Dynamic expression of cathepsin L in the black soldier fly (Hermetia illucens) gut during Escherichia coli challenge.

The black soldier fly (BSF), Hermetia illucens, has the potential to serve as a valuable resource for waste bioconversion due to the ability of the larvae to thrive in a microbial-rich environment. Being an ecological decomposer, the survival of BSF larvae (BSFL) relies on developing an efficient defense system. Cathepsin L (CTSL) is a cysteine protease that plays roles in physiological and pathological processes. In this study, the full-length of CTSL was obtained from BSF. The 1,020-bp open reading frame encoded a preprotein of 339 amino acids with a predicted molecular weight of 32 kDa. The pro-domain contained the conserved ERFNIN, GNYD, and GCNGG motifs, which are all characteristic of CTSL. Homology revealed that the deduced amino acid sequence of BSF CTSL shared 74.22-72.99% identity with Diptera flies. Immunohistochemical (IHC) analysis showed the CTSL was predominantly localized in the gut, especially in the midgut. The mRNA expression of CTSL in different larval stages was analyzed by quantitative real-time PCR (RT-qPCR), which revealed that CTSL was expressed in the second to sixth instar, with the highest expression in the fifth instar. Following an immune challenge in vivo using Escherichia coli (E. coli), CTSL mRNA was significantly up-regulated at 6 h post-stimulation. The Z-Phe-Arg-AMC was gradually cleaved by the BSFL extract after 3 h post-stimulation. These results shed light on the potential role of CTSL in the defense mechanism that helps BSFL to survive against pathogens in a microbial-rich environment.

Inhibition of cysteine protease cathepsin Lincreases the level and activity of lysosomal glucocerebrosidase.

The glucocerebrosidase (GCase) encoded by the GBA1 gene hydrolyzes glucosylceramide (GluCer) to ceramide and glucose in lysosomes. Homozygous or compound heterozygous GBA1 mutations cause the lysosomal storage disease Gaucher disease (GD) due to severe loss of GCase activity. Loss-of-function variants in the GBA1 gene are also the most common genetic risk factor for Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Restoring lysosomal GCase activity represents an important therapeutic approach for GBA1-associated diseases. We hypothesized that increasing the stability of lysosomal GCase protein could correct deficient GCase activity in these conditions. However, it remains unknown how GCase stability is regulated in the lysosome. We found that cathepsin L, a lysosomal cysteine protease, cleaves GCase and regulates its stability. In support of these data, GCase protein was elevated in the brain of cathepsin L-KO mice. Chemical inhibition of cathepsin L increased both GCase levels and activity in fibroblasts from patients with GD. Importantly, inhibition of cathepsin L in dopaminergic neurons from a patient GBA1-PD led to increased GCase levels and activity as well as reduced phosphorylated α-synuclein. These results suggest that targeting cathepsin L-mediated GCase degradation represents a potential therapeutic strategy for GCase deficiency in PD and related disorders that exhibit decreased GCase activity.

Cathepsin L prevents the accumulation of alpha-synuclein fibrils in the cell.

The deposition of α-synuclein (α-Syn) fibrils in neuronal cells has been implicated as a causative factor in Parkinson's disease (PD) and dementia with Lewy Bodies (DLB). α-Syn can be degraded by autophagy, proteasome, and chaperone-mediated autophagy, and previous studies have suggested the potency of certain cathepsins, lysosomal proteases, for α-Syn degradation. However, no studies have comprehensively evaluated all cathepsins. Here, we evaluated the efficacy of all 15 cathepsins using a cell model of α-Syn fibril propagation and found that overexpression of cathepsin L (CTSL) was the most effective in preventing the accumulation of α-Syn aggregates. CTSL-mediated degradation of α-Syn aggregates was dependent on the autophagy machinery, and CTSL itself promoted autophagy flux. Interestingly, CTSL was effective in autophagic degradation of wild-type (WT) α-Syn, but not in the case of A53T and E46K missense mutations, which are causative for familial PD. These results suggest that CTSL is a potential therapeutic strategy for sporadic PD pathology in WT α-Syn.

Trichinella spiralis cathepsin L damages the tight junctions of intestinal epithelial cells and mediates larval invasion.

