here are three major areas of research in my laboratory.
(1) Role of the adaptor protein CASP: (Supported by NSERC). It is generally thought that the immune system is able to recognize and eliminate newly arising tumor cells. One of the major mediators of the inborn or "natural" immune system is the natural killer (NK) cell. Several years ago, using a subtractive hybridization technique we discovered several novel human genes that are expressed in cytotoxic NK cells.

We were the first group to clone and characterize a human protein that contained the Sec7 domain. Sec mutants were originally discovered and characterized in yeast and involve the secretory pathway. In other organisms these proteins are now referred to as cytohesins or ARNOs. Furthermore, we discovered an adaptor protein which we named CASP (Cytohesin Associated Scaffolding Protein, a.k.a. Cybr, CYTIP, PSCDBP, B3-1) that can interact with several members of the Cytohesin/ARNO family through its coiled-coil motif. CASP is primarily expressed in hematopoietic cells with the highest expression found in cytokine-stimulated lymphocytes.

Recently, we found a novel interaction in which CASP’s carboxy PDZ binding motif (PDZbm) can directly bind with the PDZ domain of sorting nexin 27 (SNX27.  In NK-92 cells we showed that endosomal SNX27 polarizes to the immune synapse (IS) during tumor cell engagement in a distinct compartment adjacent to the cytotoxic granules. We also showed that during NK cell migration SNX27 localized to the apical membrane, opposite the uropod. CASP co-localizes with SNX27 suggesting a role for this interaction in endocytic trafficking and/or signaling.

Interestingly, we have detected low levels of the CASP binding partner, Cytohesin-1, in a complex with immunoprecipitated SNX27. The CASP–SNX27 interaction could support a mechanism where members of the Cytohesin/ARNO family are dynamically recruited to early endosomal membranes for vesicular trafficking in lymphocytes. .

A CASP-aided interaction between Cytohesin/ARNO family members and SNX27 could bring both PX and PH domains into one complex and may contribute to the rapid transition of budding vesicles to early endosomes, as shown in our proposed model (see figure) that was recently published.

(2) Tilapia Islet Cell Biology:

(Funded by JDF USA, Collaboration with Dr. Jim Wright, Pathology, University of Calgary).  Dr. Wright has successfully used transplantation of tilapia pancreatic islets to treat diabetic animals (xenograft transplantation). The major advantages are that islets in some fish occur as a discreet organ (Brockman Bodies) that can be easily extracted and contain primarily islet cells (unlike human pancreases). To use this therapy successfully in humans, it would be extremely beneficial if the insulin produced was human. My group cloned the complete tilapia insulin gene and by using site-directed mutagenesis and linker substitutions we have completed the "humanization" of this tilapia insulin gene. We have re-introduced this new hybrid gene into tilapia (transgenic) and have produced homozygous animals that secrete human insulin. We are presently studying the regulation of blood glucose levels in tilapia.

(3) Vaccine Development  (Funded by AIF. Collaboration with ImmunoVaccine Technologies IVT- Halifax, NS). Several years ago we developed at Dalhousie a single immunization birth control vaccine for seals.          This vaccine was able to elicit a strong response that lasted for more than 10 years. Using this technology we have developed potent vaccines for several animal diseases. We are presently developing vaccines that can be used to treat cancer in humans.

Tilapia

• Asra'a Abidali- GRASP interaction with Sorting Nexin 27 suggests possible rescue of CASP in knockdown models
• Matthew Nicoll- Characterization of Nile Tilapia insulin II cDNA
  

• Olga Hrytsenko, Ph.D. - Characterization of Transgenic Tilapia containing a humanized insulin gene.
• Krista Tilley, M.Sc. - Determination of B3-1 binding partners.
• Jamie Claude, M.Sc. Transgenic Tilapia (co-supervisor with Dr. J. Wright).

• Lin Liu, M.Sc. Technician UBC.
• Brian Dixon, Ph.D. Assistant Professor, University of Waterloo.
• Peter Ross, M.Sc. Scientist, DFO.
• Jim Eddington, M.Sc., Technician, Dalhousie University.
• Jun Chen, M.Sc., Technician, University of Toronto.
• Stella Lee, Ph.D., Post Doctoral Fellow, Harvard USA.
• Marc Mansour, PhD., Research Scientist, IVT.
• Olga Hrytsenko, Ph.D, Post-Doctoral Fellow .
• Adam MacNeil, Ph.D. Post-Doctoral Fellow.

MacNeil AJ, and Pohajdak B. 2009. Getting a GRASP on CASP: properties and role of the cytohesin-associated scaffolding protein in immunity.  Immunology and Cell Biol. 87: 72-80

Olga Hrytsenko, Bill Pohajdak, Bao-You Xu, Carol Morrison, Brenna vanTol and James R. Wright Jr.. 2009. Cloning and Molecular Characterization of the Glucose Transporter 1 in Tilapia (Oreochromis niloticus). General and Comparative Endocrinology (in press).

Olga Hrytsenko, Bill Pohajdak and James R. Wright Jr. 2009. Production of transgenic tilapia homozygous for a humanized insulin gene. Transgenic Res. (in press).

Olga Hrytsenko; James R Wright, Bill Pohajdak  2008  Regulation of insulin gene expression and insulin production in Nile tilapia (Oreochromis niloticus) General and Comparative Endocrinology. 155(2):328-40.

James R. Wright Jr, Jaime Snowdon, Olga Hrytsenko, Carol M. Morrison & Bill Pohajdak 2008 Immunohistochemical staining for tilapia and human insulin demonstrates that a tilapia transgenic for humanized insulin is a mosaic. Transgenic Res. 2008 Oct;17(5):991-992.

Adam J. MacNeil, Lori A. McEachern, and Bill Pohajdak  2008. Gene duplication in early vertebrates results in tissue-specific subfunctionalized adaptor proteins: CASP and GRASP.  J Mol Evol. 2008 Aug; 67(2):168-78

Adam J. MacNeil, Bill Pohajdak  2007. Polarization of endosomal SNX27 in migrating and tumor-engaged Natural Killer cells.  Biochemical and Biophysical Research Communications 361(1):146-50.

Hrytsenko O, Wright JR Jr, Morrison CM, Pohajdak B. Insulin expression in the brain and pituitary cells of tilapia  (Oreochromis niloticus). Brain Res. 2007 11(1):31-40.

Mansour M, Pohajdak B, Kast WM, Fuentes-Ortega A, Korets-Smith E,Weir GM, . 2007 Therapy of established B16-F10 melanoma tumors by a single vaccination of CTL/T helper peptides in VacciMax(R). J Transl Med Apr 23;5(1):20

Adam MacNeil, Marc Mansour and Bill Pohajdak. 2007 Sorting Nexin 27 interacts with the Cytohesin Associated Scaffolding Protein (CASP) in lymphocytes. Biochemical and Biophysical Research Communications. 359(4):848-853.

Pirouz Daftarian, Marc Mansour, Anita C. Benoit, Bill Pohajdak, David W. Hoskin, Robert G. Brown and W. Martin Kast. 2006. Eradication of established HPV 16-expressing tumors by a single administration of a vaccine composed of a liposome-encapsulated CTL-T helper fusion peptide in a water-in-oil emulsion. Vaccine 12;24(24):5235-44.
           
Pohajdak B, Mansour M, Hrytsenko O, Conlon JM, Dymond LC and Wright JR Jr 2004.   Production of Transgenic Tilapia with Brockmann bodies secreting (desThrB30) human insulin. Transgenic Res. 13 (4): 313-323.

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