Fusobacterium nucleatum is an opportunistic pathogenic bacterium that can be enriched in colorectal cancer tissues, affecting multiple stages of colorectal cancer development. The two-component system plays an important role in the regulation and expression of genes related to pathogenic resistance and pathogenicity. In this paper, we focused on the CarRS two-component system of F. nucleatum, and the histidine kinase protein CarS was recombinantly expressed and characterized. Several online software such as SMART, CCTOP and AlphaFold2 were used to predict the secondary and tertiary structure of the CarS protein. The results showed that CarS is a membrane protein with two transmembrane helices and contains 9 α-helices and 12 β-folds. CarS protein is composed of two domains, one is the N-terminal transmembrane domain (amino acids 1-170), the other is the C-terminal intracellular domain. The latter is composed of a signal receiving domain (histidine kinases, adenylyl cyclases, methyl-accepting proteins, prokaryotic signaling proteins, HAMP), a phosphate receptor domain (histidine kinase domain, HisKA), and a histidine kinase catalytic domain (histidine kinase-like ATPase catalytic domain, HATPase_c). Since the full-length CarS protein could not be expressed in host cells, a fusion expression vector pET-28a(+)-MBP-TEV-CarS_cyto was constructed based on the characteristics of secondary and tertiary structures, and overexpressed in Escherichia coli BL21-Codonplus(DE3)RIL. CarS_cyto-MBP protein was purified by affinity chromatography, ion-exchange chromatography, and gel filtration chromatography with a final concentration of 20 mg/ml. CarS_cyto-MBP protein showed both protein kinase and phosphotransferase activities, and the MBP tag had no effect on the function of CarS_cyto protein. The above results provide a basis for in-depth analysis of the biological function of the CarRS two-component system in F. nucleatum.
Humans ; Histidine Kinase/metabolism* ; Fusobacterium nucleatum/metabolism* ; Automobiles ; Protein Kinases/genetics* ; Escherichia coli/metabolism* ; Colorectal Neoplasms

Ergothioneine (ERG) is a natural antioxidant that has been widely used in the fields of food, medicine and cosmetics. Compared with traditional plant extraction and chemical synthesis approaches, microbial synthesis of ergothioneine has many advantages, such as the short production cycle and low cost, and thus has attracted intensive attention. In order to engineer an ergothioneine high-yielding Escherichia coli strain, the ergothioneine synthesis gene cluster egtABCDE from Mycobacterium smegmatis and egt1 from Schizosaccharomyces pombe were introduced into E. coli BL21(DE3) to generate a strain E1-A1 harboring the ergothioneine biosynthesis pathway. As a result, (95.58±3.2) mg/L ergothioneine was produced in flask cultures. To further increase ergothioneine yield, the relevant enzymes for biosynthesis of histidine, methionine, and cysteine, the three precursor amino acids of ergothioneine, were overexpressed. Individual overexpression of serA^T410STOP and thrA resulted in an ergothioneine titer of (134.83±4.22) mg/L and (130.26±3.34) mg/L, respectively, while co-overexpression of serA^T410STOP and thrA increased the production of ergothioneine to (144.97±5.40) mg/L. Eventually, by adopting a fed-batch fermentation strategy in 3 L fermenter, the optimized strain E1-A1-thrA-serA^* produced 548.75 mg/L and 710.53 mg/L ergothioneine in glucose inorganic salt medium and rich medium, respectively.
Culture Media ; Ergothioneine/metabolism* ; Escherichia coli/metabolism* ; Fermentation ; Histidine/metabolism* ; Metabolic Engineering

Animals ; Carrier Proteins/metabolism* ; Histidine/metabolism* ; Humans ; Methylation ; Methyltransferases/metabolism* ; Neoplasm Proteins/metabolism* ; Neoplasms/metabolism* ; Protein Processing, Post-Translational

