Tubulin code eraser CCP5 binds branch glutamates by substrate deformation.

Microtubule function is modulated by the tubulin code, diverse posttranslational modifications that are altered dynamically by writer and eraser enzymes1. Glutamylation-the addition of branched (isopeptide-linked) glutamate chains-is the most evolutionarily widespread tubulin modification2. It is introduced by tubulin tyrosine ligase-like enzymes and erased by carboxypeptidases of the cytosolic carboxypeptidase (CCP) family1. Glutamylation homeostasis, achieved through the balance of writers and erasers, is critical for normal cell function3-9, and mutations in CCPs lead to human disease10-13. Here we report cryo-electron microscopy structures of the glutamylation eraser CCP5 in complex with the microtubule, and X-ray structures in complex with transition-state analogues. Combined with NMR analysis, these analyses show that CCP5 deforms the tubulin main chain into a unique turn that enables lock-and-key recognition of the branch glutamate in a cationic pocket that is unique to CCP family proteins. CCP5 binding of the sequences flanking the branch point primarily through peptide backbone atoms enables processing of diverse tubulin isotypes and non-tubulin substrates. Unexpectedly, CCP5 exhibits inefficient processing of an abundant ß-tubulin isotype in the brain. This work provides an atomistic view into glutamate branch recognition and resolution, and sheds light on homeostasis of the tubulin glutamylation syntax.

The Activin Branch Ligand Daw Regulates the Drosophila melanogaster Immune Response and Lipid Metabolism against the Heterorhabditis bacteriophora Serine Carboxypeptidase.

Despite impressive advances in the broad field of innate immunity, our understanding of the molecules and signaling pathways that control the host immune response to nematode infection remains incomplete. We have shown recently that Transforming Growth Factor-ß (TGF-ß) signaling in the fruit fly Drosophila melanogaster is activated by nematode infection and certain TGF-ß superfamily members regulate the D. melanogaster anti-nematode immune response. Here, we investigate the effect of an entomopathogenic nematode infection factor on host TGF-ß pathway regulation and immune function. We find that Heterorhabditis bacteriophora serine carboxypeptidase activates the Activin branch in D. melanogaster adults and the immune deficiency pathway in Activin-deficient flies, it affects hemocyte numbers and survival in flies deficient for Activin signaling, and causes increased intestinal steatosis in Activin-deficient flies. Thus, insights into the D. melanogaster signaling pathways and metabolic processes interacting with H. bacteriophora pathogenicity factors will be applicable to entomopathogenic nematode infection of important agricultural insect pests and vectors of disease.

Genome-Wide Analysis of Serine Carboxypeptidase-like Genes in Soybean and Their Roles in Stress Resistance.

The serine carboxypeptidase-like (SCPL) gene family plays a crucial role in the regulation of plant growth, development, and stress response through activities such as acyltransferases in plant secondary metabolism pathways. Although SCPL genes have been identified in various plant species, their specific functions and characteristics in soybean (Glycine max) have not yet been studied. We identified and characterized 73 SCPL genes, grouped into three subgroups based on gene structure and phylogenetic relationships. These genes are distributed unevenly across 20 soybean chromosomes and show varied codon usage patterns influenced by both mutation and selection pressures. Gene ontology (GO) enrichment suggests these genes are involved in plant cell wall regulation and stress responses. Expression analysis in various tissues and under stress conditions, including the presence of numerous stress-related cis-acting elements, indicated that these genes have varied expression patterns. This suggests that they play specialized roles such as modulating plant defense mechanisms against nematode infections, enhancing tolerance to drought and high salinity, and responding to cold stress, thereby helping soybean adapt to environmental stresses. Moreover, the expression of specific GmSCPLs was significantly affected following exposure to nematode infection, drought, high salt (NaCl), and cold stresses. Our findings underscore the potential of SCPL genes in enhancing stress resistance in soybean, providing a valuable resource for future genetic improvement and breeding strategies.

SARS-CoV-2 Spike Protein Enhances Carboxypeptidase Activity of Angiotensin-Converting Enzyme 2.

