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1.
BMC Res Notes ; 14(1): 417, 2021 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-34794506

RESUMO

OBJECTIVE: Neisseria meningitidis is a Gram-negative bacterium that causes meningitis. N. meningitidis serogroup W (NmW) capsule polymerase synthesizes capsular polysaccharide of this serogroup. This enzyme could be a tool for meningococcal glycoconjugate vaccine development. Our long-term goal is to control activity of the NmW capsule polymerase for production of defined carbohydrates for vaccines. The enzyme lacks a simple, high-throughput activity assay. Here, we describe the use of high-throughput bioluminescence assays (CMP-Glo and UDP-Glo by Promega) to investigate NmW capsule polymerase activity. These assays detect free nucleotides produced during transfer of sugar from UDP-Galactose and CMP-Sialic Acid to an acceptor. Kinetic studies using NmW hydrolyzed polysaccharide (PS) acceptor are described as well as preliminary work with a sialic acid trimer (DP3) acceptor. RESULTS: In CMP-Glo kinetic studies, with constant donor (80 µM) and varied NmW hydrolyzed polysaccharide (0-2000 µg/mL), a Km of 629.2 ± 101.4 µg/mL and a Vmax of 0.8965 ± 0.05823 µM/min was obtained. Using UDP-Glo, Km and Vmax values of 13.84 ± 9.675 µM and 0.6205 ± 0.1331 µM/min were obtained with varied CMP-NeuNAc (0-80 µM) and constant acceptor (400 µg/mL) and UDP-Gal (80 µM). This is the first report of using bioluminescence assays for NmW kinetics.


Assuntos
Vacinas Meningocócicas , Neisseria meningitidis , Cinética , Sorogrupo , Transferases
2.
Biochemistry (Mosc) ; 86(8): 992-1002, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34488575

RESUMO

During protein synthesis, ribosome moves along mRNA to decode one codon after the other. Ribosome translocation is induced by a universally conserved protein, elongation factor G (EF-G) in bacteria and elongation factor 2 (EF-2) in eukaryotes. EF-G-induced translocation results in unwinding of the intramolecular secondary structures of mRNA by three base pairs at a time that renders the translating ribosome a processive helicase. Professor Alexander Sergeevich Spirin has made numerous seminal contributions to understanding the molecular mechanism of translocation. Here, we review Spirin's insights into the ribosomal translocation and recent advances in the field that stemmed from Spirin's pioneering work. We also discuss key remaining challenges in studies of translocase and helicase activities of the ribosome.


Assuntos
RNA Helicases/química , Ribossomos/fisiologia , Transferases/química , Transporte Biológico , Microscopia Crioeletrônica , Eucariotos/metabolismo , Transferência Ressonante de Energia de Fluorescência , Modelos Moleculares , Fator 2 de Elongação de Peptídeos/química , Fator G para Elongação de Peptídeos/química , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , RNA de Transferência/química , Ribossomos/química
3.
Environ Toxicol Pharmacol ; 88: 103748, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34534692

RESUMO

In the current study, Triclosan (TCS, a commonly used antimicrobial agent) induced alterations in biochemical parameters and gene expression were recorded in the larvae of Labeo rohita after 96 h exposure and 10 days recovery period to find out health status biomarkers. 96 h exposure to 0.06, 0.067 and 0.097 mg/L TCS significantly declined the levels of glucose, triglycerides, urea and uric acid and activity of alkaline phosphatase (ALP), glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT). There was a non-significant decline in the levels of cholesterol and total protein but albumin and total bilirubin showed no change. After 10 days of recovery period, trend was opposite for glucose, urea and ALP only. Decline in the expression of trypsin and pancreatic amylase and elevation in creatine kinase during exposure to TCS showed a reverse trend after recovery period. However, concentration dependent elevation of chymotrypsin persisted till the end of recovery period. Principal Component Analysis (PCA) showed association of total protein, ALP, GOT, creatine kinase and pancreatic amylase with PC1 after exposure as well as recovery period. Therefore, these can be considered as important biomolecules for identification of health status of TCS stressed fish.


