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1.
Nature ; 581(7808): 339-343, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32433613

RESUMO

Cholesterol is an essential component of mammalian cell membranes, constituting up to 50% of plasma membrane lipids. By contrast, it accounts for only 5% of lipids in the endoplasmic reticulum (ER)1. The ER enzyme sterol O-acyltransferase 1 (also named acyl-coenzyme A:cholesterol acyltransferase, ACAT1) transfers a long-chain fatty acid to cholesterol to form cholesteryl esters that coalesce into cytosolic lipid droplets. Under conditions of cholesterol overload, ACAT1 maintains the low cholesterol concentration of the ER and thereby has an essential role in cholesterol homeostasis2,3. ACAT1 has also been implicated in Alzheimer's disease4, atherosclerosis5 and cancers6. Here we report a cryo-electron microscopy structure of human ACAT1 in complex with nevanimibe7, an inhibitor that is in clinical trials for the treatment of congenital adrenal hyperplasia. The ACAT1 holoenzyme is a tetramer that consists of two homodimers. Each monomer contains nine transmembrane helices (TMs), six of which (TM4-TM9) form a cavity that accommodates nevanimibe and an endogenous acyl-coenzyme A. This cavity also contains a histidine that has previously been identified as essential for catalytic activity8. Our structural data and biochemical analyses provide a physical model to explain the process of cholesterol esterification, as well as details of the interaction between nevanimibe and ACAT1, which may help to accelerate the development of ACAT1 inhibitors to treat related diseases.


Assuntos
Microscopia Crioeletrônica , Esterol O-Aciltransferase/química , Esterol O-Aciltransferase/ultraestrutura , Ureia/análogos & derivados , Colesterol/química , Colesterol/metabolismo , Histidina/química , Histidina/metabolismo , Holoenzimas/química , Holoenzimas/ultraestrutura , Humanos , Ligantes , Modelos Moleculares , Multimerização Proteica , Eletricidade Estática , Ureia/química
2.
Nature ; 581(7808): 323-328, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32433611

RESUMO

Triacylglycerols store metabolic energy in organisms and have industrial uses as foods and fuels. Excessive accumulation of triacylglycerols in humans causes obesity and is associated with metabolic diseases1. Triacylglycerol synthesis is catalysed by acyl-CoA diacylglycerol acyltransferase (DGAT) enzymes2-4, the structures and catalytic mechanisms of which remain unknown. Here we determined the structure of dimeric human DGAT1, a member of the membrane-bound O-acyltransferase (MBOAT) family, by cryo-electron microscopy at approximately 3.0 Å resolution. DGAT1 forms a homodimer through N-terminal segments and a hydrophobic interface, with putative active sites within the membrane region. A structure obtained with oleoyl-CoA substrate resolved at approximately 3.2 Å shows that the CoA moiety binds DGAT1 on the cytosolic side and the acyl group lies deep within a hydrophobic channel, positioning the acyl-CoA thioester bond near an invariant catalytic histidine residue. The reaction centre is located inside a large cavity, which opens laterally to the membrane bilayer, providing lipid access to the active site. A lipid-like density-possibly representing an acyl-acceptor molecule-is located within the reaction centre, orthogonal to acyl-CoA. Insights provided by the DGAT1 structures, together with mutagenesis and functional studies, provide the basis for a model of the catalysis of triacylglycerol synthesis by DGAT.


Assuntos
Biocatálise , Microscopia Crioeletrônica , Diacilglicerol O-Aciltransferase/metabolismo , Diacilglicerol O-Aciltransferase/ultraestrutura , Triglicerídeos/biossíntese , Acil Coenzima A/química , Acil Coenzima A/metabolismo , Acil Coenzima A/ultraestrutura , Aciltransferases/química , Aciltransferases/metabolismo , Domínio Catalítico , Membrana Celular/química , Membrana Celular/metabolismo , Diacilglicerol O-Aciltransferase/química , Histidina/química , Histidina/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Multimerização Proteica , Especificidade por Substrato
3.
DNA Cell Biol ; 39(5): 890-899, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32282228

