A transcription factor ATF3 involves in the phagocytosis of granulocytes in oyster Crassostrea gigas.

Phagocytosis is a major cellular mechanism for mollusk granulocytes to eliminate nonself substances and dead cells, and thus to preserve the immune homeostasis. The knowledge of the regulatory mechanisms controlling phagocytic capacity is vital to understanding the immune system. In the present study, an ATF3 homolog (CgATF3) with a typical bZIP domain was identified in the Pacific oyster Crassostrea gigas. Its highly conserved bZIP domain consisted of two structural features, a basic region for DNA binding and a leucine zipper region for dimerization. Its transcript was found to be abundantly expressed in haemocytes, which was induced by Vibrio splendidus stimulation and recombinant CgTNF-2 treatment, along with an increase of its protein content in the nucleus. Moreover, CgATF3 showed a consistent and specific high expression in granulocytes, and CgATF3+ granulocytes were characterized morphologically by the largest diameter, smaller nucleus to cytoplasmic ratio, and abundant cytoplasmic granules, and functionally by a higher capacity for phagocytosis. When CgATF3 expression was inhibited by RNAi, the expression levels of CgRab1, CgRab33 and CgCathepsin L1, as well as the phagocytic rate and index of granulocytes all decreased after V. splendidus stimulation. These results together demonstrated the involvement of CgATF3 in regulating the expressions of Rabs and Cathepsin L1, as well as the phagocytosis of granulocytes in oyster C. gigas.

Hyperglycemia induced cathepsin L maturation linked to diabetic comorbidities and COVID-19 mortality.

Diabetes, a prevalent chronic condition, significantly increases the risk of mortality from COVID-19, yet the underlying mechanisms remain elusive. Emerging evidence implicates Cathepsin L (CTSL) in diabetic complications, including nephropathy and retinopathy. Our previous research identified CTSL as a pivotal protease promoting SARS-CoV-2 infection. Here, we demonstrate elevated blood CTSL levels in individuals with diabetes, facilitating SARS-CoV-2 infection. Chronic hyperglycemia correlates positively with CTSL concentration and activity in diabetic patients, while acute hyperglycemia augments CTSL activity in healthy individuals. In vitro studies reveal high glucose, but not insulin, promotes SARS-CoV-2 infection in wild-type cells, with CTSL knockout cells displaying reduced susceptibility. Utilizing lung tissue samples from diabetic and non-diabetic patients, alongside Leprdb/dbmice and Leprdb/+mice, we illustrate increased CTSL activity in both humans and mice under diabetic conditions. Mechanistically, high glucose levels promote CTSL maturation and translocation from the endoplasmic reticulum (ER) to the lysosome via the ER-Golgi-lysosome axis. Our findings underscore the pivotal role of hyperglycemia-induced CTSL maturation in diabetic comorbidities and complications.

People with diabetes are at greater risk of developing severe COVID-19 and dying from the illness, which is caused by a virus known as SARS-CoV-2. The high blood sugar levels associated with diabetes appear to be a contributing factor to this heightened risk. However, diabetes is a complex condition encompassing a range of metabolic disorders, and it is therefore likely that other factors may contribute. Previous research identified a link between an enzyme called cathepsin L and more severe COVID-19 in people with diabetes. Elevated cathepsin L levels are known to contribute to diabetes complications, such as kidney damage and vision loss. It has also been shown that cathepsin L helps SARS-CoV-2 to enter and infect cells. This raised the question of whether elevated cathepsin L is responsible for the increased COVID-19 vulnerability in patients with diabetes. To investigate, He, Zhao et al. monitored disease severity and cathepsin L levels in patients with COVID-19. This confirmed that people with diabetes had more severe COVID-19 and that higher levels of cathepsin L are linked to more severe disease. Analysis also revealed that cathepsin L activity increases as blood glucose levels increase. In laboratory experiments, cells exposed to glucose or fluid from the blood of people with diabetes were more easily infected with SARS-CoV-2, with cells genetically modified to lack cathepsin L being more resistant to infection. Further experiments revealed this was due to glucose promoting maturation and migration of cathepsin L in the cells. The findings of He, Zhao et al. help to explain why people with diabetes are more likely to develop severe or fatal COVID-19. Therefore, controlling blood glucose levels in people with diabetes may help to prevent or reduce the severity of the disease. Additionally, therapies targeting cathepsin L could also potentially help to treat COVID-19, especially in patients with diabetes, although more research is needed to develop and test these treatments.

