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Modulation Of Kras Structure And Dynamics By Kras Ubiquitination And Membrane Depolarization, Vinay Nair

Dissertations & Theses (Open Access)

KRAS, a 21 kDa small GTPase protein, functions as a molecular switch playing a key role in regulating cell proliferation. Dysregulation of KRAS signaling by oncogenic mutations leads to uncontrolled cell proliferation, a hallmark of cancer cells. Attempts to therapeutically target oncogenic KRAS have led to limited success resulting in a need to identify new mechanisms to targeting KRAS. The interaction of KRAS with its regulators, effectors, and the membrane present one such avenue. In this study, we investigated how post-translational covalent and environmental modifications could modulate these interactions of KRAS. Using computational molecular dynamics simulations, nuclear magnetic resonance spectroscopy …

Deubiquitinating Enzymes Promote Cancer Progression And Metastasis Via Regulating Protein Stability, Zhenna Xiao

Dissertations & Theses (Open Access)

Deubiquitinating enzymes (DUBs, also called deubiquitinases) are enzymes that remove monoubiquitin or polyubiquitin chains from target proteins. DUBs have critical roles in cell homeostasis and signal transduction, as they regulate protein degradation, subcellular localization, and protein-protein interaction. Deregulation of DUBs contributes substantially to tumor formation and progression, and therefore targeting DUBs may be a promising cancer therapy strategy. My dissertation focuses on identifying the DUBs of EZH2 and SNAI1, two proteins critical for cancer progression and metastasis, and establishing these DUBs as promising anti-cancer targets.

EZH2, the catalytic component of the PRC2 complex, silences gene transcription by histone methylation. High …

Phopsphorylation And Ubiquitin Modification At Dna Damage Sites In Response To Double-Strand Breaks, Atanu Paul

Dissertations & Theses (Open Access)

Genomes of all organisms are continuously damaged by numerous exogenous and endogenous sources leading to different kinds of DNA lesions, which if not repaired efficiently may trigger wide-scale genomic instability, a hallmark of cancer development. To overcome this, cells have evolved a sophisticated sensory network called the DNA damage response (DDR) comprised of a large number of distinct protein complexes categorized as sensor, mediator, transducer and effector proteins that amplify the DNA damage signal and activate cell cycle checkpoint to initiate DNA repair or trigger apoptosis where the defect is beyond repair. This intricate signaling pathway is tightly regulated by …

The Role Of Amp-Activated Protein Kinase (Ampk) In Tumorigenesis, Fei Han

Dissertations & Theses (Open Access)

AMPK plays a central role in controlling cellular and whole body energy level. Increasing studies have also discovered the diverse function of AMPK in cancer, such as autophagy and mitochondria biogenesis. However, how AMPK promotes cancer progression is still not clear. Here, we show that AMPK is essential for EGF-induced Akt activation, Glut1 expression, and glucose uptake. AMPK is also required for various stresses induced Akt activation and promote cell survival, including hypoxia and glucose deprivation. In addition, we found glucose deprivation-induced VEGF expression and secretion is also depend on AMPK, which may contribute to angiogenesis of surrounding endothelial cell …

Novel Posttranslational Modification In Lkb1 Activation And Function, Szu-Wei Lee

Dissertations & Theses (Open Access)

Cancer cells display dramatic alterations in cellular metabolism to meet their needs of increased growth and proliferation. In the last decade, cancer research has brought these pathways into focus, and one emerging issue that has come to attention is that many oncogenes and tumor-suppressors are intimately linked to metabolic regulation (Jones and Thompson, 2009). One of the key tumor-suppressors involved in metabolism is Liver Kinase B1 (LKB1). LKB1 is the major upstream kinase of the evolutionarily conserved metabolic sensor—AMP-activated protein kinase (AMPK). Activation of the LKB1/AMPK pathway provides a survival advantage for cells under energy stress. LKB1 forms a heterotrimeric …

The Role Of Traf6 Phosphorylation In Src/Traf6-Mediated Ikk, Jnk, Akt Activation And Tumorigenesis, Yun Seong Jeong

Dissertations & Theses (Open Access)

TRAF6 E3 ligase regulates numerous essential biological processes such as innate immune response, cell survival and osteoclast differentiation. Upon activation, it mediates activation of IKK/NF-κB and JNK signaling in response to engagement of toll-like receptor 4 (TLR4), interleukin-1 receptor (IL-1R), and receptor activator of NF-κB (RANK) to their cognate ligands, including lipopolysaccharide (LPS), IL-1, and RANK ligand (RANKL). Recently, TRAF6 has also been shown to be involved in Akt signaling activation upon activation of insulin-like growth factor-1 receptor (IGF-1R), in turn orchestrating cell survival and tumorigenesis. Therefore, TRAF6 is a key player for the activation of IKK, JNK and Akt …

The Role Of K63-Linked Ubiquitination Cycles In Akt Kinase Activation, Wei-Lei Yang

