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1.
Am J Hum Genet ; 108(11): 2195-2204, 2021 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-34715011

RESUMO

Human mitochondrial RNase P (mt-RNase P) is responsible for 5' end processing of mitochondrial precursor tRNAs, a vital step in mitochondrial RNA maturation, and is comprised of three protein subunits: TRMT10C, SDR5C1 (HSD10), and PRORP. Pathogenic variants in TRMT10C and SDR5C1 are associated with distinct recessive or x-linked infantile onset disorders, resulting from defects in mitochondrial RNA processing. We report four unrelated families with multisystem disease associated with bi-allelic variants in PRORP, the metallonuclease subunit of mt-RNase P. Affected individuals presented with variable phenotypes comprising sensorineural hearing loss, primary ovarian insufficiency, developmental delay, and brain white matter changes. Fibroblasts from affected individuals in two families demonstrated decreased steady state levels of PRORP, an accumulation of unprocessed mitochondrial transcripts, and decreased steady state levels of mitochondrial-encoded proteins, which were rescued by introduction of the wild-type PRORP cDNA. In mt-tRNA processing assays performed with recombinant mt-RNase P proteins, the disease-associated variants resulted in diminished mitochondrial tRNA processing. Identification of disease-causing variants in PRORP indicates that pathogenic variants in all three subunits of mt-RNase P can cause mitochondrial dysfunction, each with distinct pleiotropic clinical presentations.


Assuntos
Alelos , Pleiotropia Genética , Mitocôndrias/enzimologia , RNA Mitocondrial/genética , RNA de Transferência/genética , Ribonuclease P/genética , Adulto , Feminino , Humanos , Masculino , Linhagem
2.
Virology ; 562: 128-141, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34315103

RESUMO

Picornavirus family members cause disease in humans. Human rhinoviruses (RV), the main causative agents of the common cold, increase the severity of asthma and COPD; hence, effective agents against RVs are required. The 2A proteinase (2Apro), found in all enteroviruses, represents an attractive target; inactivating mutations in poliovirus 2Apro result in an extension of the VP1 protein preventing infectious virion assembly. Variations in sequence and substrate specificity on eIF4G isoforms between RV 2Apro of genetic groups A and B hinder 2Apro as drug targets. Here, we demonstrate that although RV-A2 and RV-B4 2Apro cleave the substrate GAB1 at different sites, the 2Apro from both groups cleave equally efficiently an artificial site containing P1 methionine. We determined the RV-A2 2Apro structure complexed with zVAM.fmk, containing P1 methionine. Analysis of this first 2Apro-inhibitor complex reveals a conserved hydrophobic P4 pocket among enteroviral 2Apro as a potential target for broad-spectrum anti-enteroviral inhibitors.

3.
Biochimie ; 185: 96-104, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33746066

RESUMO

Cancer cells exhibit an altered metabolic phenotype, consuming higher levels of the amino acid glutamine. This metabolic reprogramming depends on increased mitochondrial glutaminase activity to convert glutamine to glutamate, an essential precursor for bioenergetic and biosynthetic processes in cells. Mammals encode the kidney-type (GLS) and liver-type (GLS2) glutaminase isozymes. GLS is overexpressed in cancer and associated with enhanced malignancy. On the other hand, GLS2 is either a tumor suppressor or an oncogene, depending on the tumor type. The GLS structure and activation mechanism are well known, while the structural determinants for GLS2 activation remain elusive. Here, we describe the structure of the human glutaminase domain of GLS2, followed by the functional characterization of the residues critical for its activity. Increasing concentrations of GLS2 lead to tetramer stabilization, a process enhanced by phosphate. In GLS2, the so-called "lid loop" is in a rigid open conformation, which may be related to its higher affinity for phosphate and lower affinity for glutamine; hence, it has lower glutaminase activity than GLS. The lower affinity of GLS2 for glutamine is also related to its less electropositive catalytic site than GLS, as indicated by a Thr225Lys substitution within the catalytic site decreasing the GLS2 glutamine concentration corresponding to half-maximal velocity (K0.5). Finally, we show that the Lys253Ala substitution (corresponding to the Lys320Ala in the GLS "activation" loop, formerly known as the "gating" loop) renders a highly active protein in stable tetrameric form. We conclude that the "activation" loop, a known target for GLS inhibition, may also be a drug target for GLS2.


