Differential Bioactivation Profiles of Different GS-441524 Prodrugs in Cell and Mouse Models: ProTide Prodrugs with High Cell Permeability and Susceptibility to Cathepsin A Are More Efficient in Delivering Antiviral Active Metabolites to the Lung.

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.

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.

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.

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.

Human carboxylesterase 1A plays a predominant role in the hydrolytic activation of remdesivir in humans.

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 Km 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.

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.

Prognostic significance and oncogene function of cathepsin A in hepatocellular carcinoma.

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.

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.

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.

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.

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.

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.

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.

Cathepsin A knockdown decreases the proliferation and invasion of A549 lung adenocarcinoma cells.

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 epithelial­mesenchymal transition were analyzed by western blotting. CTSA knockdown enhanced the expression of E­cadherin, but decreased the expression of N­cadherin and ß­catenin 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.

Structural and kinetic evidence of aging after organophosphate inhibition of human Cathepsin A.

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 (KI) of 2 µM followed by VR (KI = 2.8 µM). Bimolecular rate constants for inhibition by cyclosarin and VR were 1.3 × 103 M-1sec-1 and 1.2 × 103 M-1sec-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 KIDFP = 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.

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.

Expression of Cathepsins B, D, and G by the Embryonic Stem Cell-Like Population within Human Keloid Tissues and Keloid-Derived Primary Cell Lines.

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.

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.

Mechanisms of the Ase1/PRC1/MAP65 family in central spindle assembly.

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.

How Complex Is the Concanavalin A-Carboxypeptidase Y Interaction?

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.