Peter Henklein

A novel influenza A virus mitochondrial protein that induces cell death

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.

COP9 signalosome‐specific phosphorylation targets p53 to degradation by the ubiquitin system

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, Δp53(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 Δp53(145–164) results in accumulation of endogenous p53.

SIRT1 regulates HIV transcription via Tat deacetylation.

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.

Identification of an ion channel activity of the Vpu transmembrane domain and its involvement in the regulation of virus release from HIV‐1‐infected cells

HIV‐1 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 full‐length 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.

An essential role for tripeptidyl peptidase in the generation of an MHC class I epitope

Most of the peptides presented by major histocompatibility complex (MHC) class I molecules require processing by proteasomes. Tripeptidyl peptidase II (TPPII), an aminopeptidase with endoproteolytic activity, may also have a role in antigen processing. Here, we analyzed the processing and presentation of the immunodominant human immunodeficiency virus epitope HIV-Nef(73–82) in human dendritic cells. We found that inhibition of proteasome activity did not impair Nef(73–82) epitope presentation. In contrast, specific inhibition of TPPII led to a reduction of Nef(73–82) epitope presentation. We propose that TPPII can act in combination with or independent of the proteasome system and can generate epitopes that evade generation by the proteasome-system.

Conserved P-TEFb-interacting domain of BRD4 inhibits HIV transcription

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)h. Both BRDT and fs(1)h specifically interact with P-TEFb in mammalian cells, and this interaction depends on their C-terminal domains. Overexpression of the BRD4 P-TEFb-interacting domain disrupts the interaction between the HIV transactivator Tat and P-TEFb and suppresses the ability of Tat to transactivate the HIV promoter. Incubation of cells with a synthetic peptide containing the C-terminal domain of BRD4 interferes with transactivation of the HIV promoter by the Tat protein.

Protein kinase CK2 and protein kinase D are associated with the COP9 signalosome

The COP9 signalosome (CSN) purified from human erythrocytes possesses kinase activity that phosphoryl ates proteins such as c‐Jun and p53 with consequence for their ubiquitin (Ub)‐dependent degradation. Here we show that protein kinase CK2 (CK2) and protein kinase D (PKD) co‐purify with CSN. Immunoprecipi tation and far‐western blots reveal that CK2 and PKD are in fact associated with CSN. As indicated by electron microscopy with gold‐labeled ATP, at least 10% of CSN particles are associated with kinases. Kinase activity, most likely due to CK2 and PKD, co‐immuno precipitates with CSN from HeLa cells. CK2 binds to ΔCSN3(111–403) and CSN7, whereas PKD interacts with full‐length CSN3. CK2 phosphorylates CSN2 and CSN7, and PKD modifies CSN7. Both CK2 and PKD phosphorylate c‐Jun as well as p53. CK2 phosphoryl ates Thr155, which targets p53 to degradation by the Ub system. Curcumin, emodin, DRB and resveratrol block CSN‐associated kinases and induce degradation of c‐Jun in HeLa cells. Curcumin treatment results in elevated amounts of c‐Jun–Ub conjugates. We conclude that CK2 and PKD are recruited by CSN in order to regulate Ub conjugate formation.

Screening and Characterization of Surface-Tethered Cationic Peptides for Antimicrobial Activity

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.

Transcriptional synergy between Tat and PCAF is dependent on the binding of acetylated Tat to the PCAF bromodomain

The human immunodeficiency virus (HIV) Tat protein plays an essential role in promoting efficient transcriptional elongation of viral transcripts. We report that the transcriptional co‐activator PCAF and Tat interact and synergize to activate the HIV promoter. The binding of Tat and PCAF in vitro and in vivo is dependent on the acetylated state of Lys50 of Tat and on the PCAF bromodomain. Structural analysis of the acetylated Tat peptide bound to the PCAF bromodomain defined amino acids Y47 and R53 in Tat and V763, Y802, and Y809 in PCAF as critical interaction points between the two proteins. Mutation of each of these residues in either Tat or PCAF inhibited in a cumulative manner the Tat–PCAF interaction in vitro and in vivo, and abrogated the synergistic activation of the HIV promoter by both proteins. These observations demonstrate that acetylation of Tat establishes a novel protein–protein interaction domain at the surface of Tat that is necessary for the transcriptional activation of the HIV promoter.

Hepatitis C virus mutation affects proteasomal epitope processing

The high incidence of hepatitis C virus (HCV) persistence raises the question of how HCV interferes with host immune responses. Studying a single-source HCV outbreak, we identified an HCV mutation that impaired correct carboxyterminal cleavage of an immunodominant HLA-A2-restricted CD8 cell epitope that is frequently recognized by recovered patients. The mutation, a conservative HCV nonstructural protein 3 (NS3) tyrosine to phenylalanine substitution, was absent in 54 clones of the infectious source, but present in 15/21 (71%) HLA-A2-positive and in 11/24 (46%) HLA-A2-negative patients with chronic hepatitis C. In order to analyze whether the mutation affected the processing of the HLA-A2-restricted CD8 cell epitope, mutant and wild-type NS3 polypeptides were digested in vitro with 20S constitutive proteasomes and with immunoproteasomes. The presence of the mutation resulted in impaired carboxyterminal cleavage of the epitope. In order to analyze whether impaired epitope processing affected T cell priming in vivo, HLA-A2-transgenic mice were infected with vaccinia viruses encoding either wild-type or mutant HCV NS3. The mutant induced fewer epitope-specific, IFN-gamma;-producing and fewer tetramer(+) cells than the wild type. These data demonstrate how a conservative mutation in the flanking region of an HCV epitope impairs the induction of epitope-specific CD8(+) T cells and reveal a mechanism that may contribute to viral sequence evolution in infected patients.