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The pathogenesis of osteoporosis (OP) is closely associated with the disrupted balance between osteogenesis and adipogenesis in bone marrow-derived mesenchymal stem cells (BMSCs). We analyzed published single-cell RNA sequencing (scRNA-seq) data to dissect the transcriptomic profiles of bone marrow-derived cells in OP, reviewing 56 377 cells across eight scRNA-seq datasets from femoral heads (osteoporosis or osteopenia n = 5, osteoarthritis n = 3). Seventeen genes, including carboxypeptidase M (CPM), were identified as key osteogenesis-adipogenesis regulators through comprehensive gene set enrichment, differential expression, regulon activity, and pseudotime analyses. In vitro, CPM knockdown reduced osteogenesis and promoted adipogenesis in BMSCs, while adenovirus-mediated CPM overexpression had the reverse effects. In vivo, intraosseous injection of CPM-overexpressing BMSCs mitigated bone loss in ovariectomized mice. Integrated scRNA-seq and bulk RNA sequencing analyses provided insight into the MAPK/ERK pathway's role in the CPM-mediated regulation of BMSC osteogenesis and adipogenesis; specifically, CPM overexpression enhanced MAPK/ERK signaling and osteogenesis. In contrast, the ERK1/2 inhibitor binimetinib negated the effects of CPM overexpression. Overall, our findings identify CPM as a pivotal regulator of BMSC differentiation, which provides new clues for the mechanistic study of OP., (© 2024 Federation of American Societies for Experimental Biology.)

Peptidoglycan (PG) is the main component of the bacterial cell wall; it maintains cell shape while protecting the cell from internal osmotic pressure and external environmental challenges. PG synthesis is essential for bacterial growth and survival, and a series of PG modifications are required to allow expansion of the sacculus. Endopeptidases (EPs), for example, cleave the crosslinks between adjacent PG strands to allow the incorporation of newly synthesized PG. EPs are collectively essential for bacterial growth and must likely be carefully regulated to prevent sacculus degradation and cell death. However, EP regulation mechanisms are poorly understood. Here, we used TnSeq to uncover novel EP regulators in Vibrio cholerae. This screen revealed that the carboxypeptidase DacA1 (PBP5) alleviates EP toxicity. dacA1 is essential for viability on LB medium, and this essentiality was suppressed by EP overexpression, revealing that EP toxicity both mitigates, and is mitigated by, a defect in dacA1. A subsequent suppressor screen to restore viability of ΔdacA1 in LB medium identified hypomorphic mutants in the PG synthesis pathway, as well as mutations that promote EP activation. Our data thus reveal a more complex role of DacA1 in maintaining PG homeostasis than previously assumed., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Alvarado Obando et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Recently, an autosomal recessive subtype of connective tissue disorder within the spectrum of Ehlers-Danlos syndrome (EDS), named classical-like EDS type 2 (clEDS2), was identified. clEDS2 is associated with biallelic variants in the adipocyte enhancer binding protein 1 ( AEBP1 ) gene, specifically, affecting its aortic carboxypeptidase-like protein (ACLP) isoform. We described the 15th patient (13th family) diagnosed with clEDS2. This patient presented with notable similarities in phenotype to the documented cases, along with additional characteristics such as significant prematurity and short stature. An EDS sequencing panel-based analysis revealed homozygous AEBP1: NM_001129.5:c.2923del, p.Ala975Profs*22 likely pathogenic variants, and maternally inherited heterozygous COL11A1: NM_001854.4:c.1160A>G, p.Lys387Arg variant of uncertain significance in our patient. Upon comprehensive review of all previously reported clEDS2 patients, our patient exhibited the following overlapping phenotypes, including cutaneous features: hyperextensibility, atrophic scars/delayed wound healing (100%), easy bruising (100%), excessive skin (93%); skeletal features: generalized joint hypermobility (93%), pes planus (93%), dislocation/subluxation (93%); and cardiovascular features (86%). Our patient did not display symptoms of the critical complications reported in a few individuals, including superior mesenteric artery aneurysms and ruptures, aortic root aneurysm/dissection, spontaneous pneumothoraxes, and bowel ruptures. Together, this case expands the genetic and clinical phenotypic spectrum of AEBP1-related clEDS2.

Papillary thyroid cancer (PTC) is the most prevalent endocrine cancer worldwide. Approximately 30 % of PTC patients will progress into the advanced or metastatic stage and have a relatively poor prognosis. It is well known that epithelial-mesenchymal transition (EMT) plays a pivotal role in thyroid cancer metastasis, resistance to therapy, and recurrence. Clarifying the molecular mechanisms of EMT in PTC progression will help develop the targeted therapy of PTC. The aberrant expression of some transcription factors (TFs) participated in many pathological processes of cancers including EMT. In this study, by performing bioinformatics analysis, adipocyte enhancer-binding protein 1 (AEBP1) was screened as a pivotal TF that promoted EMT and tumor progression in PTC. In vitro experiments indicated that knockout of AEBP1 can inhibit the growth and invasion of PTC cells and reduce the expression of EMT markers including N-cadherin, TWIST1, and ZEB2. In the xenograft model, knockout of AEBP1 inhibited the growth and lung metastasis of PTC cells. By performing RNA-sequencing, dual-luciferase reporter assay, and chromatin immunoprecipitation assay, Bone morphogenetic protein 4 (BMP4) was identified as a downstream target of AEBP1. Over-expression of BMP4 can rescue the inhibitory effects of AEBP1 knockout on the growth, invasion, and EMT phenotype of PTC cells. In conclusion, these findings demonstrated that AEBP1 plays a critical role in PTC progression by regulating BMP4 expression and the AEBP1-BMP4 axis may present novel therapeutic targets for PTC treatment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Human glutamate carboxypeptidase 2 (GCP2) from the M28B metalloprotease group is an important target for therapy in neurological disorders and an established tumor marker. However, its physiological functions remain unclear. To better understand general roles, we used the model organism Caenorhabditis elegans to genetically manipulate its three existing orthologous genes and evaluate the impact on worm physiology. The results of gene knockout studies showed that C. elegans GCP2 orthologs affect the pharyngeal physiology, reproduction, and structural integrity of the organism. Promoter-driven GFP expression revealed distinct localization for each of the three gene paralogs, with gcp-2.1 being most abundant in muscles, intestine, and pharyngeal interneurons, gcp-2.2 restricted to the phasmid neurons, and gcp-2.3 located in the excretory cell. The present study provides new insight into the unique phenotypic effects of GCP2 gene knockouts in C. elegans, and the specific tissue localizations. We believe that elucidation of particular roles in a non-mammalian organism can help to explain important questions linked to physiology of this protease group and in extension to human GCP2 involvement in pathophysiological processes., (© 2024 The Author(s).)