RESUMO

Lung adenocarcinoma (LUAD) is the most common subtype of nonsmall cell lung cancer, and 5-year survival rate is only 15% in recent years. This study aimed to explore the FAM83A expression and its potential functions in LUAD. Data of LUAD were downloaded from The Cancer Genome Atlas database. Expression level of FAM83A was compared between LUAD samples and adjacent normal samples. The association between FAM83A expression and clinic-pathological parameters was analyzed, as well as copy number variation and methylation status. Kaplan-Meier curve was used to visualize the relationship of FAM83A expression with survival outcomes. Finally, gene set enrichment analysis was used to identify potential signaling pathways in LUAD specimens. FAM83A expression was significantly correlated with four clinical factors in LUAD specimens, age, gender, smoking, and overall survival status (all p < 0.05). High expression level of FAM83A was negatively correlated with methylation level. Moreover, patients in low expression groups exhibited a better prognosis than those in high expressed groups, which was independent of gender (p < 0.001). Histidine metabolism pathway was significantly upregulated in FAM83A-high expressed samples than FAM83A-low expressed samples according to functional enrichment analysis. High expression of FAM83A predicted a poor prognosis in LUAD patients. Our study demonstrated that FAM83A might be a potential biomarker and meaningful therapeutic target in LUAD.


Assuntos
Adenocarcinoma de Pulmão/diagnóstico , Adenocarcinoma de Pulmão/genética , Proteínas de Neoplasias/genética , Oncogenes/genética , Adenocarcinoma de Pulmão/metabolismo , Adenocarcinoma de Pulmão/patologia , Idoso , Carcinogênese/genética , Proliferação de Células/genética , Metilação de DNA/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Histidina/metabolismo , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Prognóstico
4.
Nat Commun ; 11(1): 1757, 2020 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-32273505

RESUMO

NifB is a radical S-adenosyl-L-methionine (SAM) enzyme that is essential for nitrogenase cofactor assembly. Previously, a nitrogen ligand was shown to be involved in coupling a pair of [Fe4S4] clusters (designated K1 and K2) concomitant with carbide insertion into an [Fe8S9C] cofactor core (designated L) on NifB. However, the identity and function of this ligand remain elusive. Here, we use combined mutagenesis and pulse electron paramagnetic resonance analyses to establish histidine-43 of Methanosarcina acetivorans NifB (MaNifB) as the nitrogen ligand for K1. Biochemical and continuous wave electron paramagnetic resonance data demonstrate the inability of MaNifB to serve as a source for cofactor maturation upon substitution of histidine-43 with alanine; whereas x-ray absorption spectroscopy/extended x-ray fine structure experiments further suggest formation of an intermediate that lacks the cofactor core arrangement in this MaNifB variant. These results point to dual functions of histidine-43 in structurally assisting the proper coupling between K1 and K2 and concurrently facilitating carbide formation via deprotonation of the initial carbon radical.


Assuntos
Proteínas de Bactérias/metabolismo , Methanosarcina/metabolismo , Nitrogênio/metabolismo , Nitrogenase/biossíntese , Alanina/genética , Alanina/metabolismo , Proteínas de Bactérias/genética , Espectroscopia de Ressonância de Spin Eletrônica , Histidina/genética , Histidina/metabolismo , Ligantes , Methanosarcina/genética , Mutagênese , Nitrogenase/genética , Espectroscopia por Absorção de Raios X
5.
PLoS One ; 15(3): e0230282, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32160243

RESUMO

Cloning and expression of a desired gene is indispensable in molecular biology studies. Expression vectors, in this regard, should offer much needed flexibility and choice of cloning strategies for both in vivo and in vitro protein expression experiments. Furthermore, availability of option to choose from various reporter tags allows one to be flexible during designing of an experiment in a more relevant manner. Thus, the need of a versatile expression system cannot be ignored. Although several different expression vectors are available for gene expression in mycobacteria, they lack the required versatility of expression and the inclusion of reporter tags. We here present the construction of a set of nine E. coli-Mycobacterium shuttle plasmids, which offer a combination of three mycobacterial promoter systems (heat shock inducible-hsp60, tetracycline-, and acetamide-inducible) along with three polypeptide tags (Green Fluorescent Protein (GFP), Glutathione S-transferase (GST) and hexa-histidine tag). These vectors offer the cloning of a target gene in all the nine given vectors in parallel, thus allowing the generation of recombinant plasmids that will express the target gene from different promoters with different tags. Here, while the hexa-histidine and GST tags can be used for protein purification and pull-down experiments, the GFP-tag can be used for protein localization within the cell. Additionally, the vectors also offer the choice of positioning of the reporter tag either at the N-terminus or at the C-terminus of the expressed protein, which is achieved by cloning of the gene at any of the two blunt-end restriction enzyme sites available in the vector. We believe that these plasmids will be extremely useful in the gene expression studies in mycobacteria by offering the choices of promoters and reporters. Our work also paves the way to developing more such plasmids with other tags and promoters that may find use in mycobacterial biology.


