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The deubiquitylation of target proteins is mediated by deubiquitylating enzymes (DUB) such as OTUB1, which plays an important role in immune response, cell cycle progression, and DNA repair. Within these processes, OTUB1 reduces the ubiquitylation of target proteins in two distinct ways, either by using its catalytic DUB activity or in a noncatalytic manner by inhibiting the E2-conjugating enzyme. Here, we show that the ubiquitin-like modifier FAT10 regulates OTUB1 stability and functionality in different ways. Covalent FAT10ylation of OTUB1 resulted in its proteasomal degradation, whereas a noncovalent interaction stabilized OTUB1. We provide evidence that OTUB1 interacts directly with FAT10 and the E2-conjugating enzyme USE1. This interaction strongly stimulated OTUB1 DUB activity toward Lys-48–linked diubiquitin. Furthermore, the noncovalent interaction between FAT10 and OTUB1 not only enhanced its isopeptidase activity toward Lys-48–linked ubiquitin moieties but also strengthened its noncatalytic activity in reducing Lys-63 polyubiquitylation of its target protein TRAF3 (TNF receptor–associated factor 3). Additionally, the cellular clearance of overall polyubiquitylation by OTUB1 was strongly stimulated through the presence of FAT10. The addition of FAT10 also led to an increased interaction between OTUB1 and its cognate E2 UbcH5B, implying a function of FAT10 in the inhibition of polyubiquitylation. Overall, these data indicate that FAT10 not only plays a role in covalent modification, leading its substrates to proteasomal degradation, but also regulates the stability and functionality of target proteins by interacting in a noncovalent manner. FAT10 is thereby able to exert a major influence on ubiquitylation processes.

The ubiquitin-like modifier FAT10 (also called ubiquitin D (UBD)) interacts noncovalently with a substantial number of proteins and also gets covalently conjugated to many substrate proteins, leading to their degradation by the 26S proteasome. FAT10 comprises two loosely folded ubiquitin-like domains that are connected by a flexible linker, and this unusual structure makes it highly prone to aggregation. Here, we report methods to purify high amounts of soluble recombinant FAT10 for various uses, such as in vitro FAT10ylation assays. In addition, we describe how to generate and handle overexpressed as well as endogenous FAT10 in cellulo for use in immunoprecipitations, Western blot analyses, and FAT10 degradation studies.

The ubiquitin-like modifier FAT10 (also called ubiquitin D (UBD)) interacts noncovalently with a substantial number of proteins and also gets covalently conjugated to many substrate proteins, leading to their degradation by the 26S proteasome. FAT10 comprises two loosely folded ubiquitin-like domains that are connected by a flexible linker, and this unusual structure makes it highly prone to aggregation. Here, we report methods to purify high amounts of soluble recombinant FAT10 for various uses, such as in vitro FAT10ylation assays. In addition, we describe how to generate and handle overexpressed as well as endogenous FAT10 in cellulo for use in immunoprecipitations, Western blot analyses, and FAT10 degradation studies.

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.