RESUMEN
The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a plasma membrane chloride channel protein that regulates vertebrate fluid homeostasis. The inefficiency of wild type human CFTR protein folding/trafficking is exacerbated by genetic mutations that can cause protein misfolding in the endoplasmic reticulum (ER) and subsequent degradation. This project investigates small changes in protein sequence that can alter the thermal stability of the large multi-domain CFTR protein. We target a conserved 70-residue α-subdomain located in the first nucleotide-binding domain that hosts the common misfolding mutation ∆F508. To investigate substitutions that can stabilize this domain, we constructed chimeras between human CFTR and its closest yeast homolog Yor1p. The α-subdomain of Yor1p was replaced with that of CFTR in Saccharomyces cerevisiae. Cellular localization of green fluorescence protein-tagged Yor1p-CFTR chimeras was analyzed by fluorescence microscopy and quantitative multispectral imaging flow cytometry, steady-state protein levels were compared by SDS-PAGE and protein function probed by a phenotypic oligomycin resistance assay. The chimeras exhibited ER retention in yeast characteristic of defective protein folding/processing. Substitution of seven CFTR α-subdomain residues that are highly conserved in Yor1p and other transporters but differ in CFTR (S495P/R516K/F533L/A534P/K536G/I539T/R553K) improved Yor1p-CFTR chimera localization to the yeast plasma membrane. When introduced into human CFTR expressed in mammalian cells, the same substitutions improve the purified protein thermal stability. This stabilized human CFTR protein will be directly useful for structural and biophysical studies that have been limited by the thermal sensitivity of wild type CFTR. The insights into critical structural residues within CFTR could facilitate development of effective therapeutics for CF-causing mutations.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/química , Sustitución de Aminoácidos , Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Retículo Endoplásmico/metabolismo , Proteínas Mutantes Quiméricas/química , Proteínas de Saccharomyces cerevisiae/química , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Células CHO , Cricetulus , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Calor , Humanos , Modelos Moleculares , Proteínas Mutantes Quiméricas/genética , Proteínas Mutantes Quiméricas/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Pliegue de Proteína , Dominios y Motivos de Interacción de Proteínas , Estabilidad Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Relación Estructura-ActividadRESUMEN
Ophidian envenomation is an important health problem in Brazil and other South American countries. In folk medicine, especially in developing countries, several vegetal species are employed for the treatment of snakebites in communities that lack prompt access to serum therapy. However, the identification and characterization of the effects of several new plants or their isolated compounds, which are able to inhibit the activities of snake venom, are extremely important and such studies are imperative. Snake venom contains several organic and inorganic compounds; phospholipases A2 (PLA2s) are one of the principal toxic components of venom. PLA2s display a wide variety of pharmacological activities, such as neurotoxicity, myotoxicity, cardiotoxicity, anticoagulant, hemorrhagic, and edema-inducing effects. PLA2 inhibition is of pharmacological and therapeutic interests as these enzymes are involved in several inflammatory diseases. This review describes the results of several studies of plant extracts and their isolated active principles, when used against crude snake venoms or their toxic fractions. Isolated inhibitors, such as steroids, terpenoids, and phenolic compounds, are able to inhibit PLA2s from different snake venoms. The design of specific inhibitors of PLA2s might help in the development of new pharmaceutical drugs, more specific antivenom, or even as alternative approaches for treating snakebites.