The effects of mutations in protein interaction domain of IRF6
| Autor: | Li-Ting Kung, 龔莉婷 |
|---|---|
| Rok vydání: | 2014 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 102 Interferon regulatory factor 6 (IRF6) belongs to IRF family, which acts as a transcription factor. Several studies showed that IRF6 involves in keratinocyte differentiation and tumor suppression through regulating cell cycle and cell proliferation. Mutations in IRF6 cause van der Woude syndrome (VWS) and popliteal pterygium syndrome (PPS). The characteristic of VWS and PPS are cleft lip with or without cleft palate. Both IRF6 null mice and IRF6 R84C/+ heterozygous mice had cleft palate phenotype, indicate that IRF6 plays an important role on palate development. IRF6 contains a highly conserved DNA-binding domain and a less well-conserved protein interaction domain. Protein interaction domain of other IRF family proteins is important for dimerization, transcription activation, and nuclear translocation. However, the effects of mutations in protein interaction domain of IRF6 remain unclear. To gain in sight of its effect, three VWS hot mutation sites, R250Q, R400W, R412X, and two deletion mutations ∆290 and ∆404 have been constructed to study their biological functions. Within these mutants, R400W protein had shorter half life relative to wild type IRF6 protein (half-life [t1/2] ~ 8 hr versus 18 hr), and R412X and ∆290 proteins have shortest protein half-life for only 2 and 4 hours. Co-immunoprecipitation studies show that all of the mutants were interacting with wild type IRF6, suggesting the formation of heterodimer. Nuclear localization of IRF6 mutants was also investigated. Immunofluorescence showed nuclear accumulation of R412X and ∆290 mutants. And western blot analysis showed that all the mutant proteins could translocate into nucleus as well as wild type protein, while the protein level of R412X and ∆290 mutants were reduced in the cytosolic fraction. LMB treatment showed that CRM1-depedent nuclear export pathway was involved in the regulation of IRF6 subcellular localization. And R412X mutant has lost its nuclear export activity. This suggests that carboxyl terminal of IRF6 is important for its nuclear trafficking. Using qRT-PCR, wild type IRF6 enhanced OVOL1 expression in TE1 cells, whereas R84C and other mutants lose the transcriptional activation function. These data suggest that mutantions in IRF6 protein interation domain affect protein stability, subcellular localization, and transactivation functions. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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