In this study, we investigated whether severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike protein may modify angiotensin-converting enzyme 2 (ACE2) activity in the plasma, heart, kidney, liver, lung, and six brain regions (amygdala, brain stem, cortex, hippocampus, hypothalamus, and striatum) of diabetic and hypertensive rats. We determine ACE2 activity in the plasma and lysates of heart, kidney, liver, lung, and six brain regions. MLN-4760 inhibits ACE2 activity in the plasma and all organs. On the other hand, soluble ACE2 (sACE2) activity increased in the plasma of diabetic rats, and there was no change in the plasma of hypertensive rats. ACE2 activity was augmented in the liver, brain stem, and striatum, while it decreased in the kidney, amygdala, cortex, and hippocampus of diabetic rats. ACE2 activity increased in the kidney, liver, and lung, while it decreased in the heart, amygdala, cortex, and hypothalamus of hypertensive rats. We measured the ACE2 content via enzyme-linked immunosorbent assay and found that ACE2 protein levels increased in the heart, while it decreased in the plasma, kidney, brain stem, cortex, hippocampus, hypothalamus, and striatum of diabetic rats. ACE2 protein levels decreased in the brain stem, cortex, hippocampus, and hypothalamus of hypertensive rats. Our data showed that the spike protein enhanced ACE2 activity in the liver and lungs of diabetic rats, as well as in the heart and three of the brain regions (cortex, hypothalamus, and striatum) of hypertensive rats.

A serine carboxypeptidase-like acyltransferase catalyzes consecutive four-step reactions of hydrolyzable tannin biosynthesis in Camellia oleifera.

Hydrolyzable tannins (HTs), a class of polyphenolic compounds found in dicotyledonous plants, are widely used in food and pharmaceutical industries because of their beneficial effects on human health. Although the biosynthesis of simple HTs has been verified at the enzymatic level, relevant genes have not yet been identified. Here, based on the parent ion-fragment ion pairs in the feature fragment data obtained using UPLC-Q-TOF-/MS/MS, galloyl phenolic compounds in the leaves of Camellia sinensis and C. oleifera were analyzed qualitatively and quantitatively. Correlation analysis between the transcript abundance of serine carboxypeptidase-like acyltransferases (SCPL-ATs) and the peak area of galloyl products in Camellia species showed that SCPL3 expression was highly correlated with HT biosynthesis. Enzymatic verification of the recombinant protein showed that CoSCPL3 from C. oleifera catalyzed the four consecutive steps involved in the conversion of digalloylglucose to pentagalloylglucose. We also identified the residues affecting the enzymatic activity of CoSCPL3 and determined that SCPL-AT catalyzes the synthesis of galloyl glycosides. The findings of this study provide a target gene for germplasm innovation of important cash crops that are rich in HTs, such as C. oleifera, strawberry, and walnut.

Two different types of hydrolases co-degrade ochratoxin A in a highly efficient degradation strain Lysobacter sp. CW239.

Ochratoxin A (OTA) is a toxic secondary metabolite that widely contaminates agro-products and poses a significant dietary risk to human health. Previously, a carboxypeptidase CP4 was characterized for OTA degradation in Lysobacter sp. CW239, but the degradation activity was much lower than its host strain CW239. In this study, an amidohydrolase ADH2 was screened for OTA hydrolysis in this strain. The result showed that 50 µg/L OTA was completely degraded by 1.0 µg/mL rADH2 within 5 min, indicating ultra-efficient activity. Meanwhile, the two hydrolases (i.e., CP4 and ADH2) in the strain CW239 showed the same degradation manner, which transformed the OTA to ochratoxin α (OTα) and l-ß-phenylalanine. Gene mutants (Δcp4, Δadh2 and Δcp4-adh2) testing result showed that OTA was co-degraded by carboxypeptidase CP4 and amidohydrolase ADH2, and the two hydrolases are sole agents in strain CW239 for OTA degradation. Hereinto, the ADH2 was the overwhelming efficient hydrolase, and the two types of hydrolases co-degraded OTA in CW239 by synergistic effect. The results of this study are highly significant to ochratoxin A contamination control during agro-products production and postharvest.

Carboxypeptidase M modulates BMSCs osteogenesis-adipogenesis via the MAPK/ERK pathway: An integrated single-cell and bulk transcriptomic study.

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.

Decoding microtubule detyrosination: enzyme families, structures, and functional implications.

Microtubules are a major component of the cytoskeleton and can accumulate a plethora of modifications. The microtubule detyrosination cycle is one of these modifications; it involves the enzymatic removal of the C-terminal tyrosine of α-tubulin on assembled microtubules and the re-ligation of tyrosine on detyrosinated tubulin dimers. This modification cycle has been implicated in cardiac disease, neuronal development, and mitotic defects. The vasohibin and microtubule-associated tyrosine carboxypeptidase enzyme families are responsible for microtubule detyrosination. Their long-sought discovery allows to review and summarise differences and similarities between the two enzymes families and discuss how they interplay with other modifications and functions of the tubulin code.