Purpose: Distant metastasis is the main obstacle to treating nasopharyngeal carcinoma (NPC). Tumor distance metastasis is a complex process involving the jointly participation of multiple oncogenes, tumor suppressor genes, and metastasis-associated genes. Enough accurate prognostic genes for evaluating metastasis risk are lacking. We aimed to identify more precise biomarkers for NPC metastasis., Methods: We performed weighted gene co-expression network analysis, differentially expressed gene analysis, univariate and multivariate stepwise Cox regression, and Kaplan-Meier (K-M) survival analyses, on data obtained from RNA sequencing of 10 NPC samples and the public database, to identify key genes correlated with NPC metastasis. Wound healing assays, transwell assays, and immunohistochemistry were conducted to validate our bioinformatic conclusions. Western blotting was performed to evaluate and quantify the effect of identified EMT genes on epithelial-mesenchymal transition (EMT) of NPC., Results: Combined our own RNA sequencing data and public data, we determined carboxypeptidase vitellogenic-like protein (CPVL) as a tumor suppressor for NPC. Pathway enrichment analyses indicated that genes associated with CPVL are involved in EMT. NPC with low CPVL expression had high tumor purity and low levels of immune cells. Experimental results showed that CPVL protein predominantly expressed in cytoplasmic and membranous and it exhibited higher expression levels in NPC tissues without distant metastasis than those with distant metastasis. CPVL inhibits the migration and invasive capability of NPC cells. Overexpression of CPVL upregulates E-cadherin and ZO-1, whereas it downregulates vimentin, suggesting that CPVL suppresses tumor metastasis by inhibiting EMT., Conclusion: CPVL inhibits migration and invasion of NPC cells and is associated with tumor metastasis suppression through upregulating epithelial marker and inhibiting mesenchymal marker expression and could be a prognostic biomarker for metastasis risk evaluation in NPC., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Streptomyces lividans is an efficient host for extracellular overproduction of recombinant proteins. To enhance the overexpression strength of S. lividans, we designed several kinds of expression plasmids with different positioning of repeat promoters. The effect of repeat promoters was evaluated by measuring the accumulated amounts of a stable transglutaminase or an unstable carboxypeptidase that was secreted into the medium. Successive tandem positions of repeat promoters upstream of the normal promoter did not enhance the expression of transglutaminase. Discrete positions of repeat promoters both upstream and downstream of the normal promoter enhanced the expression of transglutaminase to 2-fold, and the downstream ones also enhanced the expression of carboxypeptidase to 1.7-fold. On the other hand, there were still some constructs of plasmids with discrete repeat promoters that did not promote the expression of the target enzymes, indicating the complexity of the mechanisms of repeat promoters working on gene expression., (© The Author(s) 2023. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Objectives: Aortic carboxypeptidase-like protein (ACLP) is an extracellular protein involved in adipogenesis, epithelial-mesenchymal transition, epithelial cell hyperplasia, and collagen fibrogenesis. This study mainly aimed to analyze the potential role of adipocyte enhancer binding protein 1 (AEBP1), the ACLP-encoding gene, as a pathological target or prognostic marker for liver fibrosis regardless of etiology., Methods: Dysregulation pattern, clinical relevance, and biological significance of AEBP1 gene in liver fibrosis were analyzed using publicly available transcriptomic profiles, different liver fibrosis mouse models, biological databases, and AEBP1 gene silencing followed by RNA sequencing in human hepatic stellate cells (HSCs)., Results: AEBP1 gene expression was upregulated and positively correlated with liver fibrogenesis independent of etiology, the protein of which was further verified in liver fibrosis mouse models induced by different pathogenic factors. A higher expression of liver AEBP1 gene had the potential to predict poor prognosis in liver fibrosis. Systematic bioinformatic analyses revealed that AEBP1 expression was HSCs-specific and associated with extracellular matrix (ECM) remodeling and its downstream mechanical-chemical signaling transition. AEBP1 knockdown by specific small interfering RNAs (siRNAs) in HSCs inhibited ECM-receptor interaction and immune-related pathways as well as HSC proliferation or activation., Conclusion: A high expression of AEBP1 was specifically associated with liver fibrosis and was related to a poor prognosis and predicted the role of AEBP1 in HSCs, providing a new insight for understanding AEBP1 in liver fibrosis., (© 2023 Chinese Medical Association Shanghai Branch, Chinese Society of Gastroenterology, Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine and John Wiley & Sons Australia, Ltd.)

Background: Colorectal cancer (CRC) is a common malignant tumor, and this study aims to explore the role and the regulatory mechanism of carboxypeptidase A6 (CPA6) in CRC cells., Methods: Specific shRNA targeting CPA6 mRNA was transfected into NCM460 and HT29 cells to down-regulate CPA expression, and expression plasmid was transfected into HCT116 cells to exogenously overexpress CPA6. The dual luciferase assay was used to detect the direct binding of miR-96-3p to CPA6 3'UTR. Phosphorylation and activation of Akt were detected using Western blot. Cells were treated with miR-96-3p mimics, Akt inhibitor (MK-2206) or agonist (SC79) for rescue experiments. The cell functions were evaluated using CCK-8, clone formation, transwell, and Western blot assays. Xenograft tumor assay was also used to analyze the effect of altered CPA6 expression on tumor growth., Results: Knockdown of CPA6 promoted the proliferation, clone formation, migration, and invasion of NCM460 and HT29 cells in vitro, and the tumor growth of nude mouse xenograft tumor in vivo. Moreover, over-expression of CPA6 significantly inhibited the malignant proliferation and invasion of HCT116 cells in vitro, and the tumor growth of xenograft tumor in vivo. Furthermore, miR-96-3p could directly regulate CPA6 expression by targeting its 3'UTR, and miR-96-3p mimics rescued the inhibitory effects of CPA6 overexpression on the malignant proliferation and invasion of CRC cells. Finally, CPA6 knockdown enhanced Akt/mTOR phosphorylation and activation, while CPA6 overexpression inhibited Akt/mTOR activation. The regulatory effect of CPA6 on Akt/mTOR signaling was naturally regulated by miR-96-3p. Akt inhibitor or agonist rescued the effects of CPA6 knockdown or overexpression on proliferation and EMT of colon cancer cells., Conclusion: CPA6 has a significant tumor suppressive effect on CRC by inhibiting the activation of Akt/mTOR signaling, and miR-96-3p negatively regulates the expression of CPA6., Competing Interests: Declaration of competing interest All authors declare that they have no conflict of interest., (Copyright © 2023. Published by Elsevier Inc.)

Angiopoietin-1 (ANG1) is a pro-angiogenic regulator that contributes to the progression of solid tumors by stimulating the proliferation, migration and tube formation of vascular endothelial cells, as well as the renewal and stability of blood vessels. However, the functions and mechanisms of ANG1 in triple-negative breast cancer (TNBC) are unclear. The clinical sample database shows that a higher level of ANG1 in TNBC is associated with poor prognosis compared to non-TNBC. In addition, knockdown of ANG1 inhibits TNBC cell proliferation and induces cell cycle G1 phase arrest and apoptosis. Overexpression of ANG1 promotes tumor growth in nude mice. Mechanistically, ANG1 promotes TNBC by upregulating carboxypeptidase A4 (CPA4) expression. Overall, the ANG1-CPA4 axis can be a therapeutic target for TNBC.

Gene duplication is a key first step in the process of expanding the functionality of a multigene family. In order to better understand the process of gene duplication and its role in the formation of new enzymes, we investigated recent duplication events in the M14 family of proteolytic enzymes. Within vertebrates, four of 23 M14 genes were frequently found in duplicate form. While AEBP1, CPXM1, and CPZ genes were duplicated once through a large-scale, likely whole-genome duplication event, the CPO gene underwent many duplication events within fish and Xenopus lineages. Bioinformatic analyses of enzyme specificity and conservation suggested a greater amount of neofunctionalization and purifying selection in CPO paralogs compared with other CPA/B enzymes. To examine the functional consequences of evolutionary changes on CPO paralogs, the four CPO paralogs from Xenopus tropicalis were expressed in Sf9 and HEK293T cells. Immunocytochemistry showed subcellular distribution of Xenopus CPO paralogs to be similar to that of human CPO. Upon activation with trypsin, the enzymes demonstrated differential activity against three substrates, suggesting an acquisition of new function following duplication and subsequent mutagenesis. Characteristics such as gene size and enzyme activation mechanisms are possible contributors to the evolutionary capacity of the CPO gene., (© 2023. The Author(s).)

Background: Studies have shown that excessive iron can lead to an increased incidence of cancer. The role of adipocyte enhancer-binding protein 1 (AEBP1) on ferroptosis is unknown. Thus, we explored the effect of AEBP1 silencing in regulation of ferroptosis in cisplatin-resistant oral cancer cells., Methods: The functions of AEBP1 silencing and sulfasalazine (SSZ) treatment were determined on oral cancer cell lines and tumor xenograft mouse models. Then we evaluated the functions of AEBP1 on cell proliferation, migration, invasion, lipid reactive oxygen species (ROS), labile iron pool (LIP) and free iron, lipid peroxidation, and expression levels of ferroptosis-related genes., Results: AEBP1 was highly expressed in oral cancer cells and tissues. AEBP1 silencing inhibited oral cancer cell proliferation, migration, and invasion after SSZ treatment. SSZ-induced ferroptosis is due to enhanced ROS level, free iron, and lipid peroxidation, which were distinctly increased by AEBP1 silencing. Meanwhile, AEBP1 silencing enhanced the effects of SSZ on levels of LIP and Fe 2+ , lipid peroxidation, as well as the expression levels of ferroptosis-related genes in the tumor xenograft mouse models. Importantly, AEBP1 silencing suppressed tumor growth in vivo. Furthermore, silencing of AEBP1 might activate the JNK/ P38 /ERK pathway., Conclusion: This research suggested that silencing of AEBP1 predisposes cisplatin-resistant oral cancer cells to ferroptosis via the JNK/p38 /ERK pathway., (© 2022. The Author(s).)

