Activity Of Cathepsin Research Articles

In vitro validation of Cathepsin S docking models

Abstract In dendritic cells, the dominant lysosomal protease involved in Ii chain degradation and antigen digestion is Cathepsin S, a cysteine protease in the papain family. Extracellularly, Cathepsin S also contributes to pathological processes in cardiovascular disease, cancer, and autoimmune arthritis: unlike most lysosomal proteases, it retains its activity at serum pH. In computational molecular docking studies comparing peptides from the literature, we found that Cathepsin S shows a pH-dependent specificity switch, with predicted dissociation constants for specific peptide sequences differing at lysosomal and serum pH. The aim of this study is to further characterize this specificity switch and to compare docking results to kinetic parameters of Cathepsin S digestion in vitro. We identified peptide sequences of interest from docking, including sequences from the Ii chain and elastin, and digested these peptides (LifeTein) using human Cathepsin S (Calbiochem), collecting samples for LC-MS analysis over 16 hour reaction times. We report the kinetic parameters of Cathepsin S catalysis at both lysosomal and serum pH. Digestion results are compared to docking models and the models most predictive of catalytic activity are identified. Further investigation is needed to determine whether pH-dependent catalytic activity and selectivity of Cathepsin S will enable the design of protein-based therapies and inhibitors optimized for their targeted microenvironments. This work was partially supported by the NIDA/NIH award UG3DA048775, the USDA National Institute of Food and Agriculture, AFRI project #2021-0858, and the department of Biological Systems Engineering at Virginia Tech.

ZIP8 impacts protease activity in macrophages in response to Streptococcus pneumoniae

Abstract Zinc (Zn) is essential for immune function and Zn deficiency increases the incidence of community acquired pneumonia. The Zn transporter ZIP8 is required by myeloid cells to defend against pathogens. Previously, we reported that S. pneumoniae infected ZIP8 knockout mice (Zip8KO) have increased bacterial burden and mortality and that Zip8KO bone marrow derived macrophages (BMDMs) have reduced phagocytic activity. Others have shown that Zn induces cathepsin expression via the TFEB pathway. However, it is unknown whether ZIP8 impacts TFEB-dependent phagolysosomal processing in macrophages. Based on this, we hypothesized that ZIP8 facilitates phagolysosomal processing and eradication of bacteria via TFEB. BMDMs were derived from wild type and Zip8KO mice and then exposed to S. pneumoniae(MOI=10) for 1 hour. At 6–18 hours post challenge we assessed cathepsin B and L mRNA and protein levels as well as total protease, cathepsin B and L activity. In conjunction, we measured p-TFEB and total TFEB proteins levels. Active TFEB and cathepsin B protein levels were significantly lower in Zip8KO BMDMs with no difference in cathepsin L. Strikingly, Zip8KO BMDMs had significantly reduced total protease, cathepsin B and cathepsin L activity which also corresponded with decreased mRNA levels. Based on these findings we predict that ZIP8 loss in macrophages impairs phagolysosomal processing and bacterial eradication in a TFEB-dependent manner. Overall, this warrants future studies given the high incidence of Zn deficiency and the rs13107325 defective ZIP8 allele in humans. National Heart, Lung and Blood Institute #1R01HL156952-01-A1 (DLK)

Abstract 126: Effect Of Icospaent Ethyl On In Vivo Atheroma Progression And Inflammatory Protease Activity

