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1.
Gene ; 705: 109-112, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31028865

RESUMO

Variants of KCNQ4 are one of the most common causes of dominantly inherited nonsyndromic hearing loss. We investigated a consanguineous family in which two individuals had prelignual hearing loss, apparently inherited in a recessive mode. Whole-exome sequencing analyses demonstrated genetic heterogeneity as variants in two different genes segregated with the phenotype in two branches of the family. Members in one branch were homozygous for a pathogenic variant of TMC1. The other two affected individuals were homozygous for a missense pathogenic variant in KCNQ4 c.872C>T; p.(Pro291Leu). These two individuals had prelingual, progressive moderate to severe hearing loss, while a heterozygous carrier had late onset mild hearing loss. Our work demonstrates that p.Pro291L variant is semi-dominantly inherited. This is the first report of semi-dominance of a KCNQ4 variant.


Assuntos
Surdez/genética , Canais de Potássio KCNQ/genética , Mutação de Sentido Incorreto , Sequenciamento Completo do Exoma/métodos , Idade de Início , Consanguinidade , Feminino , Heterogeneidade Genética , Predisposição Genética para Doença , Humanos , Leucina/genética , Masculino , Linhagem , Prolina/genética
2.
Viruses ; 11(2)2019 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-30791508

RESUMO

The Andes Orthohantavirus (ANDV), which causes the hantavirus cardiopulmonary syndrome, enters cells via integrins, and a change from leucine to proline at residue 33 in the PSI domain (L33P), impairs ANDV recognition. We assessed the association between this human polymorphism and ANDV infection. We defined susceptible and protective genotypes as "TT" (coding leucine) and "CC" (coding proline), respectively. TT was present at a rate of 89.2% (66/74) among the first cohort of ANDV cases and at 60% (63/105) among exposed close-household contacts, who remained uninfected (p < 0.05). The protective genotype (CC) was absent in all 85 ANDV cases, in both cohorts, and was present at 11.4% of the exposed close-household contacts who remained uninfected. Logistic regression modeling for risk of infection had an OR of 6.2⁻12.6 (p < 0.05) in the presence of TT and well-known ANDV risk activities. Moreover, an OR of 7.3 was obtained when the TT condition was analyzed for two groups exposed to the same environmental risk. Host genetic background was found to have an important role in ANDV infection susceptibility, in the studied population.


Assuntos
Predisposição Genética para Doença , Infecções por Hantavirus/genética , Hantavirus , Integrina alfaVbeta3/genética , Polimorfismo de Nucleotídeo Único , Adulto , Características da Família , Feminino , Genótipo , Humanos , Leucina/genética , Masculino , Prolina/genética , Estudos Prospectivos , Medição de Risco , Fatores de Risco
3.
Pestic Biochem Physiol ; 154: 1-6, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30765051

RESUMO

Ten putative resistant and two susceptible Rapistrum rugosum populations originating from Greece were studied for resistance to acetolactate synthase (ALS)-inhibiting herbicides, using dose-response assays, sequencing of als gene and in vitro ALS activity assays. The dose-response assays showed that one (P1) out of ten putative resistant populations was cross-resistant to tribenuron and imazamox, while another population (P4) was resistant to tribenuron only. All populations were susceptible to MCPA at the recommended rate. Gene sequencing of als revealed that the P4 population had a point mutation at Pro197 by His providing resistance to tribenuron, whereas the P1 had a Trp574 by Leu point mutation conferring cross-resistance to tribenuron and imazamox. The in vitro activity of the ALS enzyme indicated I50 values (tribenuron concentration required for 50% reduction of the ALS activity) ranging from 66.68 to 137.01 µM, whereas the respective value for the S populations ranged from 0.29 to 0.54 µM. These results strongly support that two R. rugosum populations evolved resistance to ALS-inhibiting herbicides due different point mutations in the als gene.


Assuntos
Acetolactato Sintase/genética , Sulfonatos de Arila/toxicidade , Brassicaceae/efeitos dos fármacos , Resistência a Herbicidas/genética , Herbicidas/toxicidade , Imidazóis/toxicidade , Substituição de Aminoácidos , Brassicaceae/enzimologia , Mutação , Proteínas de Plantas/genética , Prolina/genética , Triptofano/genética
4.
Biochemistry ; 58(6): 621-632, 2019 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-30574775

