Melanoma is one of the most common cancers in the world. For primary melanoma, early diagnosis and surgical excision are effective treatments but, despite the new targeted therapies and immunotherapies, there is still a need for more effective treatment options to improve overall survival for patients with metastatic melanoma. Chemotherapy with genotoxic agents remains the main approach for most cancers, but DNA repair pathways in cancer cells reduce their effectiveness. So disruption of key DNA repair pathways, such as nucleotide excision repair (NER), could be an effective option to combine with chemotherapy for melanoma. The structure-specific endonuclease ERCC1-XPF, which heterodimerises through the C-terminal helix-hairpin-helix (HhH)2 domains of both proteins, is essential for NER. The aim of my project was to determine the mechanism involved in regulating the level of the ERCC1-XPF heterodimer with a view to disrupting NER activity. The project started by determining the ERCC1 and XPF response in six melanoma cell lines to the chemotherapeutic cisplatin at the mRNA and protein levels. Although the mRNA and protein levels of both ERCC1 and XPF increased, there was variable consistency between the cell lines, raising the possibility that post translational modification may play an important role in the regulation of ERCC1- XPF activity. We chose to focus on ubiquitination, because it can affect a protein’s activity at both expression and activation levels and several examples of ubiquitinated DNA repair proteins were known. In the pilot study we found that ERCC1 was accumulated after proteasome inhibitor treatment and decreased by treatment with a translation inhibitor in two melanoma cell lines, suggesting that ERCC1 may be ubiquitinated. By cotransfecting His-tagged ubiquitin and non-tagged ERCC1 constructs into melanoma cells and performing an ubiquitin assay, we found that ERCC1 was degraded by the proteasome system through polyubiquitination or multiple monoubiquitination. To determine the nature of the ubiquitination type, we mutated each of the seven Lys residues on ubiquitin and carried out additional assays with ubiquitin single and combination mutants, and discovered that Lys33 was most likely involved in the proteasome dependent degradation of ERCC1. By immunoprecipitation with an antibody to linear ubiquitin from melanoma cell extracts containing a ubiquitin construct with all seven Lys residues mutated to Arg, we found that the N-Met of ubiquitin was also most likely involved in ERCC1 ubiquitination. To determine which Lys of ERCC1 is used by ubiquitin, we did another series of in vivo ubiquitin assays with full length and truncated ERCC1 constructs and found that the key amino acid is most likely within the C-terminal XPF binding domain of ERCC1. By cotransfecting the full length ERCC1 and ERCC1 truncation constructs together with full length XPF, we showed that the ubiquitination of ERCC1 was not an artefact resulting from overexpression of ERCC1 alone and that the stability of XPF was dependent on the overexpression and stability of ERCC1. We then made single lysine and lysine combination mutants in the XPF binding domain of ERCC1 and found that none of the lysines were essential for ubiquitination of ERCC1, indicating that a non- lysine amino acid might be used for ubiquitination. However, using a transfection-based NER assay in ERCC1-deficient cells, we found that ubiquitination of Lys 295 could be involved in regulating the DNA repair activity of ERCC1-XPF. The in vivo ubiquitin assay result after cotransfection of ERCC1 and XPF, which showed that XPF was dependent on the presence of ERCC1 for stability, but not vice versa, was inconsistent with previous published data suggesting that heterodimerization was essential for the stability of both proteins. Instead we hypothesised that homodimerization of ERCC1 might be another mechanism to keep ERCC1 stable and obtained evidence for this at the overexpression level by immunoprecipitation following cotransfection of Myc-tagged ERCC1 and Flag-tagged ERCC1 or ERCC1 truncations, which was supported at the endogenous expression level by size exclusion chromatography on melanoma cell extracts to identify ERCC1 in different molecular weight fractions. In the previous in vivo ubiquitin assay, we found that levels of transfected full length ERCC1 and XPF were dramatically reduced by cotransfection with the Flag-tagged ERCC1 (220-297) construct that just contains the XPF binding domain of ERCC1. This led to another hypothesis, that the ERCC1 (220-297) peptide can decrease endogenous levels of ERCC1 and XPF and so be a potential drug in combination with cisplatin chemotherapy. This hypothesis was verified in stable transgenic cell lines expressing ERCC1 (220-297) which showed reduced levels of ERCC1 and XPF and of NER and increased sensitivity to cisplatin and UV irradiation. Based on the above results and supporting bioinformatics analysis we have made the following conclusions: ERCC1 is regulated by the ubiquitin-proteasome degradation pathway through linkages most likely involving Lys33 and N-Met; the XPF binding domain is most likely the key domain for ERCC1 ubiquitination; XPF stability is dependent on the presence of ERCC1 and seems affected by ERCC1 ubiquitination; ERCC1 seems to be ubiquitinated in a non-conventional lysine-independent manner and ubiquitination of Lys 295 might be involved in the regulation of the DNA repair activity of ERCC1- XPF; homodimerization is most likely a novel mechanism to keep ERCC1 stable; the ERCC1 (220-297) peptide can destabilise both ERCC1 and XPF and could be a potential drug in combination with genotoxic therapies.

