Post-translational modification of proteins with ubiquitin plays a role in most biological processes. The key players of this system are the E3 ubiquitin ligases, which mediate substrate specific covalent attachment of ubiquitin. The SCF ubiquitin ligases are amongst best-understood E3 complexes. They are composed of Skp1, Cdc53/Cullin, Rbx1, and one of the multiple F-box proteins, which bind substrates and confer specificity to this complex. As these modular ligases share the same core components but differ in the identity of F-box proteins, the repertoire of SCF ligases present in a cell at any given time is thus governed by the abundance of different F-box proteins. Regulating F-box protein abundance is thus essential for normal cellular function and misregulation causes human diseases. In the studies presented here, we describe a novel mechanism governing maintenance of F-box protein homeostasis.

In the absence of substrates, the core SCF ligase ubiquitylates its F-box protein and targets it for degradation. Using the yeast F-box protein Met30 as a model system, we demonstrate presence of an additional mechanism for F-box protein degradation apart from autoubiquitylation. This pathway targets F-box proteins that are dissociated from Skp1, and plays a crucial role in limiting substrate shielding effects caused by association of excess F-box proteins with their substrates thereby inhibiting substrate recognition by fully assembled ligases. Characterization of this pathway shows that it is governed by the ubiquitin proteasome system and is mediated by a ligase dependent on Cdc53 but independent of Skp1 and for the first time illustrates uncoupling of Cdc53 and Skp1 function in yeast.

Furthermore, we show that Cic1 and the AAA ATPase Cdc48, along with its substrate recruiting factors Npl4/Ufd1 are involved in this mechanism of F-box protein degradation. Preliminary studies conducted to ascribe a role for these proteins in this process, suggest Cic1 as a unique factor that recruits ubiquitylated F-box proteins to Cdc48/Npl4/Ufd1 for targeting to proteasome for degradation.

Together, our findings shed light on the complexity of the ubiquitin system. They uncover a novel degradation pathway, suggest existence of yet unknown types of ligases, and introduce the concept of protein specificity factors for Cdc48.

The majority of cancer cells have a unique metabolic addiction to methionine in contrast to normal cells. This “methionine-dependent” phenotype describes the inability of cancer cells to proliferate in methionine stress conditions, where methionine has been replaced with its metabolic precursor, homocysteine, in the growth media. Methionine-dependence is implicated as a signature of cancer and upon spontaneous reversion to a methionine-independent phenotype, cells lose anchorage independent growth, a hallmark of tumorigenicity. Comparing methionine-dependent and -independent cell lines is critical for a clear interpretation of our results as we investigate the molecular mechanisms behind methionine-dependence and malignancy. For this purpose, we use the methionine-dependent, triple negative, breast cancer cell line MDA-MB-468 and derived a methionine-independent variant, MDA-MB-468res-R8. With this breast cancer cell pair we are able to compare cancer and normal-like cells due to their similar genetic backgrounds, proliferation rates, and media requirements.

Previous studies on methionine-dependence identified a reduction in the synthesis of S-adenosylmethionine (SAM) during methionine stress. SAM is the principal methyl donor in the cell and, interestingly, cancer cells have been shown to have higher methylation activity as compared to normal cells. Therefore, the studies presented in this thesis focus on the early response to methionine stress and how the resultant decrease in SAM is communicated throughout the cell. We used mass spectroscopy methods to understand the metabolic response to methionine stress in both cell lines over a 24 hour period. We observed methionine stress induced oxidative stress in both cell types resulting in a redirection of homocysteine metabolism toward synthesis of the antioxidant glutathione. Additionally, lipidomic analyses indicated a complete reprogramming of lipid synthesis in the methionine-dependent cell line that may be caused by irreversible oxidative damage and limited SAM availability for the synthesis of SAM-dependent lipids such as phospho- and sphingolipids.

To further understand how SAM levels are communicated to initiate a cellular response for protection of cell integrity, we focused on SAM as a co-factor for substrate methylation. SAM is a universal methyl donor and can serve as a co-factor for protein, lipid, chromatin, and RNA methylation events. Particularly, we focus on mRNA 5’ cap methylation and it’s influence on protein translation as a possible mechanism to communicate SAM levels. We employed both human cell lines and yeast strains to develop a methyl cap purification method for use in high-throughput RNA sequencing and have thus far identified unique, gene-specific responses to SAM depletion in regards to methyl cap stability. Continuing our efforts to fully understand this unique, metabolic requirement of cancer will allow us to shed light on an undefined molecular area essential for the development and use for cancer therapeutics in methionine-dependent cancers.