Dlgh1 coordinates actin polymerization, synaptic T cell receptor and lipid raft aggregation, and effector function in T cells.

Lipid raft membrane compartmentalization and membrane-associated guanylate kinase (MAGUK) family molecular scaffolds function in establishing cell polarity and organizing signal transducers within epithelial cell junctions and neuronal synapses. Here, we elucidate a role for the MAGUK protein, Dlgh1, in polarized T cell synapse assembly and T cell function. We find that Dlgh1 translocates to the immune synapse and lipid rafts in response to T cell receptor (TCR)/CD28 engagement and that LckSH3-mediated interactions with Dlgh1 control its membrane targeting. TCR/CD28 engagement induces the formation of endogenous Lck-Dlgh1-Zap70-Wiskott-Aldrich syndrome protein (WASp) complexes in which Dlgh1 acts to facilitate interactions of Lck with Zap70 and WASp. Using small interfering RNA and overexpression approaches, we show that Dlgh1 promotes antigen-induced actin polymerization, synaptic raft and TCR clustering, nuclear factor of activated T cell activity, and cytokine production. We propose that Dlgh1 coordinates TCR/CD28-induced actin-driven T cell synapse assembly, signal transduction, and effector function. These findings highlight common molecular strategies used to regulate cell polarity, synapse assembly, and transducer organization in diverse cellular systems

DETECTING CHANGING POLARIZATION STRUCTURES IN SAGITTARIUS A* WITH HIGH FREQUENCY VLBI

Sagittarius A* is the source of near infrared, X-ray, radio, and (sub)millimeter emission associated with the supermassive black hole at the Galactic Center. In the submillimeter regime, Sgr A* exhibits time-variable linear polarization on timescales corresponding to <10 Schwarzschild radii of the presumed 4 × 10[superscript 6] M[subscript ☉] black hole. In previous work, we demonstrated the potential for total-intensity (sub)millimeter-wavelength very long baseline interferometry (VLBI) to detect time-variable—and periodic—source structure changes in the Sgr A* black hole system using nonimaging analyses. Here, we extend this work to include full polarimetric VLBI observations. We simulate full-polarization (sub)millimeter VLBI data of Sgr A* using a hot spot model that is embedded within an accretion disk, with emphasis on nonimaging polarimetric data products that are robust against calibration errors. Although the source-integrated linear polarization fraction in the models is typically only a few percent, the linear polarization fraction on small angular scales can be much higher, enabling the detection of changes in the polarimetric structure of Sgr A* on a wide variety of baselines. The shortest baselines track the source-integrated linear polarization fraction, while longer baselines are sensitive to polarization substructures that are beam-diluted by connected-element interferometry. The detection of periodic variability in source polarization should not be significantly affected even if instrumental polarization terms cannot be calibrated out. As more antennas are included in the (sub)millimeter-VLBI array, observations with full polarization will provide important new diagnostics to help disentangle intrinsic source polarization from Faraday rotation effects in the accretion and outflow region close to the black hole event horizon.National Science Foundation (U.S.

Ancient origin of the CARD-coiled coil/Bcl10/MALT1-like paracaspase signaling complex indicates unknown critical functions

The CARD-coiled coil (CC)/Bcl10/MALT1-like paracaspase (CBM) signaling complexes composed of a CARD-CC family member (CARD-9, -10, -11, or -14), Bcl10, and the type 1 paracaspase MALT1 (PCASP1) play a pivotal role in immunity, inflammation, and cancer. Targeting MALT1 proteolytic activity is of potential therapeutic interest. However, little is known about the evolutionary origin and the original functions of the CBM complex. Type 1 paracaspases originated before the last common ancestor of planulozoa (bilaterians and cnidarians). Notably in bilaterians, Ecdysozoa (e.g., nematodes and insects) lacks Bcl10, whereas other lineages have a Bcl10 homolog. A survey of invertebrate CARD-CC homologs revealed such homologs only in species with Bcl10, indicating an ancient common origin of the entire CBM complex. Furthermore, vertebrate-like Syk/Zap70 tyrosine kinase homologs with the ITAM-binding SH2 domain were only found in invertebrate organisms with CARD-CC/Bcl10, indicating that this pathway might be related to the original function of the CBM complex. Moreover, the type 1 paracaspase sequences from invertebrate organisms that have CARD-CC/Bcl10 are more similar to vertebrate paracaspases. Functional analysis of protein-protein interactions, NF-kappa B signaling, and CYLD cleavage for selected invertebrate type 1 paracaspase and Bcl10 homologs supports this scenario and indicates an ancient origin of the CARD-CC/Bcl10/paracaspase signaling complex. By contrast, many of the known MALT1-associated activities evolved fairly recently, indicating that unknown functions are at the basis of the protein conservation. As a proof-of-concept, we provide initial evidence for a CBM- and NF-kappa B-independent neuronal function of the Caenorhabditis elegans type 1 paracaspase malt-1. In conclusion, this study shows how evolutionary insights may point at alternative functions of MALT1

