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1.
Nat Commun ; 10(1): 3197, 2019 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-31324765

RESUMO

Mitochondrial proteins are replete with phosphorylation, yet its functional relevance remains largely unclear. The presence of multiple resident mitochondrial phosphatases, however, suggests that protein dephosphorylation may be broadly important for calibrating mitochondrial activities. To explore this, we deleted the poorly characterized matrix phosphatase Pptc7 from mice using CRISPR-Cas9 technology. Strikingly, Pptc7-/- mice exhibit hypoketotic hypoglycemia, elevated acylcarnitines and serum lactate, and die soon after birth. Pptc7-/- tissues have markedly diminished mitochondrial size and protein content despite normal transcript levels, and aberrantly elevated phosphorylation on select mitochondrial proteins. Among these, we identify the protein translocase complex subunit Timm50 as a putative Pptc7 substrate whose phosphorylation reduces import activity. We further find that phosphorylation within or near the mitochondrial targeting sequences of multiple proteins could disrupt their import rates and matrix processing. Overall, our data define Pptc7 as a protein phosphatase essential for proper mitochondrial function and biogenesis during the extrauterine transition.


Assuntos
Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Proteína Fosfatase 2C/genética , Proteína Fosfatase 2C/metabolismo , Animais , Sistemas CRISPR-Cas , Clonagem Molecular , Modelos Animais de Doenças , Metabolismo Energético/genética , Metabolismo Energético/fisiologia , Feminino , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lipidômica , Masculino , Proteínas de Membrana Transportadoras/metabolismo , Erros Inatos do Metabolismo/genética , Erros Inatos do Metabolismo/patologia , Metabolômica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/ultraestrutura , Membranas Mitocondriais/metabolismo , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Mutagênese Sítio-Dirigida , Fosforilação , Proteômica
2.
Health Secur ; 17(3): 229-239, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31206321

RESUMO

Dual-use research poses a significant challenge for scientists in the biomedical field and for global health security in general. As the scientific knowledge and materials required for the development of biological agents become progressively more accessible and inexpensive, there is an increased need to understand and improve the governance of scientific research. Prevention of the misuse of facilities, equipment, agents, and scientific knowledge requires high levels of awareness of the concept of dual-use research, starting with early-career scientists and graduate students. In this study, the attitudes and level of awareness of postgraduate students in Pakistan toward the issues surrounding dual-use research were assessed through a survey containing both quantitative and qualitative questions in 32 universities in 4 provinces, federal area, and Azad Jammu and Kashmir regions of Pakistan; 933 students responded. Most (58.2%) had never heard of dual-use research of concern (DURC), while 18.5% had heard the term but were unsure of its meaning. Irrespective of prior knowledge, a higher percentage of students (68.6%) felt an obligation to report research misuse. Considering the need for DURC training, 94.1% of the respondents agreed that the principal investigator should take the responsibility to train students on DURC at the start of a research project. When experimental results having dual-use potential, 69.1% indicated they would publish with limited protocol, with 43.5% indicating they would publish the limited protocol only if there was a way for scientists to access their data. The survey results revealed limited DURC awareness among researchers across Pakistan. However, the respondents, although not formally educated about DURC, were quite aware of its impact. The information gained in this survey will be valuable in addressing country-specific awareness and training needs.


Assuntos
Pesquisa Biomédica/ética , Disseminação de Informação/ética , Estudantes/psicologia , Pesquisa Biomédica/normas , Bioterrorismo/prevenção & controle , Contenção de Riscos Biológicos/ética , Estudos Transversais , Humanos , Paquistão , Medidas de Segurança/ética , Medidas de Segurança/normas , Inquéritos e Questionários
3.
Mol Cell ; 73(4): 763-774.e10, 2019 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-30661980

RESUMO

The biosynthesis of coenzyme Q presents a paradigm for how cells surmount hydrophobic barriers in lipid biology. In eukaryotes, CoQ precursors-among nature's most hydrophobic molecules-must somehow be presented to a series of enzymes peripherally associated with the mitochondrial inner membrane. Here, we reveal that this process relies on custom lipid-binding properties of COQ9. We show that COQ9 repurposes the bacterial TetR fold to bind aromatic isoprenes with high specificity, including CoQ intermediates that likely reside entirely within the bilayer. We reveal a process by which COQ9 associates with cardiolipin-rich membranes and warps the membrane surface to access this cargo. Finally, we identify a molecular interface between COQ9 and the hydroxylase COQ7, motivating a model whereby COQ9 presents intermediates directly to CoQ enzymes. Overall, our results provide a mechanism for how a lipid-binding protein might access, select, and deliver specific cargo from a membrane to promote biosynthesis.


