Biomedical subjects

A Sharipo

Publications and source records attributed to A Sharipo.

7 recordsLinked to original sources

PubMed2002

Avoiding proteasomal processing: the case of EBNA1.

Ubiquitin/proteasome-dependent proteolysis is involved in the regulation of a large variety of cellular processes including cell cycle progression, tissue development and atrophy, flux of substrates through metabolic pathways, selective elimination of abnormal proteins and processing of intracellular antigens for major histocompatibility complex (MHC) class I-restricted T-cell responses. Many viruses tamper with this proteolytic machinery by encoding proteins that interact with various components of the pathway. A particularly interesting example of a viral protein that interferes with proteasomal processing is the Epstein-Barr virus (EBV) nuclear antigen-1 (EBNA1). EBNA1 contains an internal repeat exclusively composed of glycines and alanines that inhibits in cis the presentation of MHC class I-restricted T-cell epitopes and prevents ubiquitin/proteasome-dependent proteolysis in vitro and in vivo. The glycine-alanine repeat acts as a transferable element on a variety of proteasomal substrates and may therefore provide a new approach to the modification of cellular proteins for therapeutic purposes.

Alanine↗

PubMed2001

cis-Inhibition of proteasomal degradation by viral repeats: impact of length and amino acid composition.

The Gly-Ala repeat (GAr) of the Epstein-Barr virus nuclear antigen 1 is a cis acting inhibitor of ubiquitin-proteasome proteolysis. We have investigated the capacity of various repeats to inhibit the turnover of the proteasomal substrate IkappaBalpha. Inhibition of TNFalpha-induced degradation was achieved by insertion of octamers containing three alanines or valines, interspersed by no more then three consecutive glycines. The inhibitory activity was abolished by increasing the length of the spacer, by eliminating the spacers, or by substitution of a single hydrophobic residue with a polar or charged residue. A serine containing octamer was inactive but inhibition was partially restored by insertion of three consecutive repeats. These findings suggest a model where inhibition requires the interaction of at least three alanine residues of the GAr in a beta-strand conformation with adjacent hydrophobic binding pockets of a putative receptor.

Alanine↗

PubMed2001

Thioredoxin fold as homodimerization module in the putative chaperone ERp29: NMR structures of the domains and experimental model of the 51 kDa dimer.

BACKGROUND: ERp29 is a ubiquitously expressed rat endoplasmic reticulum (ER) protein conserved in mammalian species. Fold predictions suggest the presence of a thioredoxin-like domain homologous to the a domain of human protein disulfide isomerase (PDI) and a helical domain similar to the C-terminal domain of P5-like PDIs. As ERp29 lacks the double-cysteine motif essential for PDI redox activity, it is suggested to play a role in protein maturation and/or secretion related to the chaperone function of PDI. ERp29 self-associates into 51 kDa dimers and also higher oligomers. RESULTS: 3D structures of the N- and C-terminal domains determined by NMR spectroscopy confirmed the thioredoxin fold for the N-terminal domain and yielded a novel all-helical fold for the C-terminal domain. Studies of the full-length protein revealed a short, flexible linker between the two domains, homodimerization by the N-terminal domain, and the presence of interaction sites for the formation of higher molecular weight oligomers. A gadolinium-based relaxation agent is shown to present a sensitive tool for the identification of macromolecular interfaces by NMR. CONCLUSIONS: ERp29 is the first eukaryotic PDI-related protein for which the structures of all domains have been determined. Furthermore, an experimental model of the full-length protein and its association states was established. It is the first example of a protein where the thioredoxin fold was found to act as a specific homodimerization module, without covalent linkages or supporting interactions by further domains. A homodimerization module similar as in ERp29 may also be present in homodimeric human PDI.

Amino Acid Sequence↗

PubMed1998

Random coil conformation of a Gly/Ala-rich insert in IkappaB alpha excludes structural stabilization as the mechanism for protection against proteasomal degradation.

