RESUMO
Recent studies have shown that human solute carrier SLC19A3 (hSLC19A3) can transport pyridoxine (vitamin B6) in addition to thiamine (vitamin B1), its originally identified substrate, whereas rat and mouse orthologs of hSLC19A3 can transport thiamine but not pyridoxine. This finding implies that some amino acid residues required for pyridoxine transport, but not for thiamine transport, are specific to hSLC19A3. Here, we sought to identify these residues to help clarify the unique operational mechanism of SLC19A3 through analyses comparing hSLC19A3 and mouse Slc19a3 (mSlc19a3). For our analyses, hSLC19A3 mutants were prepared by replacing selected amino acid residues with their counterparts in mSlc19a3, and mSlc19a3 mutants were prepared by substituting selected residues with their hSLC19A3 counterparts. We assessed pyridoxine and thiamine transport by these mutants in transiently transfected human embryonic kidney 293 cells. Our analyses indicated that the hSLC19A3-specific amino acid residues of Gln86, Gly87, Ile91, Thr93, Trp94, Ser168, and Asn173 are critical for pyridoxine transport. These seven amino acid residues were found to be mostly conserved in the SLC19A3 orthologs that can transport pyridoxine but not in orthologs that are unable to transport pyridoxine. In addition, these residues were also found to be conserved in several SLC19A2 orthologs, including rat, mouse, and human orthologs, which were all found to effectively transport both pyridoxine and thiamine, exhibiting no species-dependent differences. Together, these findings provide a molecular basis for the unique functional characteristics of SLC19A3 and also of SLC19A2.
Assuntos
Aminoácidos , Proteínas de Membrana Transportadoras/metabolismo , Aminoácidos/metabolismo , Animais , Transporte Biológico , Células Epiteliais/metabolismo , Humanos , Camundongos , Ratos , Tiamina/genética , Tiamina/metabolismoRESUMO
Thiamine pyrophosphate (TPP), the substrate of Thiamine pyrophosphate kinase (TPK), is an important cofactor in carbohydrate metabolism, specifically as a cofactor of the Pyruvate dehydrogenase complex (PDH) complex. The nervous system is particularly dependent on TPP due to its reliance on glucose metabolism. In this case, a four-year-old girl had a previously unreported pathogenic variant of the gene encoding TPK (TPK1) which presented as Thiamine metabolism dysfunction syndrome 5 (THMD5; OMIM 614458). She had been diagnosed with acute disseminated encephalomyelitis and autism spectrum disorder (ASD), and initially presented with fever and agitation following vaccinations. After follow-up with genetic testing, our patient was found to have compound heterozygous pathogenic variants of TPK1. After treatment with biotin and thiamine her clinical status improved, and her ASD features resolved. The presentation of our patient was consistent with previous reports and adds to the evidence that thiamine and biotin are effective treatments of TPK1 related metabolic deficiencies. The improvement of neurobehavioral symptoms in this case was marked, highlighting the importance of early identification and therapeutic intervention in this condition.
Assuntos
Transtorno do Espectro Autista , Encefalomielite Aguda Disseminada , Humanos , Feminino , Pré-Escolar , Encefalomielite Aguda Disseminada/tratamento farmacológico , Biotina/uso terapêutico , Tiamina/uso terapêutico , Tiamina/genética , Tiamina/metabolismo , Tiamina Pirofosfato/metabolismoRESUMO
Urban garden plants are frequently affected by drought, which can hinder their growth, development, and greening effect. Previous studies have indicated that Chinese wingnut (Pterocarya stenoptera) responds to drought stress by increasing the expression of thiamine synthesis genes. In this study, it was found that exogenous thiamine can effectively alleviate the negative effects of drought stress on plants. Forward transcriptome sequencing and physiological tests were further conducted to reveal the molecular mechanism of thiamine in alleviating drought stress. Results showed that exogenous thiamine activated the expression of eight chlorophyll synthesis genes in Chinese wingnut under drought stress. Moreover, physiological indicators proved that chlorophyll content increased in leaves of Chinese wingnut with thiamine treatment under drought stress. Photosynthesis genes were also activated in Chinese wingnut treated with exogenous thiamine under drought stress, as supported by photosynthetic indicators PIabs and PItotal. Additionally, exogenous thiamine stimulated the expression of genes in the auxin-activated signaling pathway, thus attenuating the effects of drought stress. This study demonstrates the molecular mechanism of thiamine in mitigating the effects of drought stress on non-model woody plants lacking transgenic systems. This study also provides an effective method to mitigate the negative impacts of drought stress on plants.
