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1.
Eur J Endocrinol ; 181(5): K43-K53, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31539878

RESUMO

Background: IGF1 is a key factor in fetal and postnatal growth. To date, only three homozygous IGF1 gene defects leading to complete or partial loss of IGF1 activity have been reported in three short patients born small for gestational age. We describe the fourth patient with severe short stature presenting a novel homozygous IGF1 gene mutation. Results: We report a boy born from consanguineous parents at 40 weeks of gestational age with intrauterine growth restriction and severe postnatal growth failure. Physical examination revealed proportionate short stature, microcephaly, facial dysmorphism, bilateral sensorineural deafness and mild global developmental delay. Basal growth hormone (GH) fluctuated from 0.2 to 29 ng/mL, while IGF1 levels ranged from -1.15 to 2.95 SDS. IGFBP3 was normal-high. SNP array delimited chromosomal regions of homozygosity, including 12q23.2 where IGF1 is located. IGF1 screening by HRM revealed a homozygous missense variant NM_000618.4(IGF1):c.322T>C, p.(Tyr108His). The change of the highly conserved Tyr60 in the mature IGF1 peptide was consistently predicted as pathogenic by multiple bioinformatic tools. Tyr60 has been described to be critical for IGF1 interaction with type 1 IGF receptor (IGF1R). In vitro, HEK293T cells showed a marked reduction of IGF1R phosphorylation after stimulation with serum from the patient as compared to sera from age-matched controls. Mutant IGF1 was also less efficient in inducing cell growth. Conclusion: The present report broadens the spectrum of clinical and biochemical presentation of homozygous IGF1 defects and underscores the variability these patients may present depending on the IGF/IGF1R pathway activity.


Assuntos
Transtornos do Crescimento/genética , Perda Auditiva Neurossensorial/genética , Fator de Crescimento Insulin-Like I/deficiência , Mutação de Sentido Incorreto/genética , Anormalidades Múltiplas/genética , Proliferação de Células , Biologia Computacional , Simulação por Computador , Retardo do Crescimento Fetal/genética , Células HEK293 , Homozigoto , Humanos , Lactente , Fator de Crescimento Insulin-Like I/genética , Masculino , Linhagem , Polimorfismo de Nucleotídeo Único/genética , Receptores de Somatomedina/genética , Tirosina/genética
2.
Gene ; 713: 143951, 2019 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-31269464

RESUMO

Rifampicin (RIF) is still a first line of antibiotic in the treatment of bacterial diseases, in particular the Mycobacterial infections. The antimicrobial activity of RIF is attributed to its ability to inhibit transcription by binding to the ß subunit of bacterial RNA polymerase (encoded by rpoB). Continued use of this drug resulted in the emergence of RIF resistant rpoB mutations in a high frequency that compels the use of RIF almost exclusively in drug combinations. As of date, a broad array of rif mutations have been isolated and characterized by different research groups. Studies on rpoB mutations strengthen the view that the ß subunit of RNA polymerase (RNAP) is very crucial in modulating transcription thereby leading to differential gene expression. Very recently we have reported the transcriptome profile of rpoB12 mutant that provides molecular evidence that presence of rpoB12 mutation modulates the transcription of about 450 genes. Here we present a maiden report that rpoB mutations that substitute Tyr at the Rif binding pocket (RBP) of ß subunit of RNA polymerase are able to suppress the over-production of colanic acid capsular polysaccharide (Ces phenotype) in Δlon mutant of Escherichia coli. Further analyses of the rif mutants involving their growth pattern on LB at higher temperature (42 °C), LB media without NaCl, survival in LB media with acidic pH (pH - 3) and motility revealed that only rpoB12 (His526Tyr) and rpoB137 (Ser522Tyr) affected all the above mentioned physiological parameters in addition to the elicitation of Ces phenotype. These two rif mutations confer fast movement to RNAP and they bear Tyr as the substituted amino acid in the RBP. This is perhaps the first study that brings out the possible role of Tyr in the RBP and its participation in the global gene expression. This study also envisages the point that amino acid residues that share the properties of Tyr in the RBP can be employed as a tool to bring out differential gene expression which would certainly have basic and applied values for the mankind.


