Date of Award

Summer 2010

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Biochemistry & Molecular Biophysics

First Advisor

Kathryn M. Ferguson

Abstract

The work described in this dissertation comprises two distinct projects. In the first, we describe the structural and functional characterization of a family of Golgi associated cytosolic proteins, represented by Vps74 in fungi and GOLPH3 in animals, by X-ray crystallography, biophysical assays, and cellular techniques. We find that Vps74 is required for the proper steady state localization of a subset of Golgi enzymes in yeast, and that disruption of vps74 results in incomplete protein glycosylation. We further describe the crystal structures of Vps74 and GOLPH3, identifying structural motifs required both for oligomer formation and protein function. Finally, we find that both Vps74 and GOLPH3 specifically bind the Golgi enriched phospholipid, PtdIns4P. These results suggest a role for Vps74 and GOLPH3 in retrograde trafficking of components to the Golgi apparatus.

In a separate and unrelated project, we characterize several inhibitory antibodies directed against the extracellular region of the epidermal growth factor receptor (EGFR). Aberrant activation of EGFR occurs in large proportion of epithelial cancers. Consequently, this receptor is a target for anti-cancer therapeutics that inhibit its activation, including antibodies and antibody-derived molecules. We have biochemically characterized a panel of conventional inhibitory antibodies with unique properties, and have identified approximate epitopes for these antibodies on domain 3 of EGFR. Additionally, we describe the crystal structures of three unconventional single chain antibody fragments in complex with the EGFR extracellular region. These single chain antibodies bind to novel epitopes on the receptor but share key characteristics with conventional inhibitory antibodies. Our findings highlight the diversity of binding modes among anti-EGFR antibodies, and suggest opportunities for novel therapeutics.

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