Due to the lack of relevant animal models, development of effective treatments for human mitochondrial diseases has been limited. Here we establish a rapid, yeast-based assay to screen for drugs active against human inherited mitochondrial diseases affecting ATP synthase, in particular NARP (neuropathy, ataxia, and retinitis pigmentosa) syndrome. This method is based on the conservation of mitochondrial function from yeast to human, on the unique ability of yeast to survive without production of ATP by oxidative phosphorylation, and on the amenability of the yeast mitochondrial genome to site-directed mutagenesis. Our method identifies chlorhexidine by screening a chemical library and oleate through a candidate approach. We show that these molecules rescue a number of phenotypes resulting from mutations affecting ATP synthase in yeast. These compounds are also active on human cybrid cells derived from NARP patients. These results validate our method as an effective high-throughput screening approach to identify drugs active in the treatment of human ATP synthase disorders and suggest that this type of method could be applied to other mitochondrial diseases.budding yeast | drug screening | transcription profiling | NARP cybrid

Epstein-Barr virus (EBV) is tightly associated with certain human cancers, but there is as yet no specific treatment against EBV-related diseases. The EBV-encoded EBNA1 protein is essential to maintain viral episomes and for viral persistence. As such, EBNA1 is expressed in all EBV-infected cells, and is highly antigenic. All infected individuals, including individuals with cancer, have CD8+ T cells directed towards EBNA1 epitopes, yet the immune system fails to detect and destroy cells harboring the virus. EBV immune evasion depends on the capacity of the Gly-Ala repeat (GAr) domain of EBNA1 to inhibit the translation of its own mRNA in cis, thereby limiting the production of EBNA1-derived antigenic peptides presented by the major histocompatibility complex (MHC) class I pathway. Here we establish a yeast-based assay for monitoring GAr-dependent inhibition of translation. Using this assay we identify doxorubicin (DXR) as a compound that specifically interferes with the GAr effect on translation in yeast. DXR targets the topoisomerase-II–DNA complexes and thereby causes genomic damage. We show, however, that the genotoxic effect of DXR and various analogs thereof is uncoupled from the effect on GAr-mediated translation control. This is further supported by the observation that etoposide and teniposide, representing another class of topoisomerase-II–DNA targeting drugs, have no effect on GAr-mediated translation control. DXR and active analogs stimulate, in a GAr-dependent manner, EBNA1 expression in mammalian cells and overcome GAr-dependent restriction of MHC class I antigen presentation. These results validate our approach as an effective high-throughput screening assay to identify drugs that interfere with EBV immune evasion and, thus, constitute candidates for treating EBV-related diseases, in particular EBV-associated cancers.

6AP and GA are potent inhibitors of yeast and mammalian prions and also specific inhibitors of PFAR, the protein-folding activity borne by domain V of the large rRNA of the large subunit of the ribosome. We therefore explored the link between PFAR and yeast prion [PSI + ] using both PFAR-enriched mutants and site-directed methylation. We demonstrate that PFAR is involved in propagation and de novo formation of [PSI + ]. PFAR and the yeast heat-shock protein Hsp104 partially compensate each other for [PSI + ] propagation. Our data also provide insight into new functions for the ribosome in basal thermotolerance and heat-shocked protein refolding. PFAR is thus an evolutionarily conserved cell component implicated in the prion life cycle, and we propose that it could be a potential therapeutic target for human protein misfolding diseases.The infectious proteins concept was first established for the prion protein PrP in mammals with transmissible spongiform encephalopathy. In its PrP Sc prion conformation, PrP accumulates as self-propagating amyloid fibers without prion-specific nucleic acid. Proteins behaving like prions have also been identified in the budding yeast S. cerevisiae, although it has no PrP homolog. The best studied yeast prions are [PSI + ] and [URE3]: heritable amyloids of translation release factor Sup35p and nitrogen catabolism Ure2p regulator, respectively 1,2 .Hsp104p is a cell factor known to be essential to prion propagation in yeast, in collaboration with heat-shock protein chaperones such as Hsp70p and Hsp40p, characterized as prion propagation modulators 3 . Hsp104p is a hexameric, ring-shaped ATPase of the AAA+ family with disaggregase, unfoldase and translocase activities 4 . In yeast, it has a prime role in disassembling and remodeling the aggregated proteome in collaboration with Hsp70 and Hsp40, thereby providing thermotolerance 5 . Hsp104p also exhibits operational plasticity adaptable to the needs of the yeast proteome, including severing prion fibers by threading Sup35p through its hexameric pore 6 . In the current model, it thus acts as a "molecular sonicator" to transform highly aggregated dead-end fibers into low-molecular-weight oligomers that can seed new rounds of polymerization and enable efficient transmission from mother to daughter cells 1,7 . Thus, when Hsp104p is inhibited (e.g., by guanidine hydrochloride GdnHCl) or when HSP104 is deleted, cells are cured of [PSI + ] prion, as fibers form and grow but are not severed to make new seeds. Hence, the number of fibers and seeds per cell decreases as cells divide, leading to prion-free cells 1 . In addition, Hsp104p overexpression cures [PSI + ] prion, likely by complete prion fiber disaggregation to a soluble form; but the mechanism remains to be fully deciphered [7][8][9] . Metazoans lack Hsp104p orthologs; none of their AAA+ protein superfamily ATPases displays amyloid disaggregation comparable to Hsp104p 5 .