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Chronic inflammation in adipose tissue is associated with metabolic disorders such as obesity and type 2 diabetes. Novel small molecules targeting adipocyte differentiation and fat accumulation offer potential for new anti-inflammatory and anti-obesity drugs. Here we show that the marine cyclic heptapeptide stylissatin A and its analogs (SAs) inhibit membranous neuraminidase 1 (Neu1) function by interacting with lysosomal protective protein cathepsin A (PPCA). Neu1 has been less explored as a therapeutic target due to the genetic defects leading to neurodegenerative disorders. However, unlike traditional neuraminidase inhibitors, SAs don't directly bind to Neu1 but modulate the molecular chaperone activity of PPCA. SAs caused degradation of perilipin 1 around lipid droplets and inhibited fat accumulation, along with decrease in membranous Neu1. Molecular docking and molecular dynamics simulations revealed that SAs interacted with activated PPCA at the Neu1 binding site. Focusing on this newfound protein-protein interaction inhibition mechanism could lead to the development of pharmaceuticals with fewer side effects., (© 2024 Wiley-VCH GmbH.)

We assessed factors that determine the tissue-specific bioactivation of ProTide prodrugs by comparing the disposition and activation of remdesivir (RDV), its methylpropyl and isopropyl ester analogues (MeRDV and IsoRDV, respectively), the oral prodrug GS-621763, and the parent nucleotide GS-441524 (Nuc). RDV and MeRDV yielded more active metabolite remdesivir-triphosphate (RDV-TP) than IsoRDV, GS-621763, and Nuc in human lung cell models due to superior cell permeability and higher susceptivity to cathepsin A. Intravenous administration to mice showed that RDV and MeRDV delivered significantly more RDV-TP to the lung than other compounds. Nevertheless, all four ester prodrugs exhibited very low oral bioavailability (<2%), with Nuc being the predominant metabolite in blood. In conclusion, ProTides prodrugs, such as RDV and MeRDV, are more efficient in delivering active metabolites to the lung than Nuc, driven by high cell permeability and susceptivity to cathepsin A. Optimizing ProTides' ester structures is an effective strategy for enhancing prodrug activation in the lung.

Patients with sialidosis (mucolipidosis type I) type I typically present with myoclonus, seizures, ataxia, cherry-red spots, and blindness because of mutations in the neuraminidase 1 (NEU1) gene. Currently, there is no treatment for sialidosis. In this study, we developed an adeno-associated virus (AAV)-mediated gene therapy for a Neu1 knockout (Neu1 -/- ) mouse model of sialidosis. The vector, AAV9-P3-NP, included the human NEU1 promoter, NEU1 cDNA, IRES, and CTSA cDNA. Untreated Neu1 -/- mice showed astrogliosis and microglial LAMP1 accumulation in the nervous system, including brain, spinal cord, and dorsal root ganglion, together with impaired motor function. Coexpression of NEU1 and protective protein/cathepsin A (PPCA) in neonatal Neu1 -/- mice by intracerebroventricular injection, and less effective by facial vein injection, decreased astrogliosis and LAMP1 accumulation in the nervous system and improved rotarod performance of the treated mice. Facial vein injection also improved the grip strength and survival of Neu1 -/- mice. Therefore, cerebrospinal fluid delivery of AAV9-P3-NP, which corrects the neurological deficits of mice with sialidosis, could be a suitable treatment for patients with sialidosis type I. After intracerebroventricular or facial vein injection of AAV vectors, NEU1 and PPCA are expressed together. PPCA-protected NEU1 is then sent to lysosomes, where β-Gal binds to this complex to form a multienzyme complex in order to execute its function., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

A comprehensive analysis of the prognostic, diagnostic, and biological significance of miR-148a-3p and cathepsin A (CTSA) in hepatocellular carcinoma (HCC) was performed using bioinformatics algorithms with The Cancer Genome Atlas (TCGA) data. miR-148a-3p and CTSA gene expression in HCC tissues and nontumor specimens was analyzed using TCGA database with R software. CTSA staining analysis was validated using the Human Protein Atlas database. Prognostic, diagnostic, gene set enrichment, Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and immune infiltration analyses were implemented using the TCGA database with R software. Based on TCGA data and our cohort populations, CTSA expression was significantly elevated in HCC tissues compared with nontumor specimens. A significant negative correlation between miR-148a-3p and CTSA was observed in the TCGA data and our cohort population. Mechanistically, CTSA was a direct gene target of miR-148a-3p. Both CTSA and miR-148a-3p could serve as prognostic and diagnostic indicators in HCC. miR-148a-3p expression was significantly and negatively correlated with the StromalScore, ImmuneScore, and ESTIMATEScore in patients with liver cancer. miR-148a-3p mimic-mediated apoptosis and the inhibition of HCC cell growth and migration were counteracted by CTSA overexpression. The miR-148a-3p/CTSA axis was implicated in immune cell infiltration and carcinogenesis of HCC. miR-148a-3p and CTSA might be prospective molecular targets to enhance the potency of immunotherapy in HCC., (© 2022 Wiley Periodicals LLC.)

Carboxypeptidases enzymatically cleave the peptide bond of C-terminal amino acids. In humans, it is involved in enzymatic synthesis and maturation of proteins and peptides. Carboxypeptidases A and Y have difficulty hydrolyzing the peptide bond of dipeptides and some other amino acid sequences. Early investigations into different N-blocking groups concluded that larger moieties increased substrate susceptibility to peptide bond hydrolysis with carboxypeptidases. This study conclusively demonstrates that 6-aminoquinoline-N-hydroxysuccimidyl carbamate (AQC) as an N-blocking group greatly enhances substrate hydrolysis with carboxypeptidase. AQC addition to the N-terminus of amino acids and peptides also improves chromatographic peak shapes and sensitivities via mass spectrometry detection. These enzymes have been used for amino acid sequence determination prior to the advent of modern proteomics. However, most modern proteomic methods assume that all peptides are comprised of l-amino acids and therefore cannot distinguish L-from d-amino acids within the peptide sequence. The majority of existing methods that allow for chiral differentiation either require synthetic standards or incur racemization in the process. This study highlights the resistance of d-amino acids within peptides to enzymatic hydrolysis by Carboxypeptidase Y. This stereoselectivity may be advantageous when screening for low abundance peptide stereoisomers., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The prodrug tenofovir alafenamide (TAF) is a first-line antiviral agent for the treatment of chronic hepatitis B infection. TAF activation involves multiple steps, and the first step is an ester hydrolysis reaction catalyzed by hydrolases. This study was to determine the contributions of carboxylesterase 1 (CES1) and cathepsin A (CatA) to TAF hydrolysis in the human liver. Our in vitro incubation studies showed that both CatA and CES1 catalyzed TAF hydrolysis in a pH-dependent manner. At their physiologic pH environment, the activity of CatA (pH 5.2) was approximately 1,000-fold higher than that of CES1 (pH 7.2). Given that the hepatic protein expression of CatA was approximately 200-fold lower than that of CES1, the contribution of CatA to TAF hydrolysis in the human liver was estimated to be much greater than that of CES1, which is contrary to the previous perception that CES1 is the primary hepatic enzyme hydrolyzing TAF. The findings were further supported by a TAF incubation study with the CatA inhibitor telaprevir and the CES1 inhibitor bis-(p-nitrophenyl) phosphate. Moreover, an in vitro study revealed that the CES1 variant G143E (rs71647871) is a loss-of-function variant for CES1-mediated TAF hydrolysis. In summary, our results suggest that CatA may play a more important role in the hepatic activation of TAF than CES1. Additionally, TAF activation in the liver could be affected by CES1 genetic variation, but the magnitude of impact appears to be limited due to the major contribution of CatA to hepatic TAF activation. SIGNIFICANCE STATEMENT: Contrary to the general perception that carboxylesterase 1 (CES1) is the major enzyme responsible for tenofovir alafenamide (TAF) hydrolysis in the human liver, the present study demonstrated that cathepsin A may play a more significant role in TAF hepatic hydrolysis. Furthermore, the CES1 variant G143E (rs71647871) was found to be a loss-of-function variant for CES1-mediated TAF hydrolysis., (Copyright © 2022 by The American Society for Pharmacology and Experimental Therapeutics.)