Assuntos
Ativação Enzimática , Glutaminase/química , Fígado/enzimologia , Substituição de Aminoácidos , Catálise , Glutaminase/genética , Glutaminase/metabolismo , Humanos , Mutação de Sentido Incorreto , Estrutura Quaternária de Proteína , Relação Estrutura-Atividade
4.
ACS Chem Biol ; 16(4): 586-595, 2021 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-33724769

RESUMO

Classic galactosemia is caused by loss-of-function mutations in galactose-1-phosphate uridylyltransferase (GALT) that lead to toxic accumulation of its substrate, galactose-1-phosphate. One proposed therapy is to inhibit the biosynthesis of galactose-1-phosphate, catalyzed by galactokinase 1 (GALK1). Existing inhibitors of human GALK1 (hGALK1) are primarily ATP-competitive with limited clinical utility to date. Here, we determined crystal structures of hGALK1 bound with reported ATP-competitive inhibitors of the spiro-benzoxazole series, to reveal their binding mode in the active site. Spurred by the need for additional chemotypes of hGALK1 inhibitors, desirably targeting a nonorthosteric site, we also performed crystallography-based screening by soaking hundreds of hGALK1 crystals, already containing active site ligands, with fragments from a custom library. Two fragments were found to bind close to the ATP binding site, and a further eight were found in a hotspot distal from the active site, highlighting the strength of this method in identifying previously uncharacterized allosteric sites. To generate inhibitors of improved potency and selectivity targeting the newly identified binding hotspot, new compounds were designed by merging overlapping fragments. This yielded two micromolar inhibitors of hGALK1 that were not competitive with respect to either substrate (ATP or galactose) and demonstrated good selectivity over hGALK1 homologues, galactokinase 2 and mevalonate kinase. Our findings are therefore the first to demonstrate inhibition of hGALK1 from an allosteric site, with potential for further development of potent and selective inhibitors to provide novel therapeutics for classic galactosemia.


Assuntos
Inibidores Enzimáticos/uso terapêutico , Galactoquinase/antagonistas & inibidores , Galactosemias/tratamento farmacológico , Cristalografia por Raios X , Galactoquinase/química , Humanos , Conformação Proteica
6.
Biochimie ; 183: 55-62, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33596448

RESUMO

Succinyl-CoA:3-oxoacid coenzyme A transferase deficiency (SCOTD) is a rare autosomal recessive disorder of ketone body utilization caused by mutations in OXCT1. We performed a systematic literature search and evaluated clinical, biochemical and genetic data on 34 previously published and 10 novel patients with SCOTD. Structural mapping and in silico analysis of protein variants is also presented. All patients presented with severe ketoacidotic episodes. Age at first symptoms ranged from 36 h to 3 years (median 7 months). About 70% of patients manifested in the first year of life, approximately one quarter already within the neonatal period. Two patients died, while the remainder (95%) were alive at the time of the report. Almost all the surviving patients (92%) showed normal psychomotor development and no neurologic abnormalities. A total of 29 missense mutations are reported. Analysis of the published crystal structure of the human SCOT enzyme, paired with both sequence-based and structure-based methods to predict variant pathogenicity, provides insight into the biochemical consequences of the reported variants. Pathogenic variants cluster in SCOT protein regions that affect certain structures of the protein. The described pathogenic variants can be viewed in an interactive map of the SCOT protein at https://michelanglo.sgc.ox.ac.uk/r/oxct. This comprehensive data analysis provides a systematic overview of all cases of SCOTD published to date. Although SCOTD is a rather benign disorder with often favourable outcome, metabolic crises can be life-threatening or even fatal. As the diagnosis can only be made by enzyme studies or mutation analyses, SCOTD may be underdiagnosed.


