No description available.

Rab11 is a critical GTPase involved in the regulation of membrane trafficking in the endocytic pathway, and it’s misregulation is involved in a variety of human diseases including Huntington’s disease and Alzheimer’s disease. Additionally, de novo mutations (DNMs) of Rab11 have been identified in patients with developmental disorders, and interestingly several parasites, viruses, and bacteria can subvert membrane trafficking through Rab11 positive vesicles to allow for replication and evasion from the immune system. Although Rab11 is one of the best characterized Rab GTPases, hindering the capability to completely understand Rab11 regulation and its role in human disease is the lack of detail describing how Rab11 proteins are activated by their cognate guanine nucleotide exchange factors (GEFs). This thesis is therefore focused on revealing the molecular mechanisms of the GEFs responsible for the activation of Rab11: SH3BP5 and TRAPPII. To investigate the recently discovered GEF SH3BP5, we solved the 3.1Å structure of Rab11 bound to SH3BP5 and revealed a coiled coil architecture of SH3BP5 that mediates exchange through a unique Rab-GEF interaction. The structure revealed a unique rearrangement of the switch-I region of Rab11 compared to other solved Rab-GEF structures, with a constrained conformation when bound to SH3BP5. Mutational analysis of switch-I revealed the molecular determinants that allow for Rab11 selectivity over evolutionarily similar Rab GTPases, and GEF deficient mutants of SH3BP5 show greatly decreased Rab11 activation in cellular assays of active Rab11. To interrogate the highly controversial GEF TRAPPII, we recombinantly expressed and purified the 9 subunit, 427 kDa complex in Spodoptera frugiperda 9(Sf9) cells. We found that the TRAPPII complex is a GEF for both Rab1 and Rab11, and we discovered novel activity for another Rab GTPase. To interrogate the role of these GEFs in human disease, we used HDX-MS and nucleotide exchange assays to show that some DNMs destabilize Rab11 either through a complete or partial disruption of nucleotide binding. Importantly, we discovered that one of these DNMs, K13N, completely prevented SH3BP5 and TRAPPII mediated nucleotide exchange, revealing a putative mechanism of disease. Overall the work completed in this thesis leads to a greater understanding of the molecular mechanisms underlying the activation of Rab11 by its cognate GEFs. / Graduate / 2020-11-25

Heart disease is a complex and heterogeneous disease. Notably, studies have demonstrated gender differences in the expression and types of cardiovascular disease, such as dilated cardiomyopathy (DCM), a major underlying cause of heart failure. Previously we showed that loss of A-Kinase Anchoring Protein 13 (Akap13), a unique proto-oncogene and estrogen receptor modulator, resulted in enlarged embryonic hearts, defective cardiac sarcomere formation, and embryonic lethality in mice. Data have also shown cAMP-dependent Protein Kinase A (PKA) to be involved in DCM pathophysiology. Given the established role of AKAP13 in cell signaling, its ability to bind and modulate ligand-activated nuclear hormone receptors and transcription factors, and its association with actin and other cytoskeletal components, we hypothesized that a functional AKAP13 protein was required for cardiomyocyte function in the adult heart; defective function of AKAP13 could promote DCM. To this end, we established an inducible, cardiac-specific Akap13 conditional knockout (Akap13cKO) mouse model using a Cre-lox recombination strategy with two separate Cre-recombinase expressing mouse models (α-MHC-MerCreMer and Tnnt2-rtTA; TetO-Cre). Cardiac functional examination of Akap13cKO mice revealed significant biventricular dilated cardiomyopathy with compensatory hypertrophic remodeling of the left ventricle and left atrial enlargement, decreased left and right ventricular systolic function, and abnormal left ventricular diastolic function. Of note, female Akap13cKO mice displayed a more pronounced cardiac phenotype and were more likely to die post-recombination.