RESUMO

SUMO, a conserved ubiquitin-like protein, is conjugated to a multitude of cellular proteins to maintain genomic integrity and resist genotoxic stress. Studies of the SUMO E2 conjugating enzyme mutant, UBC9P123L, suggested that altered substrate specificity enhances cell sensitivity to DNA damaging agents. Using nuclear magnetic resonance chemical shift studies, we confirm that the mutation does not alter the core globular fold of UBC9, while 15N relaxation measurements demonstrate mutant-induced stabilization of distinct chemical states in residues near the active site cysteine and substrate recognition motifs. We further demonstrate that the P123L substitution induces a switch from the preferential addition of SUMO to lysine residues in unstructured sites to acceptor lysines embedded in secondary structures, thereby also inducing alterations in SUMO chain linkages. Our results provide new insights regarding the impact that structural dynamics of UBC9 have on substrate selection and specifically SUMO chain formation. These findings highlight the potential contribution of nonconsensus SUMO targets and/or alternative SUMO chain linkages on DNA damage response and chemotherapeutic sensitivity.


Assuntos
Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Domínio Catalítico , Cisteína/química , Humanos , Leucina/química , Leucina/genética , Mutação , Prolina/química , Prolina/genética , Saccharomyces cerevisiae/química , Alinhamento de Sequência , Especificidade por Substrato , Sumoilação , Enzimas de Conjugação de Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/genética
5.
Int J Biol Macromol ; 112: 169-174, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29414727

RESUMO

Peroxynitrite (ONOO-) is a reactive oxidant involved in numerous pathological conditions. Thymoquinone (TQ) is an active constituent of Nigella sativa and is reported to have anti-disease activities, but its role on ONOO- has never been investigated. This study was undertaken to investigate the role of TQ on ONOO--induced damage of histone-H2A. Our novel data showed TQ significantly inhibited ONOO--induced oxidative damage in histone-H2A. ONOO- induces UV-hypochromicity of histone-H2A, whereas TQ reversed this effect to hyperchromicity. Tyrosine fluorescence was significantly reduced by ONOO- and was significantly increased upon TQ treatment. TQ reduces ONOO--induced hydrophobicity in histone-H2A and also reduces thermal stability of ONOO--histone H2A complex. SDS-PAGE of native histone-H2A showed a single band, which disappeared when treated with ONOO- alone. This changed was retained when protein samples were treated with TQ. Similar protective effects of TQ were found when protein carbonyl contents were estimated. In conclusion, this is the first study that shows the potential of TQ against ONOO--induced damaged of histone-H2A. TQ inhibits oxidative modification of tyrosine, lysine, arginine, proline and threonine in histone-H2A. These results have importance for the development of novel therapeutic strategies for the treatment of disorders, where ONOO- plays a role.


Assuntos
Antioxidantes/química , Benzoquinonas/química , Histonas/química , Antioxidantes/farmacologia , Arginina/química , Arginina/genética , Benzoquinonas/farmacologia , Histonas/antagonistas & inibidores , Humanos , Lisina/química , Lisina/genética , Nigella sativa/química , Estresse Oxidativo/efeitos dos fármacos , Ácido Peroxinitroso/toxicidade , Prolina/química , Prolina/genética , Treonina/química , Treonina/genética , Tirosina/química , Tirosina/genética
6.
Gene ; 654: 64-68, 2018 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-29458167

RESUMO

Recurrent Aphthous Stomatitis (RAS) is a common oral inflammatory disease with unknown pathogenesis. Although the immune system alterations could be involved in predisposition of individuals to oral candidiasis, precise etiologies of RAS have not been understood yet. A recent study showed that autosomal dominant IL17F deficiency could cause chronic mucocutaneous candidiasis. Considering the inflammatory nature of interleukin (IL)-17F and RAS, this study was performed to check any disease-associated mutation in a number of patients with RAS. Sixty-two Iranian individuals with RAS were investigated in this study. After DNA extraction using a phenol-chloroform method from the whole blood, amplification was accomplished by polymerase chain reaction and the products were sequenced using a 3730 ABI sequencer. The results of sequencing revealed a missense, heterozygous mutation of IL17F, converting a threonine to proline in a patient with RAS (T79P). The Poly-phen software suggested a damaging probability predicting this substitution to have a harmful effect on IL-17F protein function. This mutation was checked in fifty healthy individuals, and was not detected in any of them. This is the first study showing that a mutation in IL-17F is associated with susceptibility to RAS. However, functional studies and further studies on more patients with RAS are required to confirm such association.


