14-3-3 proteins are among a family of phospho-binding proteins that are known to regulate many essential cellular mechanisms. By binding to sites of phosphorylation, 14-3-3s are integrated into multiple signaling pathways that govern critical processes, such as apoptosis, cell cycle progression, autophagy, glucose metabolism, and cell motility. These processes are crucial for tumorigenesis and 14-3-3 proteins are known to play a central role in facilitating cancer progression. In this study, my colleagues and I discover two novel 14-3-3 interacting proteins, ATG9A and PTOV1, that are both vital to essential cellular functions and describe various mechanisms that these two proteins regulate. ATG9A is a multi-pass transmembrane lipid scramblase that is found primarily as a homotrimer in the ER or small ATG9A vesicles. It is essential in the cellular recycling process called autophagy and is believed to act at the earliest stages of autophagy by providing the seed for the growth of the double membrane vesicle called an autophagosome. Previous work in our lab demonstrated that upon hypoxic stress, AMPK, the master nutrient-sensing kinase, phosphorylates S761 on the C-terminus of ATG9A. This triggers the binding of 14-3-3ζ to contribute to ATG9A function in hypoxia induced autophagy. Despite this revelation, the exact function of ATG9A is still poorly understood, especially in unstimulated conditions where autophagy functions at a basal level and AMPK is inactive. In this study, we sought to understand ATG9A function more broadly by identifying novel interactors of ATG9A and the role ATG9A plays in basal autophagy. To do this, we employed BioID mass spectrometry and various biochemical approaches to identify LRBA as a bona fide ATG9A interactor and autophagy regulator. Furthermore, using deuterium labeling and quantitative whole proteome mass spectrometry, and various other biochemical techniques, we show that ATG9A regulates the basal degradation of p62 and is recruited to sites of basal autophagy by active poly-ubiquitination to initiate basal autophagy. PTOV1 is an oncogenic protein that is poorly understood. Our current understanding of PTOV1 is limited to a few studies, which demonstrate that PTOV1 is highly expressed in primary prostate tumor samples and is correlated with metastasis, drug resistance, and poor clinical outcomes. In this study, we identify a mechanism by which SGK2, a poorly understood kinase, phosphorylates PTOV1 at S36 to trigger 14-3-3 binding at that site to increase PTOV1 stability in the cytosol and increase c-Jun expression. Upon SGK2 inhibition, 14-3-3 releases PTOV1 and PTOV1 is shuttled into the nucleus where HUWE1, an E3 ubiquitin ligase, ubiquitinates PTOV1 and initiates PTOV1 degradation by the proteasome. This is the first detailed mechanism of regulation identified for the poorly understood oncogene, PTOV1, and sheds light on potential therapeutic targets for cancer treatments.

Decades of research have begun to identify oncogenic mut-drivers that are responsible for driving a large percentage of cancers. These high frequency mut-drivers have therapeutics in the clinic for patient treatment. However, there is another group of low frequency mut-drivers that fail to rise above the noise of the high frequently drivers. These low frequency drivers represent a group of genes with untapped potential for new targeted therapies. However, identifying these drivers can be difficult. This study focuses on identifying new functional phosphorylations using the phospho-docking protein 14-3-3. The family of 14-3-3 proteins have been linked to many oncogenic pathways due to the diversity in their client protein interactions. One critical problem in studying 14-3-3 interactors is uncovering the docking site on the phospho-binding partner. Our work indicates that intrinsic disorder and unbiased mass spectrometry identification rate of a given phosphorylation are important for improving the selection of a 14-3-3 docking site. Using a machine learning model, we developed a tool that combines current available 14-3-3 prediction data and our observations about disorder and phosphorylation observation to predict 14-3-3 binding sites. Our publicly available tool "14-3-3-site-finder" produces a rank order list of potential 14-3-3 docking sites that could help overcome the time-consuming process of identifying the correct site. In our efforts of identifying functional phosphorylations with 14-3-3, we have observed that 14-3-3 interacts with a non-receptor tyrosine kinase, TNK1. TNK1 is a poorly characterized kinase that has essentially nothing known about its substrates, function or regulation. TNK1 has been implicated in both tumor suppressor and oncogenic roles. Particularly, a Hodgkin Lymphoma cell line is dependent on a truncated form of TNK1 for growth. Our work uncovers the first mechanism of regulation for this kinase. We found that MARK kinase phosphorylates TNK1 within the proline rich domain allowing 14-3-3 to dock on this phosphorylation. 14-3-3 binding restrains TNK1 in the cytosol and holds TNK1 in an inactive state. Upon the release of 14-3-3, TNK1 moves to a membrane fraction where it is active. One unique feature of TNK1 is that it has a c-terminal ubiquitin association domain (UBA). In vitro ubiquitin pulldowns indicate that the TNK1 UBA has no preference for linkage type or length of ubiquitin. Further, biolayer interferometry indicates that the UBA binds ubiquitin tightly. Mutation of residues within the ubiquitin:TNK1 interface prevent ubiquitin binding and decrease TNK1 activity, preventing downstream oncogenic signaling, suggesting a UBA-centric mechanism of regulation for TNK1. Finally, we developed a small molecule inhibitor, TP-5801, that selectively targets TNK1. TP-5801 prevents downstream TNK1 phosphorylation of STAT3. Further, TP-5801 prolonged the life of mice injected with TNK1 driven Ba/F3 cells. Taken together, our data reveal the first mechanism of kinase regulation for TNK1 involving 14-3-3 binding and ubiquitin association as well as the development of a TNK1 specific therapeutic