Assuntos
Acidose , Encefalopatias Metabólicas Congênitas , Coenzima A-Transferases/deficiência , Mutação de Sentido Incorreto , Transtornos do Neurodesenvolvimento , Acidose/enzimologia , Acidose/genética , Encefalopatias Metabólicas Congênitas/enzimologia , Encefalopatias Metabólicas Congênitas/genética , Coenzima A-Transferases/química , Coenzima A-Transferases/genética , Coenzima A-Transferases/metabolismo , Cristalografia por Raios X , Humanos , Corpos Cetônicos/química , Corpos Cetônicos/genética , Corpos Cetônicos/metabolismo , Transtornos do Neurodesenvolvimento/enzimologia , Transtornos do Neurodesenvolvimento/genética , Domínios Proteicos
7.
Biochimie ; 183: 100-107, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33476699

RESUMO

The folate and methionine cycles, constituting one-carbon metabolism, are critical pathways for cell survival. Intersecting these two cycles, 5,10-methylenetetrahydrofolate reductase (MTHFR) directs one-carbon units from the folate to methionine cycle, to be exclusively used for methionine and S-adenosylmethionine (AdoMet) synthesis. MTHFR deficiency and upregulation result in diverse disease states, rendering it an attractive drug target. The activity of MTHFR is inhibited by the binding of AdoMet to an allosteric regulatory domain distal to the enzyme's active site, which we have previously identified to constitute a novel fold with a druggable pocket. Here, we screened 162 AdoMet mimetics using differential scanning fluorimetry, and identified 4 compounds that stabilized this regulatory domain. Three compounds were sinefungin analogues, closely related to AdoMet and S-adenosylhomocysteine (AdoHcy). The strongest thermal stabilisation was provided by (S)-SKI-72, a potent inhibitor originally developed for protein arginine methyltransferase 4 (PRMT4). Using surface plasmon resonance, we confirmed that (S)-SKI-72 binds MTHFR via its allosteric domain with nanomolar affinity. Assay of MTHFR activity in the presence of (S)-SKI-72 demonstrates inhibition of purified enzyme with sub-micromolar potency and endogenous MTHFR from HEK293 cell lysate in the low micromolar range, both of which are lower than AdoMet. Nevertheless, unlike AdoMet, (S)-SKI-72 is unable to completely abolish MTHFR activity, even at very high concentrations. Combining binding assays, kinetic characterization and compound docking, this work indicates the regulatory domain of MTHFR can be targeted by small molecules and presents (S)-SKI-72 as an excellent candidate for development of MTHFR inhibitors.


Assuntos
Inibidores Enzimáticos/química , Metilenotetra-Hidrofolato Redutase (NADPH2)/antagonistas & inibidores , Metilenotetra-Hidrofolato Redutase (NADPH2)/química , S-Adenosilmetionina/química , Regulação Alostérica , Humanos , Domínios Proteicos
8.
Biochimie ; 183: 35-43, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32659443

RESUMO

Cobalamin, commonly known as vitamin B12, is an essential micronutrient for humans because of its role as an enzyme cofactor. Cobalamin is one of over a dozen structurally related compounds - cobamides - that are found in certain foods and are produced by microorganisms in the human gut. Very little is known about how different cobamides affect B12-dependent metabolism in human cells. Here, we test in vitro how diverse cobamide cofactors affect the function of methylmalonyl-CoA mutase (MMUT), one of two cobalamin-dependent enzymes in humans. We find that, although cobalamin is the most effective cofactor for MMUT, multiple cobamides support MMUT function with differences in binding affinity (Kd), binding kinetics (kon), and concentration dependence during catalysis (KM, app). Additionally, we find that six disease-associated MMUT variants that cause cobalamin-responsive impairments in enzymatic activity also respond to other cobamides, with the extent of catalytic rescue dependent on the identity of the cobamide. Our studies challenge the exclusive focus on cobalamin in the context of human physiology, indicate that diverse cobamides can support the function of a human enzyme, and suggest future directions that will improve our understanding of the roles of different cobamides in human biology.


Assuntos
Coenzimas/química , Metilmalonil-CoA Mutase/química , Vitamina B 12/química , Coenzimas/metabolismo , Humanos , Cinética , Metilmalonil-CoA Mutase/metabolismo , Vitamina B 12/metabolismo
9.
Adv Sci (Weinh) ; 7(22): 2001970, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33240760

RESUMO

A versatile and Lipinski-compliant DNA-encoded library (DEL), comprising 366 600 glutamic acid derivatives coupled to oligonucleotides serving as amplifiable identification barcodes is designed, constructed, and characterized. The GB-DEL library, constructed in single-stranded DNA format, allows de novo identification of specific binders against several pharmaceutically relevant proteins. Moreover, hybridization of the single-stranded DEL with a set of known protein ligands of low to medium affinity coupled to a complementary DNA strand results in self-assembled selectable chemical structures, leading to the identification of affinity-matured compounds.

