Sacsin, a multidomain molecular chaperone involved in neurodegenerative disease

Proteins are the essential biological molecules that build the machines which make the body function. Proteins must be folded into complex three dimensional shapes to work properly. Proteins that do not fold correctly are detected by ‘quality-control’ systems and are then disposed of by being degraded. This folding and controlled destruction of proteins involves a special group of proteins known as molecular chaperones. This work is focused on sacsin, a novel protein, which is able to regulate a molecular chaperone machine. Sacsin has an important role in the brain as mutations in its gene cause an inherited neuronal disease. To find out more about the biology of molecular chaperones and understand why mutations in sacsin cause disease we are investigating its precise function in cells. Sacsin’s function is being looked at through a comprehensive series of experiments. We are finding out where it’s located, what other proteins it interacts with and what happens when it’s absent. Studying sacsin is important because problems with protein folding lead to diseases where badly folded proteins accumulate; these include Alzheimer’s and Parkinson’s disease. What’s more molecular chaperones are associated with these conditions and in experimental models can slow disease progression.

Autosomal recessive spastic ataxia of Charlevoix-Saguenay is an early onset neurodegenerative disease caused by mutations in the SACS gene. SACS encodes the large multi-domain protein sacsin. Although the cellular role of sacsin is unknown it has recently been identified as a component of an ?ataxia interactome?, indicating it is in common pathways with other neuronal disease proteins. Sacsin was identified as a DnaJ/Hsp40 protein based upon the presence of a putative J-domain, suggesting it may function in conjunction with an Hsp70 molecular chaperone protein. J-domains stimulate the ATPase activity of Hsp70s and stabilize their interactions with client proteins. I have demonstrated that sacsin?s J-domain is functional in a bacterial complementation assay. I have also identified a putative ubiquitin-like (UbL) domain at the N-terminus of sacsin. The aim of this proposal is to define the cellular function of sacsin based on these findings. A number of Hsp70 co-chaperones have been shown to function in the targeting of client proteins to the ubiquitin-proteasome system (UPS). I believe that sacsin represents another link between the Hsp70 chaperone network and the UPS and that this link is important for neuronal viability. In order to examine this hypothesis we will (a) test if sacsin?s putative UbL domain binds the proteasome or ubiquitin interaction motif proteins; (b) define sacsin?s localization in tissues and cells by using immuno-staining techniques and subsequent confocal microscopy; (c) identify sacsin interacting partners by yeast genetic screens and co-immunoprecipitation; and (d) investigate the effects of sacsin knockdown, by siRNA, on the cellular processing of interacting partners. Mutations in sacsin cause an inherited spastic ataxia and given the importance of both molecular chaperones and the UPS in protein misfolding disease it seems likely that sacsin will have wider relevance for neurodegeneration.