Assuntos
Anti-Infecciosos/toxicidade , Cyprinidae , Transcriptoma/efeitos dos fármacos , Triclosan/toxicidade , Poluentes Químicos da Água/toxicidade , Animais , Cyprinidae/genética , Cyprinidae/metabolismo , Proteínas de Peixes/genética , Glucose/metabolismo , Hidrolases/genética , Larva/efeitos dos fármacos , Larva/genética , Larva/metabolismo , Transferases/genética , Triglicerídeos/metabolismo , Ureia/metabolismo , Ácido Úrico/metabolismo
4.
J Biol Chem ; 297(3): 100991, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34419450

RESUMO

Fic domain-containing AMP transferases (fic AMPylases) are conserved enzymes that catalyze the covalent transfer of AMP to proteins. This posttranslational modification regulates the function of several proteins, including the ER-resident chaperone Grp78/BiP. Here we introduce a mouse FICD (mFICD) AMPylase knockout mouse model to study fic AMPylase function in vertebrates. We find that mFICD deficiency is well tolerated in unstressed mice. We also show that mFICD-deficient mouse embryonic fibroblasts are depleted of AMPylated proteins. mFICD deletion alters protein synthesis and secretion in splenocytes, including that of IgM, an antibody secreted early during infections, and the proinflammatory cytokine IL-1ß, without affecting the unfolded protein response. Finally, we demonstrate that visual nonspatial short-term learning is stronger in old mFICD-/- mice than in wild-type controls while other measures of cognition, memory, and learning are unaffected. Together, our results suggest a role for mFICD in adaptive immunity and neuronal plasticity in vivo.


Assuntos
Citocinas/metabolismo , Aprendizagem , Transferases/metabolismo , Percepção Visual , Animais , Células Cultivadas , Camundongos , Camundongos Knockout
5.
Plant Sci ; 310: 110960, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34315585

RESUMO

The methylerythritol 4-phosphate (MEP) pathway is of paramount importance for generating plastidial isoprenoids. The first enzyme of the MEP pathway, 1-deoxy-D-xylulose-5-phosphate synthase (DXS), catalyzes a flux-controlling step. In plants the DXS gene family is composed of three distinct classes with non-redundant functions. Although the DXS1 and DXS2 subfamilies have been well characterized, the DXS3 subfamily has been considerably understudied. Here, we carried out in silico and functional analyses to better understand the DXS3 class. Our phylogenetic analysis showed high variation in copy number among the different DXS classes, with the apparent absence of DXS1 class in some species. We found that DXS3 subfamily emerged later than DXS1 and DXS2 and it is under less intense purifying selection. Furthermore, in the DXS3 subfamily critical amino acids positions in the thiamine pyrophosphate binding pocket are not conserved. We demonstrated that the DXS3 proteins from Arabidopsis, Maize, and Rice lack functional DXS activity. Moreover, the Arabidopsis DXS3 protein displayed distinctive sub-organellar chloroplast localization not observed in any DXS1 or DXS2 proteins. Co-expression analysis of the DXS3 from Arabidopsis showed that, unlike DXS1 and DXS2 proteins, it co-expresses with genes related to post-embryonic development and reproduction and not with primary metabolism and isoprenoid synthesis.


Assuntos
Plantas Geneticamente Modificadas/metabolismo , Plastídeos/metabolismo , Transferases/metabolismo , Evolução Molecular , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Filogenia , Plantas Geneticamente Modificadas/genética , Plastídeos/genética , Transferases/genética
6.
Res Vet Sci ; 139: 94-101, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34273745

RESUMO

Effects of Selenium-enriched probiotics (SP) on ochratoxin A-induced kidney injury, growth performance, antioxidant injury, selenoprotein and DNA methylation transferases (DNMTs) expression of piglets were investigated in the article. A total of 48 piglets were randomly divided into 4 groups and fed with basal diet (Con, 0.15 mg Se/kg and OTA at 0.00 mg/kg), basal diets added with OTA (OTA, 0.40 mg OTA/kg), SP and OTA (SP1, 0.15 mg Se/kg and 0.40 mg OTA/kg), SP and OTA (SP2, 0.30 mg Se/kg and 0.40 mg OTA/kg) respectively for 42 days. From each group, six piglets were randomly selected for blood collection on Days 0 and 42 and three piglets were selected for tissue collection on Day 42.The results showed that OTA at 0.40 mg /kg significantly decreased growth performance of pigs, induced the histopathological lesions of kidney and increased urea and creatine levels of serum, decreased GPx and SOD activities, and increased MDA levels. OTA decreased GPx1, GPx4 and SelS expressions, and increased TR1, DNMT 1, DNMT3a and SOCS3 expressions. Both SP1 and SP2 improved OTA-induced poor growth performance, kidney injury, poor antioxidant statues, GPx1, SelS, TR1, SOCS3, DNMT1 and DNMT3a expressions in kidney of pigs. The effects of SP2 on the above parameters changes were better than that of SP1. SP increased GPx and SOD activities and decreased MDA levels changes induced by OTA treatment. These results suggest that SP may serve as a better feed additive for piglets under mycotoxin contamination environments.