Background: Icosapent ethyl (IPE), a high purity ethyl ester of eicosapentaenoic acid (EPA), is FDA-approved to reduce cardiovascular events among patients with or at risk for cardiovascular disease with elevated triglyceride levels. However, in vivo mechanisms underlying these findings remain unclear. Here we investigated whether IPE reduces plaque progression and inflammation using intravascular molecular-structural imaging. Methods: Cholesterol-fed balloon-injured rabbits (N=16) bearing aortic atherosclerosis underwent intravascular ultrasound (IVUS) analysis of plaque burden and near-infrared fluorescence-optical coherence tomography (NIRF-OCT) molecular imaging of plaque inflammatory cathepsin protease activity (ProSense VM110 NIRF agent, 4 mg/kg/iv) at 6 weeks. Next, rabbits were randomized to control (N=8) or IPE (Vascepa, Amarin, NJ) treated group (N=8, 1 g/kg/day) milled in the diet. Four weeks later, imaging was repeated, animals were sacrificed, and histopathology analysis of dissected plaque was employed. Results: Quantitative IVUS analyses (40mm segment, 3200 slices/16 rabbits) revealed that IPE significantly suppressed atheroma progression (ΔPAV, 5.2% [2.36-12.98] vs. 9.1% [4.26-12.25] control, p=0.03). Inflammatory plaque cathepsin activity detected by NIRF-OCT was similar (ΔNIRF average plaque 17.6 [6.2- 51.5] vs. 30.5 [4.9- 55.5] nM control, p=0.19)). On fluorescence microscopy, however, plaques from IPE-treated animals exhibited reduced NIRF signal (1.16% [0.03-3.94] vs. 3.13% [0.04-12.92], p=0.005)) and fewer RAM11+ macrophages (23.5±15.6 vs. control 32.9±12.6 macrophages/section, 25 sections/group, p=0.04). In IPE-treated animals, plasma EPA levels were elevated (p<0.05), and EPA metabolite levels, particularly 5,6-diHETE, were also elevated in resected plaques (p<0.05). Conclusion: IPE reduces in vivo plaque progression and plaque cathepsin protease activity and plaque macrophages, providing novel experimental evidence supporting the anti-atherosclerosis effects of IPE.

Trimethylamine N-Oxide (TMAO) Inducing Endothelial Injury: UPLC-MS/MS-Based Quantification and the Activation of Cathepsin B-Mediated NLRP3 Inflammasome.

TMAO is a new risk biomarker for cardiovascular disease. With trimethylammonium as its main chemical skeleton, TMAO is structurally similar to many endogenous metabolites, such as acetylcholine, carnitine, phosphorylcholine, etc. The mechanism of TMAO on the pathological process of CVD is still unclear. In this study, the quantitative analysis of plasma TMAO is conducted, and the contribution of Cathepsin B and NLRP3 inflammasome during the process of TMAO-induced endothelial injury was proposed and investigated at animal and cellular levels. Immunofluorescence assay was applied to represent the protein expression of Cathepsin B and NLRP3 inflammasome located at endothelial cells. The results showed that TMAO could disrupt endothelial cells permeability to induce endothelial injury, meanwhile, TMAO could increase NLRP3 inflammasome activation and promote the activity and expression of Cathepsin B in vitro and in vivo, whereas inhibition of NLRP3 inflammasome activation by MCC950 could protect the endothelial cells from TMAO associated endothelial injury via Cathepsin B. The study reveals that TMAO can cause endothelial injury via Cathepsin B-dependent NLRP3 inflammasome, and inhibition of Cathepsin B and NLRP3 inflammasome can reduce the TMAO-induced damage. The results provide new insight into the role of TMAO in CVD, which can be a potential therapeutic target for disease treatment and drug design.

Immunopeptidomics reveals determinants of Mycobacterium tuberculosis antigen presentation on MHC class I.

CD8+ T cell recognition of Mycobacterium tuberculosis (Mtb)-specific peptides presented on major histocompatibility complex class I (MHC-I) contributes to immunity to tuberculosis (TB), but the principles that govern presentation of Mtb antigens on MHC-I are incompletely understood. In this study, mass spectrometry (MS) analysis of the MHC-I repertoire of Mtb-infected primary human macrophages reveals that substrates of Mtb's type VII secretion systems (T7SS) are overrepresented among Mtb-derived peptides presented on MHC-I. Quantitative, targeted MS shows that ESX-1 activity is required for presentation of Mtb peptides derived from both ESX-1 substrates and ESX-5 substrates on MHC-I, consistent with a model in which proteins secreted by multiple T7SSs access a cytosolic antigen processing pathway via ESX-1-mediated phagosome permeabilization. Chemical inhibition of proteasome activity, lysosomal acidification, or cysteine cathepsin activity did not block presentation of Mtb antigens on MHC-I, suggesting involvement of other proteolytic pathways or redundancy among multiple pathways. Our study identifies Mtb antigens presented on MHC-I that could serve as targets for TB vaccines, and reveals how the activity of multiple T7SSs interacts to contribute to presentation of Mtb antigens on MHC-I.