Assuntos
Engenharia Genética/métodos , Vetores Genéticos/genética , Mycobacterium/genética , Escherichia coli/genética , Genes Reporter , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Histidina/genética , Histidina/metabolismo , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Regiões Promotoras Genéticas , Ativação Transcricional
6.
Arch Anim Nutr ; 74(3): 237-255, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32159388

RESUMO

To formulate low-protein diets for blue foxes with sufficient amounts of amino acids (AA), AA digestibility and AA requirements of the animals are crucial information. Therefore, a digestibility and nitrogen (N) balance trial was conducted with 20 blue foxes to determine the macronutrient and AA digestibility and N utilisation in low-protein diets supplemented with DL-methionine (Met) and L-histidine (His). In addition, plasma urea and plasma AA were measured. The diets were designated as P24 (control), P20, P20M, P16M and P16MH and contained energy from digestible crude protein (DCP) at 24%, 20% or 16% of total dietary metabolisable energy (ME). The 20% protein level was fed with or without Met and the 16% protein level was fed with Met and with or without His. The apparent total-tract digestibility (ATTD) of crude protein linearly decreased with decreasing dietary protein level. The ATTD of dry matter, organic matter and crude carbohydrates increased when wheat starch was added as a replacement for protein. The apparent ileal digestibility (AID) and ATTD methods were compared to determine the AA digestibility. The decreasing dietary protein supply decreased the ATTD of most of the AA: threonine, tryptophan (Trp), valine, alanine (Ala), aspartic acid (Asp), glutamic acid, glycine (Gly), proline (Pro), serine (Ser) and total AA. The AID of the AA was constant between diets. Diverging AA showed higher or lower digestibility when determined in the AID or ATTD methods. Isoleucine, lysine, Met, Ala and tyrosine showed higher levels of AID. Arginine, His, cysteine (Cys), Trp, Asp, Gly, Pro and Ser showed higher levels of ATTD, which may reflect the net loss of these AA in the large intestine. Met and His supplementation improved the ATTD and AID of the AA in question, respectively, but did not affect the other variables examined. N retention did not differ between diets and renal N excretion decreased with decreasing protein level; thus N utilisation improved. It was concluded that the protein supply and AA composition in low-protein diets with supplemented Met were adequate for adult blue foxes, since the lower protein supply improved N utilisation and did not affect N retention. However, His supplementation failed to reach the designed level and therefore showed no clear results.


Assuntos
Dieta com Restrição de Proteínas/veterinária , Digestão , Raposas/fisiologia , Histidina/metabolismo , Íleo/fisiologia , Nitrogênio/metabolismo , Racemetionina/metabolismo , Ração Animal/análise , Fenômenos Fisiológicos da Nutrição Animal/efeitos dos fármacos , Animais , Suplementos Nutricionais/análise , Digestão/efeitos dos fármacos , Histidina/administração & dosagem , Masculino , Racemetionina/administração & dosagem , Distribuição Aleatória
7.
Am J Physiol Renal Physiol ; 318(4): F1030-F1040, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32150446

RESUMO

Manipulation of circulating histidine-containing dipeptides (HCD) has been shown to affect the development of diabetes and early-stage diabetic nephropathy (DN). The aim of the present study was to investigate whether such interventions, which potentially alter levels of circulating HCD, also affect the development of advanced-stage DN. Two interventions, aerobic exercise training and overexpression of the human carnosinase-1 (hCN1) enzyme, were tested. BTBR ob/ob mice were either subjected to aerobic exercise training (20 wk) or genetically manipulated to overexpress hCN1, and different diabetes- and DN-related markers were compared with control ob/ob and healthy (wild-type) mice. An acute exercise study was performed to elucidate the effect of obesity, acute running, and hCN1 overexpression on plasma HCD levels. Chronic aerobic exercise training did not affect the development of diabetes or DN, but hCN1 overexpression accelerated hyperlipidemia and aggravated the development of albuminuria, mesangial matrix expansion, and glomerular hypertrophy of ob/ob mice. In line, plasma, kidney, and muscle HCD were markedly lower in ob/ob versus wild-type mice, and plasma and kidney HCD in particular were lower in ob/ob hCN1 versus ob/ob mice but were unaffected by aerobic exercise. In conclusion, advanced glomerular damage is accelerated in mice overexpressing the hCN1 enzyme but not protected by chronic exercise training. Interestingly, we showed, for the first time, that the development of DN is closely linked to renal HCD availability. Further research will have to elucidate whether the stimulation of renal HCD levels can be a therapeutic strategy to reduce the risk for developing DN.