Targeting Caspases 3/6 and Cathepsins L/B May Decrease Laminopathy-Induced Apoptosis in Alzheimer's Disease.

Background: Laminopathy is a pathological manifestation observed in Alzheimer's disease (AD), leading to neuronal apoptosis. Objective: Our objective was to assess inhibitors of enzymes involved in laminopathy. Methods: The mRNA expression of the cathepsins L and B, caspases 3 and 6, lamins b1 and b2, granzymes A and B, and lamins A and C were extracted and analyzed from GSE5281 and GSE28146 datasets. A total of 145 ligands were selected for molecular docking. Subsequently, 10 ns and 100 ns atomistic molecular dynamics (MD) and Martini 3 were performed with NAMD for two selected ligands (PubChem id: 608841 and ChEMBL id: 550872). Results: The mRNA expression level highlighted caspase 6 and lamin A/C upregulation in the hippocampus of the AD samples, in contrast to cathepsin B, lamin b2, and caspase 3. Moreover, there was a strong correlation between the expression level of cathepsin B, lamin A/C, and caspase 6 in the AD group. The MD results suggested molecule with ChEMBL id of 550872 had higher free binding energy, while in longer simulation the molecule with PubChem id of 608841 was suggested to be more stable in complex with the receptor. Conclusions: Our findings suggest that lamins A/C, cathepsins B/L, caspase 6, and lamin B2 are associated with laminopathy as potential factors contributing to apoptosis in AD. We propose that simultaneous inhibition of caspases 6 and cathepsins L may decrease the rate of apoptosis triggered by lamin degradation. Nevertheless, further studies are required to confirm these observations due to the lack of in vivo findings.

Molecular characterization of EcCLP1, a new putative cathepsin L protease from Echinococcus canadensis.

Echinococcus granulosus sensu lato is a platyhelminth parasite and the etiological cause of cystic echinococcosis (CE), a zoonotic and neglected disease that infects animals and humans worldwide. As a part of the biological arsenal of the parasite, cathepsin L proteases are a group of proteins that are believed to be essential for parasite penetration, immune evasion, and establishment in the tissues of the host. In this work, we have cloned and sequenced a new putative cathepsin L protease from Echinococcus canadensis (EcCLP1). The bioinformatic analysis suggests that EcCLP1 could be synthesized as a zymogen and activated after proteolytic cleavage. The multiple sequence alignment with other cathepsin proteases reveals important functional conserved features like a conserved active site, an N-linked glycosylation residue, a catalytic triad, an oxyanion hole, and three putative disulfide bonds. The phylogenetic analysis suggests that EcCLP1 could indeed be a cathepsin L cysteine protease from clade 1 as it grouped with cathepsins from other species in this clade. Modeling studies suggest that EcCLP1 has two domains forming a cleft where the active site is located and an occluding role for the propeptide. The transcriptomic analysis reveals different levels of cathepsin transcript expression along the different stages of the parasite life cycle. The whole-mount immunohistochemistry shows an interesting superficial punctate pattern of staining which suggests a secretory pattern of expression. The putative cathepsin L protease characterized here may represent an interesting tool for diagnostic purposes, vaccine design, or a new pharmacological target for antiparasitic intervention.

