Winter 2012 Biomedical Seminar Series
Friday, January 27, 2012
Progress Toward the Enantioselective Synthesis of Variecolin: Synthesis of the B-ring Precursor
MARC U*Star Fellow
Summer Research Site: California Institute of Technology
Abstract: Variecolin is a tetracyclic potent anti-HIV-I natural product. An expeditious synthesis of the B-ring precursor has been achieved through a five-step process. These efforts highlight the formation of a pyrone from ethyl propenyl ether and the production of an iron complex utilizing photochemical technology. This B-ring precursor will be employed in the synthesis of the AB-ring fragment and will be ultimately used toward the total synthesis of variecolin.
Copper Redox Cycling in the Octarepeat region of Prp
MARC U*Star Fellow Summer Research Site: University of California, Santa Cruz
ABSTRACT: The prion protein or PrP is a protein that is found in all mammals and though at times capable of misfolding into the disease form, the normal function of the prion protein is still considered a mystery. The prion protein itself is composed of a folded c-terminal domain and unstructured n-terminal domain, implicated in copper coordination. It was discovered that in vivo the prion protein is susceptible to ?-cleavage, which could possibly mean that the copper binding associated with the protein could allow for copper redox cycling. A series of experiments were performed to understand the function of the copper binding n-terminus of the prion protein. By synthesizing the peptide sequence of 4octa, KKRPKPWGQ (PHGGGWGQ)4, the proteins copper binding sequence, we were able to perform a series of experiments which allowed for the octa repeat sequence of the prion protein to under go copper redox cycling. Samples containing the 4 octa-repeat sequences were mixed with 1 equivalent of Cu+2 or 4 equivalents of Cu+2 and were tested under various conditions to simulate copper redox cycling. Ascorbic acid was used in the reaction to reduce Cu+2 to Cu+ , while oxygen was used to oxidize Cu+ to Cu+2 . Continuous wave, electron paramagnetic resonance (EPR), showed that the 4octa repeat sequence is capable of copper redox cycling.
Creation and Validation of RhoQ-GFP Construct
MARC U*Star Fellow
MHIRT Program at Kings College, London, UK
Abstract: Rho proteins are a family of small GTPases that are known to play a key role in regulating cytoskeletal dynamics, which means they can affect cell polarity, apoptosis, migration, cell adhesion, and vesicle trafficking. There are 20 known members of the Rho family in mammals, and our study concerns the member RhoQ, also known as TC10 or Rasl7A. The long term aim is to study how RhoQ affects cell motility, invasion, and metastasis of metastatic breast cancer cell lines. We have successfully created a construct which encodes RhoQ fused at its N-terminus to green fluorescent protein (GFP). We have over-expressed the construct in MTLn3E (low endogenous expression of RhoQ) and MDA-MB-231 (high expression of RhoQ) breast cancer cell lines. Western blotting was used to confirm the expression of GFP-RhoQ. Furthermore, GFP-RhoQ has been visualized with fluorescence microscopy. It appears that the protein mostly distributes in vesicles around the nucleus and the cell membrane. In the near future we hope to determine how GFP-RhoQ expression affects cell morphology and cytoskeletal organization. Future directions of study also include using timelapse microscopy to visualize GFP-RhoQ to better understand what role the protein plays in cell motility. References: Abe T, Kato M, Miki H, Takenawa T, Endo T (2002). Small GTPase TC10 and its homologue RhoT induce N-WASP-mediated long process formation and neurite outgrowth. Journal of Cell Science 116, 155-168. Kanzaki M, Watson R, Hou J, Stamnes M, Saltiel A, Pessin J (2002). Small GTP-binding Protein TC10 Differentially Regulates Two Distinct Populations of Filamentous Actin in 3T3L1 Adipocytes. Molecular Biology of the Cell 13: 2334-2346. Murphy GA, Solski PA, Jillian SA, de la Ossa PP, DÂEustachio P, Der CJ et al. (1999).Cellular functions of TC10, a Rho family GTPase: regulation of morphology,signal transduction and cell growth. Oncogene 18: 3831Â3845. Neudauer C, Joberty G, Tatsis N, Macara I (1998). Distinct Celluar Effects and Interactions of the Rho-Family of GTPase TC10. Current Biology 8:21. Ridley AJ.(2001a).Rho GTPases and cell migration. J Cell Sci 114: 2713Â2722. Ridley AJ.(1997).The GTP-binding protein Rho. Int J Biochem Cell Biol 29: 1225Â1229. Rossman KL, Der CJ, Sondek J.(2005).GEF means go: turning on RHO GTPases with guanine nucleotide-exchange factors. Nat Rev Mol Cell Biol 6: 167Â180. Tong S, Liss AS, You M, Bose Jr HR (2007). The activation of TC10, a Rho small GTPase, contributes to v-Rel-mediated transformation. Oncogene 26, 2318Â2329.