Target of Myb 1 (TOM1) is an endosomal adaptor protein that plays a role in cargo membrane trafficking for degradation by serving as an alternative endosomal sorting complex required for transport component. TOM1 has also been shown to serve as a novel phosphatidylinositol 5-phosphate (PtdIns5P) effector at signaling endosomes through its VHS domain, delaying cargo degradation in a bacterial infection model. The aim of this thesis is to clarify the structural and functional basis of the autoregulation mechanism of TOM1 to switch from endosomal protein trafficking to the bacterial survival signaling pathway. Our thermal denaturation and spectroscopic studies demonstrate that PtdIns5P reduced thermostability, interhelical contacts, and conformational compaction of TOM1 VHS. The thermodynamic studies indicate that TOM1 VHS endothermically binds to PtdIns5P through two potential noncooperative binding sites, with its acyl chains playing a relevant role in the interaction. These findings suggest that, under Shigella flexneri infection, TOM1 may interact with downstream effectors in a different VHS domain conformational state, thus involving the protein in bacterial survival signaling pathways. In order to obtain molecular details for the interaction of the TOM1 VHS domain for PtdIns5P and Ubiquitin (Ub), the backbone assignment information was obtained by performing NMR experiments, which assigned backbone 1H, 13C, and 15N resonances of the TOM1 VHS domain. With this structural information, our heteronuclear single quantum coherence and molecular dynamics simulations data revealed that TOM1 VHS interacts with PtdIns5P following a fast-exchange regime, with the PtdIns5P binding site predicted to be at a region spanning α-helices 6 to 8. Further mutagenesis and lipid-protein overlay assay studies indicated that K147 plays a critical role in the binding of TOM1 VHS domain to PtdIns5P. TOM1, unexpectedly, did not bind PtdIns5P. Using truncated forms of TOM1 protein, we discovered that neither TOM1 GAT domain nor the C-terminal domain modulated TOM1 VHS's PtdIns5P binding; however, surprisingly, a linker sequence between the TOM1 VHS and GAT domains exhibited an autoinhibition role for TOM1 binding to PtdIns5P. This linker region was observed to induce local conformational changes on the structure of TOM1 VHS domain, especially around α-helices 6 and 8, which are proposed to build up the binding pocket for PtdIns5P. In order to investigate whether the linker region between TOM1 VHS and GAT domain can also regulate the Ub association of TOM1 VHS domain, the binding properties of TOM1 and its domains to Ub were explored. Unexpectedly, the binding affinity of TOM1 VHS-linker for Ub was increased about 10-fold when compared with that for the TOM1 VHS domain, suggesting that the linker enhances the avidity of TOM1 for ubiquitinated cargo. Structural analysis indicated that the linker region may cap the conventional Ub-binding site of TOM1 VHS, thus forming a more compact structure. In summary, this study uncovered a novel intramolecular modulatory mechanism in TOM1 that regulates ligand recognition by its VHS domain. By providing the molecular basis of the TOM1 interactions, we may provide cargo sorting mechanistic insights, create functionally specific mutations, and precisely manipulate TOM1 function under bacterial infection conditions, and other yet-to-be-discovered PtdIns5P-dependent signaling pathways. / Doctor of Philosophy / Membrane trafficking is a delivery network established in a cell to transport proteins (cargoes) from one intracellular place to another one to control their activity. TOM1 is a protein involved in this process, which plays a role in transporting cargoes for degradation. Defects in this trafficking pathway lead to human diseases, such as immunodeficiency and neurodegeneration diseases. TOM1 has also been shown to be beneficial for bacterial survival in human cells. However, how TOM1 switches its role form protein trafficking to bacterial pathogenesis is still unclear. In our study, we discovered an internal region of TOM1 may serve as a switch to shift the role of TOM1 in human cells. In an "on" status, TOM1 favors to transport cargoes, while in an "off" status, TOM1 is used for bacteria survival. This study provides insights in the function of TOM1 which is beneficial for the design of novel therapeutic strategies against TOM1, which will prevent the progress of bacterial infections.