Assuntos
RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas de Escherichia coli/genética , Escherichia coli/metabolismo , Mutação , Rifampina/farmacologia , Tirosina/metabolismo , Antibióticos Antituberculose/farmacologia , Farmacorresistência Bacteriana , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Fenótipo , RNA Bacteriano , Tirosina/genética
3.
Nat Commun ; 10(1): 3067, 2019 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-31296851

RESUMO

WalKR (YycFG) is the only essential two-component regulator in the human pathogen Staphylococcus aureus. WalKR regulates peptidoglycan synthesis, but this function alone does not explain its essentiality. Here, to further understand WalKR function, we investigate a suppressor mutant that arose when WalKR activity was impaired; a histidine to tyrosine substitution (H271Y) in the cytoplasmic Per-Arnt-Sim (PASCYT) domain of the histidine kinase WalK. Introducing the WalKH271Y mutation into wild-type S. aureus activates the WalKR regulon. Structural analyses of the WalK PASCYT domain reveal a metal-binding site, in which a zinc ion (Zn2+) is tetrahedrally-coordinated by four amino acids including H271. The WalKH271Y mutation abrogates metal binding, increasing WalK kinase activity and WalR phosphorylation. Thus, Zn2+-binding negatively regulates WalKR. Promoter-reporter experiments using S. aureus confirm Zn2+ sensing by this system. Identification of a metal ligand recognized by the WalKR system broadens our understanding of this critical S. aureus regulon.


Assuntos
Proteínas de Bactérias/metabolismo , Histidina Quinase/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Staphylococcus aureus/metabolismo , Zinco/metabolismo , Substituição de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Cátions Bivalentes/metabolismo , Histidina/genética , Histidina Quinase/química , Histidina Quinase/genética , Simulação de Dinâmica Molecular , Mutação , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Regulon/genética , Staphylococcus aureus/genética , Tirosina/genética
4.
Nat Commun ; 10(1): 2574, 2019 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-31189925

RESUMO

Complex conformational dynamics are essential for function of the dimeric molecular chaperone heat shock protein 90 (Hsp90), including transient, ATP-biased N-domain dimerization that is necessary to attain ATPase competence. The intrinsic, but weak, ATP hydrolyzing activity of human Hsp90 is markedly enhanced by the co-chaperone Aha1. However, the cellular concentration of Aha1 is substoichiometric relative to Hsp90. Here we report that initial recruitment of this cochaperone to Hsp90 is markedly enhanced by phosphorylation of a highly conserved tyrosine (Y313 in Hsp90α) in the Hsp90 middle domain. Importantly, phosphomimetic mutation of Y313 promotes formation of a transient complex in which both N- and C-domains of Aha1 bind to distinct surfaces of the middle domains of opposing Hsp90 protomers prior to ATP-directed N-domain dimerization. Thus, Y313 represents a phosphorylation-sensitive conformational switch, engaged early after client loading, that affects both local and long-range conformational dynamics to facilitate initial recruitment of Aha1 to Hsp90.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Chaperonas Moleculares/metabolismo , Domínios Proteicos/genética , Adenosina Trifosfatases/genética , Ácido Glutâmico/genética , Células HEK293 , Proteínas de Choque Térmico HSP90/genética , Humanos , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Fosforilação/fisiologia , Relação Estrutura-Atividade , Tirosina/genética , Tirosina/metabolismo
5.
Arch Virol ; 164(7): 1851-1855, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31055651

RESUMO

The RNA genome of human parainfluenza virus type 2 (hPIV2) is encapsidated by nucleoprotein (NP) to act as a template for RNA synthesis. We examined the importance of individual amino acids in the RNA-binding domain of hPIV2 NP for polymerase activity using a mini-replicon assay. We showed that substitution of tyrosine at amino acid position 260, located in the RNA-binding pocket of NP, severely reduced polymerase activity. The aromatic side-chain of Y260 may be required for the formation of stable contacts between nucleotides and basic amino acids, thereby affecting promoter recognition by the viral polymerase.


