Student Theses and Dissertations

Date of Award

2014

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Papavasiliou Laboratory

Abstract

Often the needs of an organism exceed the number of genes in the genome. Thus, modification of the genes, themselves, or of the gene products is necessary. This becomes particularly important in cells of the immune system, which have to combat a virtually infinite array of foreign pathogens. Blymphocytes, the mediators of humoral immunity, have developed extensive mechanisms of gene diversification collectively known as antibody diversification. Antibody diversification, a set of processes necessary for an organism to mount a specific and robust immune response, relies on Activation Induced Cytidine Deaminase (AID) to initiate two of such processes: Somatic Hypermutation (SHM) and Class Switch Recombination (CSR). AID-dependent deamination of cytidine bases within the variable region (SHM) and switch region (CSR) of the immunoglobulin locus (Ig) results in the modification of the antigen binding domain and diversity within effector function of the antibody, respectively. Though the activity of AID is known, the regulation of AID during the different stages of antibody diversification is less well understood. This question has been particularly challenging to address because of the difficulty of working with AID, which becomes insoluble when expressed in non B-cells. This thesis presents the development of a screen, which searches for interacting partners for poorly soluble proteins. This screen relies on the insolubility of the protein of interest and the ability of interacting proteins to induce solubilization via binding and masking of exposed hydrophobic domains. After validation of this screen using representative soluble and insoluble proteins, it was applied to AID and thirty putative AID binding partners were identified. A handful of these proteins were uncovered in prior interaction screens, thus underscoring the validity of this new screening approach. In addition, this thesis presents a comprehensive analysis, utilizing both in vitro and in vivo approaches, of one of the putative AID cofactors discovered in the screen, RING Finger Protein 126 (RNF126). In vitro studies revealed that RNF126 is a bona fide AID binding partner and, in addition, acts as an E3 ubiquitin ligase, modifying AID with the addition of a single ubiquitin moiety. Further, a conditional knockout model of RNF126 was generated and used to determine that RNF126 plays a role in vivo in fine-tuning AID activity during SHM and CSR. The findings presented here demonstrate the utility of a novel screening technique to search for interacting partners for insoluble proteins and, through its use, expands the list of putative AID cofactors. In addition, through a thorough analysis of a single AID binding partner, this thesis puts forth a novel mode of regulation of the potent mutating enzyme, paving the way for future research to uncover the role of mono-ubiquitinated AID during SHM and/or CSR.

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

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