Cathepsin S (CTSS) in IgA nephropathy: an exploratory study on its role as a potential diagnostic biomarker and therapeutic target.

Introduction: IgA nephropathy (IgAN), a prevalent form of glomerulonephritis globally, exhibits complex pathogenesis. Cathepsins, cysteine proteases within lysosomes, are implicated in various physiological and pathological processes, including renal conditions. Prior observational studies have suggested a potential link between cathepsins and IgAN, yet the precise causal relationship remains unclear. Methods: We conducted a comprehensive bidirectional and multivariable Mendelian randomization (MR) study using publicly available genetic data to explore the causal association between cathepsins and IgAN systematically. Additionally, immunohistochemical (IHC) staining and enzyme-linked immunosorbent assay (ELISA) were employed to evaluate cathepsin expression levels in renal tissues and serum of IgAN patients. We investigated the underlying mechanisms via gene set variation analysis (GSVA), gene set enrichment analysis (GSEA), and immune cell infiltration analysis. Molecular docking and virtual screening were also performed to identify potential drug candidates through drug repositioning. Results: Univariate MR analyses demonstrated a significant link between increased cathepsin S (CTSS) levels and a heightened risk of IgAN. This was evidenced by an odds ratio (OR) of 1.041 (95% CI=1.009-1.073, P=0.012) as estimated using the inverse variance weighting (IVW) method. In multivariable MR analysis, even after adjusting for other cathepsins, elevated CTSS levels continued to show a strong correlation with an increased risk of IgAN (IVW P=0.020, OR=1.037, 95% CI=1.006-1.069). However, reverse MR analyses did not establish a causal relationship between IgAN and various cathepsins. IHC and ELISA findings revealed significant overexpression of CTSS in both renal tissues and serum of IgAN patients compared to controls, and this high expression was unique to IgAN compared with several other primary kidney diseases such as membranous nephropathy, minimal change disease and focal segmental glomerulosclerosis. Investigations into immune cell infiltration, GSEA, and GSVA highlighted the role of CTSS expression in the immune dysregulation observed in IgAN. Molecular docking and virtual screening pinpointed Camostat mesylate, c-Kit-IN-1, and Mocetinostat as the top drug candidates for targeting CTSS. Conclusion: Elevated CTSS levels are associated with an increased risk of IgAN, and this enzyme is notably overexpressed in IgAN patients' serum and renal tissues. CTSS could potentially act as a diagnostic biomarker, providing new avenues for diagnosing and treating IgAN.

Inhibition of Cathepsin S in Autoimmune CD25KO Mouse Improves Sjögren Disease-Like Lacrimal Gland Pathology.

Purpose: CD25KO mice are a model of Sjögren disease (SjD) driven by autoreactive T cells. Cathepsin S (CTSS) is a protease crucial for major histocompatibility complex class II presentation that primes T cells. We investigated if a diet containing CTSS inhibitor would improve autoimmune signs in CD25KO mice. Methods: Four-week female CD25KO mice were randomly chosen to receive chow containing a CTSS inhibitor (R05461111, 262.5 mg/kg chow) or standard chow for 4 weeks. Cornea sensitivity was measured. Inflammatory score was assessed in lacrimal gland (LG) histologic sections. Flow cytometry of LG and ocular draining lymph nodes (dLNs) investigated expression of Th1 and Th17 cells. Expression of inflammatory, T- and B-cell, and apoptotic markers in the LG were assessed with quantitative PCR. The life span of mice receiving CTSS inhibitor or standard chow was compared. CD4+ T cells from both groups were isolated from spleens and adoptively transferred into RAG1KO female recipients. Results: Mice receiving CTSS inhibitor had better cornea sensitivity and improved LG inflammatory scores. There was a significant decrease in the frequency of CD4+ immune cells and a significant increase in the frequency of CD8+ immune cells in the dLNs of CTSS inhibitor mice. There was a significant decrease in Th1 and Th17 cells in CTSS inhibitor mice in both LGs and dLNs. Ifng, Ciita, and Casp8 mRNA in CTSS inhibitor mice decreased. Mice that received the CTSS inhibitor lived 30% longer. Adoptive transfer recipients with CTSS inhibitor-treated CD4+ T cells had improved cornea sensitivity and lower inflammation scores. Conclusions: Inhibiting CTSS could be a potential venue for the treatment of SjD in the eye and LG.

Tofacitinib Regulates Endostatin via Effects on CD147 and Cathepsin S.