Assuntos
Secas , Juglandaceae , Tiamina , Transcriptoma , Clorofila , Fotossíntese/genética , Estresse Fisiológico/genética , Tiamina/genética , Tiamina/farmacologia , Juglandaceae/genética , Juglandaceae/metabolismo , Juglandaceae/fisiologia , ChinaRESUMO
BACKGROUND: Thiamine metabolism dysfunction syndrome 5 (THMD5) is a rare inherited metabolic disorder due to thiamine pyrophosphokinase 1(TPK1) deficiency, caused by mutations in TPK1. The core symptoms of the disease is acute or subacute onset encephalopathy, ataxia, muscle hypotonia, and regression of developmental milestones in early infancy, repeatedly triggered by acute infectious illness. However, we report two brothers of THMD5 with compound heterozygous for the mutations c.614-1G > A,c.224 T > A p.(Ile75Asn), but the prognosis is quite different if thiamine suppled. According to our current knowledge, the missense variant c.224 T > A p.(Ile75Asn) was not published previously. CASE PRESENTATION: Here, we describe two affected siblings in a Chinese family, after an uneventful pregnancy to non-consanguineous and healthy parents. The older brother presented with normal development during the first 6 months of life, but developed regression of developmental milestones after, accompanied with muscle hypotonia, and chronic encephalopathy, and died at 1 year and 6 months old. The younger brother presented with acute onset encephalopathy, ataxia, muscle hypotonia, repeatedly triggered by acute infectious illness. He was compound heterozygous for the mutations c.614-1G > A,c.224 T > A p.(Ile75Asn) identified by whole exome sequencing. He was diagnosed of THMD5 when he was 11 month. Oral supplementation of thiamine 100 mg/day, the symptoms gradually disappeared. At the age of 2 years and 4 months, he stoped thiamine, his symptoms returned and were once again relieved by oral supplementation of thiamine 100 mg/day. CONCLUSIONS: THMD5 is a rare, but treatable neurodegenerative disease, the clinical phenotype ranges from mild to severe. Massive-dose of thiamine supplementation may ameliorate the course of TPK1 deficiency. When similar clinical cases appear, gene detection is particularly important, which is conducive to early diagnosis. Treatment with thiamine while awaiting the outcome of diagnostic tests may be a good choice.
Assuntos
Encefalopatias , Doenças Neurodegenerativas , Ataxia/tratamento farmacológico , Humanos , Masculino , Hipotonia Muscular , Mutação/genética , Irmãos , Tiamina Pirofosfoquinase/genética , Tiamina/genética , Tiamina/metabolismo , Tiamina/uso terapêuticoRESUMO
Horizontal acquisition of bacterial genes is presently recognized as an important contribution to the adaptation and evolution of eukaryotic genomes. However, the mechanisms underlying expression and consequent selection and fixation of the prokaryotic genes in the new eukaryotic setting are largely unknown. Here we show that genes composing the pathway for the synthesis of the essential vitamin B1 (thiamine) were lost in an ancestor of a yeast lineage, the Wickerhamiella/Starmerella (W/S) clade, known to harbor an unusually large number of genes of alien origin. The thiamine pathway was subsequently reassembled, at least twice, by multiple HGT events from different bacterial donors involving both single genes and entire operons. In the W/S-clade species Starmerella bombicola we obtained direct genetic evidence that all bacterial genes of the thiamine pathway are functional. The reconstructed pathway is composed by yeast and bacterial genes operating coordinately to scavenge thiamine derivatives from the environment. The adaptation of the newly acquired operons to the eukaryotic setting involved a repertoire of mechanisms until now only sparsely documented, namely longer intergenic regions, post-horizontal gene transfer (HGT) gene fusions fostering coordinated expression, gene relocation, and possibly recombination generating mosaic genes. The results provide additional evidence that HGT occurred recurrently in this yeast lineage and was crucial for the reestablishment of lost functions and that similar mechanisms are used across a broad range of eukaryotic microbes to promote adaptation of prokaryotic genes to their new environment.