Assuntos
Nefropatias Diabéticas/enzimologia , Dipeptidases/biossíntese , Terapia por Exercício , Glomérulos Renais/enzimologia , Músculo Esquelético/enzimologia , Obesidade/enzimologia , Animais , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/patologia , Dipeptidases/genética , Dipeptídeos/metabolismo , Modelos Animais de Doenças , Indução Enzimática , Histidina/análogos & derivados , Histidina/metabolismo , Humanos , Glomérulos Renais/patologia , Camundongos Transgênicos , Músculo Esquelético/patologia , Obesidade/complicações , Obesidade/genética , Obesidade/patologia , Fatores de Tempo
8.
Nat Commun ; 11(1): 769, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-32034139

RESUMO

Histidine is a versatile residue playing key roles in enzyme catalysis thanks to the chemistry of its imidazole group that can serve as nucleophile, general acid or base depending on its protonation state. In bacteria, signal transduction relies on two-component systems (TCS) which comprise a sensor histidine kinase (HK) containing a phosphorylatable catalytic His with phosphotransfer and phosphatase activities over an effector response regulator. Recently, a pH-gated model has been postulated to regulate the phosphatase activity of HisKA HKs based on the pH-dependent rotamer switch of the phosphorylatable His. Here, we have revisited this model from a structural and functional perspective on HK853-RR468 and EnvZ-OmpR TCS, the prototypical HisKA HKs. We have found that the rotamer of His is not influenced by the environmental pH, ruling out a pH-gated model and confirming that the chemistry of the His is responsible for the decrease in the phosphatase activity at acidic pH.


Assuntos
Histidina Quinase/química , Histidina Quinase/metabolismo , Thermotoga maritima/enzimologia , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Catálise , Cristalografia por Raios X , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Histidina/metabolismo , Histidina Quinase/genética , Concentração de Íons de Hidrogênio , Modelos Biológicos , Modelos Moleculares , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Mutação , Fosforilação , Conformação Proteica , Thermotoga maritima/genética , Transativadores/química , Transativadores/metabolismo
9.
PLoS One ; 15(2): e0229376, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32084230

RESUMO

The alpha/beta-Hydrolases (ABH) are a structural class of proteins that are found widespread in nature and includes enzymes that can catalyze various reactions in different substrates. The catalytic versatility of the ABH fold enzymes, which has been a valuable property in protein engineering applications, is based on a similar acid-base-nucleophile catalytic mechanism. In our research, we are concerned with the structure that surrounds the key units of the catalytic machinery, and we have previously found conserved structural organizations that coordinate the catalytic acid, the catalytic nucleophile and the residues of the oxyanion hole. Here, we explore the architecture that surrounds the catalytic histidine at the active sites of enzymes from 40 ABH fold families, where we have identified six conserved interactions that coordinate the catalytic histidine next to the catalytic acid and the catalytic nucleophile. Specifically, the catalytic nucleophile is coordinated next to the catalytic histidine by two weak hydrogen bonds, while the catalytic acid is directly involved in the coordination of the catalytic histidine through by two weak hydrogen bonds. The imidazole ring of the catalytic histidine is coordinated by a CH-π contact and a hydrophobic interaction. Moreover, the catalytic triad residues are connected with a residue that is located at the core of the active site of ABH fold, which is suggested to be the fourth member of a "structural catalytic tetrad". Besides their role in the stability of the catalytic mechanism, the conserved elements of the catalytic site are actively involved in ligand binding and affect other properties of the catalytic activity, such as substrate specificity, enantioselectivity, pH optimum and thermostability of ABH fold enzymes. These properties are regularly targeted in protein engineering applications, and thus, the identified conserved structural elements can serve as potential modification sites in order to develop ABH fold enzymes with altered activities.


Assuntos
Histidina/química , Hidrolases/química , Modelos Moleculares , Sítios de Ligação , Catálise , Domínio Catalítico , Histidina/metabolismo , Humanos , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Hidrolases/metabolismo , Especificidade por Substrato
10.
Int J Food Microbiol ; 321: 108548, 2020 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-32050139

RESUMO

Histamine, one of the most toxic and commonly encountered biogenic amines (BA) in food, is produced by the microbial decarboxylation of histidine. It may accumulate at high concentrations in fish and fermented food. Cheese has some of the highest histamine concentrations, the result of the histidine-decarboxylase activity of certain lactic acid bacteria (LAB). The present work describes the nucleotide sequence and transcriptional organization of the gene cluster responsible for histamine biosynthesis (the HDC cluster) in Lactobacillus vaginalis IPLA 11064 isolated from cheese. The influence of histidine availability and pH on histamine production and the expression of the HDC cluster genes is also examined. As expected, the results suggest that the production of histamine under acidic conditions improves cell survival by maintaining the cytosol at an appropriate pH. However, the transcriptional regulation of the HDC cluster is quite different from that described in other dairy histamine-producing LAB, probably due to the lack of a termination-antitermination system in the histidyl-tRNA synthetase gene (hisS).


