Objective To explore the effects of aberrant O-glycosylation modifications induced by the knockout of Cosmc or T-synthase genes on the expression profiles of miRNAs in exosomes derived from colorectal cancer cells and to reveal the molecular mechanisms of O-glycosylation in the development of colorectal cancer and identify potential biomarkers for early diagnosis and treatment.Methods This research specifically targets the Cosmc or T-synthase genes in the human colorectal cancer cell line HCT116 to create stable cell lines exhibiting abnormal O-glycosylation with CRISPR/Cas-9 gene editing technology.Exosomes originating from these colorectal cancer cells were isolated and authenticated.A microarray chip equipped with primer sequences for 16 miRNAs closely associated with colorectal cancer was employed to assess the differential expression of miRNAs within these exosomes with fluorescent quantitative polymerase chain reaction(PCR).And then,a cohort of miRNAs that exhibited significant and consistent changes in expression levels across the exosomes from both cell lines was selected.These miRNAs were further validated independently with traditional fluorescent quantitative PCR.Subsequently,data from The Cancer Genome Atlas Program(TCGA)database containing patient information on colorectal cancer was harnessed.Employing R programming language,Gene Set Enrichment Analysis(GSEA)was conducted on the upregulated miRNA to investigate the downstream pathways significantly impacted and the malignant biological behaviors they may influence.Results The absence of either Cosmc or T-synthase genes results in the dysregulation of O-glycosylation in colorectal cancer cells,leading to the exposure of Tn antigens.This,in turn,affects the expression levels of specific miRNAs in exosomes derived from these cells.Specifically,the expression of hsa-miR-125b-1-3p was downregulated,while that of hsa-miR-218-5p was upregulated.Notably,hsa-miR-218-5p were found to be closely associated with the epithelial-mesenchymal transition(EMT)process in tumor cells,which is a key mechanism in cancer progression.Conclusion It elucidates that the aberrant O-glycosylation mediated by the knockout of Cosmc or T-synthase genes significantly influences the expression of certain miRNAs in exosomes from colorectal cancer cells,potentially affect the EMT process in colorectal cancer and thereby promoting distant metastasis.Given the inherent stability and detectability advantages of colorectal cancer-derived exosomes,the altered expression levels of miRNAs within these exosomes may serve as indicators of the stated of abnormal O-glycosylation in colorectal cancer.These findings suggest that exosomal miRNAs have potential as biomarkers for monitoring disease progression and therapeutic efficacy.Consequently,this could pave the way for more personalized diagnostic and treatment strategies tailored to individual colorectal cancer patients,enhancing the precision and effectiveness of clinical management.

Objective To explore the effects of aberrant O-glycosylation modifications induced by the knockout of Cosmc or T-synthase genes on the expression profiles of miRNAs in exosomes derived from colorectal cancer cells and to reveal the molecular mechanisms of O-glycosylation in the development of colorectal cancer and identify potential biomarkers for early diagnosis and treatment.Methods This research specifically targets the Cosmc or T-synthase genes in the human colorectal cancer cell line HCT116 to create stable cell lines exhibiting abnormal O-glycosylation with CRISPR/Cas-9 gene editing technology.Exosomes originating from these colorectal cancer cells were isolated and authenticated.A microarray chip equipped with primer sequences for 16 miRNAs closely associated with colorectal cancer was employed to assess the differential expression of miRNAs within these exosomes with fluorescent quantitative polymerase chain reaction(PCR).And then,a cohort of miRNAs that exhibited significant and consistent changes in expression levels across the exosomes from both cell lines was selected.These miRNAs were further validated independently with traditional fluorescent quantitative PCR.Subsequently,data from The Cancer Genome Atlas Program(TCGA)database containing patient information on colorectal cancer was harnessed.Employing R programming language,Gene Set Enrichment Analysis(GSEA)was conducted on the upregulated miRNA to investigate the downstream pathways significantly impacted and the malignant biological behaviors they may influence.Results The absence of either Cosmc or T-synthase genes results in the dysregulation of O-glycosylation in colorectal cancer cells,leading to the exposure of Tn antigens.This,in turn,affects the expression levels of specific miRNAs in exosomes derived from these cells.Specifically,the expression of hsa-miR-125b-1-3p was downregulated,while that of hsa-miR-218-5p was upregulated.Notably,hsa-miR-218-5p were found to be closely associated with the epithelial-mesenchymal transition(EMT)process in tumor cells,which is a key mechanism in cancer progression.Conclusion It elucidates that the aberrant O-glycosylation mediated by the knockout of Cosmc or T-synthase genes significantly influences the expression of certain miRNAs in exosomes from colorectal cancer cells,potentially affect the EMT process in colorectal cancer and thereby promoting distant metastasis.Given the inherent stability and detectability advantages of colorectal cancer-derived exosomes,the altered expression levels of miRNAs within these exosomes may serve as indicators of the stated of abnormal O-glycosylation in colorectal cancer.These findings suggest that exosomal miRNAs have potential as biomarkers for monitoring disease progression and therapeutic efficacy.Consequently,this could pave the way for more personalized diagnostic and treatment strategies tailored to individual colorectal cancer patients,enhancing the precision and effectiveness of clinical management.