Assuntos
Transferência Genética Horizontal , Genes Bacterianos , Óperon , Saccharomycetales/genética , Tiamina/genética , Bactérias/genética , Tiamina/metabolismoRESUMO
Like fungi and some prokaryotes, plants use a thiazole synthase (THI4) to make the thiazole precursor of thiamin. Fungal THI4s are suicide enzymes that destroy an essential active-site Cys residue to obtain the sulfur atom needed for thiazole formation. In contrast, certain prokaryotic THI4s have no active-site Cys, use sulfide as sulfur donor, and are truly catalytic. The presence of a conserved active-site Cys in plant THI4s and other indirect evidence implies that they are suicidal. To confirm this, we complemented the Arabidopsistz-1 mutant, which lacks THI4 activity, with a His-tagged Arabidopsis THI4 construct. LC-MS analysis of tryptic peptides of the THI4 extracted from leaves showed that the active-site Cys was predominantly in desulfurated form, consistent with THI4 having a suicide mechanism in planta. Unexpectedly, transcriptome data mining and deep proteome profiling showed that barley, wheat, and oat have both a widely expressed canonical THI4 with an active-site Cys, and a THI4-like paralog (non-Cys THI4) that has no active-site Cys and is the major type of THI4 in developing grains. Transcriptomic evidence also indicated that barley, wheat, and oat grains synthesize thiamin de novo, implying that their non-Cys THI4s synthesize thiazole. Structure modeling supported this inference, as did demonstration that non-Cys THI4s have significant capacity to complement thiazole auxotrophy in Escherichia coli. There is thus a prima facie case that non-Cys cereal THI4s, like their prokaryotic counterparts, are catalytic thiazole synthases. Bioenergetic calculations show that, relative to suicide THI4s, such enzymes could save substantial energy during the grain-filling period.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Ligases , Modelos Moleculares , Plantas Geneticamente Modificadas , Tiamina , Tiazóis/metabolismo , Arabidopsis/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Catálise , Biologia Computacional , Escherichia coli/enzimologia , Escherichia coli/genética , Teste de Complementação Genética , Ligases/química , Ligases/genética , Ligases/metabolismo , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/genética , Domínios Proteicos , Tiamina/biossíntese , Tiamina/genéticaRESUMO
Beauveria bassiana is an important entomopathogenic fungus used to control a variety of insect pests. Conidia are the infective propagules of the fungus. However, some important factors that influence conidiation are still to be investigated. In this study, a mutant with decreased conidial production and hyphal growth was identified from a random T-DNA insertional library of B. bassiana. The corresponding gene (Bbthi) for this mutation encodes a putative thiazole synthase. Thiazole and pyrimidine are structural components of thiamine (vitamin B1), which is an essential nutrient for all forms of life. Disruption of Bbthi, Bbpyr, a putative pyrimidine synthetic gene, or both in B. bassiana results in a significant decrease of thiamine content. Loss of Bbthi and Bbpyr function significantly decreased the conidial production and hyphal growth, as well as disrupted the integrity of conidial cell wall. However, the defect of Bbpyr and Bbthi does not decrease the virulence of B. bassiana. Our results indicate the importance of thiamine biosynthesis in conidiation of B. bassiana, and provide useful information to produce conidia of entomopathogenic fungi for biocontrol of insect pests.
