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Tetraspanin membrane proteins encompass a functionally diverse group of proteins involved in cell adhesion, cell recognition and membrane fusion events. These proteins are characterized by a highly conserved extra-cellular domain, known as the EC-2 domain that is involeid in formation of disulfide linked oligomers and functionally diverse C-termini. We are interested in understanding the role of two retinal specific tetraspanins, peripherin-2, the product of the rds gene, and it’s non-glycosylated homologue rom-1, in the development and progression of degenerative diseases. A murine model of retinitis pigmentosa (RP) in which a 10 kb insertion of exogeneous DNA results in an rds null allele as well as a rom-1 knockout mouse suggest that although peripherin-2 and rom-1 cooperate to generate healthy photoreceptors, they are not functionally equivalent and rom-1 likely plays a subsidiary role. Peripherin-2 and rom-1 form both homo and hetero-tetramers with peripherin-2 shown to oligomerize further to form octamers. Our studies focus on the regulatory and functional role played by the C-terminus of peripherin-2 in the formation of photoreceptor cells and in the maintenance of cell stability through the organization of intra-membraneous sacs known as disks. The C-terminal domain is required in the formation of newly developing membrane evaginations destined to become disks as well as their alignment along the outer segment portion of the cell. Domains within this region are postulated to be necessary for protein and vesicle targeting, calcium dependent calmodulin binding, membrane fusion, and the regulation of this fusion through the binding of a newly identified protein, melanoregulin. Using a combination of pull-down, immuno-precipitation, proteomic, confocal and live cell imaging techniques and as well as solution NMR, we have begun to define the regulatory surface of peripehirn-2. An understanding of how this region aids in disk membrane monogenesis and in maintaining photoreceptor viability is essential for the development of viable therapeutic approaches to slow the progression of these degenerations. A second series of studies is designed to address the role of cholesterol in photoreceptor function and dysfunction during degeneration. Using a combination of membrane micro-domain isolation techniques, fluorescence anisotropy studies and functional readouts we have shown that membrane cholesterol is heterogeneously distributed within disks as a function of age and spatial distribution. Moreover this distribution results in compromised GPCR function due to changes in the membrane micro-environment and membrane fusion process. In a developing project, we have expanded our interest in cholesterol to address the role of this lipid in mineralizing tissue. Mutations within various cholesterol biosynthetic enzymes results in chondrodysplasia puntacta; punctate calcification of cartilage, leading in its’ more severe forms to mental retardation and in milder forms to skeletal abnormities including craniofacial defects. Using a combination of RNA interference techniques, membrane lipid asymmetry measurements and confocal microscopy we have focused our effort on understanding how enzymes involved in the maintenance of lipid asymmetry when devoid of a cholesterol- rich membrane environment begin to dysfunction leading to alterations in the formation of the core mineralizing component, the matrix vesicle. Through a series of collaborations we are utilizing AFM and primary cell culture models to understand how depletion of cell membrane cholesterol alters mineral formation and deposition. A new area of research is focused on mechanisms of microbial pathogenesis and immunotoxicity in collaboration with Dr. B.J. Shenker, SDM. Over the past several years, significant progress has been made in our understanding of the etiology and pathogenesis of oral infectious diseases. However, the nature and contribution of the immune system to these disorders remain unclear. We propose that the immune system plays a primary role in minimizing and/or preventing infection and immunoregulatory abnormalities contribute to the pathogenesis of and susceptibility to oral infectious disorders such as periodontal disease. Previous studies in the Shenker lab have demonstrated that the A.actinomycetemcomitans immunoinhibitory protein is a member of the family of cytolethal distending toxins; this toxin induces G2 arrest in lymphocytes and eventually leads to activation of the apoptotic cascade. The cytolethal distending toxins (Cdts) are a family of heat-labile protein cytotoxins produced by several different bacterial species including diarrheal disease-causing enteropathogens such as some Escherichia coli isolates, Campylobacter jejuni, Shigella species, Haemophilus ducreyi and Actinobacillus actinomycetemcomitan. Regardless of the microbial source of Cdt, the heterotrimeric holotoxin functions as an AB2 toxin where CdtB is the active (A) unit and the complex of CdtA and CdtC comprise the binding (B) unit. CdtA and CdtC are required for the toxin to associate with lipid microdomains within lymphocyte membranes and Cdt-mediated toxicity is dependent upon the integrity of these lipid domains. The active (A) unit, CdtB, exhibits sequence homology with inositol polyphosphate 5-phosphatases specifically within highly conserved regions corresponding to the active site. Analysis of CdtB indicates that its structure can be superimposed on inositol polyphosphate 5-phosphatase with an rmsd of 3.5Å over 183 Cα atoms. In our most recent work we report that CdtB exhibits phosphatidylinositol-3,4,5-triphosphate phosphatase (PI-3,4,5-P3) phosphatase activity similar to that of the tumor suppressor phosphatases, PTEN and SHIP1. Mutation analysis indicates that Cdt toxicity correlates with phosphatase activity; furthermore, lymphocytes treated with toxin exhibit reduced PI-3,4,5-P3 levels. Finally, lymphocyte sensitivity to Cdt-induced G2 arrest correlates with intracellular levels of PI-3,4,5-P3. Our observations suggest that CdtB, like SHIP1 and PTEN, mediates its regulatory effects by dephosphorylating PI-3,4,5-P3 and thereby modulating the activity of pleckstrin homology proteins such as Akt. Future studies are underway to assess the role of membrane association in toxin entry as well as to determine the mechanism of Cdt-B mediated toxicity. Selected Publications: Damek-Poprawa, M., E. Golub, L. Otis, G. Harrison, C. Phillips and K. Boesze-Battaglia (2006) Chondrocytes utilize a cholesterol dependent lipid translocator to externalize phosphatidylserine. Biochemistry 45:3325-3336. Boesze-Battaglia, K., D. Besack, T. McKay, A. Zekavat, L. Otis, K. Jordan-Sciutto and B.J. Shenker (2006) Cholesterol-rich membrane microdomains mediate cell cycle arrest induced by Actinobacillus actinomycetemcomitans cytolethal distending toxin. Cellular Microbiology 8:823-36. Damek-Poprawa, M., J. Krouse, C. Gretzula and K. Boesze-Battaglia (2005) A novel tetraspanin fusion protein, peripherin-2, requires a region upstream of the fusion domain for activity. J. Biol. Chem. 280:9217-24. Boesze-Battaglia, K., J. Dispoto and M.A. Kahoe (2002) Association of a photoreceptor specific tetraspanin protein, ROM-1 with triton X-100 resistant membrane rafts from rod outer segment disk membranes. J. Biol. Chem. 277:41843-41849. Albert, A. and K. Boesze-Battaglia (2005) The role of cholesterol in rod outer segment membranes. Progress in Lipid Research 44:99-124. Boesze-Battaglia, K and A.F.X. Goldberg (2002) Photoreceptor renewal: a role for peripherin/RDS. International Reviews in Cytology 217:183-225.
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