Doctor of Philosophy
Ubiquitination is the transfer of a ubiquitin molecule to protein substrates by the sequential actions of E1 activating enzymes, E2 conjugating enzymes, and E3 ligases. It is a post-translational modification that controls the fate and function of the substrate protein. Substrate specificity in the ubiquitination reaction is conferred by the E3 ligases. Sequence homology suggests the human C-terminal to LisH (CTLH) complex could be an E3 ligase; however, very little is known about this complex. In this thesis, I characterize the human CTLH complex as a multi-subunit E3 ligase and define its activity, structure, and substrates. I demonstrate that the CTLH complex is comprised of several interdependent subunits localizing to the nucleus and cytoplasm. I determine that the complex has E3 ligase activity which is dependent on its two Really Interesting New Gene (RING) domain subunits, RMND5A and MAEA. I found that the complex controls ubiquitination and degradation of muskelin. Since muskelin is a subunit of the human CTLH complex and likely serves as a substrate receptor, this finding revealed a potential autoregulation mechanism. Residues critical for controlling muskelin protein levels in the RMND5A RING domain were characterized. This demonstrated which residues are required for zinc coordination, E2 conjugating enzyme binding, and stimulation of the ubiquitination reaction. I mapped the subunit arrangement of the endogenous complex using cross-linking mass spectrometry and immunoprecipitations, which provided a clearer depiction of complex architecture. Finally, quantitative analyses of global proteomes and ubiquitin-enriched proteomes in various complex-depleted HeLa cells and affinity purification mass spectrometry of endogenous RanBPM (a core complex member) were conducted and compared to identify CTLH complex ubiquitination targets. I focused on an emerging theme from the datasets of glucose metabolism and show that the CTLH complex controls ubiquitination and inhibits activity levels of multiple glycolysis enzymes. In addition, the loss of RanBPM results in increased glycolysis and deregulated central carbon metabolism. Overall, this thesis establishes the human CTLH complex as a multi-subunit E3 ligase that regulates glucose metabolism. It also provides critical and fundamental insights into the structure, E3 ligase activity, and possible ubiquitination targets of the human CTLH complex.
Summary for Lay Audience
Cells execute timed biological processes and adapt to changes in their environment by controlling signal transduction pathways. To do so, they have evolved a system to regulate proteins by quickly adding or removing chemical tags. Ubiquitination is one important example of this. It is a reaction that occurs frequently inside a cell where a ubiquitin molecule is specifically added to a protein (a substrate). Once ubiquitinated, the substrate is either eliminated from the cell or its function is changed.
E3 ligases are proteins that determine which substrates are ubiquitinated. In this thesis, I discover that a particular group of proteins that interact continuously with each other, called the ‘CTLH complex’, is an E3 ligase in human cells. I also characterize its structure and function.
At the start of this thesis, very little was known about this complex. Therefore, a first step was developing tools to study the human CTLH complex. By doing so, I characterized basic properties of the CTLH complex, such as its composition, stability determinants, and architecture. Importantly, I demonstrated using biochemical assays that the CTLH complex has E3 ligase activity and revealed a potential autoregulation mechanism via ubiquitination of one of its components.
It was previously found that loss of the CTLH complex resulted in increased growth of cancer cells. To reveal mechanisms that regulate cancer development by the CTLH complex, I used a technique that can analyze thousands of proteins at once (mass spectrometry-based proteomics). This was done to identify which proteins are ubiquitinated by the CTLH complex. It uncovered that the human CTLH complex has several substrates, including proteins in glucose metabolism. Following up on this, I found that inactivation of the CTLH complex increased the speed of glucose metabolism in cancer cells. This helps explain why mutation of the CTLH complex can cause cancer cells to be more aggressive. Overall, this thesis sets the foundation for the study of the CTLH complex in human cells and establishes it as an E3 ligase that controls essential biological pathways via ubiquitination.
Maitland, Matthew E.R., "Uncovering the ubiquitin ligase activity and substrates of the human C-terminal to LisH (CTLH) complex" (2021). Electronic Thesis and Dissertation Repository. 7766.
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