Remdesivir, an intravenous nucleotide prodrug, has been approved for treating COVID-19 in hospitalized adults and pediatric patients. Upon administration, remdesivir can be readily hydrolyzed to form its active form GS-441524, while the cleavage of the carboxylic ester into GS-704277 is the first step for remdesivir activation. This study aims to assign the key enzymes responsible for remdesivir hydrolysis in humans, as well as to investigate the kinetics of remdesivir hydrolysis in various enzyme sources. The results showed that remdesivir could be hydrolyzed to form GS-704277 in human plasma and the microsomes from human liver (HLMs), lung (HLuMs) and kidney (HKMs), while the hydrolytic rate of remdesivir in HLMs was the fastest. Chemical inhibition and reaction phenotyping assays suggested that human carboxylesterase 1 (hCES1A) played a predominant role in remdesivir hydrolysis, while cathepsin A (CTSA), acetylcholinesterase (AchE) and butyrylcholinesterase (BchE) contributed to a lesser extent. Enzymatic kinetic analyses demonstrated that remdesivir hydrolysis in hCES1A (SHUTCM) and HLMs showed similar kinetic plots and much closed K m values to each other. Meanwhile, GS-704277 formation rates were strongly correlated with the CES1A activities in HLM samples from different individual donors. Further investigation revealed that simvastatin (a therapeutic agent for adjuvant treating COVID-19) strongly inhibited remdesivir hydrolysis in both recombinant hCES1A and HLMs. Collectively, our findings reveal that hCES1A plays a predominant role in remdesivir hydrolysis in humans, which are very helpful for predicting inter-individual variability in response to remdesivir and for guiding the rational use of this anti-COVID-19 agent in clinical settings., (Copyright © 2021. Published by Elsevier B.V.)

Cathepsin A (CTSA) is a lysosomal protease that regulates galactoside metabolism. The previous study has shown CTSA is abnormally expressed in various types of cancer. However, rarely the previous study has addressed the role of CTSA in hepatocellular carcinoma (HCC) and its prognostic value. To study the clinical value and potential function of CTSA in HCC, datasets from the Cancer Genome Atlas (TCGA) database and a 136 HCC patient cohort were analyzed. CTSA expression was found to be significantly higher in HCC patients compared with normal liver tissues, which was supported by immunohistochemistry (IHC) validation. Both gene ontology (GO) and The Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses demonstrated that CTSA co-expressed genes were involved in ATP hydrolysis coupled proton transport, carbohydrate metabolic process, lysosome organization, oxidative phosphorylation, other glycan degradation, etc. Survival analysis showed a significant reduction both in overall survival (OS) and recurrence-free survival (RFS) of patients with high CTSA expression from both the TCGA HCC cohort and 136 patients with the HCC cohort. Furthermore, CTSA overexpression has diagnostic value in distinguishing between HCC and normal liver tissue [Area under curve (AUC) = 0.864]. Moreover, Gene set enrichment analysis (GSEA) showed that CTSA expression correlated with the oxidative phosphorylation, proteasome, and lysosome, etc. in HCC tissues. These findings demonstrate that CTSA may as a potential diagnostic and prognostic biomarker in HCC., (© 2021. The Author(s).)

Mouse embryonic stem cells (mESCs) are pluripotent cells that possess the ability to self-renew and differentiate into three germ layers. Owing to these characteristics, mESCs act as important models for stem cell research and are being used in many clinical applications. Among the many cathepsins, cathepsin A (Ctsa), a serine protease, affects the function and properties of stem cells. However, studies on the role of Ctsa in stem cells are limited. Here, we observed a significant increase in Ctsa expression during mESC differentiation at protein levels. Furthermore, we established Ctsa knockdown mESCs. Ctsa knockdown led to Erk1/2 phosphorylation, which in turn inhibited the pluripotency of mESCs and induced G2/M cell cycle arrest to inhibit mESC proliferation. The knockdown also induced abnormal differentiation in mESCs and aberrant expression of differentiation markers. Furthermore, we identified inhibition of teratoma formation in nude mice. Our results suggested that Ctsa affects mESC pluripotency, proliferation, cell cycle and differentiation, and highlighted the potential of Ctsa to act as a core factor that can regulate various mESC properties. SIGNIFICANCE OF THE STUDY: Our results indicate that cathepsin A (Ctsa) affects the properties of mESCs. Inhibition of Ctsa resulted in a decrease in the pluripotency of mouse embryonic stem cells (mESCs). Further, Ctsa suppression resulted in decreased proliferation via cell cycle arrest. Moreover, Ctsa inhibition reduced differentiation abilities and formation of teratoma in mESCs. Our results demonstrated that Ctsa is an important factor controlling mESC abilities., (© 2020 John Wiley & Sons Ltd.)

Proteins incorporating artificial moieties such as fluorophores and drugs have enjoyed increasing use in chemical biology and drug development research. Preparation of such artificial protein derivatives has relied mainly on native chemical ligation in which peptide/protein thioesters chemoselectively react with N-terminal cysteine (Cys) peptides to afford protein molecules. The protein thioesters derived from expressed proteins represent thioesters that are very useful for the preparation of artificial proteins by native chemical ligation with synthetic peptides with N-terminal Cys. We recently have developed a traceless thioester-producing protocol using carboxypeptidase Y (CPaseY) which is compatible with an expressed protein. The traceless character is ensured by CPaseY-mediated hydrazinolysis of C-terminal Xaa (X)-Cys-proline (Pro)-leucine (Leu)-OH sequence followed by an auto-processing of the Cys-Pro (CP) dipeptide unit, affording the corresponding X-thioester (X-SR). However, hydrazinolysis of the amide bond in the prolyl leucine junction depends significantly on the nature of X. In the case of hydrophobic X residues, the hydrazinolysis overreacts to give several hydrazides while the reaction of hydrophilic X residues proceeds slowly. In this research, we attempted to develop an X-independent CPaseY-mediated protocol and found that the incorporation of a triple CP sequence into the C-terminal end (X-(CP) 3 -Leu-OH) allows for efficient X-SR formation in a manner that is independent of X.

Neuraminidase (NEU)1 forms a multienzyme complex with beta-galactosidase (β-GAL) and protective-protein/cathepsin (PPC) A, which cleaves sialic-acids from cell surface glycoconjugates. We investigated the role of NEU1 in the myocardium after ischemia/reperfusion (I/R). Three days after inducing I/R, left ventricles (LV) of male mice (3 months-old) displayed upregulated neuraminidase activity and increased NEU1, β-GAL and PPCA expression. Mice hypomorphic for neu1 (hNEU1) had less neuraminidase activity, fewer pro-inflammatory (Lin - CD11b + F4/80 + Ly-6C high ), and more anti-inflammatory macrophages (Lin - CD11b + F4/80 + Ly-6C low ) 3 days after I/R, and less LV dysfunction 14 days after I/R. WT mice transplanted with hNEU1-bone marrow (BM) and hNEU1 mice with WT-BM showed significantly better LV function 14 days after I/R compared with WT mice with WT-BM. Mice with a cardiomyocyte-specific NEU1 overexpression displayed no difference in inflammation 3 days after I/R, but showed increased cardiomyocyte hypertrophy, reduced expression and mislocalization of Connexin-43 in gap junctions, and LV dysfunction despite a similar infarct scar size to WT mice 14 days after I/R. The upregulation of NEU1 after I/R contributes to heart failure by promoting inflammation in invading monocytes/macrophages, enhancing cardiomyocyte hypertrophy, and impairing gap junction function, suggesting that systemic NEU1 inhibition may reduce heart failure after I/R.

In the heart, the serine carboxypeptidase cathepsin A (CatA) is distributed between lysosomes and the extracellular matrix (ECM). CatA-mediated degradation of extracellular peptides may contribute to ECM remodeling and left ventricular (LV) dysfunction. Here, we aimed to evaluate the effects of CatA overexpression on LV remodeling. A proteomic analysis of the secretome of adult mouse cardiac fibroblasts upon digestion by CatA identified the extracellular antioxidant enzyme superoxide dismutase (EC-SOD) as a novel substrate of CatA, which decreased EC-SOD abundance 5-fold. In vitro , both cardiomyocytes and cardiac fibroblasts expressed and secreted CatA protein, and only cardiac fibroblasts expressed and secreted EC-SOD protein. Cardiomyocyte-specific CatA overexpression and increased CatA activity in the LV of transgenic mice (CatA-TG) reduced EC-SOD protein levels by 43%. Loss of EC-SOD-mediated antioxidative activity resulted in significant accumulation of superoxide radicals (WT, 4.54 μmol/mg tissue/min; CatA-TG, 8.62 μmol/mg tissue/min), increased inflammation, myocyte hypertrophy (WT, 19.8 μm; CatA-TG, 21.9 μm), cellular apoptosis, and elevated mRNA expression of hypertrophy-related and profibrotic marker genes, without affecting intracellular detoxifying proteins. In CatA-TG mice, LV interstitial fibrosis formation was enhanced by 19%, and the type I/type III collagen ratio was shifted toward higher abundance of collagen I fibers. Cardiac remodeling in CatA-TG was accompanied by an increased LV weight/body weight ratio and LV end diastolic volume (WT, 50.8 μl; CatA-TG, 61.9 μl). In conclusion, CatA-mediated EC-SOD reduction in the heart contributes to increased oxidative stress, myocyte hypertrophy, ECM remodeling, and inflammation, implicating CatA as a potential therapeutic target to prevent ventricular remodeling., Competing Interests: Conflict of interest—Sven Ruf, Thomas Hübschle, and Thorsten Sadowski are employees of Sanofi-Aventis Deutschland GmbH. The other authors have not conflicts of interest., (© 2020 Hohl et al.)