Angiogenesis is critical for rheumatoid arthritis (RA) progression. The effects of tofacitinib, a JAK-STAT inhibitor used for RA treatment, on angiogenesis in RA are unclear. We, therefore, evaluated the levels of angiogenic factors in two systems of a human co-culture of fibroblast (HT1080) and monocytic (U937) cell lines treated with tofacitinib and in serum samples from RA patients before and after six months of tofacitinib treatment. Tofacitinib reduced CD147 levels, matrix metalloproteinase-9 (MMP-9) activity, and angiogenic potential but increased endostatin levels and secreted proteasome 20S activity. In vitro, tofacitinib did not change CD147 mRNA but increased miR-146a-5p expression and reduced STAT3 phosphorylation. We recently showed that CD147 regulates the ability of MMP-9 and secreted proteasome 20S to cleave collagen XVIIIA into endostatin. We show here that tofacitinib-enhanced endostatin levels are mediated by CD147, as CD147-siRNA or an anti-CD147 antibody blocked proteasome 20S activity. The correlation between CD147 and different disease severity scores supported this role. Lastly, tofacitinib reduced endostatin' s degradation by inhibiting cathepsin S activity and recombinant cathepsin S reversed this in both systems. Thus, tofacitinib inhibits angiogenesis by reducing pro-angiogenic factors and enhancing the anti-angiogenic factor endostatin in a dual effect mediated partly through CD147 and partly through cathepsin S.

Unveiling the Roles of Cysteine Proteinases F and W: From Structure to Pathological Implications and Therapeutic Targets.

Cysteine cathepsins F and W are members of the papain-like cysteine protease family, which have distinct structural features and functional roles in various physiological and pathological processes. This review provides a comprehensive overview of the current understanding of the structure, biological functions, and pathological implications of cathepsins F and W. Beginning with an introduction to these proteases, we delve into their structural characteristics and elucidate their unique features that dictate their enzymatic activities and substrate specificity. We also explore the intricate involvement of cathepsins F and W in malignancies, highlighting their role as potential biomarkers and therapeutic targets in cancer progression. Furthermore, we discuss the emerging roles of these enzymes in immune response modulation and neurological disorders, shedding light on their implications in autoimmune and neurodegenerative diseases. Finally, we review the landscape of inhibitors targeting these proteases, highlighting their therapeutic potential and challenges in clinical translation. This review brings together the diverse facets of cysteine cathepsins F and W, providing insights into their roles in health and disease and guiding future investigations for therapeutic advances.

Potential role of CTSS in AMDImmune modulatory and anti-angiogenic effects of cathepsin S knockdown in ARPE-19 cells.

We aimed to determine the role of cathepsin S (CTSS) in modulating oxidative stress-induced immune and inflammatory reactions and angiogenesis in age-related macular degeneration. Human retinal pigment epithelium cells line ARPE-19 (immature) were maintained and treated with H2O2. The expression of CTSS, inflammatory cytokines, and complement factors induced by oxidative stress was compared between cells incubated without (control) and with CTSS knockdown (using small interfering ribonucleic acid; siRNA). To evaluate the role of CTSS in angiogenesis, we assayed tube formation using human umbilical vein endothelial cells and conditioned medium from ARPE-19 cells. We also used a mouse model of laser-induced choroidal neovascularization. CTSS levels were higher in ARPE-19 cells treated with H2O2 than in control cells. Oxidative stress-induced CTSS resulted in significantly elevated transcription of nuclear factor kappa B-dependent inflammatory cytokines, complement factors C3a and C5a, membrane attack complex (C5b-9), and C3a and C5a receptors. siRNA-mediated knockdown of CTSS reduced the number of inflammatory signals. Furthermore, oxidative stress-induced CTSS regulated the expression of peroxisome proliferator-activated receptor γ and vascular endothelial growth factor A/Akt serine/threonine kinase family signaling, which led to angiogenesis. Tube formation assays and mouse models of choroidal neovascularization revealed that CTSS knockdown ameliorated angiogenesis in vitro and in vivo. The present findings suggest that CTSS modulates the complement pathway, inflammatory reactions, and neovascularization, and that CTSS knockdown induces potent immunomodulatory effects. Hence, it could be a promising target for the prevention and treatment of early- and late-stage age-related macular degeneration.

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.

Targeting cathepsin S promotes activation of OLF1-BDNF/TrkB axis to enhance cognitive function.

