Schematic of the Y2H screen and coimmunoprecipitation of selected preys show interaction with Nef.

<p>A. Schematic presentation of the split-ubiquitin Y2H screen with membrane-anchored Nef as bait. Wild type Nef myristoylation is replaced by the Ost4p transmembrane anchor and amino acids 39–76 of yeast ubiquitin (Cub) linked to the LexA-VP16 transcription factor carboxy-terminally to Nef. A human adult brain cDNA library (Dualsystems Biotech AG) cloned in pPR3-N expressing the cDNAs as fusions carboxy-terminally of amino acids 1–38 of yeast ubiquitin (Nub) and an HA-tag. Upon binding of the bait and prey both parts of the ubiquitin come together and are cleaved by the protease to activate reporter genes. If no interaction with the Nef bait protein is possible, the ubiquitin subunits stay apart and no reporter genes in the nucleus are turned on. B. Coimmunoprecipitation (CoIP) of Nef and preys. Yeast cell lysate proteins from different transfected cultures as well as a non-transfected control (NMY51) as indicated at the top were immunoprecipitated (IP) either with anti-HA or anti-LexA antibodies. For negative controls, cells were alternatively transfected with a bait expression vector coding for the SV40 large T antigen (largeT) instead of Nef. The resulting immunoprecipitates were electrophoretically separated, blotted (WB) on a PVDF-membrane as indicated at the right and detected with anti-Myc or anti-HA antibodies. The approximate molecular weights of the proteins are shown. Two additional experiments gave similar results.</p

Analysis of the bait dependency test.

<p>Bait dependency test for growth on minimal media (His^−/Ade^−/lacZ⁻) of the positive preys coexpressed with pDHB1-Nef or pDHB1-LargeT (negative control) and summarized CoIP results (last column). Only hits that passed the Nef dependency test are listed. The table is sorted by the number of hits, which is given beside the protein name. Putative Nef-interacting proteins prone to be false-positive interactors based on the components of the Y2H system itself (according to the Dualsystems support page) are written in italic.</p

Sequence analysis of Nef binding site and <i>in vitro</i> binding studies of GPM6B and Nef.

<p>A. Amino acid sequence of the Nef binding motif of CD4. B. Clustal W <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051578#pone.0051578-Larkin1" target="_blank">[70]</a> based alignment of the cytoplasmic loops of GPM6B, GPM6A and PLP1. Note, for GPM6B, GPM6A and PLP-DM20/PLP1 isoform 2, the sequences of the complete cytoplasmic loop regions are shown. Additionally, positions of the indicated proteolipid proteins with homologies to a sequence region in the cytoplasmic tail of T-cell surface glycoprotein CD4 (shown at the top) that includes the core Nef binding motif of CD4 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051578#pone.0051578-Preusser1" target="_blank">[20]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051578#pone.0051578-Preusser2" target="_blank">[21]</a> are highlighted. Positions identical to the respective CD4 residue are shaded black, those with high similarity are shaded grey and those with lower similarity are boxed. The indicated residue numbers show the beginning and the end of the amino-terminally fluoresceinylated GPM6B and PLP1 peptides used for the Nef binding studies shown in D. Numbering of GPM6B is based on UniProtKB entry Q13491-1 throughout this figure. C. Sequence of the cytoplasmatic portion of CD320/TCblR, homologies to CD4 indicated as described above. D. Fluorescence titration of 0.5 µM of fluoresceinyl-labeled peptides, GPM6B_112–127, GPM6B_AA, or PLP_193–108 with recombinant HIV-1SF2 Nef_2–210 protein (prepared as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051578#pone.0051578-Hoffmann1" target="_blank">[68]</a>). The fluorescence signals are shown as a function of the Nef_2–210 protein concentration. Values result from the fluorescence of the peptides in the presence of the indicated concentration of Nef_2–210 in comparison with a buffer control titration. Assuming a simple bimolar interaction between the peptide and Nef_2–210, the data were described by a model based solely on the law of mass action which accounts for ligand depletion <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051578#pone.0051578-Tran1" target="_blank">[71]</a>. Nonlinear curve fitting of the model to the fluorescence data (lines) yielded dissociation constants of 0.64±0.06 µM for GPM6B_112–127 and of 0.71±0.03 µM for PLP_193–108.</p

Subcellular localization of the identified hits within a model cell.

<p>Overview of the subcellular localization of the interaction partners of Nef identified via the split-ubiquitin based Y2H system based on the data given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051578#pone.0051578.s001" target="_blank">Table S1</a>. Marked in magenta are the proteins studied in more detail.</p

Analysis of subcellular localization of BAP31, CD320/TCblR, CLDN10 and GPM6B with and without Nef.

