NUB1, a Protein Involved in the Regulation of Apoptosis and Cell Cycle Progression, Interacts With AIPL1, a Protein Associated With Leber Congenital Amaurosis

Abstract

Abstract: : Purpose:We previously identified the aryl-hydrocarbon receptor interacting protein-like 1 (AIPL1) gene, mutations of which cause approximately 10% of Leber congenital amaurosis. As AIPL1 is a novel gene, it is necessary to determine its normal function as the next step toward understanding the molecular and biochemical cause of blindness in these patients. The presence of three highly conserved tetratricopeptide (TPR) motifs in the AIPL1 protein sequence suggested a role as a chaperone protein and made yeast-two hybrid studies a promising approach to determine a potential target of this activity. Methods:The bovine Aipl1 cDNA in the pGBK-T7 vector was the bait in a GAL4 yeast-two hybrid testing system to screen a bovine retinal cDNA library (W. Baehr). Prey plasmids were isolated from the positive clones of the library screening and were co-transformed with either the bait or the empty pGBK-T7 vector into the yeast cells to confirm the interactions The human ortholog was then tested to confirm that the human proteins could also interact in this system. For further confirmation, co-immunoprecipitation (Co-IP) was performed in COS cells using FLAG-AIPL1 and several controls. Results:Two independent NUB1 (NEDD8 Ultimate Buster-1) clones were identified in the yeast-two hybrid screen, and human AIPL1 also interacted with NUB1 in this system. Co-IP experiments confirmed the positive interaction of NUB1 with AIPL1, as only FLAG-AIPL1, and none of the controls, could co-immunoprecipitate endogenous NUB1 from the COS cells. Conclusion:NUB1 is a negative regulator of the NEDD8 conjugation system, which regulates many biological events, including cell cycle transition and apoptosis. Data from the current study suggest NUB1 as the target of AIPL1 chaperone activity. It is possible that mutated AIPL1 in patients interferes with the ability of AIPL1 to chaperone or translocate NUB1 into the nucleus, resulting in the accumulation of NUB1 in the cytosol. This accumulation may be the cause of cell death and retinal degeneration in these patients. Supported by the Kirchgessner Foundation, the Knights Templar Eye Foundation, the Foundation Fighting Blindness, Fight for Sight, Research Division of Prevent Blindness America, the RPB, and NIH grants EY00395 and EY03040.

Keywords: 562 retinal degenerations: hereditary • 343 chaperones • 341 cell death/apoptosis