While searching for alternative reading-frame peptides encoded by influenza A virus that are recognized by CD8+ T cells, we found an abundant immunogenic peptide encoded by the +1 reading frame of PB1. This peptide derives from a novel conserved 87-residue protein, PB1-F2, which has several unusual features compared with other influenza gene products in addition to its mode of translation. These include its absence from some animal (particularly swine) influenza virus isolates, variable expression in individual infected cells, rapid proteasome-dependent degradation and mitochondrial localization. Exposure of cells to a synthetic version of PB1-F2 induces apoptosis, and influenza viruses with targeted mutations that interfere with PB1-F2 expression induce less extensive apoptosis in human monocytic cells than those with intact PB1-F2. We propose that PB1-F2 functions to kill host immune cells responding to influenza virus infection.

We have identified a conserved region in the C-terminal domain of bromodomain-containing protein 4 (BRD4) that mediates its specific interaction with positive transcription elongation factor b (P-TEFb). This domain is highly conserved in testis-specific bromodomain protein (BRDT) and Drosophila fs (1) cyclin-dependent kinase 9

The complex of importin‐alpha and ‐beta is essential for nuclear protein import. It binds the import substrate in the cytosol, and the resulting trimeric complex moves through the nuclear pores, probably as a single entity. Importin‐alpha provides the nuclear localization signal binding site, importin‐beta the site of initial docking to the pore. Here we show that the conserved, basic N‐terminus of importin‐alpha is sufficient for importin‐beta binding and essential for protein import. The fusion product of this 41 amino acid domain to a heterologous protein if transported into the nucleus in the same way as full‐length importin‐alpha itself. Transport is dependent on importin‐beta but competed by importin‐alpha. As no additional part of importin‐alpha is needed for translocation, the movement which drives the import substrate complex into the nucleus appears to be generated between importin‐beta and structures of the nuclear pore. The domain that binds to importin‐beta appears to confer import only, but not re‐export out of the nucleus, suggesting that the return of importin‐alpha into the cytoplasm is not a simple reversal of its entry.

HIV-I Vpu catalyzes two independent functions, degradation of the virus receptor CD4 in the endoplasmic reticulum and enhancement of virus release from the cell surface. These activities are confined to distinct structural domains of Vpu, the cytoplasmic tail and the transmembrane (TM) anchor, respectively. It was recently reported that Vpu forms cationselective ion channels in lipid bilayers. Here we report that this property of Vpu is a characteristic of its TM anchor. Expression of full-length Vpu in Xenopus oocytes increases membrane conductance. The Vpu-induced conductance is selective to monovalent cations over anions, does not discriminate Na + over K + and shows marginal permeability to divalent cations. Notably, introduction of the scrambled TM sequence into fulllength Vpu abrogates its capacity to increase membrane conductance in oocytes and to promote virus release from infected cells. Reconstitution of synthetic Vpu fragments in lipid bilayers identified an ion channel activity for a sequence corresponding to the TM domain of Vpu. In contrast, a peptide with the same amino acid composition but with a scrambled sequence does not form ion channels. Our findings therefore suggest that the ability of Vpu to increase virus release from infected cells may be correlated with an ion channel activity of the TM domain, thereby providing a potential target for drug intervention based on the development of Vpu-specific channel blockers.

In higher eukaryotic cells, the p53 protein is degraded by the ubiquitin-26S proteasome system mediated by Mdm2 or the human papilloma virus E6 protein. Here we show that COP9 signalosome (CSN)-specific phosphorylation targets human p53 to ubiquitin-26S proteasome-dependent degradation. As visualized by electron microscopy, p53 binds with high affinity to the native CSN complex. p53 interacts via its N-terminus with CSN subunit 5/Jab1 as shown by far-western and pull-down assays. The CSN-specific phosphorylation sites were mapped to the core domain of p53 including Thr155. A phosphorylated peptide, Deltap53(145-164), specifically inhibits CSN-mediated phosphorylation and p53 degradation. Curcumin, a CSN kinase inhibitor, blocks E6-dependent p53 degradation in reticulocyte lysates. Mutation of Thr155 to valine is sufficient to stabilize p53 against E6-dependent degradation in reticulocyte lysates and to reduce binding to Mdm2. The p53T155V mutant accumulates in both HeLa and HL 60 cells and exhibits a mutant (PAb 240+) conformation. It induces the cyclin-dependent inhibitor p21. In HeLa and MCF-7 cells, inhibition of CSN kinase by curcumin or Deltap53(145-164) results in accumulation of endogenous p53.

