Date of Award
Doctor of Philosophy (PhD)
Microbiology, Immunology, and Biochemistry
Brenda A. Schulman, Ph.D.
Eric Enemark, Ph.D. Lawrence Pfeffer, Ph.D. Charles Rock, Ph.D. Stephen White, Ph.D.
Healthy, reproducing cells create and destroy proteins in an ordered manner. Both the concentration and localization of protein pools is important to regulate the many cellular processes necessary for the life of the cell. In order to produce new proteins, cells degrade existing materials such as proteins and organelles that are dispensable or troublesome in order to recycle their raw components. Degradation is accomplished largely through two major pathways: in bulk through processes such as autophagy and phagocytosis, or in a targeted manner through the ubiquitin-proteasome pathway. Autophagy utilizes an encompassing body to encapsulate targets and surrounding materials for decomposition in a regulated but relatively non-specific manner. The ubiquitin-proteasome pathway, however, is an exquisitely precise method of degradation capable of targeting specific pools of protein substrates. So-called ubiquitination generates a signal for degradation by the 26S proteasome machinery. In order to establish the degradation signal, the cell utilizes a cascade of 3 enzymes working in concert to organize substrates and ubiquitin. By using enzymes capable of substrate specificity, the cell can regulate large pools of proteins in a specific spatio-temporal manner.
In the present study, we employ biochemical and structure-based techniques to study proteins involved in an essential ubiquitination pathway involved in maintenance of the cell cycle. A mitotic regulator called the Anaphase Promoting Complex, also called Cyclosome (APC/C), modifies myriad substrates that control cell cycle activities. The APC/C is a ubiquitin ligase complex; it is the final enzyme in a tri-enzyme cascade and catalyzes the final step of Ub transfer from an E2 enzyme directly to substrates. A protein called Emi1 is responsible for directly binding and inhibiting the APC/C throughout interphase, when APC/C substrates are stabilized. We set out to study domains of Emi1 responsible for binding and inhibiting the APC/C and its association with E2 enzymes or substrates, with the aim of charcterizing both the mechanism of the inhibitor and the essential functional requirements of the APC/C, which are not well understood. We aim to visualize the APC/C in complex with Emi1 through electron microscopy and to accurately interpret the location and orientation of Emi1 within this density.
The APC/C is a large, ~1,200 kDa multi-protein complex, and Emi1 is a single protein of only 50 kDa. Despite its small size, even a small subdomain of Emi1 potently inhibits the APC/C through a combination of not well-characterized mechanisms. Emi1 has an APC/C recognition motif called a D-box that is typically found within APC/C substrates. Emi1’s D-box is recruited to the substrate-binding sites of APC/C and serves as a pseudo-substrate inhibitor. Emi1 also inhibits through distinct mechanisms both of the E2 enzymes that coordinate with APC/C function. An essential, folded Zinc-binding Region (ZBR) and a helical “Linker” sequence cooperate to bind and block APC/C from recruiting one of two APC-specific E2 enzymes, Ubch10. Emi1 also has a conserved C-terminal motif, with charge and sequence similar to the other APC/C- specific E2 enzyme, Ube2s. Emi1 competes directly with Ube2s for APC/C recruitment, and it is through the combination of these three mechanisms that are afforded by many motifs within Emi1, that makes Emi1 a potent APC/C inhibitor. When associated with Emi1, the APC/C is inefficient at both recruiting substrates and binding E2 enzymes, allowing for stabilization of APC/C substrates, which is important for regulation of timing of cell cycle processes.
Watson, Edmond Randall (http://orcid.org/0000-0001-7756-4099), "Understanding Structural And Functional Mechanisms of Emi1 Inhibition of the Anaphase Promoting Complex" (2016). Theses and Dissertations (ETD). Paper 372. http://dx.doi.org/10.21007/etd.cghs.2016.0397.