Assuntos
Nucleoproteínas/genética , Vírus da Parainfluenza 2 Humana/genética , RNA Replicase/metabolismo , RNA Viral/metabolismo , Motivos de Ligação ao RNA/genética , Sequência de Aminoácidos , Cristalografia por Raios X , Genoma Viral/genética , Humanos , Tirosina/genética , Replicação Viral/genética
6.
Microb Cell Fact ; 18(1): 74, 2019 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-31023316

RESUMO

BACKGROUND: Production of L-tyrosine is gaining grounds as the market size of 3,4-dihydroxyphenyl-L-alanine (L-DOPA) is expected to increase due to increasing cases of Parkinson's disease a neurodegenerative disease. Attempts to overproduce L-tyrosine for conversion to L-DOPA has stemmed on the overexpressing of critical pathway enzymes, an introduction of feedback-resistant enzymes, and deregulation of transcriptional regulators. RESULTS: An E. coli BL21 (DE3) was engineered by deleting tyrR, ptsG, crr, pheA and pykF while directing carbon flow through the overexpressing of galP and glk. TktA and PpsA were also overexpressed to enhance the accumulation of E4P and PEP. Directed evolution was then applied on HpaB to optimize its activity. Three mutants, G883R, G883A, L1231M, were identified to have improved activity as compared to the wild-type hpaB showing a 3.03-, 2.9- and 2.56-fold increase in L-DOPA production respectively. The use of strain LP-8 resulted in the production of 691.24 mg/L and 25.53 g/L of L-DOPA in shake flask and 5 L bioreactor, respectively. CONCLUSION: Deletion of key enzymes to channel flux towards the shikimate pathway coupled with the overexpression of pathway enzymes enhanced the availability of L-tyrosine for L-DOPA production. Enhancing the activity of HpaB increased L-DOPA production from glucose and glycerol. This work demonstrates that increasing the availability of L-tyrosine and enhancing enzyme activity ensures maximum L-DOPA productivity.


Assuntos
Escherichia coli/metabolismo , Glucose/metabolismo , Levodopa/biossíntese , Engenharia Metabólica , Tirosina/metabolismo , Reatores Biológicos , Escherichia coli/genética , Glicerol/metabolismo , Oxigenases de Função Mista/metabolismo , Ácido Chiquímico/metabolismo , Tirosina/genética
7.
Curr Genet ; 65(4): 829-836, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30963244

RESUMO

Constitutive heterochromatin packages long stretches of repetitive DNA sequences at the centromere and telomere, and ensures genomic integrity at these loci by preventing aberrant recombination and transcription. The chromatin scaffold of heterochromatin is dynamically regulated in the cell cycle, and inheritance of the epigenetically silenced state is dependent on a transcriptional event imposed on the underlying non-coding RNA in conjunction with the DNA replicative phase. Heterochromatin becomes transiently loosened in response to a reduction in the binding of Swi6, a heterochromatin protein, and this allows RNA polymerase II access to the underlying sequence. The derived transcripts, in turn, drive heterochromatin formation via the recruitment of other silencing factors. It remains unclear how heterochromatin becomes decompacted in a cell cycle-specific manner. Here, we describe a mechanism of heterochromatin decompaction initiated by a novel histone modification, histone H3 tyrosine 41 phosphorylation (H3Y41p). We will discuss how H3Y41p cooperates with other regulatory pathways to enforce cell cycle-dependent regulation of constitutive heterochromatin.


Assuntos
Centrômero/genética , Heterocromatina/genética , Histonas/genética , Tirosina/genética , Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Epigênese Genética/genética , Fosforilação/genética , RNA Polimerase II/genética , RNA não Traduzido/genética , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética
8.
Biochemistry ; 58(17): 2218-2227, 2019 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-30946568

RESUMO

Cysteine dioxygenase (CDO) is a nonheme iron enzyme that adds two oxygen atoms from dioxygen to the sulfur atom of l-cysteine. Adjacent to the iron site of mammalian CDO, there is a post-translationally generated Cys-Tyr cofactor, whose presence substantially enhances the oxygenase activity. The formation of the Cys-Tyr cofactor in CDO is an autocatalytic process, and it is challenging to study by traditional techniques because the cross-linking reaction is a side, uncoupled, single-turnover oxidation buried among multiple turnovers of l-cysteine oxygenation. Here, we take advantage of our recent success in obtaining a purely uncross-linked human CDO due to site-specific incorporation of 3,5-difluoro-l-tyrosine (F2-Tyr) at the cross-linking site through the genetic code expansion strategy. Using EPR spectroscopy, we show that nitric oxide (•NO), an oxygen surrogate, similarly binds to uncross-linked F2-Tyr157 CDO as in wild-type human CDO. We determined X-ray crystal structures of uncross-linked F2-Tyr157 CDO and mature wild-type CDO in complex with both l-cysteine and •NO. These structural data reveal that the active site cysteine (Cys93 in the human enzyme), rather than the generally expected tyrosine (i.e., Tyr157), is well-aligned to be oxidized should the normal oxidation reaction uncouple. This structure-based understanding is further supported by a computational study with models built on the uncross-linked ternary complex structure. Together, these results strongly suggest that the first target to oxidize during the iron-assisted Cys-Tyr cofactor biogenesis is Cys93. Based on these data, a plausible reaction mechanism implementing a cysteine radical involved in the cross-link formation is proposed.