BACKGROUND: Cathepsin S (CTSS) is a cysteine protease that played diverse roles in immunity, tumor metastasis, aging and other pathological alterations. At the cellular level, increased CTSS levels have been associated with the secretion of pro-inflammatory cytokines and disrupted the homeostasis of Ca2+ flux. Once CTSS was suppressed, elevated levels of anti-inflammatory cytokines and changes of Ca2+ influx were observed. These findings have inspired us to explore the potential role of CTSS on cognitive functions. METHODS: We conducted classic Y-maze and Barnes Maze tests to assess the spatial and working memory of Ctss-/- mice, Ctss+/+ mice and Ctss+/+ mice injected with the CTSS inhibitor (RJW-58). Ex vivo analyses including long-term potentiation (LTP), Golgi staining, immunofluorescence staining of sectioned whole brain tissues obtained from experimental animals were conducted. Furthermore, molecular studies were carried out using cultured HT-22 cell line and primary cortical neurons that treated with RJW-58 to comprehensively assess the gene and protein expressions. RESULTS: Our findings reported that targeting cathepsin S (CTSS) yields improvements in cognitive function, enhancing both working and spatial memory in behavior models. Ex vivo studies showed elevated levels of long-term potentiation levels and increased synaptic complexity. Microarray analysis demonstrated that brain-derived neurotrophic factor (BDNF) was upregulated when CTSS was knocked down by using siRNA. Moreover, the pharmacological blockade of the CTSS enzymatic activity promoted BDNF expression in a dose- and time-dependent manner. Notably, the inhibition of CTSS was associated with increased neurogenesis in the murine dentate gyrus. These results suggested a promising role of CTSS modulation in cognitive enhancement and neurogenesis. CONCLUSION: Our findings suggest a critical role of CTSS in the regulation of cognitive function by modulating the Ca2+ influx, leading to enhanced activation of the BDNF/TrkB axis. Our study may provide a novel strategy for improving cognitive function by targeting CTSS.

Machine-Learning-Assisted Rational Design of Si─Rhodamine as Cathepsin-pH-Activated Probe for Accurate Fluorescence Navigation.

High-performance fluorescent probes stand as indispensable tools in fluorescence-guided imaging, and are crucial for precise delineation of focal tissue while minimizing unnecessary removal of healthy tissue. Herein, machine-learning-assisted strategy to investigate the current available xanthene dyes is first proposed, and a quantitative prediction model to guide the rational synthesis of novel fluorescent molecules with the desired pH responsivity is constructed. Two novel Si─rhodamine derivatives are successfully achieved and the cathepsin/pH sequentially activated probe Si─rhodamine─cathepsin-pH (SiR─CTS-pH) is constructed. The results reveal that SiR─CTS-pH exhibits higher signal-to-noise ratio of fluorescence imaging, compared to single pH or cathepsin-activated probe. Moreover, SiR─CTS-pH shows strong differentiation abilities for tumor cells and tissues and accurately discriminates the complex hepatocellular carcinoma tissues from normal ones, indicating its significant application potential in clinical practice. Therefore, the continuous development of xanthene dyes and the rational design of superior fluorescent molecules through machine-learning-assisted model broaden the path and provide more advanced methods to researchers.

Polyphenol oxidase mediates (-)-epigallocatechin gallate to inhibit endogenous cathepsin activity in Apostichopus japonicus.

Apostichopus japonicus (A. japonicus) has rich nutritional value and is an important economic crop. Due to its rich endogenous enzyme system, fresh A. japonicus is prone to autolysis during market circulation and storage, resulting in economic losses. In order to alleviate this phenomenon, we investigated the effect of polyphenol oxidase (PPO) mediated (-)-epigallocatechin gallate (EGCG) on the activity and structure of endogenous cathepsin series protein (CEP) from A. japonicus. Research on cathepsin activity showed that PPO mediated EGCG could significantly reduce enzyme activity, resulting in a decrease in enzymatic reaction rate. SDS-PAGE and scanning electron microscopy results showed that PPO mediates EGCG could induce CEP aggregation to form protein aggregates. Various spectral results indicated that EGCG caused changes in the structure of CEP. Meanwhile, the conjugates formed by PPO mediated EGCG had lower thermal stability. In conclusion, PPO mediated EGCG was an effective method to inhibit the endogenous enzyme activity.

Specific photodamage on HT-29 cancer cells leads to endolysosomal failure and autophagy blockage by cathepsin depletion.

