Activity Of Cathepsin Research Articles

The activity, distribution, and colocalization of cathepsin K and matrix metalloproteases in intact and eroded dentin.

This study aimed to assess the activity, distribution, and colocalization of cathepsin K (catK) and matrix metalloproteases (MMPs) in both intact and eroded dentin in vitro. Eroded dentin was obtained by consecutive treatment with 5% citric acid (pH = 2.3) for 7days, while intact dentin remained untreated. Pulverized dentin powder (1.0g) was extracted from both intact and eroded dentin using 5mL of 50mM Tris-HCl buffer (0.2g/1mL, pH = 7.4) for 60h to measure the activity of catK and MMPs spectrofluorometrically. In addition, three 200-μm-thick dentin slices were prepared from intact and eroded dentin for double-labeling immunofluorescence to evaluate the distribution and colocalization of catK and MMPs (MMP-2 and MMP-9). The distribution and colocalization of enzymes were analyzed using inverted confocal laser scanning microscopy (CLSM), with colocalization rates quantified using Leica Application Suite Advanced Fluorescent (LAS AF) software. One-way analysis of variance (ANOVA) was used to analyze the fluorescence data related to enzyme activity (α = 0.05). The activity of catK and MMPs was significantly increased in eroded dentin compared with intact dentin. After erosive attacks, catK, MMP-2, and MMP-9 were prominently localized in the eroded regions. The colocalization rates of catK with MMP-2 and MMP-9 were 13- and 26-fold higher in eroded dentin, respectively, than in intact dentin. Erosive attacks amplified the activity of catK and MMPs in dentin while also altering their distribution patterns. Colocalization between catK and MMPs increased following erosive attacks. CatK, MMP-2, and MMP-9 likely play synergistic roles in the pathophysiology of dentin erosion.

Bioconcentration, biotransformation, and transcriptomic impact of the UV-filter 4-MBC in the manila clam Ruditapes philippinarum

Ultraviolet filters (UV-filters) are compounds extensively used in personal care products. These compounds are produced at increasing rates and discharged into marine ecosystems in unknown quantities and with no regulation, making them emerging contaminants. Among those, the UV-filter 4-Methylbenzylidene camphor (4-MBC) is used in a variety of personal care products such as sunscreens, soaps, or lipsticks. This high consumption has resulted in its presence in various environmental matrices at in concentrations ranging from ng to μg L−1. Very little is known, however, about the possible adverse effects in exposed non-target organisms. Our study presents novel data on the bioconcentration, toxicokinetics, and molecular effects of 4-MBC in a marine bivalve species of commercial interest, Ruditapes philippinarum (Manila clam). Organisms were exposed at two different concentrations (1.34 and 10.79 μg L−1) of 4-MBC for 7 days, followed by a 3-day depuration period (clean sea waters). Bioconcentration factors (BCF) were 3562 and 2229 L kg−1 for the low and high exposure concentrations, respectively, making this pollutant bioaccumulative according to REACH criteria. Up to six 4-MBC biotransformation products (BTPs)were identified, 2 of them for the first time. Transcriptomic analysis revealed between 658 and 1310 differently expressed genes (DEGs) after 4-MBC exposure. Functional and enrichment analysis of the DEGs showed the activation of the detoxification pathway to metabolize and excrete the bioconcentrated 4-MBC, which also involved energy depletion and caused an impact on the metabolism of carbohydrates and lipids and in the oxidative phosphorylation pathways. Oxidative stress and immune response were also evidenced through the activation of cathepsins and the complement system. Such elucidation of the mode of action of a ubiquitous pollutant such as 4-MBC at the molecular level is valuable both from an environmental point of view and for the sustainable production of Manila clam, one of the most cultivated mollusk species worldwide.

Evaluation of variable new antigen receptors (vNARs) as a novel cathepsin S (CTSS) targeting strategy.

