Date of Award

2015

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Cell & Molecular Biology

First Advisor

Paula M. Oliver

Abstract

Ubiquitylation of cellular proteins alters protein function and half-life to impact cell signaling and fate decisions. In T cells, ubiquitylation events, mediated by substrate-specific E3 ubiquitin ligases and deubiquitylating enzymes, can promote or limit T cell activation and alter function. For example, the catalytic E3 ligase Nedd4 is required for robust T cell activation, while a related Nedd4 family member, Itch, negatively regulates T cell signaling. Several Nedd4 family ligases are activated by binding Nedd4 family interacting protein 1 (Ndfip1) and/or Ndfip2. I have determined that ligase activity depends non-redundantly on both Ndfip1 and Ndfip2. Unlike Ndfip1, Ndfip2 is not a prominent negative regulator of T cell activation or TH2 polarization. However, loss of Ndfip2 in Ndfip1 deficient cells leads to a T cell-intrinsic expansion of pathogenic TH2 effector cells. Defining substrates of critical ubiquitylation events in activated T cells can enhance our understanding of T cell function in both health and disease. Thus, I took a targeted approach to identify new substrates of Nedd4 family catalytic E3 ligases in activated T cells, developing a proteomics workflow for unbiased quantification of differential ubiquitylation in T cells. I identified Jak1 as a substrate for Ndfip-dependent E3 ligases, and determined that Ndfip-mediated Jak1 degradation limits cytokine signaling during TCR engagement. Having successfully used proteomics for substrate-identification, I considered the utility of quantitative proteomics in globally assessing ubiquitylation in T cells. I developed a mass spectrometry-based approach pairing immunoprecipitation of “ubiquitin remnant” peptides (with di-glycine modified lysine residues) and whole proteome analysis to quantify changes in protein-specific ubiquitylation in activated CD4+ T cells. I observed dynamic changes in ubiquitylation of hundreds of proteins, including key signal transducers, during TCR stimulation. Comparing changes in ubiquitylation with changes in protein abundance in stimulated CD4+ T cells I propose to identify proteins for which ubiquitylation is tightly linked to protein abundance and/or function in a signal-dependent manner. This profiling effort, in combination with targeted studies similar to those undertaken to identify substrates of Ndfip-dependent E3 ligases, will facilitate a more global understanding of how ubiquitylation events in activated T cells finely tune the T cell response.

Available for download on Sunday, December 02, 2018

Files over 3MB may be slow to open. For best results, right-click and select "save as..."

Share

COinS