RESUMEN
BACKGROUND: Artificial genomic reference standards in a cytocentrifuge/cytospin format with well-annotated genomic data are useful for validating next-generation sequencing (NGS) on routine cytopreparations. Here, reference standards were optimized to be stained by different laboratories before DNA extraction and to contain a lower number of cells (2 × 105 ). This was done to better reflect the clinical challenge of working with insufficient cytological material. METHODS: A total of 17 worldwide laboratories analyzed customized reference standard slides (slides A-D). Each laboratory applied its standard workflow. The sample slides were engineered to harbor epidermal growth factor receptor (EGFR) c.2235_2249del15 p.E746_A750delELREA, EGFR c.2369C>T p.T790M, Kirsten rat sarcoma viral oncogene homolog (KRAS) c.38G>A p.G13D, and B-Raf proto-oncogene, serine/threonine kinase (BRAF) c.1798_1799GT>AA p.V600K mutations at various allele frequencies (AFs). RESULTS: EGFR and KRAS mutation detection showed excellent interlaboratory reproducibility, especially on slides A and B (10% and 5% AFs). On slide C (1% AF), either the EGFR mutation or the KRAS mutation was undetected by 10 of the 17 laboratories (58.82%). A reassessment of the raw data in a second-look analysis highlighted the mutations (n = 10) that had been missed in the first-look analysis. BRAF c.1798_1799GT>AA p.V600K showed a lower concordance rate for mutation detection and AF quantification. CONCLUSIONS: The data show that the detection of low-abundance mutations is still clinically challenging and may require a visual inspection of sequencing reads to detect. Genomic reference standards in a cytocentrifuge/cytospin format are a valid tool for regular quality assessment of laboratories performing molecular studies on cytology with low-AF mutations.
Asunto(s)
Biomarcadores de Tumor/genética , Citodiagnóstico/métodos , Análisis Mutacional de ADN/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Mutación , Neoplasias/diagnóstico , Receptores ErbB/genética , Humanos , Neoplasias/genética , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Reproducibilidad de los ResultadosRESUMEN
RATIONALE: Signal initiation by the high-density lipoprotein (HDL) receptor scavenger receptor class B, type I (SR-BI), which is important to actions of HDL on endothelium and other processes, requires cholesterol efflux and the C-terminal transmembrane domain. The C-terminal transmembrane domain uniquely interacts with plasma membrane (PM) cholesterol. OBJECTIVE: The molecular basis and functional significance of SR-BI interaction with PM cholesterol are unknown. We tested the hypotheses that the interaction is required for SR-BI signaling, and that it enables SR-BI to serve as a PM cholesterol sensor. METHODS AND RESULTS: In studies performed in COS-M6 cells, mutation of a highly conserved C-terminal transmembrane domain glutamine to alanine (SR-BI-Q445A) decreased PM cholesterol interaction with the receptor by 71% without altering HDL binding or cholesterol uptake or efflux, and it yielded a receptor incapable of HDL-induced signaling. Signaling prompted by cholesterol efflux to methyl-ß-cyclodextrin also was prevented, indicating that PM cholesterol interaction with the receptor enables it to serve as a PM cholesterol sensor. Using SR-BI-Q445A, we further demonstrated that PM cholesterol sensing by SR-BI does not influence SR-BI-mediated reverse cholesterol transport to the liver in mice. However, the PM cholesterol sensing does underlie apolipoprotein B intracellular trafficking in response to postprandial micelles or methyl-ß-cyclodextrin in cultured enterocytes, and it is required for HDL activation of endothelial NO synthase and migration in cultured endothelial cells and HDL-induced angiogenesis in vivo. CONCLUSIONS: Through interaction with PM cholesterol, SR-BI serves as a PM cholesterol sensor, and the resulting intracellular signaling governs processes in both enterocytes and endothelial cells.
Asunto(s)
Membrana Celular/metabolismo , Colesterol/metabolismo , Células Endoteliales/metabolismo , Enterocitos/metabolismo , Receptores Depuradores de Clase B/metabolismo , Transducción de Señal , Alanina , Animales , Apolipoproteínas B/metabolismo , Células CACO-2 , Bovinos , Membrana Celular/efectos de los fármacos , HDL-Colesterol/metabolismo , Células Endoteliales/efectos de los fármacos , Enterocitos/efectos de los fármacos , Glutamina , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Hígado/metabolismo , Masculino , Proteínas de la Membrana , Ratones , Ratones Endogámicos C57BL , Mutación , Neovascularización Fisiológica , Óxido Nítrico Sintasa de Tipo III/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Receptores Depuradores de Clase B/química , Receptores Depuradores de Clase B/genética , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Transfección , beta-Ciclodextrinas/farmacologíaRESUMEN
Scavenger receptor B type I (SR-BI) plays an important role in mediating cholesterol exchange between cells, high-density lipoprotein (HDL) cholesterol, and other lipoproteins. SR-BI in hepatocytes is essential for reverse cholesterol transport and biliary secretion of HDL cholesterol; thus, it is atheroprotective. More recently, it has been discovered that the HDL-SR-BI tandem serves other functions that also likely contribute to HDL-related cardiovascular protection. A number of the latter mechanisms, particularly in endothelial cells, involve unique direct signal initiation by SR-BI that leads to the activation of diverse kinase cascades. SR-BI signaling occurs in response to plasma membrane cholesterol flux. It requires the C-terminal PDZ-interacting domain of the receptor, which mediates direct interaction with the adaptor molecule PDZK1; and the C-terminal transmembrane domain, which directly binds membrane cholesterol. In endothelium, direct SR-BI signaling in response to HDL results in enhanced production of the antiatherogenic molecule nitric oxide; in a nitric oxide-independent manner, it serves to maintain endothelial monolayer integrity. The role of SR-BI signaling in the numerous other cellular targets of HDL, including hepatocytes, macrophages, and platelets, and the basis by which SR-BI senses plasma membrane cholesterol movement to modify cell behavior are unknown. Further understanding of signaling by SR-BI will optimize the capacity to harness the mechanisms of action of HDL-SR-BI for cardiovascular benefit.
