Conformation and Lipid Interaction of the Fusion Peptide of the Paramyxovirus PIV5 in Anionic and Negative-Curvature Membranes from Solid-State NMR

Viral fusion proteins catalyze the merger of the virus envelope and the target cell membrane through multiple steps of protein conformational changes. The fusion peptide domain of these proteins is important for membrane fusion, but how it causes membrane curvature and dehydration is still poorly understood. We now use solid-state NMR spectroscopy to investigate the conformation, topology, and lipid and water interactions of the fusion peptide of the PIV5 virus F protein in three lipid membranes, POPC/POPG, DOPC/DOPG, and DOPE. These membranes allow us to investigate the effects of lipid chain disorder, membrane surface charge, and intrinsic negative curvature on the fusion peptide structure. Chemical shifts and spin diffusion data indicate that the PIV5 fusion peptide is inserted into all three membranes but adopts distinct conformations: it is fully α-helical in the POPC/POPG membrane, adopts a mixed strand/helix conformation in the DOPC/DOPG membrane, and is primarily a β-strand in the DOPE membrane. ³¹P NMR spectra show that the peptide retains the lamellar structure and hydration of the two anionic membranes. However, it dehydrates the DOPE membrane, destabilizes its inverted hexagonal phase, and creates an isotropic phase that is most likely a cubic phase. The ability of the β-strand conformation of the fusion peptide to generate negative Gaussian curvature and to dehydrate the membrane may be important for the formation of hemifusion intermediates in the membrane fusion pathway

Distinguishing Bicontinuous Lipid Cubic Phases from Isotropic Membrane Morphologies Using ³¹P Solid-State NMR Spectroscopy

Nonlamellar lipid membranes are frequently induced by proteins that fuse, bend, and cut membranes. Understanding the mechanism of action of these proteins requires the elucidation of the membrane morphologies that they induce. While hexagonal phases and lamellar phases are readily identified by their characteristic solid-state NMR line shapes, bicontinuous lipid cubic phases are more difficult to discern, since the static NMR spectra of cubic-phase lipids consist of an isotropic ³¹P or ²H peak, indistinguishable from the spectra of isotropic membrane morphologies such as micelles and small vesicles. To date, small-angle X-ray scattering is the only method to identify bicontinuous lipid cubic phases. To explore unique NMR signatures of lipid cubic phases, we first describe the orientation distribution of lipid molecules in cubic phases and simulate the static ³¹P chemical shift line shapes of oriented cubic-phase membranes in the limit of slow lateral diffusion. We then show that ³¹P <i>T</i>₂ relaxation times differ significantly between isotropic micelles and cubic-phase membranes: the latter exhibit 2 orders of magnitude shorter <i>T</i>₂ relaxation times. These differences are explained by the different time scales of lipid lateral diffusion on the cubic-phase surface versus the time scales of micelle tumbling. Using this relaxation NMR approach, we investigated a DOPE membrane containing the transmembrane domain (TMD) of a viral fusion protein. The static ³¹P spectrum of DOPE shows an isotropic peak, whose <i>T</i>₂ relaxation times correspond to that of a cubic phase. Thus, the viral fusion protein TMD induces negative Gaussian curvature, which is an intrinsic characteristic of cubic phases, to the DOPE membrane. This curvature induction has important implications to the mechanism of virus–cell fusion. This study establishes a simple NMR diagnostic probe of lipid cubic phases, which is expected to be useful for studying many protein-induced membrane remodeling phenomena in biology

p21 deletion increased the levels of PAR, which was reduced by 3-AB treatment.