RasGAP is a GTPase activating protein (GAP) that deactivates Ras by promoting Ras-GTP hydrolysis to Ras-GDP. In Drosophila melanogaster, RasGAP is required for the long-term survival of neurons in the adult brain because mutants in the RasGAP gene (vap) show an age-related neurodegenerative phenotype, with dying neurons showing morphological features of autophagy. RasGAP was shown to have a GAP-independent role within fly neurons that is dependent on its SH2 domains. The aim of this study was to identify proteins that interact with the SH2 domains of RasGAP and to understand the roles of these proteins in neuronal survival. By using tagged RasGAP affinity purification and mass spectrometry of RasGAP protein complexes from S2 cells, Sprint, a Ras effector and putative activator of the endocytic GTPase Rab5, was identified as a novel SH2-dependent RasGAP interacting protein. The interaction between Sprint and RasGAP is phosphotyrosine-dependent, since it requires tyrosine phosphorylation of Sprint. In addition, Sprint and RasGAP interaction requires the SH2 domains of RasGAP but not Sprint or the conserved site of RasGAP tyrosine phosphorylation (pTyr363), indicating an association between these two molecules. RasGAP and Sprint co-localised with Rab5-positive early endosomes and this co-localisation depended on the SH2 domains of both RasGAP and Sprint. This study demonstrates a key role for this interaction in neurodegeneration: mutation of Sprint (or Rab5) suppressed the autophagic neuronal cell death caused by the loss of RasGAP. These results indicate that the long-term survival of adult neurons in Drosophila depends on a critical balance between Ras activation and endocytosis, and that this balance is maintained by the interplay between RasGAP and Sprint.

No description available.

No description available.

The Rho family of guanosine triphosphatases (GTPases) function as binary molecular switches, which play an important role in the regulation of actin cytoskeleton rearrangement and are involved in several critical cellular processes including cell adhesion, division and migration. Rho GTPases are specifically activated by their associated guanine nucleotide exchange factors (RhoGEFs). Dysregulation of RhoGEFs function through mutation or overexpression has been implicated in oncogenic transformation of cells and linked to several kinds of invasive and metastatic forms of cancer. T-cell lymphoma invasion and metastasis 1 (Tiam1) is a multi-domain Dbl family GEF protein and specifically activates Rho GTPase Rac1 through the catalytic Dbl homology and Pleckstrin homology (DH-PH) bi-domain. Previous works have shown that the nucleotide exchange function of the full-length Tiam1 is auto-inhibited and can be activated by N-terminal truncation, phosphorylation and protein-protein interactions. However, the molecular mechanisms of Tiam1 GEF auto-inhibition and activation have not yet been determined. In this study, the N-terminal PH-CC-Ex domain of Tiam1 is shown to directly inhibit the GEF function of the catalytic DH-PH domain in vitro. Using fluorescencebased kinetics experiments, we demonstrate that the auto-inhibition of Tiam1 GEF function occurs by a competitive inhibition model. In this model, the maximum velocity of catalytic activity remains unchanged, but the Michaelis-Menten constant of the auto-inhibited Tiam1 (the PH-PH fragment) on the substrate Rac1 is increased compared to the activated Tiam1 (the catalytic DH-PH domain alone). Through small angle X-ray scattering (SAXS), the structure of auto-inhibited Tiam1 (the PH-PH fragment) is shown to form a closed conformation in which the catalytic DH-PH domain is blocked by the N-terminal PH-CC-Ex domain. Taken together, these findings demonstrate the molecular mechanism of Tiam1 GEF autoinhibition in which the PH-CC-Ex domain of Tiam1 inhibits its GEF function by preventing the substrate Rho GTPase Rac1 from accessing the catalytic DH-PH bi-domain.

