Molecular recruitment as a basis for negative dominant inheritance? Propagation of misfolding in oligomers of IMPDH1, the mutated enzyme in the RP10 form of retinitis pigmentosa

Retinitis pigmentosa, causing progressive blindness, is genetically heterogeneous. RP10, due to a defect in inosine monophosphate dehydrogenase 1 (IMPDH1), shows autosomal dominant inheritance. Recombinantly expressed clinical mutants show unaltered kinetic behaviour. It is unclear why reportedly impaired DNA binding is important and how it would explain negative dominance. An alternative view relates to the mutant proteins' tendency to aggregate. Regarding negative dominance, a key question is whether the defective protein can subvert the function of its normal counterpart in the same cell. Potentially, the homotetrameric structure of IMPDH1 might offer a vehicle for such an effect. We have established a reliable protocol for reproducible refolding of recombinantly expressed IMPDH1 in vitro. Clinical mutants R224P and D226N both show impaired folding. For equimolar mixtures of normal and mutant enzymes, independent refolding would predict activity regain midway between pure mutant and pure normal. Under various conditions regain is close to the mutant figure, suggesting that, in hybrid tetramers, mutant subunits impose their faulty conformation on normal partners. The observed molecular recruitment is a negative counterpart of the intra-allelic complementation, also mediated via oligomeric structure and postulated many years ago by Fincham. These findings appear potentially to account for the negative dominant inheritance. This interpretation must be provisional at present, as the predominant transcript in retina is an alternatively spliced version not fully identical to that used in our study. The results nevertheless have a general significance in pointing to a mechanism for negative dominance that could be widespread.

► IMPDH1 can be successfully refolded and purified from inclusion bodies. ► Soluble IMPDH1 can be unfolded with guanidinium chloride and refolded in high yield. ► RP10 mutants R224P, D226N of IMPDH1 refold poorly and impair folding of normal IMPDH1. ► CD studies confirm faulty structure and thermal instability in IMPDH1 mutant D226N. ► Molecular recruitment by faulty subunits explains negative dominance in RP10.