<p>A. Confocal microscopy analysis of Cos-7 cells coexpressing GFP fusions of BAP31, CD320/TCblR, CLDN10 or GPM6B with Nef-DsRed. Cos-7 cells were transiently cotransfected with pNef-DsRed and pGFP-BAP31, pGFP-CD320/TCblR, pGFP-CLDN10 or pGFP-GPM6B and fixed 24 h posttransfection. Single images from the red (Nef-DsRed) and green (GFP-prey) channels were overlaid in the merged image. Yellow regions represent colocalization, details are given in the text. Arrows point out BAP31 accumulations and distinct areas of CLDN10 and Nef overlay. Scale bar: 10 µm. B. Negative controls for the colocalization images in Figure A. Confocal microscopy analysis of Cos-7 cells coexpressing GFP fusions of BAP31, CD320/TCblR, CLDN10 or GPM6B with DsRed. Cos-7 cells were transiently cotransfected with pDsRed and pGFP-BAP31, pGFP-CD320/TCblR, pGFP-CLDN10 or pGFP-GPM6B and fixed 24 h posttransfection. Single images from the red (DsRed) and green (GFP-prey) channels were overlaid in the merged image. The respective scatter grams, as well as the images and scatter grams from cotransfections with the pDsRed control vector are given.</p

Implications of ventricular arrhythmia "bursts" with normal epicardial flow, myocardial blush, and ST-segment recovery in anterior ST-elevation myocardial infarction reperfusion: A biosignature of direct myocellular injury "downstream of downstream"

Aims: Establishing epicardial flow with percutaneous coronary intervention (PCI) for ST-segment elevation myocardial infarction (STEMI) is necessary but not sufficient to ensure nutritive myocardial reperfusion. We evaluated whether adding myocardial blush grade (MBG) and quantitative reperfusion ventricular arrhythmia "bursts" (VABs) surrogates provide a more informative biosignature of optimal reperfusion in patients with Thrombolysis in Myocardial Infarction (TIMI) 3 flow and ST-segment recovery (STR). Methods and results: Anterior STEMI patients with final TIMI 3 flow had protocol-blinded analyses of simultaneous MBG, continuous 12-lead electrocardiogram (ECG) STR, Holter VABs, and day 5-14 SPECT imaging infarct size (IS) assessments. Over 20 million cardiac cycles from >4500 h of continuous ECG monitoring in subjects with STR were obtained. IS and clinical outcomes were examined in patients stratified by MBG and VABs. VABs occurred in 51% (79/154) of subjects. Microcirculation (MBG 2/3) was restored in 75% (115/154) of subjects, of whom 53% (61/115) had VABs. No VABs were observed in subjects without microvascular flow (MBG of 0). Of 115 patients with TIMI 3 flow, STR, and MBG 2/3, those with VABs had significantly larger IS (median: 23.0% vs 6.0%, p = 0.001). Multivariable analysis identified reperfusion VABs as a factor significantly associated with larger IS (p = 0.015). Conclusions: Despite restoration of normal epicardial flow, open microcirculation, and STR, concomitant VABs are associated with larger myocardial IS, possibly reflecting myocellular injury in reperfusion settings. Combining angiographic and ECG parameters of epicardial, microvascular, and cellular response to STEMI intervention provides a more predictive "biosignature" of optimal reperfusion than do single surrogate markers

Brain transcriptome-wide screen for HIV-1 Nef protein interaction partners reveals various membrane-associated proteins.

HIV-1 Nef protein contributes essentially to the pathology of AIDS by a variety of protein-protein-interactions within the host cell. The versatile functionality of Nef is partially attributed to different conformational states and posttranslational modifications, such as myristoylation. Up to now, many interaction partners of Nef have been identified using classical yeast two-hybrid screens. Such screens rely on transcriptional activation of reporter genes in the nucleus to detect interactions. Thus, the identification of Nef interaction partners that are integral membrane proteins, membrane-associated proteins or other proteins that do not translocate into the nucleus is hampered. In the present study, a split-ubiquitin based yeast two-hybrid screen was used to identify novel membrane-localized interaction partners of Nef. More than 80% of the hereby identified interaction partners of Nef are transmembrane proteins. The identified hits are GPM6B, GPM6A, BAP31, TSPAN7, CYB5B, CD320/TCblR, VSIG4, PMEPA1, OCIAD1, ITGB1, CHN1, PH4, CLDN10, HSPA9, APR-3, PEBP1 and B3GNT, which are involved in diverse cellular processes like signaling, apoptosis, neurogenesis, cell adhesion and protein trafficking or quality control. For a subfraction of the hereby identified proteins we present data supporting their direct interaction with HIV-1 Nef. We discuss the results with respect to many phenotypes observed in HIV infected cells and patients. The identified Nef interaction partners may help to further elucidate the molecular basis of HIV-related diseases