Mechanism of redox controlled protein degradation

The proteasome plays a pivotal role in degradation of intracellular proteins in eukaryotic cells. Proteasomal activity is related to numerous human diseases such as tumour development and progression as well as neurodegenerative disorders. In general, degradation by the proteasome is linked to polyubiquitination of target proteins followed by recognition through a regulatory cap, which feeds condemned proteins into the catalytic chamber of the core particle (20S proteasome). Recently, an ubiquitin-independent pathway was discovered in mammalian cells, which is regulated by protein-protein interaction between the 20S proteasome and a flavin-dependent quinone reductase (termed NQO1 in human cells). This overlooked pathway participates in the regulation of transcription factor p53, the "guardian of the genome" and thus may play a critical role in cellular processes such as transformation and programmed cell death (apoptosis). We have recently discovered a homologous regulatory system in the unicellular eukaryote Saccharomyces cerevisiae. Similar to the mammalian system, a flavin (FMN)-dependent quinone reductase (termed Lot6p) is associated with the 20S core particle of the proteasome binding with a strict 2:1 stoichiometry - that is, one 20S proteasome molecule binds two Lot6p molecules. Interestingly, the presence of the FMN cofactor is necessary for this interaction suggesting that it is required for mediating protein-protein interactions. Moreover, upon two-electron reduction of the FMN-cofactor by e. g. NADH, the Lot6p:20S proteasome complex recruits the yeast transcription factor Yap4p, a member of the leucine zipper yeast activator protein family. Binding to the Lot6p:20S proteasome complex protects Yap4p from degradation and thus leads to an accumulation of the transcription factor. Reoxidation of the FMN-cofactor by e. g. cellular quinones, results in the release of the transcription factor and relocalization to the nucleus, where Yap4p is involved in the expression of stress related genes. The first goal of our research project is to determine the scope of this interaction by screening and identifying additional targets, for example other transcription factors, of the Lot6p(reduced):20S proteasome complex. This will be followed by a detailed biochemical characterisation of the parameters involved in formation of these putative ternary complexes. The second focus revolves around the structural determinants for protein complex formation, in particular how the redox state of the cofactor governs recruitment of Yap4p and potentially other proteins. In order to achieve this understanding on a molecular level, crystallographic elucidation of pertinent protein complex structures and protein-protein interaction studies will be carried out. This approach will lead to a comprehensive understanding of the molecular basis of redox-regulated ubiquitin-independent protein degradation and establish a link between the redox state of a cell ("oxidative stress") and maintenance of its proteome. This insight will be invaluable to define the role of protein degradation by the proteasome in a variety of human dieseases related to this central cellular proces.

Human quinone reductase is an astonishing versatile enzyme that does not only participate in the detoxification of quinones but also participates in processes protecting the integrity of the cell, for example by stabilization of tumour suppressors. As if this were not enough the activity of quinone reductase is also exploited in chemotherapies for the bioactivation of quinone-based drugs. These are all very good reasons to call an active and stable quinone reductase your own! Unfortunately, this is not always the case as single nucleotide exchanges occur in the gene that result in the expression of dysfunctional quinone reductases. Two nucleotide exchanges are frequently found: cytosine in position 465 or 609 is replaced by thymine. Both of these transitions result in an altered amino acid sequence and give rise to variants with the amino acid arginine replaced by tryptophan in position 139 (R139W variant) or the amino acid proline by serine in position 187 (P187S variant). If and how these amino acid replacements lead to altered properties of the enzyme was the subject of our research project. Toward this goal the two enzyme variants were generated by recombinant protein production and purified to initiate experiments to reveal the biochemical and structural properties. It rapidly emerged that the P187S variant exhibited substantially reduced enzymatic activity. Further investigations revealed that the ability of this variant to bind the required cofactor is greatly diminished. This result was very surprising as the amino acid exchange is far away from the cofactor binding pocket and thus it was unclear how the amino acid in this remote location affects cofactor affinity. To tackle this question we determined the crystallographic structure of the P187S variant and compared it with the known structure of human quinone reductase. Surprisingly, the structure of the P187S variant was found to be very similar to that of the "wildtype" and thus did not provide a clue to rationalize the loss of function. In order to resolve this unexpected finding, we performed experiments in solution and found that the structure of the P187S variant is unstable leading to the occurrence of partially unfolded protein thus explaining the diminished enzymatic activity. This insight into the effect of a single amino acid exchange on the properties of quinone reductase will be important for the future development of small molecular chaperons that stabilise the P187S variant and hence support chemotherapeutic approaches. In contrast to the P187S variant our studies on the R139W variant have shown that this amino acid replacement does not affect the properties of the variant. Our characterisation of the P187S variant demonstrates the limitations of crystallography regarding properties of proteins and may serve as an important reference system in the field of protein biochemistry.