Lysosomal chloride transporter CLH-6 protects lysosome membrane integrity via cathepsin activation.

Lysosomal integrity is vital for cell homeostasis, but the underlying mechanisms are poorly understood. Here, we identify CLH-6, the C. elegans ortholog of the lysosomal Cl-/H+ antiporter ClC-7, as an important factor for protecting lysosomal integrity. Loss of CLH-6 affects lysosomal degradation, causing cargo accumulation and membrane rupture. Reducing cargo delivery or increasing CPL-1/cathepsin L or CPR-2/cathepsin B expression suppresses these lysosomal defects. Inactivation of CPL-1 or CPR-2, like CLH-6 inactivation, affects cargo digestion and causes lysosomal membrane rupture. Thus, loss of CLH-6 impairs cargo degradation, leading to membrane damage of lysosomes. In clh-6(lf) mutants, lysosomes are acidified as in wild type but contain lower chloride levels, and cathepsin B and L activities are significantly reduced. Cl- binds to CPL-1 and CPR-2 in vitro, and Cl- supplementation increases lysosomal cathepsin B and L activities. Altogether, these findings suggest that CLH-6 maintains the luminal chloride levels required for cathepsin activity, thus facilitating substrate digestion to protect lysosomal membrane integrity.

Abstract 3591: Does the metastatic immunoenvironment determine poor immunotherapy responses

Abstract Despite new treatments, cancer remains the second leading cause of death worldwide with breast cancer being the most common type. Of these deaths, the majority are due to the formation of distant metastases. While new immunotherapies have shown some success in primary tumors, their efficacy in combating metastases has been lacking. Thus we aim to investigate the hypothesis that immune responses at the metastatic site contribute to limitations of current therapies.Within the last decade, new microscopy technologies now enable real-time visualization of immune responses at primary and secondary sites. Here, we present use of these microscope technologies, along with the development of novel probes to detect apoptotic cancer cells, to investigate the success of checkpoint inhibitors in the primary and secondary sites. We validate, in multiple in vitro and in vivo assays (including intravital imaging), that fluorescent cyclic amphipathic peptide probes specifically label cells undergoing therapy-induced cancer death. In addition, we introduce a newly developed fluorescent, enzyme-activatable, chemokine-conjugate capable of targeting defined macrophage subsets in live tumors. This chemokine-conjugate exploits the high expression of chemokine receptors (e.g. CCR2) and the activity of cysteine cathepsins in tumor associated macrophages to selectively target and eradicate them without affecting other macrophages or immune cells (e.g., neutrophils, T cells, B cells). This research will help to elucidate the role of combination therapy on the metastatic foci and will provide the first insights into the benefit and limitations of combination therapy on secondary site immune responses. Citation Format: Nicole D. Barth, Floris J. van Dalen, Camille Duran, Utsa Karmakar, Lorena Mendive-Tapia, Takanori Kitamura, Martjin Verdoes, John S. Condeelis, Maja Oktay, Marc Vendrell, Valerie Brunton, David Entenberg. Does the metastatic immunoenvironment determine poor immunotherapy responses. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3591.

Abstract 2655: Anti-CD19 antibody-drug conjugate therapy in B cell non-Hodgkin lymphoma