Increased Prolylcarboxypeptidase Expression Can Serve as a Biomarker of Senescence in Culture.

Prolylcarboxypeptidase (PRCP, PCP, Lysosomal Pro-X-carboxypeptidase, Angiotensinase C) controls angiotensin- and kinin-induced cell signaling. Elevation of PRCP appears to be activated in chronic inflammatory diseases [cardiovascular disease (CVD), diabetes] in proportion to severity. Vascular endothelial cell senescence and mitochondrial dysfunction have consistently been shown in models of CVD in aging. Cellular senescence, a driver of age-related dysfunction, can differentially alter the expression of lysosomal enzymes due to lysosomal membrane permeability. There is a lack of data demonstrating the effect of age-related dysfunction on the expression and function of PRCP. To explore the changes in PRCP, the PRCP-dependent prekallikrein (PK) pathway was characterized in early- and late-passage human pulmonary artery endothelial cells (HPAECs). Detailed kinetic analysis of cells treated with high molecular weight kininogen (HK), a precursor of bradykinin (BK), and PK revealed a mechanism by which senescent HPAECs activate the generation of kallikrein upon the assembly of the HK-PK complex on HPAECs in parallel with an upregulation of PRCP and endothelial nitric oxide (NO) synthase (eNOS) and NO formation. The NO production and expression of both PRCP and eNOS increased in early-passage HPAECs and decreased in late-passage HPAECs. Low activity of PRCP in late-passage HPAECs was associated with rapid decreased telomerase reverse transcriptase mRNA levels. We also found that, with an increase in the passage number of HPAECs, reduced PRCP altered the respiration rate. These results indicated that aging dysregulates PRCP protein expression, and further studies will shed light into the complexity of the PRCP-dependent signaling pathway in aging.

Clinical and Molecular Characterization of a Novel Homozygous Frameshift Variant in AEBP1-Related Classical-like Ehlers Danlos Syndrome Type 2 with Comparison to Previously Reported Rare Cases.

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.

SCPL acyltransferases catalyze the metabolism of chlorogenic acid during purple coneflower seed germination.

The metabolism of massively accumulated chlorogenic acid is crucial for the successful germination of purple coneflower (Echinacea purpurea (L.) Menoch). A serine carboxypeptidase-like (SCPL) acyltransferase (chicoric acid synthase, CAS) utilizes chlorogenic acid to produce chicoric acid during germination. However, it seems that the generation of chicoric acid lags behind the decrease in chlorogenic acid, suggesting an earlier route of chlorogenic acid metabolism. We discovered another chlorogenic acid metabolic product, 3,5-dicaffeoylquinic acid, which is produced before chicoric acid, filling the lag phase. Then, we identified two additional typical clade IA SCPL acyltransferases, named chlorogenic acid condensing enzymes (CCEs), that catalyze the biosynthesis of 3,5-dicaffeoylquinic acid from chlorogenic acid with different kinetic characteristics. Chlorogenic acid inhibits radicle elongation in a dose-dependent manner, explaining the potential biological role of SCPL acyltransferases-mediated continuous chlorogenic acid metabolism during germination. Both CCE1 and CCE2 are highly conserved among Echinacea species, supporting the observed metabolism of chlorogenic acid to 3,5-dicaffeoylquinic acid in two Echinacea species without chicoric acid accumulation. The discovery of SCPL acyltransferase involved in the biosynthesis of 3,5-dicaffeoylquinic acid suggests convergent evolution. Our research clarifies the metabolism strategy of chlorogenic acid in Echinacea species and provides more insight into plant metabolism.