Endolysosomes perform a wide range of cellular functions, including nutrient sensing, macromolecule digestion and recycling, as well as plasma membrane repair. Because of their high activity in cancerous cells, endolysosomes are attractive targets for the development of novel cancer treatments. Light-activated compounds termed photosensitizers (PS) can catalyze the oxidation of specific biomolecules and intracellular organelles. To selectively damage endosomes and lysosomes, HT-29 colorectal cancer cells were incubated with nanomolar concentrations of meso-tetraphenylporphine disulfonate (TPPS2a), an amphiphilic PS taken up via endocytosis and activated by green light (522 nm, 2.1 J.cm-1). Several cellular responses were characterized by a combination of immunofluorescence and immunoblotting assays. We showed that TPPS2a photosensitization blocked autophagic flux without extensive endolysosomal membrane rupture. Nevertheless, there was a severe functional failure of endolysosomes due to a decrease in CTSD (cathepsin D, 55%) and CTSB (cathepsin B, 52%) maturation. PSAP (prosaposin) processing (into saposins) was also considerably impaired, a fact that could be detrimental to glycosphingolipid homeostasis. Therefore, photosensitization of HT-29 cells previously incubated with a low concentration of TPPS2a promotes endolysosomal dysfunction, an effect that can be used to improve cancer therapies.

Effect of accelerated aging on shelf-stability, product loss, sensory and biochemical characteristics in 2 lower quality beef cuts.

The aim of this study was to determine the impact of accelerated aging (AA) on shelf stability, product loss, sensory and biochemical characteristics of 2 lower quality beef cuts. Triceps brachii (TB) and semimembranosus (SM) were collected and fabricated from 10 USDA Choice beef carcasses and assigned to 1 of 6 treatments: 3 d cooler aged (control), 21 d cooler aged, AA 49 °C for 2 h, AA 49 °C for 3 h, AA 54 °C for 2 h, and AA 54 °C for 3 h. The results showed that AA can decrease APC counts on steak surface and in purge and redness, but increase lightness and product loss of the steaks (P < 0.01). Lower shear force was also found for AA steaks compared to those from the control (P < 0.01), with the AA 54 °C treatments being comparable to 21 d cooler aging. However, the trained sensory panel determined AA steaks were less juicy and flavorful than those from the control and 21 d cooler aged samples (P < 0.05). There was no off-flavor detected in AA steaks though lipid oxidation was higher in AA samples than those in the control steaks (P < 0.01). The AA treatments stimulated cathepsin activity (P < 0.05), which may have enhanced the solubilization of stromal proteins and led to a different troponin-T degradation pattern compared to those from the 21 d aged samples (P < 0.01). Although AA is an economical and time-efficient method to increase tenderness of lower-quality beef cuts, further research is needed to determine strategies to mitigate the decrease in juiciness from AA treatments.

Unravelling the role of cathepsins in cardiovascular diseases.

Lysosomal cathepsins as a regulatory medium have been assessed as potential therapeutic targets for the treatment of various cardiac diseases such as abdominal aortic aneurysm, hypertension, cardiomyopathy, coronary heart disease, atherosclerosis, etc. They are ubiquitous lysosomal proteases with papain-like folded protein structures that are involved in a variety of physiological processes, such as the digestion of proteins, activation of pro-inflammatory molecules, degradation of extracellular matrix components, and maturation of peptide hormones. Cathepsins are classified into three major groups: cysteine cathepsins, aspartic cathepsins, and serine-threonine cathepsins. Each of these groups is further divided into subgroups based on their substrate specificity, structural characteristics, and biochemical properties. Several studies suggest that cathepsins control the degradation of ECM components such as collagen and elastin fibres. These enzymes are highly expressed in macrophages and inflammatory cells, and their upregulation has been demonstrated to be critical in the progression of atherosclerotic lesions. Additionally, increased cathepsin activity has been linked to increased vascular inflammation and oxidative stress, both of which are associated with CVDs. Specifically, the inhibition of cathepsins may reduce the release of pro-apoptotic mediators such as caspase-3 and PARP-1, which are thought to contribute to plaque instability. The potential of cathepsins as biomarkers and therapeutic targets has also been supported by the identification of potential cathepsin inhibitors, which could be used to modulate the activities of cathepsins in a range of diseases. This review shall familiarise the readers with the role of cysteinyl cathepsins and their inhibitors in the pathogenesis of cardiovascular diseases.

Development of Improved Spectrophotometric Assays for Biocatalytic Silyl Ether Hydrolysis.