Assuntos
Cisteína Dioxigenase/química , Dipeptídeos/química , Conformação Proteica , Tirosina/análogos & derivados , Domínio Catalítico , Reagentes para Ligações Cruzadas/química , Cristalografia por Raios X , Cisteína/química , Cisteína/genética , Cisteína/metabolismo , Cisteína Dioxigenase/genética , Cisteína Dioxigenase/metabolismo , Dipeptídeos/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Humanos , Modelos Moleculares , Óxido Nítrico/química , Óxido Nítrico/metabolismo , Oxirredução , Oxigênio/química , Oxigênio/metabolismo , Ligação Proteica , Tirosina/química , Tirosina/genética , Tirosina/metabolismo
9.
Chemistry ; 25(26): 6533-6541, 2019 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-30820987

RESUMO

Selective chemical modification of proteins plays a pivotal role for the rational design of enzymes with novel and specific functionalities. In this study, a strategic combination of genetic and chemical engineering paves the way for systematic construction of biocatalysts by tuning the product spectrum of a levansucrase from Bacillus megaterium (Bm-LS), which typically produces small levan-like oligosaccharides. The implementation of site-directed mutagenesis followed by a tyrosine-specific modification enabled control of the product synthesis: depending on the position, the modification provoked either enrichment of short oligosaccharides (up to 800 % in some cases) or triggered the formation of high molecular weight polymer. The chemical modification can recover polymerization ability in variants with defective oligosaccharide binding motifs. Molecular dynamic (MD) simulations provided insights into the effect of modifying non-native tyrosine residues on product specificity.


Assuntos
Bacillus megaterium/enzimologia , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Hexosiltransferases/química , Hexosiltransferases/genética , Oligossacarídeos/metabolismo , Tirosina/química , Bacillus megaterium/química , Bacillus megaterium/genética , Bacillus megaterium/metabolismo , Reação de Cicloadição , Frutanos/química , Frutanos/metabolismo , Glicosídeo Hidrolases/metabolismo , Hexosiltransferases/metabolismo , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Oligossacarídeos/química , Especificidade por Substrato , Tirosina/genética , Tirosina/metabolismo
10.
Curr Genet ; 65(4): 953-964, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30840111

RESUMO

Trichoderma spp. are widely used as commercial biofungicides, and most commercial formulations are conidia based. Identification of genes that regulate conidiation would thus be of help in genetic reprogramming of these species to optimize sporulation. In this study, we constructed an SSH (suppression subtractive hybridization) library from RNA samples of the wild type strain and a non-conidiating mutant, M7, grown under constant illumination for 2 days. We identified several genes that are underexpressed in the mutant. Some of these genes are related to secondary metabolism, and a few could be associated with conidiation. Genes coding for the following proteins, among others, were identified: O-methyl transferase, ATPase, alpha/beta-hydrolase, WD repeat containing protein, dehydrogenase, thioesterase, translationally controlled tumour protein, and a proline-glycine-tyrosine-rich protein (PGYRP) that has been annotated in T.reesei as a signalling protein. Two of these genes, encoding Pgy1, a novel PGYRP, and Ecm33, a GPI-anchored cell wall protein, were further studied in detail by generation of deletion mutants. We demonstrate here that both these genes not only regulate radial growth and conidiation in Trichoderma virens, but are also involved in antagonism against soil-borne wide host range plant pathogens. Furthermore, deletion of ecm33 affected hydrophobicity and cell wall integrity.