Prostate cancer has the highest incidence among men in advanced countries, as well as a high mortality rate. Despite the efforts of numerous researchers to identify a gene-based therapeutic target as an effective treatment of prostate cancer, there is still a need for further research. The cathepsin gene family is known to have a close correlation with various cancer types and is highly expressed across these cancer types. This study aimed at investigating the correlation between the cathepsin A (CTSA) gene and prostate cancer. Our findings indicated a significantly elevated level of CTSA gene expression in the tissues of patients with prostate cancer when compared with normal prostate tissues. Furthermore, the knockdown of the CTSA gene in the representative prostate cancer cell lines PC3 and DU145 led to reduced proliferation and a marked reduction in anchorage-independent colony formation, which was shown to be caused by cell cycle arrest in the S phase. In addition, CTSA gene-knockdown prostate cancer cell lines showed a substantial decrease in migration and invasion, as well as a decrease in the marker genes that promote epithelial mesenchymal transition (EMT). Such phenotypic changes in prostate cancer cell lines through CTSA gene suppression were found to be mainly caused by reduced p38 MAPK protein phosphorylation; i.e. the inactivation of the p38 MAPK cell signaling pathway. Tumorigenesis was also found to be inhibited in CTSA gene-knockdown prostate cancer cell lines when a xenograft assay was carried out using Balb/c nude mice, and the p38 MAPK phosphorylation was inhibited in tumor tissues. Thus, the CTSA gene is presumed to play a key role in human prostate cancer tissues through high-level expression, and the suppression of the CTSA gene leads to the inhibition of prostate cancer cell proliferation, colony formation, and metastasis. The mechanism, by which these effects occur, was demonstrated to be the inactivation of the p38 MAPK signaling pathway., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Post-translational modifications (PTMs) refer to the chemical modifications of proteins coordinated by PTM enzymes, and they play a key role in numerous physiological and pathological processes. Herein, chimeric peptide-functionalized titanium carbide MXenes (Pep-Ti 3 C 2 ) were devised for the activity assay of PTM enzymes by integration with carboxypeptidase Y (CPY)-mediated peptide cleavage. The Pep-Ti 3 C 2 is fabricated by self-assembly of chimeric peptide probes on the surface of phospholipid-coated Ti 3 C 2 MXenes and works as the fluorescent nanoprobe for the sensing of PTM enzymes. In the presence of a target PTM enzyme, the modification groups in the peptide probes are removed along with the digestion of the peptides by CPY, thereby leading to the release of labeled fluorophores. Consequently, fluorescent analysis of PTM enzymes, including deacetylase sirtuin-1 and protein phosphatase 2C at low-nanomolar concentrations was achieved. Furthermore, the versatility of the nanoprobes was also demonstrated in simultaneous profiling of the activities of the two PTM enzymes in different cells, as well as in evaluation of the inhibition on PTMs by small molecules in complicated biological samples. Therefore, this work deploys peptide-functionalized MXenes as a generic biosensing interface for the activity assay of PTM enzymes, providing a useful tool for biochemical research and clinical diagnosis.

Human Cathepsin A (CatA) is a lysosomal serine carboxypeptidase of the renin-angiotensin system (RAS) and is structurally similar to acetylcholinesterase (AChE). CatA can remove the C-terminal amino acids of endothelin I, angiotensin I, Substance P, oxytocin, and bradykinin, and can deamidate neurokinin A. Proteomic studies identified CatA and its homologue, SCPEP1, as potential targets of organophosphates (OP). CatA could be stably inhibited by low µM to high nM concentrations of racemic sarin (GB), soman (GD), cyclosarin (GF), VX, and VR within minutes to hours at pH 7. Cyclosarin was the most potent with a kinetically measured dissociation constant (K I ) of 2 µM followed by VR (K I  = 2.8 µM). Bimolecular rate constants for inhibition by cyclosarin and VR were 1.3 × 10 3 M -1 sec -1 and 1.2 × 10 3 M -1 sec -1 , respectively, and were approximately 3-orders of magnitude lower than those of human AChE indicating slower reactivity. Notably, both AChE and CatA bound diisopropylfluorophosphate (DFP) comparably and had K I DFP  = 13 µM and 11 µM, respectively. At low pH, greater than 85% of the enzyme spontaneously reactivated after OP inhibition, conditions under which OP-adducts of cholinesterases irreversibly age. At pH 6.5 CatA remained stably inhibited by GB and GF and <10% of the enzyme spontaneously reactivated after 200 h. A crystal structure of DFP-inhibited CatA was determined and contained an aged adduct. Similar to AChE, CatA appears to have a "backdoor" for product release. CatA has not been shown previously to age. These results may have implications for: OP-associated inflammation; cardiovascular effects; and the dysregulation of RAS enzymes by OP., (Published by Elsevier Inc.)

Cathepsin A (CTSA) is a lysosomal protease that is abnormally expressed in various types of cancer; however, the function of CTSA in lung adenocarcinoma (LUAD) is unknown. The aim of the present study was to investigate the role of CTSA during LUAD development in vitro. The Cancer Genome Atlas (TCGA) database was used to analyze the expression of CTSA mRNA in LUAD tissues. CTSA was significantly upregulated in LUAD tissues compared with normal lung tissues. To explore the effect of CTSA on LUAD in vitro, LUAD A549 cells were transfected with CTSA small interfering RNA and the hallmarks of tumorigenesis were investigated using cell proliferation, cell cycle, wound healing, invasion and western blot assays. Following CTSA knockdown, proliferation of LUAD cells was decreased and an increased proportion of LUAD cells were arrested at the G0/G1 phase, with altered expression of critical cell cycle and proliferative marker proteins, including p53, p21 and proliferating cell nuclear antigen. Moreover, CTSA knockdown decreased the migration and invasion of A549 cells, as determined by wound healing, invasion, and western blotting assays. The expression levels of key proteins involved in epithelial‑mesenchymal transition were analyzed by western blotting. CTSA knockdown enhanced the expression of E‑cadherin, but decreased the expression of N‑cadherin and β‑catenin in A549 cells. To the best of our knowledge, the present study suggested for the first time it has been identified that CTSA may serve as a tumor promoter in LUAD, enhancing the malignant progression of LUAD cells by promoting cell proliferation, migration and invasion. The results suggested that CTSA may serve as a novel therapeutic target for LUAD.

High glycosidase-producing strains of Aspergillus luchuensis were isolated from 2-deoxyglucose (2-DG) resistant mutants. α-Amylase, exo-α-1,4-glucosidase, β-glucosidase and β-xylosidase activity in the mutants was ~3, ~2, ~4 and ~2.5 times higher than the parental strain RIB2604 on koji-making conditions, respectively. Citric acid production and mycelia growth of the mutants, however, approximately halved to that of the parent. Compared to the parent, the alcohol yield from rice and sweet potato shochu mash of the mutant increased ~5.7% and 3.0%, respectively. The mutant strains showed significantly low glucose assimilability despite the fructose one was almost normal, and they had a single missense or nonsense mutation in the glucokinase gene glkA . The recombinant strain that was introduced at one of the mutations, glkA Q300K, demonstrated similar but not identical phenotypes to the mutant strain. This result indicates that glkA Q300K is one of the major mutations in 2-DG resistant strains.

Cathepsin A (CatA, EC 3.4.16.5, UniProtKB P10619 ) is a human lysosomal carboxypeptidase. Counterintuitively, crystal structures of CatA and its homologues show a cluster of Glu and Asp residues binding the C-terminal carboxylic acid of the product or inhibitor. Each of these enzymes functions in an acidic environment and contains a highly conserved pair of Glu residues with side chain carboxyl group oxygens that are approximately 2.3-2.6 Å apart. In small molecules, carboxyl groups separated by ∼3 Å can overcome the repulsive interaction by protonation of one of the two groups. The p K a of one group increases (p K a ∼ 11) and can be as much as ∼6 pH units higher than the paired group. Consequently, at low and neutral pH, one carboxylate can carry a net negative charge while the other can remain protonated and neutral. In CatA, E69 and E149 form a Glu pair that is important to catalysis as evidenced by the 56-fold decrease in k cat / K m in the E69Q/E149Q variant. Here, we have measured the pH dependencies of log( k cat ), log( K m ), and log( k cat / K m ) for wild type CatA and its variants and have compared the measured p K a with calculated values. We propose a substrate-assisted mechanism in which the high p K a of E149 (>8.5) favors the binding of the carboxylate form of the substrate and promotes the abstraction of the proton from H429 of the catalytic triad effectively decreasing its p K a in a low-pH environment. We also identify a similar motif consisting of a pair of histidines in S -formylglutathione hydrolase.

