TY - JOUR
T1 - In vitro and molecular modeling analysis of two mutant desert hedgehog proteins associated with 46,XY gonadal dysgenesis
AU - Castro, Josué Joram
AU - Méndez, Juan Pablo
AU - Coral-Vázquez, Ramón Mauricio
AU - Soriano-Ursúa, Marvin Antonio
AU - Damian-Matsumura, Pablo
AU - Benítez-Granados, Jesús
AU - Rosas-Vargas, Haydee
AU - Canto, Patricia
PY - 2013/9/1
Y1 - 2013/9/1
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=84882999528&partnerID=8YFLogxK
U2 - 10.1089/dna.2013.2052
DO - 10.1089/dna.2013.2052
M3 - Artículo
C2 - 23786321
SN - 1044-5498
VL - 32
SP - 524
EP - 530
JO - DNA and Cell Biology
JF - DNA and Cell Biology
IS - 9
ER -