<p>The levels of PAR and PARP-1 were determined by Western blot in lungs of C57BL/6J (<b>A</b>), and p21^-/- mice as well as WT littermates (<b>B</b>) in response to CS. PAR levels were increased in lungs of p21^-/- mice as compared to WT mice. 3-AB treatment reduced PAR level in lungs of both p21^-/- and WT mice. Intact PARP-1 level was not altered by either p21 deficiency or 3-AB treatment. Gel pictures shown are representative of at least 3 separate mice. Fold change is indicative of the alteration of PAR and PARP-1 compared with air-exposed and vehicle (Veh)-treated WT mice after normalizing to corresponding GAPDH or β-actin. Data are shown as mean ± SEM (n = 3-13 per group).^*<i>P</i><0.05 <i>vs</i> air group; ⁺<i>P</i><0.05, ⁺⁺<i>P</i><0.01, ⁺⁺⁺<i>P</i><0.001 <i>vs</i> Veh group; ^†††<i>P</i><0.001 <i>vs</i> WT mice.</p

3-AB increases CS-induced cellular senescence, but does not affect neutrophil influx in mouse lungs.

<p>3-AB augmented CS-induced increase in SA-β-gal activity in WT, but not p21^-/- mice (<b>A</b>). p21 deletion increased the expression of PCNA in lungs as compared to WT mice exposed to CS, which was reduced by 3-AB treatment (<b>B</b>). p21 deletion attenuated CS-induced neutrophil influx in BAL fluid, which was not affected by 3-AB (<b>C</b>). Original magnification, ×200. Gel pictures shown are representative of at least 3 separate mice. Relative density ratio is indicative of results after normalizing to corresponding GAPDH. Data are shown as mean ± SEM (n = 3-4 per group). ^*<i>P</i><0.05, ^**<i>P</i><0.01 <i>vs</i> air group; ⁺<i>P</i><0.05, ⁺⁺⁺<i>P</i><0.001 <i>vs</i> Veh group; ^††<i>P</i><0.01, ^†††<i>P</i><0.001 <i>vs</i> WT mice.</p

Solid-State Nuclear Magnetic Resonance Investigation of the Structural Topology and Lipid Interactions of a Viral Fusion Protein Chimera Containing the Fusion Peptide and Transmembrane Domain

The fusion peptide (FP) and transmembrane domain (TMD) of viral fusion proteins play important roles during virus–cell membrane fusion, by inducing membrane curvature and transient dehydration. The structure of the water-soluble ectodomain of viral fusion proteins has been extensively studied crystallographically, but the structures of the FP and TMD bound to phospholipid membranes are not well understood. We recently investigated the conformations and lipid interactions of the separate FP and TMD peptides of parainfluenza virus 5 (PIV5) fusion protein F using solid-state nuclear magnetic resonance. These studies provide structural information about the two domains when they are spatially well separated in the fusion process. To investigate how these two domains are structured relative to each other in the postfusion state, when the ectodomain forms a six-helix bundle that is thought to force the FP and TMD together in the membrane, we have now expressed and purified a chimera of the FP and TMD, connected by a Gly-Lys linker, and measured the chemical shifts and interdomain contacts of the protein in several lipid membranes. The FP–TMD chimera exhibits α-helical chemical shifts in all the membranes examined and does not cause strong curvature of lamellar membranes or membranes with negative spontaneous curvature. These properties differ qualitatively from those of the separate peptides, indicating that the FP and TMD interact with each other in the lipid membrane. However, no ¹³C–¹³C cross peaks are observed in two-dimensional correlation spectra, suggesting that the two helices are not tightly associated. These results suggest that the ectodomain six-helix bundle does not propagate into the membrane to the two hydrophobic termini. However, the loosely associated FP and TMD helices are found to generate significant negative Gaussian curvature to membranes that possess spontaneous positive curvature, consistent with the notion that the FP–TMD assembly may facilitate the transition of the membrane from hemifusion intermediates to the fusion pore

A schematic model showing the role of p21-PARP-1 in acute cigarette smoke (CS)-induced DNA damage and cellular senescence.

