This dissertation was intended to identify potential roles for phosphatidylinositol-3 kinase (PI-3 kinase) in the responses of lymphocytes to activation. To understand what functions PI-3 kinase is performing in lymphocytes, experiments were performed to identify proteins that will stably associate with the p85 subunit of PI-3 kinase. Co-precipitation revealed an activation dependent association of p85 with two different phosphotyrosine containing proteins. One protein, pp36-38, is a membrane protein that interacts with PI-3 kinase, PLCγ1, and Grb2/S0S. The other associated protein was identified as the proto-oncogene c-Cbl. The interaction of p85 with cbl was shown to be mediated through the SH2 domains of p85. More importantly, the interactions of p85 with p36-38 and cbl were found to be specific for p85 isoforms. Although the SH2 domains of the α and β isoforms are highly similar in amino acid sequence, they are shown to establish distinct protein interactions in intact cells. Experiments on the cbl/PI-3 kinase complex revealed a stimulation dependent translocation into membrane and insoluble/cytoskeletal fractions of wild type, but not mutant cells. The movement of cbl did not require tyrosine phosphorylation or PI-3 kinase activity. The cbl/PI-3 kinase complex was greatly enhanced in the membrane fraction in contrast to the cytosol, where the largest concentration of cbl can be found. In addition, these complexes were found to form at the membrane in the absence of the tyrosine kinase, p56lck.

The FYVE domain is a lipid binding domain found in approximately 27 different mammalian proteins. It specifically interacts with the lipid, PI(3)P, which is enriched on early endosomes. Consequently, many FYVE domain-containing proteins localize to the endosome, however the ability of FYVE domains to target to endosomal membranes is variable, despite high sequence conservation. Here we describe the structural requirements necessary for endosomal localization and liposome avidity. As FYVE domains are lipid binding domain, many FYVE domain-containing proteins have been implicated in membrane trafficking. We performed an RNAi screen of FYVE domain-containing proteins to identify general regulators of endocytosis in Caenorhabditis elegans. In this screen, we identified the EEA1, a known regulator of endocytosis and two novel genes: WDF2 and KIAA1643. Initial characterization of WDF2 suggest that its function is conserved in humans. Of all the FYVE domain-containing proteins, we have been particularly interested in SARA (Smad Anchor for Receptor Activation); a protein implicated in the TGFβ signaling pathway. This protein contains a binding domain for the TGFβ mediated transcription factor, Smad2/3, and a FYVE domain. It was the presence of the FYVE domain, an endosomal targeting signal, in SARA that lead us to hypothesize that endocytosis might be a necessary step in TGFβ signaling. SARA localizes to the early endosome; the TGFβ receptors also internalize into these endosome. When this internalization is prohibited, there is correlative decrease in Smad2/3 phosphorylation, Smad2 nuclear translocation and TGFβ mediated transcription. Overexpression of a dominant negative SARA construct and SARA siRNA oligonucleotides inhibit TGFβ signaling. We conclude that TGFβ receptor signaling to Smad2/3 occurs on the endosome and this signaling requires SARA. Receptor mediated endocytosis has been classically thought of as an important mechanism for attenuating signaling pathways. We have redefined the role of endocytosis to include the necessary and positive regulation of specific signaling pathways. We have also extended our insights into the biological role of the endosome as a compartment specialized for the assembly and propagation of specific extracellular signals.

The function of GTP-binding proteins (G-proteins) in diverse intracellular pathways depends on their ability to switch between two forms, a GDP-bound (inactive) form and a GTP bound (active) form in a highly regulated GTPase cycle. The inactivation step of this cycle is regulated by GTPase-activating proteins (GAPs) which increase the intrinsic rate of hydrolysis of bound GTP; the activation step is regulated by a diverse family of GDP/GTP exchange factors (GEFs). A unique model system, which consists of the 13 kDa GEF Mss4 and the monomeric G protein Rab3A involved in presynaptic neurotransmission, was chosen to study the mechanism of G-protein regulation. Structure of Rab3A at high resolution The 2.0 Å crystal structure of Rab3A, bound to a non-hydrolyzable GTP-analog (GppNHp), enables a detailed description of the structural determinants that stabilize the active conformation and regulate GTPase activity within the Rab family. Although the overall structure is similar to that of GppNHp-bound Ras and other GTPases, localized but significant differences are observed in the vicinity of the conformational switch regions and the α3/β5 loop. The active conformation is stabilized primarily by extensive hydrophobic contacts between the switch I and II regions. Novel interactions with the γ phosphate, mediated by serine residues in the P-loop and switch I region, impose stereochemical constraints on the mechanism of GTP hydrolysis and provide a structural explanation for the broad range of GTPase activities within the Rab family. Residues implicated in interactions with effectors and regulatory factors map to a common face of the protein. The asymmetric distribution of charged and non-polar residues suggests a plausible orientation with respect to vesicle membranes that would position predominantly hydrophobic surfaces to interact with membrane-associated effectors and regulatory factors. Thus, the structure of Rab3A establishes a framework for understanding the molecular mechanisms underlying the function of Rab proteins in vesicle trafficking. High resolution structure of Mss4 and structure-based mutagenesis Activation of monomeric Rab GTPases, which function as ubiquitous regulators of intracellular membrane trafficking, requires the catalytic action of guanine nucleotide exchange factors. Mss4, an evolutionarily conserved Rab exchange factor, promotes nucleotide release from exocytic but not endocytic Rab GTPases. Chapter III describes the results of a high resolution crystallographic and mutational analysis of Mss4. The 1.65 Å crystal structure of Mss4 reveals a network of direct and water mediated interactions that stabilize a partially exposed structural sub-domain derived from four highly conserved but non-consecutive sequence elements. The conserved sub-domain contains the invariant cysteine residues required for Zn2+ binding as well as the residues implicated in the interaction with Rab GTPases. A strictly conserved DΦΦ motif, consisting of an invariant aspartic acid residue (Asp73) followed by two bulky hydrophobic residues (Met74 and Phe75), encodes a prominently exposed 310 helical turn in which the backbone is well ordered but the side chains of the conserved residues are highly exposed and do not engage in intramolecular interactions. Substitution of any of these residues with alariine dramatically impairs exchange activity towards Rab3A, indicating that the DΦΦ motif is a critical element of the exchange machinery. In particular, mutation of Phe75 results in a defect as severe as that observed for mutation of Asp96, which is located near the zinc binding site at the opposite end of Rab interaction epitope. Despite severe defects, however, none of the mutant proteins is catalytically dead. Taken together, the results suggest a concerted mechanism in which distal elements of the conserved Rab interaction epitope cooperatively facilitate GDP release. The basis for selective recognition of exocytic Rab family GTPases by Mss4 Rab3A is involved in Ca2+ -dependent exocytosis and neurotransmitter release. Mss4, an evolutionarily conserved Rab exchange factor, promotes nucleotide release from exocytic RabGTPase (Rab1, Rab3A, Rab8, and Rab10, Sec4 and Ypt1) but not endocytic Rab GTPases (Rab2, Rab4, Rab5, Rab6, Rab9 and Rab11). To understand the basis for selective recognition of exocytic Rab family GTPases by Mss4, a structure based mutagenesis study of Rab3A was conducted. Three residues in Rab3A (Phe51, Val61 and Thr89) were found to be critical for interaction with Mss4. Phe51 is located at the N- terminus of the switch region, adjacent to the Mg2+ and nucleotide binding site. Val61 in the β2 strand and Thr89 in the switch II region flank a triad of hydrophobic residues that is conserved in the Rab family. These residues comprise critical determinants underlying the broad specificity of Mss4 for exocytic Rab family proteins. In addition to determining the high resolution crystal structures of Rab3A and Mss4, the experiments described above identify critical structural determinants for the exchange activity of Mss4 and provide insight into the selective recognition of Mss4 by exocytic Rab GTPases.