BACKGROUND: Cathepsin L, a lysosomal enzyme, participates in diverse physiological processes. Recombinant Trichinella spiralis cathepsin L domains (rTsCatL2) exhibited natural cysteine protease activity and hydrolyzed host immunoglobulin and extracellular matrix proteins in vitro, but its functions in larval invasion are unknown. The aim of this study was to explore its functions in T. spiralis invasion of the host's intestinal epithelial cells. METHODOLOGY/PRINCIPAL FINDINGS: RNAi significantly suppressed the expression of TsCatL mRNA and protein with TsCatL specific siRNA-302. T. spiralis larval invasion of Caco-2 cells was reduced by 39.87% and 38.36%, respectively, when anti-TsCatL2 serum and siRNA-302 were used. Mice challenged with siRNA-302-treated muscle larvae (ML) exhibited a substantial reduction in intestinal infective larvae, adult worm, and ML burden compared to the PBS group, with reductions of 44.37%, 47.57%, and 57.06%, respectively. The development and fecundity of the females from the mice infected with siRNA-302-treated ML was significantly inhibited. After incubation of rTsCatL2 with Caco-2 cells, immunofluorescence test showed that the rTsCatL2 gradually entered into the cells, altered the localization of cellular tight junction proteins (claudin 1, occludin and zo-1), adhesion junction protein (e-cadherin) and extracellular matrix protein (laminin), and intercellular junctions were lost. Western blot showed a 58.65% reduction in claudin 1 expression in Caco-2 cells treated with rTsCatL2. Co-IP showed that rTsCatL2 interacted with laminin and collagen I but not with claudin 1, e-cadherin, occludin and fibronectin in Caco-2 cells. Moreover, rTsCatL2 disrupted the intestinal epithelial barrier by inducing cellular autophagy. CONCLUSIONS: rTsCatL2 disrupts the intestinal epithelial barrier and facilitates T. spiralis larval invasion.

Enhancement of Radiation Sensitivity by Cathepsin L Suppression in Colon Carcinoma Cells.

Cancer is one of the main causes of death globally. Radiotherapy/Radiation therapy (RT) is one of the most common and effective cancer treatments. RT utilizes high-energy radiation to damage the DNA of cancer cells, leading to their death or impairing their proliferation. However, radiation resistance remains a significant challenge in cancer treatment, limiting its efficacy. Emerging evidence suggests that cathepsin L (cath L) contributes to radiation resistance through multiple mechanisms. In this study, we investigated the role of cath L, a member of the cysteine cathepsins (caths) in radiation sensitivity, and the potential reduction in radiation resistance by using the specific cath L inhibitor (Z-FY(tBu)DMK) or by knocking out cath L with CRISPR/Cas9 in colon carcinoma cells (caco-2). Cells were treated with different doses of radiation (2, 4, 6, 8, and 10), dose rate 3 Gy/min. In addition, the study conducted protein expression analysis by western blot and immunofluorescence assay, cytotoxicity MTT, and apoptosis assays. The results demonstrated that cath L was upregulated in response to radiation treatment, compared to non-irradiated cells. In addition, inhibiting or knocking out cath L led to increased radiosensitivity in contrast to the negative control group. This may indicate a reduced ability of cancer cells to recover from radiation-induced DNA damage, resulting in enhanced cell death. These findings highlight the possibility of targeting cath L as a therapeutic strategy to enhance the effectiveness of RT. Further studies are needed to elucidate the underlying molecular mechanisms and to assess the translational implications of cath L knockout in clinical settings. Ultimately, these findings may contribute to the development of novel treatment approaches for improving outcomes of RT in cancer patients.

Eosinophils promote pulmonary matrix destruction and emphysema via Cathepsin L.

Patients with chronic obstructive pulmonary disease (COPD) who exhibit elevated blood eosinophil levels often experience worsened lung function and more severe emphysema. This implies the potential involvement of eosinophils in the development of emphysema. However, the precise mechanisms underlying the development of eosinophil-mediated emphysema remain unclear. In this study, we employed single-cell RNA sequencing to identify eosinophil subgroups in mouse models of asthma and emphysema, followed by functional analyses of these subgroups. Assessment of accumulated eosinophils unveiled distinct transcriptomes in the lungs of mice with elastase-induced emphysema and ovalbumin-induced asthma. Depletion of eosinophils through the use of anti-interleukin-5 antibodies ameliorated elastase-induced emphysema. A particularly noteworthy discovery is that eosinophil-derived cathepsin L contributed to the degradation of the extracellular matrix, thereby leading to emphysema in pulmonary tissue. Inhibition of cathepsin L resulted in a reduction of elastase-induced emphysema in a mouse model. Importantly, eosinophil levels correlated positively with serum cathepsin L levels, which were higher in emphysema patients than those without emphysema. Expression of cathepsin L in eosinophils demonstrated a direct association with lung emphysema in COPD patients. Collectively, these findings underscore the significant role of eosinophil-derived cathepsin L in extracellular matrix degradation and remodeling, and its relevance to emphysema in COPD patients. Consequently, targeting eosinophil-derived cathepsin L could potentially offer a therapeutic avenue for emphysema patients. Further investigations are warranted to explore therapeutic strategies targeting cathepsin L in emphysema patients.

