Disease mechanism for retinitis pigmentosa (RP11) caused by mutations in the splicing factor gene PRPF31

This study investigates the functional consequences of two mutations, A194E and A216P, in the splicing factor gene PRPF31 linked to autosomal dominant retinitis pigmentosa (RP11). Using a yeast complementation assay, we demonstrate that introduction of the human A216P mutation into the yeast orthologue PRP31p results in only partial rescue of growth at the restrictive temperature, indicating that splicing function is not fully restored. An in vivo assay of splicing function in human cells using a bovine rod opsin splicing template did not detect any defect in splicing efficiency or accuracy attributable to either mutation, suggesting that neither has a dominant negative effect on splicing. However, western analysis and immunofluorescence microscopy of mammalian cells transfected with PRPF31 revealed that both mutations substantially hinder translocation of the protein into the nucleus. The overall effect may thus be an insufficiency in splicing function, which is revealed only under conditions of elevated splicing demand. With the need to replenish disc proteins on a daily basis, such conditions will exist in rod photoreceptors and this may underlie the disease pathology

Disease mechanism for retinitis pigmentosa (RP11) caused by mutations in the splicing factor gene PRPF31

This study investigates the functional consequences of two mutations, A194E and A216P, in the splicing factor gene PRPF31 linked to autosomal dominant retinitis pigmentosa (RP11). Using a yeast complementation assay, we demonstrate that introduction of the human A216P mutation into the yeast orthologue PRP31p results in only partial rescue of growth at the restrictive temperature, indicating that splicing function is not fully restored. An in vivo assay of splicing function in human cells using a bovine rod opsin splicing template did not detect any defect in splicing efficiency or accuracy attributable to either mutation, suggesting that neither has a dominant negative effect on splicing. However, western analysis and immunofluorescence microscopy of mammalian cells transfected with PRPF31 revealed that both mutations substantially hinder translocation of the protein into the nucleus. The overall effect may thus be an insufficiency in splicing function, which is revealed only under conditions of elevated splicing demand. With the need to replenish disc proteins on a daily basis, such conditions will exist in rod photoreceptors and this may underlie the disease pathologyThis work was supported by a programme grant from the Wellcome Trust and by project grants from the UK Medical Research Council and the Foundation Fighting Blindness

Purification, characterisation and intracellular localisation of aryl hydrocarbon interacting protein-like 1 (AIPL1) and effects of mutations associated with inherited retinal dystrophies

AbstractMutations in AIPL1 are associated with Leber Congenital Amaurosis (LCA), a major cause of childhood blindness, yet the cellular function of the encoded protein has yet to be fully elucidated. In order to investigate the biochemistry of AIPL1, we have developed a system for the expression of the recombinant protein in bacteria and its subsequent purification. The secondary structure and thermostability of wild-type and mutant proteins have been examined by circular dichroism (CD) spectroscopy. Some of the variants, notably W278X and P376S, had markedly different secondary structure compositions, indicating that the proteins had not folded properly, whilst W278X and T114I were particularly thermolabile. When eukaryotic cells were transfected with the AIPL1 expression constructs, we show by immunofluorescence microscopy that wild-type protein is distributed throughout the nucleus and cytoplasm. Several of the mutants give similar results. With two of the disease-associated variants (W278X and A336Δ2), however, the protein remains in the cytoplasm in aggresome-like particles. These particles were shown to be ubiquitinated, indicating that the mutant protein had been tagged for proteosomal degradation. On this basis, we can conclude that wild-type protein is expressed in a soluble and folded manner, and that some of the disease-associated mutant proteins are nonfunctional because they are insoluble and are degraded by the cell. Other mutations appear to have a more localised effect on secondary structure, which does not result in insolubility or affect protein targeting, but reduces the stability of the protein at human body temperature

Amelogenin Processing by MMP-20 Prevents Protein Occlusion Inside Calcite Crystals

Calcite crystals were grown in the presence of full-length amelogenin and during its proteolysis by recombinant human matrix metalloproteinase 20 (rhMMP-20). Recombinant porcine amelogenin (rP172) altered the shape of calcite crystals by inhibiting the growth of steps on the {104} faces and became occluded inside the crystals. Upon co-addition of rhMMP-20, the majority of the protein was digested resulting in a truncated amelogenin lacking the C-terminal segment. In rP172-rhMMP-20 samples, the occlusion of amelogenin into the calcite crystals was drastically decreased. Truncated amelogenin (rP147) and the 25-residue C-terminal domain produced crystals with regular shape and less occluded organic material. Removal of the C-terminal diminished the affinity of amelogenin to the crystals and therefore prevented occlusion. We hypothesize that hydroxyapatite (HAP) and calcite interact with amelogenin in a similar manner. In the case of each material, full-length amelogenin binds most strongly, truncated amelogenin binds weakly, and the C-terminus alone has the weakest interaction. Regarding enamel crystal growth, the prevention of occlusion into maturing enamel crystals might be a major benefit resulting from the selective cleavage of amelogenin at the C-terminus by MMP-20. Our data have important implications for understanding the hypomineralized enamel phenotype in cases of <i>amelogenesis imperfecta</i> resulting from MMP-20 mutations and will contribute to the design of enamel inspired biomaterials

Amelogenin processing by MMP-20 prevents protein occlusion inside calcite crystals

10.1021/cg300754aCrystal Growth and Design12104897-4905CGDE