The ubiquitin-like modifier FAT10 inhibits retinal PDE6 activity and mediates its proteasomal degradation

The retina-specific chaperone aryl hydrocarbon interacting protein-like 1 (AIPL1) is essential for the correct assembly of phosphodiesterase 6 (PDE6), which is a pivotal effector enzyme for phototransduction and vision because it hydrolyzes cGMP. AIPL1 interacts with the cytokine-inducible ubiquitin-like modifier FAT10, which gets covalently conjugated to hundreds of proteins and targets its conjugation substrates for proteasomal degradation, but whether FAT10 affects PDE6 function or turnover is unknown. Here, we show that FAT10 mRNA is expressed in human retina and identify rod PDE6 as a retina-specific substrate of FAT10 conjugation. We found that AIPL1 stabilizes the FAT10 monomer and the PDE6-FAT10 conjugate. Additionally, we elucidated the functional consequences of PDE6 FAT10ylation. On the one hand, we demonstrate that FAT10 targets PDE6 for proteasomal degradation by formation of a covalent isopeptide linkage. On the other hand, FAT10 inhibits PDE6 cGMP hydrolyzing activity by noncovalently interacting with the PDE6 GAFa and catalytic domains. Therefore, FAT10 may contribute to loss of PDE6 and, as a consequence, degeneration of retinal cells in eye diseases linked to inflammation and inherited blindness-causing mutations in AIPL1

Mapping the mechanisms of retinal degeneration caused by mutations in the co-chaperone AIPL1

Mutations in the photoreceptor/pineal-expressed gene AIPL1 cause Leber congenital amaurosis (LCA), the most severe form of childhood inherited retinopathy. AIPL1 is a photoreceptor-specific co-chaperone that interacts with HSP90 via a C-terminal tetratricopeptide repeat (TPR) domain to facilitate the correct assembly and activity of retinal cGMP phosphodiesterase (PDE6). The AIPL1 N-terminal FKBP-like domain interacts directly with the isoprenyl moiety of the PDE6 catalytic subunits. We investigated the functional impact of novel LCA-associated AIPL1 variants. Our data reveal that the relative domain organization and integrity of AIPL1 is important for PDE6-mediated catalysis, with variants mapping to one domain also affecting the activity of the other independently folded domain. The functional assessment and confirmation of likely pathogenic AIPL1 variants is moreover important for the accurate diagnosis and effective triage of patients for AIPL1-targeted gene replacement therapy

The integrity and organization of the human AIPL1 functional domains is critical for its role as a HSP90-dependent co-chaperone for rod PDE6

Biallelic mutations in the photoreceptor-expressed aryl hydrocarbon receptor interacting protein-like 1 (AIPL1) are associated with autosomal recessive Leber congenital amaurosis (LCA), the most severe form of inherited retinopathy in early childhood. AIPL1 functions as a photoreceptor-specific co-chaperone that interacts with the molecular chaperone HSP90 to facilitate the stable assembly of the retinal cyclic GMP (cGMP) phosphodiesterase (PDE6) holoenzyme. In this study, we characterized the functional deficits of AIPL1 variations, some of which induce aberrant pre-mRNA AIPL1 splicing leading to the production of alternative AIPL1 isoforms. We investigated the ability of the AIPL1 variants to mediate an interaction with HSP90 and modulate the rod cGMP PDE6 stability and activity. Our data revealed that both the FK506 binding protein (FKBP)-like domain and the tetratricopeptide repeat (TPR) domain of AIPL1 are required for interaction with HSP90. We further demonstrate that AIPL1 significantly modulates the catalytic activity of heterologously expressed rod PDE6. Although the N-terminal FKBP-like domain of AIPL1 binds the farnesylated PDE6α subunit through direct interaction with the farnesyl moiety, mutations compromising the integrity of the C-terminal TPR domain of AIPL1 also failed to modulate PDE6 activity efficiently. These AIPL1 variants moreover failed to promote the HSP90-dependent stabilization of the PDE6α subunit in the cytosol. In summary, we have successfully validated the disease-causing status of the AIPL1 variations in vitro. Our findings provide insight into the mechanism underlying the co-chaperone role of AIPL1 and will be critical for ensuring an early and effective diagnosis of AIPL1 LCA patients