Dysregulated AEBP1 and COLEC12 Genes in Late-Onset Alzheimer's Disease: Insights from Brain Cortex and Peripheral Blood Analysis.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by memory and cognitive impairment, often accompanied by alterations in mood, confusion, and, ultimately, a state of acute mental disturbance. The cerebral cortex is considered a promising area for investigating the underlying causes of AD by analyzing transcriptional patterns, which could be complemented by investigating blood samples obtained from patients. We analyzed the RNA expression profiles of three distinct areas of the brain cortex, including the frontal cortex (FC), temporal cortex (TC), and entorhinal cortex (EC) in patients with AD. Functional enrichment analysis was performed on the differentially expressed genes (DEGs) across the three regions. The two genes with the most significant expression changes in the EC region were selected for assessing mRNA expression levels in the peripheral blood of late-onset AD patients using quantitative PCR (qPCR). We identified eight shared DEGs in these regions, including AEBP1 and COLEC12, which exhibited prominent changes in expression. Functional enrichment analysis uncovered a significant association of these DEGs with the transforming growth factor-ß (TGF-ß) signaling pathway and processes related to angiogenesis. Importantly, we established a robust connection between the up-regulation of AEBP1 and COLEC12 in both the brain and peripheral blood. Furthermore, we have demonstrated the potential of AEBP1 and COLEC12 genes as effective diagnostic tools for distinguishing between late-onset AD patients and healthy controls. This study unveils the intricate interplay between AEBP1 and COLEC12 in AD and underscores their potential as markers for disease detection and monitoring.

Disruption of the mast cell carboxypeptidase A3 gene does not attenuate airway inflammation and hyperresponsiveness in two mouse models of asthma.

Mast cells are effector cells known to contribute to allergic airway disease. When activated, mast cells release a broad spectrum of inflammatory mediators, including the mast cell-specific protease carboxypeptidase A3 (CPA3). The expression of CPA3 in the airway epithelium and lumen of asthma patients has been associated with a Th2-driven airway inflammation. However, the role of CPA3 in asthma is unclear and therefore, the aim of this study was to investigate the impact of CPA3 for the development and severity of allergic airway inflammation using knockout mice with a deletion in the Cpa3 gene. We used the ovalbumin (OVA)- and house-dust mite (HDM) induced murine asthma models, and monitored development of allergic airway inflammation. In the OVA model, mice were sensitized with OVA intraperitoneally at seven time points and challenged intranasally (i.n.) with OVA three times. HDM-treated mice were challenged i.n. twice weekly for three weeks. Both asthma protocols resulted in elevated airway hyperresponsiveness, increased number of eosinophils in bronchoalveolar lavage fluid, increased peribronchial mast cell degranulation, goblet cell hyperplasia, thickening of airway smooth muscle layer, increased expression of IL-33 and increased production of allergen-specific IgE in allergen-exposed mice as compared to mocktreated mice. However, increased number of peribronchial mast cells was only seen in the HDM asthma model. The asthma-like responses in Cpa3-/- mice were similar as in wild type mice, regardless of the asthma protocol used. Our results demonstrated that the absence of a functional Cpa3 gene had no effect on several symptoms of asthma in two different mouse models. This suggest that CPA3 is dispensable for development of allergic airway inflammation in acute models of asthma in mice.

[Dynamic trajectory and cell communication of different cell clusters in malignant progression of glioblastoma].

OBJECTIVE: To delve deeply into the dynamic trajectories of cell subpopulations and the communication network among immune cell subgroups during the malignant progression of glioblastoma (GBM), and to endeavor to unearth key risk biomarkers in the GBM malignancy progression, so as to provide a more profound understanding for the treatment and prognosis of this disease by integrating transcriptomic data and clinical information of the GBM patients. METHODS: Utilizing single-cell sequencing data analysis, we constructed a cell subgroup atlas during the malignant progression of GBM. The Monocle2 tool was employed to build dynamic progression trajectories of the tumor cell subgroups in GBM. Through gene enrichment analysis, we explored the biological processes enriched in genes that significantly changed with the malignancy progression of GBM tumor cell subpopulations. CellChat was used to identify the communication network between the different immune cell subgroups. Survival analysis helped in identifying risk molecular markers that impacted the patient prognosis during the malignant progression of GBM. This method ological approach offered a comprehensive and detailed examination of the cellular and molecular dynamics within GBM, providing a robust framework for understanding the disease' s progression and potential therapeutic targets. RESULTS: The analysis of single-cell sequencing data identified 6 different cell types, including lymphocytes, pericytes, oligodendrocytes, macrophages, glioma cells, and microglia. The 27 151 cells in the single-cell dataset included 3 881 cells from the patients with low-grade glioma (LGG), 10 166 cells from the patients with newly diagnosed GBM, and 13 104 cells from the patients with recurrent glioma (rGBM). The pseudo-time analysis of the glioma cell subgroups indicated significant cellular heterogeneity during malignant progression. The cell interaction analysis of immune cell subgroups revealed the communication network among the different immune subgroups in GBM malignancy, identifying 22 biologically significant ligand-receptor pairs across 12 key biological pathways. Survival analysis had identified 8 genes related to the prognosis of the GBM patients, among which SERPINE1, COL6A1, SPP1, LTF, C1S, AEBP1, and SAA1L were high-risk genes in the GBM patients, and ABCC8 was low-risk genes in the GBM patients. These findings not only provided new theoretical bases for the treatment of GBM, but also offered fresh insights for the prognosis assessment and treatment decision-making for the GBM patients. CONCLUSION: This research comprehensively and profoundly reveals the dynamic changes in glioma cell subpopulations and the communication patterns among the immune cell subgroups during the malignant progression of GBM. These findings are of significant importance for understanding the complex biological processes of GBM, providing crucial new insights for precision medicine and treatment decisions in GBM. Through these studies, we hope to provide more effective treatment options and more accurate prognostic assessments for the patients with GBM.

