Translation initiation is the main target for regulation of protein synthesis. It is a multistep process involving ribosomes, mRNAs, tRNAs, and a number of eukaryotic translation initiation factors (eIFs). Translation initiation relies on a dynamic network of interactions within a mega-dalton translation pre-initiation complex (PIC). The guanine nucleotide exchange factor (GEF) eIF2B is the target of multiple pathways regulating protein synthesis and the cellular stress response. The activity of eIF2B is regulated by phosphorylation and by small molecules, such as nucleotides and cofactors. The ?-subunit of the substrate eIF2 is phosphorylated by several stress-induced kinases. Phosphorylated eIF2-GDP (eIF2(?-P)-GDP) is an inhibitor of eIF2B, which triggers the integrated stress response (ISR). ISR dysregulation is involved in the etiology of many diseases, including metabolic disorders and cancer. Neuronal cell death in prion disease, Alzheimer's disease, and other neurodegenerative disorders is mediated by dysregulated ISR stimulation. Accordingly, ISR inhibitors have shown promise as neuroprotective agents in animal models of these diseases. However, despite its exceptional scientific and medical importance, the eIF2B function is poorly understood, which impedes rational drug design. We will employ a combination of NMR, site-directed mutagenesis, biophysical and biochemical assays to: 1. Determine how intramolecular interactions in eIF5 coordinate its interactions with other eIFs eIF5 is an integral part of the PIC, but upon start codon selection, it disengages from most of its binding partners and leaves with only eIF2-GDP. We will identify the roles of competing intramolecular interactions within eIF5 in initiation, and specifically in the rearrangement of the eIF5 interaction network. 2. Elucidate the mechanism of regeneration of the eIF2-GTPMet-tRNAi ternary complex by eIF2B We will test our model, based on the available data about the eIF2B-catalyzed process of eIF2-GTPMet- tRNAi ternary complex (TC) regeneration, that eIF2 is channeled from the PIC to eIF5 (as eIF2-GDP), to eIF2B, and then back to eIF5 and the PIC (as TC), without being released free at any stage of the cycle. We will use fluorescence and MST assays to build a complete kinetic framework of the eIF2B-catalyzed process. 3. Develop a complete in vitro reconstituted system for analysis of eIF2B activity and regulation We will develop a complete in vitro assay for eIF2B catalysis that faithfully reproduces all aspects of regulation in vivo. which will allow us to identify the mechanisms of action of any regulator of eIF2B and the steps it affects. We will use our assay to test the effects of a set of regulators and mutations in eIF2B, for which the currently used assay has failed to produce meaningful results. The proposed studies will elucidate the mechanisms of eIF2B catalysis and regulation, and allow rational design of ISR inhibitors for treatment of neurodegenerative disorders and other diseases.
Control of protein synthesis is vital for the cell and its dysregulation plays roles in a number of diseases, such as neurodegenerative and metabolic disorders, and cancer. The work outlined in this proposal will establish new targets for the treatment of prion disease, Alzheimer's Disease, and other neurodegenerative disorders.