Assuntos
Rim/lesões , Ocratoxinas , Probióticos , Selênio , Ração Animal/análise , Animais , Metilação de DNA , Rim/metabolismo , Ocratoxinas/metabolismo , Selênio/metabolismo , Selênio/farmacologia , Suínos , Transferases/metabolismo
7.
Genes (Basel) ; 12(5)2021 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-34065649

RESUMO

The aroma of grapes and derived wines has long been one of the major traits considered in the selection of grapevine varieties through the centuries. In particular, Muscat aromatic grapes have been highly appreciated and widespread since ancient times. Monoterpenes are the key compounds responsible for the Muscat flavor. A major QTL affecting monoterpene level has been found to co-localize with the 1-deoxy-D-xylulose 5-phosphate synthase (VvDXS) gene, encoding for the 1-deoxy-D-xylulose 5-phosphate synthase enzyme involved in the plastidial pathway of terpene biosynthesis. In more detail, a single nucleotide polymorphism (SNP 1822) in the coding region of the gene causes a "gain of function" mutation, which is involved in Muscat flavor. In this work, we have developed a digital PCR-based assay to target allelic variations in the VvDXS gene, SNP1822, with the aim to propose a fast and sensitive analytical tool for targeting Muscat-flavored grapevine genotypes. The assay accurately predicts the genetic structure at 1822 SNP, critical for the development of the aroma in the great majority of Muscats. In the case of grapes in which the aromatic component is due to mutations other than SNP 1822 (e.g., Chasselas Musqué and Chardonnay Muscat), further specific assays can be developed.


Assuntos
Técnicas de Genotipagem/métodos , Proteínas de Plantas/genética , Polimorfismo de Nucleotídeo Único , Transferases/genética , Vitis/genética , Mutação com Ganho de Função , Melhoramento Vegetal/métodos , Reação em Cadeia da Polimerase/métodos , Vitis/metabolismo
8.
Arch Oral Biol ; 128: 105160, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34044343

RESUMO

OBJECTIVE: To explore the role of DNA methyltransferase 1 (DNMT1) in development and progression of hypopharyngeal squamous carcinoma. DESIGN: A total of 32 hypopharyngeal squamous carcinoma biopsy samples and 20 normal tissue specimens were collected. Immunohistochemical staining, quantitative real-time polymerase chain reaction, and Western blot were performed for expression analysis. The mRNA and protein expression in the specimens and subcellular localization were analyzed. hypopharyngeal squamous carcinoma cells (FaDu) were used for small interfering RNA of DNMT1, and proliferation, cell cycle, and apoptosis were determined in the transfected cells. Furthermore, metastatic ability and methylation status of O6-methylguanine-DNAmethyl-transferase (MGMT) promoter was assessed. RESULTS: Our results showed that DNMT1 was overexpressed, while MGMT was down expressed in hypopharyngeal squamous carcinoma. DNMT1 overexpression and MGMT down expression were significantly associated with poorly differentiated tumors, lymph node metastasis, and clinical stage. DNMT1 and MGMT were majorly distributed in the nucleus. Furthermore, knockdown of DNMT1 inhibited proliferation and metastasis, induced apoptosis and G1 phase arrest in FaDu cells, and upregulated MGMT expression to reverse methylation status of MGMT promoter. CONCLUSIONS: This study for the first time demonstrated the clinical value and the role of DNMT1 and MGMT in the biological function of hypopharyngeal squamous carcinoma. This work suggested that DNMT1 might serve as a potential therapeutic target for patients with hypopharyngeal squamous carcinoma.


