Calmodulin-dependent kinase IV links Toll-like receptor 4 signaling with survival pathway of activated dendritic cells.

Microbial products, including lipopolysaccharide (LPS), an agonist of Toll-like receptor 4 (TLR4), regulate the lifespan of dendritic cells (DCs) by largely undefined mechanisms. Here, we identify a role for calcium-calmodulin–dependent kinase IV (CaMKIV) in this survival program. The pharmacologic inhibition of CaMKs as well as ectopic expression of kinase-inactive CaMKIV decrease the viability of monocyte-derived DCs exposed to bacterial LPS. The defect in TLR4 signaling includes a failure to accumulate the phosphorylated form of the cAMP response element-binding protein (pCREB), Bcl-2, and Bcl-xL. CaMKIV null mice have a decreased number of DCs in lymphoid tissues and fail to accumulate mature DCs in spleen on in vivo exposure to LPS. Although isolated Camk4(−/−) DCs are able to acquire the phenotype typical of mature cells and release normal amounts of cytokines in response to LPS, they fail to accumulate pCREB, Bcl-2, and Bcl-xL and therefore do not survive. The transgenic expression of Bcl-2 in CaMKIV null mice results in full recovery of DC survival in response to LPS. These results reveal a novel link between TLR4 and a calcium-dependent signaling cascade comprising CaMKIV-CREB-Bcl-2 that is essential for DC survival

Fusion complexes and CD4-independent Env for the induction of broad spectrum neutralizing antibodies against HIV-1

Broad spectrum neutralizing antibodies against HIV-1 are essential for the development of a humoral anti-AIDS vaccine. We used fusion complexes and CD4-independent gp120 as new immunogens to induce neutralizing antibodies blocking the infectivity of different primary isolates of HIV-1

Association Analysis Between HIV-1 Env and HLA-C Using Bimolecular Fluorescence Complementation

Introduction: HLA-C is selectively incorporated into HIV-1 envelope. We reported that viruses produced from HLA-C silenced cells are less infectious and demonstrated a specific association between HLA-C and gp120. Our purpose is to unravel the HLA-C/gp120 association to reveal new targets for the development of neutralizing antibodies and new anti-HIV compounds. Methods: Fluorescent-tagged HLA-C and gp120 were prepared and used for bimolecular fluorescence complementation (BiFC) analysis to study their association into cellular compartments. To identify gp120 domains involved in the association with HLA-C, different gp120 deletion mutants are being constructed and tested. Results: A BiFC complementation signal between HLA-C and gp120 is detectable in the endoplasmic reticulum (ER), Golgi apparatus, late endosomes and on the cell membrane. Confocal microscopy does not show a co-localization signal between gp120 and [beta]2-microglobulin ([beta]2m). Discussion: The co-localization analysis shows that HLA-C, most likely as an open conformer, interacts with gp120 early in the ER and that they remain associated up to the cell membrane, travelling together trough the Golgi apparatus and late endosomes. No gp120 and [beta]2m co-localization is evident, suggesting a competition between them for HLA-C binding

HLA-C presence increases human immunodeficiency type 1 (HIV-1) infectivity by interacting with the envelope glycoprotein gp120/41

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P-D9\u2003Human Thioesterase 8 and HIV-1 Nef interaction

HIV-1 Nef interacts with several cellular partners, among which the human thioesterase 8 (Acot8). This interaction might modulate lipid composition in membrane rafts during HIV-1 infection. The regions involved in the interaction were experimentally characterized on Nef but not much is known for Acot8. Indeed, the lack of structural information for Acot8 hampers a deep characterization of the putative interaction regions. This calls for in silico calculations and analysis. In this work we modelled the Acot8 structure in order to identify the aminoacids putatively involved in the interaction with Nef. The predictions were then validated by in vitro assays. Starting from modelling, protein-protein docking combined with molecular dynamics simulations on the Nef-Acot8 complex, we characterized the putative contact regions between the 2 proteins. Our calculations allowed the identification of 2 putative regions on Acot8 that may interact with Nef. Moreover, they show a high charge complementarity with Nef surface

Kinase independent inhibition of NFκB transcriptional activity by GRK5 through IκBα stabilization.

Members of the G protein receptor kinase (GRK) family that regulates receptor desensitization and members of the nuclear transcription factors family NF[kappa]B have been recently and convincingly demonstrated to interact, although the effects on transcription and gene expression have not yet been described. Using overexpression, knockdown (small interfering RNA) and mutagenesis experiments, we demonstrate that GRK5 couples to and stabilizes the NF[kappa]B inhibitor I[kappa]B[alpha], and inhibits NF[kappa]B activity. Studies with minigenes suggest that the N-terminal Regulation of G protein Signaling (RGS) homology (RH) domain confers GRK5 such ability. GRK5-RH domain overexpression affects NF[kappa]B dependent phenotypes, such as apoptosis protection, cytokine production and inflammation and tissue regeneration. Our results reveal a novel, unexpected role of GRK5 in NF[kappa]B transcription activity regulation that represents a possible target for diagnostic and therapeutics

The G-protein-coupled receptor kinase 5 inhibits NFκB transcriptional activity by inducing nuclear accumulation of IκBα

G-protein-coupled receptor (GPCR) kinases, GRKs, are known as serine/threonine kinases that regulate GPCR signaling, but recent findings propose functions for these kinases besides receptor desensitization. Indeed, GRK5 can translocate to the nucleus by means of a nuclear localization sequence, suggesting that this kinase regulates transcription events in the nucleus. To evaluate the effect of GRK5–IκBα interaction on NFκB signaling, we induced the overexpression and the knockdown of GRK5 in cell cultures. GRK5 overexpression causes nuclear accumulation of IκBα, leading to the inhibition of NFκB transcriptional activity. Opposite results are achieved by GRK5 knockdown through siRNA. A physical interaction between GRK5 and IκBα, rather than phosphorylative events, appears as the underlying mechanism. We identify the regulator of gene protein signaling homology domain of GRK5 (RH) and the N-terminal domain of IκBα as the regions involved in such interaction. To confirm the biological relevance of this mechanism of regulation for NFκB, we evaluated the effects of GRK5-RH on NFκB-dependent phenotypes. In particular, GRK5-RH overexpression impairs apoptosis protection and cytokine production in vitro and inflammation and tissue regeneration in vivo. Our results reveal an unexpected role for GRK5 in the regulation of NFκB transcription activity. Placing these findings in perspective, this mechanism may represent a therapeutic target for all those conditions involving excessive NFκB activity