Deacetylation of histone proteins at the HIV type 1 (HIV-1) long terminal repeat (LTR) by histone deactylases (HDACs) can promote transcriptional repression and virus latency. As such, HDAC inhibitors (HDACI) could be used to deplete reservoirs of persistent, quiescent HIV-1 proviral infection. However, the development of HDACI to purge latent HIV-1 requires knowledge of the HDAC isoforms contributing to viral latency and the development of inhibitors specific to these isoforms. In this study, we identify the HDACs responsible for HIV-1 latency in Jurkat J89GFP cells using a chemical approach that correlates HDACI isoform specificity with their ability to reactivate latent HIV-1 expression. We demonstrate that potent inhibition or knockdown of HDAC1, an HDAC isoform reported to drive HIV-1 into latency, was not sufficient to de-repress the viral LTR. Instead, we found that inhibition of HDAC3 was necessary to activate latent HIV-1. Consistent with this finding, we identified HDAC3 at the HIV-1 LTR by chromatin immunoprecipitation. Interestingly, we show that valproic acid is a weak inhibitor of HDAC3 (IC 50 ؍ 5.5 mM) relative to HDAC1 (IC 50 ؍ 170 M). Because the total therapeutic concentration of valproic acid ranges from 275 to 700 M in adults, these data may explain why this inhibitor has no effect on the decay of latent HIV reservoirs in patients. Taken together, our study suggests an important role for HDAC3 in HIV-1 latency and, importantly, describes a chemical approach that can readily be used to identify the HDAC isoforms that contribute to HIV-1 latency in other cell types.
Approaches that allow bioorthogonal and, in turn, site-specific chemical modification of proteins present considerable opportunities for modulating protein activity and stability. However, the development of such approaches that enable site-selective modification of proteins at multiple positions, including internal sites within a protein, has remained elusive. To overcome this void, we have developed an enzymatic approach for multisite clickable modification based on the incorporation of azide moieties in proteins using lipoic acid ligase (LplA). The ligation of azide moieties to the model protein, green fluorescent protein (GFP), at the N-terminus and two internal sites using lipoic acid ligase was shown to proceed efficiently with near-complete conversion. Modification of the ligated azide groups with poly(ethylene glycol) (PEG), α-d-mannopyranoside, and palmitic acid resulted in highly homogeneous populations of protein-polymer, protein-sugar, and protein-fatty acid conjugates. The homogeneity of the conjugates was confirmed by mass spectrometry (MALDI-TOF) and SDS-PAGE electrophoresis. In the case of PEG attachment, which involved the use of strain-promoted azide-alkyne click chemistry, the conjugation reaction resulted in highly homogeneous PEG-GFP conjugates in less than 30 min. As further demonstration of the utility of this approach, ligated GFP was also covalently immobilized on alkyne-terminated self-assembled monolayers. These results underscore the potential of this approach for, among other applications, site-specific multipoint protein PEGylation, glycosylation, fatty acid modification, and protein immobilization.
We present the first crystallographic insight into the interactions of an ionic liquid (IL) with an enzyme, which has widespread implications for stabilizing enzymes in IL media for biocatalysis. Structures of Bacillus subtilis lipase A (lipA) and an IL stable varaint (QM-lipA) were obtained in the presence of increasing concentrations of 1-butyl-3-methylimidazolium chloride ([BMIM][Cl]). These studies revealed that the [BMIM] cation interacts to surface residues via hydrophobic and cation-π interactions. Of specific interest was the disruption of internal stacking interactions of aromatic side chains by [BMIM], which provide structural evidence for the mechanism of enzyme denaturation by ILs. The interaction with [BMIM] and Cl ions to lipA was reduced by the stabilizing mutations Y49E and G158E in QM-lipA. Ultimately, these findings present the molecular basis for stabilizing enzymes from IL-induced inactivation as well as the selection of ILs that are less denaturing.
The Recombination Directionality Factor, Xis, is a DNA bending protein that determines the outcome of integrase-mediated site-specific recombination by redesign of higher-order protein-DNA architectures. Although the attachment site DNA of Mycobacteriophage Pukovnik is likely to contain four sites for Xis binding, Xis crystals contain five subunits in the asymmetric unit, four of which align into a Xis filament, and a fifth that is generated by an unusual domain swap. Extensive intersubunit contacts stabilize a bent filament-like arrangement with Xis monomers aligned head-to-tail. The structure implies a DNA bend of ~120°, which is in agreement with DNA bending measured in vitro. Formation of attR-containing intasomes requires only Int and Xis, distinguishing Pukovnik from lambda. Therefore, we conclude that in Pukovnik, Xis-induced DNA bending is sufficient to promote intramolecular Int-mediated bridges during intasome formation.
Due to the prevalence of biofilm-related infections, which are mediated by bacterial quorum sensing, there is a critical need for materials and coatings that resist biofilm formation. We have developed novel anti-biofilm coatings that disrupt quorum sensing in surface-associated bacteria via the immobilization of acylase in polyurethane films. Specifically, acylase from Aspergillus melleus was covalently immobilized in biomedical grade polyurethane coatings via multipoint covalent immobilization. Coatings containing acylase were enzymatically active and catalyzed the hydrolysis of the quorum sensing (QS) molecules N-butyryl-L-homoserine lactone (C4-LHL), N-hexanoyl-L-homoserine lactone (C6-LHL), and N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-LHL). In biofilm inhibition assays, immobilization of acylase led to an approximately 60% reduction in biofilm formation by Pseudomonas aeruginosa ATCC 10145 and PAO1. Inhibition of biofilm formation was consistent with a reduction in the secretion of pyocyanin, indicating the disruption of quorum sensing as the mechanism of the coating activity. Scanning electron microscopy further showed that acylase-containing coatings contained far fewer bacterial cells than control coatings that lacked acylase. Moreover, acylase-containing coatings retained 90% activity when stored dry at 37°C for 7 days and were more stable than the free enzyme in physiological conditions, including artificial urine. Ultimately, such coatings hold considerable promise for the clinical management of catheter-related infections as well as the prevention of infections in orthopedic applications (i.e., on hip and knee prostheses) and on contact lenses. Biotechnol. Bioeng. 2016;113: 2535-2543. © 2016 Wiley Periodicals, Inc.
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