<p dir="ltr">Ubiquitination and ADP-ribosylation are reversible post-translational modifications involved in various cellular activities. Pathogens like <i>Legionella pneumophila</i> and <i>Chromobacterium violaceum</i> target host ubiquitin system via modifications involving ADP-ribosylation. Specifically, <i>Legionella pneumophila</i> mediates atypical ubiquitination of host targets using the SidE effector family in a process that involves ubiquitin ADP-ribosylation on arginine 42 as an obligatory step. On the other hand, <i>Chromobacterium violaceum</i> effector CteC ADP-ribosylates threonine 66 of ubiquitin and causes overall blocking of host ubiquitin signaling. Removal of ADP-ribosylation requires (ADP-ribosyl)hydrolases, with macrodomain enzymes being a major family in this category. In the current study, a proteome-wide screening of ubiquitin interactors in the <i>Legionella</i> secreted proteome was performed, which led to the <i>Legionella</i> macrodomain effector MavL as a regulator of the SidE-mediated ubiquitination pathway by reversing the ubiquitin arginine ADP-ribosylation, likely to minimize potential detrimental effects caused by modified ubiquitin. Crystal structure of ADP-ribose-bound MavL was determined, providing structural insights into substrate recognition and catalytic mechanism. Further bioinformatical analyses reveal DUF4804 as a class of MavL-like macrodomain enzymes uniquely selective for mono-ADP-ribosylated arginine residue. The arginine-specific macrodomains are also present in eukaryotes, as exemplified by two previously uncharacterized (ADP-ribosyl)hydrolases in <i>Drosophila melanogaster</i>. Crystal structures of several proteins in this class provide insights into arginine specificity and a shared mode of ADP-ribose interaction distinct from previously characterized macrodomains. The crystal structure of NAD⁺-bound CteC was also determined, which provided insights into its ADP-ribosylation activity and its ubiquitin specificity. Collectively, the studies described here provide biochemical and structural characterizations and mechanistic insights into bacterial effectors associated with ubiquitin ADP-ribosylation.</p>

<p>  </p> <p>Ubiquitination is a post-translational modification that activates a variety of signaling pathways. The process of tagging ubiquitin (Ub) onto a substrate protein requires three proteins. First, the E1-activating protein primes Ub for attachment to the E2-conjugating enzymes. The E2-conjugating enzyme then brings Ub to E3 ligases, which also recruit the substrate proteins. The final step of this cascade is the transfer of Ub onto the substrate protein. More commonly, ubiquitinated proteins are then degraded via the proteasome. This cascade to downregulate proteins is employed as a cellular adaptation mechanism in response to various threats, including bacterial and viral pathogens. Although the Ub system exists exclusively in eukaryotes, in recent years many bacterial effector proteins and viral factors have been shown to hijack the system through highly regulated mechanisms. In my Ph.D. work, I characterized the hijacking mechanism of a protein produced by human papillomavirus (HPV) that causes downregulation of p53. Downregulation of p53 leads to the oncogenic effects of HPV infection. A strain of oncogenic HPV, HPV-16, produces the E6 protein, which forms a complex with the human ubiquitin E3 ligase, E6AP. This allows E6AP to recognize p53 for ubiquitination. Furthermore, the ability of E6 to act as an adaptor protein to target unnatural substrate proteins has been employed by medicinal chemists as the basis of <u>pro</u>teolysis <u>ta</u>rgeting <u>c</u>himeras (PROTACs). To this extent, my thesis covers three broad ideas that will add to our understanding of <strong>1) Cellular adaptor protein regulation, 2) viral adaptor protein hijacking, and 3) PROTAC ligand development.</strong></p>