10.
Am J Med Genet A ; 182(11): 2685-2693, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32808436

RESUMO

We report the case of an 11-year-old Syrian girl born to consanguineous parents, who presents an ataxic gait from early childhood. On clinical examination, she presented a severe static - kinetic cerebellar syndrome, walking without support is possible for short distances only. Strikingly, three consecutive MRIs did not show any sign of cerebellar abnormalities, but a brain positron emission tomography (PET) using [18F]-fluorodeoxyglucose (FDG) demonstrated a clear decrease in glucose metabolism in the cerebellum as well as the anterior and medial temporal lobe bilaterally. A clinical exome analysis identified a novel homozygous c.251A > G (p.Asn84Ser) likely pathogenic variant in the carbonic anhydrase 8 (CA8) gene. CA8 mutations cause cerebellar ataxia, mental retardation, and disequilibrium syndrome subtype 3 (CAMRQ3), a rare genetically autosomal recessive disorder, only described in four families, so far with the frequent observation of quadrupedal gait. The proband differed with other reported CA8 mutations by the absence of clear cerebellar signs on brain MRI and the presence of focal seizures. This report expands the clinical spectrum associated with mutations in CA8 and illustrates the possible discrepancy between (mild) neuro-radiological images (MRI) and (severe) clinical phenotype in young individuals. In contrast, the observation of clear cerebellar abnormal metabolic findings suggests that the FDG-PET scan may be used as an early marker for hereditary ataxia.


Assuntos
Biomarcadores Tumorais/genética , Ataxia Cerebelar/patologia , Homozigoto , Deficiência Intelectual/patologia , Mutação , Fenótipo , Ataxia Cerebelar/genética , Criança , Consanguinidade , Feminino , Humanos , Deficiência Intelectual/genética , Masculino , Linhagem
11.
IUCrJ ; 7(Pt 4): 693-706, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32695416

RESUMO

DHTKD1 is a lesser-studied E1 enzyme among the family of 2-oxoacid de-hydrogenases. In complex with E2 (di-hydro-lipo-amide succinyltransferase, DLST) and E3 (dihydrolipo-amide de-hydrogenase, DLD) components, DHTKD1 is involved in lysine and tryptophan catabolism by catalysing the oxidative de-carboxyl-ation of 2-oxoadipate (2OA) in mitochondria. Here, the 1.9 Šresolution crystal structure of human DHTKD1 is solved in complex with the thi-amine diphosphate co-factor. The structure reveals how the DHTKD1 active site is modelled upon the well characterized homologue 2-oxoglutarate (2OG) de-hydrogenase but engineered specifically to accommodate its preference for the longer substrate of 2OA over 2OG. A 4.7 Šresolution reconstruction of the human DLST catalytic core is also generated by single-particle electron microscopy, revealing a 24-mer cubic scaffold for assembling DHTKD1 and DLD protomers into a megacomplex. It is further demonstrated that missense DHTKD1 variants causing the inborn error of 2-amino-adipic and 2-oxoadipic aciduria impact on the complex formation, either directly by disrupting the interaction with DLST, or indirectly through destabilizing the DHTKD1 protein. This study provides the starting framework for developing DHTKD1 modulators to probe the intricate mitochondrial energy metabolism.