Genetic interaction mapping reveals functional relationships between peptidoglycan endopeptidases and carboxypeptidases.

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.

AEBP1 upregulation contributes to cervical cancer progression by facilitating cell proliferation, migration, and invasion.

BACKGROUND: Aberrant expression of adipocyte enhancer-binding protein 1 (AEBP1) has been demonstrated to be involved in the tumorigenesis and progression of numerous cancers. This study was aimed to investigate the mechanism of AEBP1 in the development of cervical cancer. METHODS: The expression of AEBP1 in cervical cancer was assessed by immunohistochemistry. The function of AEBP1 on cell proliferation, migration, and invasion was determined by methyl thiazolyl tetrazolium assay, colony formation, and transwell assay. The activation of related signaling pathway was determined by western blot. The bioinformatics analysis was performed by Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. RESULTS: Higher protein expression of AEBP1 was observed in patients with cervical cancer. Overexpressed AEBP1 promoted cell proliferation, migration, and invasion abilities in cervical cancer cells. Moreover, the research manifested that AEBP1 activated the phosphorylation of STAT3. GO and KEGG analysis showed that genes positively related to AEBP1 were highly enriched in functions like epithelial cell proliferation, muscle cell migration, myoblast migration, smooth muscle tissue development, ECM-receptor interaction, transcriptional misregulation in cancer, and proteoglycans in cancer. While genes negatively related to AEBP1 were associated with immunity, including inflammatory response, external-stimulus response, neutrophil, granulocyte, and macrophage chemotaxis. CONCLUSIONS: This study suggested that AEBP1 acts as an oncogened and might be a potential therapeutic target for the treatment of cervical cancer.

Neuropeptidomics of Genetically Defined Cell Types in Mouse Brain.

Peptidomic techniques are powerful tools to identify peptides in a biological sample. In the case of brain, which contains a complex mixture of cell types, standard peptidomics procedures reveal the major peptides in a dissected brain region. It is difficult to obtain information on peptides within a specific cell type using standard approaches, unless that cell type can be isolated. This protocol describes a targeted peptidomic approach that uses affinity chromatography to purify peptides that are substrates of carboxypeptidase E (CPE), an enzyme present in the secretory pathway of neuroendocrine cells. Many CPE products function as neuropeptides and/or peptide hormones, and therefore represent an important subset of the peptidome. Because CPE removes C-terminal Lys and Arg residues from peptide processing intermediates, organisms lacking CPE show a large decrease in the levels of the mature forms of most neuropeptides and peptide hormones, and a very large increase in the levels of the processing intermediates that contain C-terminal Lys and/or Arg (i.e., the CPE substrates). These CPE substrates can be purified on an anhydrotrypsin-agarose affinity resin, which specifically binds peptides with C-terminal basic residues. When this method is used with mice lacking CPE activity in genetically defined cell types, it allows the detection of peptides specifically produced in that cell type.

A 5.2-kb insertion in the coding sequence of PavSCPL, a serine carboxypeptidase-like enhances fruit firmness in Prunus avium.