The serine carboxypeptidase-like protein (SCPL) family plays a key part in plant growth, development and stress responses. However, the serine carboxypeptidase-like (SCPL) proteins in Brassica napus L. (B. napus) have not been reported yet. Here, we identified a total of 117 putative SCPL genes in B. napus, which were unevenly distributed on all 19 chromosomes and were divided into three groups (carboxypeptidase Ⅰ to Ⅲ) according to their phylogenetic relationships. Synteny and duplication analysis revealed that the SCPL gene family of B. napus was amplified during allopolyploidization, in which the whole genome triplication and dispersed duplication played critical roles. After the separation of Brassica and Arabidopsis lineages, orthologous gene analysis showed that many SCPL genes were lost during the evolutionary process in B. rapa, B. oleracea and B. napus. Subsequently, the analyses of the gene structure, conserved motifs, cis-element and expression patterns showed that the members in the same group were highly conserved. Furthermore, candidate gene based association study suggested the role of BnSCPL52 in controlling seed number per silique, seed weight and silique length and a CAPS marker was developed to distinguish different haplotypes. Our results provide an overview of rapeseed SCPL genes that enable us for further functional research and benefit the marker-assisted breeding in Brassica napus., (Copyright © 2022 Elsevier Masson SAS. All rights reserved.)

CCP6 is a member of cytosolic carboxypeptidases (CCPs) family, an eraser of a reversible protein posttranslational modification - polyglutamylation, and represents a potential therapeutic target. Currently, production of CCPs mainly depends on eukaryotic expression system, which is time-consuming and costly. Here, we reported that mouse origin full-length CCP6 can be successfully expressed in the soluble fraction of bacteria ArcticExpress (DE3) strain. However, the recombinant mCCP6 was initially co-purified with Cpn60 in a stoichiometric ratio of roughly 1:7 and exhibited no enzyme activity. When coupled with a step to promote the release of the substrate protein from the chaperonins by treatment with ATP/Mg 2+ /K + , the recombinant CCP6 with deglutamylation activity was obtained, though still partially associated with Cpn60. This is the first report, to our knowledge, that the successful expression and purification of active recombinant mammalian CCPs using a bacterial system was achieved., (Copyright © 2022. Published by Elsevier Inc.)

Vancomycin and β-lactams are clinically important antibiotics that inhibit the formation of peptidoglycan cross-links, but their binding targets are different. The binding target of vancomycin is d-alanine-d-alanine (d-Ala-d-Ala), whereas that of β-lactam is penicillin-binding proteins (PBPs). In this study, we revealed the divergent effects of peptidoglycan (PG) carboxypeptidase DacA on vancomycin and β-lactam resistance in Escherichia coli and Bacillus subtilis. The deletion of DacA induced sensitivity to most β-lactams, whereas it induced strong resistance toward vancomycin. Notably, both phenotypes did not have a strong association with ld-transpeptidases, which are necessary for the formation of PG 3-3 cross-links and covalent bonds between PG and an Lpp outer membrane (OM) lipoprotein. Vancomycin resistance was induced by an increased amount of decoy d-Ala-d-Ala residues within PG, whereas β-lactam sensitivity was associated with physical interactions between DacA and PBPs. The presence of an OM permeability barrier strongly strengthened vancomycin resistance, but it significantly weakened β-lactam sensitivity. Collectively, our results revealed two distinct functions of DacA, which involved inverse modulation of bacterial resistance to clinically important antibiotics, β-lactams and vancomycin, and presented evidence for a link between DacA and PBPs. IMPORTANCE Bacterial PG hydrolases play important roles in various aspects of bacterial physiology, including cytokinesis, PG synthesis, quality control of PG, PG recycling, and stress adaptation. Of all the PG hydrolases, the role of PG carboxypeptidases is poorly understood, especially regarding their impacts on antibiotic resistance. We have revealed two distinct functions of PG carboxypeptidase DacA with respect to antibiotic resistance. The deletion of DacA led to sensitivity to most β-lactams, while it caused strong resistance to vancomycin. Our study provides novel insights into the roles of PG carboxypeptidases in the regulation of antibiotic resistance and a potential clue for the development of a drug to improve the clinical efficacy of β-lactam antibiotics.

The M14 family metal carboxypeptidase genes play an important role in digestion and pathogenic infections in the gut of insects. However, the roles of these genes in Antheraea pernyi (Guérin-Méneville, 1855) remain to be analyzed. In the present study, we cloned a highly expressed M14 metal carboxypeptidase gene (ApMCP1) found in the gut and discovered that it contained a 1,194 bp open reading frame encoding a 397-amino acid protein with a predicted molecular weight of 45 kDa. Furthermore, 14 members of the M14 family metal carboxypeptidases (ApMCP1-ApMCP14) were identified in the A. pernyi genome, with typical Zn_pept domains and two Zn-anchoring motifs, and were further classified into M14A, M14B, and M14D subfamilies. Expression analysis indicated that ApMCP1 and ApMCP9 were mainly expressed in the gut. Additionally, we observed that ApMCP1 and ApMCP9 displayed opposite expression patterns after starvation, highlighting their functional divergence during digestion. Following natural infection with baculovirus NPV, their expression was significantly upregulated in the gut of A. pernyi. Our results suggest that the M14 family metal carboxypeptidase genes are conservatively digestive enzymes and evolutionarily involved in exogenous pathogenic infections., (© The Author(s) 2022. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Serine carboxypeptidase-like acyltransferases (SCPL-ATs) play a vital role in the diversification of plant metabolites. Galloylated flavan-3-ols highly accumulate in tea (Camellia sinensis), grape (Vitis vinifera), and persimmon (Diospyros kaki). To date, the biosynthetic mechanism of these compounds remains unknown. Herein, we report that two SCPL-AT paralogs are involved in galloylation of flavan-3-ols: CsSCPL4, which contains the conserved catalytic triad S-D-H, and CsSCPL5, which has the alternative triad T-D-Y. Integrated data from transgenic plants, recombinant enzymes, and gene mutations showed that CsSCPL4 is a catalytic acyltransferase, while CsSCPL5 is a non-catalytic companion paralog (NCCP). Co-expression of CsSCPL4 and CsSCPL5 is likely responsible for the galloylation. Furthermore, pull-down and co-immunoprecipitation assays showed that CsSCPL4 and CsSCPL5 interact, increasing protein stability and promoting post-translational processing. Moreover, phylogenetic analyses revealed that their homologs co-exist in galloylated flavan-3-ol- or hydrolyzable tannin-rich plant species. Enzymatic assays further revealed the necessity of co-expression of those homologs for acyltransferase activity. Evolution analysis revealed that the mutations of the CsSCPL5 catalytic residues may have taken place about 10 million years ago. These findings show that the co-expression of SCPL-ATs and their NCCPs contributes to the acylation of flavan-3-ols in the plant kingdom., (© 2022 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

The detyrosination-tyrosination cycle involves the removal and religation of the C-terminal tyrosine of α-tubulin and is implicated in cognitive, cardiac, and mitotic defects. The vasohibin-small vasohibin-binding protein (SVBP) complex underlies much, but not all, detyrosination. We used haploid genetic screens to identify an unannotated protein, microtubule associated tyrosine carboxypeptidase (MATCAP), as a remaining detyrosinating enzyme. X-ray crystallography and cryo-electron microscopy structures established MATCAP's cleaving mechanism, substrate specificity, and microtubule recognition. Paradoxically, whereas abrogation of tyrosine religation is lethal in mice, codeletion of MATCAP and SVBP is not. Although viable, defective detyrosination caused microcephaly, associated with proliferative defects during neurogenesis, and abnormal behavior. Thus, MATCAP is a missing component of the detyrosination-tyrosination cycle, revealing the importance of this modification in brain formation.

