Browsing by Subject "ADP-Ribosylation Factors"
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Item Brain Molecules under the Influence: Intracellular Regulation of Behavioral Responses Induced by Ethanol(2013-01-17) Peru Y Colón de Portugal, Raniero Leonette; Rothenfluh, AdrianAlcohol abuse is a devastating condition affecting millions of individuals. Regulation of insulin receptor (InR) signaling is critical for ethanol-induced responses and consumatory ethanol behavior. However, the precise intracellular mechanisms regulating InR signaling, which in turn, affect ethanol-induced behaviors remain unknown. I describe an InR/Arf6/S6K pathway that controls acute ethanol responses in Drosophila. I show that Arf6 mutants are hypersensitive to ethanol's intoxicating effects, and that Arf6 is specifically required in the adult nervous system to regulate na•ve ethanol sensitivity. While Arf6 functionally integrates activated Rac1 to the InR signaling, neuronal S6K, an InR effector, is a key mediator of Arf6-dependent regulation of ethanol-induced behaviors. Ethanol vapor concentrations that produce moderate sedation increase S6K-P, while doses that confer total sedation completely abate S6K-P. Arf6 mutants are completely devoid of neuronal S6K-P at baseline, suggesting that lack of S6K-P pre-sensitizes Arf6 mutants to the intoxicating effects of ethanol, and thus sedate at low physiologic ethanol concentrations. Because Arf6 has been implicated in receptor-mediated endocytosis, and signal transduction pathways are largely regulated by receptor trafficking, I propose a model in which Arf6 regulates InR signaling via endocytosis to control behavioral ethanol responses. My doctoral work on the intracellular mechanisms that govern ethanol's intoxicating effects on behavior will be described. The present dissertation is divided in four main sections: 1) Introduction, 2) results and methods, which include figures and figure legends and 3) a discussion of the results. In the introduction, I will review the scientific literature on the regulatory mechanisms of ethanol-driven behaviors performed in humans and other vertebrate species, while the central focus of this thesis is on Drosophila research. In doing so, I will also highlight current issues and problems concerning the study of ethanol's direct and candidate targets, which affect behavioral responses to ethanol. In the result and methods section, I will describe my obtained experimental data and the methodology employed. In the discussion section, I will first illustrate on the initial part of the results dealing with neuronal Rac1, Arfip, and Arf6, which through a linear genetic pathway regulate acute ethanol sedation. Second, I will explain how Arf6 GTPase may integrate the Rho to InR signaling to control behavioral ethanol responses. Third, I will illuminate on recent data showing that Arf6 via neuronal S6K mediates behavioral sensitivity to ethanol. Moreover, I will propose a model in which Arf6 plays separable but intertwined roles in InR signaling and endocytosis, in order to regulate acute ethanol behaviors.Item Identification and Characterization of a Bacterial Catalytic Scaffold with Specificity for Host Endomembrane Traffic(2013-11-20) Selyunin, Andrey S.; Sperandio, Vanessa; Cobb, Melanie H.; Yarovinsky, Felix; Alto, NealThe fidelity and specificity of information flow within a cell is controlled by scaffolding proteins that assemble and link enzymes into signaling circuits. These circuits can be inhibited by bacterial effector proteins that post-translationally modify individual pathway components. However, there is emerging evidence that pathogens directly organize higher order signaling networks through enzyme scaffolding, and the identity of the effectors or their mechanisms of action are poorly understood. Here, we used a functional screen to identify the EHEC O157:H7 type III effector EspG as a regulator of endomembrane trafficking and we report ADP-ribosylation factor (ARF) GTPases and p21-activated kinases (PAK) as its relevant host substrates. The 2.5 Å crystal structure of EspG in complex with ARF6 shows how EspG blocks GAP-assisted GTP hydrolysis, revealing a potent mechanism of GTPase signaling inhibition at membrane organelles. In addition, the 2.8 Å crystal structure of EspG in complex with the autoinhibitory Iα3-helix of PAK2 defines a previously unknown catalytic site in EspG and provides an allosteric mechanism of kinase activation by a bacterial effector. Unexpectedly, ARF and PAK are organized on adjacent surfaces of EspG, suggesting its dual role as a “catalytic scaffold” that effectively reprograms cellular events through the functional assembly of GTPase-kinase signaling complex. Bidirectional vesicular transport between ER and Golgi is mediated largely by ARF and Rab GTPases, which orchestrate vesicle fission and fusion, respectively. How their activities are coordinated to define the successive steps of the secretory pathway and preserve traffic directionality is not well understood, in part due to the scarcity of molecular tools that simultaneously