Aberrant activity of the cysteine protease Cathepsin S (CTSS) has been implicated across a wide range of pathologies. Notably in cancer, CTSS has been shown to promote tumour progression, primarily through facilitating invasion and migration of tumour cells and augmenting angiogenesis. Whilst an attractive therapeutic target, more efficacious CTSS inhibitors are required. Here, we investigated the potential application of Variable New Antigen Receptors (vNARs) as a novel inhibitory strategy. A panel of potential vNAR binders were identified following a phage display panning process against human recombinant proCTSS. These were subsequently expressed, purified and binding affinity confirmed by ELISA and SPR based approaches. Selected lead clones were taken forward and were shown to inhibit CTSS activity in recombinant enzyme activity assays. Further assessment demonstrated that our lead clones functioned by a novel inhibitory mechanism, by preventing the activation of proCTSS to the mature enzyme. Moreover, using an intrabody approach, we exhibited the ability to express these clones intracellularly and inhibit CTSS activity whilst lead clones were also noted to impede cell invasion in a tumour cell invasion assay. Collectively, these findings illustrate a novel mechanistic approach for inhibiting CTSS activity, with anti-CTSS vNAR clones possessing therapeutic potential in combating deleterious CTSS activity. Furthermore, this study exemplifies the potential of vNARs in targeting intracellular proteins, opening a range of previously "undruggable" targets for biologic-based therapy.

Investigating how CSTB affects the activity of cysteine cathepsin in the AD‐DS, EOAD, and human cellular models of trisomy Hsa21

AbstractBackgroundPeople with Down syndrome (DS) have a high genetic risk of developing Alzheimer’s disease (AD) ‐related dementia and pathology. Triplication of a human chromosome 21 (Hsa21)‐located gene APP play an important role in AD pathology. Wiseman et al. demonstrated that triplication of a gene or genes on Hsa21, other than APP exacerbates amyloid‐β pathology in an APP transgenic mouse model. Multiple lines of evidence indicate that cysteine cathepsin activity influences APP cleavage and Aβ accumulation. Cystine cathepsins are predominantly expressed in microglia in the brain and are involved in key neuroinflammation pathways. Hsa21‐located gene CYSTATIN B (CSTB) is an endogenous inhibitor of cathepsin proteases. We hypothesise that three copies of CSTB inhibit cathepsin activity and that this could contribute to altered neuroinflammation in people with DS.MethodHuman fibroblasts from individuals with DS and euploid controls, temporal cortex samples from individuals who had AD‐DS and Braak‐stage matched euploid cases of early‐onset AD (EOAD) or healthy aging and human iPSC‐derived microglia‐like cells (iMLCs: T21 and an isogenic control) were used. Western blot and immunocytochemistry were conducted to determine the abundance and subcellular localisation of CSTB. Cathepsin B (CatB) activity in human fibroblasts and temporal cortex samples was measured using an enzymatic assay (Abcam) and/or a pan‐cysteine cathepsin quenched activity‐based probe (BMV109).ResultWestern blotting confirmed that trisomy of Hsa21 resulted in an increase in the protein level of CSTB, that endogenous CSTB is predominantly localised in the cytoplasm and that its subcellular localisation is not altered by trisomy of Hsa21. Significantly lower CatB activity occurs in the temporal cortex of people who had AD‐DS compared with individuals who had EOAD. However, CatB enzymatic activity was not significantly altered in Hsa21 trisomic fibroblasts.ConclusionNext, we will determine in iMLCs, whether trisomy of chromosome 21 alters CatB activity and which genes have altered expression at baseline and after neuroinflammatory challenge.

Loss of Progranulin Results in Increased Pan‐Cathepsin Expression

AbstractBackgroundMutations in the progranulin (PGRN) encoding gene, GRN, cause familial frontotemporal dementia (FTD) and neuronal ceroid lipofuscinosis and is also implicated in Parkinson’s disease. These mutations result in decreased PGRN expression. PGRN is highly expressed in peripheral immune cells and microglia and regulates cell growth, survival, repair, and inflammation. As well, PGRN is implicated in regulating lysosome function, however, the exact role of PGRN in lysosomal function and how this contributes to inflammation and degeneration is not entirely understood. To better understand the role of PGRN in regulating lysosome function, I examined how loss of GRN impacts lysosomal and cathepsin activity.MethodUsing mouse embryonic fibroblasts (MEFs), I performed immunocytochemistry and immunoblotting assays to analyze fluorescent signal from LAMP1 (lysosomal marker) and BMV109 (marker for cathepsin activity).ResultGRN‐/‐ MEFs exhibit increased expression of pan‐cathepsin activity relative to GRN+/+ MEFs, and significantly impacts expression of LAMP1.ConclusionThe significant increase in pan‐cathepsin activity in the GRN‐/‐ MEFs confirms that PGRN loss does alter cathepsin expression, which may be a result of compensatory mechanisms happening within the cell. Further investigations will include assessing LAMP1 and BMV109 expression in microglia from from GRN‐/‐ mice, in the hopes to understand the role of PGRN in lysosomal function in immune cells of the central nervous system and the diseases in which it is implicated.