Assuntos
Genes Dominantes , Interleucina-17/genética , Mutação de Sentido Incorreto , Estomatite Aftosa/genética , Algoritmos , Estudos de Casos e Controles , Feminino , Frequência do Gene , Predisposição Genética para Doença , Genótipo , Heterozigoto , Humanos , Inflamação , Irã (Geográfico) , Masculino , Reação em Cadeia da Polimerase , Probabilidade , Prolina/genética , Análise de Sequência de DNA , Software , Treonina/genética
7.
J Inorg Biochem ; 180: 230-234, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29317104

RESUMO

Ascorbate peroxidase (APX) is a class I heme peroxidase. It has two sites for binding of substrates. One is close to the γ-heme edge and is used for oxidation of ascorbate; the other is at the δ-heme edge and is used for binding of aromatic substrates [Gumiero et al., (2010) Arch. Biochem. Biophys. 500, 13-20]. In this work, we have examined the structural factors that control binding at the δ-heme edge by replacement of Ala134 in APX with a proline residue that is more commonly found in other class II and III peroxidases. Kinetic data indicate that replacement of Ala134 by proline has only a small effect on the catalytic mechanism, or the oxidation of ascorbate or guaiacol. Chemical modification with phenylhydrazine indicates that heme accessibility close to the δ-heme edge is only minorly affected by the substitution. We conclude that the A134P mutation alone is not enough to substantially affect the reactivity of APX towards aromatic substrates bound at the δ-heme edge. The data are relevant to the recent application of APX (APEX) in cellular imaging.


Assuntos
Alanina/metabolismo , Ascorbato Peroxidases/metabolismo , Alanina/genética , Ácido Ascórbico/metabolismo , Catálise , Cromatografia Líquida de Alta Pressão , Guaiacol/metabolismo , Heme/metabolismo , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Oxirredução , Prolina/genética , Especificidade por Substrato
8.
Protein J ; 37(1): 13-20, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29119487

RESUMO

Three-dimensional (3D) domain swapping is a mechanism to form protein oligomers. It has been proposed that several factors, including proline residues in the hinge region, may affect the occurrence of 3D domain swapping. Although introducing prolines into the hinge region has been found to promote domain swapping for some proteins, the opposite effect has also been observed in several studies. So far, how proline affects 3D domain swapping remains elusive. In this work, based on a large set of 3D domain-swapped structures, we performed a systematic analysis to explore the correlation between the presence of proline in the hinge region and the occurrence of 3D domain swapping. We further analyzed the conformations of proline and pre-proline residues to investigate the roles of proline in 3D domain swapping. We found that more than 40% of the domain-swapped structures contained proline residues in the hinge region. Unexpectedly, conformational transitions of proline residues were rarely observed upon domain swapping. Our analyses showed that hinge regions containing proline residues preferred more extended conformations, which may be beneficial for the occurrence of domain swapping by facilitating opening of the exchanged segments.


Assuntos
Simulação de Dinâmica Molecular , Prolina/química , Proteínas/química , Prolina/genética , Domínios Proteicos , Proteínas/genética
9.
RNA Biol ; 15(4-5): 576-585, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-28737471

RESUMO

Accuracy in protein biosynthesis is maintained through multiple pathways, with a critical checkpoint occurring at the tRNA aminoacylation step catalyzed by aminoacyl-tRNA synthetases (ARSs). In addition to the editing functions inherent to some synthetases, single-domain trans-editing factors, which are structurally homologous to ARS editing domains, have evolved as alternative mechanisms to correct mistakes in aminoacyl-tRNA synthesis. To date, ARS-like trans-editing domains have been shown to act on specific tRNAs that are mischarged with genetically encoded amino acids. However, structurally related non-protein amino acids are ubiquitous in cells and threaten the proteome. Here, we show that a previously uncharacterized homolog of the bacterial prolyl-tRNA synthetase (ProRS) editing domain edits a known ProRS aminoacylation error, Ala-tRNAPro, but displays even more robust editing of tRNAs misaminoacylated with the non-protein amino acid α-aminobutyrate (2-aminobutyrate, Abu) in vitro and in vivo. Our results indicate that editing by trans-editing domains such as ProXp-x studied here may offer advantages to cells, especially under environmental conditions where concentrations of non-protein amino acids may challenge the substrate specificity of ARSs.


Assuntos
Aminoacil-tRNA Sintetases/genética , Aminobutiratos/metabolismo , Prolina/metabolismo , Processamento Pós-Transcricional do RNA , RNA de Transferência de Prolina/genética , Aminoacilação de RNA de Transferência , Alanina/genética , Alanina/metabolismo , Aminoacil-tRNA Sintetases/química , Aminoacil-tRNA Sintetases/metabolismo , Aminobutiratos/química , Anticódon/química , Anticódon/metabolismo , Sítios de Ligação , Códon/química , Códon/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Cinética , Simulação de Acoplamento Molecular , Mutação , Conformação de Ácido Nucleico , Prolina/genética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , RNA de Transferência de Prolina/química , RNA de Transferência de Prolina/metabolismo , Rodopseudomonas/genética , Rodopseudomonas/metabolismo , Especificidade por Substrato
10.
RNA Biol ; 15(4-5): 567-575, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-28933646