La petite protéine G Arf6 et son facteur d'échange EFA6 sont impliquées dans de nombreux processus cellulaires tels que le remodelage du cytosquelette d’actine, le transport vésiculaire et mise en place de la polarité épithéliale. Elles jouent également un rôle dans la voie d'endocytose dépendante de la clathrine. Ce travail de thèse nous a permis d’identifier différents mécanismes régulant l’interaction d’EFA6 avec ses différents partenaires. Nous avons pu mettre en évidence une interaction directe entre le domaine N-BAR de l’endophiline et le domaine Sec7 d’EFA6. Nous avons démontré que la courbure membranaire était un facteur régulant cette interaction. EFA6 est capable d’interagir et de recruter l’endophiline sur une membrane lipidique plane alors qu’en présence de vésicules courbées le complexe protéique ne se forme pas. Nous observons également que l’endophiline stimule l’activité d’échange nucléotidique d’EFA6 sur Arf6. Dans un second temps nous avons démontré, dans une étude menée par le Dr Cherfils, que l’activité catalytique d’EFA6 était régulée par une boucle de rétrocontrôle négatif exercée spécifiquement par la protéine Arf6-GTP. Celle-ci induit une diminution de l’activité d’échange d’EFA6 probablement grâce à sa capacité à interagir avec le domaine PH-C-terminal d’EFA6. Enfin, nous avons mis en évidence un repli intramoléculaire entre le domaine C-terminal et le domaine PH d’EFA6 qui semble contrôler l’interaction de cette extrémité C-terminale avec différents partenaires dont la β-arrestine et de façon surprenante la protéine Arf6 dans sa forme inactive. / The small G protein Arf6 and its exchange factor EFA6 control numerous cellular processes such as actin cytoskeleton remodeling, vesicular transport and apico-basal cell polarity. They are also involved in clathrin-dependent endocytosis. In this work we identify different mechanisms by which EFA6 interaction with its various partners is regulated. We have highlighted a direct interaction between the N-BAR domain of endophilin and the Sec7 domain of EFA6. We demonstrated that this interaction is regulated by the membrane curvature. EFA6 interacts and recruits endophilin on a flat lipid membrane whereas the protein complex does not occur in the presence of curved vesicules. We showed that endophilin stimulates the nucleotidic exchange activity of EFA6 on Arf6. Next we demonstrated that the catalytic activity of EFA6 is regulated by a negative feedback loop specifically mediated by the Arf6-GTP. We observed in the presence of Arf6-GTP a decrease of EFA6 catalytic activity and we showed that this effect was due to an interaction between Arf6-GTP and PH-C-terminal domain of EFA6. Finally we demonstrated an intramolecular folding between the C-terminal domain and the PH domain of EFA6 that controls the interaction of the C-terminus domain with various partners including β-arrestin and surprisingly the inactive GDP form of Arf6.

Doctor of Philosophy / Biochemistry Interdepartmental Program / Qize Wei / This dissertation describes the role of myosin-interacting guanine nucleotide exchange factor (MyoGEF) and centrosome/spindle pole associated protein (CSPP) in mitotic progression and cytokinesis. We have identified three mouse isoforms of CSPP, all of which interact and colocalize with MyoGEF to the central spindle in anaphase cells. The N-terminus of MyoGEF interacts with myosin whereas the C terminus interacts with the N-terminus of CSPP, forming a complex. The N-terminus of CSPP appears to be important for both localization and interaction with MyoGEF. CSPP plays a role in mitotic progression since its depletion by RNAi resulted in metaphase arrest. MyoGEF is required for completion of cytokinesis. Both MyoGEF and CSPP are phosphorylated by mitotic kinases including Plk1 and Aurora. Importantly, MyoGEF is phosphorylated at Thr-574 in mitosis by Polo-like kinase 1, and this phosphorylation is required for activation of RhoA. Thr-543 of MyoGEF is required for Plk1 binding in mitosis and phosphorylation of MyoGEF by Cdk1/cyclinB, possibly at Thr-543 may generate a Plk1 docking site, i.e., Cdk1 can phosphorylate MyoGEF at Thr-543, thereby allowing Plk1 to bind and phosphorylate MyoGEF at Thr-574. Finally, MyoGEF and CSPP are also phosphorylated by Aurora-B kinase in vitro. Taken together, we propose that Aurora-B may phosphorylate and recruit MyoGEF and CSPP to the central spindle, where phosphorylation of MyoGEF at Thr-543 promotes Polo kinase binding and additional phosphorylation of MyoGEF, leading to the activation of RhoA at the cleavage furrow.