Title: Caractérisation moléculaire d'EcCLP1, une nouvelle protéase putative de type cathepsine L d'Echinococcus canadensis. Abstract: Echinococcus granulosus sensu lato est un Plathelminthe parasite et la cause étiologique de l'échinococcose kystique (EK), une maladie zoonotique et négligée qui infecte les animaux et les humains dans le monde entier. En tant que partie de l'arsenal biologique du parasite, les protéases de type cathepsine L sont un groupe de protéines considérées comme essentielles à la pénétration du parasite, l'évasion immunitaire et son établissement dans les tissus de l'hôte. Dans ce travail, nous avons cloné et séquencé une nouvelle protéase putative de type cathepsine L d'Echinococcus canadensis (EcCLP1). L'analyse bioinformatique suggère qu'EcCLP1 pourrait être synthétisée sous forme de zymogène et activée après clivage protéolytique. L'alignement de séquences multiples avec d'autres protéases de type cathepsine révèle d'importantes caractéristiques fonctionnelles conservées telles qu'un site actif conservé, un résidu de glycosylation lié à N, une triade catalytique, un trou oxyanion et trois liaisons disulfure putatives. L'analyse phylogénétique suggère qu'EcCLP1 pourrait en effet être une protéase de type cathepsine L du clade 1 car elle se regroupe avec les cathepsines d'autres espèces de ce clade. Les études de modélisation suggèrent qu'EcCLP1 possède deux domaines formant une fente où se trouve le site actif et un rôle d'occlusion pour le propeptide. L'analyse transcriptomique révèle différents niveaux d'expression du transcrit de la cathepsine au cours des différentes étapes du cycle de vie du parasite. L'immunohistochimie de montages entiers montre un intéressant motif de coloration ponctuée superficielle qui suggère un modèle d'expression sécrétoire. La protéase putative de type cathepsine L caractérisée ici peut représenter un outil intéressant à des fins de diagnostic, de conception de vaccins ou une nouvelle cible pharmacologique pour une intervention antiparasitaire.

Novel proteolytic activation of Ebolavirus glycoprotein GP by TMPRSS2 and cathepsin L at an uncharted position can compensate for furin cleavage.

A multistep priming process involving furin and endosomal cathepsin B and L (CatB/L) has been described for the Orthoebolavirus zairense (EBOV) glycoprotein GP. Inhibition or knockdown of either furin or endosomal cathepsins, however, did not prevent virus multiplication in cell cultures. Moreover, an EBOV mutant lacking the furin cleavage motif (RRTRR→AGTAA) was able to replicate and cause fatal disease in nonhuman primates, indicating that furin cleavage may be dispensable for virus infectivity. Here, by using protease inhibitors and EBOV GP-carrying recombinant vesicular stomatitis virus (VSV) and transcription and replication-competent virus-like particles (trVLPs) we found that processing of EBOV GP is mediated by different proteases in different cell lines depending on the protease repertoire available. Endosomal cathepsins were essential for EBOV GP entry in Huh-7 but not in Vero cells, in which trypsin-like proteases and stably expressed trypsin-like transmembrane serine protease 2 (TMPRSS2) supported wild-type EBOV GP and EBOV GP_AGTAA mutant entry. Furthermore, we show that the EBOV GP_AGTAA mutant is cleaved into fusion-competent GP2 by TMPRSS2 and by CatL at a so far unknown site. Fluorescence microscopy co-localization studies indicate that EBOV GP cleavage by TMPRSS2 may occur in the TGN prior to virus release or in the late endosome at the stage of virus entry into a new cell. Our data show that EBOV GP must be proteolytically activated to support virus entry but has even greater flexibility in terms of proteases and the precise cleavage site than previously assumed.

Genetic disruption of ATAT1 causes RhoA downregulation through abnormal truncation of C/EBPß.

