Regulator of G Protein Signaling 4 (RGS4) mediates motor defects in Parkinson's disease. Small molecule RGS4 inhibitors (e.g. CCG-50014) modify buried cysteine residues, but the structural and dynamic mechanisms underpinning specificity of inhibitors for RGS4 within the RGS family are poorly understood. We used NMR and other biophysical methods to examine ligand-induced structural changes and the dynamics of unliganded RGS4 and RGS8 that allow ligand binding. NMR and fluorescence spectroscopy data reveal details of the hidden, excited conformational state of RGS4 that exposes Cys148, one of the buried cysteines bound by inhibitors. We further show that specificity of RGS4 inhibitors is driven by differential accessibility of the target cysteine compared to its equivalent in RGS8. Cys148 is buried beneath the lid at the center the α4-α7 helix bundle, and this bundle is destabilized in RGS4 compared to RGS8. Notably, helix 6 is highly destabilized in RGS4 compared to RGS8 and is likely the key mediator of access to Cys148. Our findings provide key insight into the mechanism of allosteric RGS4 inhibition and show that dynamics drive inhibitory specificity among RGS proteins.

Chemokines are important chemical signals that guide lymphocyte movement within the immune system and promote the organization and functions of germinal centers (GCs) in the secondary lymphoid tissues. Previous studies have shown that GC T cells exhibit high expression of chemokine receptor 4, CXCR4, but that these cells are unable to migrate to the ligand for this receptor, the chemokine CXCL12. This “migratory paralysis” to CXCL12 was found to be correlated with the expression of two Regulators of G-protein Signaling, RGS13 and RGS16 in the GC T cells. The objective of my research was to determine whether RGS13 and RGS16 expression were associated with CXCL12-mediated CD4+ T cell migration. Because human GC T cells are rare and vary from one individual to another, I utilized two human neoplastic CD4+ T cell lines (i.e. Hut78 and SupT1) to facilitate and standardize my research. I also confirmed my observations using primary CD4+ T cells. Hut78 cells behaved similarly to GC T cells interms of CXCL12-mediated migration and RGS13 and RGS16 expression, while SupT1 cells appeared similar to CD4+ T cells that resided outside of GCs. The effect of RGS13 and RGS16 expression in the various CD4+ T cells was examined by altering the natural levels of these genes using RNA-mediated silencing and/or gene overexpression analysis after which, I examined the ability of the cells to migrate to CXCL12. RNA-mediated silencing of RGS16-, but not RGS13-, expression in Hut78 T cells resulted in a doubling of the migration rate in response to CXCL12. Over-expression of RGS13 or RGS16 in SupT1 and primary CD4+ T cells resulted in migration that was decreased by fifty percent. Because GC T cells demonstrated decreased migration to CXCL12 signals that may help them leave the GC, I reasoned that these cells may have an increased opportunity over other CD4+ T cells to become infected by the Human Immunodeficiency Virus (HIV) trapped on Follicular Dendritic Cells in the GCs of infected subjects. Examination of GC T cells obtained from HIV-infected subjects indicated that these cells were more frequently infected by HIV than other CD4+ T cells thereby confirming my postulate. My research indicated that RGS13 and RGS16 were associated with CXCL12-mediated CD4+ T cell migration and suggests that these molecules may play an important role in HIV pathogenesis within the GC.