The endosomal trafficking system is a network of highly coordinated cellular pathways that control the growth and function of cells. The coordination of secretion and endocytosis in cells is one of the primary drivers of polarized growth, where new plasma membrane and cell wall components are deposited at the growing apex. In plants, one of the cell types exhibiting polarized growth are the root hairs. Root hairs are regulated extensions of epidermal cells called trichoblasts and are essential for anchorage, absorption of water and nutrients, and plant-microbe interactions. In this thesis, I characterize a previously undescribed protein involved in retromer function and endosomal trafficking pathways that regulate tip growth in root hairs of Arabidopsis thaliana. The large retromer complex functions in recycling receptors in endosomal trafficking pathways essential for diverse developmental programs including cell polarity, programmed cell death, and shoot gravitropism in the model plant, Arabidopsis thaliana. I have characterized VPS26C, a novel member of the large retromer complex, that is essential in maintaining root hair growth in Arabidopsis. We used Bimolecular Fluorescence Complementation (BiFC) analysis to demonstrate thatVPS26C interacts with previously characterized core retromer subunits VPS35A and VPS29. Genetic analysis also indicates that vps26c suppresses the root hair growth and cell wall organization phenotypes of a null mutant of the SNARE VTI13 that localizes to early endosomes and the vacuole membrane, indicating a crosstalk between the VPS26C-retromer and VTI13-dependent vesicular trafficking pathways. Phylogenetic analysis was used to show that VPS26C genes are present in most angiosperms but appear to be absent in monocot genomes. Moreover, using a genetic complementation assay, we have demonstrated that VPS26C shares deep conservation of biochemical function with its human ortholog (DSCR3/VPS26C). We also used an affinity purification-based proteomic analysis to identify proteins associated with VTI13 in young seedlings. Preliminary results suggest that a number of proteins linked to cell plate organization in plants are associated with the VTI13 proteome, emphasizing the potential role of this pathway in new cell wall biosynthesis/organization. Additionally, we have identified endoplasmic reticulum (ER)-body proteins, involved in plant defense response pathways, suggesting that either the VTI13 endosomal trafficking pathway is functioning in plant defense responses, or the ER-body proteins have additional independent function(s) in Arabidopsis roots that depend on VTI13. In summary, I have described a novel retromer complex essential for polarized growth in Arabidopsis. VPS26C is an ancient gene and shares sequence and functional homology between human and Arabidopsis. vps26c is a genetic suppressor of the vti13- dependent root hair growth and cell wall organization pathways. Proteomic analysis of VTI13 endosomes in young seedlings suggests that a number of proteins associated with cell plate formation are associated with VTI13 compartments, supporting the genetic analysis described here and serves as a starting point to further describe the role of this pathway in controlling polarized growth in plants.

Adaptor proteins, like Tollip and Tom1, facilitate cellular cargo sorting through their ubiquitin-binding domains. Tollip and Tom1 bind to each other through their TBD and GAT domains, respectively, whereas Tollip interacts with phosphatidylinositol-3-phosphate (PtdIns(3)P)-containing endosomal membranes. Tom1 and Tollip interaction and association with endosomes is proposed to be involved in the lysosomal degradation of polyubiquitinated cargo. Through cellular, biochemical, and biophysical techniques, we have further characterized the association of Tom1 with Tollip. Mutations in the binding interface of the Tom1 GAT and Tollip TBD complex leads to a subcellular mis-localization of both proteins, indicating that Tom1 may serve to direct Tollip to specific cellular pathways. It was determined that Tom1 inhibits the binding of Tollip to PtdIns(3)P and inhibition was reversed when mutations in the binding interface of the Tom1 GAT and Tollip TBD were present. Furthermore, it was established that, upon the binding of Tollip TBD to Tom1 GAT, ubiquitin is inhibited from binding to Tom1 GAT. It was also demonstrated that Tom1 GAT, but not Tollip TBD, can weakly bind to PtdIns(3)P. Consequently, we propose that association of Tom1 may serve to direct Tollip for involvement in specific cell signaling pathways. Gaining insight into the function of Tom1 and Tollip may lead to their use as therapeutic targets for increasing the efficiency of cargo trafficking and also for patients recovering from various cardiac injuries. / Master of Science