Abstract Previously, we have shown that overexpression of Polo-like kinase (PLK)-1 is associated with poor clinical outcomes in patients with aggressive B cell lymphomas. PLK-1 selective inhibitor, Volasertib (BI 6727), has a potent anti-tumor effect in aggressive B cell lymphomas. Unfortunately, Volasertib was withdrawn in Phase III clinical trials due to severe myelosuppression. In this study, we developed a Volasertib antibody-drug conjugate (V-ADC) using -CD19 antibody Inebilizumab to increase the targeting specificity for B-cell lymphoma cells and minimize the side effects of Volasertib. However, we found that V-ADC treatment exhibited little cytotoxic effects, comparing to free Volasertib treatment in Z138 cells, a mantle cell lymphoma (MCL) cell line. We hypothesized that the expression level/density of CD19 on B-lymphoma cells as well as its gene mutations may affect V-ADC binding affinity required for its endocytosis. We, therefore, established Z138 cell lines with overexpression of either wild-type (WT) or mutant (Mut) CD19. The cell viability at 48h post 10nM V-ADC treatment was determined to be 90%, 61%, and 69% in parent, WT, and Mut CD19-overexpressing Z138 cells, respectively. In comparison, the cell viability with 10nM free volasertib treatment ranged from 3-30% in these cell lines. Therefore, other factors, in addition to CD19 expression, may have also played an important role in V-ADC induced cytotoxicity. Upon endocytosis, the V-ADC will enter the endolysosome where cathepsin, a lysosomal enzyme, cleaves the linker between Volasertib and Inebilizumab and releases free Volasertib into the cells. Our previous works have shown that a starvation condition (culture media with 1% FBS) increased lysosomal cathepsin activity, comparing to the culture media with 10% FBS. Herein, we found that upon treatment with 10nM V-ADC, CD19-overexpressing Z138 cells cultured in media with 1% FBS exhibited markedly increased apoptotic cell death comparing to cells in media with 10% FBS (64% vs 41%, respectively). Furthermore, WT and Mut CD19-overexpressing Z138 cells exhibited 2.8- and 2.4-folds of apoptotic cell death compared to control Z138 cells at 48h post 10nM V-ADC treatment with 1% FBS. The cell apoptosis occurred via upregulation of caspase 3, but not caspase 9, with induction of cleavage of poly (ADP-ribose) polymerase (PARP) and reduction of mitochondria membrane potential. In conclusion, starvation and lysosomal cathepsin activation increased V-ADC-induced apoptotic cell death in CD19 overexpression Z138 cell lines. We are actively investigating the in vivo therapeutic effects of V-ADC in patient-derived xenograft (PDX) animal models of MCL and other aggressive B-cell lymphomas. Citation Format: Chieko Saito, Chengfeng Bi, Kai Fu. Anti-CD19 antibody-drug conjugate therapy in B cell non-Hodgkin lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2655.

Prioritized mass spectrometry increases the depth, sensitivity and data completeness of single-cell proteomics

Major aims of single-cell proteomics include increasing the consistency, sensitivity and depth of protein quantification, especially for proteins and modifications of biological interest. Here, to simultaneously advance all these aims, we developed prioritized Single-Cell ProtEomics (pSCoPE). pSCoPE consistently analyzes thousands of prioritized peptides across all single cells (thus increasing data completeness) while maximizing instrument time spent analyzing identifiable peptides, thus increasing proteome depth. These strategies increased the sensitivity, data completeness and proteome coverage over twofold. The gains enabled quantifying protein variation in untreated and lipopolysaccharide-treated primary macrophages. Within each condition, proteins covaried within functional sets, including phagosome maturation and proton transport, similarly across both treatment conditions. This covariation is coupled to phenotypic variability in endocytic activity. pSCoPE also enabled quantifying proteolytic products, suggesting a gradient of cathepsin activities within a treatment condition. pSCoPE is freely available and widely applicable, especially for analyzing proteins of interest without sacrificing proteome coverage. Support for pSCoPE is available at http://scp.slavovlab.net/pSCoPE.

CHANGES IN THE ACTIVITY OF LYSOSOMAL CYSTEINE PROTEASES IN VASCULAR WALL HOMOGENATES IN VARICOSE VEINS OF THE LOWER EXTREMITIES