Osteonecrosis of the femoral head (ONFH) involves necrosis of bone and bone marrow of the femoral head caused by ischemia with unknown etiology. Previous genetic studies on ONFH failed to produce consistent results, presumably because ONFH has various causes with different genetic backgrounds and the underlying diseases confounded the associations. Steroid-associated ONFH (S-ONFH) accounts for one-half of all ONFH, and systemic lupus erythematosus (SLE) is a representative disease underlying S-ONFH. We performed a genome-wide association study (GWAS) to identify genetic risk factors for S-ONFH in patients with SLE. We conducted a two-staged GWAS on 636 SLE patients with S-ONFH and 95 588 non-SLE controls. Among the novel loci identified, we determined S-ONFH-specific loci by comparing allele frequencies between SLE patients without S-ONFH and non-SLE controls. We also used Korean datasets comprising 148 S-ONFH cases and 37 015 controls to assess overall significance. We evaluated the functional annotations of significant variants by in silico analyses. The Japanese GWAS identified 4 significant loci together with 12 known SLE susceptibility loci. The four significant variants showed comparable effect sizes on S-ONFH compared with SLE controls and non-SLE controls. Three of the four loci, MIR4293/MIR1265 [odds ratio (OR) = 1.99, P-value = 1.1 × 10-9)], TRIM49/NAALAD2 (OR = 1.65, P-value = 4.8 × 10-8) and MYO16 (OR = 3.91, P-value = 4.9 × 10-10), showed significant associations in the meta-analysis with Korean datasets. Bioinformatics analyses identified MIR4293, NAALAD2 and MYO16 as candidate causal genes. MIR4293 regulates a PPARG-related adipogenesis pathway relevant to S-ONFH. We identified three novel susceptibility loci for S-ONFH in SLE., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Left ventricular hypertrophy (LVH) is a major risk factor for adverse cardiovascular events. Recently, a novel candidate gene encoding the carboxypeptidase X member 2 (CPXM2) was found to be associated with hypertension-induced LVH. CPXM2 belongs to the M14 family of metallocarboxypeptidases, yet it lacks detectable enzyme activity, and its function remains unknown. Here, we investigated the impact of micro (mi)RNA-29b, miRNA-195, and miRNA-497 on the posttranscriptional expression control of CPXM2. Candidate miRNAs for CPXM2 expression control were identified in silico. CPXM2 expression in rat cardiomyocytes (H9C2) was characterized via real-time PCR, Western blotting, and immunofluorescence. Direct miRNA/target mRNA interaction was analysed by dual luciferase assay. CPXM2 was expressed in H9C2 and co-localised with z-disc associated protein PDZ and LIM domain 3 (Pdlim3). Transfection of H9C2 with miRNA-29b, miRNA-195, and miRNA-497 led to decreased levels of CPXM2 mRNA and protein, respectively. Results of dual luciferase assays revealed that miRNA-29b and miRNA-497, but not miRNA-195, directly regulated CPXM2 expression on a posttranscriptional level via binding to the 3'UTR of CPXM2 mRNA. We identified two miRNAs capable of the direct posttranscriptional expression control of CPXM2 expression in rat cardiomyocytes. This novel data may help to shed more light on the-so far-widely unexplored expression control of CPXM2 and its potential role in LVH.

Background: Kallikrein is generated when the contact system is activated, subsequently cleaving high molecular weight kininogen to bradykinin (BK). BK binds to bradykinin receptor 2, causing vascular leakage. BK is inactivated by proteolysis by the plasma carboxypeptidase B2 and N (CPB2 and CPN). CPN is constitutively active but CPB2 is generated from its zymogen, proCPB2., Objectives: Determine the role of CPB2 and CPN in the regulation of vascular leakage., Methods: Mice deficient in CPB2, CPN, or both (Cpb2 -/- , Cpn -/- , and Cpb2 -/- /Cpn -/- ) were compared with wild-type mice (WT) in a model of vascular leakage caused by skin irritation. In some experiments, mice were pretreated with antibodies that prevent activation of proCPB2., Results: Skin irritation increased vascular leakage most in Cpb2 -/- /Cpn -/- , less in Cpb2 -/- and Cpn -/- , and least in WT mice. There was no difference in vascular leakage without the challenge. Antibodies inhibiting activation of proCPB2 by plasmin, but not by the thrombin/thrombomodulin complex, increased vascular leakage to the level seen in Cpb2 -/- mice. There was no change in levels of markers of coagulation and fibrinolysis., Conclusions: Bradykinin is inactivated by both CPB2 and CPN independently. Plasmin is the activator of proCPB2 in this model. Mice lacking both plasma carboxypeptidases have more vascular leak than those lacking either alone. Although BK levels were not determined, BK is the likely substrate for CPB2 and CPN in this model., (© 2021 International Society on Thrombosis and Haemostasis.)

The hospital-acquired pathogen Acinetobacter baumannii possesses a complex cell envelope that is key to its multidrug resistance and virulence. The bacterium, however, lacks many canonical enzymes that build the envelope in model organisms. Instead, A. baumannii contains a number of poorly annotated proteins that may allow alternative mechanisms of envelope biogenesis. We demonstrated previously that one of these unusual proteins, ElsL, is required for maintaining a characteristic short rod shape and for withstanding antibiotics that attack the septal cell wall. Curiously, ElsL is composed of a leaderless YkuD-family domain usually found in secreted, cell wall-modifying l,d-transpeptidases (LDTs). Here, we show that, rather than being an LDT, ElsL is actually a new class of cytoplasmic l,d-carboxypeptidase (LDC) that provides a critical step in cell wall recycling previously thought to be missing from A. baumannii. Absence of ElsL impairs cell wall integrity, morphology, and intrinsic resistance due to buildup of murein tetrapeptide precursors, toxicity of which is bypassed by preventing muropeptide recycling. Multiple pathways in the cell become sites of vulnerability when ElsL is inactivated, including l,d-cross-link formation, cell division, and outer membrane lipid homoeostasis, reflecting its pleiotropic influence on envelope physiology. We thus reveal a novel class of cell wall-recycling LDC critical to growth and homeostasis of A. baumannii and likely many other bacteria. IMPORTANCE To grow efficiently, resist antibiotics, and control the immune response, bacteria recycle parts of their cell wall. A key step in the typical recycling pathway is the reuse of cell wall peptides by an enzyme known as an l,d-carboxypeptidase (LDC). Acinetobacter baumannii, an "urgent-threat" pathogen causing drug-resistant sepsis in hospitals, was previously thought to lack this enzymatic activity due to absence of a known LDC homolog. Here, we show that A. baumannii possesses this activity in the form of an enzyme class not previously associated with cell wall recycling. Absence of this protein intoxicates and weakens the A. baumannii cell envelope in multiple ways due to the accumulation of dead-end intermediates. Several other organisms of importance to health and disease encode homologs of the A. baumannii enzyme. This work thus reveals an unappreciated mechanism of cell wall recycling, manipulation of which may contribute to enhanced treatments targeting the bacterial envelope.

Epithelial-mesenchymal transition (EMT) is involved in various tumor processes, including tumorigenesis, tumor cell migration and metastasis, tumor stemness, and therapeutic resistance. Therefore, it is important to identify the genes most associated with EMT and develop them as therapeutic targets. In this work, we first analyzed EMT hallmark gene expression profiles among 10,535 pan-cancer samples from The Cancer Genome Atlas (TCGA) and divided them into EMT high and EMT low groups according to the metagene scores. Then, we identified 12 genes that were most associated with high EMT metagene score ( R > 0.9) in 329 colon adenocarcinoma (COAD) patients. Among them, only 4 genes (AEBP1, KCNE4, GFPT2, and FAM26E) had statistically significant differences in prognosis ( P < 0.05). Next, we selected AEBP1 as a candidate and showed that AEBP1 mRNA levels and EMT biomarkers strongly coexpressed in 329 COAD samples. In addition, AEBP1 was highly expressed and associated with poor clinical outcomes and prognosis in COAD patients. Finally, to explore whether AEBP1-mediated EMT was related to the tumor microenvironment (TME), we examined AEBP1 expression levels at the single-cell levels. Our results showed that AEBP1 levels were extremely high in tumor-associated fibroblasts, which may induce EMT. AEBP1 expression was also positively correlated with the expression of fibroblast biomarkers and also with EMT metascores, suggesting that AEBP1-mediated EMT may be associated with the stimulation of fibroblast activation. Therefore, AEBP1 may be a promising target for EMT inhibition, which reduces cancer metastasis and drug resistance in COAD patients., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2021 Dandan Li et al.)

As one of the most abundant bacteria in the human oral cavity, Fusobacterium nucleatum is closely involved in various oral diseases and is also a risk factor for other diseases. The peptidases of F. nucleatum can digest exogenous peptides into amino acids to satisfy its nutrient requirements. Here, a putative F. nucleatum peptidase, termed S9Cfn, which belongs to the S9C peptidase family was identified. Enzymatic activity assays combined with mass-spectrometric analysis revealed that S9Cfn is a carboxypeptidase, but not an aminopeptidase as previously annotated. The crystal structure of the S9Cfn tetramer was solved at 2.6 Å resolution and was found to contain a pair of oligomeric pores in the center. Structural analysis, together with site-directed mutagenesis and enzymatic activity assays, revealed a substrate-entrance tunnel that extends from each oligomeric pore to the catalytic triad, adjacent to which three conserved arginine residues are responsible for substrate binding. Moreover, comparison with other S9 peptidase structures indicated drastic conformational changes of the oligomeric pores during the catalytic cycle. Together, these findings increase the knowledge of this unique type of tetrameric carboxypeptidase and provide insight into the homeostatic control of microbiota in the human oral cavity.

