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Proceedings of the National Academy of... May 2024Phytophagous insects have evolved sophisticated detoxification systems to overcome the antiherbivore chemical defenses produced by many plants. However, how these...
Phytophagous insects have evolved sophisticated detoxification systems to overcome the antiherbivore chemical defenses produced by many plants. However, how these biotransformation systems differ in generalist and specialist insect species and their role in determining insect host plant range remains an open question. Here, we show that UDP-glucosyltransferases (UGTs) play a key role in determining the host range of insect species within the genus. Comparative genomic analyses of species that differ in host plant breadth identified a relatively conserved number of UGT genes in generalist species but high levels of UGT gene pseudogenization in the specialist . CRISPR-Cas9 knockouts of the three main UGT gene clusters of revealed that UGT33 genes play an important role in allowing this species to utilize the poaceous plants maize, wheat, and rice, while UGT40 genes facilitate utilization of cotton. Further functional analyses in vivo and in vitro identified the UGT SfUGT33F32 as the key mechanism that allows generalist to detoxify the benzoxazinoid DIMBOA (2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one), a potent insecticidal phytotoxin produced by poaceous plants. However, while this detoxification capacity is conserved in several generalist species, , which specializes on plants, is unable to detoxify DIMBOA due to a nonfunctionalizing mutation in . Collectively, these findings provide insight into the role of insect UGTs in host plant adaptation, the mechanistic basis of evolutionary transitions between generalism and specialism and offer molecular targets for controlling a group of notorious insect pests.
Topics: Animals; Spodoptera; Glycosyltransferases; Host Specificity; Uridine Diphosphate; Insect Proteins; Phylogeny
PubMed: 38683998
DOI: 10.1073/pnas.2402045121 -
Viruses Mar 2024Glycosylation, a dynamic modification prevalent in viruses and higher eukaryotes, is principally regulated by uridine diphosphate (UDP)-glycosyltransferases (UGTs) in...
Glycosylation, a dynamic modification prevalent in viruses and higher eukaryotes, is principally regulated by uridine diphosphate (UDP)-glycosyltransferases (UGTs) in plants. Although UGTs are involved in plant defense responses, their responses to most pathogens, especially plant viruses, remain unclear. Here, we aimed to identify UGTs in the whole genome of () and to analyze their function in Chinese wheat mosaic virus (CWMV) infection. A total of 147 were identified in . To conduct a phylogenetic analysis, the UGT protein sequences of and were aligned. The gene structure and conserved motifs of the UGTs were also analyzed. Additionally, the physicochemical properties and predictable subcellular localization were examined in detail. Analysis of cis-acting elements in the putative promoter revealed that were involved in temperature, defense, and hormone responses. The expression levels of 20 containing defense-related cis-acting elements were assessed in CWMV-infected , revealing a significant upregulation of 8 . Subcellular localization analysis of three NbUGTs (NbUGT12, NbUGT16 and NbUGT17) revealed their predominant localization in the cytoplasm of leaves, and NbUGT12 was also distributed in the chloroplasts. CWMV infection did not alter the subcellular localization of NbUGT12, NbUGT16, and NbUGT17. Transient overexpression of , , and enhanced CWMV infection, whereas the knockdown of , and inhibited CWMV infection in . These could serve as potential susceptibility genes to facilitate CWMV infection. Overall, the findings throw light on the evolution and function of .
Topics: Nicotiana; Plant Diseases; Glycosyltransferases; Phylogeny; Disease Resistance; Gene Expression Regulation, Plant; Plant Proteins; Genome, Plant; Uridine Diphosphate; Potyvirus; Genome-Wide Association Study
PubMed: 38675832
DOI: 10.3390/v16040489 -
Microbial Cell Factories Apr 2024Isoquercitrin (quercetin-3-O-β-D-glucopyranoside) has exhibited promising therapeutic potentials as cardioprotective, anti-diabetic, anti-cancer, and anti-viral agents....