target ARF and Rab signaling. Here, we take advantage of the unique scaffolding properties of E.coli Secreted Protein G (EspG) to describe the critical role of ARF1/Rab1 spatiotemporal coordination in vesicular transport at the ER-Golgi Intermediate Compartment. Structural modeling and cellular studies show that EspG induces bidirectional traffic arrest by tethering vesicles through select ARF1-GTP/effector complexes and local inactivation of Rab1. Mechanistic insights presented in this study establish the effectiveness of a small bacterial catalytic scaffold in studying complex processes and reveal an alternative mechanism of immune regulation by an important human pathogen.Item Investigating the Mechanism and Mode of Action of Golgi Toxins(December 2023) Cervantes, Christopher Luis; Liszczak, Glen; Posner, Bruce A.; Wang, Fei; Nijhawan, DeepakAuxin-inducible forward genetics uncovered point mutations within Golgi Brefeldin A Resistant Guanine Nucleotide Exchange Factor 1 or GBF1 following lethal dose selection with a synthetic disubstituted pyrimidine toxin called Golgitox (GTOX). Resistant clones were also cross-resistant to the fungal toxin, Brefeldin A (BFA), and synthetic GBF1 inhibitor, Golgicide A (GCA). Like BFA and GCA, GTOX triggered Golgi disassembly via GBF1. Given that BFA is a reported molecular glue, we profiled Gbf1-Arf interactions in 293T cell lysates pre-treated with Golgi toxin. Both GTOX and GCA promoted GBF1-dependent interactions with Arfs 4 and 5, whereas BFA also interacted with Arf1. GBF1 domain mapping revealed that the HUS-SEC7-HDS1 domains were sufficient for promoting GTOX-dependent engagement with Arfs 4 and 5. Meanwhile, structural activity relationship studies showed that modifying the methyl group on the benzimidazole ring preserved GTOX activity and interactions between Gbf1 and Arfs 4 and 5. To assess which Arfs regulate BFA and GTOX cytotoxicities, genome-wide CRISPR/Cas9 compound enrichment screens were carried out, which identified ARF4 as being the most enriched hit. Next, we validated that ARF4 loss-of-function partially confers resistance to BFA and GTOX. Next, we asked whether GTOX preferentially interacts with ARF4-GDP versus ARF4-GTP. We found that exogenous ARF4 T31N (GDP-locked mutant) sensitized HCT116 scramble control cells 4-fold to GTOX, interacted with Gbf1 just as well as WT Arf4, but failed to rescue Arf4-mediated BiP retrieval. Collectively, these results suggest that the Gbf1-GTOX-Arf4-GDP complex is functionally inactive but deleterious to cell viability. Taken together, GTOX may act as a molecular glue to suppress GBF1 functions through downstream effector substrates like Arfs 4 and 5.Item Trimming Fat Upon Infection: Proteolytic Demyristoylation as a Novel Bacterial Pathogenic Strategy(2014-08-28) Burnaevskiy, Nikolay; Seemann, Joachim; Chook, Yuh Min; Orth, Kim; Alto, NealHost-pathogen interaction is a complex process that involves an array of molecular tools on both sides of the conflict. In the light of growing antibiotics resistance, understanding of virulence factors utilized by pathogenic bacteria is crucial for developing better treatment for infectious diseases. Shigella spp are food-borne bacterial pathogens and are one of the leading causes of bacterial dysentery worldwide. Similarly to other gram-negative pathogens, Shigella injects effector proteins into an infected cell to control its responses. Dedicated type III secretion system delivers more than twenty effectors which highjack normal cell signaling by modulating activity of host proteins. By interfering with immune signaling, cytoskeleton, and other pathways, effector proteins promote survival of the bacterial pathogen. Inability to secrete effectors severely attenuates Shigella spp, pointing to the crucial role of these proteins in pathogenesis. Therefore, gaining an insight into the mechanism of action of secreted effectors may hold a key to better understanding and treatment of infectious diseases. Here I characterized the Shigella flexneri secreted virulence factor IpaJ. I found that IpaJ is a cysteine protease with novel specificity. IpaJ cleaves a myristoylated amino-terminal glycine of host proteins and therefore irreversibly delipidates them. Although IpaJ can target multiple host proteins, it strongly favors members of ADP-ribosylation factors family (ARF) and related ARF-like (ARL) proteins. By inactivating ARF and ARL proteins, IpaJ disrupts the Golgi structure and prevents secretion through general secretory pathway. I also found that by blocking protein export from the endoplasmic reticulum, IpaJ suppresses the activation of immune signaling pathways. In summary, I characterized a novel virulence factor of Shigella spp, IpaJ. I have shown that IpaJ promotes bacterial pathogenesis and therefore may be of potential interest for therapeutic interventions. The biochemical analysis of the enzyme provides the first look at the mechanism of substrate recognition and highlights the potential use of IpaJ as a research tool to study protein N-myristoylation and the mechanisms of membrane trafficking.