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The human ether-à-go-go related gene (hERG) encodes the pore-forming voltage-gated K+ channel that is essential for cardiac repolarization. Dr. Tsushima’s laboratory has previously characterized the endogenous expression of SNARE proteins in the mammalian heart, and the interaction of the SNARE protein syntaxin 1A (STX1A) with several cardiac ion channels. Here, we utilize a multi-disciplinary approach to describe the inhibitory effect of STX1A on hERG channel function. STX1A impairs hERG channel maturation and trafficking to the plasma membrane and induces a hyperpolarizing shift in the voltage-sensitivity of steady-state inactivation. We identify the residues involved in this protein-protein interaction through the use of hERG truncation mutations. We also describe the pharmacological and temperature-mediated rescue of hERG channel trafficking in the presence of STX1A. The regulation of cardiac ion channels by SNARE proteins represents a novel biological mechanism that may have universally intrinsic implications for normal and diseased heart function.

The human ether-à-go-go related gene (hERG) encodes the pore-forming voltage-gated K+ channel that is essential for cardiac repolarization. Dr. Tsushima’s laboratory has previously characterized the endogenous expression of SNARE proteins in the mammalian heart, and the interaction of the SNARE protein syntaxin 1A (STX1A) with several cardiac ion channels. Here, we utilize a multi-disciplinary approach to describe the inhibitory effect of STX1A on hERG channel function. STX1A impairs hERG channel maturation and trafficking to the plasma membrane and induces a hyperpolarizing shift in the voltage-sensitivity of steady-state inactivation. We identify the residues involved in this protein-protein interaction through the use of hERG truncation mutations. We also describe the pharmacological and temperature-mediated rescue of hERG channel trafficking in the presence of STX1A. The regulation of cardiac ion channels by SNARE proteins represents a novel biological mechanism that may have universally intrinsic implications for normal and diseased heart function.

No description available.