The ubiquitin-proteasome system (UPS) controls the stability, activity and localisation of most of the proteome and regulates virtually all cellular processes through modification of proteins with ubiquitin. Ubiquitin conjugation is mediated by a conserved enzymatic cascade composed of E1, E2 and E3 enzymes, which cooperate to activate and transfer ubiquitin to substrate proteins. Dysfunction of the UPS is implicated in many disease states, including cancer, neurodegeneration, immune and cardiovascular disorders. Despite the central role of the UPS in cellular regulation, our understanding of the function, interactions and specificity of proteins that comprise the UPS is still limited. One approach to dissect and to study the UPS is to identify molecular probes, which can be used to specifically interrogate catalytic mechanisms and can be potentially considered as entry points for drug discovery. This work focuses on developing novel high-throughput screening methods for inhibitors of the ubiquitin-conjugating enzymes (E2s), using a unicellular organism Saccharomyces cerevisiae and in vitro technologies. S. cerevisiae is a model organism, commonly used in research as a valuable tool for genetic investigations and other high-throughput studies. In this work, we evaluated the toxicity of exogenously expressed human E2s on yeast cells and discovered that one of the E2s, Ube2U, significantly inhibited yeast growth. This inhibition was dependent on the Ube2U ubiquitin-conjugation activity, as demonstrated with a catalytically inactive Ube2U C89A control, which did not affect yeast growth. The growth defect induced by Ube2U allowed us to develop a screening setup for inhibitors of Ube2U, where the enzyme activity was coupled to cell growth readout. Potential Ube2U inhibitors would be identified as rescuers of the slow growing Ube2U-expressing yeast phenotype. Although screening methods in yeast are relatively straightforward to set up and run, the advantages of this system, namely simplicity of the detection signal and high-throughput, are limited by the fact that yeast is not a recognised large scale screening system in pharmaceutical industry, and that it is difficult to identify the target in a complex pathway such as the UPS. In vitro technologies are needed to provide the necessary structure-activity relationship for chemical optimisation. Therefore, we developed a novel, fluorescence-based, miniaturised assay technology, suitable for biochemical investigations and screening for inhibitors of a wide range of specific ubiquitination reactions within the UPS.

p53 is a key component of the protein network that regulates cell cycle progression and prevents cancer. Under non-stressed conditions, its activity is controlled by an autoregulatory feedback loop with MDM2 that maintains low levels of the p53 protein. However, in response to stress signals, p53 is triggered to become active. MDM2 has been reported to regulate p53 by a combination of mechanisms: ubiquitination using its E3-ligase capability, chaperone activity in an ATP-dependent manner and directly transrepressing p53. Because of MDM2's central role in the control of p53, it has been the target of intense drug development efforts. A family of small molecules, the Nutlins, can bind to an MDM2 pocket modulating the p53: MDM2 complex. This leads to p53 activation and growth inhibitory effects. The aim of our study was to analyse the interactome of endogenous MDM2 and to determine whether anti-cancer drugs, such as Nutlin-3, could stabilise or disrupt sets of MDM2 interactions in order to better understand the p53- dependent and independent functions of MDM2 as a signalling hub, as well as the p53-independent activity of Nutlin-3. Results show a remarkable difference in the sets of proteins found in MDM2 complexes in control and Nutlin-3 treated cells. Two proteins, TRIM25 and OTUB2, were selected from the output list for validation based on their known functions in the ubiquitin signalling network. Binding has been studied in detail and confirmed using both in cell and in vitro techniques. The data highlight potentially novel functions for MDM2 and provides insight into the on-target p53-independent activities of Nutlin-3. Additionally, and with the aim of blocking p53 ubiquitination by MDM2, I have developed probes that are able to inhibit the ubiquitylation of p53 in vitro.