Assuntos
Parede Celular/genética , Proteínas Fúngicas/genética , Microbiologia do Solo , Trichoderma/genética , Regulação Fúngica da Expressão Gênica , Biblioteca Gênica , Glicina/genética , Prolina/genética , RNA Fúngico/genética , Esporos Fúngicos/genética , Esporos Fúngicos/crescimento & desenvolvimento , Tirosina/genética
11.
Biochim Biophys Acta Proteins Proteom ; 1867(6): 654-661, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30797104

RESUMO

The human fungal pathogen Candida albicans ambiguously decodes the universal leucine CUG codon predominantly as serine but also as leucine. C. albicans has a high capacity to survive and proliferate in adverse environments but the rate of leucine incorporation fluctuates in response to different stress conditions. C. albicans is adapted to tolerate this ambiguous translation through a mechanism that combines drastic decrease in CUG usage and reduction of CUG-encoded residues in conserved positions in the protein sequences. However, in a few proteins, the residues encoded by CUG codons are found in strictly conserved positions, suggesting that this genetic code alteration might have a functional impact. One such example is Cek1, a central signaling protein kinase that contains a single CUG-encoded residue at a conserved position, whose identity might regulate the correct flow of information across the MAPK cascade. Here we show that insertion of a leucine at the CUG-encoded position decreases the stability of Cek1, apparently without major structural alterations. In contrast, incorporation of a serine residue at the CUG position induces the autophosphorylation of the conserved tyrosine residue of the Cek1 231TEY233 motif, and increases its intrinsic kinase activity in vitro. These findings show that CUG ambiguity modulates the activity of Cek1, a key kinase directly linked to morphogenesis and virulence in C. albicans.


Assuntos
Candida albicans/enzimologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Candida albicans/genética , Candida albicans/crescimento & desenvolvimento , Candida albicans/patogenicidade , Parede Celular/fisiologia , Código Genético , Leucina/genética , Leucina/metabolismo , Fosforilação , Biossíntese de Proteínas , Serina/genética , Serina/metabolismo , Transdução de Sinais , Tirosina/genética , Tirosina/metabolismo , Virulência
12.
Nucleic Acids Res ; 47(7): 3711-3727, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-30715423

RESUMO

In eukaryotes, the wobble position of tRNA with a GUN anticodon is modified to the 7-deaza-guanosine derivative queuosine (Q34), but the original source of Q is bacterial, since Q is synthesized by eubacteria and salvaged by eukaryotes for incorporation into tRNA. Q34 modification stimulates Dnmt2/Pmt1-dependent C38 methylation (m5C38) in the tRNAAsp anticodon loop in Schizosaccharomyces pombe. Here, we show by ribosome profiling in S. pombe that Q modification enhances the translational speed of the C-ending codons for aspartate (GAC) and histidine (CAC) and reduces that of U-ending codons for asparagine (AAU) and tyrosine (UAU), thus equilibrating the genome-wide translation of synonymous Q codons. Furthermore, Q prevents translation errors by suppressing second-position misreading of the glycine codon GGC, but not of wobble misreading. The absence of Q causes reduced translation of mRNAs involved in mitochondrial functions, and accordingly, lack of Q modification causes a mitochondrial defect in S. pombe. We also show that Q-dependent stimulation of Dnmt2 is conserved in mice. Our findings reveal a direct mechanism for the regulation of translational speed and fidelity in eukaryotes by a nutrient originating from bacteria.


Assuntos
DNA (Citosina-5-)-Metiltransferases/genética , Micronutrientes/genética , Biossíntese de Proteínas/genética , Proteínas de Schizosaccharomyces pombe/genética , Animais , Anticódon/genética , Asparagina/genética , DNA Mitocondrial/genética , Eucariotos/genética , Guanina/análogos & derivados , Guanina/metabolismo , Metilação , Camundongos , RNA de Transferência/genética , Ribossomos/genética , Schizosaccharomyces/genética , Tirosina/genética
13.
Chem Commun (Camb) ; 55(9): 1287-1290, 2019 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-30633261

RESUMO

Inteins change the structure and function of their host protein in a unique way and the Gp41-1 split intein is the fastest protein trans-splicing intein known to date. To design a photo-activatable variant, we have incorporated ortho-nitrobenzyl-tyrosine (ONBY) at the position of a structurally conserved phenylalanine in the Gp41-1-N fragment. Using irradiation at 365 nm, the splicing reaction was triggered with virtually unchanged rates. The partial cellular reduction of the nitro group in ONBY, previously observed during bacterial protein expression for several photo-caged amino acids, was overcome by periplasmatic expression and by using an E. coli K12(DE3) strain instead of BL21(DE3). Together, our findings provide new tools for the artificial photo-control of proteins.