Relevance. The article deals with the issue of changes in the activity of lysosomal cysteine proteases – cathepsins L, B, H in homogenates of vascu-lar wall, obtained from patients with varicose veins of the lower extremities. Due to the fact that the proteolytic processes occurring in the wall of vari-cose transformed veins currently remain poorly understood, and the incidence of varicose veins is increasing, the relevance of the topic remains at a high level. Materials and methods. The study shows the results from 22 patients divided into two groups: the first group (n=12): patients with varicose veins of the lower extremities, the second group (control) – patients who had healthy vein sections, taken during femoral-popliteal bypass surgery using au-tovena (n=10). The material for the study was homogenates of vascular wall, obtained during surgical intervention. The vessels were weighed on elec-tronic scales (Ohaus corporation, USA). The samples were crushed and placed in a glass of the Potter S homogenizer (Sartorius, Germany). Homoge-nized for 60 seconds with a rotation speed of a Teflon pestle of 1500 rpm. The activity of cathepsins B, L and H was determined spectrofluorimetrically on the RF – Zhimadzu Spectrofluorometer (Japan). The protein content was assessed using the Lowry method by the commercial set of the NPC "Eco-service" (St. Petersburg). Results. The activity of cathepsin B in the homogenate of varicose veins was increased by 35 times compared to the control group. The activity of cathepsin D in the homogenate of varicose veins was also increased 17-fold compared to the control. The activity of cathepsin H in varicose vein ho-mogenate showed a unidirectional trend - it was higher than the control values, but by a smaller number of times – by 9. Thus, in varicose vein ho-mogenates, all three studied cathepsins increase their activity, but to varying degrees. The greatest changes, according to our study, confirmed the activity of cathepsin B. Conclusions. Varicose veins of the lower extremities are accompanied by a significant increase in the activity of lysosomal cysteine proteinases – cathepsins B, L and H in the wall homogenates of the affected vein, which may indicate the involvement of LC in the pathogenesis of venous trans-formation

Autophagy and exosomes coordinately mediate quercetin's protective effects on alcoholic liver disease

Alcoholic liver disease (ALD), a spectrum of liver abnormalities induced by chronic alcohol abuse, continues to be the major cause of life-threatening liver disease in developed countries. Autophagy and exosomes were individually confirmed to be involved in the pathogenesis of ALD. Here, we sought to identify the role of autophagy and exosomes in the liver protective effects of quercetin. We observed decreased hepatic LC3II/LC3I and increased p62 level in ethanol-fed mice, and these changes were alleviated by quercetin. Meanwhile, nanoparticle tracking analysis (NTA) showed elevated serum exosomes numbers in ethanol-fed mice, which was combated by quercetin. Ethanol induced elevated LDH, ALT, and AST in HepG2 supernatant, which was alleviated by cytochalasin D (exosomes uptake inhibitor). Moreover, quercetin reduced ethanol-induced LDH and ALT elevation in vitro, and the effects of quercetin were reversed by Rab27a overexpression (induce exosomes release) or wortmannin treatment (autophagy inhibitor). Transcriptomic analysis supported that quercetin reversed the change of lysosome related genes disturbed by ethanol. Meanwhile, western blot analysis exhibited decreased hepatic expression of LAMP2 and ATPA6V1B2, and active Cathepsin B/Cathepsin B by quercetin treatment, indicating quercetin alleviated lysosome dysfunction in ethanol-fed mice. Baf A treatment or transfection of siTFEB offset quercetin's effects in ethanol-induced LDH and ALT elevation, exosomes release, and autophagy inhibition (LC3II/I and p62 accumulation). Taken together, quercetin coordinately activates autophagy and combats exosomes release by restoring lysosome function, and further mitigates ethanol-induced liver damage.

Co-infection of TYLCV and ToCV increases cathepsin B and promotes ToCV transmission by Bemisia tabaci MED.

Tomato disease is an important disease affecting agricultural production, and the combined infection of tomato chlorosis virus (ToCV) and tomato yellow leaf curl virus (TYLCV) has gradually expanded in recent years, but no effective control method has been developed to date. Both viruses are transmitted by Bemisia tabaci Mediteranean (MED). Previously, we found that after B. tabaci MED was fed on ToCV-and TYLCV-infected plants, the transmission efficiency of ToCV was significantly higher than that on plants infected only with ToCV. Therefore, we hypothesize that co-infection could enhance the transmission rates of the virus. In this study, transcriptome sequencing was performed to compare the changes of related transcription factors in B. tabaci MED co-infected with ToCV and TYLCV and infected only with ToCV. Hence, transmission experiments were carried out using B. tabaci MED to clarify the role of cathepsin in virus transmission. The gene expression level and enzyme activity of cathepsin B (Cath B) in B. tabaci MED co-infected with ToCV and TYLCV increased compared with those under ToCV infection alone. After the decrease in cathepsin activity in B. tabaci MED or cathepsin B was silenced, its ability to acquire and transmit ToCV was significantly reduced. We verified the hypothesis that the relative expression of cathepsin B was reduced, which helped reduce ToCV transmission by B. tabaci MED. Therefore, it was speculated that cathepsin has profound research significance in the control of B. tabaci MED and the spread of viral diseases.