RESUMO

High-fidelity translation and a strictly accurate proteome were originally assumed as essential to life and cellular viability. Yet recent studies in bacteria and eukaryotic model organisms suggest that proteome-wide mistranslation can provide selective advantages and is tolerated in the cell at higher levels than previously thought (one error in 6.9 × 10-4 in yeast) with a limited impact on phenotype. Previously, we selected a tRNAPro containing a single mutation that induces mistranslation with alanine at proline codons in yeast. Yeast tolerate the mistranslation by inducing a heat-shock response and through the action of the proteasome. Here we found a homologous human tRNAPro (G3:U70) mutant that is not aminoacylated with proline, but is an efficient alanine acceptor. In live human cells, we visualized mistranslation using a green fluorescent protein reporter that fluoresces in response to mistranslation at proline codons. In agreement with measurements in yeast, quantitation based on the GFP reporter suggested a mistranslation rate of up to 2-5% in HEK 293 cells. Our findings suggest a stress-dependent phenomenon where mistranslation levels increased during nutrient starvation. Human cells did not mount a detectable heat-shock response and tolerated this level of mistranslation without apparent impact on cell viability. Because humans encode ∼600 tRNA genes and the natural population has greater tRNA sequence diversity than previously appreciated, our data also demonstrate a cell-based screen with the potential to elucidate mutations in tRNAs that may contribute to or alleviate disease.


Assuntos
Alanina/metabolismo , Aminoacil-tRNA Sintetases/genética , Mutação , Prolina/metabolismo , Biossíntese de Proteínas , Processamento Pós-Transcricional do RNA , RNA de Transferência de Prolina/genética , Alanina/genética , Aminoacil-tRNA Sintetases/metabolismo , Aminoacilação , Anticódon/química , Anticódon/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Códon/química , Códon/metabolismo , Meios de Cultura/farmacologia , Escherichia coli/genética , Escherichia coli/metabolismo , Genes Reporter , Glucose/deficiência , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Plasmídeos/química , Plasmídeos/metabolismo , Prolina/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , RNA de Transferência de Prolina/metabolismo , Transfecção
11.
Oncogene ; 37(4): 489-501, 2018 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-28967904

RESUMO

Both humans and mice lacking functional growth hormone (GH) receptors are known to be resistant to cancer. Further, autocrine GH has been reported to act as a cancer promoter. Here we present the first example of a variant of the GH receptor (GHR) associated with cancer promotion, in this case lung cancer. We show that the GHRP495T variant located in the receptor intracellular domain is able to prolong the GH signal in vitro using stably expressing mouse pro-B-cell and human lung cell lines. This is relevant because GH secretion is pulsatile, and extending the signal duration makes it resemble autocrine GH action. Signal duration for the activated GHR is primarily controlled by suppressor of cytokine signalling 2 (SOCS2), the substrate recognition component of the E3 protein ligase responsible for ubiquitinylation and degradation of the GHR. SOCS2 is induced by a GH pulse and we show that SOCS2 binding to the GHR is impaired by a threonine substitution at Pro 495. This results in decreased internalisation and degradation of the receptor evident in TIRF microscopy and by measurement of mature (surface) receptor expression. Mutational analysis showed that the residue at position 495 impairs SOCS2 binding only when a threonine is present, consistent with interference with the adjacent Thr494. The latter is key for SOCS2 binding, together with nearby Tyr487, which must be phosphorylated for SOCS2 binding. We also undertook nuclear magnetic resonance spectroscopy approach for structural comparison of the SOCS2 binding scaffold Ile455-Ser588, and concluded that this single substitution has altered the structure of the SOCS2 binding site. Importantly, we find that lung BEAS-2B cells expressing GHRP495T display increased expression of transcripts associated with tumour proliferation, epithelial-mesenchymal transition and metastases (TWIST1, SNAI2, EGFR, MYC and CCND1) at 2 h after a GH pulse. This is consistent with prolonged GH signalling acting to promote cancer progression in lung cancer.


