Utility of Hybrid Transferrin Binding Protein Antigens for Protection Against Pathogenic Neisseria Species

The surface transferrin receptor proteins from Neisseria gonorrhoeae have been recognized as ideal vaccine targets due to their critical role in survival in the human male genitourinary tract. Recombinant forms of the surface lipoprotein component of the receptor, transferrin binding protein B (TbpB), can be readily produced at high levels in the Escherichia coli cytoplasm and is suitable for commercial vaccine production. In contrast, the integral outer membrane protein, transferrin binding protein A (TbpA), is produced at relatively low levels in the outer membrane and requires detergents for solubilization and stabilization, processes not favorable for commercial applications. Capitalizing on the core β-barrel structural feature common to the lipoprotein and integral outer membrane protein we engineered the lipoprotein as a scaffold for displaying conserved surface epitopes from TbpA. A stable version of the C-terminal domain of TbpB was prepared by replacing four larger exposed variable loops with short linking peptide regions. Four surface regions from the plug and barrel domains of Neisseria TbpA were transplanted onto this TbpB C-lobe scaffold, generating stable hybrid antigens. Antisera generated in mice and rabbits against the hybrid antigens recognized TbpA at the surface of Neisseria meningitidis and inhibited transferrin-dependent growth at levels comparable or better than antisera directed against the native TbpA protein. Two of the engineered hybrid antigens each elicited a TbpA-specific bactericidal antibody response comparable to that induced by TbpA. A hybrid antigen generated using a foreign scaffold (TbpB from the pig pathogen Haemophilus parasuis) displaying neisserial TbpA loop 10 was evaluated in a model of lower genital tract colonization by N. gonorrhoeae and a model of invasive infection by N. meningitidis. The loop 10 hybrid antigen was as effective as full length TbpA in eliminating N. gonorrhoeae from the lower genital tract of female mice and was protective against the low dose invasive infection by N. meningitidis. These results demonstrate that TbpB or its derivatives can serve as an effective scaffold for displaying surface epitopes of integral outer membrane antigens and these antigens can elicit protection against bacterial challenge

Structures of the cGMP-dependent protein kinase in malaria parasites reveal a unique structural relay mechanism for activation.

The cyclic guanosine-3',5'-monophosphate (cGMP)-dependent protein kinase (PKG) was identified >25 y ago; however, efforts to obtain a structure of the entire PKG enzyme or catalytic domain from any species have failed. In malaria parasites, cooperative activation of PKG triggers crucial developmental transitions throughout the complex life cycle. We have determined the cGMP-free crystallographic structures of PKG from Plasmodium falciparum and Plasmodium vivax, revealing how key structural components, including an N-terminal autoinhibitory segment (AIS), four predicted cyclic nucleotide-binding domains (CNBs), and a kinase domain (KD), are arranged when the enzyme is inactive. The four CNBs and the KD are in a pentagonal configuration, with the AIS docked in the substrate site of the KD in a swapped-domain dimeric arrangement. We show that although the protein is predominantly a monomer (the dimer is unlikely to be representative of the physiological form), the binding of the AIS is necessary to keep Plasmodium PKG inactive. A major feature is a helix serving the dual role of the N-terminal helix of the KD as well as the capping helix of the neighboring CNB. A network of connecting helices between neighboring CNBs contributes to maintaining the kinase in its inactive conformation. We propose a scheme in which cooperative binding of cGMP, beginning at the CNB closest to the KD, transmits conformational changes around the pentagonal molecule in a structural relay mechanism, enabling PKG to orchestrate rapid, highly regulated developmental switches in response to dynamic modulation of cGMP levels in the parasite

Etude du "centralspindlin complex", un élément régulateur de la cytokinese.