Background: The authors have previously shown that an embryonic stem cell-like population within keloid-associated lymphoid tissues in keloid lesions expresses components of the renin-angiotensin system that may be dysregulated. The authors hypothesized that cathepsins B, D, and G are present within the embryonic stem cell-like population in keloid lesions and contribute to bypass loops of the renin-angiotensin system., Methods: 3,3'-Diaminobenzidine immunohistochemical staining for cathepsins B, D, and G was performed on formalin-fixed paraffin-embedded sections in keloid tissue samples of 11 patients. Immunofluorescence immunohistochemical staining was performed on three of these keloid tissue samples, by co-staining with CD34, tryptase, and OCT4. Western blotting, reverse transcription quantitative polymerase chain reaction, and enzyme activity assays were performed on five keloid tissue samples and four keloid-derived primary cell lines to investigate protein and mRNA expression, and functional activity, respectively., Results: 3,3'-Diaminobenzidine immunohistochemical staining demonstrated expression of cathepsins B, D, and G in all 15 keloid tissue samples. Immunofluorescence immunohistochemical staining showed localization of cathepsins B and D to the endothelium of microvessels within the keloid-associated lymphoid tissues and localization of cathepsin G to the tryptase-positive perivascular cells. Western blotting confirmed semiquantitative levels of cathepsins B and D in keloid tissue samples and keloid-derived primary cell lines. Reverse transcription quantitative polymerase chain reaction showed quantitative transcriptional activation of cathepsins B and D in keloid tissue samples and keloid-derived primary cell lines and cathepsin G in keloid tissue samples. Enzyme activity assays demonstrated functional activity of cathepsins B and D., Conclusion: Cathepsins B, D, and G are expressed by the embryonic stem cell-like population within the keloid-associated lymphoid tissues of keloid lesions and may act to bypass the renin-angiotensin system, suggesting a potential therapeutic target using renin-angiotensin system modulators and cathepsin inhibitors.

During cytokinesis, the organization of the spindle midzone and chromosome segregation is controlled by the central spindle, a microtubule cytoskeleton containing kinesin motors and non-motor microtubule-associated proteins. The anaphase spindle elongation 1/protein regulator of cytokinesis 1/microtubule associated protein 65 (Ase1/PRC1/MAP65) family of microtubule-bundling proteins are key regulators of central spindle assembly, mediating microtubule crosslinking and spindle elongation in the midzone. Ase1/PRC1/MAP65 serves as a complex regulatory platform for the recruitment of other midzone proteins at the spindle midzone. Herein, we summarize recent advances in understanding of the structural domains and molecular kinetics of the Ase1/PRC1/MAP65 family. We summarize the regulatory network involved in post-translational modifications of Ase1/PRC1 by cyclin-dependent kinase 1 (Cdk1), cell division cycle 14 (Cdc14) and Polo-like kinase 1 (Plk1) and also highlight multiple functions of Ase1/PRC1 in central spindle organization, spindle elongation and cytokinesis during cell division., (© 2019 Cambridge Philosophical Society.)

Insufficient autophagy has been reported in idiopathic pulmonary fibrosis (IPF) lungs. Specific roles of autophagy-related proteins in lung fibrosis development remain largely unknown. Here, we investigated the role of autophagy marker protein microtubule - associated protein 1 light chain 3β (LC3B) in the development of lung fibrosis. LC3B -/- mice upon aging show smaller lamellar body profiles, increased cellularity, alveolar epithelial cell type II (AECII) apoptosis, surfactant alterations, and lysosomal and endoplasmic reticulum stress. Autophagosomal soluble N -ethylmaleimide-sensitive factor attachment protein receptor syntaxin 17 is increased in the AECII of aged LC3B -/- mice and patients with IPF. Proteasomal activity, however, remained unaltered in LC3B -/- mice. In vitro knockdown of LC3B sensitized mouse lung epithelial cells to bleomycin-induced apoptosis, but its overexpression was protective. In vivo , LC3B -/- mice displayed increased susceptibility to bleomycin-induced lung injury and fibrosis. We identified cathepsin A as a novel LC3B binding partner and its overexpression in vitro drives MLE12 cells to apoptosis. Additionally, cathepsin A is increased in the AECII of aged LC3B -/- mice and in the lungs of patients with IPF. Our study reveals that LC3B mediated autophagy plays essential roles in AECII by modulating the functions of proteins like cathepsin A and protects alveolar epithelial cells from apoptosis and subsequent lung injury and fibrosis.-Kesireddy, V. S., Chillappagari, S., Ahuja, S., Knudsen, L., Henneke, I., Graumann, J., Meiners, S., Ochs, M., Ruppert, C., Korfei, M., Seeger, W., Mahavadi, P. Susceptibility of microtubule-associated protein 1 light chain 3β (MAP1LC3B/LC3B) knockout mice to lung injury and fibrosis.

Lectin-carbohydrate interactions can be exploited in ultrasensitive biochemical recognition or medical diagnosis. For this purpose, besides the high specificity of the interactions, an appropriate methodology for their quantitative and detailed characterization is demanded. In this work, we determine the unbinding properties of the concanavalin A-carboxypeptidase Y complex, which is important for characterization of glycoproteins on the surface of biological cells. To achieve the goal, we have developed a methodology based on dynamic force spectroscopy measurements and two advanced theoretical models of force-induced unbinding. Our final results allowed excluding both, rebinding processes and the multibarrier character of the interaction potential, as possible explanations of the concanavalin A-carboxypeptidase Y unbinding mechanisms. Such characteristics as the position and height of the activation barrier and the force-free dissociation rate were determined. We hope our paper contributes to a better understanding of the unbinding processes in receptor-ligand complexes.

Yeast carboxypeptidase Y (CPY) is a serine protease with broad substrate specificity. Structurally, CPY belongs to the α/β hydrolase fold family and contains characteristic large helices, termed the V-shape helix, above the active site cavity. Four intramolecular disulfide bonds are located in and around the V-shape helix. In this study, mutant CPYs were constructed in which one of these disulfide bonds was disrupted. Mutants lacking the C193-C207 bond located at the beginning of the V-shape helix aggregated easily, while mutants lacking the C262-C268 bond located at the end of the V-shape helix displayed decreased hydrolytic activity. The results indicate that the V-shape helix is involved in CPY catalysis and in maintenance of its conformation., (© 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

Aims: Cathepsin A (CTSA) is a key regulatory enzyme for galactoside metabolism. Additionally, it has a distinct proteolytic activity and plays a role in tumour progression. CTSA is differentially expressed at the mRNA level between breast ductal carcinoma in situ (DCIS) and invasive breast carcinoma (IBC). In this study, we aimed to characterise CTSA protein expression in DCIS and evaluate its prognostic significance., Methods and Results: A large cohort of DCIS [n = 776 for pure DCIS and n = 239 for DCIS associated with IBC (DCIS/IBC)] prepared as a tissue microarray was immunohistochemically stained for CTSA. High CTSA expression was observed in 48% of pure DCIS. High expression was associated with features of poor DCIS prognosis, including younger age at diagnosis (<50 years), higher nuclear grade, hormone receptor negativity, HER2 positivity, high proliferative index and high hypoxia inducible factor 1 alpha expression. High CTSA expression was associated with shorter recurrence-free interval (RFI) (P = 0.0001). In multivariate survival analysis for patients treated with breast conserving surgery, CTSA was an independent predictor of shorter RFI (P = 0.015). DCIS associated with IBC showed higher CTSA expression than pure DCIS (P = 0.04). In the DCIS/IBC cohort, CTSA expression was higher in the invasive component than the DCIS component (P < 0.0001)., Conclusion: CTSA is not only associated with aggressive behaviour and poor outcome in DCIS but also a potential marker to predict co-existing invasion in DCIS., (© 2019 John Wiley & Sons Ltd.)