L'émergence de bactéries résistantes aux antibiotiques constitue un problème majeur de santé publique responsable d'un nombre croissant de décès, surtout dans les hôpitaux. La résistance aux aminoglycosides est principalement due à l'expression d'enzymes capables de les modifier, comme les aminoglycosides phosphotransférases (APH).Le premier volet de ce travail de thèse vise à mieux comprendre les bases moléculaires des interactions protéine-ligands et de la catalyse enzymatique d'une de ces enzymes, l'APH(2”)-IVa. La spécificité de substrats a été caractérisée en détails pour différents aminoglycosides par des méthodes thermodynamiques, de mesures cinétiques à l'état stationnaire et transitoire, par amarrage moléculaire et cristallographie aux rayons X. La seconde partie de cette étude consiste à développer et optimiser des inhibiteurs allostériques de ces enzymes capables de restaurer l'efficacité des aminoglycosides. Pour cela, une cavité, potentiellement impliquée dans la dynamique de l'APH(2”)-IVa, a été identifiée à partir de simulations de dynamique moléculaire. Celle-ci a servi de cible pour cribler, in silico, 12 000 composés issus de la banque de données Zinc. Ainsi, 14 composés ont été testés in vitro pour leur capacité à diminuer l'activité enzymatique d'APH. Parmi ces derniers, une molécule s'est révélée être un inhibiteur non-compétitif de l'APH(2”)-IVa. Une étude des relations structure-fonction a permis de déterminer les groupements les plus favorables à l'inhibition et d'identifier un composé plus efficace. L'utilisation de ces deux molécules permet de restaurer, par exemple, la sensibilité à la sisomicine d'une souche d'E. faecium exprimant cette enzyme. Cette étude fournit des bases au développement de thérapies combinant un aminoglycoside et un inhibiteur des enzymes d'inactivation constituant une stratégie pour lutter contre la résistance aux antibiotiques dans un contexte thérapeutique. / Emergence of multi-drug resistant bacteria leads to increasing fatal issues especially in hospitals. Resistance to aminoglycoside antibiotics is mainly due to the expression of modifying enzymes, such as aminoglycoside phosphotransferases (APH). The first aim of this project was to elucidate the molecular basis of protein-ligand interactions and catalysis of one of these enzymes, the APH(2”)-IVa. Promiscuity of aminoglycoside substrates has thus been characterized in details using thermodynamics, transient and steady state kinetics, molecular docking and X-ray crystallography techniques.The second part aimed to develop and optimize allosteric inhibitors of these enzymes able to counterbalance aminoglycoside resistance. For this purpose, a small cavity, potentially involved in APH dynamics, was identified from molecular dynamic simulations. This cavity was used as a target to virtually screen 12 000 compounds of the Zinc database. The efficiency of the 14 high-ranked molecules to inhibit APH was evaluated in vitro and lead to the identification of a non-competitive inhibitor of APH(2”)-IVa. Structure-activity relationships highlighted the most favourable substituents for APH inhibition and permitted to obtain a more potent compound. The two molecules were able to restore, for example, sisomicin susceptibility of an E. faecium strain, expressing this enzyme.This study provides a basis for the development of combined chemotherapies (antibiotic with enzyme inhibitor) which may overcome antibiotic resistance in a clinical context.

Thesis (Ph.D.); Submitted to Iowa State Univ., Ames, IA (US); 12 Dec 2003. / Published through the Information Bridge: DOE Scientific and Technical Information. "IS-T 2604" Jason Alan Gruenhagen. 12/12/2003. Report is also available in paper and microfiche from NTIS.