Assuntos
Escherichia coli K12/metabolismo , Inteínas/genética , Nitrobenzenos , Engenharia de Proteínas/métodos , Tirosina/análogos & derivados , Tirosina/genética , Inteínas/efeitos da radiação , Cinética , Mutação , Nitrobenzenos/efeitos da radiação , Trans-Splicing/genética , Tirosina/efeitos da radiação , Raios Ultravioleta
15.
Nat Cell Biol ; 21(2): 179-189, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30664791

RESUMO

Loss of TGF-ß tumour suppressive response is a hallmark of human cancers. As a central player in TGF-ß signal transduction, SMAD4 (also known as DPC4) is frequently mutated or deleted in gastrointestinal and pancreatic cancer. However, such genetic alterations are rare in most cancer types and the underlying mechanism for TGF-ß resistance is not understood. Here we describe a mechanism of TGF-ß resistance in ALK-positive tumours, including lymphoma, lung cancer and neuroblastoma. We demonstrate that, in ALK-positive tumours, ALK directly phosphorylates SMAD4 at Tyr 95. Phosphorylated SMAD4 is unable to bind to DNA and fails to elicit TGF-ß gene responses and tumour suppressing responses. Chemical or genetic interference of the oncogenic ALK restores TGF-ß responses in ALK-positive tumour cells. These findings reveal that SMAD4 is tyrosine-phosphorylated by an oncogenic tyrosine kinase during tumorigenesis. This suggests a mechanism by which SMAD4 is inactivated in cancers and provides guidance for targeted therapies in ALK-positive cancers.


Assuntos
Quinase do Linfoma Anaplásico/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias/genética , Proteína Smad4/genética , Fator de Crescimento Transformador beta/farmacologia , Quinase do Linfoma Anaplásico/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Perfilação da Expressão Gênica/métodos , Células HEK293 , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias/metabolismo , Neoplasias/patologia , Fosforilação , Proteína Smad4/metabolismo , Transplante Heterólogo , Tirosina/genética , Tirosina/metabolismo
16.
Biochim Biophys Acta Mol Basis Dis ; 1865(3): 661-677, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30611859

RESUMO

Mutations in cardiac myosin binding protein C (cMyBPC) are a major cause of hypertrophic cardiomyopathy (HCM). In particular, a single amino acid substitution of tyrosine to serine at residue 237 in humans (residue 235 in mice) has been linked to HCM with strong disease association. Although cMyBPC truncations, deletions and insertions, and frame shift mutations have been studied, relatively little is known about the functional consequences of missense mutations in cMyBPC. In this study, we characterized the functional and structural effects of the HCM-causing Y235S mutation by performing mechanical experiments and molecular dynamics simulations (MDS). cMyBPC null mouse myocardium was virally transfected with wild-type (WT) or Y235S cMyBPC (KOY235S). We found that Y235S cMyBPC was properly expressed and incorporated into the cardiac sarcomere, suggesting that the mechanism of disease of the Y235S mutation is not haploinsufficiency or poison peptides. Mechanical experiments in detergent-skinned myocardium isolated from KOY235S hearts revealed hypercontractile behavior compared to KOWT hearts, evidenced by accelerated cross-bridge kinetics and increased Ca2+ sensitivity of force generation. In addition, MDS revealed that the Y235S mutation causes alterations in important intramolecular interactions, surface conformations, and electrostatic potential of the C1 domain of cMyBPC. Our combined in vitro and in silico data suggest that the Y235S mutation directly disrupts internal and surface properties of the C1 domain of cMyBPC, which potentially alters its ligand-binding interactions. These molecular changes may underlie the mechanism for hypercontractile cross-bridge behavior, which ultimately results in the development of cardiac hypertrophy and in vivo cardiac dysfunction.


Assuntos
Cardiomiopatia Hipertrófica/genética , Proteínas de Transporte/química , Proteínas de Transporte/genética , Mutação de Sentido Incorreto , Contração Miocárdica/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Cardiomiopatia Hipertrófica/metabolismo , Proteínas de Transporte/fisiologia , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Knockout , Proteínas Mutantes/fisiologia , Mutação de Sentido Incorreto/fisiologia , Miocárdio/metabolismo , Domínios Proteicos/genética , Sarcômeros/genética , Sarcômeros/metabolismo , Serina/genética , Tirosina/genética
17.
J Anim Sci ; 97(2): 587-595, 2019 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-30535023

