Molecular characterization of the interaction between peripherin-2 and opsins in rod and cone photoreceptors

The tetraspanin peripherin-2 is a glyco-membrane protein exclusively expressed in the outer segments of rod and cone photoreceptors. Mutations in peripherin-2 are associated with retinal disorders characterized by Degeneration of rod or cone cells. Previous unpublished work identified peripherin-2 as a potential novel part of the protein complex comprising the B-subunit of the cyclic nucleotide-gated channel (CNGB1a and the light detector rhodopsin. In the first part of this study, using a combination of protein biochemical and FRET approaches in transfected HEK293 cells and in virally transduced murine rod outer segments, it could be demonstrated that peripherin-2 simultaneously binds to both, CNGB1a and rhodopsin. The interaction between peripherin-2 and rhodopsin was not described in previous studies. The binding domain mediating the peripherin-2/rhodopsin interaction could be narrowed down to the fourth transmembrane domain (TM4) of peripherin-2. Finally, the data revealed that the G266D point mutation in TM4 of peripherin-2 that is linked to a rod degenerative disease selectively disrupts the peripherin-2/rhodopsin interaction. To analyze if peripherin-2 also binds to cone opsins in the second part of this study, a similar experimental approach was conducted as used for the investigation of the peripherin-2/rhodopsin interaction. In this context, it was unveiled that peripherin-2 binds to both, short wavelength-and medium wavelength-sensitive cone opsin (S-opsin and M-opsin, respectively) in transfected HEK293 cells and in outer segments of transduced murine cones. Co-immunoprecipitation and quantitative FRET analysis revealed that binding of peripherin-2 to M-opsin was stronger than the peripherin-2/S-opsin interaction. This result was supported by transmission electron microscopy studies using gold particles coupled to opsin- and peripherin-2-specific antibodies. Finally, quantitative FRET analysis in transfected HEK293 cells and in transduced cone outer segments demonstrated that the V268I Point mutation in TM4 of peripherin-2 associated with a degenerative cone disease significantly attenuates the peripherin-2/M-opsin interaction. Taken together, this study provides a proof-of-principle for FRET-based analysis of protein-protein interactions in the outer segments of rod and cone photoreceptors. This approach led to the identification of hitherto unknown Protein complexes between peripherin-2 and opsins suggesting a novel physiological role of peripherin-2 in rods and cones. Finally, Analysis of disease-linked point mutations unveiled the molecular determinants of the peripherin-2/opsin interaction. These results might contribute to understanding the differential penetrance of certain point mutations in rods and cones

Molecular characterization of the interaction between peripherin-2 and opsins in rod and cone photoreceptors

The tetraspanin peripherin-2 is a glyco-membrane protein exclusively expressed in the outer segments of rod and cone photoreceptors. Mutations in peripherin-2 are associated with retinal disorders characterized by Degeneration of rod or cone cells. Previous unpublished work identified peripherin-2 as a potential novel part of the protein complex comprising the B-subunit of the cyclic nucleotide-gated channel (CNGB1a and the light detector rhodopsin. In the first part of this study, using a combination of protein biochemical and FRET approaches in transfected HEK293 cells and in virally transduced murine rod outer segments, it could be demonstrated that peripherin-2 simultaneously binds to both, CNGB1a and rhodopsin. The interaction between peripherin-2 and rhodopsin was not described in previous studies. The binding domain mediating the peripherin-2/rhodopsin interaction could be narrowed down to the fourth transmembrane domain (TM4) of peripherin-2. Finally, the data revealed that the G266D point mutation in TM4 of peripherin-2 that is linked to a rod degenerative disease selectively disrupts the peripherin-2/rhodopsin interaction. To analyze if peripherin-2 also binds to cone opsins in the second part of this study, a similar experimental approach was conducted as used for the investigation of the peripherin-2/rhodopsin interaction. In this context, it was unveiled that peripherin-2 binds to both, short wavelength-and medium wavelength-sensitive cone opsin (S-opsin and M-opsin, respectively) in transfected HEK293 cells and in outer segments of transduced murine cones. Co-immunoprecipitation and quantitative FRET analysis revealed that binding of peripherin-2 to M-opsin was stronger than the peripherin-2/S-opsin interaction. This result was supported by transmission electron microscopy studies using gold particles coupled to opsin- and peripherin-2-specific antibodies. Finally, quantitative FRET analysis in transfected HEK293 cells and in transduced cone outer segments demonstrated that the V268I Point mutation in TM4 of peripherin-2 associated with a degenerative cone disease significantly attenuates the peripherin-2/M-opsin interaction. Taken together, this study provides a proof-of-principle for FRET-based analysis of protein-protein interactions in the outer segments of rod and cone photoreceptors. This approach led to the identification of hitherto unknown Protein complexes between peripherin-2 and opsins suggesting a novel physiological role of peripherin-2 in rods and cones. Finally, Analysis of disease-linked point mutations unveiled the molecular determinants of the peripherin-2/opsin interaction. These results might contribute to understanding the differential penetrance of certain point mutations in rods and cones