Targeted screening of baicalein inhibitor with controlling lipid and protein oxidation for quality preservation of grass carp fillets

In this study, targeted screening of candidate polyphenols was conducted by docking methods and inhibition assay. Baicalein (BCL) was identified as the target polyphenol, with IC50 value and docking score of 27.16 μmol/L and −7 kcal/mol, respectively. During the chilled storage of grass carp fillets at 4 °C, baicalein has been shown to reduce the degree of lipid oxidation by inhibiting the activity of lipase and lipoxygenase effectively and enhancing the activity of antioxidant enzymes, which is manifested by a decrease in thiobarbituric acid values and peroxide value. Baicalein has also been proved to have the ability to reduce protein oxidation by decreasing carbonyl content and sulfhydryl content of grass carp protein. Furthermore, baicalein can significantly eliminate the degree of protein hydrolysis of grass carp fillets by regulating the activity of cathepsin B and cathepsin L. The results suggest that baicalein expects to become a potential natural fresh-keeping agent for aquatic product industry and provides some new insights into understanding of the mechanism of fish quality preservation.

Stable chlorine dioxide combined with slightly acidic electrolyzed water: an effective way to delay protein deterioration of refrigerated large yellow croaker (Pseudosciaena crocea)

SummaryFish is susceptible to spoilage for enzymatic and microbial activities during storage, proteolysis of MPs by endogenous proteases played a crucial influence in the early postmortem decomposition of myosin. This study aimed to investigate the impact of stable chlorine dioxide (ClO2) combined with slightly acidic electrolyzed water (SAEW) on myofibrillar proteins (MPs) and endogenous proteases alterations in large yellow croaker (Pseudosciaena crocea) during cold storage. The results of Fourier‐transform infrared spectroscopy (FT‐IR) and intrinsic fluorescence intensity (IFI) showed that the ClO2 + SAEW treatment kept the structural stability of protein in fillets during cold storage. Moreover, ClO2 + SAEW treatment dramatically inhibited protein degradation and oxidation. The results of light microscopy further confirmed the results. Meanwhile, there was a tendency to first increase and then decrease on the activities of calpain and cathepsin B, L. According to the correlation analysis and principal component analysis, the activities of endogenous proteases (cathepsin B, D, L, and calpain) were significant (P < 0.01) positively correlated to myofibrillar fragmentation index and trichloroacetic acid‐soluble peptide, and were significant (P < 0.01) negative correlated with the contents of total sulfhydryl and Ca2+‐ATPase. The endogenous proteases activities and protein deterioration were strongly associated. Therefore, endogenous protease was affected by ClO2 and SAEW, which delayed protein degradation in large yellow croaker, especially ClO2 combined with SAEW treatment.

Lysosomal Membrane Permeabilization Sensitizes Ctss-Hyperactive Tumors to BCL2-Targeting Therapies