Assuntos
Queijo/microbiologia , Citosol/química , Histamina/biossíntese , Lactobacillus/fisiologia , Animais , Queijo/análise , Regulação Bacteriana da Expressão Gênica , Histidina/análise , Histidina/metabolismo , Histidina Descarboxilase/genética , Concentração de Íons de Hidrogênio , Lactobacillus/genética , Lactobacillus/isolamento & purificação , Lactobacillus/metabolismo , Viabilidade Microbiana
11.
Yakugaku Zasshi ; 140(1): 51-62, 2020.
Artigo em Japonês | MEDLINE | ID: mdl-31902886

RESUMO

The author has described two new functions of endothelial cells for efficient delivery of drugs to tissues. First, it was indicated that tight junction (TJ)-associated protein, claudin-1, exerts potent paracellular barrier function in cultured mouse lung microvascular endothelial cells (LMECs). This barrier was instantly and reversibly opened by reduction of TJ proteins expression via histamine H1 and H2 receptors. Histamine was biosynthesized by l-histidine decarboxylase from uptaken l-histidine, and biotransformed by type B of monoamine oxidase, suggesting that histamine concentration is controlled in rat brain MECs (BMECs) and LMECs. Moreover, uptake of l-histidine into BMECs and LMECs markedly increased with addition of ZnSO4. Second, it was suggested that drug-metabolizing enzymes such as CYP and flavin-containing monooxygenase exist in vascular endothelial cells exposed to blood and to aerobic conditions. These cells have the same ability to metabolize drugs as hepatocytes, demonstrating that vascular endothelial cells are a metabolic barrier against tissue transfer of drugs. From these results, it was suggested that reversible opening of TJ and selective inhibition of drug metabolism in vascular endothelial cells may be efficient delivery strategies of drugs to tissues. Finally, I hope that this research will lead to development of new drugs and possible re-evaluation of discontinued drugs.


Assuntos
Sistemas de Liberação de Medicamentos , Células Endoteliais/enzimologia , Células Endoteliais/fisiologia , Animais , Células Cultivadas , Claudina-1/genética , Claudina-1/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Células Endoteliais/metabolismo , Expressão Gênica , Histamina/biossíntese , Histidina/metabolismo , Camundongos , Oxigenases de Função Mista/metabolismo , Ratos , Receptores Histamínicos H1 , Receptores Histamínicos H2
12.
IET Nanobiotechnol ; 14(1): 67-72, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31935680

RESUMO

To effective capture and universal enrichment of His-tagged protein, polyacrylic acid (PAA) brushes were used to encapsulate Fe3O4 nanoparticles, connect NTA, and Ni2+ to prepare magnetic beads. These materials provide many advantages, such as excellent stability, tuneable particle size, and a surface for further functionalisation with biomolecules. His-tagged green fluorescence protein (GFP) was separated efficiently, and the binding capacity of Fe3O4/MPS@PAA/NTA-Ni2+ was 93.4 mg/g. Compared with High-Affinity Ni-NTA Resin and Ni-NTA Magnetic Agarose Beads, Fe3O4/MPS@PAA/NTA-Ni2+ nanocomposites exhibited higher separation efficiency and binding capacity towards His-tagged GFP. Moreover, the selectivity and recyclability of them for the target proteins were maintained well after six cycles. This study would widen the application of PAA in constructing multifunctional nanocomposites for biomedical fields.