Persistent Asymmetric Structure of Sagittarius A^* on Event Horizon Scales

The Galactic Center black hole Sagittarius A^* (Sgr A^*) is a prime observing target for the Event Horizon Telescope (EHT), which can resolve the 1.3 mm emission from this source on angular scales comparable to that of the general relativistic shadow. Previous EHT observations have used visibility amplitudes to infer the morphology of the millimeter-wavelength emission. Potentially much richer source information is contained in the phases. We report on 1.3 mm phase information on Sgr A^* obtained with the EHT on a total of 13 observing nights over four years. Closure phases, which are the sum of visibility phases along a closed triangle of interferometer baselines, are used because they are robust against phase corruptions introduced by instrumentation and the rapidly variable atmosphere. The median closure phase on a triangle including telescopes in California, Hawaii, and Arizona is nonzero. This result conclusively demonstrates that the millimeter emission is asymmetric on scales of a few Schwarzschild radii and can be used to break 180° rotational ambiguities inherent from amplitude data alone. The stability of the sign of the closure phase over most observing nights indicates persistent asymmetry in the image of Sgr A^* that is not obscured by refraction due to interstellar electrons along the line of sight

Computational Modeling and Simulations of Protein-Drug and Protein-Protein Complexes: as potential target for therapeutics development

The main objective of my thesis is to illustrate the potential of computational modeling techniques in determining decisive protein-protein interactions and protein-ligand interactions of two relevant macromolecular biological systems associated to human diseases. Computational tools such as homology modeling, molecular docking, molecular dynamics simulations and the developed protocols implemented for the preparation, simulation and analysis of each biological system are presented. The first contribution is the simulation of modeling of protein-peptide-protein complexes related to adaptive immune system and multiple sclerosis disease. Investigation of molecular similarity between self-peptide and two microbial peptides for the complexes with respect to molecular recognition mechanism is presented. The second contribution is the investigation of protein-ligand interactions of biological systems associated to Alzheimer’s disease. Computational results are compared with experiments to evidence the origin and degree of selective inhibition displayed by 2-Phenylbenzofurans ligands against butyrylcholinesterase (BChE) protein. The final contribution is on the application of a priori knowledge gathered on protein-ligand interactions in designing ligands with specific structural modifications that display an improved inhibitory activity against BChE protein. In conclusion, therapeutical perspectives and application of hybrid computational approaches to design and develop of potential drugs are discussed

Resolved magnetic-field structure and variability near the event horizon of Sagittarius A*

Near a black hole, differential rotation of a magnetized accretion disk is thought to produce an instability that amplifies weak magnetic fields, driving accretion and outflow. These magnetic fields would naturally give rise to the observed synchrotron emission in galaxy cores and to the formation of relativistic jets, but no observations to date have been able to resolve the expected horizon-scale magnetic-field structure. We report interferometric observations at 1.3-millimeter wavelength that spatially resolve the linearly polarized emission from the Galactic Center supermassive black hole, Sagittarius A*. We have found evidence for partially ordered magnetic fields near the event horizon, on scales of ~6 Schwarzschild radii, and we have detected and localized the intrahour variability associated with these fields