Classical Ehlers-Danlos syndrome (cEDS) is a heritable connective tissue disorder mainly caused by pathogenic variants in COL5A1 or COL5A2, encoding type V collagen. Its diagnosis, based on clinical criteria and molecular confirmation, can be challenging. We report the molecular and clinical characteristics of 168 probands (72 clinically evaluated at our center) and 65 relatives with a clinical presentation of cEDS. Type V collagen defects were found in 145 probands, 121 (83.5%) were located in COL5A1 and 24 (16.5%) in COL5A2. Although 85.6% of molecularly confirmed patients presented the two major clinical criteria (generalized joint hypermobility, hyperextensible skin with atrophic scarring), significant inter- and intrafamilial phenotypic variability was noted. COL5A2 variants often caused a more severe phenotype. Vascular complications were rare in individuals with type V collagen defects (1.4%). Among the 72 probands clinically evaluated in our center, the mutation detection rate was 82.0%. The majority (68.1%) harbored COL5A1/COL5A2 defects. Yet, 13.9% harbored a defect in another gene (COL1A1, PLOD1, TNXB, AEBP1) highlighting important clinical overlap and the need for molecular confirmation of the diagnosis as this has implications regarding follow-up and genetic counseling. Eighteen percent of the 72 probands remained molecularly unexplained and a COL5A1 variant of unknown significance was identified in 6.9%., (© 2021 Wiley Periodicals LLC.)

Bacillus subtilis CW14, isolated from fresh elk droppings in Beijing Zoo, is a Gram-positive, conferred Generally Recognized as Safe (GRAS) bacterium with the capacity of ochratoxin A (OTA) detoxification. The genome sequence of the CW14 strain showed a size of 4,287,522 bp with 44.06% GC content. It was predicted many putative enzymes involved in degrading mycotoxin by analyzing the signal peptides and the transmembrane regions. Nine extracellular enzymes were predicted relating to OTA detoxification, including four D-Ala-D-Ala carboxypeptidases, two hydrolases, two amidases, and one lactamase. Indeed, two of the carboxypeptidase genes dacA and dacB, expressed in Escherichia coli, were verified contributing to OTA detoxification. DacA and OTA were mixed incubated for 24 h, and the degradation rate reached 71.3%. After purification, the concentration of recombinant DacA protein was 0.5 mg/mL. Bacillus subtilis CW14 and its carboxypeptidases may be used as OTA detoxification agents in food and feed industry production., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: Glioma is the most common malignant tumor of the brain in adult patients. The standardized treatment protocol is based on surgical therapy, supplemented with radiotherapy and chemotherapy. However, the prognosis is still unsatisfied. Chemoresistance is one of the most important reason for the poor prognosis of glioma patients. It has confirmed that glioma stem cell (GSC) is one of the reasons for chemoresistance., Methods: In this study, three datasets (GSE23806, COSMIC, and TCGA) were used to perform the analysis to search for the key genes related to GSC, temozolomide (TMZ) resistance, and prognosis. The key gene for further research was selected by reviewing the previous studies. The selected gene investigated the relation between expression levels and clinical characteristics in both TCGA and CGGA dataset. The bioinformatics analysis was performed by Gene Ontology (GO) analysis. The survival analysis was performed by Kaplan-Meier survival analysis., Results: AE binding protein 1 (AEBP1) was selected for further analysis. AEBP1 was overexpressed in GSCs and TMZ resistance cells. In both TCGA and CGGA dataset, the results showed that the expression level of AEBP1 was increased in glioblastoma (GBM) samples, IDH wild-type samples, and MGMT promoter unmethylated samples. Meanwhile, AEBP1 expression was positively related to several GSC markers. GO analysis showed that AEBP1 was related to immune response, cell adhesion, apoptotic process, inflammatory response, positive regulation of cell proliferation, angiogenesis, response to drug, and response to hypoxia. The survival analysis showed that the overexpressed level of AEBP1 was correlated with short survival time in both glioma and GBM patients., Conclusion: In summary, AEBP1 was related with GSC-induced TMZ resistance. Our study showed that AEBP1 might be an oncogene and a new effective therapeutic target for the treatment of glioma., Competing Interests: The authors declare no conflict of interest., (Copyright © 2021 Kuanyu Wang et al.)

MicroRNAs (miRNAs) control various biological processes by repressing target mRNAs. In plants, miRNAs mediate target gene repression via both mRNA cleavage and translational repression. However, the mechanism underlying this translational repression is poorly understood. Here, we found that Arabidopsis thaliana HYPONASTIC LEAVES1 (HYL1), a core component of the miRNA processing machinery, regulates miRNA-mediated mRNA translation but not miRNA biogenesis when it localized in the cytoplasm. Cytoplasmic HYL1 localizes to the endoplasmic reticulum and associates with ARGONAUTE1 (AGO1) and ALTERED MERISTEM PROGRAM1. In the cytoplasm, HYL1 monitors the distribution of AGO1 onto polysomes, binds to the mRNAs of target genes, represses their translation, and partially rescues the phenotype of the hyl1 null mutant. This study uncovered another function of HYL1 and provides insight into the mechanism of plant gene regulation., (© American Society of Plant Biologists 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The integrity of the cell envelope of E. coli relies on the concerted activity of multi-protein machineries that synthesize the peptidoglycan (PG) and the outer membrane (OM). Our previous work found that the depletion of lipopolysaccharide (LPS) export to the OM induces an essential PG remodeling process involving LD-transpeptidases (LDTs), the glycosyltransferase function of PBP1B and the carboxypeptidase PBP6a. Consequently, cells with defective OM biogenesis lyse if they lack any of these PG enzymes. Here we report that the morphological defects, and lysis associated with a ldtF mutant with impaired LPS transport, are alleviated by the loss of the predicted OM-anchored lipoprotein ActS (formerly YgeR). We show that ActS is an inactive member of LytM-type peptidoglycan endopeptidases due to a degenerated catalytic domain. ActS is capable of activating all three main periplasmic peptidoglycan amidases, AmiA, AmiB, and AmiC, which were previously reported to be activated only by EnvC and/or NlpD. Our data also suggest that in vivo ActS preferentially activates AmiC and that its function is linked to cell envelope stress., (© 2021 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

Caldicellulosiruptor bescii is the most thermophilic, cellulolytic bacterium known and has the native ability to utilize unpretreated plant biomass. Cellulase A (CelA) is the most abundant enzyme in the exoproteome of C. bescii and is primarily responsible for its cellulolytic ability. CelA contains a family 9 glycoside hydrolase and a family 48 glycoside hydrolase connected by linker regions and three carbohydrate-binding domains. A truncated version of the enzyme (TM1) containing only the endoglucanase domain is thermostable and actively degrades crystalline cellulose. A catalytically active TM1 was successfully produced via the attachment of the PelB signal peptide (P-TM1), which mediates post-translational secretion via the SecB-dependent translocation pathway. We sought to enhance the extracellular secretion of TM1 using an alternative pathway, the signal recognition particle (SRP)-dependent translocation pathway. The co-translational extracellular secretion of TM1 via the SRP pathway (D-TM1) resulted in a specific activity that was 4.9 times higher than that associated with P-TM1 overexpression. In batch fermentations, the recombinant Escherichia coli overexpressing D-TM1 produced 1.86 ± 0.06 U/ml of TM1 in the culture medium, showing a specific activity of 1.25 ± 0.05 U/mg cell, 2.7- and 3.7-fold higher than the corresponding values of the strain overexpressing P-TM1. We suggest that the TM1 secretion system developed in this study can be applied to enhance the capacity of E. coli as a microbial cell factory for the extracellular secretion of this as well as a variety proteins important for commercial production., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society of Industrial Microbiology and Biotechnology.)

Plant serine carboxypeptidase-like acyltransferases (SCPL-AT) have similar structural characteristics and high homology compared to the serine carboxypeptidase. They can transfer the acyl from acyl glucose esters to many natural products, participate in the acylation modification of plant secondary metabolites, enrich the structural diversity of natural products, and improve the physicochemical properties such as water solubility and stability of compounds. This review summarizes the structural characteristics, catalytic mechanism, functional characterization, and biocatalytic applications of SCPL-AT from plants. This will help to promote the functional characterization of these acyltransferase genes and the biosynthesis of useful plant secondary metabolites by synthetic biotechnology.

Background: Brassinosteroid-insensitive 1 suppressor 1 (BRS1) is a serine carboxypeptidase that mediates brassinosteroid signaling and participates in multiple developmental processes in Arabidopsis. However, little is known about the precise role of BRS1 in this context., Results: In this study, we analyzed transcriptional and proteomic profiles of Arabidopsis seedlings overexpressing BRS1 and found that this gene was involved in both cold stress responses and redox regulation. Further proteomic evidence showed that BRS1 regulated cell redox by indirectly interacting with cytosolic NADP + -dependent isocitrate dehydrogenase (cICDH). One novel alternative splice form of BRS1 was identified in over-expression mutants brs1-1D, which may confer a new role in plant development and stress responses., Conclusions: This study highlights the role of BRS1 in plant redox regulation and stress responses, which extends our understanding of extracellular serine carboxypeptidases.