Reported herein is the development of assays for the spectrophotometric quantification of biocatalytic silicon-oxygen bond hydrolysis. Central to these assays are a series of chromogenic substrates that release highly absorbing phenoxy anions upon cleavage of the sessile bond. These substrates were tested with silicatein, an enzyme from a marine sponge that is known to catalyse the hydrolysis and condensation of silyl ethers. It was found that, of the substrates tested, tert-butyldimethyl(2-methyl-4-nitrophenoxy)silane provided the best assay performance, as evidenced by the highest ratio of enzyme catalysed reaction rate compared with the background (uncatalysed) reaction. These substrates were also found to be suitable for detailed enzyme kinetics measurements, as demonstrated by their use to determine the Michaelis-Menten kinetic parameters for silicatein.

Radiosensitizing effect of a novel CTSS inhibitor by enhancing BRCA1 protein stability in triple-negative breast cancer cells.

Triple-negative breast cancer (TNBC) patients harboring wild-type breast cancer susceptibility gene 1 (BRCA1) account for most TNBC patients but lack adequate targeted therapeutic options. Although radiotherapy (RT) is the primary treatment modality for TNBC patients, radioresistance is one of the major challenges. RT-induced increase in cathepsin S (CTSS) causes radioresistance through suppressing BRCA1-mediated apoptosis of tumor cells, which was induced by CTSS-mediated degradation of BRCA1. Targeting CTSS may provide a novel therapeutic opportunity for TNBC patients. Publicly available data and human tissue microarray slides were analyzed to investigate the relationship between CTSS and BRCA1 in breast cancer patients. A CTSS enzyme assay and in silico docking analysis were conducted to identify a novel CTSS inhibitor. RO5461111 was used first to confirm the concept of targeting CTSS for radiosensitizing effects. The MDA-MB-231 TNBC cell line was used for in vitro and in vivo assays. Western blotting, promoter assay, cell death assay, clonogenic survival assay, and immunohistochemistry staining were conducted to evaluate novel CTSS inhibitors. CTSS inhibitors were further evaluated for their additional benefit of inhibiting cell migration. A novel CTSS inhibitor, TS-24, increased BRCA1 protein levels and showed radiosensitization in TNBC cells with wild-type BRCA1 and in vivo in a TNBC xenograft mouse model. These effects were attributed by BRCA1-mediated apoptosis facilitated by TS-24. Furthermore, TS-24 demonstrated the additional effect of inhibiting cell migration. Our study suggests that employing CTSS inhibitors for the functional restoration of BRCA1 to enhance RT-induced apoptosis may provide a novel therapeutic opportunity for TNBC patients harboring wild-type BRCA1.

Ruthenium-Cathepsin Inhibitor Conjugates for Green Light-Activated Photodynamic Therapy and Photochemotherapy.

Dysregulated cathepsin activity is linked to various human diseases including metabolic disorders, autoimmune conditions, and cancer. Given the overexpression of cathepsin in the tumor microenvironment, cathepsin inhibitors are promising pharmacological agents and drug delivery vehicles for cancer treatment. In this study, we describe the synthesis and photochemical and biological assessment of a dual-action agent based on ruthenium that is conjugated with a cathepsin inhibitor, designed for both photodynamic therapy (PDT) and photochemotherapy (PCT). The ruthenium-cathepsin inhibitor conjugate was synthesized through an oxime click reaction, combining a pan-cathepsin inhibitor based on E64d with the Ru(II) PCT/PDT fragment [Ru(dqpy)(dppn)], where dqpy = 2,6-di(quinoline-2-yl)pyridine and dppn = benzo[i]dipyrido[3,2-a:2',3'-c]phenazine. Photochemical investigations validated the conjugate's ability to release a triazole-containing cathepsin inhibitor for PCT and to generate singlet oxygen for PDT upon exposure to green light. Inhibition studies demonstrated the conjugate's potent and irreversible inactivation of purified and intracellular cysteine cathepsins. Two Ru(II) PCT/PDT agents based on the [Ru(dqpy)(dppn)] moiety were evaluated for photoinduced cytotoxicity in 4T1 murine triple-negative breast cancer cells, L929 fibroblasts, and M0, M1, and M2 macrophages. The cathepsin inhibitor conjugate displayed notable selectivity for inducing cell death under irradiation compared to dark conditions, mitigating toxicity in the dark observed with the triazole control complex [Ru(dqpy)(dppn)(MeTz)]2+ (MeTz = 1-methyl-1H-1,2,4-triazole). Notably, our lead complex is among a limited number of dual PCT/PDT agents activated with green light.

