RESUMEN
BACKGROUND: The Melanocortin 1 Receptor (MC1R) contributes to pigmentation, an important risk factor for developing melanoma. Evaluating SNPs in MC1R and association with race/ethnicity, skin type, and perceived cancer risk in a New Mexico (NM) population will elucidate the role of MC1R in a multicultural population. METHODS: We genotyped MC1R in 191 NMs attending a primary care clinic in Albuquerque. We obtained individuals' self-identified race/ethnicity, skin type, and perceived cancer risk. We defined genetic risk as carriage of any one or more of the nine most common SNPs in MC1R. RESULTS: We found that one MC1R SNP, R163Q (rs885479), was identified in 47.6% of self-identified Hispanics and 12.9% of non-Hispanic whites (NHW), making Hispanics at higher "genetic risk" (as defined by carrying one of the MC1R common variants). When we deleted R163Q from analyses, Hispanics were no longer at higher genetic risk (33.3%) compared with NHW (48.3%), consistent with melanoma rates, tanning ability, and lower perceived risk. Hispanics had a perceived risk significantly lower than NHW and a nonsignificant better tanning ability than NHW. CONCLUSIONS: The R163Q variant in MC1R may not be a risk factor for melanoma among NM Hispanics. This suggestion points to the need to carefully interpret genetic risk factors among specific populations. IMPACT: Genetic risk cannot be extrapolated from Northern European populations directly to non-European populations.
Asunto(s)
Receptor de Melanocortina Tipo 1/genética , Variación Genética , Genotipo , Humanos , New MexicoRESUMEN
Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels that play a central role in neuronal and neuromuscular signal transduction. Here, we have developed FANG ligands, fibronectin antibody-mimetic nicotinic acetylcholine receptor-generated ligands, using mRNA display. We generated a 1 trillion-member primary e10FnIII library to target a stabilized α1 nicotinic subunit (α211). This library yielded 270000 independent potential protein binding ligands. The lead sequence, α1-FANG1, represented 25% of all library sequences, showed the highest-affinity binding, and competed with α-bungarotoxin (α-Btx). To improve this clone, a new library based on α1-FANG1 was subjected to heat, protease, binding, off-rate selective pressures, and point mutations. This resulted in α1-FANG2 and α1-FANG3. These proteins bind α211 with KD values of 3.5 nM and 670 pM, respectively, compete with α-Btx, and show improved subunit specificity. α1-FANG3 is thermostable ( Tm = 62 °C) with a 6 kcal/mol improvement in folding free energy compared with that of the parent α1-FANG1. α1-FANG3 competes directly with the α-Btx binding site of intact neuromuscular heteropentamers [(α1)2ß1γδ] in mammalian culture-derived cellular membranes and in Xenopus laevis oocytes expressing these nAChRs. This work demonstrates that mRNA display against a monomeric ecto-domain of a pentamer has the capability to select ligands that bind that subunit in both a monomeric and a pentameric context. Overall, our work provides a route to creating a new family of stable, well-behaved proteins that specifically target this important receptor family.
Asunto(s)
Bungarotoxinas/metabolismo , Fibronectinas/metabolismo , Receptores Nicotínicos/metabolismo , Animales , Fibronectinas/genética , Biblioteca de Genes , Humanos , Ligandos , Ratones , Mutación Puntual , Unión Proteica , Subunidades de Proteína/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Termodinámica , XenopusRESUMEN
Identification of positive staining is often qualitative and subjective. This is particularly troublesome in pigmented melanoma lesions, because melanin is difficult to distinguish from the brown stain resulting from immunohistochemistry (IHC) using horse radish peroxidase developed with 3,3'-Diaminobenzidine (HRP-DAB). We sought to identify and quantify positive staining, particularly in melanoma lesions. We visualized G-protein coupled estrogen receptor (GPER) expression developed with HRP-DAB and counterstained with Azure B (stains melanin) in melanoma tissue sections (nâ=â3). Matched sections (nâ=â3), along with 22 unmatched sections, were stained only with Azure B as a control. Breast tissue (nâ=â1) was used as a positive HRP-DAB control. Images of the stained tissues were generated using a Nuance Spectral Imaging Camera. Analysis of the images was performed using the Nuance Spectral Imaging software and SlideBook. Data was analyzed using a Kruskal-Wallis one way analysis of variance (ANOVA). We showed that a pigmented melanoma tissue doubly stained with anti-GPER HRP-DAB and Azure B can be unmixed using spectra derived from a matched, Azure B-only section, and an anti-GPER HRP-DAB control. We unmixed each of the melanoma lesions using each of the Azure B spectra, evaluated the mean intensity of positive staining, and examined the distribution of the mean intensities (Pâ=â.73; Kruskal-Wallis). These results suggest that this method does not require a matched Azure B-only stained control tissue for every melanoma lesion, allowing precious tissues to be conserved for other studies. Importantly, this quantification method reduces the subjectivity of protein expression analysis, and provides a valuable tool for accurate evaluation, particularly for pigmented tissues.