Gene therapy corrects the neurological deficits of mice with sialidosis.

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 ß-Gal binds to this complex to form a multienzyme complex in order to execute its function.

Contributions of Cathepsin A and Carboxylesterase 1 to the Hydrolysis of Tenofovir Alafenamide in the Human Liver, and the Effect of CES1 Genetic Variation on Tenofovir Alafenamide Hydrolysis.

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.

Enhanced carboxypeptidase efficacies and differentiation of peptide epimers.

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.

Identification of the prognostic, diagnostic, and biological significance of the miR-148a-3p/cathepsin A axis in hepatocellular carcinoma.

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.

Cathepsin A contributes to left ventricular remodeling by degrading extracellular superoxide dismutase in mice.

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.

Cathepsin A regulates pluripotency, proliferation and differentiation in mouse embryonic stem cells.

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.

Chimeric Peptides Self-Assembling on Titanium Carbide MXenes as Biosensing Interfaces for Activity Assay of Post-translational Modification Enzymes.

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-Ti3C2) were devised for the activity assay of PTM enzymes by integration with carboxypeptidase Y (CPY)-mediated peptide cleavage. The Pep-Ti3C2 is fabricated by self-assembly of chimeric peptide probes on the surface of phospholipid-coated Ti3C2 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.

Neuraminidase-1 promotes heart failure after ischemia/reperfusion injury by affecting cardiomyocytes and invading monocytes/macrophages.

Neuraminidase (NEU)1 forms a multienzyme complex with beta-galactosidase (ß-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, ß-GAL and PPCA expression. Mice hypomorphic for neu1 (hNEU1) had less neuraminidase activity, fewer pro-inflammatory (Lin-CD11b+F4/80+Ly-6Chigh), and more anti-inflammatory macrophages (Lin-CD11b+F4/80+Ly-6Clow) 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.

Suppression of cathepsin a inhibits growth, migration, and invasion by inhibiting the p38 MAPK signaling pathway in prostate cancer.

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.

Susceptibility of microtubule-associated protein 1 light chain 3ß (MAP1LC3B/LC3B) knockout mice to lung injury and fibrosis.

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 3ß (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 3ß (MAP1LC3B/LC3B) knockout mice to lung injury and fibrosis.

Isolation methods of high glycosidase-producing mutants of Aspergillus luchuensis and its mutated genes.

High glycosidase-producing strains of Aspergillus luchuensis were isolated from 2-deoxyglucose (2-DG) resistant mutants. α-Amylase, exo-α-1,4-glucosidase, ß-glucosidase and ß-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.

Sequence-Independent Traceless Method for Preparation of Peptide/Protein Thioesters Using CPaseY-Mediated Hydrazinolysis.

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.

Paired Carboxylic Acids in Enzymes and Their Role in Selective Substrate Binding, Catalysis, and Unusually Shifted pKa Values.

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 Šapart. In small molecules, carboxyl groups separated by ∼3 Šcan overcome the repulsive interaction by protonation of one of the two groups. The pKa of one group increases (pKa ∼ 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 kcat/Km in the E69Q/E149Q variant. Here, we have measured the pH dependencies of log(kcat), log(Km), and log(kcat/Km) for wild type CatA and its variants and have compared the measured pKa with calculated values. We propose a substrate-assisted mechanism in which the high pKa 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 pKa in a low-pH environment. We also identify a similar motif consisting of a pair of histidines in S-formylglutathione hydrolase.

The prognostic significance of lysosomal protective protein (cathepsin A) in breast ductal carcinoma in situ.

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.

Characterization, expression signatures and microbial binding analysis of cathepsin A in turbot, Scophthalmus maximus L.(SmCTSA).

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.

Structural basis for the binding of tryptophan-based motifs by δ-COP.

Coatomer consists of two subcomplexes: the membrane-targeting, ADP ribosylation factor 1 (Arf1):GTP-binding ßγδζ-COP F-subcomplex, which is related to the adaptor protein (AP) clathrin adaptors, and the cargo-binding αß'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.

Dolichyl pyrophosphate phosphatase-mediated N-glycosylation defect dysregulates lipid homeostasis in Saccharomyces cerevisiae.

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.

Label-Free Homogeneous Electrochemical Sensing Platform for Protein Kinase Assay Based on Carboxypeptidase Y-Assisted Peptide Cleavage and Vertically Ordered Mesoporous Silica Films.

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.

Serine carboxypeptidase SCPEP1 and Cathepsin A play complementary roles in regulation of vasoconstriction via inactivation of endothelin-1.

The potent vasoconstrictor peptides, endothelin 1 (ET-1) and angiotensin II control adaptation of blood vessels to fluctuations of blood pressure. Previously we have shown that the circulating level of ET-1 is regulated through its proteolytic cleavage by secreted serine carboxypeptidase, cathepsin A (CathA). However, genetically-modified mouse expressing catalytically inactive CathA S190A mutant retained about 10-15% of the carboxypeptidase activity against ET-1 in its tissues suggesting a presence of parallel/redundant catabolic pathway(s). In the current work we provide direct evidence that the enzyme, which complements CathA action towards ET-1 is a retinoid-inducible lysosomal serine carboxypeptidase 1 (Scpep1), a CathA homolog with previously unknown biological function. We generated a mouse strain devoid of both CathA and Scpep1 activities (DD mice) and found that in response to high-salt diet and systemic injections of ET-1 these animals showed significantly increased blood pressure as compared to wild type mice or those with single deficiencies of CathA or Scpep1. We also found that the reactivity of mesenteric arteries from DD mice towards ET-1 was significantly higher than that for all other groups of mice. The DD mice had a reduced degradation rate of ET-1 in the blood whereas their cultured arterial vascular smooth muscle cells showed increased ET-1-dependent phosphorylation of myosin light chain 2. Together, our results define the biological role of mammalian serine carboxypeptidase Scpep1 and suggest that Scpep1 and CathA together participate in the control of ET-1 regulation of vascular tone and hemodynamics.

Molecular Cloning, Characterization, and Expression Analysis of Cathepsin A in the Chinese Giant Salamander Andrias davidianus.

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.