Final Report Summary - METAMETA (Metastability of proteins during tumor metastasis) The key goal of the MetaMeta program was to gain deeper understanding of how unstable cellular proteins contribute to the metastasis and how the Heat shock protein (HSP)70 family is involved in tumor protein stabilization. ERC support allowed us to make key discoveries in clarifying these aspects of tumor biology. We uncovered a novel principle of the reorganization of the subcellular architecture during proteostasis stress. The mechanism of the reorganization is mediated by the reversible association of HSP70 and the ubiquitin ligase CHIP. Under stress, misfolded proteins in the cytosol sequester HSP70, which frees CHIP for interaction with cellular membranes. At new cellular locations, access to compartment-specific substrates enables CHIP to participate in the degradation of new proteins leading to the reorganization of the respective organelles. We could affect this regulatory loop by inhibiting the generation of one particular lipid by phospholipase D. Clinical use of phospholipase D inhibitors to weaken the stress adaptability would open new perspectives of controlling tumor metastasis.While working with murine metastatic melanoma, we discovered a new subset of metastable proteins in mammalian cells, the cofactor-free flavoproteins. We found that the same HSP70-associated protein quality control (PQC) machinery can recognize not only mutant but also wild-type proteins if the latter fail to associate with the vitamin B2-derived cofactor FAD. Our data indicate that relative insufficiency of apoprotein degradation caused by the vitamin B2 shortage can aggravate protein aggregation, which suggests a novel means to destabilize the tumor proteome. We succeeded in identifying the composition of wild-type apoprotein NQO1 aggregates. A number of chaperones and co-chaperones from the HSP70 superfamily was found among apo-NQO1 interactors, which underscores the importance of HSP70 members in the turnover of the metastable proteome.Furthermore, we discovered a link between PQC and mRNA modifying machinery during proteostasis stress. We identified a set of proteins assembled by the naked coding region of an mRNA in the heat-shocked cytosol of tumor cells. The complex which methylates adenine was among the most enriched proteins on the RNA. Downregulation of the methylase sensitized human tumor cells to heat shock and affected clearance of an aggregating protein. Our results reveal how the RNA modifying machinery participates in the metastable proteome sequestration into granules during misfolding stress.In the frame of the MetaMeta, we established an in vitro approach to streamline the mechanistic analysis of the transcriptional regulation of HSP70 genes in metastatic cells. The promoter DNA regions around the transcription start sites were used to isolate promoter binding complexes in tumors. Composition of the complexes from normoxic versus hypoxic conditions was analyzed by means of quantitative mass spectrometry. The hypoxia-specific and promoter-specific massive recruitment of chromatin-remodelling complexes is one of the several discoveries we made using this novel technology, which will advance our understanding of the HSP70 family role during tumor metastasis.
