Abstract
Mutations in retinitis pigmentosa 2 (RP2) account for 10-20% of X-linked retinitis pigmentosa (RP) cases. The encoded RP2 protein is implicated in ciliary trafficking of myristoylated and prenylated proteins in photoreceptor cells. To date >70 mutations in RP2 have been identified. How these mutations disrupt the function of RP2 is not fully understood. Here we report a novel in-frame 12-bp deletion (c.357_368del, p.Pro120_Gly123del) in zebrafish rp2 The mutant zebrafish shows reduced rod phototransduction proteins and progressive retinal degeneration. Interestingly, the protein level of mutant Rp2 is almost undetectable, whereas its mRNA level is near normal, indicating a possible post-translational effect of the mutation. Consistent with this hypothesis, the equivalent 12-bp deletion in human RP2 markedly impairs RP2 protein stability and reduces its protein level. Furthermore, we found that a majority of the RP2 pathogenic mutations (including missense, single-residue deletion, and C-terminal truncation mutations) severely destabilize the RP2 protein. The destabilized RP2 mutant proteins are degraded via the proteasome pathway, resulting in dramatically decreased protein levels. The remaining non-destabilizing mutations T87I, R118H/R118G/R118L/R118C, E138G, and R211H/R211L are suggested to impair the interaction between RP2 and its protein partners (such as ARL3) or with as yet unknown partners. By utilizing a combination of in silico, in vitro, and in vivo approaches, our work comprehensively indicates that loss of RP2 protein structural stability is the predominating pathogenic consequence for most RP2 mutations. Our study also reveals a role of the C-terminal domain of RP2 in maintaining the overall protein stability.
Highlights
Mutations in retinitis pigmentosa 2 (RP2) account for 10 –20% of X-linked retinitis pigmentosa (RP) cases
We previously reported the generation of rp2 knock-out zebrafish by transcription activator-like effector nuclease (TALEN) technology that resulted in a frameshift mutation (c.359_363del, named del5) causing null allele of rp2 [7]
We examined the localization of Rp2 in WT and del12 mutant retinas at the age of 3 months under dark- and light-adapted conditions
Summary
We previously reported the generation of rp knock-out zebrafish by TALEN technology that resulted in a frameshift mutation (c.359_363del, named del5) causing null allele of rp2 [7]. Using the del mutation, which showed protein levels of ϳ30% of WT in cultured cells (in vitro) but Ͻ10% in zebrafish (in vivo), as a reference, we predicted that patients carrying these mutations should have very low levels of RP2 protein (Fig. 4C) These results indicate that for more than half of the missense and small in-frame deletion mutations, loss of RP2 proteins might be the main cause of disease. This prompted us that the C-terminal domain might play a role in stabilizing the RP2 protein To verify this hypothesis, we constructed a series of C-terminaltruncated RP2 mutants (1– 42, 1–192, 1–251, 1–300, 1–330 and 1–340) to mimic the truncation mutations (Fig. 6A) and examined their expression levels in ARPE-19 cells. Ylmaleimide-sensitive factor, vesicle fusing ATPase) [23], PKD2 (polycystin 2, transient receptor potential cation channel) [40], or unknown new partners might play a causal role for developing retinitis pigmentosa in patients carrying T87I and R211H/R211L mutations
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