Involvement of cathepsin L in the degradation and degeneration of postovulatory follicle of the medaka ovary†.

Cathepsin L plays physiological and pathological roles in immune responses, cancer, metamorphosis, and oogenesis in several species. However, the function of Cathepsin L in medaka ovaries remains unclear. Therefore, here, we examined the physiological functions of Cathepsin L in the medaka ovaries. Cathepsin L mRNA transcripts and proteins were found to be constitutively expressed in the ovaries of Oryzias latipes over a 24-h spawning cycle. Expression was localized within the oocyte cytoplasm of growing follicles and the follicle layer of preovulatory and postovulatory follicles. Moreover, the active form of Cathepsin L was highly expressed in the follicle layer of periovulatory follicles and the ovaries 2-6 h after ovulation. Recombinant Cathepsin L was activated under acidic conditions and exhibited enzymatic activity in acidic and neutral pH conditions. However, extracellular matrix proteins were degraded by recombinant Cathepsin L under acidic, not neutral pH conditions. Cathepsin L was secreted from preovulatory follicles, while active recombinant Cathepsin L was detected in the conditioned medium of a medaka cell line, OLHNI-2. Mechanistically, recombinant Cathepsin L activates recombinant urokinase-type plasminogen activator-1, which is expressed within the follicle layers post-ovulation. Meanwhile, the treatment of medakas with an E-64 or anti-Cathepsin L antibody effectively blocked follicular layer degeneration and degradation after ovulation, whereas in vitro ovulation was not inhibited by either. Collectively, the findings of this study indicate that although Cathepsin L does not impact ovulation in medakas, it contributes to the degeneration and degradation of the follicle layers following ovulation via activation of urokinase-type plasminogen activator-1, and not via the degradation of extracellular matrix proteins.

The analysis of cathepsin L that mediates cellular SARS-CoV-2 infection leading to COVID-19 in head and neck squamous cell carcinoma.

The vulnerability of the oral cavity to SARS-CoV-2 infection is well-known, and cancer patients are at a higher risk of COVID-19, emphasizing the need to prioritize this patient population. Head and neck squamous cell carcinoma (HNSCC) is one of the most common malignant cancers associated with early metastasis and poor prognosis. It has been established that cancerous tissues express Cathepsin L (CTSL), a proteinase that regulates cancer progression and SARS-CoV-2 entry. Therefore, it is essential to evaluate the correlation between disease outcomes and CTSL expression in cancer tissues and predict the susceptibility of cancer patients to SARS-CoV-2. In this study, we used transcriptomic and genomic data to profile CTSL expression in HNSCC and developed a CTSL signature that could reflect the response of HNSCC patients to chemotherapy and immunotherapy. Additionally, we investigated the relationship between CTSL expression and immune cell infiltration and established CTSL as a potential carcinogenic factor for HNSCC patients. These findings could aid in understanding the mechanisms underlying the increased susceptibility of HNSCC patients to SARS-CoV-2 and contribute to the development of therapy for both HNSCC and COVID-19.

Identification and genetic characterisatin of cathepsin L in Demodex.

