|Institution:||University of Manchester|
|Keywords:||Prions; Autophagy; Chronological ageing; Actin cytoskeleton; Oxidative stress; Yeast|
|Full text PDF:||http://www.manchester.ac.uk/escholar/uk-ac-man-scw:308633|
Prions are infectious protein entities capable ofself-replication. Prions are the causal agents behind thetransmissible spongiform encephalopathies causing neurodegenerationand death in affected organisms. Prions have been identified inyeast with the best-characterized prions being [PSI+] and [PIN+],whose respective native proteins are the Sup35 translationtermination factor and Rnq1 (function unknown). Autophagy is acellular housekeeping mechanism mediating the degradation ofdamaged proteins and superfluous organelles. It is a highlysequential process regulated by autophagy related genes (ATGs).Autophagy has also been implicated in the clearance ofamyloidogenic proteins including prions. However, the mechanisticbasis underlying this activity is poorly understood, and a keyobjective of this project was to characterize how autophagyprevents spontaneous prion formation. Our study found that thedeletion of core ATGs correlated with an increase in de novo [PSI+]and [PIN+] formation as well as Sup35 aggregation. Enhancement ofautophagic flux through spermidine treatment attenuated theincreased levels of de novo [PSI+] formation in mutants thatnormally show elevated levels of [PSI+] formation. Defectiveautophagy correlated with increased oxidatively damaged Sup35 in anatg1 mutant whereas anaerobic growth abrogated the increased [PSI+]formation in the atg1 mutant to wild-type levels. Our data suggestthat autophagy serves a protective role in the clearance ofoxidatively damaged Sup35 proteins that otherwise has a higherpropensity towards [PSI+] prion formation. We also investigated therole of prion formation and autophagy during yeast chronologicalageing which is the time that non-dividing cells remain viable.Prion diseases are associated with advanced age which correlateswith a decline in cellular protective mechanisms includingautophagy. Our study found an age dependent increase in thefrequency of de novo [PSI+] formation with chronological age ofyeast cells, more so in an atg1 mutant relative to the wild-type.Autophagy competent cells carrying the [PSI+] and [PIN+] prionsalso had improved chronological lifespan relative to prion freecells and atg1 cells. Cells carrying the [PSI+] prion elicitedelevated autophagic flux that may promote improved lifespan thussuggesting a beneficial role of the [PSI+] prion duringchronological ageing. The actin cytoskeleton provides thestructural framework essential for a multitude of cellularprocesses to occur. We investigated the role of the Arp2/3 complexresponsible for branching of actin filaments towards prionformation. Knockout mutants of the nucleation promoting factors ofthe Arp2/3 complex, in particular the abp1 mutant, showed reducedde novo [PSI+] formation and Sup35 aggregation under basal andoxidative stress conditions. Similarly, treatment with latrunclinA, an actin monomer-sequestering drug also abrogated de novo [PSI+]formation. Colocalization studies revealed that Sup35 often doesnot colocalize with Rnq1, a marker for theAdvisors/Committee Members: HIGH, STEPHEN S, Grant, Christopher, High, Stephen.