Microtubule acetylation has been shown to regulate actin filament dynamics by modulating signaling pathways that control actin organization, although the precise mechanisms remain unknown. In this study, we found that the downregulation of microtubule acetylation via the disruption ATAT1 (which encodes α-tubulin N-acetyltransferase 1) inhibited the expression of RhoA, a small GTPase involved in regulating the organization of actin filaments and the formation of stress fibers. Analysis of RHOA promoter and chromatin immunoprecipitation assays revealed that C/EBPß is a major regulator of RHOA expression. Interestingly, the majority of C/EBPß in ATAT1 knockout (KO) cells was found in the nucleus as a 27-kDa fragment (referred to as C/EBPßp27) lacking the N-terminus of C/EBPß. Overexpression of a gene encoding a C/EBPßp27-mimicking protein via an N-terminal deletion in C/EBPß led to competitive binding with wild-type C/EBPß at the C/EBPß binding site in the RHOA promoter, resulting in a significant decrease of RHOA expression. We also found that cathepsin L (CTSL), which is overexpressed in ATAT1 KO cells, is responsible for C/EBPßp27 formation in the nucleus. Treatment with a CTSL inhibitor led to the restoration of RHOA expression by downregulation of C/EBPßp27 and the invasive ability of ATAT1 KO MDA-MB-231 breast cancer cells. Collectively, our findings suggest that the downregulation of microtubule acetylation associated with ATAT1 deficiency suppresses RHOA expression by forming C/EBPßp27 in the nucleus through CTSL. We propose that CTSL and C/EBPßp27 may represent a novel therapeutic target for breast cancer treatment. [BMB Reports 2024; 57(6): 293-298].

Cathepsins L and B target HIF1α for oxygen-independent proteolytic cleavage.

The oxygen-labile transcription factor called hypoxia-inducible factor (HIF) is responsible for the cellular and organismal adaptive response to reduced oxygen availability. Deregulation of HIF is associated with the pathogenesis of major human diseases including cardiovascular disease and cancer. Under normoxia, the HIFα subunit is hydroxylated on conserved proline residues within the oxygen-dependent degradation domain (ODD) that labels HIFα for proteasome-mediated degradation. Despite similar oxygen-dependent degradation machinery acting on HIF1α and HIF2α, these two paralogs have been shown to exhibit unique kinetics under hypoxia, which suggests that other regulatory processes may be at play. Here, we characterize the protease activity found in rabbit reticulocytes that specifically cleaves the ODD of HIF1α but not HIF2α. Notably, the cleavage product is observed irrespective of the oxygen-dependent prolyl-hydroxylation potential of HIF1α, suggesting independence from oxygen. HIF1α M561T substitution, which mimics an evolutionary substitution that occurred during the duplication and divergence of HIF1α and HIF2α, diminished the cleavage of HIF1α. Protease inhibitor screening suggests that cysteine proteases cathepsins L and B preferentially cleave HIF1αODD, thereby revealing an additional layer of differential HIF regulation.

Cathepsin L induces cellular senescence by upregulating CUX1 and p16INK4a.

Cathepsin L (CTSL) has been implicated in aging and age-related diseases, such as cardiovascular diseases, specifically atherosclerosis. However, the underlying mechanism(s) is not well documented. Recently, we demonstrated a role of CUT-like homeobox 1 (CUX1) in regulating the p16INK4a-dependent cellular senescence in human endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) via its binding to an atherosclerosis-associated functional SNP (fSNP) rs1537371 on the CDKN2A/B locus. In this study, to determine if CTSL, which was reported to proteolytically activate CUX1, regulates cellular senescence via CUX1, we measured the expression of CTSL, together with CUX1 and p16INK4a, in human ECs and VSMCs undergoing senescence. We discovered that CUX1 is not a substrate that is cleaved by CTSL. Instead, CTSL is an upstream regulator that activates CUX1 transcription indirectly in a process that requires the proteolytic activity of CTSL. Our findings suggest that there is a transcription factor in between CTSL and CUX1, and cleavage of this factor by CTSL can activate CUX1 transcription, inducing endothelial senescence. Thus, our findings provide new insights into the signal transduction pathway that leads to atherosclerosis-associated cellular senescence.

Functional characterization of a cystatin A from the bat Myotis davidii.

