Statins Disrupt CCR5 and RANTES Expression Levels in CD4(+) T Lymphocytes In Vitro and Preferentially Decrease Infection of R5 Versus X4 HIV-1

BACKGROUND: Statins have previously been shown to reduce the in vitro infection of human immunodeficiency virus type 1 (HIV-1) through modulation of Rho GTPase activity and lipid raft formation at the cell surface, as well as by disrupting LFA-1 incorporation into viral particles. PRINCIPLE FINDINGS: Here we demonstrate that treatment of an enriched CD4(+) lymphocyte population with lovastatin (Lov), mevastatin (Mev) and simvastatin (activated and non-activated, Sim(A) and Sim(N), respectively) can reduce the cell surface expression of the CC-chemokine receptor CCR5 (P<0.01 for Sim(A) and Lov). The lowered CCR5 expression was associated with down-regulation of CCR5 mRNA expression. The CC-chemokine RANTES protein and mRNA expression levels were slightly increased in CD4(+) enriched lymphocytes treated with statins. Both R5 and X4 HIV-1 were reduced for their infection of statin-treated cells; however, in cultures where statins were removed and where a decrease in CCR5 expression was observed, there was a preferential inhibition of infection with an R5 versus X4 virus. CONCLUSIONS: The results indicate that the modulation of CC-chemokine receptor (CCR5) and CC-chemokine (RANTES) expression levels should be considered as contributing to the anti-viral effects of statins, preferentially inhibiting R5 viruses. This observation, in combination with the immunomodulatory activity exerted by statins, suggests they may possess more potent anti-HIV-1 activity when applied during the early stages of infection or in lowering viral transmission. Alternatively, statin treatment could be considered as a way to modulate immune induction such as during vaccination protocols

SUMOylation of DEC1 Protein Regulates Its Transcriptional Activity and Enhances Its Stability

Differentiated embryo-chondrocyte expressed gene 1 (DEC1, also known as sharp2, stra13, or BHLHB2) is a mammalian basic helix-loop-helix protein that is involved in many aspects of gene regulation through acting as a transcription factor. Changes in DEC1 expression levels have been implicated in the development of cancers. Using COS-7 cell, we showed that DEC1 can be modified by the small ubiquitin-like modifiers, SUMO1, 2 and 3. Two major SUMOylation sites (K159 and K279) were identified in the C-terminal domain of DEC1. Substitution of either K159 or K279 with arginine reduced DEC1 SUMOylation, but substitution of both K159 and K279 abolished SUMOylation, and more protein appeared to be retained in the cytoplasm compared to wild-type DEC1. The expression of DEC1 was up-regulated after serum starvation as previously reported, but at the same time, serum starvation also led to more SUMOylation of DEC1. In MCF-7 cells SUMOylation also stabilized DEC1 through inhibiting its ubiquitination. Moreover, SUMOylation of DEC1 promoted its repression of CLOCK/BMAL1-mediated transcriptional activity through recruitment of histone deacetylase1. These findings suggested that posttranslational modification of DEC1 in the form of SUMOylation may serve as a key factor that regulates the function of DEC1 in vivo

Delivery setup compatible for in vitro impedance measurements and exposures to electromagnetic fields: nanosecond pulse electric fields and radiofrequency signals

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Real-time study of the effects of radiofrequency fields at the cellular and molecular levels: the results of the Geronimo project

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Herpes simplex virus enhances chemokine function through modulation of receptor trafficking and oligomerization

