RESUMEN
The enzyme 17beta-hydroxysteroid dehydrogenase type 10 (HSD10), also known as amyloid beta-peptide-binding alcohol dehydrogenase (ABAD), has been implicated in the development of Alzheimer's disease. This protein, a member of the short-chain dehydrogenase/reductase family of enzymes, has been shown to bind beta-amyloid and to participate in beta-amyloid neurotoxicity. We have determined the crystal structure of human ABAD/HSD10 complexed with NAD(+) and an inhibitory small molecule. The inhibitor occupies the substrate-binding site and forms a covalent adduct with the NAD(+) cofactor. The crystal structure provides a basis for the design of potent, highly specific ABAD/HSD10 inhibitors with potential application in the treatment of Alzheimer's disease.
Asunto(s)
3-Hidroxiacil-CoA Deshidrogenasas/antagonistas & inhibidores , 3-Hidroxiacil-CoA Deshidrogenasas/química , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/enzimología , 3-Hidroxiacil-CoA Deshidrogenasas/genética , 3-Hidroxiacil-CoA Deshidrogenasas/metabolismo , Secuencia de Aminoácidos , Péptidos beta-Amiloides/metabolismo , Secuencia de Bases , Dominio Catalítico , Cristalografía por Rayos X , ADN Complementario/genética , Diseño de Fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Técnicas In Vitro , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , NAD/química , Conformación Proteica , Estructura Cuaternaria de Proteína , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Endothelial lipase (EL) plays a pivotal role in HDL metabolism. We sought to characterize EL and its interaction with HDL as well as its natural variants genetically, functionally and structurally. We screened our biethnic population sample (nâ=â802) for selected missense mutations (nâ=â5) and identified T111I as the only common variant. Multiple linear regression analyses in Hispanic subjects revealed an unexpected association between T111I and elevated LDL-C (p-valueâ=â0.012) and total cholesterol (p-valueâ=â0.004). We examined lipase activity of selected missense mutants (nâ=â10) and found different impacts on EL function, ranging from normal to complete loss of activity. EL-HDL lipidomic analyses indicated that EL has a defined remodeling of HDL without exhaustion of the substrate and a distinct and preference for several fatty acids that are lipid mediators and known for their potent pro- and anti-inflammatory properties. Structural studies using homology modeling revealed a novel α/ß motif in the C-domain, unique to EL. The EL dimer was found to have the flexibility to expand and to bind various sizes of HDL particles. The likely impact of the all known missense mutations (nâ=â18) on the structure of EL was examined using molecular modeling and the impact they may have on EL lipase activity using a novel structure-function slope based on their structural free energy differences. The results of this multidisciplinary approach delineated the impact of EL and its variants on HDL. Moreover, the results suggested EL to have the capacity to modulate vascular health through its role in fatty acid-based signaling pathways.
Asunto(s)
Lipasa/genética , Lipasa/metabolismo , Mutación Missense , Alelos , Secuencia de Aminoácidos , Colesterol/sangre , Colesterol/metabolismo , HDL-Colesterol/sangre , HDL-Colesterol/metabolismo , Colorado , Activación Enzimática , Estudios de Asociación Genética , Genotipo , Proteoglicanos de Heparán Sulfato/química , Proteoglicanos de Heparán Sulfato/metabolismo , Hispánicos o Latinos/genética , Hidrólisis , Inflamación/genética , Inflamación/metabolismo , Lipasa/química , Modelos Biológicos , Modelos Moleculares , Datos de Secuencia Molecular , Fosfolipasas/metabolismo , Polimorfismo de Nucleótido Simple , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Alineación de Secuencia , Transducción de Señal , Relación Estructura-ActividadRESUMEN
BACKGROUND: Influenza A viral surface protein, hemagglutinin, is the major target of neutralizing antibody response and hence a main constituent of all vaccine formulations. But due to its marked evolutionary variability, vaccines have to be reformulated so as to include the hemagglutinin protein from the emerging new viral strain. With the constant fear of a pandemic, there is critical need for the development of anti-viral strategies that can provide wider protection against any Influenza A pathogen. An anti-viral approach that is directed against the conserved regions of the hemaggutinin protein has a potential to protect against any current and new Influenza A virus and provide a solution to this ever-present threat to public health. METHODOLOGY/PRINCIPAL FINDINGS: Influenza A human hemagglutinin protein sequences available in the NCBI database, corresponding to H1, H2, H3 and H5 subtypes, were used to identify highly invariable regions of the protein. Nine such regions were identified and analyzed for structural properties like surface exposure, hydrophilicity and residue type to evaluate their suitability for targeting an anti-peptide antibody/anti-viral response. CONCLUSION/SIGNIFICANCE: This study has identified nine conserved regions in the hemagglutinin protein, five of which have the structural characteristics suitable for an anti-viral/anti-peptide response. This is a critical step in the design of efficient anti-peptide antibodies as novel anti-viral agents against any Influenza A pathogen. In addition, these anti-peptide antibodies will provide broadly cross-reactive immunological reagents and aid the rapid development of vaccines against new and emerging Influenza A strains.