Les cellules de Schwann (CS) sont les cellules gliales myélinisantes du Système Nerveux Périphérique (SNP). Il existe une communication étroite entre ces cellules et les axones auxquels elles s’associent et ce dès les stades les plus précoces de leur développement. Elles migrent tout en se divisant le long des axones; cette division migratoire est suivie d’une deuxième division post-migratoire dans le but d’établir un ratio 1:1 avec les axones pour ensuite les myéliniser. Ce travail vise à analyser, in vivo, le comportement des CS chez le poisson zèbre au cours de leurs divisions.Nous avons remarqué que les CS se divisent parallèlement aux axones le long du nerf de la Ligne Latérale Postérieure (PLL). En analysant les deux mutants has et nok, nous avons montré que les gènes de polarité apicale aPKC et pals1 ne sont pas requis pour la migration et la division des CS, ni pour leur capacité à myéliniser. Nous avons mis en évidence, en analysant le mutant cassiopeia qui présente des défauts d’organisation du fuseau mitotique et en utilisant l’agent pharmacologique le nocodazole, que l’assemblage du fuseau mitotique au cours de la division des CS est essentiel pour la myélinisation.En parallèle, nous avons analysé le rôle du gène rgs4 (regulator of G-protein Signaling 4) dans le développement du SNP chez le poisson zèbre. Nous avons généré un mutant stable rgs4 par la technique CRISPR/Cas9 et montré un rôle de ce gène dans le développement du ganglion de la PLL et des motoneurones, et ce en agissant en amont de la voie PI3K/Akt/mTOR.Contrairement à l’inhibition pharmacologique qui suggère un rôle de rgs4 dans la myélinisation périphérique, le mutant ne présente pas de défauts de myéline. / Schwann cells (SCs) are the myelinating glial cells of the Peripheral Nervous System (PNS). They derive from neural crest cells during development, then migrate and divide along the axons of the peripheral nerves. This migratory division is followed by a post-migratory division in order to radially sort the axons in a 1:1 ratio and wrap them with a myelin sheath. This work provides an analysis of the polarity of SC divisions, in vivo, in intact zebrafish embryos.We showed that SCs divide parallel to the axons along the Posterior Lateral Line nerve (PLL). By analyzing the two mutants has and nok, we revealed that the apical polarity genes aPKC and pals1, are neither required for the migration and division of SCs, nor for their capacity to myelinate. By studying the cassiopeia mutant that shows defects in mitotic spindle, we revealed that the assembly of the mitotic spindle is essential for SC myelination.We have also analysed the role of rgs4 (regulator of G-protein Signaling 4) in PNS development. We generated a stable rgs4 mutant using the CRISPR/Cas9 technology. We showed that rgs4 plays an essentiel role in PLLg and motoneurons development by acting upstream of PI3K/Akt/mTOR pathway. Pharmacological analysis suggested a role for rgs4 in peripheral myelination, however, the rgs4 mutant do not show any myelin defects.

Gαq signaling is implicated in a number of physiological processes that include platelet activation, cardiovascular development and smooth muscle function. Historically, Gαq is known to function by activating its effector, phospholipase Cβ. Desensitization of Gαq signaling is mediated by G-protein coupled receptor kinases (GRK) such as GRK2 that phosphorylates the activated receptor and also sequesters activated Gαq and Gβγ subunits. Our crystal structure of Gαq-GRK2-Gβγ complex shows that Gαq forms effector-like interactions with the regulator of G-protein signaling (RGS) homology domain of GRK2 involving the classic effector-binding site of Gα subunits, raising the question if GRK2 can itself be a Gáq effector and initiate its own signaling cascade. In the structure, Gα and Gβγ subunits are completely dissociated from one another and the orientation of activated Gαq with respect to the predicted cell membrane is drastically different from its position in the inactive Gαβγ heterotrimer. Recent studies have identified a novel Gαq effector, p63RhoGEF that activates RhoA. Our crystal structure of the Gαq-p63RhoGEF-RhoA complex reveals that Gαq interacts with both the Dbl homology (DH) and pleckstrin homology (PH) domains of p63RhoGEF with its C-terminal helix and its effector-binding site, respectively. The structure predicts that Gαq relieves auto-inhibition of the catalytic DH domain by the PH domain. We show that Gαq activates p63RhoGEF-related family members, Trio and Kalirin, revealing several conduits by which RhoA is activated in response to Gq-coupled receptors. The Gαq effector-site interaction with p63RhoGEF/GRK2 does not overlap with the Gαq-binding site of RGS2/RGS4 that function as GTPase activating proteins (GAPs). This suggests that activated G proteins, effectors, RGS proteins, and activated receptors can form high-order complexes at the cell membrane. We confirmed the formation of RGS-Gαq-effector complexes and our results suggest that signaling pathways initiated by GRK2 and p63RhoGEF are regulated by RGS proteins via both allosteric and GAP mechanisms. Our structural studies of Gαq signaling provide insight into protein-protein interactions that induce profound physiological changes. Understanding such protein interfaces is a key step towards structure-based drug design that can be targeted to treat diseases concerned with impaired Gαq signaling. / text

