Epigenetic silencing of HIV-1 by the histone H3 lysine 27 methyltransferase enhancer of Zeste 2

Latent HIV proviruses are silenced as the result of deacetylation and methylation of histones located at the viral long terminal repeat (LTR). Inhibition of histone deacetylases (HDACs) leads to the reemergence of HIV-1 from latency, but the contribution of histone lysine methyltransferases (HKMTs) to maintaining HIV latency remains uncertain. Chromatin immunoprecipitation experiments using latently infected Jurkat T-cell lines demonstrated that the HKMT enhancer of Zeste 2 (EZH2) was present at high levels at the LTR of silenced HIV proviruses and was rapidly displaced following proviral reactivation. Knockdown of EZH2, a key component of the Polycomb repressive complex 2 (PRC2) silencing machinery, and the enzyme which is required for trimethyl histone lysine 27 (H3K27me3) synthesis induced up to 40% of the latent HIV proviruses. In contrast, there was less than 5% induction of latent proviruses following knockdown of SUV39H1, which is required for H3K9me3 synthesis. Knockdown of EZH2 also sensitized latent proviruses to external stimuli, such as T-cell receptor stimulation, and slowed the reversion of reactivated proviruses to latency. Similarly, cell populations that responded poorly to external stimuli carried HIV proviruses that were enriched in H3K27me3 and relatively depleted in H3K9me3. Treating latently infected cells with the HKMT inhibitor 3-deazaneplanocin A, which targets EZH2, led to the reactivation of silenced proviruses, whereas chaetocin and BIX01294 showed only minimal reactivation activities. These findings suggest that PRC2-mediated silencing is an important feature of HIV latency and that inhibitors of histone methylation may play a useful role in induction strategies designed to eradicate latent HIV pools.     

Glucose uptake is limiting in T cell activation and requires CD28-mediated Akt-dependent and independent pathways

T cell activation potently stimulates cellular metabolism to support the elevated energetic and biosynthetic demands of growth, proliferation, and effector function. We show that glucose uptake is limiting in T cell activation and that CD28 costimulation is required to allow maximal glucose uptake following TCR stimulation by up-regulating expression and promoting the cell surface trafficking of the glucose transporter Glut1. Regulation of T cell glucose uptake and Glut1 was critical, as low glucose prevented appropriate T cell responses. Additionally, transgenic expression of Glut1 augmented T cell activation, and led to accumulation of readily activated memory-phenotype T cells with signs of autoimmunity in aged mice. To further examine the regulation of glucose uptake, we analyzed CD28 activation of Akt, which appeared necessary for maximal glucose uptake of stimulated cells and which we have shown can promote Glut1 cell surface trafficking. Consistent with a role for Akt in Glut1 trafficking, transgenic expression of constitutively active myristoylated Akt increased glucose uptake of resting T cells, but did not alter Glut1 protein levels. Therefore, CD28 appeared to promote Akt-independent up-regulation of Glut1 and Akt-dependent Glut1 cell surface trafficking. In support of this model, coexpression of Glut1 and myristoylated Akt transgenes resulted in a synergistic increase in glucose uptake and accumulation of activated T cells in vivo that were largely independent of CD28. Induction of Glut1 protein and Akt regulation of Glut1 trafficking are therefore separable functions of CD28 costimulation that cooperate to promote glucose metabolism for T cell activation and proliferation.     

A Catalytically Essential Motif in External Loop 5 of the Bacterial Oligosaccharyltransferase PglB

Asparagine-linked glycosylation is a post-translational protein modification that is conserved in all domains of life. The initial transfer of a lipid-linked oligosaccharide (LLO) onto acceptor asparagines is catalyzed by the integral membrane protein oligosaccharyltransferase (OST). The previously reported structure of a single-subunit OST enzyme, the Campylobacter lari protein PglB, revealed a partially disordered external loop (EL5), whose role in catalysis was unclear. We identified a new and functionally important sequence motif in EL5 containing a conserved tyrosine residue (Tyr²⁹³) whose aromatic side chain is essential for catalysis. A synthetic peptide containing the conserved motif can partially but specifically rescue in vitro activity of mutated PglB lacking Tyr²⁹³. Using site-directed disulfide cross-linking, we show that disengagement of the structurally ordered part of EL5 is an essential step of the glycosylation reaction, probably by allowing sequon binding or glyco-product release. Our findings define two distinct mechanistic roles of EL5 in OST-catalyzed glycosylation. These functions, exerted by the two halves of EL5, are independent, because the loop can be cleaved by specific proteolysis with only slight reduction in activity.     

Protease profiling using a fluorescent domino peptide cocktail

Five hexapeptides were prepared containing, in a domino-type arrangement, all 25 possible dipeptides between (1) aromatic, (2) hydrophobic, (3) positively charged, (4) negatively charged, and (5) small and polar amino acids. The peptides were fluorescence labeled at the N-terminus with a (7-coumaryl)oxyacetyl group, allowing the selective detection of N-terminal cleavage products. The five peptides were used as a cocktail reagent in an HPLC analysis. The cocktail produced specific cleavage patterns, or fingerprints, for a variety of proteases. This domino peptide cocktail can be used as a general reagent for protease identification and functional profiling.     

Enhanced sensitivity detection of protein immobilization by fluorescent interference on oxidized silicon

We present a silicon chip-based approach for the enhanced sensitivity detection of surface-immobilized fluorescent molecules. Green fluorescent protein (GFP) is bound to the silicon substrate by a disuccinimidyl terephtalate-aminosilane immobilization procedure. The immobilized organic layers are characterized by surface analysis techniques, like ellipsometry, atomic force microscopy (AFM) and X-ray induced photoelectron spectroscopy. We obtain a 20-fold enhancement of the fluorescent signal, using constructive interference effects in a fused silica dielectric layer, deposited before immobilization onto the silicon. Our method opens perspectives to increase by an order of magnitude the fluorescent response of surface immobilized DNA- or protein-based layers for a variety of biosensor applications.