Fruit firmness is an important trait in sweet cherry breeding because it directly positively influences fruit transportability, storage and shelf life. However, the underlying genes responsible and the molecular mechanisms that control fruit firmness remain unknown. In this study, we identified a candidate gene, PavSCPL, encoding a serine carboxypeptidase-like protein with natural allelic variation, that controls fruit firmness in sweet cherry using map-based cloning and functionally characterized PavSCPL during sweet cherry fruit softening. Genetic analysis revealed that fruit firmness in the 'Rainier' × 'Summit' F1 population was controlled by a single dominant gene. Bulked segregant analysis combined with fine mapping narrowed the candidate gene to a 473-kb region (7418778-7 891 914 bp) on chromosome 6 which included 72 genes. The candidate gene PavSCPL, and a null allele harbouring a 5244-bp insertion in the second exon that completely inactivated PavSCPL expression and resulted in the extra-hard-flesh phenotype, were identified by RNA-sequencing analysis and gene cloning. Quantitative RT-PCR analysis revealed that the PavSCPL expression level was increased with fruit softening. Virus-induced gene silencing of PavSCPL enhanced fruit firmness and suppressed the activities of certain pectin-degrading enzymes in the fruit. In addition, we developed functional molecular markers for PavSCPL and the Pavscpl5.2-k allele that co-segregated with the fruit firmness trait. Overall, this research identified a crucial functional gene for fruit firmness. The results provide insights into the genetic control and molecular mechanism of the fruit firmness trait and present useful molecular markers for molecular-assisted breeding for fruit firmness in sweet cherry.

A new and promiscuous α/ß hydrolase from Acinetobacter tandoii DSM 14970 T inactivates the mycotoxin ochratoxin A.

The presence of ochratoxin A (OTA) in food and feed represents a serious concern since it raises severe health implications. Bacterial strains of the Acinetobacter genus hydrolyse the amide bond of OTA yielding non-toxic OTα and L-ß-phenylalanine; in particular, the carboxypeptidase PJ15_1540 from Acinetobacter sp. neg1 has been identified as an OTA-degrading enzyme. Here, we describe the ability to transform OTA of cell-free protein extracts from Acinetobacter tandoii DSM 14970 T, a strain isolated from sludge plants, and also report on the finding of a new and promiscuous α/ß hydrolase (ABH), with close homologs highly distributed within the Acinetobacter genus. ABH from A. tandoii (AtABH) exhibited amidase activity against OTA and OTB mycotoxins, as well as against several carboxypeptidase substrates. The predicted structure of AtABH reveals an α/ß hydrolase core composed of a parallel, six-stranded ß-sheet, with a large cap domain similar to the marine esterase EprEst. Further biochemical analyses of AtABH reveal that it is an efficient esterase with a similar specificity profile as EprEst. Molecular docking studies rendered a consistent OTA-binding mode. We proposed a potential procedure for preparing new OTA-degrading enzymes starting from promiscuous α/ß hydrolases based on our results. KEY POINTS: • AtABH is a promiscuous αß hydrolase with both esterase and amidohydrolase activities • AtABH hydrolyses the amide bond of ochratoxin A rendering nontoxic OTα • Promiscuous αß hydrolases are a possible source of new OTA-degrading enzymes.

Disease-Associated Neurotoxic Astrocyte Markers in Alzheimer Disease Based on Integrative Single-Nucleus RNA Sequencing.

Alzheimer disease (AD) is an irreversible neurodegenerative disease, and astrocytes play a key role in its onset and progression. The aim of this study is to analyze the characteristics of neurotoxic astrocytes and identify novel molecular targets for slowing down the progression of AD. Single-nucleus RNA sequencing (snRNA-seq) data were analyzed from various AD cohorts comprising about 210,654 cells from 53 brain tissue. By integrating snRNA-seq data with bulk RNA-seq data, crucial astrocyte types and genes associated with the prognosis of patients with AD were identified. The expression of neurotoxic astrocyte markers was validated using 5 × FAD and wild-type (WT) mouse models, combined with experiments such as western blot, quantitative real-time PCR (qRT-PCR), and immunofluorescence. A group of neurotoxic astrocytes closely related to AD pathology was identified, which were involved in inflammatory responses and pathways related to neuron survival. Combining snRNA and bulk tissue data, ZEP36L, AEBP1, WWTR1, PHYHD1, DST and RASL12 were identified as toxic astrocyte markers closely related to disease severity, significantly elevated in brain tissues of 5 × FAD mice and primary astrocytes treated with Aß. Among them, WWTR1 was significantly increased in astrocytes of 5 × FAD mice, driving astrocyte inflammatory responses, and has been identified as an important marker of neurotoxic astrocytes. snRNA-seq analysis reveals the biological functions of neurotoxic astrocytes. Six genes related to AD pathology were identified and validated, among which WWTR1 may be a novel marker of neurotoxic astrocytes.