12.
Nat Commun ; 11(1): 2813, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32499479

RESUMO

5'-aminolevulinate synthase (ALAS) catalyzes the first step in heme biosynthesis, generating 5'-aminolevulinate from glycine and succinyl-CoA. Inherited frameshift indel mutations of human erythroid-specific isozyme ALAS2, within a C-terminal (Ct) extension of its catalytic core that is only present in higher eukaryotes, lead to gain-of-function X-linked protoporphyria (XLP). Here, we report the human ALAS2 crystal structure, revealing that its Ct-extension folds onto the catalytic core, sits atop the active site, and precludes binding of substrate succinyl-CoA. The Ct-extension is therefore an autoinhibitory element that must re-orient during catalysis, as supported by molecular dynamics simulations. Our data explain how Ct deletions in XLP alleviate autoinhibition and increase enzyme activity. Crystallography-based fragment screening reveals a binding hotspot around the Ct-extension, where fragments interfere with the Ct conformational dynamics and inhibit ALAS2 activity. These fragments represent a starting point to develop ALAS2 inhibitors as substrate reduction therapy for porphyria disorders that accumulate toxic heme intermediates.


Assuntos
5-Aminolevulinato Sintetase/química , Regulação Enzimológica da Expressão Gênica , 5-Aminolevulinato Sintetase/deficiência , 5-Aminolevulinato Sintetase/genética , Acil Coenzima A/química , Catálise , Domínio Catalítico , Cristalografia por Raios X , Doenças Genéticas Ligadas ao Cromossomo X/genética , Heme/química , Humanos , Cinética , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Protoporfiria Eritropoética/genética , Especificidade por Substrato
13.
Am J Hum Genet ; 106(2): 256-263, 2020 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-32004446

RESUMO

We report an inborn error of metabolism caused by TKFC deficiency in two unrelated families. Rapid trio genome sequencing in family 1 and exome sequencing in family 2 excluded known genetic etiologies, and further variant analysis identified rare homozygous variants in TKFC. TKFC encodes a bifunctional enzyme involved in fructose metabolism through its glyceraldehyde kinase activity and in the generation of riboflavin cyclic 4',5'-phosphate (cyclic FMN) through an FMN lyase domain. The TKFC homozygous variants reported here are located within the FMN lyase domain. Functional assays in yeast support the deleterious effect of these variants on protein function. Shared phenotypes between affected individuals with TKFC deficiency include cataracts and developmental delay, associated with cerebellar hypoplasia in one case. Further complications observed in two affected individuals included liver dysfunction and microcytic anemia, while one had fatal cardiomyopathy with lactic acidosis following a febrile illness. We postulate that deficiency of TKFC causes disruption of endogenous fructose metabolism leading to generation of by-products that can cause cataract. In line with this, an affected individual had mildly elevated urinary galactitol, which has been linked to cataract development in the galactosemias. Further, in light of a previously reported role of TKFC in regulating innate antiviral immunity through suppression of MDA5, we speculate that deficiency of TKFC leads to impaired innate immunity in response to viral illness, which may explain the fatal illness observed in the most severely affected individual.


Assuntos
Catarata/etiologia , Cerebelo/anormalidades , Deficiências do Desenvolvimento/etiologia , Mutação , Malformações do Sistema Nervoso/etiologia , Fósforo-Oxigênio Liases/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Alelos , Sequência de Aminoácidos , Catarata/patologia , Cerebelo/patologia , Pré-Escolar , Deficiências do Desenvolvimento/patologia , Feminino , Homozigoto , Humanos , Lactente , Masculino , Malformações do Sistema Nervoso/patologia , Linhagem , Fenótipo , Fosforilação , Homologia de Sequência , Sequenciamento Completo do Exoma
14.
Genet Med ; 22(1): 199-209, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31462754

RESUMO

PURPOSE: Mitochondrial DNA (mtDNA) depletion syndrome (MDDS) encompasses a group of genetic disorders of mtDNA maintenance. Mutation of RRM2B is an uncommon cause of infantile-onset encephalomyopathic MDDS. Here we describe the natural history of this disease. METHODS: Multinational series of new genetically confirmed cases from six pediatric centers. RESULTS: Nine new cases of infantile-onset RRM2B deficiency, and 22 previously published cases comprised a total cohort of 31 patients. Infants presented at a mean of 1.95 months with truncal hypotonia, generalized weakness, and faltering growth. Seizures evolved in 39% at a mean of 3.1 months. Non-neurological manifestations included respiratory distress/failure (58%), renal tubulopathy (55%), sensorineural hearing loss (36%), gastrointestinal disturbance (32%), eye abnormalities (13%), and anemia (13%). Laboratory features included elevated lactate (blood, cerebrospinal fluid (CSF), urine, magnetic resonance (MR), spectroscopy), ragged-red and cytochrome c oxidase-deficient fibers, lipid myopathy, and multiple oxidative phosphorylation enzyme deficiencies in skeletal muscle. Eight new RRM2B variants were identified. Patients with biallelic truncating variants had the worst survival. Overall survival was 29% at 6 months and 16% at 1 year. CONCLUSIONS: Infantile-onset MDDS due to RRM2B deficiency is a severe disorder with characteristic clinical features and extremely poor prognosis. Presently management is supportive as there is no effective treatment. Novel treatments are urgently needed.