In Vivo Analysis of Disease-Associated Point Mutations Unveils Profound Differences in mRNA Splicing of Peripherin-2 in Rod and Cone Photoreceptors

Point mutations in peripherin-2 (PRPH2) are associated with severe retinal degenerative disorders affecting rod and/or cone photoreceptors. Various disease-causing mutations have been identified, but the exact contribution of a given mutation to the clinical phenotype remains unclear. Exonic point mutations are usually assumed to alter single amino acids, thereby influencing specific protein characteristics;however, they can also affect mRNA splicing. To examine the effects of distinct PRPH2 point mutations on mRNA splicing and protein expression in vivo, we designed PRPH2 minigenes containing the three coding exons and relevant intronic regions of human PRPH2. Minigenes carrying wild type PRPH2 or PRPH2 exon 2 mutations associated with rod or cone disorders were expressed in murine photoreceptors using recombinant adeno-associated virus (rAAV) vectors. We detect three PRPH2 splice isoforms in rods and cones: correctly spliced, intron 1 retention, and unspliced. In addition, we show that only the correctly spliced isoform results in detectable protein expression. Surprisingly, compared to rods, differential splicing leads to lower expression of correctly spliced and higher expression of unspliced PRPH2 in cones. These results were confirmed in qRT-PCR experiments from FAC-sorted murine rods and cones. Strikingly, three out of five cone disease-causing PRPH2 mutations profoundly enhanced correct splicing of PRPH2, which correlated with strong upregulation of mutant PRPH2 protein expression in cones. By contrast, four out of six PRPH2 mutants associated with rod disorders gave rise to a reduced PRPH2 protein expression via different mechanisms. These mechanisms include aberrant mRNA splicing, protein mislocalization, and protein degradation. Our data suggest that upregulation of PRPH2 levels in combination with defects in the PRPH2 function caused by the mutation might be an important mechanism leading to cone degeneration. By contrast, the pathology of rod-specific PRPH2 mutations is rather characterized by PRPH2 downregulation and impaired protein localization

Agonist-mediated switching of ion selectivity in TPC2 differentially promotes lysosomal function

Ion selectivity is a defining feature of a given ion channel and is considered immutable. Here we show that ion selectivity of the lysosomal ion channel TPC2, which is hotly debated (Calcraft et al., 2009;Guo et al., 2017;Jha et al., 2014;Ruas et al., 2015;Wang et al., 2012), depends on the activating ligand. A high-throughput screen identified two structurally distinct TPC2 agonists. One of these evoked robust Ca2+-signals and non-selective cation currents, the other weaker Ca2+-signals and Na+-selective currents. These properties were mirrored by the Ca2+ mobilizing messenger, NAADP and the phosphoinositide, PI(3,5)P-2, respectively. Agonist action was differentially inhibited by mutation of a single TPC2 residue and coupled to opposing changes in lysosomal pH and exocytosis. Our findings resolve conflicting reports on the permeability and gating properties of TPC2 and they establish a new paradigm whereby a single ion channel mediates distinct, functionally-relevant ionic signatures on demand

Global, regional, and national burden of colorectal cancer and its risk factors, 1990–2019: a systematic analysis for the Global Burden of Disease Study 2019