L’introduction commence avec la description de toxines d’origines bactérienne et végétale, en particulier la toxine Shiga ainsi que les toxines de la même famille (chapitre 9.1.2). Les petites molécules inhibitrices de ces toxines sont ensuite résumées dans le chapitre 9.1.3, en particulier le composé Retro-2. L’efficacité de ces toxines à atteindre leurs cibles reposant sur le trafic intracellulaire, un aperçu général de l’endocytose et du trafic endosomal sont présentés (chapitre 9.2). Puis, l’entrée de la voie rétrograde est décrite (chapitre 9.2.5), avec un intérêt particulier porté sur la clathrine, le rétromère et GPP130, une protéine qui circule de manière continue entre le Golgi, la membrane plasmique et les endosomes. Les protéines SNARE, en particulier la syntaxine-5 et le syntaxine-16, sont ensuite introduites (chapitre 9.2.6). Après une brève section sur les micro-ARNs de la famille 199 (chapitre 9.3), l’introduction se termine avec la description des techniques clés utilisées au cours de mon travail, tels que la chimie click bio-orthogonale, la synchronisation du trafic antérograde par rétention grâce à des hameçons spécifiques (RUSH), et la ligation par proximité basé sur des anticorps (chapitre 9.4).Ci-inclus, mon article en cours de soumission ouvre la partie résultats (chapitre 10.1), dans laquelle je présente l’intérêt de la chimie click bio-orthogonale pour identifier les cibles cellulaires de Retro-2. Je décris un des candidats potentiels, Sec16A, et illustre comment grâce à la technique de RUSH, perturber la fonction de Sec16A conduit à la relocalisation partielle de la syntaxin-5 au niveau du reticulum endoplasmique via l’inhibition du transport antérograde de la syntaxine-5. La seconde partie de l’article décrit comment la relocalisation de la syntaxine-5 induit l’inhibition du trafic de la toxine Shiga des endosomes au TGN. Je présente une nouvelle interaction entre la syntaxine-5 et la protéine TGN GPP130, qui ont déjà été caractérisées en relation avec le trafic de la toxine Shiga. Mon travail connecte à la fois les facteurs de trafic avec le trafic rétrograde au niveau de l’interface endosome-TGN. De manière frappante, cette interaction est très probablement basée sur une fonction non-SNARE de la syntaxine-5 car le domaine de fixation sur GPP130 est structurellement non lié à toute fonction SNARE.En collaboration avec Juan Francisco Aranda et Carlos Fernandez aux Etats-Unis, nous avons placés des micro-ARNs dans un contexte de régulation endogène du trafic rétrograde de la toxine Shiga (chapitre 11.2). Une discussion plus approfondie sera apportée dans le chapitre 12.Enfin, une vue d’ensemble des projets en cours est apportée dans la section des perspectives (chapitre 12), dans laquelle les collaborations plus approfondies sont mises en lumière.Mots clés : transport rétrograde, toxine Shiga, toxine de la famille Shiga, STxB, syntaxin-5, Sec16A, GPP130, Retro-2, Retro-2.1, chimie click sans cuivre, identification des cibles de petites molécules, spétrométrie de masse, function non-SNARE, inhibition du trafic antérograde, miARN, miR199, rétromère, VPS26 / The introduction of my PhD manuscript starts with describing plant and bacterial toxins (chapter 9.1), in particular Shiga toxin and Shiga-like toxins (SLTs) (chapter 9.1.2). Small molecule inhibitors of these toxins are summarized afterwards in chapter 9.1.3, notably the Retro-2 compound. Since these toxins rely on intracellular trafficking to reach their molecular targets, a general overview of endocytosis and endosomal trafficking is provided (chapter 9.2). Next, the retrograde route entry is presented (chapter 9.2.5), with focus on clathrin, the retromer and GPP130, a protein that constantly cycles between Golgi, plasma membrane, and endosomes. SNARE proteins, particularly syntaxin-5 and syntaxin-16, are then introduced (chapter 9.2.6). After a brief section of the micro RNA family 199 (chapter 9.3), the introduction finishes with the description of some salient techniques that were used in my work, such as - bio-orthogonal Click-Chemistry, anterograde trafficking synchronization with the retention using selective hooks (RUSH) assay, and the antibody-based proximity ligation assay (chapter 10.6.1, 0, 10.11.1).Herein, my submitted publication opens the results part (chapter 11.1), in which I present the utility of biorthogonal click chemistry for the search of the cellular targets of Retro-2, a small molecule inhibitor that was previously shown to protect cells and animals against Shiga toxin and ricin. I describe that Sec16A is a likely cellular target candidate, and illustrate using the RUSH approach how interfering with Sec16A functions leads to the partial relocalization of syntaxin-5 to the endoplasmic reticulum (ER) by slowing-down its anterograde transport. The second part of the paper describes how syntaxin-5 relocalization causes the inhibition of Shiga toxin trafficking from endosomes to the TGN. I present a novel interaction between syntaxin-5 and the Golgi protein GPP130, which both have been already described in relation to Shiga toxin trafficking. My work connects both trafficking factors in retrograde trafficking at the endosomes-TGN interface. Strikingly, I demonstrate that this interaction is most probably based on a non-SNARE function of syntaxin-5.In collaboration with Juan Francisco Aranda and Carlos Fernandez in the US, we put micro RNAs into an endogenous regulation context of Shiga toxin retrograde trafficking (chapter 11.2). An extended discussion will be given in chapter 12.Last, a general outlook of ongoing projects is given in the perspectives section (chapter 13), in which further collaborations are highlighted.Keywords: Retrograde transport, Shiga toxin, Shiga-like toxin (SLT), STxB, syntaxin-5, Sec16A, GPP130, Retro-2, Retro-2.1, azide-functionalized Retro-2, copper-free click chemistry, small molecule target identification, mass spectrometry, non-SNARE function, anterograde trafficking inhibition, miRNA, miR199, retromer, VPS26