Hepatocellular carcinoma (HCC) is the most common primary malignant tumor of the liver and the third most common cause of cancer-related death worldwide. HCC is caused by infection of hepatitis B/C virus and liver dysfunctions, such as alcoholic liver disease, nonalcoholic fatty liver disease, and cirrhosis. Amino acids are organic substances containing amine and carboxylic acid functional groups. There are over 700 kinds of amino acids in nature, but only about 20 of them are used to synthesize proteins in cells. Liver is an important organ for protein synthesis, degradation and detoxification as well as amino acid metabolism. In the liver, there are abundant non-essential amino acids, such as alanine, aspartate, glutamate, glycine, and serine and essential amino acids, such as histidine and threonine. These amino acids are involved in various cellular metabolisms, the synthesis of lipids and nucleotides as well as detoxification reactions. Understanding the role of amino acids in the pathogenesis of liver and the effects of amino acid intake on liver disease can be a promising strategy for the prevention and treatment of liver disease. In this review, we describe the biochemical properties and functions of amino acids and to review how they have been applied to treatment of liver diseases.
Alanine ; Amino Acids ; Amino Acids, Essential ; Aspartic Acid ; Carcinoma, Hepatocellular ; Fibrosis ; Glutamic Acid ; Glycine ; Hepatitis ; Histidine ; Liver Diseases ; Liver Diseases, Alcoholic ; Liver ; Metabolism ; Non-alcoholic Fatty Liver Disease ; Nucleotides ; Serine ; Therapeutic Uses ; Threonine

Glimepiride, a third generation sulfonylurea, is an antihyperglycemic agent widely used to treat type 2 diabetes mellitus. In this study, an untargeted urinary metabolomic analysis was performed to identify endogenous metabolites affected by glimepiride administration. Urine samples of twelve healthy male volunteers were collected before and after administration of 2 mg glimepiride. These samples were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and then subjected to multivariate data analysis including principal component analysis and orthogonal partial least squares discriminant analysis. Through this metabolomic profiling, we identified several endogenous metabolites such as adenosine 3′, 5′-cyclic monophosphate (cAMP), quercetin, tyramine, and urocanic acid, which exhibit significant metabolomic changes between pre- and posturine samples. Among these, cAMP, which is known to be related to insulin secretion, was the most significantly altered metabolite following glimepiride administration. In addition, the pathway analysis showed that purine, tyrosine, and histidine metabolism was affected by pharmacological responses to glimepiride. Together, the results suggest that the pharmacometabolomic approach, based on LC-MS/MS, is useful in understanding the alterations in biochemical pathways associated with glimepiride action.
Adenosine ; Diabetes Mellitus, Type 2 ; Histidine ; Humans* ; Insulin ; Least-Squares Analysis ; Male ; Mass Spectrometry* ; Metabolism ; Metabolomics* ; Principal Component Analysis ; Quercetin ; Statistics as Topic ; Tyramine ; Tyrosine ; Urocanic Acid ; Volunteers

The rapid increase in the prevalence of metabolic syndrome, which is associated with a state of elevated systemic oxidative stress and inflammation, is expected to cause future increases in the prevalence of diabetes and cardiovascular diseases. Oxidation of polyunsaturated fatty acids and sugars produces reactive carbonyl species, which, due to their electrophilic nature, react with the nucleophilic sites of certain amino acids. This leads to formation of protein adducts such as advanced glycoxidation/lipoxidation end products (AGEs/ALEs), resulting in cellular dysfunction. Therefore, an effective reactive carbonyl species and AGEs/ALEs sequestering agent may be able to prevent such cellular dysfunction. There is accumulating evidence that histidine containing dipeptides such as carnosine (beta-alanyl-L-histidine) and anserine (beta-alanyl-methyl-L-histidine) detoxify cytotoxic reactive carbonyls by forming unreactive adducts and are able to reverse glycated protein. In this review, 1) reaction mechanism of oxidative stress and certain chronic diseases, 2) interrelation between oxidative stress and inflammation, 3) effective reactive carbonyl species and AGEs/ALEs sequestering actions of histidine-dipeptides and their metabolism, 4) effects of carnosinase encoding gene on the effectiveness of histidine-dipeptides, and 5) protective effects of histidine-dipeptides against progression of metabolic syndrome are discussed. Overall, this review highlights the potential beneficial effects of histidine-dipeptides against metabolic syndrome. Randomized controlled human studies may provide essential information regarding whether histidine-dipeptides attenuate metabolic syndrome in humans.
Amino Acids ; Anserine ; Carbohydrates ; Cardiovascular Diseases ; Carnosine ; Chronic Disease ; Dipeptides ; Fatty Acids, Unsaturated ; Histidine ; Humans ; Inflammation ; Metabolism ; Oxidative Stress ; Prevalence ; Sequestering Agents

Bacterial Outer Membrane Proteins ; chemistry ; genetics ; metabolism ; Escherichia coli Proteins ; chemistry ; genetics ; metabolism ; Glutamic Acid ; genetics ; metabolism ; Histidine ; genetics ; Hydrogen-Ion Concentration ; Ion Channel Gating ; genetics ; Lipid Bilayers ; metabolism ; Mutant Proteins ; chemistry ; genetics ; metabolism ; Mutation ; Porins ; chemistry ; genetics ; metabolism

