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.

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.

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.

No description available.

Eukaryotic translation initiation is a complex and highly regulated process involving the ribosome, mRNA and proteins called eukaryotic initiation factors (eIFs). The overall aim of translation initiation is to position the ribosome at the initiation codon of the mRNA. eIF2, in its GTP-bound conformation, binds the initiator tRNA (Met-tRNAiMet) and delivers it to the 40S ribosomal subunit. When the anticodon of the tRNA is bound to the initiation codon, the GTP on eIF2 is hydrolysed to GDP. The guanine nucleotide exchange factor (GEF) eIF2B regenerates eIF2-GTP. eIF2 and eIF2B are multisubunit/multidomain protein complexes. Because information regarding the interface between each complex is limited, particularly the interface on the eIF2γ subunit, which binds the guanine-nucleotides and Met-tRNAiMet, interactions between the minimal GEF domain of eIF2Bε, εGEF, and eIF2 were mapped using mutagenesis and an in vitro cysteine cross-linking approach, with the cross-linker Mts-Atf-Biotin. Site-directed mutagenesis (SDM) was used to mutate five N-terminal and five C-terminal surface-exposed εGEF residues to cysteines. The mutant alleles were analysed in Saccharomyces cerevisiae and it was found that the gcd6-R574C allele was lethal and the gcd6-T572C was Gcd-. Further gcd6-R574 mutant alleles were also found to be lethal in yeast but expressed in vivo.εGEF-R574C has dramatically reduced GEF activity in vitro and binding assays showed that this mutant has significantly reduced affinity for eIF2. The εGEF-T572C and εGEF-S576C mutants also have severe and minor eIF2-binding defects respectively, while the C-terminal εGEF-Cys mutants have slightly reduced affinity for eIF2. The N-terminal εGEF-Cys mutants cross-link specifically to eIF2γ, while the C-terminal εGEF-Cys mutants interact predominantly with eIF2β. From the data obtained in this study, we propose a new model for eIF2B-mediated guanine-nucleotide exchange that reduces the importance of eIF2β and suggests εGEF resembles other GEFs in binding primarily to its G protein partner eIF2γ.

Les petites protéines G Arf ont besoin d'un facteur d'échange nucléotidique (GEF) afin de passer de leur forme inactive liée au GDP à leur forme active liée au GTP. GBF1 est la GEF pour Arf1 qui assure, notamment, le recrutement du complexe manteau COPI impliqué dans le transport entre le Golgi précoce et le réticulum endoplasmique. Il a été récemment montré que GBF1 est impliqué dans la livraison de l'Adipose TriGlycéride Lipase (ATGL) sur les corps lipidiques (LDs). ATGL est une enzyme qui catalyse l'hydrolyse des triglycérides en diglycérides. Les travaux présentés dans cette thèse ont eu pour objectif d'étudier et de caractériser l'interaction entre GBF1 et la lipase ATGL. Par des expériences de co-immunoprécipitation dans les cellules de mammifère, les domaines des deux protéines impliquées dans l'interaction ont été identifiés. Par des expériences de pulldown utilisant les protéines exprimées chez E. coli, j'ai montré que ces interactions sont directes. Afin d'approfondir l'étude de l'interaction entre GBF1 et ATGL, j'ai construit des outils permettant l'étude biochimique de GBF1 en purifiant plusieurs de ses domaines. J'ai tout d'abord cherché à mettre au point un test d'activité pour GBF1 afin de tester l'influence de protéines partenaires, dont ATGL, sur son activité. Malgré la purification de différents fragments de GBF1 contenant le domaine Sec7, aucun n'a présenté une activité avec Arf1Δ17 en solution. Le domaine N-terminal de la protéine, avec et sans une mutation empêchant une interaction intramoléculaire, ainsi que les domaines HDS1 et HDS2 de GBF1 ont également été purifiés / Small G proteins Arf require assistance from a Guanine nucleotide exchange factor (GEF) in order to switch between GDP- and GTP-bound forms. GBF1 is the Arf1 GEF that mediates COPI coat complex recruitment to early secretory pathway membranes. COPI is a protein that coats vesicles transporting proteins from the cis side of the Golgi complex back to the rough endoplasmic reticulum. GBF1 was recently shown to mediate delivery of Adipose TriGlyceride Lipase (ATGL) to the surface of lipid droplets (LDs). ATGL is an enzyme catalyzing the initial step in triglyceride hydrolysis in LDs. Thus, the aim of this work was to study interactions between GBF1 and ATGL. By co-immunoprecipitation experiments in mammalian cells, the domains of two proteins involved in the interaction have been identified. By pulldown assays using proteins expressed in bacteria, I showed that these interactions are direct. To further study of the GBF1-ATGL interaction, I developed tools for the biochemical study of GBF1, by purifying several of its domains. I first tried to develop a kinetic essay for GBF1 to test the influence of interacting partners, including ATGL, on its activity. Despite the purification of various GBF1 fragments containing the Sec7 domain, none have activity with Arf1Δ17 in solution. The N-terminal domain of the protein, with and without a mutation disrupting an intramolecular interaction, and the HDS1 and HDS2 domains of GBF1 were also purified.