Assuntos
Histidina/metabolismo , Nanopartículas de Magnetita/química , Ácido Nitrilotriacético/análogos & derivados , Compostos Organometálicos/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Resinas Acrílicas/química , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/isolamento & purificação , Histidina/química , Nanocompostos/química , Ácido Nitrilotriacético/química , Ácido Nitrilotriacético/metabolismo , Compostos Organometálicos/metabolismo , Proteínas Recombinantes de Fusão/química
13.
Biochem Biophys Res Commun ; 523(3): 733-738, 2020 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-31948765

RESUMO

Histidine kinase (HK) of two-component signal transduction system (TCS) is a potential drug target for treating bacterial infections, and most HKs are bifunctional. We have previously identified the HXXXT motif of HK in HisKA subfamily to perform the phosphatase activity, but the specific working mechanism of the threonine is not well understood. In this paper, we use the phosphate group analog BeF3- to capture the enzymatic intermediates between HK853 and RR468 from Thermotoga maritima during dephosphorylation, and demonstrate that the T264 site is essential for populating capable near attack conformers (NAC) between enzyme and substrate to facilitate catalysis. Importantly, mutations at this site can modulate the phosphatase activity of HK. Our results help to understand the TCS signal transduction mechanisms and provide a reference for drug design.


Assuntos
Histidina Quinase/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Thermotoga maritima/enzimologia , Motivos de Aminoácidos , Histidina/metabolismo , Histidina Quinase/química , Simulação de Dinâmica Molecular , Monoéster Fosfórico Hidrolases/química , Conformação Proteica , Especificidade por Substrato , Thermotoga maritima/química , Thermotoga maritima/metabolismo
14.
Appl Biochem Biotechnol ; 190(3): 949-965, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31630339

RESUMO

Alzheimer's disease (AD) is related to the anomalous binding that occurs between amyloid-ß peptide (Aß) and copper ion, through imidazole ring of histidine (His), as stated in the literature. It is also known that high-affinity metal ion chelators can be pharmacologically used as a possible therapeutic approach. In this work, we tested the ability "in vitro" of chitosan (Chi) to reduce Aß aggregation and Thioflavin T binding assay indicated that chitosan has affinity for Aß and interferes in its aggregation. We also tested the ability of Chi to uptake copper ions in the presence of Aß or His. Equilibrium data reveals that chitosan acted as an effective chelating agent competing with Aß and histidine for copper binding. The addition of histidine or Aß in the system promotes an unfolding of chitosan chains, as verified by small-angle X-ray scattering. Extended X-ray absorption fine structure and XPS spectra show that new copper interactions with groups containing nitrogen in the presence of histidine may occur. These results can help understanding fundamental chemical interactions among species detected in AD and biopolymers, opening up possibilities for new treatment approaches for this disease.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Quitosana/metabolismo , Cobre/metabolismo , Histidina/metabolismo , Doença de Alzheimer/metabolismo , Benzotiazóis/química , Biopolímeros/metabolismo , Fluorescência , Humanos
15.
Spectrochim Acta A Mol Biomol Spectrosc ; 224: 117468, 2020 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-31425861

RESUMO

Low-frequency vibrations of l-histidine in a neat solid state and its monohydrochloride monohydrate are both investigated using THz spectroscopy and DFT calculations. The molecular motions in those modes are further quantitatively decomposed into a number of submotions and discussed in terms of their contributions to a mode. The results show significant differences in the averaged contribution percentage of intermolecular motions and the dihedral angle distortions of the imidazole ring between these two crystals. Those phenomena are interpreted from the viewpoint of their hydrogen-bond configurations.


Assuntos
Histidina/química , Teoria da Densidade Funcional , Histidina/metabolismo , Modelos Moleculares , Espectroscopia Terahertz/métodos , Termodinâmica
16.
Appl Environ Microbiol ; 86(5)2020 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-31862725

RESUMO

The biosynthesis of histidine, a proteinogenic amino acid, has been extensively studied due to its importance in bacterial growth and survival. Histidinol-phosphate phosphatase (Hol-Pase), which is responsible for the penultimate step of histidine biosynthesis, is generally the last enzyme to be characterized in many bacteria because its origin and evolution are more complex compared to other enzymes in histidine biosynthesis. However, none of the enzymes in histidine biosynthesis, including Hol-Pase, have been characterized in Pseudomonas aeruginosa, which is an important opportunistic Gram-negative pathogen that can cause serious human infections. In our previous work, a transposon mutant of P. aeruginosa was found to display a growth defect on glucose-containing minimal solid medium. In this study, we found that the growth defect was due to incomplete histidine auxotrophy caused by PA0335 inactivation. Subsequently, PA0335 was shown to encode Hol-Pase, and its function and enzymatic activity were investigated using genetic and biochemical methods. In addition to PA0335, the roles of 12 other predicted genes involved in histidine biosynthesis in P. aeruginosa were examined. Among them, hisC2 (PA3165), hisH2 (PA3152), and hisF2 (PA3151) were found to be dispensable for histidine synthesis, whereas hisG (PA4449), hisE (PA5067), hisF1 (PA5140), hisB (PA5143), hisI (PA5066), hisC1 (PA4447), and hisA (PA5141) were essential because deletion of each resulted in complete histidine auxotrophy; similar to the case for PA0335, hisH1 (PA5142) or hisD (PA4448) deletion caused incomplete histidine auxotrophy. Taken together, our results outline the histidine synthesis pathway of P. aeruginosa IMPORTANCE Histidine is a common amino acid in proteins. Because it plays critical roles in bacterial metabolism, its biosynthetic pathway in many bacteria has been elucidated. However, the pathway remains unclear in Pseudomonas aeruginosa, an important opportunistic pathogen in clinical settings; in particular, there is scant knowledge about histidinol-phosphate phosphatase (Hol-Pase), which has a complex origin and evolution. In this study, P. aeruginosa Hol-Pase was identified and characterized. Furthermore, the roles of all other predicted genes involved in histidine biosynthesis were examined. Our results illustrate the histidine synthesis pathway of P. aeruginosa The knowledge obtained from this study may help in developing strategies to control P. aeruginosa-related infections. In addition, some enzymes of the histidine synthesis pathway from P. aeruginosa might be used as elements of histidine synthetic biology in other industrial microorganisms.