Mucosal immune system is one of the most vital components in the innate immunity and constitutes the first line of host defense against bacterial infections, especially for the teleost, which live in the pathogen-rich aquatic environment. Cathepsins, a superfamily of hydrolytic enzymes produced and enclosed within lysosomes, play multiple roles at physiological and pathological states. In this regard, we sought here to identify Cathepsin A in turbot (SmCTSA), characterize its mucosal expression patterns following Vibrio anguillarum and Streptococcus iniae infections in mucosal tissues, and explore its binding ability with three microbial ligands for the first time. The SmCTSA was 2631 bp long containing a 1422 bp open reading frame (ORF) that encoded 473 amino acids. Phylogenetic analysis revealed that SmCTSA showed the closest relationship to half-smooth tongue sole (Cynoglossus semilaevis). In addition, SmCTSA was ubiquitously expressed in all examined healthy tissues, with high expression levels in head kidney (HK) and intestine, while the lowest expression level in blood. Moreover, SmCTSA was significantly differentially expressed at least two timepoints in each mucosal tissue, suggesting its potential important roles in innate immune responses of turbot. Finally, in vitro assays showed that recombinant SmCTSA bound Lipopolysaccharide (LPS) with high affinity, and lipoteichoic acid (LTA) and peptidoglycan (PGN) with relatively low affinity. This study provides valuable data for understanding the roles of ctsa in the host defense against bacterial infections., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Cathepsin A (CTSA) is serine carboxypeptidase, an important protease in the lysosome. In this study, the full complementary DNA (cDNA) sequence of CTSA in Chinese giant salamanders Andrias davidianus was cloned, and its sequence features were analyzed. Tissue expression patterns of CTSA in healthy and Aeromonas hydrophila-infected salamanders were also investigated. The full cDNA sequence of salamander CTSA was 1,620 base pairs in length, encoding 472 amino acids. Salamander CTSA shared high sequence identities with other vertebrates' CTSAs, ranging from 62.7% to 68.9%. In healthy salamanders, CTSA was highly expressed in spleen, followed by brain, intestine, and stomach. After A. hydrophila infection, salamander CTSA was significantly upregulated in lung, heart, muscle, and kidney; was downregulated in liver, spleen, and intestine; and exhibited no significant changes in stomach and skin, indicating that salamander CTSA might play defense roles in multiple tissues during bacterial infection. These results provide a solid basis for further study of the immune function of amphibian CTSA. Received September 18, 2016; accepted June 18, 2017.

Accurate chromosome segregation relies on the bipolar mitotic spindle. In many eukaryotes, spindle formation is driven by the plus-end-directed motor kinesin-5 that generates outward force to establish spindle bipolarity. Its inhibition leads to the emergence of monopolar spindles with mitotic arrest. Intriguingly, simultaneous inactivation of the minus-end-directed motor kinesin-14 restores spindle bipolarity in many systems. Here we show that in fission yeast, three independent pathways contribute to spindle bipolarity in the absence of kinesin-5/Cut7 and kinesin-14/Pkl1. One is kinesin-6/Klp9 that engages with spindle elongation once short bipolar spindles assemble. Klp9 also ensures the medial positioning of anaphase spindles to prevent unequal chromosome segregation. Another is the Alp7/TACC-Alp14/TOG microtubule polymerase complex. Temperature-sensitive alp7cut7pkl1 mutants are arrested with either monopolar or very short spindles. Forced targeting of Alp14 to the spindle pole body is sufficient to render alp7cut7pkl1 triply deleted cells viable and promote spindle assembly, indicating that Alp14-mediated microtubule polymerization from the nuclear face of the spindle pole body could generate outward force in place of Cut7 during early mitosis. The third pathway involves the Ase1/PRC1 microtubule cross-linker that stabilizes antiparallel microtubules. Our study, therefore, unveils multifaceted interplay among kinesin-dependent and -independent pathways leading to mitotic bipolar spindle assembly., (© 2017 Yukawa et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

We isolated and characterised a cDNA encoding the lysosomal protective protein (serine protease) cathepsin A (CTSA) from rock bream (Oplegnathus fasciatus). The full-length rock bream CTSA (RbCTSA) cDNA (1814 bp) contains an open reading frame of 1419 bp that encodes 472 amino acids. Alignment of multiple CTSA protein sequences revealed that the active site serine and histidine residues were well-conserved among the other CTSA sequences. RbCTSA is highly expressed in the peripheral blood leukocytes, kidney, spleen, liver, intestine, gill, heart, brain, stomach, and eye. RbCTSA expression was also examined in several tissues, including whole kidneys and spleens, under bacterial and viral challenge. In general, all of the examined tissues that were infected with Edwardsiella tarda, Streptococcus iniae, or red sea bream iridovirus (RSIV) exhibited significant upregulation of RbCTSA expression compared to the controls. Our results reveal that RbCTSA may be involved in the immune responses of rock bream., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Presented herein is a simple, robust, and label-free homogeneous electrochemical sensing platform constructed for the detection of protein kinase activity and inhibition by integration of carboxypeptidase Y (CPY)-assisted peptide cleavage reaction and vertically ordered mesoporous silica films (MSFs). In this sensing platform, the substrate peptide composed of kinase-specific recognized sequence and multiple positively charged arginine (R) residues was ingeniously designed. In the presence of protein kinase, the substrate peptide was phosphorylated and then immediately resisted CPY cleavage. The phosphorylated peptide could be effectively adsorbed on the negatively charged surface of MSFs modified indium-tin oxide (ITO) electrode (MSFs/ITO) by noncovalent electrostatic attraction. The adsorbed peptide was subsequently used as a hamper to prevent the diffusion of electroactive probe (FcMeOH) to the electrode surface through the vertically aligned nanopores, resulting in a detectable reduction of electrochemical signal. As demonstrated for the feasibility and universality of the sensing platform, both protein kinase A (PKA) and casein kinase II (CK2) were selected as the models, and the detection limits were determined to be 0.083 and 0.095 UmL -1 , respectively. This sensing platform had the merits of simplicity, easy manipulation, and improved phosphorylation and cleavage efficiency, which benefited from homogeneous solution reactions without sophisticated modification or immobilization procedures. In addition, given the key role of inhibition and protein kinase activity detection in cell lysates, this proposed sensing platform showed great potential in kinase-related bioanalysis and clinical biomedicine.

Aim: To investigate the capability of salvianolic acid B (Sal B) to protect hepatocytes from hydrogen peroxide (H 2 O 2 )/carbon tetrachloride (CCl 4 )-induced lysosomal membrane permeabilization., Methods: Cell Counting Kit-8 assay was used to measure cell viability. Apoptosis and death were assayed through flow cytometry. BrdU incorporation was used to detect cell proliferation. Serum alanine aminotransferase activity and liver malondialdehyde (MDA) content were measured. Liver histopathological changes were evaluated using hematoxylin-eosin staining. Lysosomal membrane permeability was detected with LysoTracker Green-labeled probes and acridine orange staining. The levels of protein carbonyl content (PCC), cathepsins (Cat)B/D, and lysosome-associated membrane protein 1 (LAMP1) were evaluated through western blotting. Cytosol CatB activity analysis was performed with chemiluminescence detection. The mRNA level of LAMP1 was evaluated through quantitative real-time polymerase chain reaction., Results: Results indicated that H 2 O 2 induced cell injury/death. Sal B attenuated H 2 O 2 -induced cell apoptosis and death, restored the inhibition of proliferation, decreased the amount of PCC, and stabilized the lysosome membrane by increasing the LAMP1 protein level and antagonizing CatB/D leakage into the cytosol. CCl 4 also triggered hepatocyte death. Furthermore, Sal B effectively rescued hepatocytes by increasing LAMP1 expression and by reducing lysosomal enzyme translocation to the cytosol., Conclusion: Sal B protected mouse embryonic hepatocytes from H 2 O 2 /CCl 4 -induced injury/death by stabilizing the lysosomal membrane., Competing Interests: Conflict-of-interest statement: The authors declare that there are no conflicts of interest.