Pulmonary surfactant is a critical surface-active substance consisting of dipalmitoylphosphatidylcholine (DPPtdCho) and key apoproteins that are produced and secreted into the airspace from alveolar type II epithelial cells. Deficiency of the surfactant leads to severe lung atelectasis, ventilatory impairment, and gas-exchange abnormalities. The generation of DPPtdCho in cells occurs via two integral routes: the de novo and remodeling pathways. The interplay between these pathways has not been investigated. Overexpression of the remodeling enzyme, acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT1), in epithelia decreases de novo PtdCho synthesis without significantly altering cellular phospholipid mass; this occurs through increased degradation of cholinephosphotransferase (CPT1), the terminal enzyme of the de novo pathway. CPT1 is degraded by multi-ubuiquitination and trafficking via the lysosomal pathway. When expressed in lung epithelia, CPT1 mutants harboring arginine substitutions at multiple carboxyl-terminal lysine residues exhibited proteolytic resistance to effects of LPCAT1 overexpression. Cellular expression of these CPT1 mutants also restores de novo PtdCho synthesis to levels normally observed in lung epithelia. Further studies demonstrate that the SCF (Skip-Cullen-F-box) ubiquitin E3 ligase component, β-TrCP, was sufficient to degrade CPT1. Similar to CPT1, LPCAT1 levels are also regulated at the level of protein stability. However, LPCAT1 is a polyubiquitinated enzyme processed within the proteasome. Similar to CPT1, β-TrCP is the putative E3 ubiquitin ligase subunit responsible for LPCAT1 ubiquitination. β-TrCP appears to dock and ubiquitinate LPCAT1 within its amino-terminus. Collectively, these observations indicate the presence of cross-talk between the phospholipid remodeling and de novo pathways; this involves tight regulation by site-specific ubiquitination of indispensable regulatory enzymes catalyzed by SCF ubiquitin E3 ligase members that mechanistically provide homeostatic control of cellular phospholipid content.

The IkappaB kinase epsilon (IKKepsilon, IKKi, IKBKE) is both a regulator of innate immunity and a breast cancer oncogene that is amplified and overexpressed in ~30% of breast cancers. IKKepsilon promotes malignant transformation through the activation of NF-kappaB signaling. In addition, breast cancers that harbor amplifications in the IKBKE gene are dependent on IKKepsilon protein expression for survival. IKKepsilon has been characterized as a non-canonical inhibitor of kappaB kinase (IKK) that activates both the interferon response pathway and NF-kappaB signaling in innate immunity. In this dissertation, I explore both the regulation and effectors of the IKKepsilon kinase in the context of malignant transformation. I found that IKKepsilon is modified and regulated by K63-linked polyubiquitination, a proteasome- and degradation-independent form of ubiquitination, at Lysine 30 and Lysine 401. This modification is essential for IKKepsilon-induced kinase function and IKKepsilon-mediated NF-kappaB activation and malignant transformation. Furthermore, I identified TRAF2 as the K63 ubiquitin E3 ligase that associates with and modifies IKKepsilon. I also found that TBK1, a close family member of IKKepsilon, is also regulated by K63-linked ubiquitination. In collaborative work, we used an unbiased positional scanning peptide library screen to identify two novel downstream targets of IKKepsilon phosphorylation in the context of cancer. Specifically, we found IKKepsilon phosphorylates the tumor suppressor CYLD at Serine 418. CYLD phosphorylation at Ser418 downregulates its deubiquitinase activity and is necessary for IKKepsilon-driven transformation. IKKepsilon also phosphorylates TRAF2 at Serine 11. This activity promotes K63-linked TRAF2 ubiquitination, NF-kappaB activation and is also essential for IKKepsilon-transformation. In addition, breast cancer cells that depend on IKKepsilon expression for survival are also dependent on TRAF2. Together, these observations define an oncogenic network that promotes NF-kappaB-mediated cell transformation through the K63-linked ubiquitination of IKKepsilon and subsequent phosphorylation of two novel substrates, TRAF2 and CYLD.