The gram-negative bacterial cell envelope is made up of an outer membrane (OM), an inner membrane (IM) that surrounds the cytoplasm, and a periplasmic space between the two membranes containing peptidoglycan (PG or murein). PG is an elastic polymer that forms a mesh-like sacculus around the IM, protecting cells from turgor and environmental stress conditions. In several bacteria, including Escherichia coli , the OM is tethered to PG by an abundant OM lipoprotein, Lpp (or Braun's lipoprotein), that functions to maintain the structural and functional integrity of the cell envelope. Since its discovery, Lpp has been studied extensively, and although l,d-transpeptidases, the enzymes that catalyze the formation of PG-Lpp linkages, have been earlier identified, it is not known how these linkages are modulated. Here, using genetic and biochemical approaches, we show that LdtF (formerly yafK ), a newly identified paralog of l,d-transpeptidases in E. coli , is a murein hydrolytic enzyme that catalyzes cleavage of Lpp from the PG sacculus. LdtF also exhibits glycine-specific carboxypeptidase activity on muropeptides containing a terminal glycine residue. LdtF was earlier presumed to be an l,d-transpeptidase; however, our results show that it is indeed an l,d-endopeptidase that hydrolyzes the products generated by the l,d-transpeptidases. To summarize, this study describes the discovery of a murein endopeptidase with a hitherto unknown catalytic specificity that removes the PG-Lpp cross-links, suggesting a role for LdtF in the regulation of PG-OM linkages to maintain the structural integrity of the bacterial cell envelope., Competing Interests: The authors declare no competing interest.

The detyrosination/retyrosination cycle is the most common post-translational modification of α-tubulin. Removal of the conserved C-terminal tyrosine of α-tubulin by a still elusive tubulin tyrosine carboxypeptidase, and religation of this tyrosine by a tubulin tyrosine ligase (TTL), are probably common to all eukaryotes. Interestingly, for plants, the only candidates qualifying as potential TTL homologs are the tubulin tyrosine ligase-like 12 proteins. To get insight into the biological functions of these potential TTL homologs, we cloned the rice TTL-like 12 protein (OsTTLL12) and generated overexpression OsTTLL12-RFP lines in both rice and tobacco BY-2 cells. We found, unexpectedly, that overexpression of this OsTTLL12-RFP increased the relative abundance of detyrosinated α-tubulin in both coleoptile and seminal root, correlated with more stable microtubules. This was independent of the respective orientation of cortical microtubule, and followed by correspondingly changing growth of coleoptiles and seminal roots. A perturbed organization of phragmoplast microtubules and disoriented cell walls were further characteristics of this phenotype. Thus, the elevated tubulin detyrosination in consequence of OsTTLL12 overexpression affects structural and dynamic features of microtubules, followed by changes in the axiality of cell plate deposition and, consequently, plant growth., (© 2020 The Authors. Journal of Integrative Plant Biology published by John Wiley & Sons Australia, Ltd on behalf of Institute of Botany, Chinese Academy of Sciences.)

Vasohibin-1 (VASH1) is a VEGF-inducible endothelium-derived angiogenesis inhibitor, and vasohibin-2 (VASH2), its homolog, exhibits proangiogenic activity. VASH2 is expressed by various cancer cells and accelerates tumor angiogenesis and progression. VASH2 was recently shown to exhibit tubulin carboxypeptidase (TCP) activity related to microtubule functions. Paclitaxel (PTX), an effective chemotherapeutic agent that is widely used to treat ovarian cancer, inhibits microtubule depolymerization and may interact with VASH2. We herein established several VASH2 knockout ovarian cancer cell lines using the CRISPR/Cas9 genome editing system to examine the intracellular tubulin detyrosination status and PTX chemosensitivity. The knockout of VASH2 did not affect the proliferation or sphere-forming activity of ovarian cancer cells in vitro. A Western blot analysis of VASH2 knockout cells revealed the weak expression of detyrosinated tubulin and upregulated expression of cyclin B1. The knockout of VASH2 significantly increased chemosensitivity to PTX, but not to cisplatin in ovarian cancer cell lines. The knockout of VASH2 reduced TCP activity and increased cyclin B1 expression, resulting in increased PTX chemosensitivity in ovarian cancer cells. The inhibition of angiogenesis and regulation of microtubule activity may be achieved in ovarian cancer treatment strategies targeting VASH2., (© 2021 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

High Fischer ratio oligopeptides have better conditioning effects on chronic diseases caused by long-term sub-health. At present, the enzymatic method for producing high Fischer ratio oligopeptides has a low yield, complicated purification, and a high cost. The use of exopeptidases with specific catalytic activity for aromatic amino acids in the preparation of high Fischer ratio oligopeptides is an important means to solve this problem. The carboxypeptidase from Aspergillus niger M00988 was cloned, which has good specificity for hydrophobic amino acids. Mutations at important substrate binding sites 135, 160, and 206 were performed to study important factors affecting the enzyme-specific recognition of aromatic groups. The results showed that the steric hindrance of amino acid residues at position 135 and the effects of positions 160 and 206 on the binding force of the enzyme to the substrate have important effects on the specific recognition of aromatic groups by the enzyme. Therefore, the S135 G, Y160S, and Y206S mutant enzymes have good application prospects in the preparation of high Fischer ratio oligopeptides with Chlorella powder. The obtained oligopeptides' Fischer ratio reached 31.45, 38.42, and 36.54, respectively. Compared with the original enzyme, the Fischer ratio increased by 2.58 %, 25.31 %, and 19.18 %, respectively., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Cigarette smoking is a risk factor for developing chronic obstructive pulmonary disease and protein aggresome formation is considered to be a hallmark event for the disease. Since dysfunction of lysosome-mediated protein degradation leads to enhanced accumulation of misfolded proteins and subsequent aggresome formation, we examined the effect of cigarette smoke extract (CSE) on ESCRT-mediated sorting in S. cerevisiae as this process is necessary for the functioning of the vacuole, the lysosomal equivalent in yeast. An operational ESCRT pathway is essential for ion homeostasis and our observation that exposure to CSE caused increased sensitivity to LiCl indicated CSE-induced impairment of ESCRT function. To confirm the inhibition of ESCRT function, the targeting of carboxypeptidase S (CPS), which reaches the vacuole lumen via the ESCRT pathway, was examined. Treatment with CSE resulted in the mislocalization of GFP-tagged CPS to the vacuolar membrane, instead of the vacuolar lumen, confirming defective functioning of the ESCRT machinery in CSE-treated cells. Further analysis revealed that CSE-treatment inhibited the recruitment of the ESCRT-0 component, Vps27, to the endosome surface, which is a key event is for the functioning of the ESCRT pathway. This lack of endosomal recruitment of Vps27 most likely results from a depletion of the endosomally-enriched lipid, phosphatidylinositol 3-phosphate (PI3-P), which is the target of Vps27. This is supported by our observation that the presence of excess leucine, a known activator of the lipid kinase responsible for the generation of PI3-P, Vps34, in the medium can rescue the CSE-induced ESCRT misfunctioning. Thus, the current study provides an insight into CSE-induced aggresome formation as it documents that CSE treatment compromises vacuolar degradation due to an impairment of the ESCRT pathway, which likely stems from the inhibition of Vps34. It also indicates that leucine has the potential to attenuate the CSE-induced accumulation of misfolded proteins., Competing Interests: Declaration of Competing Interest The authors declare no competing interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Glioblastoma (GBM) is the most lethal primary brain tumor in adults with a median survival of around 15 months. A potential treatment strategy involves targeting glioma stem-like cells (GSCs) that are able to initiate, maintain, and repopulate the tumor mass. Here, we identify ACT001, a parthenolide derivative, targeting GSCs through regulation of adipocyte enhancer binding protein 1 (AEBP1) signaling. Methods: The effects of ACT001 on cell survival of normal human astrocytes (NHA) and patient-derived glioma stem-like cells (GSCs) were evaluated. RNA-Seq were performed to detect differentially expressed genes. ACT001 efficacy as a single agent or in combination with SHP-2 inhibitor SHP099 was assessed using a GSC orthotopic xenograft model. Results: GSCs exhibit high response to ACT001 in compared with normal human astrocytes. AEBP1 is a putative target of ACT001 by RNA-Seq analysis, which expression associates with prognosis of GBM patients. Knockdown of AEBP1 inhibits GSC proliferation and glioma sphere formation. Treatment with ACT001 or PI3K inhibitor or AEBP1 depletion would impair AKT phosphorylation and GSC proliferation, whereas constitutive AKT activation rescues ACT001 treatment or AEBP1 depletion-inhibited cell proliferation. Moreover, ACT001 blocks TGF-β-activated AEBP1/AKT signaling in GSCs. ACT001 exhibits antitumor activity either as a single agent or in combination with SHP099, which provides significant survival benefits for GSC tumor xenograft-bearing animals. Conclusions: Our data demonstrate AEBP1 as a new druggable target in GBM and ACT001 as a potential therapeutic option for improving the clinical treatment of GBM in combination with SHP099., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Cytosolic carboxypeptidases (CCPs) comprise a unique subfamily of M14 carboxypeptidases and are erasers of the reversible protein posttranslational modification- polyglutamylation. Potent inhibitors for CCPs may serve as leading compounds targeting imbalanced polyglutamylation. However, no efficient CCP inhibitor has yet been reported. Here, we showed that 2-phosphonomethylpentanedioic acid (2-PMPA), a potent inhibitor of the distant M28 family member glutamate carboxypeptidase II (GCPII), rather than the typical M14 inhibitor 2-benzylsuccinic acid, could efficiently inhibit CCP activities. 2-PMPA inhibited the recombinant Nna1 (a.k.a. CCP1) for hydrolyzing a synthetic peptide in a mixed manner, with Ki and Ki' being 0.11 μM and 0.24 μM respectively. It inhibited Nna1 for deglutamylating tubulin, the best-known polyglutamylated protein, with an IC 50 of 0.21 mM. Homology modeling predicted that the R-form of 2-PMPA is more favorable to bind Nna1, unlike that GCPII prefers to S-form. This work for the first time identified a potent inhibitor for CCP family., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Synthetic promoters are vital for genetic engineering-based strategies for crop improvement, but effective methodologies for their creation and systematic testing are lacking. We report here on the comparative analysis of the promoters pro-SmAMP1 and pro-SmAMP2 from Stellaria media ANTIMICROBIAL PEPTIDE1 ( AMP1 ) and ANTIMICROBIAL PEPTIDE2 ( AMP2 ). These promoters are more effective than the well-known Cauliflower mosaic virus 35S promoter. Although these promoters share about 94% identity, the pro-SmAMP1 promoter demonstrated stronger transient expression of a reporter gene in Agrobacterium infiltration of Nicotiana benthamiana leaves, while the pro-SmAMP2 promoter was more effective for the selection of transgenic tobacco ( Nicotiana tabacum ) cells when driving a selectable marker. Using the cap analysis of gene expression method, we detected no differences in the structure of the transcription start sites for either promoter in transgenic plants. For both promoters, we used fine-scale deletion analysis to identify 160 bp-long sequences that retain the unique properties of each promoter. With the use of chimeric promoters and directed mutagenesis, we demonstrated that the superiority of the pro-SmAMP1 promoter for Agrobacterium -mediated infiltration is caused by the proline-inducible ACTCAT cis -element strictly positioned relative to the TATA box in the core promoter. Surprisingly, the ACTCAT cis -element not only activated but also suppressed the efficiency of the pro-SmAMP1 promoter under proline stress. The absence of the ACTCAT cis -element and CAANNNNATC motif (negative regulator) in the pro-SmAMP2 promoter provided a more constitutive gene expression profile and better selection of transgenic cells on selective medium. We created a new synthetic promoter that enjoys high effectiveness both in transient expression and in selection of transgenic cells. Intact promoters with differing properties and high degrees of sequence identity may thus be used as a basis for the creation of new synthetic promoters for precise and coordinated gene expression.