Hyperactivity of the cysteine protease cathepsin S (CTSS) -either through Y132 mutations or amplification/overexpression- is a recurrent alteration in follicular lymphoma (FL) and promotes tumor growth by inducing a supportive immune microenvironment (Bararia et al, 2020). Of note, patients with CTSS-hyperactive FL had better outcomes with standard therapies, suggesting that CTSS-hyperactivity can sensitize tumors to treatment. CTSS hyperactivity has also been reported in other B cell lymphomas (BCLs) (Dheilly et al, 2020) and solid cancers (Olson & Joyce, 2015). CTSS is mainly localized intralysosomally but can be released into the cytosol by lysosomal membrane permeabilization (LMP). Low level LMP can occur spontaneously (e.g., during cell division) and can be enhanced by treatment. Unlike other cathepsins, cytosolically released CTSS maintains its enzymatic activity at non-acidic pH. Thus, we aimed to (i) identify the determinants of the cytosolic CTSS activity, (ii) determine its impact on the regulation of apoptosis, and (iii) study LMP as a therapeutic approach for CTSS-hyperactive tumors. First, we accrued biochemical, functional, and clinical data supporting the role of cystatin B (CSTB) as a clinically relevant endogenous CTSS inhibitor in BCLs. Through unbiased and complementary proteomics (BioID2 labelling and co-IP followed by LC-MS/MS) we identified CSTB as a direct CTSS-interacting protein (8-fold enriched in the BCL cell line Karpas422 engineered to express CTSS wild type (WT) or Y132D vs CTSS knock-out (KO), P=0.0002). Single-cell RNA-Seq of primary FL (N=10) showed significantly higher CSTB expression in FL cells compared to normal B cells ( P=0.004). Moreover, shRNA mediated knock-down (k/d) of CSTB increased the overall cathepsin activity in BCL cell lines (N=8) by 1.5 to 5.5-fold, most notably in CTSS-hyperactive cells ( Fig A, top). We next employed LMP-inducing tool compounds (LLOMe) and clinically used drugs or analogs (desipramine, hexamethylene amiloride) to release cathepsins into the cytosol. CTSS-hyperactive Karpas422 were significantly more sensitive to LMP-inducing treatments compared to native cells (1.5 to 10-fold reduction of IC50). Importantly, CTSS hyperactivity and CSTB k/d increased LMP-mediated cell killing ( Fig A, bottom). Thus, the cytosolic CTSS/CSTB interaction determines the net cytosolic cathepsin activity and sensitivity of cells to undergo LMP-induced cell death. Next, we hypothesized that LMP-induced cytosolic CTSS hyperactivity could prime BCLs towards apoptosis. We used BH3 profiling to functionally quantify the dependencies and interactions of BCL2 family members in BCLs with and without CTSS hyperactivity. In Karpas422 cells expressing CTSS Y132D, LMP increased overall apoptotic priming and dependencies on the anti-apoptotic proteins MCL-1 (delta priming >30 % at 10 µM, P=0.04), BCL-xL (>45 % at 10 µM, P=0.0006) and BCL2 (> 45 % at 0.5 and 1 µM, P=0.0001). We hypothesized that BCL2 family members are proteolytically cleaved by cytosolic CTSS. Indeed, e.g., BCL2 protein levels were 2.5 to 3.5-fold lower in LLOMe-treated Karpas422 cells with CTSS-hyperactivity compared to CTSS KO, and CSTB k/d further decreased BCL2 levels. To validate CTSS-mediated cleavage of BCL2, we purified FLAG-tagged BCL2 and CTSS WT and Y132D. CTSS WT efficiently cleaved BCL2 in vitro <1 hour at the top ranked predicted cleavage site and the reaction rate increased 1.3-fold for CTSS Y132D. Finally, we hypothesized that LMP sensitizes cells to BCL2-targeting therapies ( Fig B). The combination of LLOMe-induced LMP and the BCL2 inhibitor venetoclax (VEN) showed increased cytotoxicity in CTSS-hyperactive Karpas422 cells compared to monotherapy and CSTB k/d enhanced this phenotype ( Fig A, bottom). We assessed cathepsin activities and generated dose-response curves for VEN with and without LLOMe-induced LMP in 15 primary CLL samples. Thereof, 12 samples had intermediate or high cathepsin activities and LLOMe-induced LMP increased their sensitivity to VEN, including a VEN-resistant sample in which the IC50 decreased to <3 nM. In summary, we show that CSTB is a functionally relevant inhibitor that determines the net activity of LMP-released cytosolic CTSS. Furthermore, LMP-inducing therapies may be a promising approach to sensitize CTSS-hyperactive tumors towards apoptosis by proteolytic cleavage of BCL2 family members.