BACKGROUND
Isoquercitrin (quercetin-3-O-β-D-glucopyranoside) has exhibited promising therapeutic potentials as cardioprotective, anti-diabetic, anti-cancer, and anti-viral agents. However, its structural complexity and limited natural abundance make both bulk chemical synthesis and extraction from medical plants difficult. Microbial biotransformation through heterologous expression of glycosyltransferases offers a safe and sustainable route for its production. Despite several attempts reported in microbial hosts, the current production levels of isoquercitrin still lag behind industrial standards.
RESULTS
Herein, the heterologous expression of glycosyltransferase UGT78D2 gene in Bacillus subtilis 168 and reconstruction of UDP-glucose (UDP-Glc) synthesis pathway led to the synthesis of isoquercitrin from quercetin with titers of 0.37 g/L and 0.42 g/L, respectively. Subsequently, the quercetin catabolism blocked by disruption of a quercetin dioxygenase, three ring-cleavage dioxygenases, and seven oxidoreductases increased the isoquercitrin titer to 1.64 g/L. And the hydrolysis of isoquercitrin was eliminated by three β-glucosidase genes disruption, thereby affording 3.58 g/L isoquercitrin. Furthermore, UDP-Glc pool boosted by pgi (encoding glucose-6-phosphate isomerase) disruption increased the isoquercitrin titer to 10.6 g/L with the yield on quercetin of 72% and to 35.6 g/L with the yield on quercetin of 77.2% in a 1.3-L fermentor.
CONCLUSION
The engineered B. subtilis strain developed here holds great potential for initiating the sustainable and large-scale industrial production of isoquercitrin. The strategies proposed in this study provides a reference to improve the production of other flavonoid glycosides by engineered B. subtilis cell factories.
Topics: Quercetin; Metabolic Engineering; Bacillus subtilis; Uridine Diphosphate
PubMed: 38641799
DOI: 10.1186/s12934-024-02390-5 -
Biochemical and Biophysical Research... Jun 2024Polymerization of nucleotides under prebiotic conditions simulating the early Earth has been extensively studied. Several independent methods have been used to verify...
Polymerization of nucleotides under prebiotic conditions simulating the early Earth has been extensively studied. Several independent methods have been used to verify that RNA-like polymers can be produced by hot wet-dry cycling of nucleotides. However, it has not been shown that these RNA-like polymers are similar to biological RNA with 3'-5' phosphodiester bonds. In the results described here, RNA-like polymers were generated from 5'-monophosphate nucleosides AMP and UMP. To confirm that the polymers resemble biological RNA, ribonuclease A should catalyze hydrolysis of the 3'-5' phosphodiester bonds between pyrimidine nucleotides to each other or to purine nucleotides, but not purine-purine nucleotide bonds. Here we show AFM images of specific polymers produced by hot wet-dry cycling of AMP, UMP and AMP/UMP (1:1) solutions on mica surfaces, before and after exposure to ribonuclease A. AMP polymers were unaffected by ribonuclease A but UMP polymers disappeared. This indicates that a major fraction of the bonds in the UMP polymers is indeed 3'-5' phosphodiester bonds. Some of the polymers generated from the AMP/UMP mixture also showed clear signs of cleavage. Because ribonuclease A recognizes the ester bonds in the polymers, we show for the first time that these prebiotically produced polymers are in fact similar to biological RNA but are likely to be linked by a mixture of 3'-5' and 2'-5' phosphodiester bonds.
Topics: RNA; Ribonuclease, Pancreatic; Uridine Monophosphate; Microscopy, Atomic Force; Hot Temperature; Polymers; Adenosine Monophosphate; Hydrolysis; Polymerization
PubMed: 38640739
DOI: 10.1016/j.bbrc.2024.149938 -
Nucleic Acids Research May 2024It has been proposed that coronavirus nsp15 mediates evasion of host cell double-stranded (ds) RNA sensors via its uracil-specific endoribonuclease activity. However,...