Kinesins form a family of proteins that convert the energy from ATP hydrolysis to mechanical force for displacement of their cargoes along microtubules tracts. In Humans there are 40 kinesins, 13 of which are essentials for mitosis. MKLP-1 is a mitotic kinesin belonging to kinesin sub-family 6, which is essential for cytokinesis. In cells, MKLP-1 together with MgcRacGAP (a GTPase activating protein) contributes to the formation of the centralspindlin heterotetrameric complex. This MKLP-1/MgcRacGAP complex is an important regulatory component which participates to the formation of the midbody matrix during cell cleavage. MKLP1 has in its motor domain a large insert of 80 amino acid in the loop 6 compared to the other kinesins ; this longer loop 6 is an caracteristic of the kinesin sub-family 6 (MPP1, MKLP-2...). To probe the role of the MKLP1 loop 6 in the mechanochemical cycle, I compared the MKLP1 ATPase activity of wild type with that a mutant in which the native Loop 6 (80 residues) was replaced by the conventionnal loop 6 of Kinesin heavy Chain (5 residues). In addition, I screen four librairies of small molecules in order to identify ATPase inhibitors of the MKLP-1 motor domain. This screen contributed to the identification of a non-specific MKLP-1 inhibitor that prevent the kinesin/tubulin association. Furthermore, in order to understand the microtubules-bundling activity of the centralspindlin complex, I studied the microtubule interaction pattern for differents MKLP-1 and MgcRacGAP constructs. The results allowed me to purpose an putative interaction model.Les kinésines constituent une famille protéique convertissant l'énergie d'hydrolyse de l'ATP en un travail mécanique permettant un déplacement de l'enzyme le long des microtubules. Chez l'homme, une quarantaine de kinésines sont actuellement recensées et 13 d'entre elles sont nommées kinésines mitotiques en raison de leurs implications essentielles au bon déroulement de la Mitose. La protéine MKLP-1, kinésine de la sous-famille 6, est l'une de ces kinésines mitotiques. In vivo, elle s'associe à la protéine MgcRacGAP (protéine activatrice de petites protéines G) pour former un complexe hétérotétramérique : le « centralspindlin complex ». Ce complexe est un élément important de la régulation de la cytokinèse et participe notamment à la formation du « corps intermédiaire » durant la division cellulaire. MKLP-1 possède à la différence des autres kinésines, une large insertion de 80 acides aminés dans la boucle L6 de son domaine moteur ; cette insertion est une caractéristique unique des kinésines de la sous-famille 6 (MKLP-2, MPP1...). Afin de sonder le rôle de la boucle L6 au cours des processus catalytiques, j'ai effectué la caractérisation enzymatique du domaine moteur sauvage de MKLP1 et d'un mutant où la boucle native L6 est remplacée par la boucle L6 de la kinésine conventionnelle KHC (5 acides aminés). J'ai aussi réalisé un criblage de chimiothéques sur l'activité ATPasique du domaine moteur. J'ai ainsi pu mettre en évidence un nouvel inhibiteur de la reconnaissance kinésine/tubuline. De plus, pour éclaircir les mécanismes mis en jeu par le « centralspindlin complex » dans l'agrégation des microtubules, j'ai entrepris pour différentes constructions de MKLP-1 et de MgcRacGAP, l'étude de leurs profils d'interactions avec la tubuline. J'ai ainsi pu établir un modèle de fixation du « centralspindlin complex » avec les microtubules

Etude du "centralspindlin complex", un élément régulateur de la cytokinese.