Qufeng tongluo decoction decreased proteinuria in diabetic mice by protecting podocytes via promoting autophagy

BackgroundDiabetic kidney disease (DKD) is one of diabetic complications, which has become the leading cause of end-stage kidney disease. In addition to angiotensin-converting enzyme inhibitor/angiotensin II receptor blocker(ACEI/ARB) and sodium-glucose cotransporter-2 inhibitor (SGLT2i), traditional Chinese medicine (TCM) is an effective alternative treatment for DKD. In this study, the effect of Qufeng Tongluo (QFTL) decoction in decreasing proteinuria has been observed and its mechanism has been explored based on autophagy regulation in podocyte. MethodsIn vivo study, db/db mice were used as diabetes model and db/m mice as blank control. Db/db mice were treated with QFTL decoction, rapamycin, QFTL + 3-Methyladenine (3-MA), trehalose, chloroquine (CQ) and QFTL + CQ. Mice urinary albumin/creatinine (UACR), nephrin and autophagy related proteins (LC3 and p62) in kidney tissue were detected after intervention of 9 weeks. Transcriptomics was operated with the kidney tissue from model group and QFTL group. In vitro study, mouse podocyte clone-5 (MPC-5) cells were stimulated with hyperglycemic media (30 mmol/L glucose) or cultured with normal media. High-glucose-stimulated MPC-5 cells were treated with QFTL freeze-drying powder, rapamycin, CQ, trehalose, QFTL+3-MA and QFTL + CQ. Cytoskeletal actin, nephrin, ATG-5, ATG-7, Beclin-1, cathepsin L and cathepsin B were assessed. mRFP-GFP-LC3 was established by stubRFP-sensGFP-LC3 lentivirus transfection. ResultsQFTL decoction decreased the UACR and increased the nephrin level in kidney tissue and high-glucose-stimulated podocytes. Autophagy inhibitors, including 3-MA and chloroquine blocked the effects of QFTL decoction. Further study showed that QFTL decoction increased the LC3 expression and relieved p62 accumulation in podocytes of db/db mice. In high-glucose-stimulated MPC-5 cells, QFTL decoction rescued the inhibited LC3 and promoted the expression of ATG-5, ATG-7, and Beclin-1, while had no effect on the activity of cathepsin L and cathepsin B. Results of transcriptomics also showed that 51 autophagy related genes were regulated by QFTL decoction, including the genes of ATG10, SCOC, ATG4C, AMPK catalytic subunit, PI3K catalytic subunit, ATG3 and DRAM2. ConclusionQFTL decoction decreased proteinuria and protected podocytes in db/db mice by regulating autophagy.

Downregulation of TRPC6 regulates ERK1/2 to prevent sublytic C5b‑9 complement complex‑induced podocyte injury through activating autophagy.

Idiopathic membranous nephropathy (IMN) is a common glomerular disease, in which 50-60% of patients can progress to end-stage renal disease within 10-20 years, seriously endangering human health. Podocyte injury is the direct cause of IMN. Sublytic C5b-9 complement complex induces damage in podocytes' structure and function. In sublytic C5b-9 treated podocytes, the expression of canonical transient receptor potential 6 (TRPC6) is increased. However, the specific mechanism of TRPC6 in sublytic C5b-9 treated podocytes is unclear. The present study aimed to reveal the effect and mechanism of TRPC6 on sublytic C5b-9-induced podocytes. Normal human serum was stimulated using zymosan to form C5b-9. A lactate dehydrogenase release assay was used to examine C5b-9 cytotoxicity in podocytes. The RNA and protein expression levels were analyzed using reverse transcription-quantitative PCR, western blotting and immunofluorescent assay, respectively. Cell Counting Kit-8 assay and flow cytometry were carried out to test the viability and apoptosis of podocytes, respectively. Transmission electron microscopy was used to observe autophagic vacuole. F-actin was tested through phalloidin staining. Sublytic C5b-9 was deposited and TRPC6 expression was boosted in podocytes stimulated through zymosan activation serum. Knockdown of TRPC6 raised the viability and reduced the apoptosis rate of sublytic C5b-9-induced podocytes. Meanwhile, transfection of small-interfering (si)TRPC6 facilitated autophagy progression and enhanced the activation of cathepsin B/L in sublytic C5b-9-induced podocytes. The phosphorylation level of ERK1/2 was receded in siTRPC6 and sublytic C5b-9 co-treated podocytes. Moreover, the addition of the ERK1/2 activator partially reversed the effect of TRPC6 inhibition on sublytic C5b-9-induced podocytes. TRPC6 knockdown reduced the damage of sublytic C5b-9 to podocytes by weakening the ERK1/2 phosphorylation level to activate autophagy. These results indicated that targeting TRPC6 reduced the injury of sublytic C5b-9 on podocytes.