Funding: F Carvalho and E Fernandes acknowledge support from Fundação para a Ciência e a Tecnologia, I.P. (FCT), in the scope of the project UIDP/04378/2020 and UIDB/04378/2020 of the Research Unit on Applied Molecular Biosciences UCIBIO and the project LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy i4HB; FCT/MCTES through the project UIDB/50006/2020. J Conde acknowledges the European Research Council Starting Grant (ERC-StG-2019-848325). V M Costa acknowledges the grant SFRH/BHD/110001/2015, received by Portuguese national funds through Fundação para a Ciência e Tecnologia (FCT), IP, under the Norma Transitória DL57/2016/CP1334/CT0006.proofepub_ahead_of_prin

AAV vectors for FRET-based analysis of protein-protein interactions in photoreceptor outer segments

Fluorescence resonance energy transfer (FRET) is a powerful method for the detection and quantification of stationary and dynamic protein-protein interactions. Technical limitations have hampered systematic in vivo FRET experiments to study protein-protein interactions in their native environment. Here, we describe a rapid and robust protocol that combines adeno-associated virus (AAV) vector-mediated in vivo delivery of genetically encoded FRET partners with ex vivo FRET measurements. The method was established on acutely isolated outer segments of murine rod and cone photoreceptors and relies on the high co-transduction efficiency of retinal photoreceptors by co-delivered AAV vectors. The procedure can be used for the systematic analysis of protein-protein interactions of wild type or mutant outer segment proteins in their native environment. Conclusively, our protocol can help to characterize the physiological and pathophysiological relevance of photoreceptor specific proteins and, in principle, should also be transferable to other cell types

Protein expression of PRPH2 mutants linked to adRP.

<p>(A-F) Immunohistology of retinas transduced with PRPH2 minigenes containing single point mutations as indicated under the control of the hRHO promoter. Scale bar represents 20 μm. (G) Western blot analysis from membrane preparations of four pooled murine retinas from four animals transduced with the PRPH2 minigenes shown in (A-F) on P14. All retinas were collected three weeks post injection. The arrowhead indicates a degradation band detected at 42 kDa. Ctrl, protein lysates from non-injected control retinas. (H) Semi-quantitative analysis of the results shown in (G). For quantification, three technical replicates were conducted and PRPH2 expression was normalized to the ATPase expression. All data are given as mean values and error bars represent the SEM. Statistical analysis was performed using one-way ANOVA followed by the Dunett’s test. *, p< 0.05; **, p< 0.01; ***, p< 0.001. n.s., not significant.</p

Impaired targeting of truncated PRPH2.

<p>Immunohistology of transduced murine retinas showing rod- (B) and cone-specific (C) expression of a truncated version of PRPH2. (A) Truncated PRPH2 contains only exon 1 and a downstream stop codon (indicated by “X”) mimicking translation from unspliced PRPH2 mRNA and PRPH2 mRNA with intron 1 retention. Staining for B1a and M-ops was used to label rod and cone photoreceptors, respectively. Truncated PRPH2 is not transported to outer segments and is almost exclusively present in inner segments and somata of photoreceptors. Scale bar represents 20 μm.</p

Splice analysis of PRPH2 WT and mutant minigenes in rods and cones.

<p>(A) Representative RT-PCR from cDNA generated from total RNA three weeks post injection from retinas injected with wild-type and mutant rP-mg (left) or cP-mg (right) on P14. Ctrl, control containing the cDNA from non-transduced retina. The single bands of the relevant splice products are numbered (1–4) and highlighted by arrowheads. (B) Schematic representation of the detected splice variants using primers binding to the 3’-end of citrine and to the 5’-end of exon 3 as indicated by the arrows. The numbers of the constructs correspond to the bands marked in (A). (C-E) Semi-quantitative analysis of the relative intensities of the unspliced (C), intron 1 retention (D), and correctly spliced (E) PRPH2 transcripts. For each PRPH2 minigene, the mean percentage of the intensities of these three variants relative to the total intensity (given as sum of the single intensities) was calculated from five RT-PCR analyses conducted with a variable number of cycles (25–27 for rods and 30–32 for cones, respectively). Significance test of the rod or cone mutants to the corresponding wild type (one-way ANOVA followed by Dunett’s test) was performed for rP-mg and cP-mg, respectively. All data were shown as mean values and the error bars represent the standard error of the mean (SEM). *, p< 0.05; **, p< 0.01; ***, p< 0.001. DS, splice donor site.</p