Assuntos
Proteínas de Bactérias/genética , Histidina/metabolismo , Histidinol-Fosfatase/genética , Pseudomonas aeruginosa/genética , Proteínas de Bactérias/metabolismo , Vias Biossintéticas , Histidinol-Fosfatase/metabolismo , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/metabolismo
17.
J Biol Chem ; 295(5): 1212-1224, 2020 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-31882536

RESUMO

The natural resistance-associated macrophage protein (Nramp) family encompasses transition metal and proton cotransporters that are present in many organisms from bacteria to humans. Recent structures of Deinococcus radiodurans Nramp (DraNramp) in multiple conformations revealed the intramolecular rearrangements required for alternating access of the metal-binding site to the external or cytosolic environment. Here, using recombinant proteins and metal transport and cysteine accessibility assays, we demonstrate that two parallel cytoplasm-accessible networks of conserved hydrophilic residues in DraNramp, one lining the wide intracellular vestibule for metal release and the other forming a narrow proton transport pathway, are essential for metal transport. We further show that mutagenic or posttranslational modifications of transmembrane helix (TM) 6b, which structurally links these two pathways, impede normal conformational cycling and metal transport. TM6b contains two highly conserved histidines, His232 and His237 We found that different mutagenic perturbations of His232, just below the metal-binding site along the proton exit route, differentially affect DraNramp's conformational state, suggesting that His232 serves as a pivot point for conformational changes. In contrast, any replacement of His237, lining the metal exit route, locked the transporter in a transport-inactive outward-closed state. We conclude that these two histidines, and TM6b more broadly, help trigger the bulk rearrangement of DraNramp to the inward-open state upon metal binding and facilitate return of the empty transporter to an outward-open state upon metal release.


Assuntos
Proteínas de Transporte de Cátions/química , Deinococcus/química , Histidina/química , Metais/metabolismo , Sequência de Aminoácidos/genética , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Cobalto/química , Cobalto/metabolismo , Deinococcus/genética , Deinococcus/metabolismo , Histidina/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Transporte de Íons , Manganês/química , Manganês/metabolismo , Metais/química , Modelos Moleculares , Mutação , Conformação Proteica , Processamento de Proteína Pós-Traducional/genética , Prótons
18.
Nat Commun ; 10(1): 5465, 2019 11 29.
Artigo em Inglês | MEDLINE | ID: mdl-31784535

RESUMO

Liquid-liquid phase separation (LLPS) of intrinsically disordered proteins (IDPs) is involved in both intracellular membraneless organelles and extracellular tissues. Despite growing understanding of LLPS, molecular-level mechanisms behind this process are still not fully established. Here, we use histidine-rich squid beak proteins (HBPs) as model IDPs to shed light on molecular interactions governing LLPS. We show that LLPS of HBPs is mediated though specific modular repeats. The morphology of separated phases (liquid-like versus hydrogels) correlates with the repeats' hydrophobicity. Solution-state NMR indicates that LLPS is a multistep process initiated by deprotonation of histidine residues, followed by transient hydrogen bonding with tyrosine, and eventually by hydrophobic interactions. The microdroplets are stabilized by aromatic clustering of tyrosine residues exhibiting restricted molecular mobility in the nano-to-microsecond timescale according to solid-state NMR experiments. Our findings provide guidelines to rationally design pH-responsive peptides with LLPS ability for various applications, including bioinspired protocells and smart drug-delivery systems.