Assuntos
Beauveria/genética , Proteínas Fúngicas/genética , Genes Fúngicos/fisiologia , Esporos Fúngicos/fisiologia , Tiamina/biossíntese , Beauveria/metabolismo , Parede Celular/fisiologia , Proteínas Fúngicas/metabolismo , Tiamina/genéticaRESUMO
Thiamine pyrophosphokinase (TPK) converts thiamine (vitamin B1) into thiamine pyrophosphate (TPP), an essential cofactor for many important enzymes. TPK1 mutations lead to a rare disorder: episodic encephalopathy type thiamine metabolism dysfunction. Yet, the molecular mechanism of the disease is not entirely clear. Here we report an individual case of episodic encephalopathy, with familial history carrying a novel homozygous TPK1 mutation (p.L28S). The L28S mutation leads to reduced enzymatic activity, both in vitro and in vivo, without impairing thiamine binding and protein stability. Thiamine supplementation averted encephalopathic episodes and restored the patient's developmental progression. Biochemical characterization of reported TPK1 missense mutations suggested reduced thiamine binding as a new disease mechanism. Importantly, many disease mutants are directly or indirectly involved in thiamine binding. Thus, our study provided a novel rationale for thiamine supplementation, so far the major therapeutic intervention in TPK deficiency.
Assuntos
Encefalopatias/genética , Tiamina Pirofosfoquinase/deficiência , Tiamina Pirofosfoquinase/genética , Tiamina/genética , Sequência de Aminoácidos/genética , Encefalopatias/fisiopatologia , Pré-Escolar , China , Feminino , Homozigoto , Humanos , Masculino , Mutação de Sentido Incorreto/genética , Linhagem , Ligação Proteica , Estabilidade Proteica , Tiamina Pirofosfoquinase/química , Tiamina/metabolismo , Tiamina Pirofosfato/genética , Tiamina Pirofosfato/metabolismoRESUMO
Thiamine metabolism dysfunction syndrome-4 (THMD4) includes episodic encephalopathy, often associated with a febrile illness, causing transient neurologic dysfunction and a slowly progressive axonal polyneuropathy. Until now only two mutations (G125S and S194P) have been reported in the SLC25A19 gene as causative for this disease and a third mutation (G177A) as related to the Amish lethal microcephaly. In this work, we describe the clinical and molecular features of a patient carrying a novel mutation (c.576G>C; Q192H) on SLC25A19 gene. Functional studies on this mutation were performed explaining the pathogenetic role of c.576G>C in affecting the translational efficiency and/or stability of hMTPPT protein instead of the mRNA expression. These findings support the pathogenetic role of Q192H (c.576G>C) mutation on SLC25A19 gene. Moreover, despite in other patients the thiamine supplementation leaded to a substantial improvement of peripheral neuropathy, our patient did not show a clinical improvement.
Assuntos
Predisposição Genética para Doença , Microcefalia/genética , Proteínas de Transporte da Membrana Mitocondrial/genética , Deficiência de Tiamina/genética , Adolescente , Encefalopatias/genética , Encefalopatias/fisiopatologia , Humanos , Masculino , Microcefalia/fisiopatologia , Proteínas de Transporte da Membrana Mitocondrial/química , Mutação , Conformação Proteica , RNA Mensageiro/genética , Tiamina/genética , Tiamina/metabolismo , Deficiência de Tiamina/fisiopatologiaRESUMO
Thiamine pyrophosphate (TPP) riboswitch is a cis-regulatory element in the noncoding region of mRNA. The aptamer domain of TPP riboswitch detects the high abundance of coenzyme thiamine pyrophosphate (TPP) and modulates the gene expression for thiamine synthetic gene. The mechanistic understanding in recognition of TPP in aptamer domain and ligand-induced compactness for folding of expression platform are most important to designing novel modulators. To understand the dynamic behavior of TPP riboswitch upon TPP binding, molecular dynamics simulations were performed for 400 ns in both apo and TPP bound forms of thiM riboswitch from E. coli and analyzed in terms of eRMSD-based Markov state modeling and residual fluctuation network. Markov state models show good correlations in transition probability among metastable states from simulated trajectory and generated models. Structural compactness in TPP bound form is observed which is correlated with SAXS experiment. The importance of junction of P4 and P5 is evident during dynamics, which correlates with FRET analysis. The dynamic nature of two sensor forearms is due to the flexible P1 helix, which is its intrinsic property. The transient state in TPP-bound form was observed in the Markov state model, along with stable states. We believe that this transient state is responsible to assist the influx and outflux of ligand molecule by creating a solvent channel around the junction region of P4 and P5 and such a structure was anticipated in FRET analysis. The dynamic nature of riboswitch is dependent on the interaction between residues on distal loops L3 and L5/P3 and junction P4 and P5, J3/2 which stabilize the J2/4. It helps in the transfer of allosteric information between J2/4 and P3/L5 tertiary docking region through the active site residues. Understanding such information flow will benefit in highlighting crucial residues in highly dynamic and kinetic systems. Here, we report the residues and segments in riboswitch that play vital roles in providing stability and this can be exploited in designing inhibitors to regulate the functioning of riboswitches.
