EVIDENCE FOR ADAPTER-MEDIATED SUBSTRATE SELECTION IN ENDOPLASMIC RETICULUM ASSOCIATED DEGRADATION

Abstract

Viruses have evolved a multitude of mechanisms, which allow immune evasion in both initial and persistent infection. Understanding the intricacies of these pathways is essential to our future ability to combat primary and reactive viral infections. The murine gamma-2 herpesvirus 68 (γHV68) encodes a protein mK3, which targets Major Histocompatibility Complex (MHC) class I heavy chains for ubiquitin-dependent proteasome degradation. MK3 is able to target and ubiquitinate MHC class I by binding to Endoplasmic Reticulum (ER) resident proteins tapasin, Transporter associated with antigen processing (TAP) 1 and TAP2 that are subunits in the complex known as the peptide-loading complex (PLC). The aforementioned characteristics of mK3 make this novel protein an excellent vehicle to study MHC class I assembly, immune evasion, and ER associated degradation (ERAD). Deepening our understanding of class I assembly and viral immune evasion will impact both the fields of immunology and virology. The homology between γHV68 and many of the human γ-herpesviruses makes this an indispensable model to clarify mechanisms that can then be applied to a broader spectrum of viruses. ERAD, an emerging field of study, is known to play a key role in numerous cellular housekeeping pathways as well as a number of disease states. Illuminating the mechanisms implicated in the mK3-mediated ubiquitination of MHC class I, specifically requirements for substrate recognition and degradation, will yield an increased understanding of cellular pathways involved in ERAD. The studies in this dissertation aim to expand our understanding of the relationship between mK3 and adapter proteins TAP/tapasin as well as mK3 and mK3-targeted substrates. The results show that TAP/tapasin act as adapter proteins by recruiting substrates for mK3. Further, mK3 ubiquitinates TAP/tapasin-associated substrates as long as the substrates have a tail greater than 6aa in length and the tail possesses an ubiquitin acceptor residue (lysine, serine or threonine). These studies also confirm that location of a protein within the PLC will determine the substrate’s susceptibility to mK3-mediated degradation. In the field of ubiquitin ligases and ERAD, these studies lend support to the concept of adapter mediated substrate recruitment.