Peptide segments of multiple glycine and alanine residues prevent the proteolytic degradation of ubiquitinated proteins by the proteasome. The structure of a Gly/Ala-rich insert in IkappaB alpha was probed by nuclear magnetic resonance (NMR) spectroscopy, comparing IkappaB alpha samples with and without Gly/Ala-rich insert. Narrow 1H-NMR resonances at chemical shifts indicative of random coil conformations were observed in the difference spectrum. circular dichroism (CD) measurements further confirm that the mechanism of protection against proteolytic degradation is not based on structural transition or stabilization caused by the Gly/Ala-rich segment. In addition, most of the N- and C-terminal residues outside the ankyrin repeats in wild-type IkappaB alpha were found to be flexibly disordered.

Alanine↗

PubMed1998

Oligomerization properties of ERp29, an endoplasmic reticulum stress protein.

ERp29, a novel and ubiquitously expressed endoplasmic reticulum (ER) stress-inducible protein, was recently isolated and cDNA cloned in our laboratory. Using size exclusion chromatography and chemical cross-linking we have assessed the oligomerization properties of ERp29. Purified ERp29 in solution as well as in rat hepatoma cells self-associates predominantly into homodimers. Labeling of the cells with [35S]methionine with subsequent cross-linking and immunoprecipitation showed that ERp29 interacts with a number of ER proteins, one of which was previously identified as BiP/GRP78. Secondary structure prediction and fold recognition methods indicate that the native conformation of ERp29 resembles the thioredoxin fold, a structural motif characteristic of a number of enzymes with the redox function, including protein disulfide isomerase (with which ERp29 shares limited sequence similarity). Dimerization of the protein is suggested to be advantageous for the protein binding potential of ERp29.

Amino Acid Sequence↗

PubMed1998

A minimal glycine-alanine repeat prevents the interaction of ubiquitinated I kappaB alpha with the proteasome: a new mechanism for selective inhibition of proteolysis.

The Epstein-Barr virus nuclear antigen 1 contains a glycine-alanine repeat that inhibits in cis MHC class I-restricted presentation. We report here that insertion of a minimal glycine-alanine repeat motif in different positions of I kappaB alpha protects this NF-kappaB inhibitor from signal-induced degradation dependent on ubiquitin-proteasome, and decreases its basal turnover in vivo resulting in constitutive dominant-negative mutants. The chimeras are phosphorylated and ubiquitinated in response to tumor necrosis factor alpha, but are then released from NF-kappaB and fail to associate with the proteasome. This explains how functionally competent I kappaB alpha is protected from proteasomal disruption and identifies the glycine-alanine repeat as a new regulator of proteolysis.

Amino Acid Sequence↗

PubMed1997

Inhibition of ubiquitin/proteasome-dependent protein degradation by the Gly-Ala repeat domain of the Epstein-Barr virus nuclear antigen 1.

The Epstein-Barr virus (EBV) encoded nuclear antigen (EBNA) 1 is expressed in latently infected B lymphocytes that persist for life in healthy virus carriers and is the only viral protein regularly detected in all EBV associated malignancies. The Gly-Ala repeat domain of EBNA1 was shown to inhibit in cis the presentation of major histocompatibility complex (MHC) class I restricted cytotoxic T cell epitopes from EBNA4. It appears that the majority of antigens presented via the MHC I pathway are subject to ATP-dependent ubiquitination and degradation by the proteasome. We have investigated the influence of the repeat on this process by comparing the degradation of EBNA1, EBNA4, and Gly-Ala containing EBNA4 chimeras in a cell-free system. EBNA4 was efficiently degraded in an ATP/ubiquitin/proteasome-dependent fashion whereas EBNA1 was resistant to degradation. Processing of EBNA1 was restored by deletion of the Gly-Ala domain whereas insertion of Gly-Ala repeats of various lengths and in different positions prevented the degradation of EBNA4 without appreciable effect on ubiquitination. Inhibition was also achieved by insertion of a Pro-Ala coding sequence. The results suggest that the repeat may affect MHC I restricted responses by inhibiting antigen processing via the ubiquitin/proteasome pathway. The presence of regularly interspersed Ala residues appears to be important for the effect.

Animals↗