Owing to difficulties in obtaining functional gene sequences, molecular pathogenic mechanisms in Demodex have been understudied. In this study, overlap extension PCR was used to obtain the sequences of cathepsin L (CatL), a pathogenicity-related gene, to provide a foundation for subsequent functional research. Demodex folliculorum and Demodex brevis mites were obtained from the face skin of Chinese individuals, and Demodex canis mites were isolated from the skin lesions of a dog. RNA was extracted and used to synthesise double-stranded cDNA. PCR amplification, cloning, sequencing, and bioinformatics analysis of CatL were performed. CatL gene sequences of 1005, 1008, and 1008 bp were successfully amplified for D. brevis, D. folliculorum, and D. canis, respectively. These sequences showed 99.9 or 100% identity with templates previously obtained by RNA-seq. The Maximum Likelihood (ML) phylogenetic tree showed that D. folliculorum clustered with D. canis first, then with D. brevis, and finally with other Acariformes mite species. The three Demodex species had nine similar motifs to those of Sarcoptes scabies, Dermatophagoides pteronyssinus, and Dermatophagoides farinae, and motifs 10-13 were valuable for identification. CatL proteins of Demodex species were predicted to be approximately 38 kDa, be located in lysosomes, have a signal peptide but no transmembrane region, and have two functional domains, I29 and Pept_C1. However, interspecific differences were observed in secondary and tertiary protein structures. In conclusion, we successfully obtained CatL sequences of three Demodex species by overlap extension PCR, which creates conditions for further pathogenic mechanism studies.

Comparative analysis of two cathepsin L genes in Asiatic hard clam (Meretrix meretrix): Similar in sequence features, different in expression profiles.

Cathepsin L is widely found in eukaryotes and prokaryotes, and it plays important roles in innate immunity. In the present study, we cloned two cathepsin L genes (designated as MmCTSL1 and MmCTSL2, respectively) from Asiatic hard clam (Meretrix meretrix). The complete sequence of MmCTSL1 cDNA contained a 5' untranslated region (UTR) of 31 bp, a 3' UTR of 228 bp with a poly (A) tail, and an open reading frame (ORF) of 1005 bp encoding 334 amino acids with predicted molecular weight of 37.5 kDa and theoretical isoelectric point of 5.27, and contained a signal peptide (from M1 to A16), a protease inhibitor I29 family domain (from W27 to F87), and a papain family cysteine protease domain (from L118 to T333). The complete sequence of MmCTSL2 cDNA contained a 5' UTR of 50 bp, a 3' UTR of 162 bp with a poly (A) tail, and an ORF of 996 bp encoding a polypeptide of 331 amino acids with predicted molecular weight of 36.8 kDa and theoretical isoelectric point of 7.07. It contained a signal peptide (from M1 to A16), a protease inhibitor I29 family domain (from W30 to F89), and a papain family cysteine protease domain (from L115 to T330). Real-time quantitative PCR analysis demonstrated that MmCTSL1 and MmCTSL2 were widely expressed in all the tested tissues, including adductor muscle, foot, gill, hemocytes, hepatopancreas and mantle, with the highest mRNA expression level in hepatopancreas and hemocytes, respectively. After Vibrio splendidus challenge, the mRNA expression levels of MmCTSL1 and MmCTSL2 in hemocytes and hepatopancreas were both significantly up-regulated with different expression profiles. In hemocytes, the expression levels of MmCTSL1 and MmCTSL2 reached their respective peaks (3.4-fold and 13.0-fold compared with the control, respectively) at 12 h after bacterial challenge, and MmCTSL2 responds earlier than MmCTSL1. In hepatopancreas, the expression levels of MmCTSL1 and MmCTSL2 reached their respective peaks at 6 h (9.0-fold compared with the control) and 24 h (2.8-fold compared with the control) after bacterial challenge, meaning that MmCTSL1 responds earlier than MmCTSL2. At the same time, whether in hepatopancreas or hemocytes, MmCTSL1 persist for a while after the bacterial challenge peak, while MmCTSL2 would quickly return to the initial level after the bacterial challenge peak. These results indicate that cathepsin L may be involved in the immune process of hard clam against V. splendidus with different potential roles.

Cathepsin L was involved in vascular aging by mediating phenotypic transformation of vascular cells.

Vascular media and adventitia-induced remodeling plays an important role in vascular aging. However, the mechanism remains unclear. This study aims to investigate the mechanisms underlying vascular aging. Transcriptome analysis revealed that the expression of cathepsin L (CTSL) significantly decreased in arteries of old mice (24 months old) compared with that in arteries of young mice (4 months old), which was confirmed by immunohistochemistry and Western blot. The expression of CTSL in adventitia fibroblasts (AFs) and vascular smooth muscle cells (VSMCs) of aged mice was lower than that of young mice. Compared with wild-type control mice, CTSL knockout (CTSL - /-) mice had increased collagen deposition (fibrosis) and decreased telomerase activity and LC3Ⅱ/ LC3Ⅰratio. The expression of mammalian target of rapamycin (mTOR) and osteopontin (OPN) increased in aortas of CTSL-/-mice compared with that in aortas of wild-type control mice. In vitro, lentivirus-mediated CTSL knockdown induced VSMCs senescence and AFs transformed into myofibroblasts (MFs). Rapamycin, a mTOR inhibitor, inhibited CTSL deficiency induced VSMCs senescence, osteopontin (OPN) secretion and AFs migration. In conclusion, the decreased level of CTSL with age may participate in vascular aging by promoting the phenotypic transformation of vascular cells.