An Unusual Two-Domain Thyropin from Tick Saliva: NMR Solution Structure and Highly Selective Inhibition of Cysteine Cathepsins Modulated by Glycosaminoglycans.

The structure and biochemical properties of protease inhibitors from the thyropin family are poorly understood in parasites and pathogens. Here, we introduce a novel family member, Ir-thyropin (IrThy), which is secreted in the saliva of Ixodes ricinus ticks, vectors of Lyme borreliosis and tick-borne encephalitis. The IrThy molecule consists of two consecutive thyroglobulin type-1 (Tg1) domains with an unusual disulfide pattern. Recombinant IrThy was found to inhibit human host-derived cathepsin proteases with a high specificity for cathepsins V, K, and L among a wide range of screened cathepsins exhibiting diverse endo- and exopeptidase activities. Both Tg1 domains displayed inhibitory activities, but with distinct specificity profiles. We determined the spatial structure of one of the Tg1 domains by solution NMR spectroscopy and described its reactive center to elucidate the unique inhibitory specificity. Furthermore, we found that the inhibitory potency of IrThy was modulated in a complex manner by various glycosaminoglycans from host tissues. IrThy was additionally regulated by pH and proteolytic degradation. This study provides a comprehensive structure-function characterization of IrThy-the first investigated thyropin of parasite origin-and suggests its potential role in host-parasite interactions at the tick bite site.

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.

Whole genome identification of olive flounder (Paralichthys olivaceus) cathepsin genes: Provides insights into its regulation on biotic and abiotic stresses response.

Cathepsins are major lysosomal enzymes involved in essential physiological processes, including protein degradation, tissue differentiation, and innate or adaptive responses. Several kinds of cathepsins have been reported in teleost fishes, but no characterization have been performed for the inflammatory response of cathepsin family in olive flounder until now. In our current study, a total of 17 cathepsins in olive flounder were systematically identified and characterized. Phylogenetic analysis clearly indicated that the cathepsin genes was highly conserved. Analysis of structure and motifs exhibited high sequence similarity of cathepsin genes in olive flounder. Expression profiles of cathepsin genes in different tissues and developmental stages showed that cathepsins were temporally and spatially specific. RNA-seq analysis of bacteria and temperature stresses revealed that members of cathepsin were involved in inflammatory responses. Collectively, our findings would provide a further reference for understanding the molecular mechanisms of cathepsins in olive flounder.

Rational design of humanized antibody inhibitors for cathepsin S.

Cathepsin S (CTSS) is involved in pathogenesis of many human diseases. Inhibitors blocking its protease activity hold therapeutic potential. In comparison to small-molecule inhibitors, monoclonal antibodies capable of inhibiting CTSS enzymatic activity may possess advantageous pharmacological properties. Here we designed and produced inhibitory antibodies targeting human CTSS by genetically fusing the propeptide of procathepsin S (proCTSS) with antibodies in clinic. The resulting antibody fusions in full-length or fragment antigen-binding format could be stably expressed and potently inhibit CTSS proteolytic activity in high specificity. These fusion antibodies not only demonstrate a new approach for facile synthesis of antibody inhibitors against CTSS, but also represent novel anti-CTSS therapeutic candidates.

Cathepsin V drives lung cancer progression by shaping the immunosuppressive environment and adhesion molecules cleavage.

Cathepsin V (CTSV) is a cysteine cathepsin protease that plays a crucial role in extracellular matrix degradation. CTSV is correlated with poor prognosis in various cancers, but the underlying mechanism remains elusive. Here, we observed that CSTV is upregulated in lung cancer and is a poor prognosis factor for lung cancer. CTSV acts as a driver in the metastasis of lung cancer both in vitro and in vivo. CTSV promotes lung cancer metastasis by downregulating adhesion molecules, including fibronectin, E-cadherin, and N-cadherin. Our data revealed that CTSV functions by mediating the fragmentation of fibronectin, E-cadherin, and N-cadherin in cleavage, remodeling the extracellular matrix (ECM). The rationally designed antibody targeting CTSV blocks its cleaving ability towards fibronectin, E-cadherin, and N-cadherin, suppressing migration and invasion. Furthermore, we found that CTSV expression is negatively correlated with immune cell infiltration and immune scores and inhibits T cell activity. Targeting CTSV with specific antibodies effectively suppressed lung cancer metastasis in a mouse model. Our study demonstrates the critical role of CTSV in the immunity and metastasis of lung cancer, suggesting that the CTSV-targeting approach is a promising strategy for lung cancer.