Myotis davidii cystatin A (MdCSTA), a stefin A-like from the Chinese native bat species M. davidii, was expressed as a recombinant protein and functionally characterized as a strong inhibitor of the cysteine proteases papain, human cathepsins L and B and the tick cathepsin L-like BmCL1. Despite the highly conserved amino acid sequences among stefins A from different vertebrates, MdCSTA presents a Methionine-2 residue at the N-terminal region and the second binding loop (pos 73-79) that differs from human stefin A (HsCSTA) and might be related to the lower inhibition constant (Ki) value presented by this inhibitor in comparison to human stefin A inhibition to cathepsin B. Therefore, to investigate the importance of these variable regions in cathepsin B inhibition, recombinant stefins A MdCSTA and HsCSTA containing mutations at the second amino acid residue and second binding loop were expressed and evaluated in kinetic assays. Enzymatic inhibition assays with cathepsin B revealed that switching the amino acid residues at position 2 and second binding loop region between bat and human CSTAs improved the HsCSTA's and reduced MdCSTA's inhibitory activity. Additionally, molecular docking analysis estimated lower energy values for the complex between MdCSTA-cathepsin B, in comparison to human CSTA-cathepsin B, while the mutants presented intermediate values, suggesting that other regions might contribute to the higher inhibitory activity against cathepsin B by MdCSTA. In conclusion, MdCSTA, the first bat's stefin A-like inhibitor to be functionally characterized, presented a higher inhibitory activity against cathepsin B in comparison to the human inhibitor, which is partially related to the glutamine-rich second binding loop and Met-2. Further structural analysis should be performed to elucidate potential inhibitor effects on cysteine proteinases.

Cellular depletion of major cathepsin proteases reveals their concerted activities for lysosomal proteolysis.

Proteins delivered by endocytosis or autophagy to lysosomes are degraded by exo- and endoproteases. In humans 15 lysosomal cathepsins (CTS) act as important physiological regulators. The cysteine proteases CTSB and CTSL and the aspartic protease CTSD are the most abundant and functional important lysosomal proteinases. Whereas their general functions in proteolysis in the lysosome, their individual substrate, cleavage specificity, and their possible sequential action on substrate proteins have been previously studied, their functional redundancy is still poorly understood. To address a possible common role of highly expressed and functional important CTS proteases, we generated CTSB-, CTSD-, CTSL-, and CTSBDL-triple deficient (KO) human neuroblastoma-derived SH-SY5Y cells and CTSB-, CTSD-, CTSL-, CTSZ and CTSBDLZ-quadruple deficient (KO) HeLa cells. These cells with a combined cathepsin deficiency exhibited enlarged lysosomes and accumulated lipofuscin-like storage material. The lack of the three (SH-SY5Y) or four (HeLa) major CTSs caused an impaired autophagic flux and reduced degradation of endocytosed albumin. Proteome analyses of parental and CTS-depleted cells revealed an enrichment of cleaved peptides, lysosome/autophagy-associated proteins, and potentially endocytosed membrane proteins like the amyloid precursor protein (APP), which can be subject to endocytic degradation. Amino- and carboxyterminal APP fragments accumulated in the multiple CTS-deficient cells, suggesting that multiple CTS-mediated cleavage events regularly process APP. In summary, our analyses support the idea that different lysosomal cathepsins act in concert, have at least partially and functionally redundant substrates, regulate protein degradation in autophagy, and control cellular proteostasis, as exemplified by their involvement in the degradation of APP fragments.

Silencing Ditylenchus destructor cathepsin L-like cysteine protease has negative pleiotropic effect on nematode ontogenesis.