Characteristics of cathepsin members and expression responses to poly I:C challenge in Pacific cod (Gadus macrocephalus).

Cathepsins are major lysosomal enzymes that participate in necessary physiological processes, including protein degradation, tissue differentiation, and innate or adaptive immune responses. According to their proteolytic activity, vertebrate cathepsins are classified as cysteine proteases (cathepsins B, C, F, H, K, L, O, S, V, W, and X or Z), aspartic proteases (cathepsin D and E), and serine proteases (cathepsin A and G). Several cathepsins were reported in teleosts, however, no cathepsin gene has been identified from Pacific cod so far. In the present study, a total of 13 cathepsin genes were identified for Pacific cod. The evolutionary path of each cathepsin gene was demonstrated via analysis of phylogenetic trees, multiple alignments, conserved domains, motif compositions, and tertiary structures. Tissue distribution analysis showed that all cathepsin genes were ubiquitously expressed in eight healthy tissues but they exhibited diverse levels of expression. Several cathepsin genes were found to be highly expressed in the kidney, spleen, head kidney and liver, whereas low or modest levels were detected in the gills, skin, intestines, and heart. Temporal-specific expression of cathepsins in early developmental stages of Pacific cod were also conducted. CTSK, S, F, and Z were highly expressed at 1 dph and 5 dph and decreased later, while CTSL, L1, and L.1 transcript levels gradually increased in a time-dependent manner. Additionally, the expression profiles of cathepsin genes in Pacific cod were evaluated in the spleen and liver after poly I:C challenge. The results indicated that all cathepsin genes were significantly upregulated upon poly I:C stimulation, suggesting that they play key roles in antiviral immune responses in Pacific cod. Our findings establish a foundation for future exploration of the molecular mechanisms of cathepsins in modulating antiviral immunity in Pacific cod.

Fasciola hepatica Cathepsin L Zymogens: Immuno-Proteomic Evidence for Highly Immunogenic Zymogen-Specific Conformational Epitopes to Support Diagnostics Development.

Fasciola hepatica, the common liver fluke and causative agent of zoonotic fasciolosis, impacts on food security with global economic losses of over $3.2 BN per annum through deterioration of animal health, productivity losses, and livestock death and is also re-emerging as a foodborne human disease. Cathepsin proteases present a major vaccine and diagnostic target of the F. hepatica excretory/secretory (ES) proteome, but utilization in diagnostics of the highly antigenic zymogen stage of these proteins is surprisingly yet to be fully exploited. Following an immuno-proteomic investigation of recombinant and native procathepsins ((r)FhpCL1), including mass spectrometric analyses (DOI: 10.6019/PXD030293), and using counterpart polyclonal antibodies to a recombinant mutant procathepsin L (anti-rFhΔpCL1), we have confirmed recombinant and native cathepsin L zymogens contain conserved, highly antigenic epitopes that are conformationally dependent. Furthermore, using diagnostic platforms, including pilot serum and fecal antigen capture enzyme-linked immunosorbent assay (ELISA) tests, the diagnostic capacities of cathepsin L zymogens were assessed and validated, offering promising efficacy as markers of infection and for monitoring treatment efficacy.

Recombinant Cathepsin L of Tribolium castaneum and Its Potential in the Hydrolysis of Immunogenic Gliadin Peptides.

