Ulrich Schubert

Rapid degradation of a large fraction of newly synthesized proteins by proteasomes

MHC class I molecules function to present peptides eight to ten residues long to the immune system. These peptides originate primarily from a cytosolic pool of proteins through the actions of proteasomes, and are transported into the endoplasmic reticulum, where they assemble with nascent class I molecules. Most peptides are generated from proteins that are apparently metabolically stable. To explain this, we previously proposed that peptides arise from proteasomal degradation of defective ribosomal products (DRiPs). DRiPs are polypeptides that never attain native structure owing to errors in translation or post-translational processes necessary for proper protein folding. Here we show, first, that DRiPs constitute upwards of 30% of newly synthesized proteins as determined in a variety of cell types; second, that at least some DRiPs represent ubiquitinated proteins; and last, that ubiquitinated DRiPs are formed from human immunodeficiency virus Gag polyprotein, a long-lived viral protein that serves as a source of antigenic peptides.

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.

Membrane interactions and alignment of structures within the HIV-1 Vpu cytoplasmic domain: effect of phosphorylation of serines 52 and 56.

The cytoplasmic domain of the HIV‐1 accessory protein Vpu is involved in the binding and degradation of the viral receptor CD4. In order to analyze previous structural models in the context of membrane environments, regions of VpuCYTO incorporating particular conformational features have been synthesized and labelled with 15N at selected backbone amides. Well‐oriented proton‐decoupled 15N solid‐state NMR spectra with 15N chemical shifts at the most upfield position indicate that the amphipathic helix within [15N‐Leu 45]‐Vpu27–57 strongly interacts with mechanically aligned POPC bilayers and adopts an orientation parallel to the membrane surface. No major changes in the topology of this membrane‐associated amphipathic helix were observed upon phosphorylation of serine residues 52 and 56, although this modification regulates biological function of Vpu. In contrast, [15N‐Ala 62]‐Vpu51–81 exhibits a pronounced 15N chemical shift anisotropy.

Use of Synthetic Peptides for Structural and Functional Analyses of Viruses Like HIV

ABSTRACT Efficient solid-state synthesis now affords ready access, particularly in situations in which recombinant protocols usually fail, to high-quality synthetic peptides that are equivalent to many full-length biologically active systems or domains thereof, including posttranslational modifications such as phosphorylation and acetylation. Here we survey recent applications of such peptides to the investigations of human immunodeficiency viruses (HIV), with the focus on the regulatory peptides Vpu, Vpr, and Tat, as well as the protease, the Gag peptides nucleocapsid, matrix, and p6 from HIV-1. The importance of the full-length synthetic viral peptides for molecular characterization that involves both detailed biochemical and structural studies at the atomic level and their potential for therapeutic applications are emphasized.