Doctor of Philosophy (PhD)
Genetics, Cell Biology & Anatomy
Dr. Hamid Band
Endocytic recycling is a fundamental cellular process that allows the precise regulation of the membrane components and receptors at the cell surface. Recent studies have established that the C-terminal Eps15 homology domain-containing (EHD) proteins function as key regulators of this process. Four highly-conserved members of the EHD protein family in mammals, EHD1-EHD4, play shared as well as unique roles in endocytic trafficking. Studies presented here demonstrate a critical role of EHD1 in the normal ocular development in mice. Ehd1 knockout mice generated in our laboratory displayed gross ocular phenotypes including the anophthalmia, microphthalmia, and congenital cataracts. Hematoxylin and eosin (H&E) staining revealed defects in the Ehd1 mutant mice that included smaller lens, lack of lens, and persistence of the lens-stalk and hyaloid vasculature. By deleting Ehd1 specifically in the lens (Ehd1 CKO), my studies provide evidence that EHD1 expression in the lens precursor cells within the surface ectoderm is necessary for the normal lens development. Ehd1 CKO mice recapitulated the major ocular phenotypes of the Ehd1-null mice, and exhibited reduced proliferation and increased cell death within the lens epithelium as well as a disorganized corneal endothelium. These data suggest the important roles of EHD1 in the overall process of eye development.
EHD proteins are characterized by the presence of a C-terminal Eps15 homology (EH) domain. EH-domains are known to mediate interactions with proteins containing sequences with core Asn-Pro-Phe (NPF), Asp-Pro-Phe (DPF) or Gly-Pro-Phe (GPF) tri-peptide motifs. Such interactions are thought to facilitate EHD protein functions, but only a small number of such partners has been identified. To identify novel EH domain-NPF mediated interaction partners, I carried out a proteome-wide bioinformatics analysis to categorize proteins that possess single or multiple putative EH domain-binding motifs and generated a 9-mer peptide library corresponding to motifs containing the N/D/G-P-F sequence together with 6 C-terminal residues found in known EHD protein interaction partners as well as a subset of the previously uncharacterized proteins identified by the bioinformatics analysis selected based on their potential roles in endocytic traffic and related cellular processes. I developed a quantitative high-throughput fluorescence polarization-based competition assay using the EH domain of EHD1 as a prototype and a fluorescent peptide corresponding to a known EHD1 binding motif on MICAL-L1 protein. Using this assay, and the unlabeled peptides in the library in a competition assay, I determined the interaction affinities of the putative peptide motifs for binding to the EH domain of EHD1. This approach helped identify a large number of new EHD interacting partners of which selected candidates were validated using a GST-EH domain pull-down assay. These studies helped markedly expand the potential EHD protein interactome, and implicate the newly identified candidates in multiple cellular functions of EHD1 and potentially other family members.
Arya, Priyanka, "Functional characterization of the roles of endocytic recycling regulator EHD1 using in vivo and in vitro analyses" (2015). Theses & Dissertations. 28.