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Amino acids constitute the major organic form of transported nitrogen in plants, elements for protein synthesis, and precursors of many plant secondary metabolites, such as lignin, hormones, and flavonoids. Furthermore, amino acid metabolism lies at the crossroad of carbon and nitrogen metabolism. The Arabidopsis glutamine dumper1 (gdu1) mutant secretes glutamine from hydathodes, a phenotype caused by the overexpression of Glutamine Dumper1 (GDU1). GDU1 is a small transmembrane protein presents only in higher plants. The gdu1-1D mutant shows a pleiotropic phenotype: perturbed amino acid metabolism, tolerance to exogenous toxic concentrations of amino acids, elevated amino acid export, and activated stress/defense responses, lesions, and smaller rosettes. The biochemical function of GDU1 remains elusive. To better elucidate the biological processes leading to the complex Gdu1D phenotype, two approaches were conducted: (1) An ethyl methanesulfonate suppressor screening of the Gdu1D phenotype, which led to the isolation of intragenic mutations in GDU1 and mutations in the ubiquitin ligase LOG2 (Loss Of Gdu1D 2). Study of the intragenic mutations in GDU1 helped to characterize its structure-function relationships. Characterization of LOG2 showed that LOG2 interacts with GDU1 and is necessary for the Gdu1D phenotype. (2) The responses of the plant to the dexamethasone-induced expression of GDU1 were studied over time. This experiment identified major signaling pathways contributing to different components of the Gdu1D phenotype and the early events triggered by the perturbation of amino acid homeostasis. Our results showed that GDU1 overexpression first increases amino acid export, which is followed by amino acid imbalance and stress responses. This study sheds light on how amino acid imbalance interacts with various plant signaling pathways and stress responses, and suggests that LOG2 is involved in this process. / Ph. D.

Ischemic heart disease is a leading cause of death worldwide. Ubiquitin (UB), an evolutionary conserved protein, is found in all eukaryotic cells. Previous work has shown that treatment of mice with exogenous UB (eUB) reduces inflammatory response and preserves heart function 3 days following ischemia/reperfusion injury (I/R). This study investigated the long-term (28 days post-I/R) cardioprotective potential of eUB using a mouse model of myocardial I/R; and tested the hypothesis that eUB modulates phenotype and function of macrophages (key cells involved in inflammation post-I/R) using thioglycolate-elicited mouse peritoneal macrophages. Heart function measured at 3, 7, 14 and 28 days post-I/R using echocardiography showed that eUB improves heart function throughout the observation period, and decreases I/R-mediated increase in left ventricular dilation at 3, 14 and 28 days timepoints. Myocardial fibrosis, hypertrophy and apoptosis were lower in eUB-treated hearts 28 days post-I/R. These changes in the heart associated with decreased expression of fibrosis-related proteins (collagen-1α1 and MMP-2) and hypertrophy-related protein (MYH-7B) in UB-treated hearts. Activation of GSK3β (pro-apoptotic kinase) was lower (vs. Sham), while activation of anti-apoptotic kinases, ERK1/2 (vs. I/R) and Akt (vs. Sham), was higher in eUB-treated hearts 28 days post-I/R. Serum levels of IL-6, IL-2 and G-CSF were lower in I/R+UB vs. I/R group 28 days post-I/R. In peritoneal macrophages, eUB induced cytoskeleton reorganization in M1-polarized (IFNγ treatment for 72 hours; 100U/mL) and M2-polarized (IL-4 treatment for 72 hours; 20ng/mL) cells. eUB decreased secretion of IL-1β and TNFα in M1-polarized macrophages, while it decreased secretion of TNFα, IL-10 and GM-CSF in M2-polarized macrophages. Efferocytosis was lower in eUB-treated M2-polarized macrophages, which was reversed by CXCR4 receptor antagonist (AMD3100). eUB enhanced migration of M1-polarized macrophages, while it decreased the migration of M2-polarized macrophages. AMD3100 negated the effects of eUB on M1-polarized macrophage migration. eUB decreased activation of STAT1 and FAK, while increasing activation of ERK1/2 in M1-polarized macrophages. In M2-polarized macrophages, eUB decreased Akt activation. Thus, UB treatment preserves heart function and decreases adverse cardiac remodeling 28 days post-I/R. In polarized macrophages, eUB reduces secretion of inflammatory cytokines, and alters phenotype and function of M1- and M2-polarized macrophages.