Cry41-Related Mutants against Myzus persicae Based on Its Interaction with Cathepsin B

Cry toxins produced by Bacillus thuringiensis (Bt) are toxic to Lepidoptera, Coleoptera, and Diptera, but display very low activity against aphids. Recently, Cry41-related toxin was found to show moderate toxicity against Myzus persicae with the 50% lethal concentration (LC50) of 32.7 μg/mL, and its mode of action was proposed to be the acceleration of the apoptosis of aphid cells by enhancing Cathepsin B activity. This study focused on constructing Cry41-related mutants against M. persicae based on its interaction with Cathepsin B. First, eight key interacting residues in Cry41-related toxin were identified using alanine scanning and site-directed saturation mutagenesis. Subsequently, the positive mutant Cry41-7M protein (mutations of Gly48, Ile59, Lys364, Gln367, Gln377, Tyr378, and Ser400 to Tyr, Ala, Arg, Lys, Lys, Lys, and Ala in Cry41-related toxin, respectively) and the negative mutant Cry41-6A protein (mutations of Gly48, Lys364, Gln367, Gln377, and Tyr378 to Ala and mutation of Pro453 to Glu in Cry41-related toxin) were constructed, expressed, and purified. We then found that Cry41-7M protein performed slightly stronger than Cry41-related toxin in enhancing the enzymatic activity of Cathepsin B, whereas Cry41-6A protein did not affect Cathepsin B activity. A further bioassay showed that the mortality caused by Cry41-7M protein (LC50 = 19.144 μg/mL) was marginally higher than that of Cry41-related toxin, while Cry41-6A protein caused a decreased mortality (LC50 = 42.478 μg/mL). These results are expected to open new avenues for improving Cry aphidicidal activity.

Determination of the autophagic flux in murine and human peripheral blood mononuclear cells.

The autophagy lysosomal system (ALS) is crucial for cellular homeostasis, contributing to maintain whole body health and alterations are associated with diseases like cancer or cardiovascular diseases. For determining the autophagic flux, inhibition of lysosomal degradation is mandatory, highly complicating autophagy measurement in vivo. To overcome this, herein blood cells were used as they are easy and routinely to isolate. Within this study we provide detailed protocols for determination of the autophagic flux in peripheral blood mononuclear cells (PBMCs) isolated from human and, to our knowledge the first time, also from murine whole blood, extensively discussing advantages and disadvantages of both methods. Isolation of PBMCs was performed using density gradient centrifugation. To minimize changes on the autophagic flux through experimental conditions, cells were directly treated with concanamycin A (ConA) for 2h at 37°C in their serum or for murine cells in serum filled up with NaCl. ConA treatment decreased lysosomal cathepsins activity and increased Sequestosome 1 (SQSTM1) protein and LC3A/B-II:LC3A/B-I ratio in murine PBMCs, while transcription factor EB was not altered yet. Aging further enhanced ConA-associated increase in SQSTM1 protein in murine PBMCs but not in cardiomyocytes, indicating tissue-specific differences in autophagic flux. In human PBMCs, ConA treatment also decreased lysosomal activity and increased LC3A/B-II protein levels, demonstrating successful autophagic flux detection in human subjects. In summary, both protocols are suitable to determine the autophagic flux in murine and human samples and may facilitate a better mechanistic understanding of altered autophagy in aging and disease models and to further develop novel treatment strategies.