RESUMO

The keratin-associated proteins (KAPs) are important constituents of wool fibers. Of the many mammalian KAP genes (KRTAPs) identified, KRTAP20-1 has been described in humans, but it has not been described in any other species. A search of the sheep genome using the human KRTAP20-1 sequence revealed a homologous open reading frame on chromosome 1, which would encode a high glycine-tyrosine KAP. PCR-single-stranded conformational polymorphism (PCR-SSCP) analysis identified 8 different banding patterns representing 8 unique DNA sequences (named A to H). The sequences had highest similarity to the human KRTAP20-1 sequence, and this suggests that they are variants of ovine KRTAP20-1. Among these variants, a 12-bp insertion/deletion and 6 single nucleotide poly- morphisms (SNPs), including one 5' untranslated region (UTR) SNP, one 3' UTR SNP, and 2 nonsynonymous SNPs, were detected. Variant A was found to be associated with a decrease in mean fiber diameter, fiber diameter standard deviation, and prickle factor, whereas variant C was associated with increased greasy fleece weight and decreased wool yield. These associations persisted after adjusting for the effect of 2 nearby KRTAPs (KRTAP6-3 and KRTAP22-1) that have also been reported to associate with these wool traits. This suggests that variation in KRTAP20-1 affects wool yield and mean fiber diameter-associated traits, and that this effect is unlikely to be the result of the clustering of these KRTAPs on chromosome 1.


Assuntos
Genoma/genética , Queratinas/genética , Ovinos/genética , Animais , Sequência de Bases , Glicina/genética , Fenótipo , Filogenia , Reação em Cadeia da Polimerase/veterinária , Polimorfismo de Nucleotídeo Único , Polimorfismo Conformacional de Fita Simples , Alinhamento de Sequência/veterinária , Ovinos/fisiologia , Tirosina/genética ,
18.
Biomed Pharmacother ; 109: 701-707, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30551522

RESUMO

BACKGROUND: We have previously reported that 5-Aza-2-deoxycytidine (5-Aza-cdR) can repress protein serine/threonine phosphatase-1γ (PP1γ) expression and activity in the mouse hippocampus and affect the behaviour of mice in a water maze. It is well known that the phosphorylation of N-methyl-d-aspartate receptor 2B subunit (NR2B) plays a role in behaviour. In this study, we examined whether 5-Aza-cdR affects NR2B phosphorylation at tyrosine 1472 (p-Y1472 NR2B) and whether it affected the responses of the mice in a passive avoidance test. METHODS: 5-Aza-cdR (10 µM) was administered to mice via intracerebroventricular injection (i.c.v). The learning and memory behaviour of the mice were evaluated by measuring their response in a step-down type passive avoidance test 24 h after the injection. The mRNA level of NR2B was measured by real-time PCR. NR2B and p-Y1472 NR2B protein expression in the mouse hippocampus was detected by western blot and immunofluorescence. CDK5 activity was detected by the ADP-Glo™ + CDK5/p35 Kinase Enzyme System. To further clarify whether the 5-Aza-cdR effects on behaviour were dependent on cellular proliferation or not, the effect of 5-Aza-cdR on the expression level of NR2B, the phosphorylation level of p-Y1472 NR2B, cell viability and the cell cycle were analysed using the immortalized mouse hippocampal neuronal cells neural cell line HT22 treated with 10 µM 5-Aza-cdR compared with an untreated control group. RESULTS: After injection with 5-Aza-cdR, the behaviour of the mice in the step-down test was improved, while their phosphorylation level of p-Y1472 NR2B was increased and their CDK5 activity was decreased in the hippocampus. In vitro experiments showed 10 µM 5-Aza-cdR increased the p-Y1472 NR2B phosphorylation level with inhibition of cell viability and cell cycle arrest. CONCLUSIONS: Our results suggested that the effect of 5-Aza-cdR on behaviour may be related to the increase in phosphorylation of p-Y1472 NR2B in the hippocampus.


