Molecular Determinants in tRNA D-arm Required for Inhibition of HIV-1 Gag Membrane Binding

Plasma-membrane-specific localization of Gag, an essential step in HIV-1 particle assembly, is regulated by the interaction of the Gag MA domain with PI(4,5)P₂ and tRNA-mediated inhibition of non-specific or premature membrane binding. Different tRNAs inhibit PI(4,5)P₂-independent membrane binding to varying degrees in vitro; however, the structural determinants for this difference remain unknown. Here we demonstrate that membrane binding of full-length Gag synthesized in vitro using reticulocyte lysates is inhibited when RNAs that contain the anticodon arm of tRNA^Pro, but not that of tRNA^Lys3, are added exogenously. In contrast, in the context of a liposome binding assay in which the effects of tRNAs on purified MA were tested, full-length tRNA^Lys3 showed greater inhibition of MA membrane binding than full-length tRNA^Pro. While transplantation of the D loop sequence of tRNA^Lys3 into tRNA^Pro resulted in a modest increase in the inhibitory effect relative to WT tRNA^Pro, replacing the entire D arm sequence with that of tRNA^Lys3 was necessary to confer the full inhibitory effects upon tRNA^Pro. Together, these results demonstrate that the D arm of tRNA^Lys3 is a major determinant of strong inhibition of MA membrane binding and that this inhibitory effect requires not only the D loop, which was recently reported to contact the MA highly basic region, but the loop sequence in the context of the D arm structure.     

Asymmetric structure and domain binding interfaces of human tyrosyl‐tRNA synthetase studied by molecular dynamics simulations

Human tyrosyl‐tRNA synthetase (HsTyrRS) is composed of two structural modules: N‐terminal catalytic core and an EMAP II‐like C‐terminal domain. The structures of these modules are known, but no crystal structure of the full‐length HsTyrRS is currently available. An all‐atom model of the full‐length HsTyrRS was developed in this work. The structure, dynamics, and domain binding interfaces of HsTyrRS were investigated by extensive molecular dynamics (MD) simulations. Our data suggest that HsTyrRS in solution consists of a number of compact asymmetric conformations, which differ significantly by their rigidity, internal mobility, orientation of C‐terminal modules, and the strength of interdomain binding. Interfaces of domain binding obtained in MD simulations are in perfect agreement with our previous coarse‐grained hierarchical rotations technique simulations. Formation of the hydrogen bonds between R93 residue of the ELR cytokine motif and the residues A340 and E479 in the C‐module was observed. This observation supports the idea that the lack of cytokine activity in the full‐length HsTyrRS is explained by interactions between N‐modules and C‐modules, which block the ELR motif. Copyright © 2013 John Wiley & Sons, Ltd.     

Regulation of the human prostacyclin receptor gene by the cholesterol-responsive SREBP1

Prostacyclin and its prostacyclin receptor, the I Prostanoid (IP), play essential roles in regulating hemostasis and vascular tone and have been implicated in a range cardio-protective effects but through largely unknown mechanisms. In this study, the influence of cholesterol on human IP [(h)IP] gene expression was investigated in cultured vascular endothelial and platelet-progenitor megakaryocytic cells. Cholesterol depletion increased human prostacyclin receptor (hIP) mRNA, hIP promoter-directed reporter gene expression, and hIP-induced cAMP generation in all cell types. Furthermore, the constitutively active sterol-response element binding protein (SREBP)1a, but not SREBP2, increased hIP mRNA and promoter-directed gene expression, and deletional and mutational analysis uncovered an evolutionary conserved sterol-response element (SRE), adjacent to a known functional Sp1 element, within the core hIP promoter. Moreover, chromatin immunoprecipitation assays confirmed direct cholesterol-regulated binding of SREBP1a to this hIP promoter region in vivo, and immunofluorescence microscopy corroborated that cholesterol depletion significantly increases hIP expression levels. In conclusion, the hIP gene is directly regulated by cholesterol depletion, which occurs through binding of SREBP1a to a functional SRE within its core promoter. Mechanistically, these data establish that cholesterol can regulate hIP expression, which may, at least in part, account for the combined cardio-protective actions of low serum cholesterol through its regulation of IP expression within the human vasculature.     

Molecular mimicry of human endothelial cell antigen by autoantibodies to nonstructural protein 1 of dengue virus

The pathogenesis of dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS), both serious complications of dengue virus (DV) infection, remains unclear. In this study, we found that anti-DV NS1 (nonstructural protein 1) polyclonal antibodies cross-reacted with human umbilical vein endothelial cells (HUVECs). We further identified a complex-specific mAb, DB16-1, which could recognize DV NS1 and cross-react with HUVECs and human blood vessels. The target protein of DB16-1 was further purified by immunoaffinity chromatography. LC-MS/MS analysis and co-immunoprecipitation revealed that the target protein of DB16-1 was human LYRIC (lysine-rich CEACAM1 co-isolated). Our newly generated anti-LYRIC mAbs bound to HUVECs in a pattern similar to that of DB16-1. The B-cell epitope of DB16-1 displayed a consensus motif, Lys-X-Trp-Gly (KXWG), which corresponded to amino acid residues 116-119 of DV NS1 and mimicked amino acid residues 334-337 in LYRIC. Moreover, the binding activity of DB16-1 in NS1 of DV-2 and in LYRIC disappeared after the KXWG epitope was deleted in each. In conclusion, DB16-1 targeted the same epitope in DV NS1 and LYRIC protein on human endothelial cells, suggesting that it might play a role in the pathogenesis of DHF/DSS. Future studies on the role of the anti-NS1 antibody in causing vascular permeability will undoubtedly be performed on sera collected from individuals before, during, and after the endothelial cell malfunction phase of a dengue illness.