Wheat gliadins contain a large amount of glutamine- and proline-rich peptides which are not hydrolyzed by human digestive peptidases and can cause autoimmune celiac disease and other forms of gluten intolerance in predisposed people. Peptidases that efficiently cleave such immunogenic peptides can be used in enzyme therapy. The stored product insect pest Tribolium castaneum efficiently hydrolyzes gliadins. The main digestive peptidase of T. castaneum is cathepsin L, which is from the papain C1 family with post-glutamine cleavage activity. We describe the isolation and characterization of T. castaneum recombinant procathepsin L (rpTcCathL1, NP_001164001), which was expressed in Pichia pastoris cells. The activation of the proenzyme was conducted by autocatalytic processing. The effects of pH and proenzyme concentration in the reaction mixture on the processing were studied. The mature enzyme retained high activity in the pH range from 5.0 to 9.0 and displayed high pH-stability from 4.0 to 8.0 at 20 °C. The enzyme was characterized according to electrophoretic mobility under native conditions, activity and stability at various pH values, a sensitivity to various inhibitors, and substrate specificity, and its hydrolytic effect on 8-, 10-, 26-, and 33-mer immunogenic gliadins peptides was demonstrated. Our results show that rTcCathL1 is an effective peptidase that can be used to develop a drug for the enzyme therapy of various types of gluten intolerance.

RNA Interference-Mediated Knockdown of Bombyx mori Haemocyte-Specific Cathepsin L (Cat L)-Like Cysteine Protease Gene Increases Bacillus thuringiensis kurstaki Toxicity and Reproduction in Insect Cadavers.

The silkworm's Cat L-like gene, which encodes a lysosomal cathepsin L-like cysteine protease, is thought to be part of the insect's innate immunity via an as-yet-undetermined mechanism. Assuming that the primary function of Cat L-like is microbial degradation in mature phagosomes, we hypothesise that the suppression of the Cat L-like gene expression would increase Bacillus thuringiensis (Bt) bacteraemia and toxicity in knockdown insects. Here, we performed a functional analysis of Cat L-like in larvae that were fed mulberry leaves contaminated with a commercial biopesticide formulation based on Bt kurstaki (Btk) (i.e., Dipel) to investigate its role in insect defence against a known entomopathogen. Exposure to sublethal doses of Dipel resulted in overexpression of the Cat L-like gene in insect haemolymph 24 and 48 h after exposure. RNA interference (RNAi)-mediated suppression of Cat L-like expression significantly increased the toxicity of Dipel to exposed larvae. Moreover, Btk replication was higher in RNAi insects, suggesting that Cat L-like cathepsin may be involved in a bacterial killing mechanism of haemocytes. Finally, our results confirm that Cat L-like protease is part of the antimicrobial defence of insects and suggest that it could be used as a target to increase the insecticidal efficacy of Bt-based biopesticides.

Molecular characterization and determination of the biochemical properties of cathepsin L of Trichinella spiralis.

Cathepsin L is an important cysteine protease, but its function in T. spiralis remains unclear. The aim of this research was to explore the biological characteristics of T. spiralis cathepsin L (TsCatL) and its role in T. spiralis-host interactions. Bioinformatic analysis revealed the presence of the cysteine protease active site residues Gln, Cys, His and Asn in mature TsCatL, as well as specific motifs of cathepsin L similar to ERFNIN and GYLND in the prepeptide of TsCatL. Molecular docking of mature TsCatL and E64 revealed hydrophobic effects and hydrogen bonding interactions. Two domains of TsCatL (TsCatL2) were cloned and expressed, and recombinant TsCatL2 (rTsCatL2) was autocatalytically cleaved under acidic conditions to form mature TsCatL. TsCatL was transcribed and expressed in larvae and adults and located in the stichosome, gut and embryo. Enzyme kinetic tests showed that rTsCatL2 degraded the substrate Z-Phe-Arg-AMC under acidic conditions, which was inhibited by E64 and PMSF and enhanced by EDTA, L-cysteine and DTT. The kinetic parameters of rTsCatL2 were a Km value of 48.82 µM and Vmax of 374.4 nM/min at pH 4.5, 37 °C and 5 mM DTT. In addition, it was shown that rTsCatL2 degraded haemoglobin, serum albumin, immunoglobulins (mouse IgG, human IgG and IgM) and extracellular matrix components (fibronectin, collagen I and laminin). The proteolytic activity of rTsCatL2 was host specific and significantly inhibited by E64. rTsCatL2 possesses the natural activity of a sulfhydryl-containing cysteine protease, and TsCatL is an important digestive enzyme that seems to be important for the nutrient acquisition, immune evasion and invasion of Trichinella in the host.

Effects of vitamin C and D on the mRNA expression of angiotensin converting enzyme 2 receptor, cathepsin L, and transmembrane serine protease in the mouse lungs.

