RESUMO
Endoplasmic reticulum (ER) stress is emerging as a potential contributor to the onset of type 2 diabetes by making cells insulin-resistant. However, our understanding of the mechanisms by which ER stress affects insulin response remains fragmentary. Here we present evidence that the ER stress pathway acts via a conserved signaling mechanism involving the protein kinase PERK to modulate cellular insulin responsiveness. Insulin signaling via AKT reduces activity of FOXO transcription factors. In some cells, PERK can promote insulin responsiveness. However, we found that PERK also acts oppositely via phosphorylation of FOXO to promote FOXO activity. Inhibition of PERK improves cellular insulin responsiveness at the level of FOXO activity. We suggest that the protein kinase PERK may be a promising pharmacological target for ameliorating insulin resistance.
Assuntos
Estresse do Retículo Endoplasmático/fisiologia , Fatores de Transcrição Forkhead/metabolismo , Resistência à Insulina/fisiologia , eIF-2 Quinase/metabolismo , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Células Hep G2 , Humanos , Células MCF-7 , Proteína Oncogênica v-akt/metabolismo , Fosforilação , Transporte ProteicoRESUMO
Using a combination of exome sequencing and linkage analysis, we investigated an English family with two affected siblings in their 40s with recessive Charcot-Marie Tooth disease type 2 (CMT2). Compound heterozygous mutations in the immunoglobulin-helicase-µ-binding protein 2 (IGHMBP2) gene were identified. Further sequencing revealed a total of 11 CMT2 families with recessively inherited IGHMBP2 gene mutations. IGHMBP2 mutations usually lead to spinal muscular atrophy with respiratory distress type 1 (SMARD1), where most infants die before 1 year of age. The individuals with CMT2 described here, have slowly progressive weakness, wasting and sensory loss, with an axonal neuropathy typical of CMT2, but no significant respiratory compromise. Segregating IGHMBP2 mutations in CMT2 were mainly loss-of-function nonsense in the 5' region of the gene in combination with a truncating frameshift, missense, or homozygous frameshift mutations in the last exon. Mutations in CMT2 were predicted to be less aggressive as compared to those in SMARD1, and fibroblast and lymphoblast studies indicate that the IGHMBP2 protein levels are significantly higher in CMT2 than SMARD1, but lower than controls, suggesting that the clinical phenotype differences are related to the IGHMBP2 protein levels.
Assuntos
Doença de Charcot-Marie-Tooth/genética , Exoma/genética , Modelos Moleculares , Mutação de Sentido Incorreto/genética , Fenótipo , Adulto , Sequência de Bases , Doença de Charcot-Marie-Tooth/patologia , Mapeamento Cromossômico , Feminino , Haplótipos/genética , Humanos , Dados de Sequência Molecular , Linhagem , Mapeamento de Interação de Proteínas , Análise de Sequência de DNA , Nervo Sural/patologiaRESUMO
Mutations in immunoglobulin µ-binding protein 2 (Ighmbp2) cause distal spinal muscular atrophy type 1 (DSMA1), an autosomal recessive disease that is clinically characterized by distal limb weakness and respiratory distress. However, despite extensive studies, the mechanism of disease-causing mutations remains elusive. Here we report the crystal structures of the Ighmbp2 helicase core with and without bound RNA. The structures show that the overall fold of Ighmbp2 is very similar to that of Upf1, a key helicase involved in nonsense-mediated mRNA decay. Similar to Upf1, domains 1B and 1C of Ighmbp2 undergo large conformational changes in response to RNA binding, rotating 30° and 10°, respectively. The RNA binding and ATPase activities of Ighmbp2 are further enhanced by the R3H domain, located just downstream of the helicase core. Mapping of the pathogenic mutations of DSMA1 onto the helicase core structure provides a molecular basis for understanding the disease-causing consequences of Ighmbp2 mutations.