It has been proposed that coronavirus nsp15 mediates evasion of host cell double-stranded (ds) RNA sensors via its uracil-specific endoribonuclease activity. However, how nsp15 processes viral dsRNA, commonly considered as a genome replication intermediate, remains elusive. Previous research has mainly focused on short single-stranded RNA as substrates, and whether nsp15 prefers single-stranded or double-stranded RNA for cleavage is controversial. In the present work, we prepared numerous RNA substrates, including both long substrates mimicking the viral genome and short defined RNA, to clarify the substrate preference and cleavage pattern of SARS-CoV-2 nsp15. We demonstrated that SARS-CoV-2 nsp15 preferentially cleaved pyrimidine nucleotides located in less thermodynamically stable areas in dsRNA, such as AU-rich areas and mismatch-containing areas, in a nicking manner. Because coronavirus genomes generally have a high AU content, our work supported the mechanism that coronaviruses evade the antiviral response mediated by host cell dsRNA sensors by using nsp15 dsRNA nickase to directly cleave dsRNA intermediates formed during genome replication and transcription.
Topics: RNA, Double-Stranded; SARS-CoV-2; RNA, Viral; Viral Nonstructural Proteins; Humans; Endoribonucleases; Virus Replication; Substrate Specificity; Genome, Viral; COVID-19
PubMed: 38634805
DOI: 10.1093/nar/gkae290 -
Accounts of Chemical Research May 2024In 1960, Weber prophesied that "There are many ways in which the properties of the excited state can be utilized to study points of ignorance of the structure and...
In 1960, Weber prophesied that "There are many ways in which the properties of the excited state can be utilized to study points of ignorance of the structure and function of proteins". This has been realized, illustrating that an intrinsic and highly responsive fluorophore such as tryptophan can alter the course of an entire scientific discipline. But what about RNA and DNA? Adapting Weber's protein photophysics prophecy to nucleic acids requires the development of intrinsically emissive nucleoside surrogates as, unlike Trp, the canonical nucleobases display unusually low emission quantum yields, which render nucleosides, nucleotides, and oligonucleotides practically dark for most fluorescence-based applications.Over the past decades, we have developed emissive nucleoside surrogates that facilitate the monitoring of nucleoside-, nucleotide-, and nucleic acid-based transformations at a nucleobase resolution in real time. The premise underlying our approach is the identification of minimal atomic/structural perturbations that endow the synthetic analogs with favorable photophysical features while maintaining native conformations and pairing. As illuminating probes, the photophysical parameters of such isomorphic nucleosides display sensitivity to microenvironmental factors. Responsive isomorphic analogs that function similarly to their native counterparts in biochemical contexts are defined as isofunctional.Early analogs included pyrimidines substituted with five-membered aromatic heterocycles at their 5 position and have been used to assess the polarity of the major groove in duplexes. Polarized quinazolines have proven useful in assembling FRET pairs with established fluorophores and have been used to study RNA-protein and RNA-small-molecule binding. Completing a fluorescent ribonucleoside alphabet, composed of visibly emissive purine (A, G) and pyrimidine (U, C) analogs, all derived from thieno[3,4-]pyrimidine as the heterocyclic nucleus, was a major breakthrough. To further augment functionality, a second-generation emissive RNA alphabet based on an isothiazolo[4,3-]pyrimidine core (A, G, U, and C) was fabricated. This single-atom "mutagenesis" restored the basic/coordinating nitrogen corresponding to N7 in the purine skeleton and elevated biological recognition.The isomorphic emissive nucleosides and nucleotides, particularly the purine analogs, serve as substrates for diverse enzymes. Beyond polymerases, we have challenged the emissive analogs with metabolic and catabolic enzymes, opening optical windows into the biochemistry of nucleosides and nucleotides as metabolites as well as coenzymes and second messengers. Real-time fluorescence-based assays for adenosine deaminase, guanine deaminase, and cytidine deaminase have been fabricated and used for inhibitor discovery. Emissive cofactors (e.g., SAM), coenzymes (e.g., NAD), and second messengers (e.g., c-di-GMP) have been enzymatically synthesized, using NTPs and native enzymes. Both their biosynthesis and their transformations can be fluorescently monitored in real time.Highly isomorphic and isofunctional emissive surrogates can therefore be fabricated and judiciously implemented. Beyond their utility, side-by-side comparison to established analogs, particularly to 2-aminopurine, the workhorse of nucleic acid biophysics over 5 decades, has proven prudent as they refined the scope and limitations of both the new analogs and their predecessors. Challenges, however, remain. Associated with such small heterocycles are relatively short emission wavelengths and limited brightness. Recent advances in multiphoton spectroscopy and further structural modifications have shown promise for overcoming such barriers.