Glycosylation Analysis of Urinary Peptidome Highlights IGF2 Glycopeptides in Association with CKD

Chronic kidney disease (CKD) is prevalent in 10% of world's adult population. The role of protein glycosylation in causal mechanisms of CKD progression is largely unknown. The aim of this study was to identify urinary O-linked glycopeptides in association to CKD for better characterization of CKD molecular manifestations. Urine samples from eight CKD and two healthy subjects were analyzed by CE-MS/MS and glycopeptides were identified by a specific software followed by manual inspection of the spectra. Distribution of the identified glycopeptides and their correlation with Age, eGFR and Albuminuria were evaluated in 3810 existing datasets. In total, 17 O-linked glycopeptides from 7 different proteins were identified, derived primarily from Insulin-like growth factor-II (IGF2). Glycosylation occurred at the surface exposed IGF2 Threonine 96 position. Three glycopeptides (DVStPPTVLPDNFPRYPVGKF, DVStPPTVLPDNFPRYPVG and DVStPPTVLPDNFPRYP) exhibited positive correlation with Age. The IGF2 glycopeptide (tPPTVLPDNFPRYP) showed a strong negative association with eGFR. These results suggest that with aging and deteriorating kidney function, alterations in IGF2 proteoforms take place, which may reflect changes in mature IGF2 protein. Further experiments corroborated this hypothesis as IGF2 increased plasma levels were observed in CKD patients. Protease predictions, considering also available transcriptomics data, suggest activation of cathepsin S with CKD, meriting further investigation.

The Interplay of Glycosaminoglycans and Cysteine Cathepsins in Mucopolysaccharidosis.

Mucopolysaccharidosis (MPS) consists of a group of inherited lysosomal storage disorders that are caused by a defect of certain enzymes that participate in the metabolism of glycosaminoglycans (GAGs). The abnormal accumulation of GAGs leads to progressive dysfunctions in various tissues and organs during childhood, contributing to premature death. As the current therapies are limited and inefficient, exploring the molecular mechanisms of the pathology is thus required to address the unmet needs of MPS patients to improve their quality of life. Lysosomal cysteine cathepsins are a family of proteases that play key roles in numerous physiological processes. Dysregulation of cysteine cathepsins expression and activity can be frequently observed in many human diseases, including MPS. This review summarizes the basic knowledge on MPS disorders and their current management and focuses on GAGs and cysteine cathepsins expression in MPS, as well their interplay, which may lead to the development of MPS-associated disorders.

Cathepsin B maturation plays a critical role in leptin-induced hepatic cancer cell growth through activation of NLRP3 inflammasomes

Leptin, an adipose tissue-derived hormone, exhibits potent tumor promoting effects through various mechanisms. Cathepsin B, a member of the lysosomal cysteine proteases, has been shown to modulate the growth of cancer cells. In this study, we have investigated the role of cathepsin B signaling in leptin-induced hepatic cancer growth. Leptin treatment caused significant increase in the levels of active cathepsin B through the axis of endoplasmic reticulum stress and autophagy induction without significant effects on pre- and pro-forms of cathepsin B. Interestingly, inhibition of cathepsin B signaling by gene silencing or treatment with a selective pharmacological inhibitor (CA-074) prevented leptin-enhanced viability of hepatic cancer cell and suppressed progression of cell cycle, indicating the critical role of cathepsin B in leptin-induced hepatic cancer growth. We have further observed that maturation of cathepsin B is required for NLRP3 inflammasomes activation, which is implicated in the growth of hepatic cancer cell. The crucial roles of cathepsin B maturation in leptin-induced hepatic cancer growth and NLRP3 inflammasomes activation were confirmed in an in vivo HepG2 tumor xenograft model. Taken together, these results demonstrate that cathepsin B signaling plays a pivotal role in leptin-induced hepatic cancer cell growth by activating NLRP3 inflammasomes.

Effect of pH-Shifting Process on the Cathepsin Activity, Muddy Off-Odor Compounds' Content and Gelling Properties of Isolated Protein from Silver Carp.

