THE ROLE OF ENDOPLASMIC RETICULUM PROTEIN MISFOLDING IN CELL DEATH AND DISEASE

In many human diseases defective proteins are made that damage the cell, so-called protein misfolding. In response to this, our cells reduce the rate at which they make new proteins in order to allow correction or destruction of these abnormal components. When the defect is corrected, protein synthesis increases once again.

The reduction of protein synthesis requires a protein called PERK, while the recovery involves a protein called GADD34. We have previously shown how mutations in the GADD34 gene protect against kidney damage in an animal model of protein misfolding. This led us to hypothesise that by developing drugs either to block GADD34 function or activate PERK, we might be able to protect tissues in diseases where protein misfolding is important, such as diabetes and stroke.

To help us identify potential drug targets, we are now studying how GADD34 and PERK are controlled by other components of the cell. We are using two main approaches: First, by studying how GADD34 and PERK are regulated in cultured human and mouse cells; and second, using genetically modified fruit flies (Drosophila melanogaster) to allow rapid identification of genes involved in the this system.

Protein mis-folding in the endoplasmic reticulum (ER) is central to the pathogenesis of many human diseases (Marciniak & Ron, 2006 Physiol. Rev. 86:1133). Increased protein flux through the ER (ER stress) activates mechanisms that balance protein-folding capacity with protein load. These are referred to as the unfolded protein response (UPR). The UPR combines inhibition of protein translation with a transcriptional increase of ER chaperones. Translational inhibition follows phosphorylation of eIF2 by the ER stress-sensing kinase PERK; subsequent up-regulation of GADD34, an eIF2 phosphatase, reverses this inhibition. My previous studies into ER stress-induced cell death revealed an unexpected link between GADD34-dependent recovery of protein synthesis and the causation of tissue damage (Marciniak et al., 2004 Genes Dev. 18:3066). This suggested that inhibition of GADD34 or augmenting PERK activity might protect against ER stress-induced cell death in human disease.

Aims: 1. To characterise the post-translational modulation of PERK and GADD34 in order to identify novel factors that regulate the UPR.
2. To use Drosophila models to identify pathways linking ER stress and cell death.

Methods: 1. I will use techniques that I have previously developed (Marciniak et al., 2006 J Cell Biol. 172: 201) to study the PERK-inactivating factors in cell lysates. In particular I will use specific phosphatase inhibitors to determine pharmacologically the class(es) of phosphatase involved. I will over-express and knockdown individual phosphatase catalytic subunits in cultured 293T cells and assess the effect on PERK phosphatase activity. The effects of cellular stresses on PERK-inactivating activity will be studied. Biochemical fractionation will be used to isolate for identification the PERK-inactivating factors involved. Using a highly sensitive assay I have developed to measure eIF2 phosphatase activity, I will study the effects of GADD34 phosphorylation on its activity, both by in vitro phosphorylation and dephosphorylation of recombinant GADD34/PP1 holoenzyme. The effect of candidate GADD34-interactors will similarly be assessed. 2. I have generated Drosophila models of PERK/GADD34 signalling. Flies expressing PERK in the eye show a dramatic atrophic eye phenotype that is dependent on PERK catalytic activity and is rescued by co-expression of GADD34. Using these flies I will test candidate PERK and GADD34-modifying genes. Also, by crossing these flies with an existing 3000 fly P-element library I will screen for novel PERK and GADD34-interacting genes. Pathways involved will be manipulated using genetic and pharmacological tools in order to dissect their involvement in cellular survival and death during protein aggregation within the ER.