Assuntos
Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , Carcinoma de Células Escamosas/genética , Proliferação de Células , DNA , DNA (Citosina-5-)-Metiltransferase 1 , Metilação de DNA , Guanina/análogos & derivados , Humanos , Metiltransferases , Transferases
9.
Nat Commun ; 12(1): 3007, 2021 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-34021140

RESUMO

Small heat shock proteins (sHsps) bind unfolding proteins, thereby playing a pivotal role in the maintenance of proteostasis in virtually all living organisms. Structural elucidation of sHsp-substrate complexes has been hampered by the transient and heterogeneous nature of their interactions, and the precise mechanisms underlying substrate recognition, promiscuity, and chaperone activity of sHsps remain unclear. Here we show the formation of a stable complex between Arabidopsis thaliana plastid sHsp, Hsp21, and its natural substrate 1-deoxy-D-xylulose 5-phosphate synthase (DXPS) under heat stress, and report cryo-electron microscopy structures of Hsp21, DXPS and Hsp21-DXPS complex at near-atomic resolution. Monomeric Hsp21 binds across the dimer interface of DXPS and engages in multivalent interactions by recognizing highly dynamic structural elements in DXPS. Hsp21 partly unfolds its central α-crystallin domain to facilitate binding of DXPS, which preserves a native-like structure. This mode of interaction suggests a mechanism of sHsps anti-aggregation activity towards a broad range of substrates.


Assuntos
Arabidopsis/metabolismo , Proteínas de Choque Térmico Pequenas/química , Proteínas de Choque Térmico Pequenas/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Microscopia Crioeletrônica , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Proteínas de Choque Térmico Pequenas/genética , Resposta ao Choque Térmico , Modelos Moleculares , Dobramento de Proteína , Transferases/química , Transferases/metabolismo
10.
Biomolecules ; 11(5)2021 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-33947162

RESUMO

Nowadays, enzyme-mediated processes offer an eco-friendly and efficient alternative to the traditional multistep and environmentally harmful chemical processes. Herein we report the enzymatic synthesis of cladribine by a novel 2'-deoxyribosyltransferase (NDT)-based combined biocatalyst. To this end, Lactobacillus delbrueckii NDT (LdNDT) was successfully immobilized through a two-step immobilization methodology, including a covalent immobilization onto glutaraldehyde-activated biomimetic silica nanoparticles followed by biocatalyst entrapment in calcium alginate. The resulting immobilized derivative, SiGPEI 25000-LdNDT-Alg, displayed 98% retained activity and was shown to be active and stable in a broad range of pH (5-9) and temperature (30-60 °C), but also displayed an extremely high reusability (up to 2100 reuses without negligible loss of activity) in the enzymatic production of cladribine. Finally, as a proof of concept, SiGPEI 25000-LdNDT-Alg was successfully employed in the green production of cladribine at mg scale.


Assuntos
Cladribina/metabolismo , Lactobacillus delbrueckii/enzimologia , Transferases/química , Transferases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Biocatálise , Estabilidade Enzimática , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Glutaral/química , Química Verde , Concentração de Íons de Hidrogênio , Dióxido de Silício/química , Temperatura
11.
Plant Biotechnol J ; 19(9): 1878-1886, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33949064

RESUMO

To what degree can the lignin subunits in a monocot be derived from monolignol ferulate (ML-FA) conjugates? This simple question comes with a complex set of variables. Three potential requirements for optimizing ML-FA production are as follows: (1) The presence of an active FERULOYL-CoA MONOLIGNOL TRANSFERASE (FMT) enzyme throughout monolignol production; (2) Suppression or elimination of enzymatic pathways competing for monolignols and intermediates during lignin biosynthesis; and (3) Exclusion of alternative phenolic compounds that participate in lignification. A 16-fold increase in lignin-bound ML-FA incorporation was observed by introducing an AsFMT gene into Brachypodium distachyon. On its own, knocking out the native p-COUMAROYL-CoA MONOLIGNOL TRANSFERASE (BdPMT) pathway that competes for monolignols and the p-coumaroyl-CoA intermediate did not change ML-FA incorporation, nor did partial loss of CINNAMOYL-CoA REDUCTASE1 (CCR1) function, which reduced metabolic flux to monolignols. However, stacking AsFMT into the Bdpmt-1 mutant resulted in a 32-fold increase in ML-FA incorporation into lignin over the wild-type level.