Assuntos
Proteínas de Ligação a DNA/química , Atrofia Muscular Espinal/genética , Mutação de Sentido Incorreto , RNA Helicases/química , Síndrome do Desconforto Respiratório do Recém-Nascido/genética , Fatores de Transcrição/química , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Modelos Moleculares , Ligação Proteica , Estrutura Terciária de Proteína , RNA/química , RNA Helicases/genética , Transativadores/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Ferulic acid (FA) exhibits antioxidant and anti-inflammatory properties, making it valuable for numerous industrial applications. Traditionally, FA is produced by the alkaline hydrolysis of γ-oryzanol, which is typically associated with wastewater generation. Recently, an increasing demand of natural FA necessitates its green production via enzymatic hydrolysis of γ-oryzanol, a mixture comprising triterpene alcohol ferulates and phytosteryl ferulates. Thus far, γ-oryzanol can be hydrolyzed by only four commercial cholesterol esterases with low yields. Herein, we report a recombinant cholesterol esterase from Mustela putorius furo (MPFCE) for the enzymatic hydrolysis of γ-oryzanol. The enzyme yielded 25.5% FA, which is the highest reported through enzymatic means thus far. The hydrolysis profile revealed that the enhanced yield primarily resulted from the near-complete hydrolysis of phytosteryl ferulates, together with slight hydrolysis of triterpene alcohol ferulates. MPFCE serves as a potential candidate for the enzymatic production of FA through targeted hydrolysis of γ-oryzanol.
Assuntos
Fenilpropionatos , Esterol Esterase , Fenilpropionatos/metabolismo , Fenilpropionatos/química , Hidrólise , Esterol Esterase/genética , Esterol Esterase/metabolismo , Esterol Esterase/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Saccharomycetales/genética , Saccharomycetales/enzimologia , Saccharomycetales/metabolismo , Ácidos Cumáricos/metabolismo , Ácidos Cumáricos/química , Expressão Gênica , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/químicaRESUMO
INTRODUCTION: This study aimed to evaluate the LITE (Lifestyle Intervention for TEenagers) group programme, a family-based behavioural lifestyle intervention for overweight and obese adolescents. METHODS: We conducted a two-arm randomised controlled trial that recruited overweight and obese adolescents who attended a tertiary care weight management clinic. Participants were randomised to the LITE programme or usual care. The primary outcome assessed was body mass index (BMI) z-score. Secondary outcomes of anthropometric measurements, metabolic profile, parenting and adolescents' perception of family support were measured at baseline, three months and six months. Feasibility and acceptability of the LITE programme were also evaluated. RESULTS: 61 adolescents were enrolled, with 31 in the LITE programme and 30 in usual care. At three months, participants in the programme had a greater reduction in weight (-0.18 ± 2.40 kg vs. 1.48 ± 1.97 kg; p = 0.107), waist circumference (-1.0 ± 3.1 cm vs. 2.4 ± 2.7 cm; p = 0.016), waist-height ratio (-0.01 ± 0.02 vs. 0.01 ± 0.02; p = 0.040) and systolic blood pressure (-3.8 ± 13.7 vs. 5.7 ± 13.1; p = 0.119) compared to the usual care group. There was no significant difference in BMI z-score. At six months, there were significant improvements in adolescents' perception of family support for eating habits in the LITE group compared to the usual care group. The LITE programme had a good attendance rate of 67.7% and was well received. CONCLUSION: The LITE programme showed feasibility and short-term clinical effectiveness in improving some clinical outcomes and improved adolescents' perception of family support.
Assuntos
Obesidade Infantil , Adolescente , Terapia Comportamental , Índice de Massa Corporal , Humanos , Estilo de Vida , Obesidade Infantil/terapia , Projetos PilotoRESUMO
The second harmonic generation of antiferromagnetic and dielectric multilayers is analysed by using a conventional nonlinear optics approach and transfer matrix formalism. The theoretical modelling of the multilayers is configured in Voigt geometry in order to observe second harmonic transmission and reflection through the film system, with the assumption of weak nonlinearity and no depletion of incident waves. With these, some of the linear and second harmonic transmissions and reflections are calculated numerically and shown graphically.
RESUMO
In this issue of Structure, Romano and colleagues show that RNase AS specifically hydrolyses adenylate-containing RNA and affects mycobacterial virulence. This study reveals the structural basis underlying the substrate specificity of this enzyme.