Objective: Our study was aimed at investigating the mechanistic consequences of the upregulation of adipocyte enhancer-binding protein 1 ( AEBP1 ) in glioblastoma (GBM)., Methods: The expression of AEBP1 in GBM was assessed by bioinformatics analysis and qRT-PCR; the effects of AEBP1 on GBM cell proliferation, migration, invasion, and tumor growth in vitro and in vivo were detected by a CCK-8 assay, colony formation assay, scratch assay, Transwell assay, and subcutaneous tumor formation, respectively. The activation of related signaling pathways was monitored using western blot., Results: Tumor-related databases and bioinformatics analysis revealed that AEBP1 was highly expressed in GBM and indicated poor outcome of patients; its high expression that was also confirmed in GBM tissues and cell lines was closely related to the tumor size. The results of in vitro experiments showed that AEBP1 could significantly promote GBM cell proliferation, migration, and invasion; in vivo experiments suggested that AEBP1 could contribute to the growth of GBM tumors. AEBP1 could upregulate the level of IκBα phosphorylation, decrease IκBα expression, activate the NF- κ B signaling pathway, and promote the expression of downstream oncogenes., Conclusion: Upregulated AEBP1 in GBM promotes GBM cell proliferation, migration, and invasion and facilitates tumor growth in vivo by activating the classical NF- κ B pathway., Competing Interests: The authors declare that there are no conflicts of interest regarding the publication of this paper., (Copyright © 2020 Kai Guo et al.)

Ciliary microtubules are subject to post-translational modifications that act as a "Tubulin Code" to regulate motor traffic, binding proteins and stability. In humans, loss of CCP1, a cytosolic carboxypeptidase and tubulin deglutamylating enzyme, causes infantile-onset neurodegeneration. In C. elegans, mutations in ccpp-1, the homolog of CCP1, result in progressive degeneration of neuronal cilia and loss of neuronal function. To identify genes that regulate microtubule glutamylation and ciliary integrity, we performed a forward genetic screen for suppressors of ciliary degeneration in ccpp-1 mutants. We isolated the ttll-5(my38) suppressor, a mutation in a tubulin tyrosine ligase-like glutamylase gene. We show that mutation in the ttll-4, ttll-5, or ttll-11 gene suppressed the hyperglutamylation-induced loss of ciliary dye filling and kinesin-2 mislocalization in ccpp-1 cilia. We also identified the nekl-4(my31) suppressor, an allele affecting the NIMA (Never in Mitosis A)-related kinase NEKL-4/NEK10. In humans, NEK10 mutation causes bronchiectasis, an airway and mucociliary transport disorder caused by defective motile cilia. C. elegans NEKL-4 localizes to the ciliary base but does not localize to cilia, suggesting an indirect role in ciliary processes. This work defines a pathway in which glutamylation, a component of the Tubulin Code, is written by TTLL-4, TTLL-5, and TTLL-11; is erased by CCPP-1; is read by ciliary kinesins; and its downstream effects are modulated by NEKL-4 activity. Identification of regulators of microtubule glutamylation in diverse cellular contexts is important to the development of effective therapies for disorders characterized by changes in microtubule glutamylation. By identifying C. elegans genes important for neuronal and ciliary stability, our work may inform research into the roles of the tubulin code in human ciliopathies and neurodegenerative diseases., Competing Interests: The authors have declared that no competing interests exist.

Background: Classical Ehlers-Danlos syndrome (cEDS) is a connective tissue disorder mainly caused by heterozygous COL5A1 or COL5A2 variants encoding type V collagen and rarely by the p.(Arg312Cys) missense substitution in COL1A1 encoding type I collagen. The current EDS nosology specifies that minimal suggestive criteria are marked skin hyperextensibility plus atrophic scarring together with either generalized joint hypermobility or at least three minor criteria comprising additional cutaneous and articular signs. To reach a final diagnosis, molecular testing is required. Herein, we report on a 3-year-old female who came to our attention with an inconclusive next generation sequencing (NGS) panel comprising all cEDS-associated genes., Methods: Despite the patient did not formally fulfill the nosological criteria because the skin was only slightly hyperextensible, we made a cEDS diagnosis, mainly for the presence of typical atrophic scars. We investigated COL5A1 intragenic deletions/duplications by Multiplex Ligation-dependent Probe Amplification (MLPA), excluded the recessive classical-like EDS type 2 by AEBP1 Sanger analysis, and retested COL5A1 with the Sanger method., Results: Molecular analyses revealed the novel COL5A1 c.3369&#95;3431dup p.(Glu1124&#95;Gly1144dup) intermediate-sized duplication with a predicted dominant negative effect that was missed both by NGS and MLPA., Conclusions: This report highlights that some cEDS patients might not display overt skin hyperextensibility and the importance of clinical expertise to make such a diagnosis in patients with an incomplete presentation. Our results also exemplify that NGS is not a fool-proof technology and that Sanger sequencing achieves the diagnostic goal when there is a sufficiently clear phenotypic indication., (© 2020 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.)