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Antígenos CD36/metabolismo , Enfermedades Cardiovasculares/prevención & control , Colesterol/metabolismo , Lipoproteínas HDL/metabolismo , Transducción de Señal , Animales , Transporte Biológico , Antígenos CD36/química , Enfermedades Cardiovasculares/etiología , Enfermedades Cardiovasculares/metabolismo , Humanos , Conformación Proteica , Relación Estructura-ActividadRESUMEN
The enzyme complexes involved in mitochondrial oxidative phosphorylation are organized into higher ordered assemblies termed supercomplexes. Subunits e and g (Su e and Su g, respectively) are catalytically nonessential subunits of the F1F0-ATP synthase whose presence is required to directly support the stable dimerization of the ATP synthase complex. We report here that Su g and Su e are also important for securing the correct organizational state of the cytochrome bc1-cytochrome oxidase (COX) supercomplex. Mitochondria isolated from the Delta su e and Delta su g null mutant strains exhibit decreased levels of COX enzyme activity but appear to have normal COX subunit protein levels. An altered stoichiometry of the cytochrome bc1-COX supercomplex was observed in mitochondria deficient in Su e and/or Su g, and a perturbation in the association of Cox4, a catalytically important subunit of the COX complex, was also detected. In addition, an increase in the level of the TIM23 translocase associated with the cytochrome bc1-COX supercomplex is observed in the absence of Su e and Su g. Together, our data highlight that a further level of complexity exists between the oxidative phosphorylation supercomplexes, whereby the organizational state of one complex, i.e. the ATP synthase, may influence that of another supercomplex, namely the cytochrome bc1-COX complex.
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Complejo III de Transporte de Electrones/química , Complejo IV de Transporte de Electrones/química , Proteínas de Transporte de Membrana/química , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimología , Cromatografía en Gel , Dimerización , Electroforesis en Gel Bidimensional , Electroforesis en Gel de Poliacrilamida , Mitocondrias/metabolismo , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Modelos Biológicos , Oxígeno/química , Fosforilación , Estructura Terciaria de ProteínaRESUMEN
Circulating levels of high-density lipoprotein (HDL) cholesterol are inversely related to the risk of cardiovascular disease, and HDL and the HDL receptor scavenger receptor class B type I (SR-BI) initiate signaling in endothelium through src that promotes endothelial NO synthase activity and cell migration. Such signaling requires the C-terminal PDZ-interacting domain of SR-BI. Here we show that the PDZ domain-containing protein PDZK1 is expressed in endothelium and required for HDL activation of endothelial NO synthase and cell migration; in contrast, endothelial cell responses to other stimuli, including vascular endothelial growth factor, are PDZK1-independent. Coimmunoprecipitation experiments reveal that Src interacts with SR-BI, and this process is PDZK1-independent. PDZK1 also does not regulate SR-BI abundance or plasma membrane localization in endothelium or HDL binding or cholesterol efflux. Alternatively, PDZK1 is required for HDL/SR-BI to induce Src phosphorylation. Paralleling the in vitro findings, carotid artery reendothelialization following perivascular electric injury is absent in PDZK1-/- mice, and this phenotype persists in PDZK1-/- mice with genetic reconstitution of PDZK1 expression in liver, where PDZK1 modifies SR-BI abundance. Thus, PDZK1 is uniquely required for HDL/SR-BI signaling in endothelium, and through these mechanisms, it is critically involved in the maintenance of endothelial monolayer integrity.
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Células Endoteliales/citología , Células Endoteliales/metabolismo , Lipoproteínas HDL/metabolismo , Proteínas de la Membrana/metabolismo , Receptores Depuradores de Clase B/metabolismo , Animales , Aorta/citología , Bovinos , Movimiento Celular/fisiología , Células Cultivadas , Activación Enzimática/fisiología , Proteínas de la Membrana/genética , Ratones , Ratones Mutantes , Óxido Nítrico Sintasa de Tipo II/metabolismo , Óxido Nítrico Sintasa de Tipo III , Factor de Transcripción STAT1/fisiología , Túnica Íntima/citología , Túnica Íntima/metabolismoRESUMEN
The F(1)F(0)-ATP synthase enzyme is located in the inner mitochondrial membrane, where it forms dimeric complexes. Dimerization of the ATP synthase involves the physical association of the neighboring membrane-embedded F(0)-sectors. In yeast, the F(0)-sector subunits g and e (Su g and Su e, respectively) play a key role in supporting the formation of ATP synthase dimers. In this study we have focused on Su g to gain a better understanding of the function and the molecular organization of this subunit within the ATP synthase complex. Su g proteins contain a GXXXG motif (G is glycine, and X is any amino acid) in their single transmembrane segment. GXXXG can be a dimerization motif that supports helix-helix interactions between neighboring transmembrane segments. We demonstrate here that the GXXXG motif is important for the function and in particular for the stability of Su g within the ATP synthase. Using site-directed mutagenesis and cross-linking approaches, we demonstrate that Su g and Su e interact, and our findings emphasize the importance of the membrane anchor regions of these proteins for their interaction. Su e also contains a conserved GXXXG motif in its membrane anchor. However, data presented here would suggest that an intact GXXXG motif in Su g is not essential for the Su g-Su e interaction. We suggest that the GXXXG motif may not be the sole basis for a Su g-Su e interaction, and possibly these dimerization motifs may enable both Su g and Su e to interact with another mitochondrial protein.