Assuntos
Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , RNA Bacteriano/metabolismo , Riboswitch , Tiamina Pirofosfato/metabolismo , Escherichia coli/química , Escherichia coli/genética , Cadeias de Markov , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , RNA Bacteriano/química , RNA Bacteriano/genética , Tiamina/genéticaRESUMO
Parasites often have reduced genomes as their own genes become redundant when utilizing their host as a source of metabolites, thus losing their own de novo production of metabolites. Primate malaria parasites can synthesize vitamin B1 (thiamine) de novo but rodent malaria and other genome-sequenced apicomplexans cannot, as the three essential genes responsible for this pathway are absent in their genomes. The unique presence of functional thiamine synthesis genes in primate malaria parasites and their sequence similarities to bacterial orthologues, have led to speculations that this pathway was horizontally acquired from bacteria. Here we show that the genes essential for the de novo synthesis of thiamine are found also in avian Plasmodium species. Importantly, they are also present in species phylogenetically basal to all mammalian and avian Plasmodium parasites, i.e. Haemoproteus. Furthermore, we found that these genes are expressed during the blood stage of the avian malaria infection, indicating that this metabolic pathway is actively transcribed. We conclude that the ability to synthesize thiamine is widespread among haemosporidians, with a recent loss in the rodent malaria species.
Assuntos
Vias Biossintéticas/genética , Genoma de Protozoário , Haemosporida/genética , Plasmodium/genética , Tiamina/biossíntese , Animais , Aves/parasitologia , Malária/sangue , Malária Aviária/parasitologia , Filogenia , Plasmodium/fisiologia , Primatas/parasitologia , Roedores/parasitologia , Tiamina/genéticaRESUMO
Vitamin B1, which consists of the vitamers thiamin and its phosphorylated derivatives, is an essential micronutrient for all living organisms because it is required as a metabolic cofactor in several enzymatic reactions. Genetic diversity of vitamin B1 biosynthesis and accumulation has not been investigated in major crop species other than rice and potato. We analyzed cassava germplasm for accumulation of B1 vitamers. Vitamin B1 content in leaves and roots of 41 cassava accessions showed significant variation between accessions. HPLC analyses of B1 vitamers revealed distinct profiles in cassava leaves and storage roots, with nearly equal relative levels of thiamin pyrophosphate and thiamin monophosphate in leaves, but mostly thiamin pyrophosphate in storage roots. Unusually, the cassava genome has two genes encoding the 4-amino-2-methyl-5-hydroxymethylpyrimidine phosphate synthase, THIC (MeTHIC1 and MeTHIC2), both of which carry a riboswitch in the 3'-UTR, as well as the adenylated thiazole synthase, THI1 (MeTHI1a and MeTHI1b). The THIC and THI1 genes are expressed at very low levels in storage roots compared with the accumulation of vitamin B1, indicating only limited biosynthesis de novo therein. In leaves, vitamin B1 content is negatively correlated with THIC and THI1 expression levels, suggesting post-transcriptional regulation of THIC by the riboswitch present in the 3'-UTR of the THIC mRNA and regulation of THI1 by promoter activity or alternative post-transcriptional mechanisms.