PhD (Biochemistry) / Department of Biochemistry / Malaria is a parasitic disease that accounts for more than 660 thousand deaths annually, mainly in children. Malaria is caused by five Plasmodium species P. ovale, P. vivax, P. malariae, P. falciparum and P. knowlesi. The most lethal cause of cerebral malaria is P. falciparum. The parasites have been shown to up-regulate some of their heat shock proteins (Hsp) in response to stress. Heat shock protein 70 (called DnaK in prokaryotes) is one of the most prominent groups of chaperones whose role is central to protein homeostasis and determines the fate of proteins. Six Hsp70 genes are represented on the genome of P. falciparum. The Hsp70 genes encode for proteins that are localised in different sub-cellular compartments. Of these two occur in the cytosol, PfHsp70-z and PfHsp70-1; two occur in the endoplasmic reticulum, PfHsp70-2 and PfHsp70-y; one in the mitochondria, PfHsp70-3 and one exported to the red blood cell cytosol, PfHsp70-x. PfHsp70-1 is a well characterized canonical Hsp70 involved in prevention of protein aggregation and facilitates protein folding. Little is known about PfHsp70-z. PfHsp70-z was previously shown to be an essential protein implicated in the folding of proteins possessing asparagine rich repeats. However, based on structural evidence PfHsp70-z belongs to the Hsp110 family of proteins and is thought to serve as a nucleotide exchange factor (NEF) of PfHsp70-1. The main aim of this study is to elucidate the functional roles of PfHsp70-z as a chaperone and its interaction with PfHsp70-1. In the current study, PfHsp70-z was cloned and expressed in E. coli JM109 cells. This was followed by its purification using nickel chromatography. The expression of PfHsp70-z in parasites cultured in vitro was investigated and its association with PfHsp70-1 was explored using a co-immuno precipitation assay. PfHsp70-z expression in malaria parasites is up regulated by heat stress and the protein is heat stable based on investigations conducted using Circular Dichroism. Furthermore, the direct interaction between recombinant forms of PfHsp70-z and PfHsp70-1 were investigated using slot blot and surface plasmon resonance assays. PfHsp70-z was observed to exhibit ATPase activity. In addition, the direct interaction between PfHsp70-z and PfHsp70-1 is promoted by ATP. Based on limited proteolysis and tryptophan fluorescence analyses, PfHsp70-z binds ATP to assume a unique structural conformation compared to the conformation of the protein bound to ADP or in nucleotide-free state. PfHsp70-z was able to suppress the heat-induced aggregation of malate dehydrogenase and luciferase in vitro. Interestingly, while ATP appears to modulate the conformation of PfHsp70-z, the chaperone function of PfHsp70-z was not influenced by ATP. Altogether, these findings suggest that Characterization of Heat Shock Protein 70-z (PfHsp70-z) from Plasmodium falciparum iii PfHsp70-z serves as an effective peptide substrate holding chaperone. In addition, PfHsp70-z may also serve as the sole nucleotide exchange factor of PfHsp70-1. The broad spectrum of functions of this protein, could explain this PfHsp70-z is an essential protein in malaria parasite survival. This is the first study to show that PfHsp70-z possess independent chaperone activity and that it interacts with its cytosolic counterpart, PfHsp70-1 in a nucleotide dependent fashion. Furthermore, the study shows that PfHsp70-z is a heat stable molecule and that it is capable of forming high order oligomers.