The human immunodeficiency virus (HIV) Tat protein is acetylated by the transcriptional coactivator p300, a necessary step in Tat-mediated transactivation. We report here that Tat is deacetylated by human sirtuin 1 (SIRT1), a nicotinamide adenine dinucleotide-dependent class III protein deacetylase in vitro and in vivo. Tat and SIRT1 coimmunoprecipitate and synergistically activate the HIV promoter. Conversely, knockdown of SIRT1 via small interfering RNAs or treatment with a novel small molecule inhibitor of the SIRT1 deacetylase activity inhibit Tat-mediated transactivation of the HIV long terminal repeat. Tat transactivation is defective in SIRT1-null mouse embryonic fibroblasts and can be rescued by expression of SIRT1. These results support a model in which cycles of Tat acetylation and deacetylation regulate HIV transcription. SIRT1 recycles Tat to its unacetylated form and acts as a transcriptional coactivator during Tat transactivation.

There is an urgent need to coat the surfaces of medical devices, including implants, with antimicrobial agents to reduce the risk of infection. A peptide array technology was modified to permit the screening of short peptides for antimicrobial activity while tethered to a surface. Cellulose-amino-hydroxypropyl ether (CAPE) linker chemistry was used to synthesize, on a cellulose support, peptides that remained covalently bound during biological assays. Among 122 tested sequences, the best surface-tethered 9-, 12-, and 13-mer peptides were found to be highly antimicrobial against bacteria and fungi, as confirmed using alternative surface materials and coupling strategies as well as coupling through the C and N termini of the peptides. Structure-activity modeling of the structural features determining the activity of tethered peptides indicated that the extent and positioning of positive charges and hydrophobic residues were influential in determining activity.

A reversible structural unlocking reaction, in which the closepacked van der Waals interactions break cooperatively, has been found for the villin headpiece subdomain (HP35) using triplet-triplet-energy transfer to monitor conformational fluctuations from equilibrium. Unlocking is associated with an unfavorable enthalpy change (ΔH 0 ¼ 35 AE 4 kJ∕mol) which is nearly compensated in free energy by the entropy change (ΔS 0 ¼ 112 AE 20 J · mol −1 · K −1 ). The unlocking reaction has a time constant of about 1 μs at 5°C and is enthalpy-limited with an activation energy of 32 AE 1 kJ∕mol and a large Arrhenius preexponential factor of A ¼ 7.5 × 10 11 s −1 . In the unlocked state a fast local conformational fluctuation with a time constant of 170 ns and a low activation barrier of 17 AE 1 kJ∕mol leads to unfolding of the Cterminal helix and to its undocking from the core. On a much slower time scale, global unfolding occurs from the unlocked state. These results suggest that native protein structures are locked into conformations with low amplitude motions. Large scale motions and global unfolding require an initial structural unlocking step leading to a state with properties of a dry molten globule. The experiments additionally yielded information on the dynamics of loop formation between different positions in unfolded HP35. Comparison of the results with dynamics in unstructured model peptides indicates slightly decelerated kinetics of local loop formation in the Cterminal region which points at residual, nonrandom structure. Dynamics of long-range loop formation, in contrast, are not influenced by residual structure, which argues against unfolded state properties as molecular origin for ultrafast folding of HP35.dry molten globule | native-state fluctuations | protein dynamics | protein folding | unfolded state dynamics N ative states of proteins are known to be close-packed but almost nothing is known about at which stage of the folding process packing occurs. Shaknovich and Finkelstein proposed in 1989 (1) that heat-induced protein unfolding begins with a slight expansion of the protein, such that at least some of the close-packing interactions are broken but water cannot yet penetrate into the interior. This dry molten globule state was postulated to be located on the unfolded side of the major folding barrier but it was argued that it should not be experimentally detectable for water soluble proteins (1). Later, GdmCl-induced unfolding experiments on ribonuclease A (2) and dihydrofolate reductase (DHFR) (3) monitored by time resolved NMR spectroscopy observed rapid formation of an intermediate located on the native side of the major unfolding barrier with properties resembling those of the predicted dry molten globule. The observation of an intact hydrogen-bonding network in the unfolding intermediate of ribonuclease A supported the dry nature of this state (4). Recently, FRET-detected unfolding kinetics of monellin showed rapid expansion of the native state prior to global unfolding (5), which is also in line w...