Assuntos
Lipídeos de Membrana/metabolismo , Membranas Mitocondriais/enzimologia , Proteínas Mitocondriais/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Ubiquinona/biossíntese , Sítios de Ligação , Cardiolipinas/metabolismo , Cristalografia , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica em alfa-Hélice , Transporte Proteico , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Relação Estrutura-Atividade , Triptofano , Ubiquinona/química , Ubiquinona/genética
4.
Mol Cell ; 63(4): 608-620, 2016 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-27499294

RESUMO

The UbiB protein kinase-like (PKL) family is widespread, comprising one-quarter of microbial PKLs and five human homologs, yet its biochemical activities remain obscure. COQ8A (ADCK3) is a mammalian UbiB protein associated with ubiquinone (CoQ) biosynthesis and an ataxia (ARCA2) through unclear means. We show that mice lacking COQ8A develop a slowly progressive cerebellar ataxia linked to Purkinje cell dysfunction and mild exercise intolerance, recapitulating ARCA2. Interspecies biochemical analyses show that COQ8A and yeast Coq8p specifically stabilize a CoQ biosynthesis complex through unorthodox PKL functions. Although COQ8 was predicted to be a protein kinase, we demonstrate that it lacks canonical protein kinase activity in trans. Instead, COQ8 has ATPase activity and interacts with lipid CoQ intermediates, functions that are likely conserved across all domains of life. Collectively, our results lend insight into the molecular activities of the ancient UbiB family and elucidate the biochemical underpinnings of a human disease.


Assuntos
Comportamento Animal , Ataxia Cerebelar/enzimologia , Cerebelo/enzimologia , Proteínas Mitocondriais/deficiência , Músculo Esquelético/enzimologia , Ubiquinona/deficiência , Animais , Células COS , Ataxia Cerebelar/genética , Ataxia Cerebelar/fisiopatologia , Ataxia Cerebelar/psicologia , Cerebelo/fisiopatologia , Cerebelo/ultraestrutura , Chlorocebus aethiops , Modelos Animais de Doenças , Tolerância ao Exercício , Feminino , Predisposição Genética para Doença , Células HEK293 , Humanos , Metabolismo dos Lipídeos , Masculino , Aprendizagem em Labirinto , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Modelos Moleculares , Atividade Motora , Força Muscular , Músculo Esquelético/fisiopatologia , Fenótipo , Ligação Proteica , Conformação Proteica , Proteômica/métodos , Reconhecimento Psicológico , Teste de Desempenho do Rota-Rod , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Convulsões/enzimologia , Convulsões/genética , Convulsões/fisiopatologia , Relação Estrutura-Atividade , Fatores de Tempo , Transfecção , Ubiquinona/química , Ubiquinona/genética
5.
Proc Natl Acad Sci U S A ; 111(44): E4697-705, 2014 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-25339443

RESUMO

Coenzyme Q (CoQ) is an isoprenylated quinone that is essential for cellular respiration and is synthesized in mitochondria by the combined action of at least nine proteins (COQ1-9). Although most COQ proteins are known to catalyze modifications to CoQ precursors, the biochemical role of COQ9 remains unclear. Here, we report that a disease-related COQ9 mutation leads to extensive disruption of the CoQ protein biosynthetic complex in a mouse model, and that COQ9 specifically interacts with COQ7 through a series of conserved residues. Toward understanding how COQ9 can perform these functions, we solved the crystal structure of Homo sapiens COQ9 at 2.4 Å. Unexpectedly, our structure reveals that COQ9 has structural homology to the TFR family of bacterial transcriptional regulators, but that it adopts an atypical TFR dimer orientation and is not predicted to bind DNA. Our structure also reveals a lipid-binding site, and mass spectrometry-based analyses of purified COQ9 demonstrate that it associates with multiple lipid species, including CoQ itself. The conserved COQ9 residues necessary for its interaction with COQ7 comprise a surface patch around the lipid-binding site, suggesting that COQ9 might serve to present its bound lipid to COQ7. Collectively, our data define COQ9 as the first, to our knowledge, mammalian TFR structural homolog and suggest that its lipid-binding capacity and association with COQ7 are key features for enabling CoQ biosynthesis.


Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Metabolismo dos Lipídeos/fisiologia , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Ubiquinona/biossíntese , Animais , Proteínas de Transporte/genética , Cristalografia por Raios X , Humanos , Proteínas de Membrana/genética , Camundongos , Camundongos Mutantes , Proteínas Mitocondriais/genética , Oxigenases de Função Mista , Estrutura Terciária de Proteína , Ubiquinona/genética
6.
J Am Chem Soc ; 135(39): 14473-5, 2013 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-24041082

RESUMO

In the biological fixation of halide ions, several enzymes have been found to catalyze alkyl transfer from S-adenosylmethionine to halide ions. It proves possible to measure the rates of reaction of the trimethylsulfonium ion with I(-), Br(-), Cl(-), F(-), HO(-), and H2O in water at elevated temperatures. Comparison of the resulting second-order rate constants, extrapolated to 25 °C, with the values of k(cat)/K(m) reported for fluorinase and chlorinase indicates that these enzymes enhance the rates of alkyl halide formation by factors of 2 × 10(15)- and 1 × 10(17)-fold, respectively. These rate enhancements, achieved without the assistance of cofactors, metal ions, or general acid-base catalysis, are the largest that have been reported for an enzyme that acts on two substrates.


Assuntos
Proteínas de Bactérias/metabolismo , Halogênios/metabolismo , Micromonosporaceae/enzimologia , Oxirredutases/metabolismo , S-Adenosilmetionina/metabolismo , Streptomyces/enzimologia , Alquilação , Ânions/química , Ânions/metabolismo , Halogênios/química , Água/química , Água/metabolismo
7.
J Org Chem ; 77(6): 2907-10, 2012 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-22352783

RESUMO

The final step in the degradation of heparin sulfate involves the enzymatic hydrolysis of its 2-sulfamido groups. To evaluate the power of the corresponding sulfamidases as catalysts, we examined the reaction of N-neopentyl sulfamate at elevated temperatures and found it to undergo specific acid catalyzed hydrolysis even at alkaline pH. A rate constant of 10(-16) s(-1) was calculated using the Eyring equation for water attack on the N-protonated species at pH 7, 25 °C. As a model for the pH neutral reaction, a rate constant for hydroxide attack on (CH(3))(3)CCH(2)N(+)H(2)SO(3)(-) at pH 7, 25 °C was calculated to be 10(-19) s(-1). The corresponding rate enhancement (k(cat)/k(non)) produced by the N-sulfamidase of F. heparinum is approximately 10(16)-fold, which is somewhat larger than those generated by most hydrolytic enzymes but considerably smaller than those generated by S-O cleaving sulfatases.


Assuntos
Hidrolases/química , Ácidos Sulfônicos/química , Água/química , Catálise , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Estrutura Molecular
8.
J Am Chem Soc ; 134(1): 525-31, 2012 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-22087808

RESUMO

As benchmarks for judging the catalytic power of sulfate monoesterases, we sought to determine the rates of spontaneous hydrolysis of unactivated alkyl sulfate monoesters by S-O bond cleavage. Neopentyl sulfate proved to be unsuitable for this purpose, since it was found to undergo hydrolysis by a C-O bond cleaving mechanism with rearrangement of its carbon skeleton. Instead, we examined the temperature dependence of the spontaneous hydrolyses of aryl sulfate monoesters, which proceed by S-O cleavage. Extrapolation of a Bronsted plot [log(k(25)(N)) = (-1.81 ± 0.09) pK(a)(LG) + (3.6 ± 0.7)] based on the rate constants at 25 °C for hydrolysis of a series of sulfate monoesters to a pK(a)(LG) value of 16.1, typical of an aliphatic alcohol, yields k(25)(N) = 3 × 10(-26) s(-1). Comparison of that value with established k(cat) values of bacterial sulfatases indicates that these enzymes produce rate enhancements (k(cat)/k(uncat)) of up to 2 × 10(26)-fold for the hydrolysis of sulfate monoesters. These rate enhancements surpass by several orders of magnitude the ~10(21)-fold rate enhancements that are generated by phosphohydrolases, the most powerful biological catalysts previously known. The hydrolytic rates of phosphate and sulfate monoesters are compared directly, and the misleading impression that the two classes of ester are of similar reactivity is dispelled.


Assuntos
Biocatálise , Oxigênio/química , Sulfatases/metabolismo , Enxofre/química , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Sulfatos/química , Sulfatos/metabolismo , Temperatura
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