IFNT stimulates lysosomal function via type I IFN signaling pathway in pregnant bovine leukocytes.

Interferon tau (IFNT) is important in establishing pregnancy in ruminants. Secreted IFNT in the uterus induces the expression of an interferon-stimulated gene (ISG) in uterine tissues and peripheral blood leukocytes (PBLs). In our previous study, increased lysosome and lysosomal cathepsin (CTS) activity and mRNA expression were observed in PBLs of pregnant cows on day 18 of pregnancy. However, the mechanism of IFNT stimulation in PBLs is unclear. Here, we explored the IFNT-mediated lysosomal activation mechanisms in PBLs during early pregnancy in dairy cows. PBLs collected from the peripheral blood of Holstein cows on day 18 post-artificial insemination (AI), after confirmation of their pregnancy status, were used to detect the expression of lysosomal-associated membrane protein (LAMP) 1, 2, CTSB and CTSK. Expression of all genes was significantly higher in PBLs of pregnant cows than in non-pregnant cows. In vitro IFN-mediated stimulation of PBLs collected from cows that did not undergo AI significantly increased lysosomal acidification and expression of LAMP1 and 2, as well as the activities of CTSB and CTSK. Immunodetection analysis showed an increase in LAMP1 and CTSK levels in the PBLs of day 18 pregnant cows. JAK inhibitor significantly decreased lysosomal acidification, CTSK activity, LAMP1, 2, and CTSK expression in the presence of IFNT. These results suggest that IFNT regulates lysosomal function via a type 1IFN-mediated pathway in PBLs during pregnancy recognition.

Loss of Progranulin Results in Increased Pan-Cathepsin Activity

Mutations in the progranulin (PGRN) encoding gene, GRN, cause familial frontotemporal dementia (FTD) and neuronal ceroid lipofuscinosis (NCL) and PGRN is also implicated in Parkinson’s disease (PD). These mutations result in decreased PGRN expression. PGRN is highly expressed in peripheral immune cells and microglia and regulates cell growth, survival, repair, and inflammation. As well, PGRN is implicated in regulating lysosome function, however, the exact role of PGRN in lysosomal function and how this contributes to inflammation and degeneration is not entirely understood. To better understand the role of PGRN in regulating lysosome function, this study examined how loss of GRN impacts lysosomal and cathepsin activity. Using mouse embryonic fibroblasts (MEFs), immunocytochemistry and immunoblotting assays were performed to analyze fluorescent signal from LAMP1 (lysosomal marker) and BMV109 (marker for pan-cathepsin activity). GRN-/- MEFs exhibit increased expression of pan-cathepsin activity relative to GRN+/+ MEFs, and significantly impacts expression of LAMP1. The significant increase in pan-cathepsin activity in the GRN-/- MEFs confirms that PGRN loss does alter cathepsin expression, which may be a result of compensatory mechanisms happening within the cell. Further investigations will include assessing LAMP1 and BMV109 expression in microglia from GRN-/- mice, in the hopes of understanding the role of PGRN in lysosomal function in immune cells of the central nervous system and the diseases in which it is implicated.

Does reduced cathepsin activity contribute to skin and lung fibrosis in patients with systemic sclerosis and related disorders?

Does reduced cathepsin activity contribute to skin and lung fibrosis in patients with systemic sclerosis and related disorders?

Evolutionary Adaptation of Protein Turnover in White Muscle of Stenothermal Antarctic Fish: Elevated Cold Compensation at Reduced Thermal Responsiveness.