Assuntos
Proteínas de Transporte/genética , Regulação Neoplásica da Expressão Gênica/genética , Neoplasias Pulmonares/genética , Transdução de Sinais/genética , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Animais , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Estudos de Coortes , Biologia Computacional , Análise Mutacional de DNA , Progressão da Doença , Transição Epitelial-Mesenquimal/genética , Feminino , Células HEK293 , Humanos , Pulmão/patologia , Neoplasias Pulmonares/patologia , Espectroscopia de Ressonância Magnética , Masculino , Camundongos , Fosforilação , Polimorfismo de Nucleotídeo Único , Prolina/genética , Ligação Proteica/genética , Domínios Proteicos/genética , Proteólise , Treonina/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
12.
Biotechnol J ; 13(3): e1700479, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29024569

RESUMO

Human butyrylcholinesterase (BChE), predominantly tetramers with a residence time of days, offers the potential to scavenge organophosphorus pesticides and chemical warfare agents. Efficient assembly of human BChE into tetramers requires an association with proline-rich peptide chaperones. In this study, the incorporation of different proline-rich peptide chaperones into BChE is investigated computationally and experimentally. First, the authors applied molecular dynamic (MD) simulations to interpret the interactions between proline-rich chaperones with human BChE tetramer domains. The P24 chaperone which contains 24 prolines, promoted the association of BChE tetramer with a 74% simulated helicity of BChE subunits, whereas the control without chaperone and BChE with an 8-proline chaperone (P8) complex exhibited 55.8 and 60.6% predicted helicity, respectively. The interaction of proline-rich chaperones with BChE subunits (B-P) provides a conduit to facilitate the interactions between BChE subunits (B-B) of the complex, which is mainly attributed to hydrophobic interactions and hydrogen-bond binding. Experimental assessment of these two proline-rich chaperones plus a 14-proline chaperone (P14) was performed and confirmed that P24 has superior capability to facilitate recombinant BChE (rBChE) tetramerization with >60% rBChE tetramer in P24-transfected rBChE cells, whereas P14- and P8-transfected rBChE cells had 44 and 33% rBChE tetramer, respectively. The rBChE control had 14% tetramer. Finally, we developed a stable rBChE tetramer expression system in CHO cells by enriching P24 expression in rBChE expressing cells. Overall, our simulations provided a design concept for identifying proline-rich peptides that promote the rBChE tetramerization in CHO cells.


Assuntos
Butirilcolinesterase/química , Células CHO , Chaperonas Moleculares/química , Proteínas Recombinantes/química , Animais , Butirilcolinesterase/genética , Cricetulus , Chaperonas Moleculares/genética , Prolina/química , Prolina/genética , Multimerização Proteica , Proteínas Recombinantes/genética
13.
Int J Biol Macromol ; 107(Pt B): 1641-1649, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29024683

RESUMO

Dextransucrase (DSR, EC2.4.5.1) from Leuconostoc mesenteroides 0326 which catalyzes sucrose to produce dextran, are popularly used in food and medicine industries. However, its poor kinetic stability especially at higher temperatures is a limiting factor for developing industrial applications. The aim of this study is to improve the enzyme activity and thermal stability of dextransucrase by single, double and triple mutations of proline and lysine. In this work, Pro-473, Pro-678, and Pro-856 were selected as engineering targets and individually replaced with serine. Lys-378, Lys-725, and Lys-955 were replaced with threonine, respectively. Mutant P473S/P856S (MW: 170kDa) was selected as the highest enzymatic activity mutant from fourteen mutants. Specifically, the mutant P473S/P856S showed a significant increase in thermal inactivation with a 7.4-fold increase in half-life at 35°C and a 2-fold increase in catalytic efficiency compared with the wild-type. The results of structural simulation are demonstrated that the new intramolecular hydrogen bonds in mutated enzymes increased structural stability, thereby increasing the thermal stability. The thermal stable mutants have an enormous application potential in food and pharmaceutical industry.


Assuntos
Glucosiltransferases/genética , Leuconostoc mesenteroides/enzimologia , Lisina/genética , Mutagênese Sítio-Dirigida , Prolina/genética , Temperatura Ambiente , Dextranos/química , Estabilidade Enzimática , Escherichia coli/metabolismo , Glucosiltransferases/química , Ligações de Hidrogênio , Concentração de Íons de Hidrogênio , Cinética , Modelos Moleculares , Proteínas Mutantes/química , Mutação/genética , Espectroscopia de Prótons por Ressonância Magnética
14.
Georgian Med News ; (285): 86-92, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30702076

