Regulation of the ubiquitin-proteasome system : characterization of viral and cellular stabilization signals

The ubiquitin-proteasome system plays a fundamental role in virtually every cellular process. Degradation of endogenous proteins by this system is the major source for antigenic peptides that are presented to MHC class I-restricted cytotoxic T cells. The, Epstein-Barr virus (EBV) nuclear antigen-1 (EBNA1) contains a long glycine-alanine (GA) repeat that inhibits proteasomal processing, resulting in a blockage of antigen presentation. The GA repeat acts in cis and can be functionally transferred to other proteins. The aim of the work described in this thesis was to characterize the protective effect of this viral stabilization signal and to identify similar cellular stabilization signals.

To evaluate the inhibitory activity of the GA repeat, we used green fluorescent protein (GFP)-based reporters that were targeted for ubiquitin/proteasome-dependent proteolysis by various degrons. Introducing GA repeats of increasing length resulted in enhanced protection of the fluorescent reporter from proteolysis. When provided with a strong degradation signal even EBNA1 could be efficiently degraded. This study showed that a balance between the strength of the degradation signal and the length of the repeat determines the GA repeat-dependent stabilization effect.

Next, we tested the ability of the GA repeat to prevent degradation of the tumor suppressor p53 because inactivation of p53 by accelerated degradation is a common event in tumor development. P53-GA repeat chimeras were protected from degradation and showed improved growth inhibitory activity in tumor cells with impaired endogenous p53 activity, suggesting that insertion of the GA repeat could provide a convenient strategy for the stabilization of potential therapeutic proteins.

We used the aforementioned GFP reporters to test the protective effect of the GA repeat in the yeast Saccharomyces cerevisiae. Expression of proteins carrying GA repeats required the generation of yeast codon-optimized recombinant GA (rGA) repeats. We found that introduction of rGA repeats in the GFP substrates resulted in stabilization of the proteins in mammalian and yeast cells, indicating that the protective signal targets a conserved mechanism in the ubiquitin-proteasome system.

The yeast DNA-repair protein Rad23 is long-lived despite the fact that it is ubiquitinated and interacts with the proteasome. We investigated whether Rad23 contains domains that can protect it from proteasomal degradation. Disruption of the UBA2 domain converted Rad23 into a short-lived protein that is targeted for proteasomal degradation through its ubiquitin-like domain. Insertion of the UBA2 domain from Rad23 or its human homologue HHR23A prevented the degradation of destabilized GFP reporters without causing a general inhibition of proteolysis. We suggest that the Rad23 UBA2 domain functions as a novel cis-acting stabilization signal that confers protection against proteasomal degradation.

List of scientific papers

I. Dantuma NP, Heessen S, Lindsten K, Jellne M, Masucci MG (2000). Inhibition of proteasomal degradation by the gly-Ala repeat of Epstein-Barr virus is influenced by the length of the repeat and the strength of the degradation signal. Proc Natl Acad Sci U S A. 97(15): 8381-5.
https://pubmed.ncbi.nlm.nih.gov/10890896

II. Heessen S, Leonchiks A, Issaeva N, Sharipo A, Selivanova G, Masucci MG, Dantuma NP (2002). Functional p53 chimeras containing the Epstein-Barr virus Gly-Ala repeat are protected from Mdm2- and HPV-E6-induced proteolysis. Proc Natl Acad Sci U S A. 99(3): 1532-7.
https://pubmed.ncbi.nlm.nih.gov/11805282

III. Heessen S, Dantuma NP, Tessarz P, Jellne M, Masucci MG (2003). Inhibition of ubiquitin/proteasome-dependent proteolysis in Saccharomyces cerevisiae by a Gly-Ala repeat. [Submitted]

IV. Heessen S, Masucci MG, Dantuma NP (2003). The UBA2 domain functions as an intrinsic stabilization signal that protects Rad23 from proteasomal degradation. [Manuscript]