Assuntos
Aprendizagem da Esquiva/fisiologia , Decitabina/farmacologia , Hipocampo/metabolismo , Memória/fisiologia , Receptores de N-Metil-D-Aspartato/metabolismo , Tirosina/metabolismo , Animais , Aprendizagem da Esquiva/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Hipocampo/efeitos dos fármacos , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Aprendizagem em Labirinto/fisiologia , Memória/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos ICR , Tempo de Reação/efeitos dos fármacos , Tempo de Reação/fisiologia , Receptores de N-Metil-D-Aspartato/agonistas , Receptores de N-Metil-D-Aspartato/genética , Tirosina/genética
19.
Protein Sci ; 28(1): 100-110, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30056630

RESUMO

Peroxiredoxins efficiently remove hydroperoxides and peroxynitrite in pro- and eukaryotes. However, isoforms of one subfamily of peroxiredoxins, the so-called Prx6-type enzymes, usually have very low activities in standard peroxidase assays in vitro. In contrast to other peroxiredoxins, Prx6 homologues share a conserved histidyl residue at the bottom of the active site. Here we addressed the role of this histidyl residue for redox catalysis using the Plasmodium falciparum homologue PfPrx6 as a model enzyme. Steady-state kinetics with tert-butyl hydroperoxide (tBuOOH) revealed that the histidyl residue is nonessential for Prx6 catalysis and that a replacement with tyrosine can even increase the enzyme activity four- to six-fold in vitro. Stopped-flow kinetics with reduced PfPrx6WT , PfPrx6C128A , and PfPrx6H39Y revealed a preference for H2 O2 as an oxidant with second order rate constants for H2 O2 and tBuOOH around 2.5 × 107 M-1 s-1 and 3 × 106 M-1 s-1 , respectively. Differences between the oxidation kinetics of PfPrx6WT , PfPrx6C128A , and PfPrx6H39Y were observed during a slower second-reaction phase. Our kinetic data support the interpretation that the reductive half-reaction is the rate-limiting step for PfPrx6 catalysis in steady-state measurements. Whether the increased activity of PfPrx6H39Y is caused by a facilitated enzyme reduction because of a destabilization of the fully folded enzyme conformation remains to be analyzed. In summary, the conserved histidyl residue of Prx6-type enzymes is non-essential for catalysis, PfPrx6 is rapidly oxidized by hydroperoxides, and the gain-of-function mutant PfPrx6H39Y might provide a valuable tool to address the influence of conformational changes on the reactivity of Prx6 homologues.


Assuntos
Substituição de Aminoácidos , Histidina/química , Peroxirredoxina VI/química , Plasmodium falciparum/enzimologia , Proteínas de Protozoários/química , Tirosina/química , Domínio Catalítico , Ativação Enzimática/genética , Mutação com Ganho de Função , Histidina/genética , Peróxido de Hidrogênio/química , Cinética , Oxirredução , Peroxirredoxina VI/genética , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Tirosina/genética
20.
Blood ; 133(4): 356-365, 2019 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-30366922

RESUMO

The frequent von Willebrand factor (VWF) variant p.Phe2561Tyr is located within the C4 domain, which also harbors the platelet GPIIb/IIIa-binding RGD sequence. To investigate its potential effect on hemostasis, we genotyped 865 patients with coronary artery disease (CAD), 915 with myocardial infarction (MI), and 417 control patients (Ludwigshafen Risk and Cardiovascular Health Study) and performed functional studies of this variant. A univariate analysis of male and female carriers of the Tyr2561 allele aged 55 years or younger revealed an elevated risk for repeated MI (odds ratio, 2.53; 95% confidence interval [CI], 1.07-5.98). The odds ratio was even higher in females aged 55 years or younger, at a value of 5.93 (95% CI, 1.12-31.24). Cone and plate aggregometry showed that compared with Phe2561, Tyr2561 was associated with increased platelet aggregate size both in probands' blood and with the recombinant variants. Microfluidic assays revealed that the critical shear rate for inducing aggregate formation was decreased to 50% by Tyr2561 compared with Phe2561. Differences in C-domain circular dichroism spectra resulting from Tyr2561 suggest an increased shear sensitivity of VWF as a result of altered association of the C domains that disrupts the normal dimer interface. In summary, our data emphasize the functional effect of the VWF C4 domain for VWF-mediated platelet aggregation in a shear-dependent manner and provide the first evidence that a functional variant of VWF plays a role in arterial thromboembolism.


Assuntos
Alelos , Mutação com Ganho de Função/genética , Predisposição Genética para Doença , Infarto do Miocárdio/genética , Tirosina/genética , Fator de von Willebrand/genética , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Razão de Chances , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Ligação Proteica , Conformação Proteica , Fatores de Risco , Fator de von Willebrand/química
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