RESUMO

The aim of this study was to determine the role of Pro12Ala polymorphism of the PPAR-γ gene, associated with lipid and carbohydrate metabolism, in the genesis of gestational weight gain (GWG). We performed anthropometry, the determination of GWG, the study of PPAR-γ Pro12Ala polymorphism, the concentration of markers of lipid and carbohydrate metabolism in 97 pregnant women with normal body weight before pregnancy (55.2±4.8 kg, body mass index 20.3±1.5 kg/m2). The recommended GWG was diagnosed in 33 (34.0±4.8%), insufficient in 19 (19.6±4.0%), and excessive in 45 (46.4±5.1%) patients, according to the recommendations of the Institute of Medicine (USA, 2009) and the Order of the Ministry of Health of Ukraine № 417 (2011). The PPAR-γ Pro12Ala polymorphism was determined by a molecular genetic study. "Statistica 6.0" was used. The frequency of Ala-allele carriers is 0.28. We studied that GWG is 1.6 times higher in women with PPAR-γ Pro12Ala polymorphism, than the Pro/Pro genotype (p<0.05). The frequency of Ala-alleles carriers in a group of excessive GWG 2.6 times (OR 3.2; 95% Cl 1.1-9.3; p<0.05) higher compared to normal weight gain patients. In pregnant women with Ala12 polymorphism concentration of triglycerides (p<0.05), cholesterol (p<0.05) are increased and no differences in glucose level (p>0.05), insulin level (p>0.05), and HOMO-IR index (p>0.05) compared to women with Pro/Pro genotype. The insulin concentration in PPAR-γ Ala12 carriers at the end of pregnancy was 1.4 times higher than the early terms (p<0.01) on the background of normoglycemia and the normal HOMO-IR index. The modified transcriptional activity of PPAR-γ gene, associated with lipid and carbohydrate metabolism, has a direct effect on the weight gain during pregnancy. Excessive GWG can serve as a marker for the maternal genotype and genetic predisposition to the development of metabolic diseases after delivery.


Assuntos
Ganho de Peso na Gestação/genética , PPAR gama/genética , Polimorfismo de Nucleotídeo Único , Alanina/genética , Glicemia/análise , Colesterol/sangue , Feminino , Genótipo , Humanos , Insulina/sangue , Gravidez , Prolina/genética , Triglicerídeos/sangue
15.
Brain ; 140(10): 2706-2721, 2017 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-28969384

RESUMO

Mutations in glucocerebrosidase 1 (GBA1) represent the most prevalent risk factor for Parkinson's disease. The molecular mechanisms underlying the link between GBA1 mutations and Parkinson's disease are incompletely understood. We analysed two aged (24-month-old) Gba1 mouse models, one carrying a knock-out mutation and the other a L444P knock-in mutation. A significant reduction of glucocerebrosidase activity was associated with increased total alpha-synuclein accumulation in both these models. Gba1 mutations alone did not alter the number of nigral dopaminergic neurons nor striatal dopamine levels. We then investigated the effect of overexpression of human alpha-synuclein in the substantia nigra of aged (18 to 21-month-old) L444P Gba1 mice. Following intraparenchymal injections of human alpha-synuclein carrying viral vectors, pathological accumulation of phosphorylated alpha-synuclein occurred within the transduced neurons. Stereological counts of nigral dopaminergic neurons revealed a significantly greater cell loss in Gba1-mutant than wild-type mice. These results indicate that Gba1 deficiency enhances neuronal vulnerability to neurodegenerative processes triggered by increased alpha-synuclein expression.


Assuntos
Dopamina/metabolismo , Glucosilceramidase/genética , Mutação/genética , Neurônios/patologia , Substância Negra/patologia , alfa-Sinucleína/metabolismo , Fatores Etários , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Glucosilceramidase/deficiência , Humanos , Leucina/genética , Camundongos , Camundongos Transgênicos , Neurônios/metabolismo , Prolina/genética , Desempenho Psicomotor/fisiologia , Olfato/genética , Substância Negra/metabolismo , Transdução Genética , Tirosina 3-Mono-Oxigenase/metabolismo , beta-N-Acetil-Hexosaminidases/metabolismo
16.
J Neurosci ; 37(46): 11271-11284, 2017 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-29038237