MALT1 protease function – from ubiquitin binding to substrate cleavage

The protease MALT1 has a key function in the activation of lymphocytes and the regulation of the immune response, by promoting the activation of pro-inflammatory transcription factors such as NF-B. Dysregulation of MALT1 is implicated in immunodeficiency, autoimmune diseases, lymphomagenesis and non-lymphoid malignancies. Therefore, MALT1 proteolytic activity has appeared as a possible pharmaceutical target, however, the precise mechanism of MALT1 protease activation and the role of individual substrate cleavage events remain incompletely defined. Thus, the aims of this study are to firstly elucidate the molecular mechanism of MALT1 activation and second, to explore the role of the MALT1-dependent cleavage of one particular substrate namely, A20. The protease activity of MALT1 is tightly controlled by conjugation of monoubiquitin to its third auto-inhibitory Ig-like domain, but the mechanism governing the release of the protease domain by a single ubiquitin moiety remains unknown. Here, we identify the Ig3 domain of MALT1 as a novel ubiquitin-binding domain, responsible for MALT1 monoubiquitination, which is essential for MALT1 proteolytic activity and lymphocyte activation. Furthermore, we reveal an allosteric communication from the monoubiquitination site through the protease-Ig3 interaction surface to the catalytic active site of the protease domain. One of the first substrates of MALT1 that has been identified is A20, a potent anti-inflammatory protein. A20 is a well-described negative regulator of the NF-B signaling pathway downstream of different pro-inflammatory stimuli and a regulator of cell death. Although MALT1-dependent A20 cleavage is generally thought to promote NF-B activity, the functional role of A20 cleavage remains controversial and not well defined. This is due to the fact that the originally described single cleavage site in A20 is not conserved among species and only a small proportion of cellular A20 undergoes cleavage. Here, we demonstrate that MALT1 cleaves A20 at a total of four distinct sites in B and T lymphocytes, including three novel cleavage sites with unusual sequence properties, which are conserved in the mouse and other species. The cleavage fragments lost their capacity to regulate the NF-B pathway, but are stable within the cell, suggesting that they retain an unknown physiological function in lymphocytes. Collectively, our findings provide fundamentally new insights into the mechanism of MALT1 protease activation and its cleavage site specificity, and suggest that MALT1-dependent A20 cleavage has roles that go beyond the enhancement of NF-B activity. -- La protéase MALT1 a une fonction clé dans l’activation des lymphocytes et la régulation de la réponse immunitaire, en favorisant l’activation de facteurs de transcription pro- inflammatoires comme le NF-B. La dérégulation de MALT1 est impliquée dans l’immunodéficience, les maladies auto-immunes, la lymphomagénèse et les tumeurs malignes non-lymphoides. Par conséquent, l’activité protéolytique de MALT1 est apparue comme une cible thérapeutique possible, cependant le mécanisme précis de l’activation de la protéase MALT1 et le rôle du clivage de chacun de ses substrats restent en partie incompris. Ainsi les objectifs de cette étude sont d’abord d’élucider le mécanisme moléculaire d’activation de MALT1 puis d’explorer le rôle du clivage dépendant de MALT1 d’un substrat en particulier nommé A20. L’activité protéase de MALT1 est étroitement contrôlée par la conjugaison d’une monoubiquitine à son troisième domaine auto-inhibiteur de type Ig, mais le mécanisme régissant la libération du domaine protéase par un seul fragment d'ubiquitine reste inconnu. Ici, nous avons identifié le domaine Ig3 de MALT1 comme un nouveau domaine de liaison à l’ubiquitine, responsable de la monoubiquitination de MALT1, qui est essentielle pour son activité protéolytique et l’activation lymphocytaire. De plus, nous avons révélé une transmission allostérique du site de monoubiquitination à travers la surface d’interaction protéase-Ig3 au site catalytique actif du domaine protéase. L’un des premiers substrats de MALT1 qui a été identifié est A20, une protéine anti-inflammatoire puissante. A20 est un régulateur négatif bien décrit de la voie de signalisation NF-B en aval de différents stimuli pro-inflammatoires et un régulateur de la mort cellulaire. Bien que le clivage de A20 dépendant de MALT1 soit généralement vu comme un promoteur de l’activité de NF-B, le rôle fonctionnel du clivage de A20 reste controversé et mal défini. Cela est dû au fait que le site de clivage originellement découvert de A20 n’est pas conservé chez les autres espèces et que seulement une petite partie de la protéine cellulaire est clivée. Ici, nous démontrons que MALT1 clive A20 sur un total de quatre sites distincts dans les lymphocytes B et T. Nous avons également identifié trois nouveaux sites de clivage avec des motifs inhabituels, sites qui sont conservés chez la souris et d’autres espèces. Les fragments issus du clivage ont perdu leur capacité à réguler la voie NF-B, mais sont stables dans la cellule, suggérant qu’ils gardent une fonction physiologique inconnue dans les lymphocytes. Ensemble, nos résultats apportent de nouvelles informations fondamentales sur le mécanisme d’activation de la protéase MALT1 et sur la spécificité de ses sites de clivage, et suggèrent que le clivage de A20 dépendant de MALT1 a un rôle qui va au- delà d’une simple activation de NF-B

The impact of the SZ effect on cm-wavelength (1-30 GHz) observation of galaxy cluster radio relics

(Abridged) Radio relics in galaxy clusters are believed to be associated with powerful shock fronts that originate during cluster mergers, and are a testbed for the acceleration of relativistic particles in the intracluster medium. Recently, radio relic observations have pushed into the cm-wavelength domain (1-30 GHz) where a break from the standard synchrotron power-law spectrum has been found, most noticeably in the famous 'Sausage' relic. In this paper, we point to an important effect that has been ignored or considered insignificant while interpreting these new high-frequency radio data, namely the contamination due to the Sunyaev-Zel'dovich (SZ) effect that changes the observed synchrotron flux. Even though the radio relics reside in the cluster outskirts, the shock-driven pressure boost increases the SZ signal locally by roughly an order of magnitude. The resulting flux contamination for some well-known relics are non-negligible already at 10 GHz, and at 30 GHz the observed synchrotron fluxes can be diminished by a factor of several from their true values. Interferometric observations are not immune to this contamination, since the change in the SZ signal occurs roughly at the same length scale as the synchrotron emission, although there the flux loss is less severe than single-dish observations. We present a simple analytical approximation for the synchrotron-to-SZ flux ratio, based on a theoretical radio relic model that connects the non-thermal emission to the thermal gas properties, and show that by measuring this ratio one can potentially estimate the relic magnetic fields or the particle acceleration efficiency.Comment: Updated to the accepted version. Includes major text modifications and a correction to the numerical coefficient in Eq. 15. Results and conclusions are unchange