Programmed cell death (PCD) is an active process by which organisms coordinate the controlled destruction of cells. In tomato, the protein kinase Adi3 (AvrPto-dependent Pto-interacting kinase 3), acts as a negative regulator of PCD and shares important functional homologies with the mammalian anti-apoptotic AGC kinase PBK/Akt. Adi3 was originally identified as an interactor of the complex formed by the tomato resistance protein Pto and the Pseudomonas syringae pv. tomato (Pst) effector protein AvrPto. The complex formed by AvrPto and Pto causes a resistance response characterized by a rapid form of PCD that limits the spread of Pst and prevents the onset of the tomato bacterial speck disease. In an effort to characterize the mechanisms by which Adi3 regulates PCD, we identified Adi3 interacting partners in a Y2H screen. Here, I describe the interaction of Adi3 with two interacting partners identified: the Sucrose Non-fermenting (SNF1) kinase complex (SnRK) which is a eukaryotic master regulator of energy homeostasis and the E3 RING Ubiquitin ligase AdBiL. Using a combination of in vitro and in vivo approaches I found that AdBiL is an active ubiquitin ligase that ubiquitinates Adi3. Interestingly, Adi3 was found to be degraded in a proteasome-dependent manner suggesting ubiquitination could play a role in its degradation. On the other hand, Adi3 was found to inhibit the SnRK complex by directly interacting with its catalytic subunit as well as by phosphorylating the regulatory subunit SlGal83 at Ser26. SlGal83 is phosphorylated at multiple sites in vivo, and this phosphorylation state, as well as its intracellular localization was found to depend on a myristoylation signal present at its N-terminus. Phosphorylation at Ser26 by Adi3 was found to alter the localization of this subunit in a myristoylation-dependent manner. The interactions studied in this dissertation provide additional evidence on the functional homologies shared by Adi3 and PKB. In addition, the regulatory control of SnRK activity and cellular localization offers a novel connection between pathways involved in energy homeostasis and pathogen-mediated PCD.

The Large Tumor Suppressor 1 (LATS1) is a serine/threonine kinase and tumor suppressor found down-regulated in a broad spectrum of human cancers. It is a central player of the emerging Hippo-LATS tumour suppressor pathway, which plays important roles in cell proliferation, apoptosis, and stem cell differentiation. Despite the ample data supporting a role of LATS1 in tumour suppression, how LATS1 is regulated at the molecular level remains largely unknown. In this study, we have identified Itch, a HECT class E3 ubiquitin ligase, as a novel binding partner of LATS1. Itch can complex with LATS1 both in vitro and in vivo through the PPxY motifs of LATS1 and the WW domains of Itch. Significantly, we found that over-expression of Itch promoted LATS1 degradation by polyubiquitination through the 26S proteasome pathway. On the other hand, knockdown of endogenous Itch by shRNAs provoked stabilization of endogenous LATS1 proteins. Finally, through several functional assays, we also revealed that change of Itch abundance alone is sufficient for altering LATS1-mediated downstream signaling, negative regulation of cell proliferation, and induction of apoptosis. Together, our study identifies E3 ubiquitin ligase Itch as the first negative regulator of LATS1 and presents for the first time a possibility of targeting LATS1/Itch interaction as a therapeutic strategy in cancer. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2011-04-26 22:25:46.008

Le Ca[indice supérieur 2+] intracellulaire est impliqué dans un grand nombre de processus biologiques chez toutes les cellules de l’organisme. Chez les cellules non-excitables, les protéines TRPC, pour transient receptor potentiel canonical, situées à la membrane plasmique, sont des canaux calciques impliqués dans l’entrée de calcium. TRPC6 est particulièrement étudiée vu son implication potentielle dans plusieurs problèmes pathologiques. La glomérulosclérose focale et segmentale (FSGS) qui est une maladie dérégulant le système de filtration du rein, l’hypertension artérielle pulmonaire idiopathique (IPAH), caractérisé par une élévation anormale et sporadique de la pression sanguine au niveau des artères pulmonaires, ainsi que dans certains cancers. Ses modes et mécanismes d’activation ainsi que sa régulation sont encore aujourd’hui très peu connus, malgré les nombreuses recherches menées. Le but de la présente étude est d'investiguer la régulation de TRPC6 via son ubiquitination. L’ubiquitination des protéines, processus étudié depuis longtemps, a été démontré pour moduler l’activité de plusieurs protéines et réguler leur localisation cellulaire et membranaire. Nos travaux démontrent, que l’état d'ubiquitination de TRPC6 est modulé et favorisé par une stimulation avec des agonistes du canal TRPC6 , tel que le CCh et la Tg. L’utilisation d’inhibiteurs des différentes voies de dégradation utilisant l’ubiquitine comme moyen de régulation, nous permet de démontrer que l’ubiquitination de TRPC6 se fait suivant son internalisation. Dans un second temps, nous nous sommes intéressés à l’implication de Rabex-5, un facteur d’échange de nucléotide guanylique (GEF) de Rab5 qui se lie aux protéines membranaire ubiquitinylées. Nous démontrons une interaction entre TRPC6 et Rabex-5. Cette interaction n’a aucune influence sur l’activité de TRPC6. De plus, nous démontrons que l’activité GEF de Rabex-5 n’est pas nécessaire à cette interaction et n’influence pas l’activité du canal TRPC6 . Cette étude a permis de déterminer que TRPC6 est un canal ubiquitinylé, que cette ubiquitination se fait suivant son internalisation et est modulée par certains agonistes, tel que le CCh et la Tg. Sans que cette dernière ne soit impliquée dans l’activité fonctionnelle du canal, nous démontrons également une interaction entre TRPC6 et Rabex-5, une protéine localisée au niveau des endosomes précoces. Cette étude est un premier pas vers la compréhension de la régulation via l’ubiquitination, pour le canal TRPC6.