Vitamins (Vit) C and D are widely used as immunogenic supplements among severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infected patients. The SAR-CoV-2 virus enters into the pulmonary endothelial cells through attachment to angiotensin converting enzyme 2 receptor (Ace2) and the proteolytic activity of Cathepsin L (Ctsl) and transmembrane serine protease 2 (Tmprss2) enzymes. This study aimed to determine the influence of Vit C and D on the mRNA expression of Ace2, Tmprss2, and Ctsl genes in the mouse lungs. Vitamins C and D were administrated to different groups of mice through intra-peritoneal route in doses equivalent to human for 30 days. Then, the mRNA expression of SARS-CoV-2 entry gene was analyzed using qRT-PCR. It is found that Vit D, but not C, upregulated significantly (P < 0.05) the mRNA expression of Ace2 by more than six folds, while downregulated the expression of Ctsl and Tmprss2 genes by 2.8 and 2.2 folds, respectively. It can be concluded from this study that Vit D alters the mRNA expression of Ace2, Tmprss, and Ctsl genes in the mouse lungs. This finding can help us in understanding, at least in part, the molecular influence of Vit D on genes involved in the entry of SARS-CoV-2 into the cells.

Bioactivity of orange-spotted grouper (Epinephelus coioides) cathepsin L: Proteolysis of bacteria and regulation of the innate immune response.

Cathepsin L (CTSL) is a cysteine endopeptidase involved in protein degradation mainly in lysosomes. Following activation in an acidic environment, it plays a key role in a variety of physiological, immunological, and pathological processes. The biological function of CTSL in teleost remains unclear. Immunohistochemical analysis revealed that CTSL was expressed mainly in lymphoid organs, head kidney, trunk kidney, and liver, which particularly was expressed in leukocyte-like cells. We performed two forms of recombinant CTSL (rCTSL and rTCTSL) derived from orange-spotted grouper (Epinephelus coioides) to elucidate the role of CTSL in teleost innate immunity, based on differences in immune-related gene expression. We determined that rCTSL has a proteolytic function whereas rTCTSL does not. Under CTSL activation, we observed increases in IL-1ß, IL-6, IL-12, IFNγ, CCL-1, CCL-3, epinecidin-1, lysozyme, and IgM. The bacteriolytic activity of rCTSL was more pronounced against Gram-positive bacteria than Gram-negative bacteria. Our findings indicate CTSL plays multiple roles in the reactions of innate immunity.

Cathepsin L involved in the freezing resistance of murine normal hatching embryos and dormant embryos.

The cryopreservation of mammalian embryos is an important technology in embryo engineering. The discovery and application of the embryo's own high freezing resistance factors are the main methods to improve the utilization of mammalian embryos in cryopreservation. Cathepsin L gene expression in the frozen and thawed dormant embryos displayed a significant difference from those normal hatched ones. The aim of the present study was to dig out the potential role of Cathepsin L in anti-freezing capacity of murine blastocysts by investigating the location and expression of Cathepsin L in frozen and thawed both activated and dormant hatching blastocysts. Different concentrations of Cathepsin L recombinant protein and E-64d were then respectively added into the embryo cryoprotectant and pre-cryo culture medium. Our results found that down-regulation of Cathepsin L improves the freezing resistance of murine normal hatching embryos by reducing apoptosis. Cathepsin L inhibitors can be used to improve the efficiency of cryopreservation and recovery of blastocysts in vitro. Our study provides a theoretical basis for the further development and application of Cathepsin L.

The unfavorable clinical outcome of COVID-19 in smokers is mediated by H3K4me3, H3K9me3 and H3K27me3 histone marks.

Smoking could predispose individuals to a more severe COVID-19 by upregulating a particular gene known as mdig, which is mediated through a number of well-known histone modifications. Smoking might regulate the transcription-activating H3K4me3 mark, along with the transcription-repressing H3K9me3 and H3K27me3 marks, in a way to favor SARS-CoV-2 entry by enhancing the expression of ACE2, NRP1 and NRP2, AT1R, CTSD and CTSL, PGE2 receptors 2-4, SLC6A20 and IL-6, all of which interact either directly or indirectly with important receptors, facilitating viral entry in COVID-19.

Lay abstract The role of smoking in development of several respiratory diseases has been clearly established. A significant proportion of these deleterious effects is mediated through epigenetic mechanisms, particularly histone modifications. Recent evidence indicates that smoking induces the expression of a mediator known as mdig, which in turn alters the transcription of several key proteins that have been implicated in development of COVID-19.