Assuntos
Manihot/genética , Tiamina/genética , Tiamina/metabolismo , Cromatografia Líquida de Alta Pressão , Manihot/metabolismo , Especificidade de Órgãos , Folhas de Planta/metabolismo , Raízes de Plantas/metabolismo , Tiamina/biossínteseRESUMO
The methylotrophic yeast Pichia pastoris is widely used for production of recombinant proteins. Here we characterize a vitamin-sensitive regulatory sequence, which can be controlled independently of the main culture medium compounds such as carbon, nitrogen, or phosphor source. The THI11 promoter (PTHI11 ) sequence derives from a gene involved in biosynthesis of thiamine. For characterization, a P. pastoris strain expressing recombinant human serum albumin under control of PTHI11 was grown in the controlled environment of a bioreactor. The thiamine sensitivity of PTHI11 was proven and specified in batch cultures containing different amounts of extracellular thiamine. Under non-repressing conditions PTHI11 offers a constitutive expression pattern with growth rate dependent product formation. Furthermore, promoter activity and thus product formation can be repressed for a desired period of time by supplementing the culture with a pre-defined amount of exogenous thiamine. Once a threshold of biomass is reached, PTHI11 driven expression starts autonomously without external intervention. Based on these findings a tailor-made process strategy was developed and experimentally verified. Additionally, we compared the THI11 promoter with the commonly used GAP promoter. In conclusion, the THI11 promoter is a versatile and easy to control regulatory sequence which enables the realization of novel protein production strategies. Biotechnol. Bioeng. 2016;113: 2633-2643. © 2016 Wiley Periodicals, Inc.
Assuntos
Pichia/fisiologia , Regiões Promotoras Genéticas/genética , Engenharia de Proteínas/métodos , Proteínas Recombinantes/biossíntese , Sequências Reguladoras de Ácido Ribonucleico/genética , Tiamina/genética , Melhoramento Genético/métodos , Proteínas Recombinantes/genéticaRESUMO
Thiamine pyrophosphate (TPP) is a critical cofactor and its biosynthesis is under the control of TPP availability. Here we disrupted a predicted thiA gene of the fungus Aspergillus nidulans and demonstrated that it is essential for synthesizing cellular thiamine. The thiamine riboswitch is a post-transcriptional mechanism for TPP to repress gene expression and it is located on A. nidulans thiA pre-messenger RNA. The thiA riboswitch was not fully derepressed under thiamine-limited conditions, and fully derepressed under environmental stressors. Upon exposure to hypoxic stress, the fungus accumulated more ThiA and NmtA proteins, and more thiamine than under aerobic conditions. The thiA gene was required for the fungus to upregulate hypoxic branched-chain amino acids and ethanol fermentation that involve enzymes containing TPP. These findings indicate that hypoxia modulates thiA expression through the thiamine riboswitch, and alters cellular fermentation mechanisms by regulating the activity of the TPP enzymes.
Assuntos
Aspergillus nidulans/enzimologia , Proteínas Fúngicas/genética , Riboswitch/genética , Tiamina/biossíntese , Hipóxia Celular , Fermentação , Proteínas Fúngicas/biossíntese , Regulação Fúngica da Expressão Gênica , Estresse Fisiológico/genética , Tiamina/genética , Tiamina Pirofosfato/biossíntese , Tiamina Pirofosfato/genéticaRESUMO
Energy coupling factor (ECF) transporters take up micronutrients in Bacteria and Archaea. They consist of a membrane-embedded S-component that provides substrate specificity and a three-subunit ECF module that couples ATP hydrolysis to transport. The S-components ThiT (for thiamin) and NiaX (for niacin) from Lactococcus lactis form complexes with the same ECF module. Here, we assayed the uptake of thiamin and niacin in Escherichia coli cells expressing the transporter genes. We demonstrate that the two different S-components compete for the ECF module, and that competition is more efficient in the presence of the transported substrate. The data suggest that binding and release of the S-components is a step in the transport cycle.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Lactococcus lactis/metabolismo , Niacina/metabolismo , Tiamina/metabolismo , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/genética , Transporte Biológico Ativo/fisiologia , Proteínas de Transporte/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Hidrólise , Lactococcus lactis/genética , Niacina/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tiamina/genéticaRESUMO
