RESUMO
Using the energy of ATP hydrolysis, ABC transporters catalyze the trans-membrane transport of molecules. In bacteria, these transporters partner with a high-affinity substrate-binding protein (SBP) to import essential micronutrients. ATP binding by Type I ABC transporters (importers of amino acids, sugars, peptides, and small ions) stabilizes the interaction between the transporter and the SBP, thus allowing transfer of the substrate from the latter to the former. In Type II ABC transporters (importers of trace elements, e.g. vitamin B12, heme, and iron-siderophores) the role of ATP remains debatable. Here we studied the interaction between the Yersinia pestis ABC heme importer (HmuUV) and its partner substrate-binding protein (HmuT). Using real-time surface plasmon resonance experiments and interaction studies in membrane vesicles, we find that in the absence of ATP the transporter and the SBP tightly bind. Substrate in excess inhibits this interaction, and ATP binding by the transporter completely abolishes it. To release the stable docked SBP from the transporter hydrolysis of ATP is required. Based on these results we propose a mechanism for heme acquisition by HmuUV-T where the substrate-loaded SBP docks to the nucleotide-free outward-facing conformation of the transporter. ATP binding leads to formation of an occluded state with the substrate trapped in the trans-membrane translocation cavity. Subsequent ATP hydrolysis leads to substrate delivery to the cytoplasm, release of the SBP, and resetting of the system. We propose that other Type II ABC transporters likely share the fundamentals of this mechanism.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Heme/metabolismo , Hemeproteínas/metabolismo , Modelos Moleculares , Yersinia pestis/metabolismo , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/genética , Trifosfato de Adenosina/química , Apoenzimas/química , Apoenzimas/genética , Apoenzimas/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Transporte/química , Proteínas de Transporte/genética , Membrana Celular/química , Membrana Celular/metabolismo , Dimerização , Heme/química , Proteínas Ligantes de Grupo Heme , Hemeproteínas/química , Hemeproteínas/genética , Holoenzimas/química , Holoenzimas/genética , Holoenzimas/metabolismo , Hidrólise , Proteínas Imobilizadas/química , Proteínas Imobilizadas/genética , Proteínas Imobilizadas/metabolismo , Cinética , Simulação de Acoplamento Molecular , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes , Ressonância de Plasmônio de SuperfícieRESUMO
PURPOSE: To describe our experience with a large cohort (411 patients from 288 families) of various forms of skeletal dysplasia who were molecularly characterized. METHODS: Detailed phenotyping and next-generation sequencing (panel and exome). RESULTS: Our analysis revealed 224 pathogenic/likely pathogenic variants (54 (24%) of which are novel) in 123 genes with established or tentative links to skeletal dysplasia. In addition, we propose 5 genes as candidate disease genes with suggestive biological links (WNT3A, SUCO, RIN1, DIP2C, and PAN2). Phenotypically, we note that our cohort spans 36 established phenotypic categories by the International Skeletal Dysplasia Nosology, as well as 18 novel skeletal dysplasia phenotypes that could not be classified under these categories, e.g., the novel C3orf17-related skeletal dysplasia. We also describe novel phenotypic aspects of well-known disease genes, e.g., PGAP3-related Toriello-Carey syndrome-like phenotype. We note a strong founder effect for many genes in our cohort, which allowed us to calculate a minimum disease burden for the autosomal recessive forms of skeletal dysplasia in our population (7.16E-04), which is much higher than the global average. CONCLUSION: By expanding the phenotypic, allelic, and locus heterogeneity of skeletal dysplasia in humans, we hope our study will improve the diagnostic rate of patients with these conditions.
Assuntos
Exoma/genética , Heterogeneidade Genética , Predisposição Genética para Doença , Anormalidades Musculoesqueléticas/genética , Alelos , Proteínas Sanguíneas/genética , Hidrolases de Éster Carboxílico , Estudos de Coortes , Exorribonucleases/genética , Feminino , Proteínas Fetais/genética , Efeito Fundador , Genética Populacional , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Proteínas de Membrana/genética , Anormalidades Musculoesqueléticas/classificação , Anormalidades Musculoesqueléticas/patologia , Proteínas de Neoplasias/genética , Proteínas Oncogênicas/genética , Fenótipo , Receptores de Superfície Celular/genética , Proteína Wnt3A/genéticaRESUMO
BACKGROUND: Respiratory infections are known to exacerbate wheezing in many asthmatic patients. We aimed to use molecular methods for the fast detection of Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila in respiratory specimens from asthmatic patients in Kuwait. METHODS: We used uniplex PCR assays to detect the three atypical bacteria in clinical specimens from 235 asthmatic and non-asthmatic patients in Kuwait. A regression analysis was used to identify the risk factors related to the bacterial type. Group comparisons for similarity were conducted and correlation coefficients were calculated using SPSS statistical software. RESULTS: The detection limits using uniplex PCR for C. pneumoniae, L. pneumophila and M. pneumoniae were approximately 1pg, 2.4fg and 12pg of DNA, respectively. M. pneumoniae PCR positivity was more common in asthmatic patients (15%) than in non-asthmatic subjects (9%) (P<0.05). A marked difference was observed between patients with acute asthma exacerbation (11%) and patients with chronic (stable) asthma (7%) among Kuwaiti patients; these percentages were 16% for non-Kuwaiti acute asthma patients and 14% for non-Kuwaiti chronic asthma patients (P<0.201). There was a weak positive correlation between asthma severity and PCR positivity for M. pneumoniae. The PCR results for C. pneumoniae and L. pneumoniae were found to be statistically insignificant. CONCLUSIONS: The results of this study suggest that infection with M. pneumoniae may be related to the exacerbation of asthma symptoms and could possibly be a factor that induces wheezing.