme activity, functions as a key regulator for antibacterial and antiviral immunity in teleost fish. Upon lipopolysaccharide (LPS) stimulation, knockdown of calpain2a promotes the upregulation of inflammatory cytokines. Mechanistically, calpain2a interacts with TRAF6 and reduces the protein level of TRAF6 by hydrolyzing. After loss of enzymatic activity, mutant calpain2a competitively inhibits dimer formation and auto-ubiquitination of TRAF6. Knockdown of calpain2a also promotes cellular antiviral response. Mutant calpain2a lacking hydrolase activity represses ubiquitination of IFN regulatory factor (IRF) 3/7 from TRAF6. Taken together, these findings classify calpain2a is a negative regulator of innate immune responses by targeting TRAF6 in teleost fish.

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

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

Proprotein Convertase Furin Regulates Melanogenesis via the Notch Signaling Pathway.

Furin is a calcium-dependent serine protease found in almost all mammals. It plays an important role in embryogenesis, tissue homeostasis, tumors pathogenesis, viral infectious diseases, and neurodegenerative diseases. However, whether furin directly regulates melanin synthesis and transport has rarely been evaluated yet. The present study aimed to investigate furin potential function and mechanisms in melanogenesis. Short hairpin RNAs targeting furin gene (sh-furin RNAs) were used to inhibit furin gene expression in human melanoma cell line MNT-1 cells. Then, intracellular melanin content was measured using a sodium hydroxide method. Extracellular melanin content was measured determining cell culture medium absorbance at 450 nm. Levodopa (L-DOPA) oxidation rate was measured to assess the tyrosinase activity. Quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting (WB) were performed to measure melanogenesis-related genes and Notch pathway-related genes expression levels. Human primary melanocytes (MCs) were extracted from foreskin tissues and were stimulated with a furin inhibitor. Then, the extracellular and intracellular melanin content, tyrosinase activity and molecules related to melanogenesis and the Notch pathway expression were measured in MCs with or without a furin inhibitor. Additionally, morpholino technology was used to inhibit furin in zebrafish. Zebrafish pigmentary phenotypes in the control group and furin inhibition group were observed with a stereo microscope. Then, MCs number in the tail and head of the zebrafish were counted using Image J software (version 1.53t, National Institute of Health, Bethesda, MD, USA). Meanwhile, melanin content, tyrosinase activity, and molecules related to melanogenesis and the Notch pathway expression levels were measured. Subsequently, valproic acid (VPA), a Notch pathway agonist, was used in MNT-1 melanoma cells treated with or without sh-furin lentiviral vectors for rescue experiments. Furin inhibition enhanced intracellular and extracellular melanin content, and cellular tyrosinase activity in MNT-1 cells and MCs. Additionally, furin inhibition increased melanin synthesis-associated and transport-associated proteins expression levels while inhibiting Notch pathway-relevant proteins. After using VPA to activate the Notch pathway in MNT-1 cells transfected with a sh-furin RNA, the biological effects resulting from furin knockdown were reversed. In addition, the results of in vivo experiments using morpholino to knock down furin gene in zebrafish further confirmed that furin knockdown regulated melanogenesis and impaired the Notch pathway. This study clarified that furin affected the synthesis and transport of melanin via Notch pathway. Notch pathway may be a potential therapeutic target for pigmented skin diseases.