published_or_final_version / Botany / Doctoral / Doctor of Philosophy

published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

Sulfur, an essential element required by the cell for the biosynthesis of crucial compounds, such as amino acids, co-factors and vitamins, is found most abundantly in the form of sulfate. In order to cross the lipid bilayer for cell usage, sulfate, being a negatively charged ion, requires an energetically `friendly' passageway. This could be accomplished via an ion channel that allows the flow of sulfate into the cell along its concentration gradient, or via an active transport system that allows sulfate to be taken up by the cell when it is in scarcity in the environment, against its concentration gradient, like the ABC transporter and sodium or proton-coupled symporters. The subject of this dissertation, CysZ, is a bacterial sulfate transport protein that does not belong to any known superfamily of canonical transporters or channels with no distinguishing features that could hint at its functional mechanism. We have undertaken a structural and functional approach to further understanding the role of CysZ in sulfate transport. In this dissertation, we report the structure of CysZ, its functional role in sulfate uptake and have suggested a mechanistic hypothesis for the passage of sulfate through CysZ along with the exciting future directions that might follow.

Thesis (Ph.D.)--University of Delaware, 2009. / Principal faculty advisors: N. Carolyn Schanen and Mary C. Farach-Carson, Dept. of Biological Sciences. Includes bibliographical references.

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (p. 290-314). Also available in print.

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (p. 290-314). Also available online.

APPL BAR-PH heterodimer and APPL2 BAR-PH homodimer The APPL (Adaptor protein containing PH domain, PTB domain and Leucine zipper) family are adaptor proteins with only two isoforms, APPL1 and APPL2. They bind to early endosomes with a small GTPase, Rab5, and mediate the interactions between various receptors and downstream signaling components, thus functioning in many signaling pathways evoked by adiponectin, insulin, FSH, EGF, and so on. However, evidences have shown APPL1 and APPL2 should perform some opposite functions, which cannot be simply explained by the functional differences attributed to their PTB domains. We hypothesize that the heterodimerization of APPL1 and APPL2’s BAR domains may account for their opposing functions. The crystal structure of APPL BAR-PH heterodimer was solved to resolution 2.8 Å. Its overall structure exhibits crescent shape with a larger curvature radius of 76 Å, compared to 55 Å of the APPL1 BAR-PH homodimer. And the crystal structure APPL2 BAR-PH homodimer was solve to resolution 3.3 Å. The overall structure of APPL2 BAR-PH homodimer is very similar to that of APPL BAR-PH heterodimer, but shows greater difference in curvature to the APPL1 BAR-PH homodimer structure. The concave side of APPL BAR-PH heterodimer and APPL2 BAR-PH homodimer all possess less positive charge than the APPL1 BAR-PH homodimer. Structural analysis reveals that leucine patches at the dimer interface may account for the formation of dimeric curve with certain curvature. Consequently, APPL2 BAR is able to enforce the curvature reduction to APPL1 BAR upon heterodimerization. In conclusion, the alterations of curvature and electrostasis are responsible for the modulation of endosome binding specificity and can elucidate the opposite roles of APPL1 and APPL2. LanCL2 LanCL2 is a member of Lanthionine synthetase component C-like family. In human, LanCL2 binds to lanthionine derivatives and glutathione, participating in keeping intracellular reducing state. By binding to absiscic acid (ABA), LanCL2 is indispensible for the ABA-stimulated adipogenesis, insulin release, glucose homeostasis, and inflammatory response. It is also implicated in anticancer drug resistance. All these functions underscore the importance of LanCL2 in the diseases like diabetes, inflammation, and cancer. The crystal structure of LanCL2 was solved to resolution 1.8 Å. The overall structure displays canonical double-layer α-barrel. The major differences from LanCL1 lay in the loops on the barrel top, which are implicated in various substrate bindings. A zinc-coordinating pocket was found among the loops, with conserved amino acid residues of distinct conformation. The electrostatic surface shows remarkable differences compared to that of LanCL1, suggesting that it may contribute to distinct substrate binding profile. Future implications APPL proteins and LanCL proteins are all involved in the regulation of metabolism, such as glucose uptake, fatty acid oxidation, and insulin secretion, and play roles in diseases like obesity and type 2 diabete. Structural and functional studies of these proteins can provide insights into the molecular mechanisms and clues for related therapeutic approaches. / published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

Thesis (Ph. D.)--University of Hong Kong, 2008.

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 145-170). Also available in print.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1982. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.