Assuntos
Proteínas de Ciclo Celular/genética , Pseudo-Obstrução Intestinal/genética , Distrofia Muscular Oculofaríngea/genética , Mutação de Sentido Incorreto , Ribonucleotídeo Redutases/genética , Proteínas de Ciclo Celular/química , Feminino , Humanos , Lactente , Recém-Nascido , Pseudo-Obstrução Intestinal/mortalidade , Masculino , Modelos Moleculares , Distrofia Muscular Oculofaríngea/mortalidade , Oftalmoplegia/congênito , Prognóstico , Conformação Proteica , Ribonucleotídeo Redutases/química , Análise de Sobrevida
15.
J Inherit Metab Dis ; 42(5): 809-817, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31177572

RESUMO

The first step in branched-chain amino acid (BCAA) catabolism is catalyzed by the two BCAA transferase isoenzymes, cytoplasmic branched-chain amino acid transferase (BCAT) 1, and mitochondrial BCAT2. Defects in the second step of BCAA catabolism cause maple syrup urine disease (MSUD), a condition which has been far more extensively investigated. Here, we studied the consequences of BCAT2 deficiency, an ultra-rare condition in humans. We present genetic, clinical, and functional data in five individuals from four different families with homozygous or compound heterozygous BCAT2 mutations which were all detected following abnormal biochemical profile results or familial mutation segregation studies. We demonstrate that BCAT2 deficiency has a recognizable biochemical profile with raised plasma BCAAs and, in contrast with MSUD, low-normal branched-chain keto acids (BCKAs) with undetectable l-allo-isoleucine. Interestingly, unlike in MSUD, none of the individuals with BCAT2 deficiency developed acute encephalopathy even with exceptionally high BCAA levels. We observed wide-ranging clinical phenotypes in individuals with BCAT2 deficiency. While one adult was apparently asymptomatic, three individuals had presented with developmental delay and autistic features. We show that the biochemical characteristics of BCAT2 deficiency may be amenable to protein-restricted diet and that early treatment may improve outcome in affected individuals. BCAT2 deficiency is an inborn error of BCAA catabolism. At present, it is unclear whether developmental delay and autism are parts of the variable phenotypic spectrum of this condition or coincidental. Further studies will be required to explore this.


Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/diagnóstico , Erros Inatos do Metabolismo dos Aminoácidos/genética , Aminoácidos de Cadeia Ramificada/sangue , Encéfalo/patologia , Mitocôndrias/patologia , Proteínas da Gravidez/deficiência , Transaminases/deficiência , Adolescente , Adulto , Encéfalo/diagnóstico por imagem , Criança , Pré-Escolar , Feminino , Homozigoto , Humanos , Imageamento por Ressonância Magnética , Masculino , Antígenos de Histocompatibilidade Menor/genética , Mutação , Fenótipo , Proteínas da Gravidez/genética , Transaminases/genética
16.
Nat Commun ; 10(1): 2210, 2019 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-31101807

RESUMO

The core machinery for de novo biosynthesis of iron-sulfur clusters (ISC), located in the mitochondria matrix, is a five-protein complex containing the cysteine desulfurase NFS1 that is activated by frataxin (FXN), scaffold protein ISCU, accessory protein ISD11, and acyl-carrier protein ACP. Deficiency in FXN leads to the loss-of-function neurodegenerative disorder Friedreich's ataxia (FRDA). Here the 3.2 Å resolution cryo-electron microscopy structure of the FXN-bound active human complex, containing two copies of the NFS1-ISD11-ACP-ISCU-FXN hetero-pentamer, delineates the interactions of FXN with other component proteins of the complex. FXN binds at the interface of two NFS1 and one ISCU subunits, modifying the local environment of a bound zinc ion that would otherwise inhibit NFS1 activity in complexes without FXN. Our structure reveals how FXN facilitates ISC production through stabilizing key loop conformations of NFS1 and ISCU at the protein-protein interfaces, and suggests how FRDA clinical mutations affect complex formation and FXN activation.