Assuntos
Brachypodium , Brachypodium/genética , Lignina , Proteínas de Plantas/genética , Transferases
12.
FASEB J ; 35(5): e21575, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33826776

RESUMO

Staphylopine (StP) and other nicotianamine-like metallophores are crucial for many pathogens to acquire the transition metals from hosts during invasion. CntL from Staphylococcus aureus (SaCntL) catalyzes the condensation of the 2-aminobutyrate (Ab) moiety of S-adenosylmethionine (SAM) with D-histidine in the biosynthesis of StP. Here, we report the crystal structures of SaCntL in complex with either SAM or two products. The structure of SaCntL consists of an N-terminal four-helix bundle (holding catalytic residue E84) and a C-terminal Rossmann fold (binding the substrates). The sequence connecting the N- and C-terminal domains (N-C linker) in SaCntL was found to undergo conformational alternation between open and closed states. Our structural and biochemical analyses suggested that this intrinsically dynamic interdomain linker forms an additional structural module that plays essential roles in ligand diffusion, recognition, and catalysis. We confirmed that SaCntL stereoselectively carries out the catalysis of D-His but not its enantiomer, L-His, and we found that the N-C linker and active site of SaCntL could accommodate both enantiomers. SaCntL is likely able to bind L-His without catalysis, and as a result, L-His could show inhibitory effects toward SaCntL. These findings provide critical structural and mechanistic insights into CntL, which facilitates a better understanding of the biosynthesis of nicotianamine-like metallophores and the discovery of inhibitors of this process.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Imidazóis/metabolismo , Staphylococcus aureus/enzimologia , Transferases/química , Transferases/metabolismo , Sítios de Ligação , Catálise , Domínio Catalítico , Cristalografia por Raios X , Ligantes , Modelos Moleculares , Conformação Proteica
13.
Plant Biotechnol J ; 19(5): 878-896, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33811433

RESUMO

The cytokinins, which are N6 -substituted adenine derivatives, control key aspects of crop productivity. Cytokinin levels are controlled via biosynthesis by isopentenyl transferase (IPT), destruction by cytokinin oxidase/dehydrogenase (CKX), and inactivation via glucosylation by cytokinin glucosyl transferases (CGTs). While both yield components and tolerance to drought and related abiotic stressors have been positively addressed via manipulation of IPT and/or CKX expression, much less attention has been paid to the CGTs. As naming of the CGTs has been unclear, we suggest COGT, CNGT, CONGT and CNOGT to describe the O-, N- and dual function CGTs. As specific CGT mutants of both rice and arabidopsis showed impacts on yield components, we interrogated the wheat genome database, IWGSC RefSeq v1.0 & v2.0, to investigate wheat CGTs. Besides providing unambiguous names for the 53 wheat CGTs, we show their expression patterns in 70 developmental tissues and their response characteristics to various stress conditions by reviewing more than 1000 RNA-seq data sets. These revealed various patterns of responses and showed expression generally being more limited in reproductive tissues than in vegetative tissues. Multiple cis-regulatory elements are present in the 3 kb upstream of the start codons of the 53 CGTs. Elements associated with abscisic acid, light and methyl jasmonate are particularly over-represented, indicative of the responsiveness of CGTs to the environment. These data sets indicate that CGTs have potential value for wheat improvement and that these could be targeted in TILLING or gene editing wheat breeding programmes.


Assuntos
Citocininas , Triticum , Regulação da Expressão Gênica de Plantas , Homeostase , Melhoramento Vegetal , Transferases , Triticum/genética
14.
J Biol Chem ; 296: 100679, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33872599