Genetically encoded Ca(2+) indicators (GECI) are important for the measurement of Ca(2+) in vivo. GCaMP2, a widely-used GECI, has recently been iteratively improved. Among the improved variants, GCaMP3 exhibits significantly better fluorescent intensity. In this study, we developed a new GECI called GCaMPJ and determined the crystal structures of GCaMP3 and GCaMPJ. GCaMPJ has a 1.5-fold increase in fluorescence and 1.3-fold increase in calcium affinity over GCaMP3. Upon Ca(2+) binding, GCaMP3 exhibits both monomeric and dimeric forms. The structural superposition of these two forms reveals the role of Arg-376 in improving monomer performance. However, GCaMPJ seldom forms dimers under conditions similar to GCaMP3. St ructural and mutagenesis studies on Tyr-380 confirmed its importance in blocking the cpEGFP β-barrel holes. Our study proposes an efficient tool for mapping Ca(2+) signals in intact organs to facilitate the further improvement of GCaMP sensors.
Calcium ; chemistry ; metabolism ; Calmodulin ; chemistry ; genetics ; metabolism ; Crystallography, X-Ray ; Dimerization ; Green Fluorescent Proteins ; chemistry ; genetics ; metabolism ; Histidine ; chemistry ; genetics ; metabolism ; Hydrogen-Ion Concentration ; Myosin-Light-Chain Kinase ; chemistry ; genetics ; metabolism ; Peptide Fragments ; chemistry ; genetics ; metabolism ; Protein Structure, Tertiary ; Recombinant Fusion Proteins ; biosynthesis ; chemistry ; genetics

Two component system is a signal transduction system. It typically consists of a sensor histitine kinase and a cognate response regulator (RR) component. The activity of RR is regulated by a phosphorylation dependent mechanism. In recent years, the existence of atypical response regulators (ARRs), which rely on a phosphorylation independent mechanism to regulate their activity, have been recognized. ARRs are involved in the regulation of bacterial growth and development, antibiotic biosynthesis, iron transport, among others. Here we review the recent advances in the understanding of the structure and function of atypical response regulators, by using JadR1, a regulator in jadomycin biosynthesis in Streptomyces, as an example to elucidate the novel mechanism used by ARR to fine-tune its activity.
Bacterial Proteins ; genetics ; Gene Expression Regulation, Bacterial ; Gene Expression Regulation, Developmental ; Genes, Regulator ; genetics ; Histidine Kinase ; Isoquinolines ; metabolism ; Naphthoquinones ; metabolism ; Phosphorylation ; Protein Kinases ; genetics ; RNA-Binding Proteins ; genetics ; Signal Transduction ; Streptomyces ; metabolism ; Transcription Factors ; genetics

Human NUDT16 (hNUDT16) is a decapping enzyme initially identified as the human homolog to the Xenopus laevis X29. As a metalloenzyme, hNUDT16 relies on divalent cations for its cap-hydrolysis activity to remove m⁷GDP and m²²⁷GDP from RNAs. Metal also determines substrate specificity of the enzyme. So far, only U8 small nucleolar RNA (snoRNA) has been identified as the substrate of hNUDT16 in the presence of Mg²(+). Here we demonstrate that besides U8, hNUDT16 can also actively cleave the m⁷GDP cap from mRNAs in the presence of Mg²(+) or Mn²(+). We further show that hNUDT16 does not preferentially recognize U8 or mRNA substrates by our cross-inhibition and quantitative decapping assays. In addition, our mutagenesis analysis identifies several key residues involved in hydrolysis and confirms the key role of the REXXEE motif in catalysis. Finally an investigation into the subcellular localization of hNUDT16 revealed its abundance in both cytoplasm and nucleus. These findings extend the substrate spectrum of hNUDT16 beyond snoRNAs to also include mRNA, demonstrating the pleiotropic decapping activity of hNUDT16.
Amino Acid Motifs ; Biocatalysis ; Cell Nucleus ; enzymology ; Consensus Sequence ; Cytoplasm ; enzymology ; metabolism ; Guanosine Diphosphate ; metabolism ; Histidine ; metabolism ; Humans ; Hydrolysis ; Luciferases ; genetics ; Magnesium ; metabolism ; Manganese ; metabolism ; Mutagenesis ; Mutation ; Pyrophosphatases ; antagonists & inhibitors ; chemistry ; genetics ; metabolism ; RNA Caps ; chemistry ; metabolism ; pharmacology ; RNA, Small Nucleolar ; chemistry ; metabolism ; pharmacology