RESUMO

Engagement of integrins by the extracellular matrix initiates signaling cascades that drive a variety of cellular functions, including neuronal migration and axonal pathfinding in the brain. Multiple lines of evidence link the ITGB3 gene encoding the integrin ß3 subunit with the serotonin (5-HT) system, likely via its modulation of the 5-HT transporter (SERT). The ITGB3 coding polymorphism Leu33Pro (rs5918, PlA2) produces hyperactive αvß3 receptors that influence whole-blood 5-HT levels and may influence the risk for autism spectrum disorder (ASD). Using a phenome-wide scan of psychiatric diagnoses, we found significant, male-specific associations between the Pro33 allele and attention-deficit hyperactivity disorder and ASDs. Here, we used knock-in (KI) mice expressing an Itgb3 variant that phenocopies the human Pro33 variant to elucidate the consequences of constitutively enhanced αvß3 signaling to the 5-HT system in the brain. KI mice displayed deficits in multiple behaviors, including anxiety, repetitive, and social behaviors. Anatomical studies revealed a significant decrease in 5-HT synapses in the midbrain, accompanied by decreases in SERT activity and reduced localization of SERTs to integrin adhesion complexes in synapses of KI mice. Inhibition of focal adhesion kinase (FAK) rescued SERT function in synapses of KI mice, demonstrating that constitutive active FAK signaling downstream of the Pro32Pro33 integrin αvß3 suppresses SERT activity. Our studies identify a complex regulation of 5-HT homeostasis and behaviors by integrin αvß3, revealing an important role for integrins in modulating risk for neuropsychiatric disorders.SIGNIFICANCE STATEMENT The integrin ß3 Leu33Pro coding polymorphism has been associated with autism spectrum disorders (ASDs) within a subgroup of patients with elevated blood 5-HT levels, linking integrin ß3, 5-HT, and ASD risk. We capitalized on these interactions to demonstrate that the Pro33 coding variation in the murine integrin ß3 recapitulates the sex-dependent neurochemical and behavioral attributes of ASD. Using state-of-the-art techniques, we show that presynaptic 5-HT function is altered in these mice, and that the localization of 5-HT transporters to specific compartments within the synapse, disrupted by the integrin ß3 Pro33 mutation, is critical for appropriate reuptake of 5-HT. Our studies provide fundamental insight into the genetic network regulating 5-HT neurotransmission in the CNS that is also associated with ASD risk.


Assuntos
Encéfalo/fisiologia , Mutação com Ganho de Função/genética , Variação Genética/genética , Integrina beta3/genética , Prolina/genética , Serotonina/genética , Animais , Feminino , Técnicas de Introdução de Genes/métodos , Humanos , Integrina beta3/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Prolina/metabolismo , Ligação Proteica/fisiologia , Serotonina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina/genética , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo
17.
Neurodegener Dis ; 17(6): 292-303, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29035885

RESUMO

BACKGROUND: Amyotrophic lateral sclerosis (ALS) shows a strong genetic basis, with SOD1, FUS, TARDBP, and C9ORF72 being the genes most frequently involved. This has allowed identification of asymptomatic mutation carriers, which may be of help in understanding the molecular changes preceding disease onset. OBJECTIVES: We studied the cellular expression of FUS protein and the effect of heat-shock- and dithiothreitol-induced stress in fibroblasts from FUS P525L mutation carriers, healthy controls, and patients with sporadic ALS. METHODS: Western blots and immunocytochemistry were performed to study the subcellular localization of FUS protein. Control and stressed cells were double stained with FUS and the stress marker TIA-R. RESULTS: Fibroblasts from healthy controls and sporadic ALS cases showed a prominent nuclear FUS expression. In the 2 FUS P525L mutation carriers, instead, most cells showed a protein localization in both nucleus and cytoplasm, or exclusively in the cytoplasm. Stress prompted the formation of cytoplasmic granules in all subjects and in sporadic ALS FUS mislocalization to the cytoplasm. Cytoplasmic FUS was recruited into stress granules, which showed a time-dependent decrease in all subjects. However, in the FUS P525L fibroblasts, the granules persisted longer, and they were more numerous than those detected in the cells from controls and sporadic ALS patients. CONCLUSIONS: We show that in fibroblasts of FUS P525L mutation carriers, FUS mislocalized to the cytoplasm where it redistributed into stress granules with likely a dose effect, i.e. a higher number of cells with granules, which persist longer, than in controls and ALS cases. These data represent an early molecular change occurring before ALS onset, suggesting a transient preaggregative state.


Assuntos
Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/patologia , Fibroblastos/metabolismo , Mutação/genética , Transporte Proteico/genética , Proteína FUS de Ligação a RNA/genética , Esclerose Amiotrófica Lateral/fisiopatologia , Núcleo Celular/metabolismo , Células Cultivadas , Grânulos Citoplasmáticos/metabolismo , Feminino , Seguimentos , Humanos , Leucina/genética , Masculino , Condução Nervosa/genética , Prolina/genética , Pele/citologia , Frações Subcelulares/metabolismo , Frações Subcelulares/patologia , Fatores de Tempo , Tubulina (Proteína)/metabolismo
18.
J Neurol ; 264(12): 2387-2393, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28993872

