RESUMO
Congenital heart malformations, including those of the great vessels, are among the most common human birth defects. The goal of this study was to identify the significance of a de novo mosaic PTPN12 partial deletion identified in a newborn with an interrupted aortic arch type A, ventricular septal defect, and pyloric stenosis. PTPN12, a downstream target of the RAS pathway, has a known role in endothelial cell adhesion and migration. Neither genetic nor genomic variants in PTPN12 have been described in a human patient; therefore, we evaluated the effect of ptpn12 in a mouse conditional knockout and zebrafish knockdown model to determine the significance of a loss in gene expression. Observed loss of ptpn12 expression in zebrafish resulted in abnormal branchial arch and tail vasculature patterns, with reduced blood flow throughout the animal. This phenotype was supported by anomalous vasculature in a conditional Ptpn12 mouse knockout. Given the novel co-occurrence of interrupted aortic arch type A, ventricular septal defect, and partial deletion of PTPN12 in the patient, as well as vascular phenotypes in Ptpn12 mouse and ptpn12 zebrafish models, it is likely that PTPN12 has a significant role in cardiovascular development and vessel formation during human embryonic development. Furthermore, the partial deletion of PTPN12 lead to interrupted aortic arch type A in this child and may represent a novel condition caused by a null mutation in the RAS pathway.
Assuntos
Aorta Torácica/anormalidades , Aorta Torácica/patologia , Mosaicismo , Proteína Tirosina Fosfatase não Receptora Tipo 12/genética , Deleção de Sequência , Adulto , Sequência de Aminoácidos , Angiografia , Animais , Aorta Torácica/diagnóstico por imagem , Aorta Torácica/embriologia , Criança , Hibridização Genômica Comparativa , Sequência Conservada , Desenvolvimento Embrionário , Técnicas de Silenciamento de Genes , Humanos , Hibridização in Situ Fluorescente , Lactente , Recém-Nascido , Larva , Masculino , Camundongos , Dados de Sequência Molecular , Neovascularização Fisiológica , Fenótipo , Proteína Tirosina Fosfatase não Receptora Tipo 12/química , Alinhamento de Sequência , Tomografia Computadorizada por Raios X , Peixe-Zebra/embriologiaRESUMO
(13)C-Cholesterol was produced with high efficiency by a genetically engineered yeast strain. The method produces â¼ 1 mg of cholesterol per gram of glucose using 100 ml of culture medium. Uniform 94% enrichment where the most abundant product is the fully enriched isotopomer (u-(13)C(27)) is obtained using (u-(13)C(6), 99%) glucose medium. High enrichment is very important for relaxation experiments, but for NMR applications where carbon-carbon couplings are measured, this is problematic. A good compromise between sensitivity and cost consists in diluting (u-(13)C(6), 25%) with natural-abundance glucose. With a 2:3 ratio, the maximal amount of singlets can be obtained in 1 dimensional (D) carbon and 2D heteronuclear single-quantum correlation (HSQC) spectra with 6× intensity increase relative to natural-abundance samples. The use of (1-(13)C(1)-glucose, 99%) or (2-(13)C(1)-glucose, 99%) as isotope sources allows the labeling of the cholesterol in multiple mostly nonvicinal positions and reach 45× intensity increase. As an alternative, the dilution of (u-(13)C(6), 99%) glucose can be used to simultaneously enrich eleven pairs of (13)C up to â¼ 1,000× natural-abundance probability, which should be very beneficial to double-quantum NMR experiments including the INADEQUATE and related pulse sequences. The flexibility of the method and the potential to adapt the culture protocol to specific needs should find many applications in chemistry and biology and in different fields of NMR and MS.
Assuntos
Isótopos de Carbono/química , Colesterol/química , Espectroscopia de Ressonância Magnética/métodos , Estrutura MolecularRESUMO
Sterols are major lipids in eukaryotes and differ in their specific structure between species. Both cholesterol and ergosterol can form liquid ordered domains in artificial membranes. We reasoned that substituting the main sterol ergosterol by cholesterol in yeast should permit domain formation and discriminate between physical and sterol structure-dependent functions. Using a cholesterol-producing yeast strain, we show that solute transporters for tryptophan and arginine are functional, whereas the export of weak organic acids via Pdr12p, a multi-drug resistance family member, is not. The latter reveals a sterol function that is probably dependent upon a precise sterol structure. We present a series of novel yeast strains with different sterol compositions as valuable tools to characterize sterol function and use them to refine the sterol requirements for Pdr12p. These strains will also be improved hosts for heterologous expression of sterol-dependent proteins and safe sources to obtain pure cholesterol and other sterols.
Assuntos
Colesterol/biossíntese , Colesterol/genética , Ergosterol , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Triptofano/genética , Triptofano/metabolismoRESUMO
Sterols and sphingolipids are limited to eukaryotic cells, and their interaction has been proposed to favor formation of lipid microdomains. Although there is abundant biophysical evidence demonstrating their interaction in simple systems, convincing evidence is lacking to show that they function together in cells. Using lipid analysis by mass spectrometry and a genetic approach on mutants in sterol metabolism, we show that cells adjust their membrane composition in response to mutant sterol structures preferentially by changing their sphingolipid composition. Systematic combination of mutations in sterol biosynthesis with mutants in sphingolipid hydroxylation and head group turnover give a large number of synthetic and suppression phenotypes. Our unbiased approach provides compelling evidence that sterols and sphingolipids function together in cells. We were not able to correlate any cellular phenotype we measured with plasma membrane fluidity as measured using fluorescence anisotropy. This questions whether the increase in liquid order phases that can be induced by sterol-sphingolipid interactions plays an important role in cells. Our data revealing that cells have a mechanism to sense the quality of their membrane sterol composition has led us to suggest that proteins might recognize sterol-sphingolipid complexes and to hypothesize the coevolution of sterols and sphingolipids.
Assuntos
Membrana Celular/fisiologia , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/fisiologia , Esfingolipídeos/metabolismo , Esteróis/metabolismo , Anisotropia , Transporte Biológico/efeitos dos fármacos , Cafeína/farmacologia , Membrana Celular/efeitos dos fármacos , Análise por Conglomerados , Perfilação da Expressão Gênica , Mutação/genética , Fenótipo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Sirolimo/farmacologia , Ácido Sórbico/farmacologia , Esfingolipídeos/química , Esteróis/biossíntese , Esteróis/químicaRESUMO
Natamycin is a polyene antibiotic that is commonly used as an antifungal agent because of its broad spectrum of activity and the lack of development of resistance. Other polyene antibiotics, like nystatin and filipin are known to interact with sterols, with some specificity for ergosterol thereby causing leakage of essential components and cell death. The mode of action of natamycin is unknown and is investigated in this study using different in vitro and in vivo approaches. Isothermal titration calorimetry and direct binding studies revealed that natamycin binds specifically to ergosterol present in model membranes. Yeast sterol biosynthetic mutants revealed the importance of the double bonds in the B-ring of ergosterol for the natamycin-ergosterol interaction and the consecutive block of fungal growth. Surprisingly, in strong contrast to nystatin and filipin, natamycin did not change the permeability of the yeast plasma membrane under conditions that growth was blocked. Also, in ergosterol containing model membranes, natamycin did not cause a change in bilayer permeability. This demonstrates that natamycin acts via a novel mode of action and blocks fungal growth by binding specifically to ergosterol.