Protein turnover is highly energy consuming and overall relates to an organism's growth performance varying largely between species, e.g., due to pre-adaptation to environmental characteristics such as temperature. Here, we determined protein synthesis rates and capacity of protein degradation in white muscle of the cold stenothermal Antarctic eelpout (Pachycara brachycephalum) and its closely related temperate counterpart, the eurythermal common eelpout (Zoarces viviparus). Both species were exposed to acute warming (P. brachycephalum, 0 °C + 2 °C day-1; Z. viviparus, 4 °C + 3 °C day-1). The in vivo protein synthesis rate (Ks) was monitored after injection of 13C-phenylalanine, and protein degradation capacity was quantified by measuring the activity of cathepsin D in vitro. Untargeted metabolic profiling by nuclear magnetic resonance (NMR) spectroscopy was used to identify the metabolic processes involved. Independent of temperature, the protein synthesis rate was higher in P. brachycephalum (Ks = 0.38-0.614 % day-1) than in Z. viviparus (Ks= 0.148-0.379% day-1). Whereas protein synthesis remained unaffected by temperature in the Antarctic species, protein synthesis in Z. viviparus increased to near the thermal optimum (16 °C) and tended to fall at higher temperatures. Most strikingly, capacities for protein degradation were about ten times higher in the Antarctic compared to the temperate species. These differences are mirrored in the metabolic profiles, with significantly higher levels of complex and essential amino acids in the free cytosolic pool of the Antarctic congener. Together, the results clearly indicate a highly cold-compensated protein turnover in the Antarctic eelpout compared to its temperate confamilial. Constant versus variable environments are mirrored in rigid versus plastic functional responses of the protein synthesis machinery.

The Golgi stacking protein GRASP55 is targeted by the natural compound prodigiosin

BackgroundThe bacterial secondary metabolite prodigiosin has been shown to exert anticancer, antimalarial, antibacterial and immunomodulatory properties. With regard to cancer, it has been reported to affect cancer cells but not non-malignant cells, rendering prodigiosin a promising lead compound for anticancer drug discovery. However, a direct protein target has not yet been experimentally identified.MethodsWe used mass spectrometry-based thermal proteome profiling in order to identify target proteins of prodigiosin. For target validation, we employed a genetic knockout approach and electron microscopy.ResultsWe identified the Golgi stacking protein GRASP55 as target protein of prodigiosin. We show that prodigiosin treatment severely affects Golgi morphology and functionality, and that prodigiosin-dependent cytotoxicity is partially reduced in GRASP55 knockout cells. We also found that prodigiosin treatment results in decreased cathepsin activity and overall blocks autophagic flux, whereas co-localization of the autophagosomal marker LC3 and the lysosomal marker LAMP1 is clearly promoted. Finally, we observed that autophagosomes accumulate at GRASP55-positive structures, pointing towards an involvement of an altered Golgi function in the autophagy-inhibitory effect of this natural compound.ConclusionTaken together, we propose that prodigiosin affects autophagy and Golgi apparatus integrity in an interlinked mode of action involving the regulation of organelle alkalization and the Golgi stacking protein GRASP55.8brCwiZ4E3PVMoFvaLMguTVideo

Thiol Cathepsins and Oxidative Modification of Stallion’s Seminal Plasma Proteins with Normal and Low Percentage of Live Spermatozoa Post Cryopreservation

Background: Stallion sperms are very sensitive to cryopreservation. Free radicals generated during cryopreservation lead to spermatozoa membrane damage, decline in their viability and change the activity of cysteine cathepsins. On the other hand, cathepsins may involve in the utilization of oxidatively modified proteins. The present study was conducted to assess cysteine cathepsins activity and level of oxidatively modified proteins in seminal plasma of stallions with a low ( less than 30%) and normal ( greater than 30%) percentage of live spermatozoa post cryopreservation Methods: A total of 33 breeding stallions comprising 6 stallions of the Budennovskaya breed, 22 Arabian breed, 4 Soviet Heavy-Draft and 1 Bashkir breed. We assessed standard quality parameters in fresh and cryopreserved sperm, as well as cysteine cathepsins B,L,H activity, the level of oxidatively modified proteins in stallions seminal plasma with a low ( less than 30%) and normal ( greater than 30%) percentage of live spermatozoa post cryopreservation. Result: The group with a low percentage of live spermatozoa was characterized by decreased sperm quality, increased level of aldehyde dinitrophenylhydrazones (p=0.045) and ketone dinitrophenylhydrazones (p=0.049) of a neutral nature, as well as increased activity of cathepsin L (p=0.029). Evaluation of activity of cathepsin L and the concentration of oxidatively modified proteins in the seminal plasma can serve as a marker in the selection of stallions for sperm cryopreservation.