Kinesins form a family of proteins that convert the energy from ATP hydrolysis to mechanical force for displacement of their cargoes along microtubules tracts. In Humans there are 40 kinesins, 13 of which are essentials for mitosis. MKLP-1 is a mitotic kinesin belonging to kinesin sub-family 6, which is essential for cytokinesis. In cells, MKLP-1 together with MgcRacGAP (a GTPase activating protein) contributes to the formation of the centralspindlin heterotetrameric complex. This MKLP-1/MgcRacGAP complex is an important regulatory component which participates to the formation of the midbody matrix during cell cleavage. MKLP1 has in its motor domain a large insert of 80 amino acid in the loop 6 compared to the other kinesins ; this longer loop 6 is an caracteristic of the kinesin sub-family 6 (MPP1, MKLP-2...). To probe the role of the MKLP1 loop 6 in the mechanochemical cycle, I compared the MKLP1 ATPase activity of wild type with that a mutant in which the native Loop 6 (80 residues) was replaced by the conventionnal loop 6 of Kinesin heavy Chain (5 residues). In addition, I screen four librairies of small molecules in order to identify ATPase inhibitors of the MKLP-1 motor domain. This screen contributed to the identification of a non-specific MKLP-1 inhibitor that prevent the kinesin/tubulin association. Furthermore, in order to understand the microtubules-bundling activity of the centralspindlin complex, I studied the microtubule interaction pattern for differents MKLP-1 and MgcRacGAP constructs. The results allowed me to purpose an putative interaction model.Les kinésines constituent une famille protéique convertissant l'énergie d'hydrolyse de l'ATP en un travail mécanique permettant un déplacement de l'enzyme le long des microtubules. Chez l'homme, une quarantaine de kinésines sont actuellement recensées et 13 d'entre elles sont nommées kinésines mitotiques en raison de leurs implications essentielles au bon déroulement de la Mitose. La protéine MKLP-1, kinésine de la sous-famille 6, est l'une de ces kinésines mitotiques. In vivo, elle s'associe à la protéine MgcRacGAP (protéine activatrice de petites protéines G) pour former un complexe hétérotétramérique : le « centralspindlin complex ». Ce complexe est un élément important de la régulation de la cytokinèse et participe notamment à la formation du « corps intermédiaire » durant la division cellulaire. MKLP-1 possède à la différence des autres kinésines, une large insertion de 80 acides aminés dans la boucle L6 de son domaine moteur ; cette insertion est une caractéristique unique des kinésines de la sous-famille 6 (MKLP-2, MPP1...). Afin de sonder le rôle de la boucle L6 au cours des processus catalytiques, j'ai effectué la caractérisation enzymatique du domaine moteur sauvage de MKLP1 et d'un mutant où la boucle native L6 est remplacée par la boucle L6 de la kinésine conventionnelle KHC (5 acides aminés). J'ai aussi réalisé un criblage de chimiothéques sur l'activité ATPasique du domaine moteur. J'ai ainsi pu mettre en évidence un nouvel inhibiteur de la reconnaissance kinésine/tubuline. De plus, pour éclaircir les mécanismes mis en jeu par le « centralspindlin complex » dans l'agrégation des microtubules, j'ai entrepris pour différentes constructions de MKLP-1 et de MgcRacGAP, l'étude de leurs profils d'interactions avec la tubuline. J'ai ainsi pu établir un modèle de fixation du « centralspindlin complex » avec les microtubules

Etude du "centralspindlin complex" (un élément régulateur essentiel de la cytokinèse)

Les kinésines constituent une famille protéique convertissant l'énergie d'hydrolyse de l'ATP en un travail mécanique permettant un déplacement de l'enzyme le long des microtubules. Chez l'homme, une quarantaine de kinésines sont actuellement recensées et 13 d'entre elles sont nommées kinésines mitotiques en raison de leurs implications essentielles au bon déroulement de la Mitose. La protéine MKLP-1, kinésine de la sous-famille 6, est l'une de ces kinésines mitotiques. In vivo, elle s'associe à la protéine MgcRacGAP (protéine activatrice de petites protéines G) pour former un complexe hétérotétramérique : le centralspindlin complex . Ce complexe est un élément important de la régulation de la cytokinèse et participe notamment à la formation du corps intermédiaire durant la division cellulaire. MKLP-1 possède à la différence des autres kinésines, une large insertion de 80 acides aminés dans la boucle L6 de son domaine moteur ; cette insertion est une caractéristique unique des kinésines de la sous-famille 6 (MKLP-2, MPP1...). Afin de sonder le rôle de la boucle L6 au cours des processus catalytiques, j'ai effectué la caractérisation enzymatique du domaine moteur sauvage de MKLP1 et d'un mutant où la boucle native L6 est remplacée par la boucle L6 de la kinésine conventionnelle KHC (5 acides aminés). J'ai aussi réalisé un criblage de chimiothéques sur l'activité ATPasique du domaine moteur. J'ai ainsi pu mettre en évidence un nouvel inhibiteur de la reconnaissance kinésine/tubuline. De plus, pour éclaircir les mécanismes mis en jeu par le centralspindlin complex dans l'agrégation des microtubules, j'ai entrepris pour différentes constructions de MKLP-1 et de MgcRacGAP, l'étude de leurs profils d'interactions avec la tubuline. J'ai ainsi pu établir un modèle de fixation du centralspindlin complex avec les microtubules.Kinesins form a family of proteins that convert the energy from ATP hydrolysis to mechanical force for displacement of their cargoes along microtubules tracts. In Humans there are 40 kinesins, 13 of which are essentials for mitosis. MKLP-1 is a mitotic kinesin belonging to kinesin sub-family 6, which is essential for cytokinesis. In cells, MKLP-1 together with MgcRacGAP (a GTPase activating protein) contributes to the formation of the centralspindlin heterotetrameric complex. This MKLP-1/MgcRacGAP complex is an important regulatory component which participates to the formation of the midbody matrix during cell cleavage. MKLP1 has in its motor domain a large insert of 80 amino acid in the loop 6 compared to the other kinesins ; this longer loop 6 is an caracteristic of the kinesin sub-family 6 (MPP1, MKLP-2 ). To probe the role of the MKLP1 loop 6 in the mechanochemical cycle, I compared the MKLP1 ATPase activity of wild type with that a mutant in which the native Loop 6 (80 residues) was replaced by the conventionnal loop 6 of Kinesin heavy Chain (5 residues). In addition, I screen four librairies of small molecules in order to identify ATPase inhibitors of the MKLP-1 motor domain. This screen contributed to the identification of a non-specific MKLP-1 inhibitor that prevent the kinesin/tubulin association. Furthermore, in order to understand the microtubules-bundling activity of the centralspindlin complex, I studied the microtubule interaction pattern for differents MKLP-1 and MgcRacGAP constructs. The results allowed me to purpose an putative interaction model.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

