Pannexin 1 (Panx1) is a metabolite channel enriched in the brain and known to localize to the cell surface, where it is involved in a variety of neuronal processes including cell proliferation and differentiation. The mechanisms through which Panx1 is trafficked or stabilized at the surface, however, are not fully understood. The proximal Panx1 C-terminus (Panx1CT), upstream of a caspase-cleavage site has been demonstrated to be required for Panx1 cell-surface expression. We discovered a previously unreported putative leucine-rich repeat (LRR) motif within the proximal Panx1CT. I investigated the involvement of this putative LRR motif on Panx1 localization and oligomerization. Deletion of the putative LRR motif or uniquely the highly conserved segment of the putative LRR motif resulted in a significant loss of Panx1 cell surface expression. Finally, ectopic expression of Panx1-EGFP in HEK293T cells increased cell proliferation, which was not recapitulated by a Panx1 deletion mutant lacking the putative LRR motif. Overall the findings presented in this thesis provide new insights into the molecular determinants of Panx1 trafficking and oligomerization. / Graduate / 2020-02-14

Candida albicans is an opportunistic fungal pathogen that may cause mucutaneous infection and/or disseminated candidasis if the host defense system is impaired (such as those in HIV patients). Cell surface of C. albicans is the frontier where initial interplay between host-pathogen takes place and therefore is of great importance in understanding the mechanism of hostpathogen interaction. MALDI-TOF-MS analysis of intact fungal cells yielded mass signatures for rapid species differentiation, strain grouping and yeast morphogenesis monitoring. Cell surface biotinylations at low temperature (4°C), enzymatic digestion of the intact fungal cell surface proteins ("whole cell shaving"), biotin-avidin affinity enrichment of biotinylated peptides, liquid chromatography mass spectrometry (LC-MS) based proteomic approach were employed for unambiguous identification of cell wall/cell wall associated proteins and the exposed peptide segments of these proteins. SILAC (Stable Isotope Labeling by Amino acids in Cell Culture) based CWP quantification analyses were performed to monitor CWP accumulation level change in response to hyphae induction. Information on surface exposed peptide segments and regulation of cell wall/surface protein during morphogenesis provided new candidates to the pool of potential peptide targets for protective vaccine development. A New type of "fluorous" (fluorinated alkane) affinity gained popularity due to its low level nonspecific protein/peptide binding. Fluorous labeling reagents that target primary amine groups in proteins/peptides were synthesized and characterized. The acid labile linker in the labeling reagents allows cleavage of the bulky fluorous tag moiety and the long oligo ethylene glycol (OEG) spacer after fluorous affinity purification. Upon collision induced decomposition, the labeled peptide ion yielded a characteristic fragment that could be retrieved from the residual portion of fluorous affinity tag, and serve as a marker to indicate that the relevant peptide had been successfully labeled. Results showed that both the protein/peptide labeling and affinity enrichment/separation process were highly efficient.