title: In vitro and molecular modeling analysis of two mutant desert hedgehog proteins associated with 46,XY gonadal dysgenesis.
abstract: Mutations of Desert hedgehog (DHH) have been associated to 46,XY pure gonadal dysgenesis (PGD) and to mixed gonadal dysgenesis (MGD); however, there have been no functional studies of mutations described in DHH. To determine if mutations p.L162P and Δ1086delG yield functional impairment, we performed in vitro and in silico analysis of both DHH mutants. In complementary DNA of DHH, we performed site-directed mutagenesis, which was confirmed by DNA sequencing. Protein extracts were obtained from HEK293cells transfected with different constructs and analyzed by Western blot; besides, densitometric analysis of chemiluminescent signals was performed. In addition, the structure of the wt-DHH and its two mutant proteins was inferred using in silico protein molecular modeling. In the Western blot analysis, we observed the absence of signal for p.L162P in DHH-N and a diminished signal for Δ1086delG in DHH-C, when compared to wt-DHH. Protein modeling showed notable conformational changes for the side chains of p.L162P, while the secondary structure was drastically modified in Δ1086delG, when compared to wt-DHH. To our knowledge, this is the first study focused to determine by in vitro studies, the effect of two specific mutations in DHH associated with 46,XY PGD and MGD. Our results suggest that both mutations have a deleterious effect on the expression of the DHH mutant proteins.
authors.affiliation: División de Investigación Biomédica, Subdirección de Enseñanza e Investigación, Centro Médico Nacional 20 de Noviembre, Instituto de Seguridad y Servicios Sociales de los Trabajadores del Estado, México, D.F., México.
authors.forename: Josué Joram
authors.initials: JJ
authors.lastname: Castro
authors.forename: Juan Pablo
authors.initials: JP
authors.lastname: Méndez
authors.forename: Ramón Mauricio
authors.initials: RM
authors.lastname: Coral-Vázquez
authors.forename: Marvin Antonio
authors.initials: MA
authors.lastname: Soriano-Ursúa
authors.forename: Pablo
authors.initials: P
authors.lastname: Damian-Matsumura
authors.forename: Jesús
authors.initials: J
authors.lastname: Benítez-Granados
authors.forename: Haydee
authors.initials: H
authors.lastname: Rosas-Vargas
authors.forename: Patricia
authors.initials: P
authors.lastname: Canto
mesh_terms: D019943:Amino Acid Substitution; D019295:Computational Biology; D020022:Genetic Predisposition to Disease; D006061:Gonadal Dysgenesis, 46,XY / Q000235:genetics* / Q000378:metabolism; D006060:Gonadal Dysgenesis, Mixed / Q000235:genetics* / Q000378:metabolism; D057809:HEK293 Cells; D053823:Hedgehog Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D055550:Protein Stability; D017433:Protein Structure, Secondary
chemical_list: C509245:DHH protein, human; D053823:Hedgehog Proteins