Carboxypeptidase Y from Saccharomyces cerivisiae was characterized for its site specific N-glycosylation through mass spectrometry. The N-glycopeptides were derived using non specific proteases and are analysed directly on liquid chromatography coupled to ion trap mass spectrometer in tandem mode. The evaluation of glycan fragment ions and the Y 1 ions (peptide+HexNAc) +n revealed the glycan sequence and the corresponding site of attachment. We observed the microheterogeneity in N-glycans such as Man 11-15 GlcNAc 2 at Asn 13 , Man 8-12 GlcNAc 2 at Asn 87 , Man 9-14 GlcNAc 2 at Asn 168 and phosphorylated Man 12-17 GlcNAc 2 as well as Man 11-16 GlcNAc 2 at Asn 368 . The presence of N-glycans with Man <18 GlcNAc 2 indicated that in vacuoles the steady release of mannose/phospho mannose residues from glycans occurs initially at Asn 13 or Asn 168 followed by at Asn 368 . However, glycans at Asn 87 which comprises Man 8-12 residues as reported earlier remain intact suggesting its inaccessibility for a similar processing. This in turn indicates the interaction of the glycan at Asn 87 with the polypeptide chain implicating it in the folding of the protein., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Vasoactive and mitogenic peptide, endothelin-1 (ET-1) plays an important role in physiology of the ocular tissues by regulating the growth of corneal epithelial cells and maintaining the hemodynamics of intraocular fluids. We have previously established that ET-1 can be degraded in vivo by two lysosomal/secreted serine carboxypeptidases, Cathepsin A (CathA) and Serine Carboxypeptidase 1 (Scpep1) and that gene-targeted CathAS190A /Scpep1-/- mice, deficient in CathA and Scpep1 have a prolonged half-life of circulating ET-1 associated with systemic hypertension. In the current work we report that starting from 6 months of age, ~43% of CathAS190A /Scpep1-/- mice developed corneal clouding that eventually caused vision impairment. Histological evaluation of these mice demonstrated a selective fibrotic thickening and vacuolization of the corneas, resembling human hyperproliferative vesicular corneal stromal dystrophy and coexisting with a peculiar thickening of the skin epidermis. Moreover, we found that cultured corneal epithelial cells, skin fibroblasts and vascular smooth muscle cells derived from CathA/Scpep1-deficient mice, demonstrated a significantly higher proliferative response to treatment with exogenous ET-1, as compared with cells from wild type mice. We also detected increased activation level of ERK1/2 and AKT kinases involved in cell proliferation in the ET-1-treated cultured cells from CathA/Scpep1 deficient mice. Together, results from our experimental model suggest that; in normal tissues the tandem of serine carboxypeptidases, Scpep1 and CathA likely constitutes an important part of the physiological mechanism responsible for the balanced elimination of heightened levels of ET-1 that otherwise would accumulate in tissues and consequently contribute to development of the hyper-proliferative corneal dystrophy and abnormal skin thickening.

Proteins are frequently processed by proteases in cell signaling pathways to perform their biological functions in response to environmental stimuli. Identification of the exact cleavage sites provides necessary information for the study of their biological functions. Although proteomic approaches for profiling of protein N-termini have been developed extensively in the past few years, the N-terminal profiling strategy has its inherent disadvantages. Therefore, C-terminal profiling approaches might be a complementary approach for the identification of protein cleavages although it has similar shortcomings as N-terminal profiling methods. In this protocol, we describe an approach, termed ProC-TEL: Profiling of Protein C-Termini by Enzymatic Labeling, for affinity labeling of protein C-termini for a protein or proteome. This method uses the transpeptidase activity of carboxypeptidase Y to label protein C-termini with an affinity biotin tag for subsequent isolation with avidin beads and identification by mass spectrometer. It is complementary to the N-terminal profiling approaches and can be used to identify proteolytic cleavages for a specific protease or in cell signaling events, such as apoptosis.

The lysosomal carboxypeptidase A, Cathepsin A (CathA), is a serine protease with two distinct functions. CathA protects β -galactosidase and sialidase Neu1 against proteolytic degradation by forming a multienzyme complex and activates sialidase Neu1. CathA deficiency causes the lysosomal storage disease, galactosialidosis. These patients present with a broad range of clinical phenotypes, including growth retardation, and neurological deterioration along with the accumulation of the vasoactive peptide, endothelin-1, in the brain. Previous in vitro studies have shown that CathA has specific activity against vasoactive peptides and neuropeptides, including endothelin-1 and oxytocin. A mutant mouse with catalytically inactive CathA enzyme ( CathA S 190 A ) shows increased levels of endothelin-1. In the present study, we elucidated the involvement of CathA in learning and long-term memory in 3-, 6-, and 12-month-old mice. Hippocampal endothelin-1 and oxytocin accumulated in CathA S 190 A mice, which showed learning impairments as well as long-term and spatial memory deficits compared with wild-type littermates, suggesting that CathA plays a significant role in learning and in memory consolidation through its regulatory role in vasoactive peptide processing., Competing Interests: The authors declare that they have no competing interests.

The endoplasmic reticulum (ER) has numerous biological functions including protein synthesis, protein folding, and lipid synthesis. The CAX4 gene encodes dolichyl pyrophosphate (Dol-PP) phosphatase, which is involved in protein N-glycosylation. In cax4Δ cells, the N-glycosylation of the vacuolar carboxypeptidase (CPY) was severely affected, and expression of the ER chaperone Kar2p was elevated, which resulted in UPR activation as an adaptive response. The cax4Δ cell growth was reduced, and this could be attributed to the formation of clumped aggregates, high vesiculation of the intracellular membrane, and plasma membrane alterations were depicted using DiOC6 fluorescence. In the cax4 deletion strain, the transcription factors INO2 and INO4 were upregulated, and the negative regulator OPI1 was concomitantly down regulated, which led to the derepression of the phospholipid genes CHO2, OPI3, PSD1, and PSD2 and resulted in increased phospholipid levels. However, the TAG, SE, and LD levels were significantly reduced, and FFA, sterol, and DAG levels were increased. These findings could be attributed to the derepression of the TAG and SE lipases TGL3, TGL4, TGL5, YEH1, and YEH2 and the repression of LRO1, DGA1, ARE1, and ARE2 in cax4Δ cells. Interestingly, the overexpression of SEC59 or CAX4 in cax4Δ cells prevented the ER stress and growth defect, and restored normal level of phospholipids, neutral lipids, and LDs. The current study revealed the disruption of N-glycosylation-induced ER stress, altered lipid homeostasis accounts for pleiotropic phenotype. Thus, CAX4 regulates membrane biogenesis by coordinating lipid homeostasis with protein quality control., (Copyright © 2016. Published by Elsevier B.V.)

Cathepsin A (CTSA) is a lysosomal carboxypeptidase present at the cell surface and secreted outside the cell. Additionally, CTSA binds to β-galactosidase and neuraminidase 1 to protect them from degradation. CTSA has gained attention as a drug target for the treatment of cardiac hypertrophy and heart failure. Here, we investigated the impact of CTSA on the murine cardiac proteome in a mouse model of cardiomyocyte-specific human CTSA overexpression using liquid chromatography-tandem mass spectrometry in conjunction with an isotopic dimethyl labeling strategy. We identified up to 2000 proteins in each of three biological replicates. Statistical analysis by linear models for microarray data (limma) found >300 significantly affected proteins (moderated p-value ≤0.01), thus establishing CTSA as a key modulator of the cardiac proteome. CTSA strongly impaired the balance of the proteolytic system by upregulating several proteases such as cathepsin B, cathepsin D, and cathepsin Z while down-regulating numerous protease inhibitors. Moreover, cardiomyocyte-specific human CTSA overexpression strongly reduced the levels of numerous antioxidative stress proteins, i.e., peroxiredoxins and protein deglycase DJ-1. In vitro, using cultured rat cardiomyocytes, ectopic overexpression of CTSA resulted in accumulation of reactive oxygen species. Collectively, our proteomic and functional data strengthen an association of CTSA with the cellular oxidative stress response.