Dissertations & Theses (Open Access)

Akt (also known as protein kinase B) serves a central regulator in PI3K/Akt signaling pathways to regulate numerous physiological functions including cell proliferation, survival and metabolism. Akt activation requires the binding of Akt to phospholipid PIP3 on the plasma membrane and subsequent phosphorylation of Akt by its kinases. Growth factor-mediated membrane recruitment of Akt is a crucial step for Akt activation. However, the mechanism of Akt membrane translocation is unclear. Protein ubiquitination is a significant posttranslational modification that controls many biological functions such as protein trafficking and signaling activation. Therefore, we hypothesize that ubiquitination may be involved in Akt signaling …

Cop 9 Signalosome Subunit 6 Stabilizes Cop1, A Novel E3 Ubiquitin Ligase For 14-3-3Σ, Hyun Ho Choi

Dissertations & Theses (Open Access)

14-3-3σ, a gene upregulated by p53 in response to DNA damage, exists as part of a positive-feedback loop which activates p53 and is a human cancer epithelial marker downregulated in various cancer types. 14-3-3σ levels are critical for maintaining p53 activity in response to DNA damage and regulating signal mediator such as Akt. Here, we identify Mammalian Constitutive Photomorphogenic 1 (COP1) as a novel E3 ubiquitin ligase for targeting 14-3-3σ through proteasome degradation. We show for the first time that COP9 signalosome subunit 6 (CSN6) associates with COP1 and is involved in 14-3-3σ ubiquitin-mediated degradation. Mechanistic studies show that CSN6 …

Regulation Of Set1-Mediated Methylation Of Dam1, John A. Latham

Dissertations & Theses (Open Access)

Eukaryotic genomes exist within a dynamic structure named chromatin in which DNA is wrapped around an octamer of histones forming the nucleosome. Histones are modified by a range of posttranslational modifications including methylation, phosphorylation, and ubiquitination, which are integral to a range of DNA-templated processes including transcriptional regulation. A hallmark for transcriptional activity is methylation of histone H3 on lysine (K) 4 within active gene promoters. In S. cerevisiae, H3K4 methylation is mediated by Set1 within the COMPASS complex. Methylation requires prior ubiquitination of histone H2BK123 by the E2-E3 ligases Rad6 and Bre1, as well as the Paf1 transcriptional …

14-3-3sigma Negatively Regulates The Stability And Subcellular Localization Of Cop1, Chun-Hui Su

Dissertations & Theses (Open Access)

Mammalian constitutive photomorphogenic 1 (COP1), a p53 E3 ubiquitin ligase, is a key negative regulator for p53. DNA damage leads to the translocation of COP1 to the cytoplasm, but the underlying mechanism remains unknown. We discovered that 14-3-3σ controlled COP1 subcellular localization and protein stability. Investigation of the underlying mechanism suggested that, upon DNA damage, 14-3-3σ bound to phosphorylated COP1 at S387, resulting in COP1 translocation to the cytoplasm and cytoplasmic COP1 ubiquitination and proteasomal degradation. 14-3-3σ targeted COP1 for degradation to prevent COP1-mediated p53 degradation, p53 ubiquitination, and p53 transcription repression. COP1 expression promoted cell proliferation, cell transformation, and …

Inhibition Of Deubiquitinase Activity And Ubiquitination Of Jak2 Blocks Cytokine Signaling And Induces Tumor Cell Apoptosis, Vaibhav Kapuria

Dissertations & Theses (Open Access)

The Jak-stat pathway is critical for cellular proliferation and is commonly found to be deregulated in many solid tumors as well as hematological malignancies. Such findings have spurred the development of novel therapeutic agents that specifically inhibit Jak2 kinase, thereby suppressing tumor cell growth. Tyrphostin AG490, the first described Jak2 inhibitor, displays poor pharmacology and requires high concentrations for anti-tumor activities. Our research group screened a small library of AG490 structural analogues and identified WP1130 as a potent inhibitor of Jak2 signaling. However, unlike AG490, WP1130 did not directly inhibit Jak2 kinase activity. Our results show that WP1130 induces rapid …

The Ubiquitin Ligase Ube4b Is Required For Efficient Epidermal Growth Factor Receptor Degradation, Natalie Sirisaengtaksin

Dissertations & Theses (Open Access)

The length of time that integral membrane proteins reside on the plasma membrane is regulated by endocytosis, a process that can inactivate these proteins by removing them from the membrane and may ultimately result in their degradation. Proteins are internalized and pass through multiple distinct intracellular compartments where targeting decisions determine their fate. Membrane proteins initially enter early endosomes, and subsequently late endosomes/multivesicular bodies (MVBs), before being degraded in the lysosome. The MVB is a subset of late endosomes characterized by the appearance of small vesicles in its luminal compartment. These vesicles contain cargo proteins sorted from the limiting membrane …