The chalcone synthase (CHS) superfamily of the type III polyketide synthases (PKSs) generates backbones of a variety of plant secondary metabolites. Benzalacetone synthase (BAS) catalyzes a condensation reaction of decarboxylation between the substrates of 4-coumaric coenzyme A and malonyl coenzyme A to generate benzylidene acetone, whose derivatives are series of compounds with various biological activities. A BAS gene Pcpks2 and a bifunctional CHS/BAS PcPKSI were isolated from medicinal plant P. cuspidatum. Crystallographic and structure-based mutagenesis studies indicate that the functional diversity of the CHS-superfamily enzymes is principally derived from small modifications of the active site architecture. In order to obtain an understanding of the biosynthesis of polyketides in P. cuspidatum, which has been poorly described, as well as of its activation mechanism, PcPKS2 was overexpressed in Escherichia coli as a C-terminally poly-His-tagged fusion protein, purified to homogeneity and crystallized, which is helpful for the clarification of the catalytic mechanism of the enzyme and lays the foundation for its genetic engineering manipulation.
Butanones ; Catalytic Domain ; Crystallization ; Fallopia japonica ; enzymology ; Polyketide Synthases ; genetics ; metabolism

Resveratrol is a natural phytoalexin with special pharmacological and health functions. Stilbene synthase (STS) is a key and rate-limiting enzyme in the biosynthesis of resveratrol that is present only in a limited number of plants. The content of resveratrol from Polygonum cuspidatum is more than 1000 times higher than grapes and peanuts. We speculate that the catalytic ability of different STS may be one of the reasons causing differences in the content of resveratrol. To verify the above speculation, Vitis vinifera stilbene synthase gene (VvSTS) was amplified according to overlap PCR protocol with genomic DNA as template. VvSTS and PcSTS (PcPKS5) were analyzed through heterologous expression in Escherichia coli. The expression products were purified with Ni-NTA sepharose affinity chromatography and desalted through PD-10 column. The molecular weight of the two fusion proteins was about 43 kDa. Enzyme reaction and product analysis showed that the two products were resveratrol. The enzyme kinetic analysis showed that the catalyze efficiency (Kcat/Km) of PcPKS5 was 2.4 times of the VvSTS. Our findings confirms that STS from certain plants has much higher catalytic capability.
Acyltransferases ; metabolism ; Fallopia japonica ; enzymology ; Recombinant Fusion Proteins ; biosynthesis ; Stilbenes ; metabolism ; Vitis ; enzymology

Glycosyltransferases (GTs) catalyze the transfer of a sugar residue of an activated sugar donor to an acceptor molecule. Many families 1 GTs utilize an uridine diphosphate (UDP) activated sugar as donor in the glycosylation reaction, and most of these belong to a group of GTs referred to as the UGTs. The relationship between the degree of amino acid sequence identity and substrate specificity of the plant UGTs is highly complicated, and the prediction of substrate specificity based on phylogenetic analyses need to be improved by more biochemical characterization. This review summarizes the three dimensional structures of plant UGTs published in the Protein Data Bank (PDB), including the detailed substrate interactions with the sugar and receptor binding pockets and mutational analyses of some critical amino acids. It will be helpful for biochemical characterization the substrate specificity of the individual UGT, and lay the foundation for the enzymatic and genetic manipulation of plant UGTs in the future.
Amino Acid Sequence ; Glycosylation ; Glycosyltransferases ; chemistry ; Phylogeny ; Plant Proteins ; chemistry ; Plants ; enzymology ; Protein Structure, Tertiary ; Substrate Specificity ; Uridine Diphosphate ; chemistry

Plant type III polyketide synthase (PKS) generates backbones of a variety of plant secondary metabolites with diverse functions, and has long been models to elucidate the relationship between the three-dimensional structure and function. More than 80 type IIII PKS crystal structures with different functions have been reported in Protein Data Bank, including the crystal structures of the well-studied Chalcone Synthase of plant type III PKS, as well as the 6 other kinds of PKSs in the family, which are critical for understanding the structural basis for diverse starter molecule selectivity, polyketide chain length and the cyclization reaction. Structure-based analysis and site-directed mutagenesis are foundation for the investigation of enzyme engineering, genetic and metabolic engineering. This review summarized 7 plant-specific type III PKS in the aspects of their crystal structures and functions.
Acyltransferases ; chemistry ; genetics ; physiology ; Amino Acid Sequence ; Catalysis ; Chalcones ; Crystallization ; Flavanones ; Genetic Engineering ; Metabolic Engineering ; Molecular Sequence Data ; Plant Proteins ; chemistry ; genetics ; physiology ; Plants ; enzymology ; genetics ; Protein Structure, Secondary ; Substrate Specificity

Objective: To investigate the safety of transgenic human lung adenocarcinoma cell line SPC-A-1/IL-2 as tumor vaccine. Methods: IL-2 gene was introduced into human lung adenocarcinoma cell line SPC-A-1 and was expressed stably on the basis of the construction of retroviral packing cell line PA317/pLIL-2SN.The mutagenesis of both the DNA and supernatant of SPC-A-1/IL-2 in the and in vitro was tested by means of genetic toxicological techniques.Results:The result indicated that mutagenesis of both the DNA and the supernatant of transgenic cell SPC-A-1/IL-2 was not observed. Conclusion: The initial experiment suggested that the application of transgenic cell SPC-A-l/IL-2 as tumor vaccine was bisically safe and reliable.