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M. Acharya, L. Huang, M. A. Walter; Prkc Apoptosis Wt1 Regulator Is a Novel Pitx2-Interacting Protein That Regulates Pitx2 Transcriptional Activity in Ocular Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1653.
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Mutations in the homeobox transcription factor PITX2 result in Axenfeld-Reiger syndrome (ARS) which is associated with anterior segment dysgenesis and an increased risk of glaucoma. To understand the pathogenesis of the defects resulting from PITX2 mutations, it is essential to know the normal functions of PITX2 and its interaction with the network of proteins in the eye. Our goal therefore, is looking for novel PITX2-interacting proteins (PIPs) and studies their role in ARS pathogenesis.
Yeast two-hybrid (Y2H) screening was performed using c-DNA library from a human trabecular meshwork (HTM) primary cell line. Identification of a single PIP was further confirmed by retransformation assay with yeast cells and nickel pull-down assay in vitro. As part of characterizing the type of interaction we mapped the domain of interactions in both the PIP and PITX2, analysed subcellular localization and effect of PITX2- mutations on the nature of protein-protein interaction.
After Y2H screening of approximately 1X106 clones, one putative PIP was identified. This novel PIP, named PRKC apoptosis WT1 regulator (PAWR) is reportedly a pro-apoptotic protein capable of selectively inducing apoptosis primarily in cancer cells. Human PAWR is a 340 amino acid protein alternatively known as prostate apoptotic response-4 (PAR-4). The PAWR gene is approximately 99 kb long, and is located on chromosome 12q21 with 7 exons and a transcript length of 1.9 kb. We established that the homeodomain and the adjacent inhibitory domain in PITX2 interact with the C-terminal leucine-zipper domain of PAWR. However, mutations located in the homeodomain of PITX2 have been found to have no effect on this interaction. In ocular cells distribution of endogenous PAWR and PITX2 was found in nucleus where PAWR was found to inhibit PITX2 transcriptional activity.
We discovered a novel PIP involved in inducing apoptosis from Y2H screening of a HTM c-DNA library that regulates PITX2 transcriptional activity in ocular cells. This information might shed some new light in understanding ARS and associated glaucoma pathogenesis since apoptosis of retinal ganglion cells is observed in glaucoma and PAWR acts as an inducer of apoptosis.
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