Assuntos
Liases de Carbono-Enxofre/ultraestrutura , Ataxia de Friedreich/patologia , Proteínas de Ligação ao Ferro/ultraestrutura , Proteínas Ferro-Enxofre/ultraestrutura , Mitocôndrias/ultraestrutura , Liases de Carbono-Enxofre/isolamento & purificação , Liases de Carbono-Enxofre/metabolismo , Microscopia Crioeletrônica , Ataxia de Friedreich/genética , Ferro/metabolismo , Proteínas de Ligação ao Ferro/isolamento & purificação , Proteínas de Ligação ao Ferro/metabolismo , Proteínas Ferro-Enxofre/isolamento & purificação , Proteínas Ferro-Enxofre/metabolismo , Mitocôndrias/metabolismo , Modelos Moleculares , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Enxofre/metabolismo , Zinco/metabolismo
17.
Commun Biol ; 2: 188, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31123712

RESUMO

Human soluble epoxide hydrolase (hsEH) is an enzyme responsible for the inactivation of bioactive epoxy fatty acids, and its inhibition is emerging as a promising therapeutical strategy to target hypertension, cardiovascular disease, pain and insulin sensitivity. Here, we uncover the molecular bases of hsEH inhibition mediated by the endogenous 15-deoxy-Δ12,14-Prostaglandin J2 (15d-PGJ2). Our data reveal a dual inhibitory mechanism, whereby hsEH can be inhibited by reversible docking of 15d-PGJ2 in the catalytic pocket, as well as by covalent locking of the same compound onto cysteine residues C423 and C522, remote to the active site. Biophysical characterisations allied with in silico investigations indicate that the covalent modification of the reactive cysteines may be part of a hitherto undiscovered allosteric regulatory mechanism of the enzyme. This study provides insights into the molecular modes of inhibition of hsEH epoxy-hydrolytic activity and paves the way for the development of new allosteric inhibitors.


Assuntos
Epóxido Hidrolases/antagonistas & inibidores , Prostaglandina D2/análogos & derivados , Regulação Alostérica , Sequência de Aminoácidos , Substituição de Aminoácidos , Domínio Catalítico/genética , Cristalografia por Raios X , Cisteína/química , Inibidores Enzimáticos/farmacologia , Estabilidade Enzimática/efeitos dos fármacos , Epóxido Hidrolases/química , Epóxido Hidrolases/genética , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Prostaglandina D2/farmacologia , Domínios Proteicos , Alinhamento de Sequência , Solubilidade
18.
Pathology ; 51(3): 274-280, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30853107

RESUMO

Genetic defects on 6-pyruvoyl-tetrahydropterin synthase (PTPS) are the most prevalent cause of hyperphenylalaninaemia not due to phenylalanine hydrolyase deficiency (phenylketonuria). PTPS catalyses the second step of tetrahydrobiopterin (BH4) cofactor biosynthesis, and its deficiency represents the most common form of BH4 deficiency. Untreated PTPS deficiency results in depletion of the neurotransmitters dopamine, catecholamine and serotonin causing neurological symptoms. We archived reported missense variants of the PTS gene. Common in silico algorithms were used to predict the effects of such variants, and substantial proportions (up to 19%) of the variants were falsely classified as benign or uncertain. We have determined the crystal structure of the human PTPS hexamer, allowing another level of interpretation to understand the potential deleterious consequences of the variants from a structural perspective. The in silico and structure approaches appear to be complimentary and may provide new insights that are not available from each alone. Information from the protein structure suggested that the variants affecting amino acid residues required for interaction between monomeric subunits of the PTPS hexamer were those misclassified as benign by in silico algorithms. Our findings illustrate the important utility of 3D protein structure in interpretation of variants and also current limitations of in silico prediction algorithms. However, software to analyse mutation in the perspective of 3D protein structure is far less readily available than other in silico prediction tools.