Toxoplasma gondii Induces Pyroptosis in Human Placental Trophoblast and Amniotic Cells by Inducing ROS Production and Activation of Cathepsin B and NLRP1/NLRP3/NLRC4/AIM2 Inflammasome

Toxoplasma gondii infection in pregnant women may cause fetal anomalies; however, the underlying mechanisms remain unclear. The current study investigated whether T. gondii induces pyroptosis in human placental cells and the underlying mechanisms. Human placental trophoblast (BeWo and HTR-8/SVneo) and amniotic (WISH) cells were infected with T. gondii, and then reactive oxygen species (ROS) production, cathepsin B (CatB) release, inflammasome activation, and pyroptosis induction were evaluated. The molecular mechanisms of these effects were investigated by treating the cells with ROS scavengers, a CatB inhibitor, or inflammasome-specific siRNA. T. gondii infection induced ROS generation and CatB release into the cytosol in placental cells but decreased mitochondrial membrane potential. T. gondii-infected human placental cells and villi exhibited NLRP1, NLRP3, NLRC4, and AIM2 inflammasome activation and subsequent pyroptosis induction, as evidenced by increased expression of ASC, cleaved caspase-1, and mature IL-1β and gasdermin D cleavage. In addition to inflammasome activation and pyroptosis induction, adverse pregnancy outcome was shown in a T. gondii-infected pregnant mouse model. Administration of ROS scavengers, CatB inhibitor, or inflammasome-specific siRNA into T. gondii-infected cells reversed these effects. Collectively, these findings show that T. gondii induces NLRP1/NLRP3/NLRC4/AIM2 inflammasome-dependent caspase-1-mediated pyroptosis via induction of ROS production and CatB activation in placental cells. This mechanism may play an important role in inducing cell injury in congenital toxoplasmosis.

PH-gated nanoparticles selectively regulate lysosomal function of tumour-associated macrophages for cancer immunotherapy

Tumour-associated macrophages (TAMs), as one of the most abundant tumour-infiltrating immune cells, play a pivotal role in tumour antigen clearance and immune suppression. M2-like TAMs present a heightened lysosomal acidity and protease activity, limiting an effective antigen cross-presentation. How to selectively reprogram M2-like TAMs to reinvigorate anti-tumour immune responses is challenging. Here, we report a pH-gated nanoadjuvant (PGN) that selectively targets the lysosomes of M2-like TAMs in tumours rather than the corresponding organelles from macrophages in healthy tissues. Enabled by the PGN nanotechnology, M2-like TAMs are specifically switched to a M1-like phenotype with attenuated lysosomal acidity and cathepsin activity for improved antigen cross-presentation, thus eliciting adaptive immune response and sustained tumour regression in tumour-bearing female mice. Our findings provide insights into how to specifically regulate lysosomal function of TAMs for efficient cancer immunotherapy.

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 2-6h 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.

Katepsin B İnhibitörü olan CA074'ün Serebral İskemi Modelinde Apoptoz ve Hücre Ölümü Üzerine Etkisi

Lysosomes and cathepsins, the most common hydrolytic enzymes in lysosomes, are available in the different models of cell death as necrosis and apoptosis. This study investigated the effect of cathepsin B-selective inhibitor CA-074 on apoptotic and necrotic neuronal cell death. Focal cerebral ischemia which has been formed by occlusion of the three-vessel consisting permanent middle cerebral artery occlusion and temporary bilateral common carotid artery occlusion for 60 minutes was selected as ischemia model. Two sets of rats were used in this study. The rats in the first set were used formeasurement of sulfhydryl groups in the lysosomal membrane, lysosomal integrity, cathepsins B and L activities and caspase-3 activity. The rats in the second set were used as histological study including "hematoxylin and eosin" for the detection of necrotic neuronal deathand "TUNEL" staining for the detection of apoptotic neuronal death. 4 mg/kg CA-074 was administered intravenouslyin the treatment group. CA-074 has substantially reduced levels of cathepsins B and L compared to ischemia and solvent groups (respectively, p<0.05 and p<0.01). Similarly, CA-074 has reduced increase in caspase-3 activity compared to ischemia and solvent groups (p<0.05). While the number of eosinophilic (necrotic) and apoptotic neurons has highly increased in post-ischemic cerebral tissue in middle cerebral artery feeding area (p<0.001), CA-074 could only reduce significantly the number of apoptotic neurons (p<0.05). CA-074 has reduced apoptotic neuronal death by inhibiting caspase and cathepsin activity. It may be useful that CA074 is used with other therapeutic drugs in stroke patients.