RESUMO

Polyprenyl groups, products of isoprenoid metabolism, are utilized in peptidoglycan biosynthesis, protein N-glycosylation, and other processes. These groups are formed by cis-prenyltransferases, which use allylic prenyl pyrophosphates as prenyl-donors to catalyze the C-prenylation of the general acceptor substrate, isopentenyl pyrophosphate. Repetition of this reaction forms (Z,E-mixed)-polyprenyl pyrophosphates, which are converted later into glycosyl carrier lipids, such as undecaprenyl phosphate and dolichyl phosphate. MM_0014 from the methanogenic archaeon Methanosarcina mazei is known as a versatile cis-prenyltransferase that accepts both isopentenyl pyrophosphate and dimethylallyl pyrophosphate as acceptor substrates. To learn more about this enzyme's catalytic activity, we determined the X-ray crystal structures of MM_0014 in the presence or absence of these substrates. Surprisingly, one structure revealed a complex with O-prenylglycerol, suggesting that the enzyme catalyzed the prenylation of glycerol contained in the crystallization buffer. Further analyses confirmed that the enzyme could catalyze the O-prenylation of small alcohols, such as 2-propanol, expanding our understanding of the catalytic ability of cis-prenyltransferases.


Assuntos
Biocatálise , Methanosarcina/enzimologia , Prenilação , Transferases/metabolismo , 2-Propanol/metabolismo , Cinética , Modelos Moleculares , Conformação Proteica , Transferases/química
15.
J Bacteriol ; 203(13): e0014921, 2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-33875545

RESUMO

Lipoprotein diacylglyceryl transferase (Lgt) catalyzes the first step in the biogenesis of Gram-negative bacterial lipoproteins which play crucial roles in bacterial growth and pathogenesis. We demonstrate that Lgt depletion in a clinical uropathogenic Escherichia coli strain leads to permeabilization of the outer membrane and increased sensitivity to serum killing and antibiotics. Importantly, we identify G2824 as the first-described Lgt inhibitor that potently inhibits Lgt biochemical activity in vitro and is bactericidal against wild-type Acinetobacter baumannii and E. coli strains. While deletion of a gene encoding a major outer membrane lipoprotein, lpp, leads to rescue of bacterial growth after genetic depletion or pharmacologic inhibition of the downstream type II signal peptidase, LspA, no such rescue of growth is detected after Lgt depletion or treatment with G2824. Inhibition of Lgt does not lead to significant accumulation of peptidoglycan-linked Lpp in the inner membrane. Our data validate Lgt as a novel antibacterial target and suggest that, unlike downstream steps in lipoprotein biosynthesis and transport, inhibition of Lgt may not be sensitive to one of the most common resistance mechanisms that invalidate inhibitors of bacterial lipoprotein biosynthesis and transport. IMPORTANCE As the emerging threat of multidrug-resistant (MDR) bacteria continues to increase, no new classes of antibiotics have been discovered in the last 50 years. While previous attempts to inhibit the lipoprotein biosynthetic (LspA) or transport (LolCDE) pathways have been made, most efforts have been hindered by the emergence of a common mechanism leading to resistance, namely, the deletion of the gene encoding a major Gram-negative outer membrane lipoprotein lpp. Our unexpected finding that inhibition of Lgt is not susceptible to lpp deletion-mediated resistance uncovers the complexity of bacterial lipoprotein biogenesis and the corresponding enzymes involved in this essential outer membrane biogenesis pathway and potentially points to new antibacterial targets in this pathway.


Assuntos
Escherichia coli/metabolismo , Lipoproteínas/metabolismo , Transferases/metabolismo , Animais , Antibacterianos/farmacologia , Ácido Aspártico Endopeptidases , Proteínas de Bactérias , Escherichia coli/genética , Feminino , Deleção de Genes , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Camundongos , Peptidoglicano/metabolismo , Transferases/química , Transferases/genética , Escherichia coli Uropatogênica/genética , Escherichia coli Uropatogênica/metabolismo
16.
Vet Res ; 52(1): 37, 2021 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-33663572

RESUMO

Fowl cholera caused by Pasteurella multocida exerts a massive economic burden on the poultry industry. Lipopolysaccharide (LPS) is essential for the growth of P. multocida genotype L1 strains in chickens and specific truncations to the full length LPS structure can attenuate bacterial virulence. Here we further dissected the roles of the outer core transferase genes pcgD and hptE in bacterial resistance to duck serum, outer membrane permeability and virulence in ducks. Two P. multocida mutants, ΔpcgD and ΔhptE, were constructed, and silver staining confirmed that they all produced truncated LPS profiles. Inactivation of pcgD or hptE did not affect bacterial susceptibility to duck serum and outer membrane permeability but resulted in attenuated virulence in ducks to some extent. After high-dose inoculation, ΔpcgD showed remarkably reduced colonization levels in the blood and spleen but not in the lung and liver and caused decreased injuries in the spleen and liver compared with the wild-type strain. In contrast, the ΔhptE loads declined only in the blood, and ΔhptE infection caused decreased splenic lesions but also induced severe hepatic lesions. Furthermore, compared with the wild-type strain, ΔpcgD was significantly attenuated upon oral or intramuscular challenge, whereas ΔhptE exhibited reduced virulence only upon oral infection. Therefore, the pcgD deletion caused greater virulence attenuation in ducks, indicating the critical role of pcgD in P. multocida infection establishment and survival.


