Yeast two-hybrid analysis of a human trabecular meshwork cDNA library identified EFEMP2 as a novel PITX2 interacting protein.
Mol Vis
; 18: 2182-9, 2012.
Article
en En
| MEDLINE
| ID: mdl-22919265
ABSTRACT
PURPOSE:
Mutations in the homeobox transcription factor paired-like homeodomain transcription factor 2 (PITX2) cause Axenfeld-Reiger syndrome (ARS), which is associated with anterior segment dysgenesis (ASD) and glaucoma. To understand ARS pathogenesis, it is essential to know the normal functions of PITX2 and the proteins with which PITX2 interacts in the eye. Therefore, we used a unique cDNA library that we created from human trabecular meshwork (TM) primary cells to discover PITX2-interacting proteins (PIPs).METHODS:
A human TM cDNA library was created from primary cells in the ProQuest Two-Hybrid prey vector pEXP-AD502. Human PITX2A and PITX2C isoforms were used independently as "bait" to identify novel PIPs. A total of 1.25×106 clones were screened by yeast two-hybrid (Y2H) analyses. PIPs obtained from each Y2H experiment were confirmed by yeast retransformation and mammalian co-immunoprecipitation assays.RESULTS:
EGF-containing fibulin-like extracellular matrix protein 2 (EFEMP2) was identified by both PITX2A and PITX2C isoforms as a novel PIP from Y2H analyses. EFEMP2 is 443 amino acids long with six epidermal growth factor (EGF)-like modules and one fibulin-like module. The PITX2-interaction domain in EFEMP2 lies between the second EGF-like module and the COOH-terminal fibulin-like module. Co-immunoprecipitation assays in COS-7 cells confirmed the interaction between PITX2 and EFEMP2.CONCLUSIONS:
We discovered EFEMP2 as a novel PITX2-interacting protein. Further, our cDNA library made from human TM primary cells is a unique and effective resource to identify novel interacting proteins for glaucoma and ASD candidates. This resource could be used both for discovery and validation of interactomes identified from in silico analysis.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Malla Trabecular
/
Factores de Transcripción
/
Proteínas de la Matriz Extracelular
/
Proteínas de Homeodominio
Tipo de estudio:
Diagnostic_studies
/
Prognostic_studies
Límite:
Animals
/
Humans
Idioma:
En
Revista:
Mol Vis
Asunto de la revista:
BIOLOGIA MOLECULAR
/
OFTALMOLOGIA
Año:
2012
Tipo del documento:
Article
País de afiliación:
Canadá