We report a novel method for sequential on-line C-terminal sequencing of peptides, which combines carboxypeptidase Y (CPY) digestion with on-line derivatization and optically gated capillary electrophoresis with laser-induced fluorescence detection (OGCE-LIF). Various factors that may affect the C-terminal sequencing were investigated and optimized. High repeatability of on-line derivatization and the sequential OGCE-LIF assay of amino acids (AAs) was achieved with relative standard deviation (RSD) (n=20) less than 1.5% and 3.2% for migration time and peak height, respectively. A total of 13 AAs was efficiently separated in the present study, indicating that the method can be used for sequencing of peptides consisting of the 13 AAs studied. Using two synthesized N-terminally blocked peptides as test examples, we show that the present method can on-line monitor the released AAs with a temporal resolution of 50s during the entire CPY digestion process. The rates of AA release as a function of digestion time were easily measured; thus, the AA sequence of the peptide was determined with just one OGCE assay. Our study indicates the present approach is an effective, reliable, and convenient method for rapid analysis of the C-terminal sequence of peptides, with potential application in peptide analysis and proteome research., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Neuraminidase-1 (NEU1) is the predominant sialidase expressed in human airway epithelia and lung microvascular endothelia where it mediates multiple biological processes. We tested whether the NEU1-selective sialidase inhibitor, C9-butyl-amide-2-deoxy-2,3-dehydro-N-acetylneuraminic acid (C9-BA-DANA), inhibits one or more established NEU1-mediated bioactivities in human lung cells. We established the IC50 values of C9-BA-DANA for total sialidase activity in human airway epithelia, lung microvascular endothelia and lung fibroblasts to be 3.74 µM, 13.0 µM and 4.82 µM, respectively. In human airway epithelia, C9-BA-DANA dose-dependently inhibited flagellin-induced, NEU1-mediated mucin-1 ectodomain desialylation, adhesiveness for Pseudomonas aeruginosa and shedding. In lung microvascular endothelia, C9-BA-DANA reversed NEU1-driven restraint of cell migration into a wound and disruption of capillary-like tube formation. NEU1 and its chaperone/transport protein, protective protein/cathepsin A (PPCA), were differentially expressed in these same cells. Normalized NEU1 protein expression correlated with total sialidase activity whereas PPCA expression did not. In contrast to eukaryotic sialidases, C9-BA-DANA exerted far less inhibitory activity for three selected bacterial neuraminidases (IC50 > 800 µM). Structural modeling of the four human sialidases and three bacterial neuraminidases revealed a loop between the seventh and eighth strands of the β-propeller fold, that in NEU1, was substantially shorter than that seen in the six other enzymes. Predicted steric hindrance between this loop and C9-BA-DANA could explain its selectivity for NEU1. Finally, pretreatment of mice with C9-BA-DANA completely protected against flagellin-induced increases in lung sialidase activity. Our combined data indicate that C9-BA-DANA inhibits endogenous and ectopically expressed sialidase activity and established NEU1-mediated bioactivities in human airway epithelia, lung microvascular endothelia, and fibroblasts in vitro and murine lungs in vivo., (© The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Aims: Type 2 diabetes (T2D) is an independent risk factor for atrial fibrillation (AF) and stroke. The serine protease cathepsin A (CatA) is up-regulated in diabetes and plays an important role in the degradation of extracellular peptides. This study sought to delineate the role of CatA for the development of atrial remodelling under diabetic conditions., Methods and Results: Zucker Diabetic Fatty rats (ZDF) were treated with vehicle (n = 20) or CatA-inhibitor (SAR; 50 mg/kg; n = 20), and compared with age-matched non-diabetic littermates (Ctr, n = 20). Left-atrial (LA) emptying function [magnetic resonance imaging (MRI)] and atrial electrophysiological parameters were measured before sacrifice for histological and biochemical analysis. The impact of enhanced cardiac CatA expression on atrial remodelling was determined using CatA-transgenic mice. At the age of 9.5 months, atrial tissues of ZDF rats showed increased CatA gene expression and CatA-activity, along with increased AF-susceptibility and impaired LA-emptying function. CatA-inhibition reduced CatA-activity in ZDF comparable to Ctr values and decreased LA-fibrosis formation and connexin 43 lateralization. This was associated with shorter median duration of LA-tachyarrhythmia (12.0 ± 1.7 vs. 1.2 ± 0.47 s, P < 0.01) induced by burst pacing and diminished regions of slow conduction. Cardiac MRI revealed better LA-emptying function parameters (active per cent emptying: 29 ± 1 vs. 23 ± 2%, P < 0.01) after CatA-inhibition. CatA-inhibition reduced LA bradykinin-degrading activity in ZDF. Transgenic mice overexpressing CatA demonstrated enhanced atrial fibrosis formation and increased AF-susceptibility., Conclusion: T2D leads to arrhythmogenic atrial remodelling in ZDF rats. CatA-inhibition reduces LA bradykinin-degrading activity in ZDF and suppresses the development of atrial structural changes and AF-promotion, implicating CatA as an important mediator for AF-substrate in T2D., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2016. For permissions please email: journals.permissions@oup.com.)

Background: Myocardial infarction (MI) is a major cause of heart failure. The carboxypeptidase cathepsin A is a novel target in the treatment of cardiac failure. We aim to show that recently developed inhibitors of the protease cathepsin A attenuate post-MI heart failure., Methods: Mice were subjected to permanent left anterior descending artery (LAD) ligation or sham operation. 24 h post-surgery, LAD-ligated animals were treated with daily doses of the cathepsin A inhibitor SAR1 or placebo. After 4 weeks, the three groups (sham, MI-placebo, MI-SAR1) were evaluated., Results: Compared to sham-operated animals, placebo-treated mice showed significantly impaired cardiac function and increased plasma BNP levels. Cathepsin A inhibition prevented the increase of plasma BNP levels and displayed a trend towards improved cardiac functionality. Proteomic profiling was performed for the three groups (sham, MI-placebo, MI-SAR1). More than 100 proteins were significantly altered in placebo-treated LAD ligation compared to the sham operation, including known markers of cardiac failure as well as extracellular/matricellular proteins. This ensemble constitutes a proteome fingerprint of myocardial infarction induced by LAD ligation in mice. Cathepsin A inhibitor treatment normalized the marked increase of the muscle stress marker CA3 as well as of Igγ 2b and fatty acid synthase. For numerous further proteins, cathepsin A inhibition partially dampened the LAD ligation-induced proteome alterations., Conclusions: Our proteomic and functional data suggest that cathepsin A inhibition has cardioprotective properties and support a beneficial effect of cathepsin A inhibition in the treatment of heart failure after myocardial infarction.

Introduction: Galactosialidosis is a rare lysosomal storage disease caused by a combined deficiency of GM1 β-galactosidase (β-gal) and neuraminidase secondary to a defect of a lysosomal enzyme protective protein/cathepsin A (PPCA) and mutation in CTSA gene. Three subtypes are recognized: early infantile, late infantile, and juvenile/adult. There is no specific therapy for patients with galactosialidosis at this time., Objectives: The aim of this study was to determine the chaperone effect of N-octyl-4-epi-β-valienamine (NOEV) on β-gal proteins in skin fibroblasts of PPCA-deficit patients., Methods: β-Gal and neuraminidase activities were measured for the diagnosis of the patients with galactosialidosis. Western blotting for PPCA protein and direct sequencing for CTSA gene were performed. Cultured skin fibroblast were treated with NOEV., Results: We report four novel patients with galactosialidosis: one had the early infantile form and the other three had the juvenile/adult form. We found that NOEV stabilized β-gal activity in lysate from cultured skin fibroblasts from these patients. Treatment with NOEV significantly enhanced β-gal activity in cultured skin fibroblasts in the absence of PPCA., Conclusions: Our results indicate the possibility that NOEV chaperone therapy might have a beneficial effect, at least in part, for patients with galactosialidosis., (Copyright © 2015 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.)

Background/aims: Hypoxia has recently been proposed as one of the most important factors in progressive renal injury. Hypoxia-induced vascular endothelial growth factor (VEGF) expression may play a critical role in maintaining peritubular capillary endothelium in renal disease. This study was designed to investigate the effect and underlying mechanism of all-trans retinoic acid (ATRA) on hypoxia-induced injury in NRK52E cells., Methods: For mimicking hypoxia, cells were treated with 100 µM of cobalt chloride (CoCl2). The cell viability, expression of VEGF, p65, transforming growth factor-β2 (TGF-β2) and serine carboxypeptidase 1 (Scpep1), and nuclear factor of kappaB (NF-x03BA;B) activities after ATRA treatment were determined by MTT, western blot and electrophoretic mobility shift assay. Co-immunoprecipitation analysis was performed to demonstrate whether Scpep1 interacted with TGF-β2., Results: It was found that CoCl2 triggered hypoxia injury and significantly reduced cell viability. ATRA pretreatment increased the cell survival rate. Under hypoxic conditions, the expression of VEGF, p65 and TGF-β2 increased. Addition of ATRA significantly attenuated the expression of VEGF, p65 and TGF-β2. There was a corresponding variation of NF-x03BA;B/DNA binding activities. In addition, ATRA stimulated Scpep1 expression under normoxic and hypoxia condition. Furthermore, TGF-β2 interacted with Scpep1., Conclusions: This study indicated that ATRA may attenuate hypoxia-induced injury in NRK52E cells via inhibiting NF-x03BA;B/VEGF and TGF-β2/VEGF pathway., (© 2016 The Author(s) Published by S. Karger AG, Basel.)