Assuntos
Proteínas de Bactérias/genética , Infecções por Pasteurella/veterinária , Pasteurella multocida/fisiologia , Pasteurella multocida/patogenicidade , Doenças das Aves Domésticas/microbiologia , Transferases/genética , Animais , Proteínas de Bactérias/metabolismo , Patos , Lipopolissacarídeos/metabolismo , Infecções por Pasteurella/microbiologia , Pasteurella multocida/genética , Transferases/metabolismo
17.
PLoS One ; 16(3): e0248000, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33705438

RESUMO

CUL9 is a non-canonical and poorly characterized member of the largest family of E3 ubiquitin ligases known as the Cullin RING ligases (CRLs). Most CRLs play a critical role in developmental processes, however, the role of CUL9 in neuronal development remains elusive. We determined that deletion or depletion of CUL9 protein causes aberrant formation of neural rosettes, an in vitro model of early neuralization. In this study, we applied mass spectrometric approaches in human pluripotent stem cells (hPSCs) and neural progenitor cells (hNPCs) to identify CUL9 related signaling pathways that may contribute to this phenotype. Through LC-MS/MS analysis of immunoprecipitated endogenous CUL9, we identified several subunits of the APC/C, a major cell cycle regulator, as potential CUL9 interacting proteins. Knockdown of the APC/C adapter protein FZR1 resulted in a significant increase in CUL9 protein levels, however, CUL9 does not appear to affect protein abundance of APC/C subunits and adapters or alter cell cycle progression. Quantitative proteomic analysis of CUL9 KO hPSCs and hNPCs identified protein networks related to metabolic, ubiquitin degradation, and transcriptional regulation pathways that are disrupted by CUL9 deletion in both hPSCs. No significant changes in oxygen consumption rates or ATP production were detected in either cell type. The results of our study build on current evidence that CUL9 may have unique functions in different cell types and that compensatory mechanisms may contribute to the difficulty of identifying CUL9 substrates.


Assuntos
Células-Tronco Pluripotentes/metabolismo , Transdução de Sinais , Transferases/metabolismo , Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Citocromos c/metabolismo , Edição de Genes , Humanos , Proteômica/métodos
18.
FEBS J ; 288(18): 5430-5445, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33755328

RESUMO

A subset of flavoproteins has a covalently attached flavin prosthetic group enzymatically attached via phosphoester bonding. In prokaryotes, this is catalysed by alternative pyrimidine biosynthesis E (ApbE) flavin transferases. ApbE-like domains are present in few eukaryotic taxa, for example the N-terminal domain of fumarate reductase (FRD) of Trypanosoma, a parasitic protist known as a tropical pathogen causing African sleeping sickness. We use the versatile reverse genetic tools available for Trypanosoma to investigate the flavinylation of glycosomal FRD (FRDg) in vivo in the physiological and organellar context. Using direct in-gel fluorescence detection of covalently attached flavin as proxy for activity, we show that the ApbE-like domain of FRDg has flavin transferase activity in vivo. The ApbE domain is preceded by a consensus flavinylation target motif at the extreme N terminus of FRDg, and serine 9 in this motif is essential as flavin acceptor. The preferred mode of flavinylation in the glycosome was addressed by stoichiometric expression and comparison of native and catalytically inactive ApbE domains. In addition to the trans-flavinylation activity, the ApbE domain catalyses the intramolecular cis-flavinylation with at least fivefold higher efficiency. We discuss how the higher efficiency due to unusual fusion of the ApbE domain to its substrate protein FRD may provide a selective advantage by faster FRD biogenesis during rapid metabolic adaptation of trypanosomes. The first 37 amino acids of FRDg, including the consensus motif, are sufficient as flavinylation target upon fusion to other proteins. We propose FRDg(1-37) as 4-kDa heat-stable, detergent-resistant fluorescent protein tag and suggest its use as a new tool to study glycosomal protein import.


