Access Type

Open Access Dissertation

Date of Award

January 2015

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Cancer Biology

First Advisor

Sokol V. Todi

Abstract

Protein Quality Control (PQC) comprises cellular pathways that regulate the turnover of short-lived, misfolded proteins. A main component of PQC is Endoplasmic Reticulum (ER)-Associated Degradation (ERAD), which controls the degradation of proteins synthesized in the ER. Aberrations in ERAD have been linked to malignancies such as sarcomas, breast, and pancreatic carcinomas, as well as neurodegenerative disease. The machinery in this system is complex and while significant progress has been made to understand ERAD, it is not clear how the different components come together, or how they are regulated. HRD1 is a resident ubiquitin ligase that has been proposed as a metastasis suppressor. My goal is to understand how HRD1 is regulated during normal states and in disease, particularly because my own work suggests complex mechanisms that regulate this protein and, more generally, ERAD.

We found that HRD1, an E3 ubiquitin ligase that functions in ERAD, along with other PQC components are differentially expressed in various cancer cell lines and in different mouse tissues, suggesting that specific types of ubiquitin chains are formed in ERAD under varying conditions. These findings led us to wonder whether HRD1 makes different types of ubiquitin-ubiquitin linkages with different E2 ubiquitin-conjugases. Through in vitro reconstituted systems, we found that HRD1 and the ubiquitin-conjugase Ube2G2 make only K48-linked polyubiquitin. This type of chain is classically tied to protein degradation by the proteasome. However HRD1, in the presence of another conjugase Ube2J1, forms unconventional K11-, K29-, and K33-linked polyubiquitin chains. These in vitro findings suggested that ERAD substrates are processed differently under specific conditions. In cells, HRD1 makes at least K48- and K63-linked polyubiquitin chains with Ube2G2 and Ube2J1, and the direct interaction of HRD1 with VCP/p97, while not required for polyubiquitin chain formation by this E3 ligase, may determine some types of ubiquitin chains formed by HRD1 with either E2 conjugase. Our findings suggest that HRD1 has specific partners in different tissues with potentially different ERAD outcomes. My work has broad implications in pathophysiology; it may emphasize specific sites of therapeutic intervention, and will provide significant clues into the functional balance of protein quality control in normal and disease states.

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