Ditylenchus destructor is a migratory plant-parasitic nematode that severely harms many agriculturally important crops. The control of this pest is difficult, thus efficient strategies for its management in agricultural production are urgently required. Cathepsin L-like cysteine protease (CPL) is one important protease that has been shown to participate in various physiological and pathological processes. Here we decided to characterize the CPL gene (Dd-cpl-1) from D. destructor. Analysis of Dd-cpl-1 gene showed that Dd-cpl-1 gene contains a signal peptide, an I29 inhibitor domain with ERFNIN and GNFD motifs, and a peptidase C1 domain with four conserved active residues, showing evolutionary conservation with other nematode CPLs. RT-qPCR revealed that Dd-cpl-1 gene displayed high expression in third-stage juveniles (J3s) and female adults. In situ hybridization analysis demonstrated that Dd-cpl-1 was expressed in the digestive system and reproductive organs. Silencing Dd-cpl-1 in 1-cell stage eggs of D. destructor by RNAi resulted in a severely delay in development or even in abortive morphogenesis during embryogenesis. The RNAi-mediated silencing of Dd-cpl-1 in J2s and J3s resulted in a developmental arrest phenotype in J3 stage. In addition, silencing Dd-cpl-1 gene expression in female adults led to a 57.43% decrease in egg production. Finally, Dd-cpl-1 RNAi-treated nematodes showed a significant reduction in host colonization and infection. Overall, our results indicate that Dd-CPL-1 plays multiple roles in D. destructor ontogenesis and could serve as a new potential target for controlling D. destructor.

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.

Designing and Developing Serological Test for the Diagnosis of Human Fascioliasis Using a New Recombinant Multi-epitope.

PURPOSE: Fascioliasis is a common parasitic disease in humans and herbivores which is caused by Fasciola hepatica and Fasciola gigantica and has a worldwide distribution. Serological tests such as the enzyme-linked immunosorbent assay (ELISA) technique play a prominent role in the fast diagnosis of the disease. However, there are diagnostic limitations, including cross-reactivity with other worms, which decline the specificity of the results. This study aimed to evaluate the structure of a recombinant multi-epitope antigen produced from linear and conformational B-cell epitopes of three parasitic proteins with sera of individuals with fasciolosis, healthy controls, and those with other diseases to gain accurate sensitivity and specificity. METHODS: After designing the multi-epitope structure of cathepsin L1, FhTP16.5, and SAP-2 antigens and then synthesizing, cloning, and expressing, the extracted purified protein was evaluated by indirect ELISA to detect IgG antibodies against Fasciola hepatica parasite among the sera of 39 serum samples of Fasciola hepatica, 35 healthy individual samples, and 20 samples of other types of parasitic diseases. The synthesized multi-epitope produced from cathepsin L1, FhTP16.5, and SAP-2 antigens was evaluated using the indirect ELISA. RESULTS: The analysis of the samples mentioned for IgG antibody diagnosis against Fasciola hepatica showed 97.43% (95% confidence interval, 94.23-100%) sensitivity and 100% (95% confidence interval, 97-100%) specificity. CONCLUSION: The recombinant B-cell multi-epitope with high antigenic potency may increase the specificity of epitopic peptides and ultimately help improve and develop indirect ELISA commercial kits for the diagnosis of fascioliasis in humans.

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.

Cathepsin X deficiency alters the processing and localisation of cathepsin L and impairs cleavage of a nuclear cathepsin L substrate.

Proteases function within sophisticated networks. Altering the activity of one protease can have sweeping effects on other proteases, leading to changes in their activity, structure, specificity, localisation, stability, and expression. Using a suite of chemical tools, we investigated the impact of cathepsin X, a lysosomal cysteine protease, on the activity and expression of other cysteine proteases and their inhibitors in dendritic cells. Among all proteases examined, cathepsin X gene deletion specifically altered cathepsin L levels; pro-cathepsin L and its single chain accumulated while the two-chain form was unchanged. This effect was recapitulated by chemical inhibition of cathepsin X, suggesting a dependence on its catalytic activity. We demonstrated that accumulation of pro- and single chain cathepsin L was not due to a lack of direct cleavage by cathepsin X or altered glycosylation, secretion, or mRNA expression but may result from changes in lysosomal oxidative stress or pH. In the absence of active cathepsin X, nuclear cathepsin L and cleavage of the known nuclear cathepsin L substrate, Lamin B1, were diminished. Thus, cathepsin X activity selectively regulates cathepsin L, which has the potential to impact the degree of cathepsin L proteolysis, the nature of substrates that it cleaves, and the location of cleavage.

Inhibition of cysteine protease cathepsin L increases 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.

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.