Objective: Prolylcarboxypeptidase (PRCP) is both involved in the Kallikrein-Kinin system (KKS) and renin-angiotensin-aldosterone system (RAAS). This study aimed to determine the genetic impact of PRCP gene polymorphisms on essential hypertension (EH) in an isolated population from a remote region of China., Methods: A haplotype-based study was investigated in 346 EH patients and 346 normal subjects and all samples were Hani minority residents in Southwest China. A total of 11 tag single nucleotide polymorphisms (SNPs) in PRCP gene were tested by polymerase chain reaction-restriction fragment length polymorphism method., Results: Single site analysis found that PRCP gene 3'UTR SNP rs3750931 was associated with EH. The minor allele G of rs3750931 was more prevalent in the EH patients compared to control subjects after Bonferroni correction ( p  < 0.05). Moreover, the rs3750931 G allele carriers showed higher average blood pressure (BP) level among the subjects. The H2 (GAGCACTAACA) haplotype without rs3750931 G allele showed the protective effect for EH (OR = 0.68, 95 CI 0.54-0.85, p  = 0.001)., Conclusion: The present study indicated PRCP gene rs3750931 was associated with the risk of EH. This SNP G allele could be considered as one of risk markers for EH in Hani population.

Higher plants can continuously form new organs by the sustained activity of pluripotent stem cells. These stem cells are embedded in meristems, where they produce descendants, which undergo cell proliferation and differentiation programs in a spatiotemporally-controlled manner. Under certain conditions, pluripotency can be reestablished in descending cells and this reversion in cell fate appears to be actively suppressed by the existing stem cell pool. Mutation of the putative carboxypeptidase ALTERED MERISTEM PROGRAM1 (AMP1) in Arabidopsis causes defects in the suppression of pluripotency in cells normally programmed for differentiation, giving rise to unique hypertrophic phenotypes during embryogenesis as well as in the shoot apical meristem. A role of AMP1 in the miRNA-dependent control of translation has recently been established, however, how this activity is connected to its developmental functions is not resolved. Here we identify members of the cytochrome P450 clade CYP78A to act in parallel with AMP1 to control cell fate in Arabidopsis. Mutation of CYP78A5 and its close homolog CYP78A7 in a cyp78a5,7 double mutant caused suspensor-to-embryo conversion and ectopic stem cell pool formation in the shoot meristem, phenotypes characteristic for amp1. The tissues affected in the mutants showed pronounced expression levels of AMP1 and CYP78A5 in wild type. A comparison of mutant transcriptomic responses revealed an intriguing degree of overlap and highlighted alterations in protein lipidation processes. Moreover, we also found elevated protein levels of selected miRNA targets in cyp78a5,7. Based on comprehensive genetic interaction studies we propose a model in which both enzyme classes act on a common downstream process to sustain cell fate decisions in the early embryo and the shoot apical meristem., Competing Interests: The authors have declared that no competing interests exist.

Aortic carboxypeptidase-like protein (ACLP) is a collagen-binding extracellular matrix protein that has important roles in wound healing and fibrosis. ACLP contains thrombospondin repeats, a collagen-binding discoidin domain, and a catalytically inactive metallocarboxypeptidase domain. Recently, mutations in the ACLP-encoding gene, AE-binding protein 1 ( AEBP1 ), have been discovered, leading to the identification of a new variant of Ehlers-Danlos syndrome causing connective tissue disruptions in multiple organs. Currently, little is known about the mechanisms of ACLP secretion or the role of post-translational modifications in these processes. We show here that the secreted form of ACLP contains N -linked glycosylation and that inhibition of glycosylation results in its intracellular retention. Using site-directed mutagenesis, we determined that glycosylation of Asn-471 and Asn-1030 is necessary for ACLP secretion and identified a specific N-terminal proteolytic ACLP fragment. To determine the contribution of secreted ACLP to extracellular matrix mechanical properties, we generated and mechanically tested wet-spun collagen ACLP composite fibers, finding that ACLP enhances the modulus (or stiffness), toughness, and tensile strength of the fibers. Some AEBP1 mutations were null alleles, whereas others resulted in expressed proteins. We tested the hypothesis that a recently discovered 40-amino acid mutation and insertion in the ACLP discoidin domain regulates collagen binding and assembly. Interestingly, we found that this protein variant is retained intracellularly and induces endoplasmic reticulum stress identified with an XBP1-based endoplasmic reticulum stress reporter. Our findings highlight the importance of N -linked glycosylation of ACLP for its secretion and contribute to our understanding of ACLP-dependent disease pathologies., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Vishwanath et al.)

Serine/Threonine Protein Kinases (STPKs) phosphorylates target proteins thereby regulates various important cellular signal transduction pathways such as cell division and cell wall synthesis. It has been demonstrated that the STPKs regulate peptidoglycan biosynthesis by phosphorylating penicillin binding proteins (PBPs). We extensively characterized both PknI (STPK) and DacB2 (PBP) roles individually as well as combining by genetic knockout and phenotypic characterization studies. In the present study, we analyzed the role of PknI and DacB2 in cell division and virulence. The double knockout (DKO) strain growth was reduced under stress conditions like acidic pH, nutrient depletion media and low oxygen availability conditions. We also found that the DKO growth was significantly reduced in macrophage cell line and it was hypersensitive to oxidative and nitrosative stress condition. The DKO strain significantly attenuated in guinea pig model which was measured by reduced bacillary load, gross pathological and histopathological damages. Overall, these results clearly demonstrated that both PknI and DacB2 together play an important role in cell division under stress conditions, the DKO strain significantly attenuated both in vitro and in vivo models., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Mycobacterial peptidoglycan (PG) is an unsolved puzzle due to its complex structure and involvement of multiple enzymes in the process of its remodelling. dd-Carboxypeptidases are low molecular mass penicillin-binding proteins (LMM-PBPs) that catalyzes the cleavage of terminal d-Ala of muramyl pentapeptide branches and thereby helps in the PG remodelling process. Here, we have assigned the function of a putative LMM-PBP, MSMEG&#95;2432 of Mycobacterium smegmatis , by showing that it exhibits both dd-CPase and β-lactamase activities. Like conventional dd-CPase (PBP5 from E. coli ), upon ectopic complementation in a deformed seven PBP deletion mutant of E. coli , MSMEG&#95;2432 has manifested its ability to restore ~75 % of the cell population to their normal rod shape. Further, in vitro dd-CPase assay has confirmed its ability to release terminal d-Ala from the synthetic tripeptide and the peptidoglycan mimetic pentapeptide substrates ending with d-Ala-d-Ala. Also, elevated resistance against penicillins and cephalosporins upon ectopic expression of MSMEG&#95;2432 suggests the presence of β-lactamase activity, which is further confirmed in vitro through nitrocefin hydrolysis assay. Moreover, it is found apparent that D169A substitution in MSMEG&#95;2432 influences both of its in vivo and in vitro dd-CPase and β-lactamase activities. Thus, we infer that MSMEG&#95;2432 is a dual function enzyme that possesses both dd-CPase and β-lactamase activities.

GS5 encoding a serine carboxypeptidase-like protein positively regulates grain size and weight through the regulation of grain width and filling and is helpful in improving cereal yields. Grain width variation determined by GS5 is associated with cell number and size, but the actual underlying mechanism is still unclear. Two orthologs of GS5, TtGS5-3A-G and TtGS5-3G-G, were cloned from the Triticum timopheevi accession no. CWI17006. To identify the proteins that interacted with TtGS5-3A-G and TtGS5-3G-G in premature grains, we performed pull-down assays followed by liquid chromatography-mass spectrometry/mass spectrometry analysis. The analyses revealed 18 proteins were present in both the TtGS5-3A-G and TtGS5-3G-G interactomes. Among five candidates selected, only Annexin D1 interacted with both TtGS5-3A-G and TtGS5-3G-G in yeast. Annexin D1, TtGS5-3A-G, and TtGS5-3G-G were located on the cytoplasmic membranes of Arabidopsis protoplasts and onion epidermal cells, and interactions between Annexin D1 and TtGS5-3A-G, as well as TtGS5-3G-G, were shown by bimolecular fluorescence complementation assays. Annexin D1 was expressed widely in different tissues, and it co-expressed with TtGS5-3A-G/TtGS5-3G-G at the grain enlargement phase. These results indicated that Annexin D1 interacted with TtGS5-3A-G and TtGS5-3G-G in premature grains. Together with the structural similarities of Annexin D1 to known fiber elongation factors, we proposed that TtGS5 might regulate the cell size by interacting with Annexin D1. The results provide significant new information for understanding the roles that GS5 plays in regulating grain size, which may be useful in improving crop yields.