Assuntos
Mycobacterium tuberculosis/patogenicidade , Ribonucleases/química , Ribonucleases/metabolismo , AnimaisRESUMO
The SOSS1 complex comprising SOSSA, SOSSB1, and SOSSC senses single-stranded DNA (ssDNA) and promotes repair of DNA double-strand breaks (DSBs). But how SOSS1 is assembled and recognizes ssDNA remains elusive. The crystal structure of the N-terminal half of SOSSA (SOSSAN) in complex with SOSSB1 and SOSSC showed that SOSSAN serves as a scaffold to bind both SOSSB1 and SOSSC for assembly of the SOSS1 complex. The structures of SOSSAN/B1 in complex with a 12 nt ssDNA and SOSSAN/B1/C in complex with a 35 nt ssDNA showed that SOSSB1 interacts with both SOSSAN and ssDNA via two distinct surfaces. Recognition of ssDNA with a length of up to nine nucleotides is mediated solely by SOSSB1, whereas neither SOSSC nor SOSSAN are critical for ssDNA binding. These results reveal the structural basis of SOSS1 assembly and provide a framework for further study of the mechanism governing longer ssDNA recognition by the SOSS1 complex during DSB repair.
Assuntos
DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Células HeLa , Humanos , Modelos Moleculares , Ligação Proteica , Estabilidade Proteica , Relação Estrutura-AtividadeRESUMO
The type III secretion system (T3SS) of Pseudomonas aeruginosa is a key virulence determinant whose expression is induced by polyamine signals from mammalian host. SpuD and SpuE were postulated to be spermidine-preferential binding proteins, which regulate the polyamine content in this bacterial pathogen. In this study, we found that SpuD is a putrescine-preferential binding protein, while SpuE binds to spermidine exclusively. We have determined the crystal structures of SpuD in free form and in complex with putrescine and SpuE in free form and in complex with spermidine. Upon ligand binding, SpuD and SpuE undergo an "open-to-closed" conformational switch with the resultant closed ligand-bound forms, SpuD-putrescine and SpuE-spermidine, similar to their Escherichia coli counterparts PotF-putrescine and PotD-spermidine, respectively. Structural comparison suggested that two aromatic residues, Trp271 of SpuE and Phe273 of SpuD in segment II region, are the key structural determinants for putrescine/spermidine recognition specificity. Mutagenesis combined with isothermal titration calorimetry showed that substitution of Trp271 by Phe enabled SpuE to gain substantial binding affinity for putrescine, while replacement of Phe273 by Trp reduced the binding affinity of SpuD toward putrescine by 250-fold. Altogether, these results revealed the molecular mechanism governing polyamine recognition specificity by SpuD and SpuE and provide the basis for further structural and functional studies of polyamine signal importation system in P. aeruginosa.
Assuntos
Proteínas Periplásmicas de Ligação/química , Proteínas Periplásmicas de Ligação/metabolismo , Poliaminas/metabolismo , Pseudomonas aeruginosa/metabolismo , Sistemas de Secreção Bacterianos/fisiologia , Cristalografia por Raios X/métodos , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Ligantes , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Modelos Moleculares , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Putrescina/metabolismo , Espermidina/metabolismo , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
The diffusible signal factor (DSF)-dependent quorum sensing (QS) system adopts a novel protein-protein interaction mechanism to autoregulate the production of signal DSF. Here, we present the crystal structures of DSF synthase RpfF and its complex with the REC domain of sensor protein RpfC. RpfF is structurally similarity to the members of the crotonase superfamily and contains an N-terminal α/ß spiral core domain and a C-terminal α-helical region. Further structural and mutational analysis identified two catalytic glutamate residues, which is the conserved feature of the enoyl-CoA hydratases/dehydratases. A putative substrate-binding pocket was unveiled and the key roles of the residues implicated in substrate binding were verified by mutational analysis. The binding of the REC domain may lock RpfF in an inactive conformation by blocking the entrance of substrate binding pocket, thereby negatively regulating DSF production. These findings provide a structural model for the RpfC-RpfF interaction-mediated QS autoinduction mechanism.