Assuntos
Mutação , Fenilcetonúrias/genética , Fósforo-Oxigênio Liases/deficiência , Fósforo-Oxigênio Liases/genética , Humanos , Fenilcetonúrias/metabolismo , Fósforo-Oxigênio Liases/metabolismo , Conformação Proteica
19.
Kidney Int ; 95(5): 1138-1152, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30885509

RESUMO

Mutations in leucine-rich-repeats and immunoglobulin-like-domains 2 (LRIG2) or in heparanase 2 (HPSE2) cause urofacial syndrome, a devastating autosomal recessive disease of functional bladder outlet obstruction. It has been speculated that urofacial syndrome has a neural basis, but it is unknown whether defects in urinary bladder innervation are present. We hypothesized that urofacial syndrome features a peripheral neuropathy of the bladder. Mice with homozygous targeted Lrig2 mutations had urinary defects resembling those found in urofacial syndrome. There was no anatomical blockage of the outflow tract, consistent with a functional bladder outlet obstruction. Transcriptome analysis revealed differential expression of 12 known transcripts in addition to Lrig2, including 8 with established roles in neurobiology. Mice with homozygous mutations in either Lrig2 or Hpse2 had increased nerve density within the body of the urinary bladder and decreased nerve density around the urinary outflow tract. In a sample of 155 children with chronic kidney disease and urinary symptoms, we discovered novel homozygous missense LRIG2 variants that were predicted to be pathogenic in 2 individuals with non-syndromic bladder outlet obstruction. These observations provide evidence that a peripheral neuropathy is central to the pathobiology of functional bladder outlet obstruction in urofacial syndrome, and emphasize the importance of LRIG2 and heparanase 2 for nerve patterning in the urinary tract.


Assuntos
Glucuronidase/genética , Glicoproteínas de Membrana/genética , Doenças do Sistema Nervoso Periférico/genética , Obstrução do Colo da Bexiga Urinária/genética , Bexiga Urinária/inervação , Doenças Urológicas/genética , Animais , Criança , Análise Mutacional de DNA , Facies , Feminino , Perfilação da Expressão Gênica , Homozigoto , Humanos , Masculino , Camundongos , Camundongos Knockout , Mutação de Sentido Incorreto , Doenças do Sistema Nervoso Periférico/patologia , Bexiga Urinária/patologia , Obstrução do Colo da Bexiga Urinária/patologia , Doenças Urológicas/patologia
20.
Medchemcomm ; 10(2): 209-220, 2019 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-30881609

RESUMO

The mitochondrial acyl carrier protein (human ACPM, yeast Acp1) is an essential mitochondrial protein. Through binding of nascent acyl chains on the serine (S112)-bound 4'-phosphopantetheine (4'-PP) cofactor, ACPM is involved in mitochondrial fatty acid synthesis and lipoic acid biogenesis. Recently, yeast Acp1 was found to interact with several mitochondrial complexes, including the iron-sulfur (Fe-S) cluster biosynthesis and respiratory complexes, via the binding to LYRM proteins, a family of proteins involved in assembly/stability of complexes. Importantly, the interaction of LYRM proteins with Acp1 was shown to be essential in maintaining integrity of mitochondrial complexes. In human, recent structures show that ACPM binding to LYRM proteins involves acyl chains attached to the 4'-PP cofactor. Here, we performed an detailed characterization of the mitochondrial interactome of human ACPM by mass spectrometry (MS) and demonstrate the crucial role of the 4'-PP cofactor in most of ACPM interactions. Specifically, we show that ACPM interacts with endogenous Fe-S cluster complex components through binding of the LYRM protein ISD11/LYRM4. Using knockdown experiments, we further determine that ACPM is essential for the stability of mitochondrial respiratory complexes I, II and III, as well as the Fe-S cluster biosynthesis complex. Finally, using native MS and a top-down MS approach, we show that C14, C16 and C18 3-keto-acyl chains on ACPM are implicated in binding to ISD11 through analysis of the recombinant ACPM-ISD11 complex. Taken together, our data provide novel understanding of the role of 4'-PP- and long acyl chains-dependent interactions in human ACPM function.

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