Topics: Fluorescent Dyes; Nucleosides; RNA; DNA
PubMed: 38613490
DOI: 10.1021/acs.accounts.4c00042 -
BMC Pediatrics Apr 2024Dilated cardiomyopathy (DCMP) is characterized by the enlargement and weakening of the heart and is a major cause of heart failure in children. Infection and nutritional...
BACKGROUND
Dilated cardiomyopathy (DCMP) is characterized by the enlargement and weakening of the heart and is a major cause of heart failure in children. Infection and nutritional deficiencies are culprits for DCMP. Zinc is an important nutrient for human health due to its anti-oxidant effect that protects cell against oxidative damage. This case-control study aimed to investigate the relationship between dietary intake of zinc and selenium and the risk of DCMP in pediatric patients.
METHODS
A total of 36 DCMP patients and 72 matched controls were recruited, and their dietary intakes were assessed via a validated food frequency questionnaire. We used chi-square and sample T-test for qualitative and quantitative variables, respectively. Logistic regression analysis was applied to assess the relationship between selenium and zinc intake with the risk of DCMP.
RESULTS
After fully adjusting for confounding factors, analyses showed that selenium (OR = 0.19, CI = 0.057-0.069, P trend < 0.011) and zinc (OR = 0.12, CI = 0.035-0.046, P trend < 0.002) intake were strongly associated with 81% and 88% lower risk of pediatric DCMP, respectively.
CONCLUSIONS
This study highlights the protective role of adequate dietary intake of selenium and zinc in decreasing the risk of DCMP in children. Malnutrition may exacerbate the condition and addressing these micronutrient deficiencies may improve the cardiac function. Further studies are recommended to detect the underlying mechanisms and dietary recommendations for DCMP prevention.
Topics: Humans; Child; Selenium; Case-Control Studies; Cardiomyopathy, Dilated; Deoxycytidine Monophosphate; Zinc; Malnutrition
PubMed: 38605385
DOI: 10.1186/s12887-024-04706-1 -
Bioorganic Chemistry Jun 2024Finding potent inhibitors of O-GlcNAc transferase (OGT) has proven to be a challenge, especially because the diversity of published inhibitors is low. The large majority...
Finding potent inhibitors of O-GlcNAc transferase (OGT) has proven to be a challenge, especially because the diversity of published inhibitors is low. The large majority of available OGT inhibitors are uridine-based or uridine-like compounds that mimic the main interactions of glycosyl donor UDP-GlcNAc with the enzyme. Until recently, screening of DNA-encoded libraries for discovering hits against protein targets was dedicated to a few laboratories around the world, but has become accessible to wider public with the recent launch of the DELopen platform. Here we report the results and follow-up of a DNA-encoded library screening by using the DELopen platform. This led to the discovery of two new hits with structural features not resembling UDP. Small focused libraries bearing those two scaffolds were made, leading to low micromolar inhibition of OGT and elucidation of their structure-activity relationship.