RESUMO

The mutation of vesicle-associated membrane protein-associated protein B (VAPB) was proved to cause family amyotrophic lateral sclerosis (FALS). Only two mutations of VAPB associated with ALS have been reported (p.Pro56Ser and p.Thr46Ile). Here we reported a Chinese Han FALS family caused by a novel VAPB point mutation. The clinical materials of one Chinese Han FALS family were collected. The genetic analysis was carried out by target sequencing and further verified by Sanger sequencing. One novel mutation of c.167C>A (p.Pro56His) on VAPB was found in the proband. The age at onset of the proband was 48 with the onset symptoms of weakness in the right arm, followed by progressive limb and trunk weakness with decreased deep-tendon reflexes, muscular cramps and fasciculation. But the disease duration was more than 15 years. He was under the tracheotomy for 1 year at last visit. Electromyography showed widespread acute and chronic neurogenic damages. His mother presented weakness in her limbs in 50 s and died 15 years later. One of his younger sisters diagnosed as ALS for 6 years also carried the same mutation. She presented the similar symptoms on 41. No dominant upper motor neuron sign was showed. The clinical features were similar to the patients carrying the known mutation of p.Pro56Ser. A novel mutation of VAPB was found in one Chinese Han FALS pedigree. The affected patients presented a much slower progression and the lesions were limited in lower motor neurons.


Assuntos
Esclerose Amiotrófica Lateral/genética , Mutação/genética , Proteínas de Transporte Vesicular/genética , Esclerose Amiotrófica Lateral/complicações , Grupo com Ancestrais do Continente Asiático , Proteína C9orf72/genética , Eletromiografia , Saúde da Família , Feminino , Testes Genéticos , Histidina/genética , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Prolina/genética
19.
J Alzheimers Dis ; 60(2): 651-661, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28922155

RESUMO

The Golgi apparatus (GA) is a highly dynamic organelle involved in the processing and sorting of cellular proteins. In Alzheimer's disease (AD), it has been shown to decrease in size and become fragmented in neocortical and hippocampal neuronal subpopulations. This fragmentation and decrease in size of the GA in AD has been related to the accumulation of hyperphosphorylated tau. However, the involvement of other pathological factors associated with the course of the disease, such as the extracellular accumulation of amyloid-ß (Aß) aggregates, cannot be ruled out, since both pathologies are present in AD patients. Here we use the P301S tauopathy mouse model to examine possible alterations of the GA in neurons that overexpress human tau (P301S mutated gene) in neocortical and hippocampal neurons, using double immunofluorescence techniques and confocal microscopy. Quantitative analysis revealed that neurofibrillary tangle (NFT)-bearing neurons had important morphological alterations and reductions in the surface area and volume of the GA compared with NFT-free neurons. Since in this mouse model there are no Aß aggregates typical of AD, the present findings support the idea that the progressive accumulation of phospho-tau is associated with structural alterations of the GA, and that these changes may occur in the absence of Aß pathology.


Assuntos
Córtex Cerebral/patologia , Células Piramidais/ultraestrutura , Tauopatias/patologia , Proteínas tau/genética , Proteínas tau/metabolismo , Animais , Proteínas de Transporte/metabolismo , Modelos Animais de Doenças , Complexo de Golgi , Humanos , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Emaranhados Neurofibrilares/patologia , Fosfopiruvato Hidratase/metabolismo , Fosforilação/genética , Prolina/genética , Células Piramidais/patologia , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Serina/genética , Sialoglicoproteínas/metabolismo , Tauopatias/genética
20.
Mol Med Rep ; 16(4): 4973-4979, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28791342

RESUMO

TNF ligand superfamily member 10 (TRAIL) is a member of the tumor necrosis factor superfamily. The present study was performed in an effort to increase the expression of soluble (s)TRAIL by rebuilding the gene sequence of TRAIL. Three principles based on the codon bias of Escherichia coli were put forward to design the rebuild strategy. Relying on these three principles, a P7R mutation near the N­terminal region of sTRAIL, named TRAIL­Mu, was designed. TRAIL­Mu was subsequently cloned into the PTWIN1 plasmid and expressed in E. coli BL21 (DE3). Using a high­level expression system and a three­step purification method, soluble TRAIL­Mu protein reached ~90% of total cellular protein and purity was >95%, demonstrating success in overcoming inclusion body formation. The cytotoxic effect of TRAIL­Mu was evaluated by sulforhodamine B assay in the MD­MB­231, A549, NCI­H460 and L02 cell lines. The results demonstrated that TRAIL­Mu exerted stronger antitumor effects on TRAIL­sensitive tumor cell lines, and was able to partially reverse the resistance of a TRAIL­resistant tumor cell line. In addition, TRAIL­Mu exhibited no notable biological effects in a normal liver cell line. The novel TRAIL variant generated in the present study may be useful for the mass production of this important protein for therapeutic purposes.


Assuntos
Substituição de Aminoácidos , Arginina/genética , Códon , Mutação , Prolina/genética , Ligante Indutor de Apoptose Relacionado a TNF/genética , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Humanos , Plasmídeos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/isolamento & purificação , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia
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