In order for G protein signaling to occur, the G protein heterotrimer must be assembled from its nascent polypeptides. The most difficult step in this process is the formation of the Gβγ dimer from the free subunits since both are unstable in the absence of the other. Recent studies have shown that phosducin-like protein (PhLP1) works as a co-chaperone with the cytosolic chaperonin complex (CCT) to fold Gβ and mediate its interaction with Gγ. However, these studies did not address questions concerning the scope of PhLP1 and CCT-mediated Gβγ assembly, which are important questions given that there are four Gβs that form various dimers with 12 Gγs and a 5th Gβ that dimerizes with the four regulator of G protein signaling (RGS) proteins of the R7 family. The data presented in Chapter 2 shows that PhLP1 plays a vital role in the assembly of Gγ2 with all four Gβ1-4 subunits and in the assembly of Gβ2 with all twelve Gγ subunits, without affecting the specificity of the Gβγ interactions. The results of Chapter 3 show that Gβ5-RGS7 assembly is dependent on CCT and PhLP1, but the apparent mechanism is different from that of Gβγ. PhLP1 seems to stabilize the interaction of Gβ5 with CCT until Gβ5 is folded, after which it is released to allow Gβ5 to interact with RGS7. These findings point to a general role for PhLP1 in the assembly of all Gβγ combinations, and suggest a CCT-dependent mechanism for Gβ5-RGS7 assembly that utilizes the co-chaperone activity of PhLP1 in a unique way. Chapter 4 discusses PhLP2, a recently discovered essential protein, and member of the Pdc family that does not play a role in G protein signaling. Several studies have indicated that PhLP2 acts as a co-chaperone with CCT in the folding of actin, tubulin, and several cell cycle and pro-apoptotic proteins. In a proteomics screen for PhLP2A interacting partners, α-tubulin, 14-3-3, elongation factor 1α, and ribosomal protein L3 were found. Further proteomics studies indicated that PhLP2A is a phosphoprotein that is phosphorylated by CK2 at threonines 47 and 52.

Major histocompatibility complex molecules (MHCII) are cell surface glycoproteins that present extracellular antigens to CD4+ T lymphocytes and initiate adaptive immune responses. Apart from their protective role, overexpression of MHCII contributes to autoimmune disorders where the immune system attacks our own tissues. Autoimmune diseases are characterized by self-reactive responses to autoantigens, promoting tissue damage, inflammation mediated by proinflammatory cytokines, autoreactive lymphocytes, and autoantibodies. MHCII molecules are tightly regulated at the level of transcription by Class II transactivator (CIITA). CIITA associates with an enhanceosome complex at MHCII promoters and regulates the expression of MHCII. It is thus crucial to understand the regulation of CIITA expression in order to regulate MHCII in autoimmune diseases. Our lab has shown that the 19S ATPases of the 26S proteasome associate with MHCII and CIITA promoters and play important roles in gene transcription, regulate covalent modifications to histones, and are involved in the assembly of activator complexes in mammalian cells. The mechanisms by which the proteasome influences transcription remain unclear. Here, we define novel roles of the 19S ATPases Sug1, S7, and S6a in expression of CIITApIV genes. These ATPases are recruited to CIITApIV promoters and coding regions, interact with the elongation factor PTEFb, and with Ser5 phosphorylated RNA Pol II. Both the generation of CIITApIV transcripts and efficient recruitment of RNA Pol II to CIITApIV are negatively impacted by knockdown of 19S ATPases. Alternatively, inflammation is also suppressed via the Regulator of G-protein signaling 10 (RGS10) in microglial cells which express high levels of RGS10 and promote homeostasis in the central nervous system. However, chronic activation of microglial cells leads to release of cytokines which cause neuroinflammation. Our investigation of roles played by RGS10 in chronically activated microglial cells indicates that RGS10 binds to promoters of IL-1β, and TNF-α and regulates these genes, while the molecular mechanism remains to be investigated. Together, our observations indicate roles for the UPS in modulating gene expression and for RGS10 in regulating proinflammatory cytokines in microglial cells, each of which provides novel therapeutic targets to combat inflammation in autoimmune and neurodegenerative diseases.

5 Abstract The thesis is focused on the role of regulator of G-protein signaling NtRGS1 in control of growth and cell proliferation of tobacco cell line BY-2. The protein NtRGS1 is an important candidate for being plant G-protein coupled receptor. Heterotrimeric G-proteins are involved in key signaling mechanisms in eukaryotic cells. Basic principles of this type of signaling are well conserved between plants and animals and related higher taxa. Outstanding difference of plant G-protein system is altered enzymatic activity of Gα subunit of the G-protein heterotrimer. These alterations correlate with chimeric structure and function of investigated NtRGS1 protein. The interaction of Gα and NtRGS1 is absolutely essential for running of heterotrimeric G-protein signaling in plants. Truncated versions of NtRGS1 fused to GFP were crated in the aim of protein characterization. The truncated proteins were investigated in respect of analysis of the role of NtRGS1 domains in protein targeting. Dynamic changes in NtRGS1 and selected truncated versions induced by experimental application of nutrition, especially sugars were described. Expression if Gα and NtRGS1 were investigated simultaneously. Influence of modulation of Gα and NtRGS1 expression on growth parameters of tobacco cell line BY-2 were described. Key words:...