Assuntos
Flavoproteínas/genética , Succinato Desidrogenase/genética , Transferases/genética , Trypanosoma brucei brucei/genética , Dinitrocresóis/metabolismo , Flavoproteínas/química , Humanos , Domínios Proteicos/genética , Transporte Proteico/genética , Pirimidinas/biossíntese , Succinato Desidrogenase/química , Trypanosoma brucei brucei/patogenicidade , Tripanossomíase Africana/genética , Tripanossomíase Africana/parasitologia , Triptofano/análogos & derivados , Triptofano/genética
19.
Biochemistry ; 60(12): 929-939, 2021 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-33660509

RESUMO

The thiamin diphosphate-dependent enzyme 1-deoxy-d-xylulose 5-phosphate synthase (DXPS) catalyzes the formation of DXP from pyruvate (donor) and d-glyceraldehyde 3-phosphate (d-GAP, acceptor). DXPS is essential in bacteria but absent in human metabolism, highlighting it as a potential antibacterial drug target. The enzyme possesses unique structural and mechanistic features that enable development of selective inhibition strategies and raise interesting questions about DXPS function in bacterial pathogens. DXPS distinguishes itself within the ThDP enzyme class by its exceptionally large active site and random sequential mechanism in DXP formation. In addition, DXPS displays catalytic promiscuity and relaxed acceptor substrate specificity, yet previous studies have suggested a preference for pyruvate as the donor substrate when d-GAP is the acceptor substrate. However, such donor specificity studies are potentially hindered by a lack of knowledge about specific, alternative donor-acceptor pairs. In this study, we exploited the promiscuous oxygenase activity of DXPS to uncover alternative donor substrates for DXPS. Characterization of glycolaldehyde, hydroxypyruvate, and ketobutyrate as donor substrates revealed differences in stabilization of enzyme-bound intermediates and acceptor substrate usage, illustrating the influence of the donor substrate on reaction mechanism and acceptor specificity. In addition, we found that DXPS prevents abortive acetyl-ThDP formation from a DHEThDP carbanion/enamine intermediate, similar to transketolase, supporting the potential physiological relevance of this intermediate on DXPS. Taken together, these results offer clues toward alternative roles for DXPS in bacterial pathogen metabolism.


Assuntos
Bactérias/metabolismo , Transferases/metabolismo , Bactérias/enzimologia , Domínio Catalítico , Modelos Moleculares , Especificidade por Substrato , Transferases/química
20.
Eur J Med Chem ; 215: 113272, 2021 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-33607457

RESUMO

Antibiotic resistance is one of the most challenging global health issues and presents an urgent need for the development of new antibiotics. In this regard, phospho-MurNAc-pentapeptide translocase (MraY), an essential enzyme in the early stages of peptidoglycan biosynthesis, has emerged as a promising new antibiotic target. We recently reported the crystal structures of MraY in complex with representative members of naturally occurring nucleoside antibiotics, including muraymycin D2. However, these nucleoside antibiotics are synthetically challenging targets, which limits the scope of medicinal chemistry efforts on this class of compounds. To gain access to active muraymycin analogs with reduced structural complexity and improved synthetic tractability, we prepared and evaluated cyclopentane-based muraymycin analogs for targeting MraY. For the installation of the 1,2-syn-amino alcohol group of analogs, the diastereoselective isocyanoacetate aldol reaction was explored. The structure-activity relationship analysis of the synthesized analogs suggested that a lipophilic side chain is essential for MraY inhibition. Importantly, the analog 20 (JH-MR-23) showed antibacterial efficacy against Staphylococcus aureus. These findings provide insights into designing new muraymycin-based MraY inhibitors with improved chemical tractability.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Ciclopentanos/farmacologia , Transferases/antagonistas & inibidores , Uridina/análogos & derivados , Uridina/farmacologia , Antibacterianos/síntese química , Arginina/análogos & derivados , Arginina/farmacologia , Ciclopentanos/síntese química , Ensaios Enzimáticos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Staphylococcus aureus/efeitos dos fármacos , Relação Estrutura-Atividade
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