Coatomer consists of two subcomplexes: the membrane-targeting, ADP ribosylation factor 1 (Arf1):GTP-binding βγδζ-COP F-subcomplex, which is related to the adaptor protein (AP) clathrin adaptors, and the cargo-binding αβ'ε-COP B-subcomplex. We present the structure of the C-terminal μ-homology domain of the yeast δ-COP subunit in complex with the WxW motif from its binding partner, the endoplasmic reticulum-localized Dsl1 tether. The motif binds at a site distinct from that used by the homologous AP μ subunits to bind YxxΦ cargo motifs with its two tryptophan residues sitting in compatible pockets. We also show that the Saccharomyces cerevisiae Arf GTPase-activating protein (GAP) homolog Gcs1p uses a related WxxF motif at its extreme C terminus to bind to δ-COP at the same site in the same way. Mutations designed on the basis of the structure in conjunction with isothermal titration calorimetry confirm the mode of binding and show that mammalian δ-COP binds related tryptophan-based motifs such as that from ArfGAP1 in a similar manner. We conclude that δ-COP subunits bind Wxn(1-6)[WF] motifs within unstructured regions of proteins that influence the lifecycle of COPI-coated vesicles; this conclusion is supported by the observation that, in the context of a sensitizing domain deletion in Dsl1p, mutating the tryptophan-based motif-binding site in yeast causes defects in both growth and carboxypeptidase Y trafficking/processing.

Tenofovir alafenamide fumarate (TAF) is an oral phosphonoamidate prodrug of the HIV reverse transcriptase nucleotide inhibitor tenofovir (TFV). Previous studies suggested a principal role for the lysosomal serine protease cathepsin A (CatA) in the intracellular activation of TAF. Here we further investigated the role of CatA and other human hydrolases in the metabolism of TAF. Overexpression of CatA or liver carboxylesterase 1 (Ces1) in HEK293T cells increased intracellular TAF hydrolysis 2- and 5-fold, respectively. Knockdown of CatA expression with RNA interference (RNAi) in HeLa cells reduced intracellular TAF metabolism 5-fold. Additionally, the anti-HIV activity and the rate of CatA hydrolysis showed good correlation within a large set of TFV phosphonoamidate prodrugs. The covalent hepatitis C virus (HCV) protease inhibitors (PIs) telaprevir and boceprevir potently inhibited CatA-mediated TAF activation (50% inhibitory concentration [IC50] = 0.27 and 0.16 μM, respectively) in vitro and also reduced its anti-HIV activity in primary human CD4(+) T lymphocytes (21- and 3-fold, respectively) at pharmacologically relevant concentrations. In contrast, there was no inhibition of CatA or any significant effect on anti-HIV activity of TAF observed with cobicistat, noncovalent HIV and HCV PIs, or various prescribed inhibitors of host serine proteases. Collectively, these studies confirm that CatA plays a pivotal role in the intracellular metabolism of TAF, whereas the liver esterase Ces1 likely contributes to the hepatic activation of TAF. Moreover, this work demonstrates that a wide range of viral and host PIs, with the exception of telaprevir and boceprevir, do not interfere with the antiretroviral activity of TAF., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Several important classes of antifungal agents, including the azoles, act by blocking ergosterol biosynthesis. It was recently reported that the azoles cause massive disruption of the fungal vacuole in the prevalent human pathogen Candida albicans. This is significant because normal vacuolar function is required to support C. albicans pathogenicity. This study examined the impact of the morpholine antifungals, which inhibit later steps of ergosterol biosynthesis, on C. albicans vacuolar integrity. It was found that overexpression of either the ERG2 or ERG24 gene, encoding C-8 sterol isomerase or C-14 sterol reductase, respectively, suppressed C. albicans sensitivity to the morpholines. In addition, both erg2Δ/Δ and erg24Δ/Δ mutants were hypersensitive to the morpholines. These data are consistent with the antifungal activity of the morpholines depending upon the simultaneous inhibition of both Erg2p and Erg24p. The vacuoles within both erg2Δ/Δ and erg24Δ/Δ C. albicans strains exhibited an aberrant morphology and accumulated large quantities of the weak base quinacrine, indicating enhanced vacuolar acidification compared with that of control strains. Both erg mutants exhibited significant defects in polarized hyphal growth and were avirulent in a mouse model of disseminated candidiasis. Surprisingly, in a mouse model of vaginal candidiasis, both mutants colonized mice at high levels and induced a pathogenic response similar to that with the controls. Thus, while targeting Erg2p or Erg24p alone could provide a potentially efficacious therapy for disseminated candidiasis, it may not be an effective strategy to treat vaginal infections. The potential value of drugs targeting these enzymes as adjunctive therapies is discussed., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

A green method was employed for synthesizing peptide-templated nanoclusters without requiring strong reducing agents. Using synthetic peptide-gold nanoclusters as fluorescence probes, a novel assay for detecting protein kinase is developed based on phosphorylation against carboxypeptidase Y digestion.

An effective assay method for monitoring protein kinase activity and screening inhibitors is greatly beneficial to kinase-related drug discovery, early diagnosis of diseases, and therapeutic effect evaluation. Herein, we develop a simple electrochemical method for detecting the activity of casein kinase II (CK2) based on phosphorylation against carboxypeptidase Y (CPY) digestion triggered signal amplification, where CK2 catalyzed phosphorylation event protects the substrate peptide from the digestion of CPY, maintains the repulsive force of the substrate peptide towards the redox probe, and results in a weak electrochemical signal. Whereas, without phosphorylation, the substrate peptide is digested by CPY and a strong electrochemical signal is obtained. The detection feasibility is demonstrated for the assay of CK2 activity with low detection limit of 0.047unit/mL. Moreover, the biosensor was used for the analysis of kinase inhibition. Based on the electrochemical signal dependent inhibitor concentration, the IC50 value of ellagic acid was estimated to be 39.77nM. The proposed method is also successfully applied to analyze CK2 activity in cell lysates, proving the applicability in complex biological samples., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: Endoplasmic reticulum (ER)-associated degradation (ERAD) is a pathway by which misfolded or improperly assembled proteins in the ER are directed to degradation. The cytoplasmic peptide:N-glycanase (PNGase) is a deglycosylating enzyme that cleaves N-glycans from misfolded glycoproteins during the ERAD process. The mutant form of yeast carboxypeptidase Y (CPY*) is an ERAD model substrate that has been extensively studied in yeast. While a delay in the degradation of CPY* in yeast cells lacking the cytoplasmic PNGase (Png1 in yeast) was evident, the in vivo action of PNGase on CPY* has not been detected., Methods: We constructed new ERAD substrates derived from CPY*, bearing epitope tags at both N- and C-termini and examined the degradation intermediates observed in yeast cells with compromised proteasome activity., Results: The occurrence of the PNGase-mediated deglycosylation of intact CPY* and its degradation intermediates was evident. A major endoproteolytic reaction on CPY* appears to occur between amino acid 400 and 404., Conclusions: The findings reported herein clearly indicate that PNGase indeed releases N-glycans from CPY* during the ERAD process in vivo., General Significance: This report implies that the PNGase-mediated deglycosylation during the ERAD process may occur more abundantly than currently envisaged., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Carboxypeptidase Y is widely used in peptide sequencing and mass spectrometry. PRC1 coding for proteinase C from Saccharomyces cerevisiae was expressed in Pichia pastoris GS115 as procarboxypeptidase Y with a yield of ~605 mg/l in shake-flasks after 168 h induction with 1 % (v/v) methanol. This precursor of carboxypeptidase Y was cleaved by endogenous proteinases of P. pastoris and released into the fermentation broth as active carboxypeptidase Y within 2 weeks at 10 °C, which facilitated the preparation of mature carboxypeptidase Y. The recombinant enzyme was purified. It was optimally active at 30 °C and pH 6.0, with an optimal activity of ~305 U/mg using benzyloxycarbonyl-L-phenylalanyl-L-leucine as substrate. This is the first report about high-level expression and activation of carboxypeptidase Y in P. pastoris.

The ubiquitous distribution of lysosomes and their heterogeneous protein composition reflects the versatility of these organelles in maintaining cell homeostasis and their importance in tissue differentiation and remodeling. In lysosomes, the degradation of complex, macromolecular substrates requires the synergistic action of multiple hydrolases that usually work in a stepwise fashion. This catalytic machinery explains the existence of lysosomal enzyme complexes that can be dynamically assembled and disassembled to efficiently and quickly adapt to the pool of substrates to be processed or degraded, adding extra tiers to the regulation of the individual protein components. An example of such a complex is the one composed of three hydrolases that are ubiquitously but differentially expressed: the serine carboxypeptidase, protective protein/cathepsin A (PPCA), the sialidase, neuraminidase-1 (NEU1), and the glycosidase β-galactosidase (β-GAL). Next to this 'core' complex, the existence of sub-complexes, which may contain additional components, and function at the cell surface or extracellularly, suggests as yet unexplored functions of these enzymes. Here we review how studies of basic biological processes in the mouse models of three lysosomal storage disorders, galactosialidosis, sialidosis, and GM1-gangliosidosis, revealed new and unexpected roles for the three respective affected enzymes, Ppca, Neu1, and β-Gal, that go beyond their canonical degradative activities. These findings have broadened our perspective on their functions and may pave the way for the development of new therapies for these lysosomal storage disorders.