Ubiquilin (UBQLN) is a member of type2 ubiquitin-like (UBL) protein family characterized by an UBL domain at the N-terminus and an ubiquitin associated (UBA) domain at the C-terminus. This protein has been shown to play an important role in the regulation of the levels, aggregation and degradation of various neurodegenerative disease-associated proteins. However, the specific functions and mechanisms of UBQLN regulation still remain to be elucidated. In this study, we investigate the effect of UBQLN expression on α-Amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid receptor (AMPAR) degradation and the underlying molecular mechanisms. We show that UBQLN overexpression decreases AMPAR levels in neurons and also reduces GluA1 expression in HEK 293T cells. Moreover, our results indicate that UBQLN can form a complex with GluA1, and this interaction is related to the ubiquitination of AMPARs. In addition, we find a higher expression of UBQLN2 in Alzheimer’s disease (AD) patient brains, which might be a potential pathological mechanism of GluA1 reduction in AD. Given the crucial effect of UBQLN in AMPAR regulation, UBQLN may play an important role in synaptic transmission, brain functions as well as neurodegenerative diseases. / 2018-07-21T00:00:00Z

>Magister Scientiae - MSc / P53 is one of the most important tumour suppressor proteins in the body which protects the cell against the tumourigenic effects of DNA damage by initiating processes such as apoptosis, senescence and cell cycle arrest. Regulation of p53 is key — so that the abovementioned processes are not initiated inappropriately. The principle negative regulator of p53 is Murine Double Minute 2 (MDM2), a RING finger-containing protein which catalyses the attachment of lysine48-linked poly-ubiquitin chains, targeting it for degradation by the 26S proteasome. It has been found to work in conjunction with the MDM2 homologue MDMX. Retinoblastoma Binding Protein 6 (RBBP6) is a RING finger-containing protein known to play a role in mRNA 3’-end processing, as well as interacting with p53 and another crucial tumour suppressor, pRb. It has previously been shown to cooperate with MDM2 in the ubiquitination and degradation of p53 in vivo and acts as a scaffold. The objectives of this project are to investigate the proposed role of RBBP6 in the MDM2-catalysed ubiquitination of p53 using a fully in vitro ubiquitination system. Due to the difficulty of expressing full length RBBP6 in bacteria, a shortened version, dubbed "R3" was used which includes the RING finger domain but excludes the domain identified in earlier studies as the p53-binding domain. Proteins required to set up the fully in vitro p53 ubiquitination assays – including E1 and E2 enzymes, MDM2, R3, p53 and ubiquitin - were all successfully expressed in bacteria. The active 26S proteasome was successfully purified out of human cell lysates using antibodies targeting the α2-subunit. Cloning, expression and purification results showed that p53, MDM2 and R3 were not very stable proteins to work with — with degradation being initiated almost immediately after expression and purification which progressed during the downstream processing of the proteins. Although levels of intact protein were not always high, they were sufficient for in vitro assays. MDM2 and GST-R3 were both capable of poly-ubiquitinating p53 independently in "partially in vitro" assays using human cell lysate. The fully in vitro ubiquitination of p53 using MDM2 and R3 was established based on the well-known MDM2/MDMX system. When acting together R3 and MDM2 was shown to produce poly-ubiquitination which is lysine-48 linked and recognised by the 26S proteasome leading to degradation. When the proteasome inhibitor MG132 was added, the poly-ubiquitinated p53 was rescued from degradation. R3 was also shown to successfully poly-ubiquitinate p53 independently of MDM2 and also interact with p53 in vitro. These results suggest R3 to be of the same order of importance as that of MDM2 — which is known to be the most important regulator of p53. It would also rule out the proposed model of RBBP6 functioning as a scaffold as it is able to poly-ubiquitinate p53 independent of MDM2. These results allow us to better understand the mechanism in which p53 is down-regulated by E3s. / National Research Foundation (NRF)