Silver carp (Hypophthalmichthys molitrix) is a potential source for making surimi products. However, it has the disadvantages of bony structures, high level of cathepsines and muddy off-odor which is mainly caused by geosmin (GEO) and 2-methylisoborneol (MIB). These disadvantages make the conventional water washing process of surimi inefficient (low protein recovery rate, and high residual muddy off-odor). Thus, the effect of the pH-shifting process (acid-isolating process and alkali-isolating process) on the cathepsins activity, GEO content, MIB content, and gelling properties of the isolated proteins (IPs) was investigated, comparing it with surimi obtained through the conventional cold water washing process (WM). The alkali-isolating process greatly boosted the protein recovery rate from 28.8% to 40.9% (p < 0.05). In addition, it removed 84% GEO and 90% MIB. The acid-isolating process removed about 77% GEO and 83% MIB. The acid-isolated protein (AC) displayed the lowest elastic modulus (G'), the highest TCA-peptide content (90.89 ± 4.65 mg/g) and the highest cathepsin L activity (65.43 ± 4.91 U/g). The AC modori (60 °C for 30 min) gel also demonstrated the lowest breaking force (226.2 ± 19.5 g) and breaking deformation (8.3 ± 0.4 mm), indicating that proteolysis caused by the cathepsin deteriorated the gel quality of AC. The setting (40 °C for 30 min) considerably increased the breaking force (386.4 ± 15.7 g) and breaking deformation (11.6 ± 0.2 mm) of the gel made from the alkali-isolated protein (AK) (p < 0.05). In AC and AK gel, a clearly visible cross-linking protein band with a molecular weight greater than MHC was seen, demonstrating the presence of endogenous trans-glutaminase (TGase) activity, that improved the gel quality of AK. In conclusion, the alkali-isolating process was an effective alternative method for making water-washed surimi from silver carp.

Hederacolchiside A1 Suppresses Autophagy by Inhibiting Cathepsin C and Reduces the Growth of Colon Cancer.

While autophagy degrades non-functional or unnecessary cellular components, producing materials for synthesizing cellular components, it can also provide energy for tumor development. Hederacolchiside A1 (HA1) derived from anemone raddeana has anticancer effects on several carcinomas by inducing apoptosis or exhibiting cytotoxicity, but the relationship with autophagy has not been studied. We investigated the association between HA1 and autophagy and evaluated its anticancer effect on colon cancer. HA1 induced accumulation of the autophagy-related markers LC3B and SQSTM1, with distinct vacuolar formation, unlike other autophagy inhibitors; the effects were similar to those of chloroquine. In addition, HA1 decreased the expression and proteolytic activity of lysosomal protein cathepsin C, reduced the growth of colon cancer cells in vitro, and inhibited tumor growth in vivo. It also reduced the expression of Ki-67 and cathepsin C in mouse tissues and reduced the growth of spheroids and organoids composed of cancer cells. Taken together, these results imply that HA1 regulates cell growth and autophagy and has potential as a promising therapeutic agent in colon cancer.

Gemcitabine promotes autophagy and lysosomal function through ERK- and TFEB-dependent mechanisms

Gemcitabine is a first-line treatment agent for pancreatic ductal adenocarcinoma (PDAC). Contributing to its cytotoxicity, this chemotherapeutic agent is primarily a DNA replication inhibitor that also induces DNA damage. However, its therapeutic effects are limited owing to chemoresistance. Evidence in the literature points to a role for autophagy in restricting the efficacy of gemcitabine. Autophagy is a catabolic process in which intracellular components are delivered to degradative organelles lysosomes. Interfering with this process sensitizes PDAC cells to gemcitabine. It is consequently inferred that autophagy and lysosomal function need to be tightly regulated to maintain homeostasis and provide resistance to environmental stress, such as those imposed by chemotherapeutic drugs. However, the mechanism(s) through which gemcitabine promotes autophagy remains elusive, and the impact of gemcitabine on lysosomal function remains largely unexplored. Therefore, we applied complementary approaches to define the mechanisms triggered by gemcitabine that support autophagy and lysosome function. We found that gemcitabine elicited ERK-dependent autophagy in PDAC cells, but did not stimulate ERK activity or autophagy in non-tumoral human pancreatic epithelial cells. Gemcitabine also promoted transcription factor EB (TFEB)-dependent lysosomal function in PDAC cells. Indeed, treating PDAC cells with gemcitabine caused expansion of the lysosomal network, as revealed by Lysosome associated membrane protein-1 (LAMP1) and LysoTracker staining. More specific approaches have shown that gemcitabine promotes the activity of cathepsin B (CTSB), a cysteine protease playing an active role in lysosomal degradation. We showed that lysosomal function induced by gemcitabine depends on TFEB, the master regulator of autophagy and lysosomal biogenesis. Interfering with TFEB function considerably limited the clonogenic growth of PDAC cells and hindered the capacity of TFEB-depleted PDAC cells to develop orthotopic tumors.