<p>CS exposure causes DNA damage including double-strand break (DSB). CS exposure also increases the level of p21, and p21 gene deletion augments PARP-1 activity and the levels of non-homologous end joining (NHEJ) proteins to repair damaged DNA. Consequently, CS-induced cellular senescence is attenuated by p21 deletion.</p

NMR Studies of the Interaction between Human Programmed Cell Death 5 and Human p53

Human programmed cell death 5 (PDCD5) is a protein playing a significant role in regulating both the apoptotic and paraptotic cell deaths. Resent findings show that PDCD5 is a positive regulator of Tip60 and also has a potential ability to interact with p53. Here we aim to experimentally characterize the nature of the interactions between PDCD5 and the p53 N-terminal domain (NTD) and to depict the binding mode between two proteins. The interprotein binding interfaces were determined by NMR experiments performed with PDCD5 and various fragments of p53 NTD. The binding affinity was investigated using the NMR titration experiments. Analysis revealed that the PDCD5 binding site on p53 is localized within residues 41–56 of p53 TAD2 subdomain while p53 binds preferentially to the positively charged surface region around the C-terminals of helices α3 and α5 and the N-terminal of helix α4 of PDCD5. The binding is mainly mediated through electrostatic interactions. The present data suggested a model for the interaction between PDCD5 and the p53 NTD

IL-13-induced YY1 expression is regulated by AKT pathway.

<p>MRC-5 cells were transfected with AKT and pCDNA1 control plasmids with electroporation. (A). At 24 h after transfection, the cells were starved for 24 h. IL-13 (30 ng/ml) was added to the cells for 12 h, The cells were lyzed, and the levels of YY1, AKT, p-AKT and β-actin were determined by Western blot. (B). YY1 and β-actin expression from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119039#pone.0119039.g004" target="_blank">Fig. 4A</a> were scanned and were conducted a densitometric analysis with Image J. YY1 expression was normalized to β-actin. Scanned data were analyzed by T-test. ** indicate p value <0.01. (C) After MRC-5 cells were starved for 24 h without serum, cells were pre-treated with or without wortmannin (10 μM) for 1 h, and then treated with or without IL-13 (30 ng/ml) for 12 h in serum-free DMEM. Whole cell extracts were subjected to Western blot analysis for determining the levels of YY1, α-SMA, p-AKT, collagen and β-actin. (D). the mRNA expression of YY1 was detected by quantitative PCR. GAPDH mRNA expression was used as an internal control. The data are presented with standard errors derived from at least three independent experiments, each performed in triplicate; n = 3 and <i>**p</i> < 0.01.</p

Influence of age on daily rhythms of plasma 5HT in air or CS-exposed mice.

<p>Adult mice (8–10 weeks old at start of CS exposure) were exposed to air or CS for 3 days (young), 10 days (young) and 6 months (middle aged). Plasma levels of 5HT were determined by ELISA. Gray shading indicates the dark phase (ZT12 and ZT18). Data from air- and CS-exposed mice are shown as mean ± SEM (n = 3–7 mice per group). Data were analyzed with non-linear regression (multi-order polynomial) analyses. *<i>P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001 significant compared to 3 days air- (ZT0–ZT18) or CS- (ZT6) exposed mice; ^{+ +}<i>P</i><0.01; ^{+ + +}<i>P</i><0.001 significant compared to 3 days air-exposed mice (ZT0–ZT18); ^{# #}<i>P</i><0.01; ^{# # #}<i>P</i><0.001 significant compared to 10 days air- (ZT0) or CS- (ZT6) exposed mice.</p

Daily rhythms of plasma CORT and 5HT in mice exposed to chronic (6 month) air or CS.

<p>Mice were exposed to chronic CS for 6 months and plasma samples were collected every 6-h interval (ZT0, ZT6, ZT12, ZT18 and ZT24) after 24 h of last CS exposure. Plasma levels of (A) CORT and (B) 5HT and were determined by ELISA. Gray shading indicates the dark phase (ZT12 to ZT24). Data from air- and CS-exposed mice are shown as mean ± SEM (n = 3–4 mice per group). Data were analyzed with non-linear regression (multi-order polynomial) analyses. *<i>P</i><0.05 significant compared to air-exposed mice at ZT0; *<i>P</i><0.05 significant compared to CS-exposed mice at ZT24; ***<i>P</i><0.001 significant compared to CS-exposed mice at ZT18.</p