Glycoprotein G (gG) from herpes simplex virus 1 and 2 (HSV-1 and HSV-2, important human neurotropic pathogens) is the first viral chemokine-binding protein found to potentiate chemokine function. Here we show that gG attaches to cell surface glycosaminoglycans and induces lipid raft clustering, increasing the incorporation of CXCR4 receptors into these microdomains. gG induces conformational rearrangements in CXCR4 homodimers and changes their intracellular partners, leading to sustained, functional chemokine/receptor complexes at the surface. This results in increased chemotaxis dependent on the cholesterol content of the plasma membrane and receptor association to Src-kinases and phosphatidylinositol-3-kinase signalling pathways, but independent of clathrin-mediated endocytosis. Furthermore, using electron microscopy, we show that such enhanced functionality is associated with the accumulation of low-order CXCR4 nanoclusters. Our results provide insights into basic mechanisms of chemokine receptor function and into a viral strategy of immune modulation.This work was funded by the Spanish Ministry of Science and Innovation (SAF2009-07857 and SAF2012-38957) and Red Española de Esclerosis Múltiple (Instituto de Salud Carlos III, RD07/0060/0014). N.M.-M. was funded by a studentship from Consejo Superior de Investigaciones Científicas and by Fundación Severo Ochoa. A.V.-B. was supported by a postdoctoral fellowship from Consejo Superior de Investigaciones Científicas.Peer Reviewe

BRET analysis of GPCR oligomerization: newer does not mean better

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A rigorous experimental framework for detecting protein oligomerization using bioluminescence resonance energy transfer

Bioluminescence resonance energy transfer (BRET), which relies on nonradiative energy transfer between luciferase-coupled donors and GFP-coupled acceptors, is emerging as a useful tool for analyzing the quaternary structures of cell-surface molecules. Conventional BRET analyses are generally done at maximal expression levels and single acceptor/donor ratios. We show that under these conditions substantial energy transfer arises from random interactions within the membrane. The dependence of BRET efficiency on acceptor/donor ratio at fixed surface density, or expression level at a defined acceptor/donor ratio, can nevertheless be used to correctly distinguish between well-characterized monomeric and oligomeric proteins, including a very weak dimer. The pitfalls associated with the nonrigorous treatment of BRET data are illustrated for the case of G protein–coupled receptors (GPCRs) proposed to form homophilic and/or mixed oligomers on the basis of previous, conventional BRET experiments

Potent and Broad Anti-HIV-1 Activity Exhibited by a Glycosyl-Phosphatidylinositol-Anchored Peptide Derived from the CDR H3 of Broadly Neutralizing Antibody PG16▿†‡

PG9 and PG16 are two recently isolated quaternary-specific human monoclonal antibodies that neutralize 70 to 80% of circulating HIV-1 isolates. The crystal structure of PG16 shows that it contains an exceptionally long CDR H3 that forms a unique stable subdomain that towers above the antibody surface to confer fine specificity. To determine whether this unique architecture of CDR H3 itself is sufficient for epitope recognition and neutralization, we cloned CDR H3 subdomains derived from human monoclonal antibodies PG16, PG9, b12, E51, and AVF and genetically linked them to a glycosyl-phosphatidylinositol (GPI) attachment signal. Each fusion gene construct is expressed and targeted to lipid rafts of plasma membranes through a GPI anchor. Moreover, GPI-CDR H3(PG16, PG9, and E51), but not GPI-CDR H3(b12 and AVF), specifically neutralized multiple clades of HIV-1 isolates with a great degree of potency when expressed on the surface of transduced TZM-bl cells. Furthermore, GPI-anchored CDR H3(PG16), but not GPI-anchored CDR H3(AVF), specifically confers resistance to HIV-1 infection when expressed on the surface of transduced human CD4+ T cells. Finally, the CDR H3 mutations (Y100HF, D100IA, and G7) that were previously shown to compromise the neutralization activity of antibody PG16 also abolished the neutralization activity of GPI-CDR H3(PG16). Thus, we conclude that the CDR H3 subdomain of PG16 neutralizes HIV-1 when targeted to the lipid raft of the plasma membrane of HIV-1-susceptible cells and that GPI-CDR H3 can be an alternative approach for determining whether the CDR H3 of certain antibodies alone can exert epitope recognition and neutralization