Dans les années 2000, la Dre Johanne Tremblay et son équipe identifient un gène régulé négativement par le calcium extracellulaire dans les glandes parathyroïdiennes de rat hypertendu (SHR). Initialement nommé Hypertension-related calcium regulated gene (HCaRG), puis COMM domain-containing 5 (COMMD5), ce gène codant pour une petite protéine de 24,67 kDa fait partie d’une famille de 10 protéines ayant une structure carboxy-terminale homologue nommée domaine COMM. De nombreux rôles ont été associés à cette famille de protéines et l’analyse expressionnelle dans différents types de cancers montre une modulation, laissant penser qu’elles auraient un rôle oncogénique ou suppresseur de tumeurs. Les études ont démontré que COMMD5 entraine une maturation des jonctions cellulaires, une diminution de la prolifération et favorise la migration cellulaire. La surexpression de COMMD5 dans les tubules proximaux de rein accélère la réparation suite à un dommage aigu en limitant d’une part la prolifération tout en favorisant la migration et la re-différenciation cellulaire. Partant de ces observations, nous avons focalisé nos études sur le développement du carcinome à cellules rénales, une pathologie affectant 300 000 personnes chaque année dans le monde. L’hypothèse que nous avons émise était que COMMD5 puisse potentiellement jouer un rôle anti-oncogénique dans le cancer du rein en contrôlant la prolifération et la différenciation cellulaires. Afin de vérifier notre hypothèse, nous avons étudié le rôle de COMMD5 dans le maintien de l’intégrité épithéliale des cellules via la régulation de la transition épithélio-mésenchymateuse (EMT) et le contrôle du récepteur du facteur de croissance épidermique (EGFR). Nos résultats ont démontré que COMMD5 est diminuée dans les carcinomes rénaux et est corrélée avec la survie des patients. La présence du facteur de transcription induit par l’hypoxie 1 (HIF1α), exprimé dans la majorité des tumeurs solides rénales a induit une diminution de COMMD5. La perte de COMMD5 dans les cellules de tubules proximaux de reins humains (HK-2) a favorisé la dé-différenciation et la tumorigénicité des cellules, médiées par l’activation de la transition épithélio-mésenchymateuse. De plus, cette perte de COMMD5 a entrainé également une réorganisation du cytosquelette d’actine ayant pour conséquence la dérégulation endosomale du récepteur de l’EGF et favorisant une activation prolongée. Dans les carcinomes rénaux, la surexpression de COMMD5 a diminué la prolifération cellulaire suivie d’une re-différenciation grâce à deux mécanismes. D’une part, COMMD5 a régulé négativement la protéine HIF1α, induisant ainsi une transition mésenchymo-épithéliale (MET), tout en séquestrant le facteur de transcription SNAIL dans le cytoplasme. D’autre part, COMMD5 contrôle négativement l’expression transcriptionnelle des récepteurs ErbB par une hyperméthylation de leurs promoteurs. Dans son ensemble, les résultats innovant de cette thèse démontrent que COMMD5 est un gène ayant des caractéristiques anti-oncogéniques en contrôlant la différenciation cellulaire via le mécanisme de transition épithélio-mésenchymateux et la régulation de l’expression des récepteurs ErbB. / In the 2000s, Dr. Johanne Tremblay and her team identified a gene that was negatively regulated by extracellular calcium in hypertensive rat parathyroid glands. Originally named Hypertension-related calcium regulated gene (HCaRG) and renamed COMM Domaincontaining 5 (COMMD5), this gene encoding a small protein of 24.67 kDa is part of a family of 10 proteins sharing a homologous structure in the carboxy-terminal position named COMM domain. Many roles have been associated and expressional analysis of different types of cancer shows a modulation suggesting that they have an oncogenic or tumor suppressor roles. Studies have shown that COMMD5 induces maturation of the cell junctions, decreased cell proliferation and promotes migration. The overexpression of COMMD5 in the renal proximal tubules accelerates repair by promoting cell proliferation and ultimately induces cell migration and redifferentiation after acute injury. Based on these observations, we focused on the development of renal cell carcinoma, a disease affecting 300,000 people each year worldwide. Our hypothesis is that COMMD5 plays a tumor suppressor role in kidney cancer by controlling cell proliferation and differentiation. To test our hypothesis, we investigated the role of COMMD5 in maintaining the epithelial integrity of cells through the regulation of epithelial to mesenchymal transition (EMT) and the control of epidermal growth factor receptors (EGFR). The results showed that COMMD5 is decreased in kidney carcinomas resulting of a great negative indicator of the survival prognostic. The presence of hypoxia-inducible factor 1a (HIF1α), expressed in the majority of solid tumors, leads to a decrease of COMMD5 in the proximal tubule cells (HK-2). Inhibition of COMMD5 promotes dedifferentiation and tumorigenicity of cells mediated by epithelial to mesenchymal transition. The loss of COMMD5 induces a reorganization of the actin cytoskeleton resulting in endosomal dysregulation of the EGFR receptor and promoting its activation. In renal cell carcinoma, COMMD5 overexpression decreases cell proliferation and induces their redifferentiation by two mechanisms: firstly, COMMD5 induces an inhibition of the HIF1α protein expression resulting in a mesenchymal to epithelial transition and sequesters the SNAIL transcription factor in the cytoplasm; secondly, COMMD5 negatively regulates the transcriptional expression of the ErbB receptors. Taken together, these results of this thesis show that COMMD5 is a gene showing tumor suppressor characteristics by controlling cellular differentiation and by regulating the expression of ErbB receptors.

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