Soybean cyst nematode (SCN), a major pathogen of soybean worldwide, causes huge losses in soybean production. Various approaches including cloning of genes to combat this devastating disease help to better understand the cellular function and immune responses of plants. Membrane fusion genes are the important regulatory parts of vesicular transport system, which works through packaging of intracellular compounds and delivering them to apoplast or nematode feeding sites to induce an incompatible reaction. The incompatible nature of membrane fusion proteins such as SNAP25, Munc18, Syntaxin, Synaptobrevin, NSF, Synaptotagmin and alpha-SNAP are conserved in eukaryotes and regulate the intracellular function to combat abiotic and biotic stress in plants. Overexpression of these genes in G. max [Williams 82(PI518671)] which is a susceptible cultivar of soybean to nematodes resulted in a reduction of the SCN population providing further insights of molecular and genetic approaches to solve the SCN problems in agriculture.

Characterization of phosphoproteins-including detection, identification of phosphoproteins and identification of phosphorylation sites-is mostly done with radiolabeling and proteomic techniques. Three main topics related to phosphoprotein characterization are included in this dissertation. First, large-scale characterization of the CHO (Chinese hamster ovary) cell phosphoproteome was done using two dimensional gel electrophoresis (2DE) separation, visualization of phosphoproteins by radiolabeling or a phosphoprotein specific dye, followed by MALDI-TOF identification. Because radiolabeling of phosphoproteins is very sensitive and straightforward to quantify, such analysis can give a clear picture of the relative phosphosphorylation of proteins present in a sample. But there are also limitations to this approach, such as the inability of 2DE to separate hydrophobic, acidic and large proteins and the poor detection limits of common protein stains such as Coomassie stain. Additionally, it is difficulty to excise the right spots for identification because of the low abundance of phosphoproteins which have been visualized by radiolabeling. Furthermore, there are problems associated with metabolic radiolabeling. A second topic of the dissertation is the development of a novel strong cation exchange monolithic column for MudPIT (multidimensional protein identification technology) and phosphopeptide isolation. This column, a poly(AMPS-co-PEGDA) monolith containing as high as 40% AMPS, has several favorable features, such as high binding capacity, extraordinarily high resolution, and high peak capacity, making it ideal for resolving complex peptide samples. Application of this novel column to isolate model phosphopeptides was shown. More general use of this column in MudPIT (strong cation exchange column followed by reverse-phased MS/MS) is probably somewhat limited, due to the hydrophobicity of the AMPS monomer. A better monolith could be obtained if a more hydrophilic monomer was used. In the third area of the dissertation, several individual protein phosphorylation sites were analyzed, employing different strategies. Phosphorylation sites of one multiply phosphorylated tryptic peptide from CK2-phosphorylated phosducin-like protein (PhLP) was well characterized using enrichment with a MonoTip® TiO Pipette Tip. Analysis of syntaxin 1a phosphorylation by AMPK (AMP-activated protein kinase) was done by peptide level mapping for potential phosphopeptides after its unsuccessful trial with enrichment using the MonoTip® TiO Pipette Tip. Several criteria such as existence of non-phosphorylated forms of potential phosphopeptides, controls and reasonable retention times were used to rule out false positives. Phosphorylation of syntaxin 1a by AMPK was narrowed down to tryptic peptide T32 with evidence from different sources. Three phosphorylation sites of syntaxin 4 by AMPK were identified within the same peptide (Q65QVTILATPLPEESMK80). Further pinpointing of phosphorylation site(s) for syntaxin 1a by AMPK and further confirmation of these phosphorylation sites in syntaxin 4 by AMPK are required in vivo. The role of phosphorylation in syntaxin 4 by AMPK is the next step toward elucidation of AMPK activation and regulation of the glucose uptake mechanism.