Assuntos
Bico/metabolismo , Decapodiformes/metabolismo , Histidina/metabolismo , Proteínas Intrinsicamente Desordenadas/metabolismo , Tirosina/metabolismo , Animais , Bico/química , Materiais Biocompatíveis , Biopolímeros/química , Biopolímeros/metabolismo , Coloides/química , Coloides/metabolismo , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Proteínas Intrinsicamente Desordenadas/química , Espectroscopia de Ressonância Magnética , Microscopia , Engenharia de Proteínas/métodos , Espalhamento a Baixo Ângulo
19.
Microb Cell Fact ; 18(1): 212, 2019 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-31830990

RESUMO

BACKGROUND: Histamine is a key mediator of the anti-inflammatory activity conferred by the probiotic organism Lactobacillus reuteri ATCC PTA 6475 in animal models of colitis and colorectal cancer. In L. reuteri, histamine synthesis and secretion requires L-histidine decarboxylase and a L-histidine/histamine exchanger. Chloride channel (ClC)-family proton/chloride antiporters have been proposed to act as electrochemical shunts in conjunction with amino acid decarboxylase systems, correcting ion imbalances generated by decarboxylation through fixed ratio exchange of two chloride ions for one proton. This family is unique among transporters by facilitating ion flux in either direction. Here we examine the histidine decarboxylase system in relation to ClC antiporters in the probiotic organism Lactobacillus reuteri. RESULTS: In silico analyses reveal that L. reuteri possesses two ClC transporters, EriC and EriC2, as well as a complete histidine decarboxylase gene cluster (HDC) for the synthesis and export of histamine. When the transport activity of either proton/chloride antiporter is disrupted by genetic manipulation, bacterial histamine output is reduced. Using fluorescent reporter assays, we further show that ClC transporters affect histamine output by altering intracellular pH and membrane potential. ClC transport also alters the expression and activity of two key HDC genes: the histidine decarboxylase (hdcA) and the histidine/histamine exchanger (hdcP). CONCLUSIONS: Histamine production is a potentially beneficial feature for intestinal microbes by promoting long-term colonization and suppression of inflammation and host immune responses. ClC transporters may serve as tunable modulators for histamine production by L. reuteri and other gut microbes.


Assuntos
Canais de Cloreto/metabolismo , Histidina/metabolismo , Lactobacillus reuteri/metabolismo , Transporte Biológico , Concentração de Íons de Hidrogênio , Potenciais da Membrana
20.
Amino Acids ; 51(10-12): 1667-1680, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31712921

RESUMO

The unique capability of proton buffering is the rationale for using histidine (HIS) as a component of solutions for induction of cardiac arrest and myocardial protection in cardiac surgery. In humans, infusion of cardioplegic solution may increase blood plasma HIS from ~ 70 to ~ 21,000 µM. We examined the effects of a large intravenous dose of HIS on ammonia and amino acid concentrations and energy status of the body. Rats received 198 mM HIS intravenously (20 ml/kg) or vehicle. Samples of blood plasma, urine, liver, and soleus (SOL) and extensor digitorum longus (EDL) muscles were analysed at 2 or 24 h after treatment. At 2 h after HIS load, we found higher HIS concentration in all examined tissues, higher urea and ammonia concentrations in blood and urine, lower ATP content and higher AMP/ATP ratio in the liver and muscles, higher concentrations of almost all examined amino acids in urine, and lower glycine concentration in blood plasma, liver, and muscles when compared with controls. Changes in other amino acids were tissue dependent, markedly increased alanine and glutamate in the blood and the liver. At 24 h, the main findings were lower ATP concentrations in muscles, lower concentrations of branched-chain amino acids (BCAA; valine, leucine, and isoleucine) in blood plasma and muscles, and higher carnosine content in SOL when compared with controls. It is concluded that a load of large HIS dose results in increased ammonia levels and marked alterations in amino acid and energy metabolism. Pathogenesis is discussed in the article.


Assuntos
Nucleotídeos de Adenina/metabolismo , Aminoácidos/metabolismo , Amônia/metabolismo , Histidina/metabolismo , Administração Intravenosa , Aminoácidos/química , Animais , Soluções Cardioplégicas/química , Carnosina/metabolismo , Metabolismo Energético , Histidina/administração & dosagem , Histidina/análise , Ácidos Cetoglutáricos/metabolismo , Masculino , Especificidade de Órgãos , Ratos , Ratos Wistar , Distribuição Tecidual , Ureia/metabolismo
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