Ubiquitylation regulates CD4+ T cell activation and effector differentiation to shape the immune response / Claire E. O'Leary.

Format:

Book

Manuscript

Thesis/Dissertation

Author/Creator:

O'Leary, Claire E., author.

Contributor:

Oliver, Paula M., degree supervisor.

Burkhardt, Janis, degree committee member.

Marks, Michael, degree committee member.

Maltzman, Jonathan S., degree committee member.

Wherry, E. John, degree committee member.

University of Pennsylvania. Department of Cell and Molecular Biology.

Language:

English

Subjects (All):

Penn dissertations--Cell and molecular biology.

Cell and molecular biology--Penn dissertations.

Local Subjects:

Penn dissertations--Cell and molecular biology.

Cell and molecular biology--Penn dissertations.

Physical Description:

xi, 156 leaves : illustrations (some color) ; 29 cm

Production:

[Philadelphia, Pennsylvania] : University of Pennsylvania, 2015.

Summary:

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.

Notes:

Ph. D. University of Pennsylvania 2015.

Department: Cell and Molecular Biology.

Supervisor: Paula M. Oliver.

Includes bibliographical references.

OCLC:

946217300