The TenA protein family occurs in prokaryotes, plants and fungi; it has two subfamilies, one (TenA_C) having an active-site cysteine, the other (TenA_E) not. TenA_C proteins participate in thiamin salvage by hydrolysing the thiamin breakdown product amino-HMP (4-amino-5-aminomethyl-2-methylpyrimidine) to HMP (4-amino-5-hydroxymethyl-2-methylpyrimidine); the function of TenA_E proteins is unknown. Comparative analysis of prokaryote and plant genomes predicted that (i) TenA_E has a salvage role similar to, but not identical with, that of TenA_C and (ii) that TenA_E and TenA_C also have non-salvage roles since they occur in organisms that cannot make thiamin. Recombinant Arabidopsis and maize TenA_E proteins (At3g16990, GRMZM2G080501) hydrolysed amino-HMP to HMP and, far more actively, hydrolysed the N-formyl derivative of amino-HMP to amino-HMP. Ablating the At3g16990 gene in a line with a null mutation in the HMP biosynthesis gene ThiC prevented its rescue by amino-HMP. Ablating At3g16990 in the wild-type increased sensitivity to paraquat-induced oxidative stress; HMP overcame this increased sensitivity. Furthermore, the expression of TenA_E and ThiC genes in Arabidopsis and maize was inversely correlated. These results indicate that TenA_E proteins mediate amidohydrolase and aminohydrolase steps in the salvage of thiamin breakdown products. As such products can be toxic, TenA_E proteins may also pre-empt toxicity.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Hidrolases/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Tiamina/metabolismo , Zea mays/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Herbicidas/farmacologia , Hidrolases/genética , Proteínas Ferro-Enxofre/genética , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Paraquat/farmacologia , Tiamina/genética , Zea mays/genéticaRESUMO
Anemia of inflammation (AI) is common in patients with infection, autoimmune diseases, cancer, and chronic kidney disease. Unless the underlying condition can be reversed, treatment options are limited to erythropoiesis-stimulating agents with or without intravenous iron therapy, modalities that are not always effective and can cause serious adverse effects. Hepcidin, the iron regulatory hormone, has been identified as a pathogenic factor in the development of AI. To explore new therapeutic options for AI and other iron-related disorders caused by hepcidin excess, we developed a cell-based screen to identify hepcidin antagonists. Of the 70,000 small molecules in the library, we identified 14 compounds that antagonized the hepcidin effect on ferroportin. One of these was fursultiamine, a Food and Drug Administration (FDA)-approved thiamine derivative. Fursultiamine directly interfered with hepcidin binding to its receptor, ferroportin, by blocking ferroportin C326 thiol residue essential for hepcidin binding. Consequently, fursultiamine prevented hepcidin-induced ferroportin ubiquitination, endocytosis, and degradation in vitro and allowed continuous cellular iron export despite the presence of hepcidin, with IC(50) in the submicromolar range. Thiamine, the fursultiamine metabolite, and benfotiamine, another thiamine derivative, did not interfere with the effect of hepcidin on ferroportin. Other FDA-approved thiol-reactive compounds were at least 1000-fold less potent than fursultiamine in antagonizing hepcidin. In vivo, fursultiamine did not reproducibly antagonize the effect of hepcidin on serum iron, likely because of its rapid conversion to inactive metabolites. Fursultiamine is a unique antagonist of hepcidin in vitro that could serve as a template for the development of drug candidates that inhibit the hepcidin-ferroportin interaction.
Assuntos
Peptídeos Catiônicos Antimicrobianos/antagonistas & inibidores , Ensaios de Triagem em Larga Escala/métodos , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/metabolismo , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Linhagem Celular , Endocitose/efeitos dos fármacos , Endocitose/genética , Fursultiamina/farmacologia , Células HEK293 , Hepcidinas , Humanos , Ferro/metabolismo , Distúrbios do Metabolismo do Ferro/tratamento farmacológico , Distúrbios do Metabolismo do Ferro/genética , Distúrbios do Metabolismo do Ferro/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/genética , Proteólise/efeitos dos fármacos , Receptores de LDL/genética , Receptores de LDL/metabolismo , Tiamina/genética , Tiamina/metabolismo , Ubiquitinação/efeitos dos fármacos , Ubiquitinação/genéticaRESUMO
Vitamin B(1) is essential for all organisms being well recognized as a necessary cofactor for key metabolic pathways such as glycolysis, and was more recently implicated in DNA damage responses. Little is known about the enzyme responsible for the formation of the pyrimidine moiety (4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate (HMP-P) synthase). We report a structure-function study of the HMP-P synthase from yeast, THI5p. Our crystallographic structure shows that THI5p is a mix between periplasmic binding proteins and pyridoxal 5'-phosphate-dependent enzymes. Mutational and yeast complementation studies identify the key residues for HMP-P biosynthesis as well as the use of pyridoxal 5'-phosphate as a substrate rather than as a cofactor. Furthermore, we could show that iron binding to HMP-P synthase is essential for the reaction.
