AIPL1 blocking peptide

PURPOSE. AIPL1 mutations cause the severe inherited blindness Leber congenital amaurosis (LCA). The similarity of AIPL1 to tetratricopeptide repeat (TPR) cochaperones that interact with the chaperone Hsp90 and the ability of AIPL1 to suppress the aggregation of NUB1 fragments in a chaperone-like manner suggest that AIPL1 might function as part of a chaperone heterocomplex facilitating retinal protein maturation. In this study the interaction of AIPL1 with molecular chaperones is revealed and functionally characterized. METHODS. AIPL1-interacting proteins were identified using a yeast two-hybrid system, and the effect of AIPL1 pathogenic mutations and sequence requirements mediating the identified interactions were investigated. The interactions were validated by a comprehensive set of biochemical assays, and the ability of the AIPL1-binding partners to cooperate with AIPL1 in the suppression of NUB1 fragment aggregation was assessed. RESULTS. AIPL1 interacts with the molecular chaperones Hsp90 and Hsp70. Mutations within the TPR domain of AIPL1 or removal of the chaperone TPR acceptor site abolished the interactions. Importantly, LCA-causing mutations in AIPL1 also compromised these interactions, suggesting that the essential function of AIPL1 in photoreceptors may involve the interaction with Hsp90 and Hsp70. Examination of the role of these chaperones in AIPL1 chaperone activity demonstrated that AIPL1 cooperated with Hsp70, but not with Hsp90, to suppress the formation of NUB1 inclusions. CONCLUSIONS. These findings suggest that AIPL1 may cooperate with both Hsp70 and Hsp90 within a retina-specific chaperone heterocomplex and that the specialized role of AIPL1 in photoreceptors may therefore be facilitated by these molecular chaperones.

Mutations in the aryl hydrocarbon receptor-interacting protein-like 1 (AlPL1) gene have been found in patients with Leber congenital amaurosis (LCA), a severe, early-onset form of retinal degeneration. To determine the normal function of AlPL1 and to better understand how mutations in this gene cause disease, we performed a yeast two-hybrid screen to identify AlPL1-interacting proteins in the retina. One of the identified interacting proteins corresponds to NUB1 (NEDD8 Ultimate Buster 1), which is thought to control many biological events, especially cell cycle progression, by downregulating NEDD8 expression. The AlPL1-NUB1 interaction was verified by co-immunoprecipitation studies in Y79 retinoblastoma cells, demonstrating that this interaction occurs within cells that share a number of features with retinal progenitor cells. Furthermore, we examined the localization of the AlPL1 protein within developing and adult retinas, and found that AlPL1 is present in the developing photoreceptor layer of the human retina and within the photoreceptors of the adult retina. Similar to AlPL1, NUB1 is also expressed in the developing and adult retina. Therefore, it is possible that the early-onset form of retinal degeneration seen in LCA patients with AlPL1 mutations may be due to a defect in the regulation of cell cycle progression during photoreceptor maturation. These data raise the possibility that AlPL1 is important for appropriate photoreceptor formation during development and/or survival following differentiation.