FWF - Redox Controlled - 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.