Topics: N-Acetylglucosaminyltransferases; Enzyme Inhibitors; Structure-Activity Relationship; DNA; Humans; Drug Discovery; Small Molecule Libraries; Molecular Structure; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Uridine Diphosphate
PubMed: 38604018
DOI: 10.1016/j.bioorg.2024.107321 -
Clinical Journal of Gastroenterology Jun 2024Hepatitis C virus (HCV) reactivation has been reported to be caused due to several anticancer drugs and immunosuppressive agents; however, HCV reactivation after steroid...
Hepatitis C virus (HCV) reactivation has been reported to be caused due to several anticancer drugs and immunosuppressive agents; however, HCV reactivation after steroid monotherapy has rarely been reported. Here, we report the case of a 65-year-old Japanese man with HCV infection who developed HCV reactivation after the administration of prednisolone (PSL) for 6 days for sudden deafness. In the patient history, the positivity for anti-HCV antibody was observed, but serum level of HCV RNA was not measured. Two months after PSL administration, the patient experienced an alanine aminotransferase (ALT) flare and the serum level of HCV RNA was observed to be 6.2 log IU/mL; then, the patient was admitted to our hospital for hepatitis treatment. Based on the clinical course and laboratory findings, the patient was diagnosed with HCV reactivation. Although the ALT levels decreased spontaneously during follow-up, they did not drop to normal range; subsequently, sofosbuvir and ledipasvir treatments were started. A sustained virological response 24 weeks after the end of treatment was achieved. This case study suggests that HCV reactivation with hepatitis flare can occur even after a steroid monotherapy, and doctors should pay attention to HCV reactivation when administering PSL for patients with HCV infection.
Topics: Humans; Male; Aged; Prednisolone; Hearing Loss, Sudden; Virus Activation; Antiviral Agents; Hepacivirus; Hepatitis C; Sofosbuvir; Fluorenes; Benzimidazoles; Alanine Transaminase; RNA, Viral; Glucocorticoids
PubMed: 38587568
DOI: 10.1007/s12328-024-01944-9 -
Journal of Microbiology and... May 2024Glucosylation is a well-known approach to improve the solubility, pharmacological, and biological properties of flavonoids, making flavonoid glucosides a target for...
Glucosylation is a well-known approach to improve the solubility, pharmacological, and biological properties of flavonoids, making flavonoid glucosides a target for large-scale biosynthesis. However, the low yield of products coupled with the requirement of expensive UDP-sugars limits the application of enzymatic systems for large-scale. is a Gram-positive and generally regarded as safe (GRAS) bacteria frequently employed for the large-scale production of amino acids and bio-fuels. Due to the versatility of its cell factory system and its non-endotoxin producing properties, it has become an attractive system for the industrial-scale biosynthesis of alternate products. Here, we explored the cell factory of for efficient glucosylation of flavonoids using apigenin as a model flavonoid, with the heterologous expression of a promiscuous glycosyltransferase, YdhE from and the endogenous overexpression of genes encoding UDP-glucose pyrophosphorylase and encoding phosphoglucomutase involved in the synthesis of UDP-glucose to create a cell factory system capable of efficiently glucosylation apigenin with a high yield of glucosides production. Consequently, the production of various apigenin glucosides was controlled under different temperatures yielding almost 4.2 mM of APG1(apigenin-4'-O-β-glucoside) at 25°C, and 0.6 mM of APG2 (apigenin-7-O-β-glucoside), 1.7 mM of APG3 (apigenin-4',7-O-β-diglucoside) and 2.1 mM of APG4 (apigenin-4',5-O-β-diglucoside) after 40 h of incubation with the supplementation of 5 mM of apigenin and 37°C. The cost-effective developed system could be used to modify a wide range of plant secondary metabolites with increased pharmacokinetic activities on a large scale without the use of expensive UDP-sugars.
Topics: Corynebacterium glutamicum; Apigenin; Metabolic Engineering; Glucosides; Glycosylation; Bacillus licheniformis; Uridine Diphosphate Glucose; Bacterial Proteins; UTP-Glucose-1-Phosphate Uridylyltransferase; Glycosyltransferases
PubMed: 38563097
DOI: 10.4014/jmb.2401.01017