TULP1 and TUB Are Required for Specific Localization of PRCD to Photoreceptor Outer Segments.

Photoreceptor disc component (PRCD) is a small protein which is exclusively localized to photoreceptor outer segments, and is involved in the formation of photoreceptor outer segment discs. Mutations in PRCD are associated with retinal degeneration in humans, mice, and dogs. The purpose of this work was to identify PRCD-binding proteins in the retina. PRCD protein-protein interactions were identified when implementing the Ras recruitment system (RRS), a cytoplasmic-based yeast two-hybrid system, on a bovine retina cDNA library. An interaction between PRCD and tubby-like protein 1 (TULP1) was identified. Co-immunoprecipitation in transfected mammalian cells confirmed that PRCD interacts with TULP1, as well as with its homolog, TUB. These interactions were mediated by TULP1 and TUB highly conserved C-terminal tubby domain. PRCD localization was altered in the retinas of TULP1- and TUB-deficient mice. These results show that TULP1 and TUB, which are involved in the vesicular trafficking of several photoreceptor proteins from the inner segment to the outer segment, are also required for PRCD exclusive localization to photoreceptor outer segment discs.

PRCD is concentrated at the base of photoreceptor outer segments and is involved in outer segment disc formation.

Mutations of the photoreceptor disc component (PRCD) gene are associated with rod-cone degeneration in both dogs and humans. Prcd is expressed in the mouse eye as early as embryonic day 14. In the adult mouse retina, PRCD is expressed in the outer segments of both rod and cone photoreceptors. Immunoelectron microscopy revealed that PRCD is located at the outer segment rim and that it is highly concentrated at the base of the outer segment. Prcd-knockout mice present with progressive retinal degeneration, starting at 20 weeks of age and onwards. This process is reflected by a significant and progressive reduction of both scotopic and photopic electroretinographic responses and by thinning of the retina, and specifically of the outer nuclear layer, indicating photoreceptor loss. Electron microscopy revealed severe damage to photoreceptor outer segments, which is associated with immigration of microglia cells to the Prcd-knockout retina and accumulation of vesicles in the inter-photoreceptor space. Phagocytosis of photoreceptor outer segment discs by the retinal pigmented epithelium is severely reduced. Our data show that Prcd-knockout mice serve as a good model for retinal degeneration caused by PRCD mutations in humans. Our findings in these mice support the involvement of PRCD in outer segment disc formation of both rod and cone photoreceptors. Furthermore, they suggest a feedback mechanism which coordinates the rate of photoreceptor outer segment disc formation, shedding and phagocytosis. This study has important implications for understanding the function of PRCD in the retina, as well as for future development of treatment modalities for PRCD deficiency in humans.

Pigmented Facial Contact Dermatitis to Benzyl Salicylate: A Comparative Histopathological and Immunohistochemical Study of the Involved Skin and the Positive Patch Test Site.

Pigmented contact dermatitis (PCD) is a noneczematous variant of allergic contact dermatitis, and benzyl salicylate is one of its causes. This type of PCD shows nonlichenoid interface dermatitis with pigment incontinence. We aimed to characterize the earliest histopathological changes of this reaction. A 51-year-old man presented with persistent facial eruption composed of hyperpigmented and hypopigmented macules due to exposure to benzyl salicylate present in his aftershave. The biopsies obtained from hyperpigmented and hypopigmented macules, and from the positive patch test site to benzyl salicylate, showed a nonlichenoid focal vacuolar interface dermatitis with mononuclear cells in the papillary dermis and around the pilosebaceous units, along with melanophages. A MART-1 immunostain showed intact melanocytes in all 3 biopsies. A Fontana-Masson stain demonstrated intact melanin in the basal cell layer of a facial hyperpigmented macule and the patch test site, but melanin was reduced in the biopsy taken from a hypopigmented facial macule. There were more epidermal and dermal CD1a+ Langerhans cells in the patch test biopsy than in the other 2 biopsies. Most of the mononuclear cells were CD3+. The CD4+ to CD8+ ratio was approximately 1:1 in the facial macules; yet, CD4+ cells outnumbered CD8+ cells in the patch test biopsy. There were a few TIA-1+ cells in all 3 biopsies. In conclusion, the earliest histopathological and immunophenotypical events in PCD due to benzyl salicylate are similar to those of longer-standing lesions, i.e., a nonlichenoid focal interface dermatitis involving the epidermis and pilosebaceous unit, along with dermal melanophages.

Ceramide kinase-like (CERKL) interacts with neuronal calcium sensor proteins in the retina in a cation-dependent manner.

PURPOSE: CERKL encodes for a ceramide kinase (CERK)-like protein. CERKL mutations are associated with severe retinal degeneration. Several studies have been conducted to prove a biochemical similarity between CERK and CERKL enzymatic activities. However, so far there has been no evidence that CERKL phosphorylates ceramide or any other lipid substrate in vitro or in vivo. The purpose of this work was to characterize CERKL's function by identification of CERKL-interacting proteins in the mammalian retina. METHODS: CERKL-interacting proteins were identified implementing the Ras-recruitment system (RRS) on a bovine retina cDNA library. Co-immunoprecipitation (co-IP) in transfected cells and in photoreceptor outer segments was used to verify the identified interactions. Serial deletion constructs were used to map the interacting sites. CERKL's kinase activity was tested by a CERK activity assay. RESULTS: We identified an interaction between CERKL and several neuronal calcium sensor (NCS) proteins, including guanylate cyclase activating protein 1 (GCAP1), GCAP2, and recoverin. These interactions were confirmed by co-IP experiments in transfected mammalian cells. Moreover, the interaction between endogenous CERKL and GCAP2 was confirmed by co-IP in photoreceptor outer segments. We found that CERKL-GCAP interaction is cation dependent and is mediated by CERKL's N-terminal region and by GCAPs cation-binding domains (EF-hands 2-4). CONCLUSIONS: This study, which is the first to describe the interactions of CERKL with other retinal proteins, links CERKL to proteins involved in the photoresponse and Ca(2+) signaling, providing important clues for future research required in this direction.