Assuntos
Ferro/química , Fosfotransferases (Aceptor do Grupo Fosfato)/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Tiamina/biossíntese , Cristalografia por Raios X , Ferro/metabolismo , Mutação , Fosfotransferases (Aceptor do Grupo Fosfato)/genética , Fosfotransferases (Aceptor do Grupo Fosfato)/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Pirimidinas/química , Pirimidinas/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Relação Estrutura-Atividade , Tiamina/química , Tiamina/genéticaRESUMO
BACKGROUND: Biotin-thiamine-responsive basal ganglia disease (BTRBGD) is a rare treatable autosomal recessive neurometabolic disorder characterized by progressive encephalopathy that eventually leads to severe disability and death if not treated with biotin and thiamine. BTRBGD is caused by mutations in the SLC19A3 gene on chromosome 2q36.6, encoding human thiamine transporter 2 (hTHTR2). Episodes of BTRBGD are often triggered by febrile illness. CASE REPORT: The patient was 2 years 10 months old male child presented with fever and progressive acute encephalopathy associated with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) virus infection. MRI revealed bilateral symmetrical high signal involving both basal ganglia and medial thalami which is swollen with central necrosis, initially diagnosed as acute necrotizing encephalomyelitis with increased severity. Genetic analysis revealed BTRBGD. CONCLUSION: BTRBGD requires high index of suspicion in any patient presenting with acute encephalopathy, characteristic MRI findings (that are difficult to differentiate from necrotizing encephalopathy), regardless of the existence of a proven viral infection.
Assuntos
Doenças dos Gânglios da Base/complicações , Doenças dos Gânglios da Base/diagnóstico , COVID-19/complicações , Encefalopatia Aguda Febril/diagnóstico , Encefalopatia Aguda Febril/etiologia , Gânglios da Base , Doenças dos Gânglios da Base/virologia , Biotina/genética , Encéfalo/metabolismo , COVID-19/virologia , Pré-Escolar , Testes Genéticos , Humanos , Imageamento por Ressonância Magnética , Masculino , Proteínas de Membrana Transportadoras/genética , Mutação , SARS-CoV-2/patogenicidade , Tiamina/genéticaRESUMO
AIMS: Release of hydrogen sulfide by fermenting yeast is a potential problem in wine production, because of its strong organoleptic impact. To identify the genetic determinants of sulfide production, we compared the transcriptomes of two wine yeast strains with similar oenological properties, but with very different levels of sulfide production, UDC522 (high sulfide producer) and P29 (low producer). METHODS AND RESULTS: Oenological microfermentations were sampled at the peak production of sulfide. Transcription profiles of the two strains were analysed by three methods, a cDNA-based array, an oligonucleotide-based array and qRT-PCR analysis of selected transcripts. Less than 10% of yeast genes showed significant differences between the two strains. High sulfide production correlated with a general overexpression of thiamine biosynthesis genes, whereas genes linked to the catabolism of sulfur-containing compounds (like amino acids) showed no significant expression differences between both strains. CONCLUSIONS: Our data suggest a relationship between the thiamine biosynthetic pathway and sulfide production during wine fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides a first hint which indicates that for some yeast strains, biosynthesis of thiamine (and perhaps of other sulfur-containing compounds) may be more relevant than the general nitrogen metabolism in explaining sulfide production by some yeast strains during vinification, defining new targets for genetic improvement of wine yeast strains.