Interaction Mechanism Between the HSV-1 Glycoprotein B and the Antimicrobial Peptide Amyloid-β

International audienceBackground: Unravelling the mystery of Alzheimer’s disease (AD) requires urgent resolution given the worldwide increase of the aging population. There is a growing concern that the current leading AD hypothesis, the amyloid cascade hypothesis, does not stand up to validation with respect to emerging new data. Indeed, several paradoxes are being discussed in the literature, for instance, both the deposition of the amyloid-β peptide (Aβ) and the intracellular neurofibrillary tangles could occur within the brain without any cognitive pathology. Thus, these paradoxes suggest that something more fundamental is at play in the onset of the disease and other key and related pathomechanisms must be investigated. Objective: The present study follows our previous investigations on the infectious hypothesis, which posits that some pathogens are linked to late onset AD. Our studies also build upon the finding that Aβ is a powerful antimicrobial agent, produced by neurons in response to viral infection, capable of inhibiting pathogens as observed in in vitro experiments. Herein, we ask what are the molecular mechanisms in play when Aβ neutralizes infectious pathogens? Methods: To answer this question, we probed at nanoscale lengths with FRET (Förster Resonance Energy Transfer), the interaction between Aβ peptides and glycoprotein B (responsible of virus-cell binding) within the HSV-1 virion Results: The experiments show an energy transfer between Aβ peptides and glycoprotein B when membrane is intact. No energy transfer occurs after membrane disruption or treatment with blocking antibody. Conclusion: We concluded that Aβ insert into viral membrane, close to glycoprotein B, and participate in virus neutralization

Mémoires du général Bon Thiébault,

I. 1769-1795. 9. éd. 1896.--II. 1795-1799. 8. éd. 1897.--III. 1799-1806. 7. éd. 1895.--IV. 1806-1813. 6. éd. 1896.--V. 1813-1820. 6. éd. 1897.Mode of access: Internet

Disabling a Type I-E CRISPR-Cas Nuclease with a Bacteriophage-Encoded Anti-CRISPR Protein

CRISPR (clustered regularly interspaced short palindromic repeat)-Cas adaptive immune systems are prevalent defense mechanisms in bacteria and archaea. They provide sequence-specific detection and neutralization of foreign nucleic acids such as bacteriophages and plasmids. One mechanism by which phages and other mobile genetic elements are able to overcome the CRISPR-Cas system is through the expression of anti-CRISPR proteins. Over 20 different families of anti-CRISPR proteins have been described, each of which inhibits a particular type of CRISPR-Cas system. In this work, we determined the structure of type I-E anti-CRISPR protein AcrE1 by X-ray crystallography. We show that AcrE1 binds to the CRISPR-associated helicase/nuclease Cas3 and that the C-terminal region of the anti-CRISPR protein is important for its inhibitory activity. We further show that AcrE1 can convert the endogenous type I-E CRISPR system into a programmable transcriptional repressor