Mitochondrial chaperones mortalin and Tid1 in protein degradation

Project Details

Description

DESCRIPTION (provided by applicant): Protein misfolding and aggregation are associated with aging, as well as a variety of human diseases, including Parkinson's disease, cystic fibrosis, amyotrophic lateral sclerosis (ALS), short chain acyl-CoA dehydrogenase (SCAD) deficiency and hypertrophic cardiomyopathy. Mitochondrial proteins are at increased risk of protein misfolding and aggregation as they are located in proximity to the respiratory chain, which is a powerful source of reactive oxygen species (ROS). When exposed to elevated ROS, mitochondrial proteins are highly susceptible to oxidative damage and conformational defects. The surveillance system that oversees mitochondrial protein quality control is composed of ATP-dependent proteases, which degrade misfolded and damaged proteins, as well as molecular chaperones, which mediate protein folding and facilitate protein degradation. Within the mitochondrial matrix, the ATP-dependent proteases Lon and ClpXP selectively degrade normal and abnormal proteins in response to metabolic demands and changing environmental conditions. The matrix chaperones Mortalin and Tid1, function in concert to fold nascent polypeptides and to assist Lon- and ClpXP- mediated proteolysis. Interestingly, our preliminary results show for the first time, that Mortalin and Tid1 also mediate protein disaggregation. The aims of this project are- (1) to elucidate the functions of Mortalin and Tid1 in protein disaggregation, reactivation and refolding, as well as the interplay between these chaperones and the mitochondrial ATP-dependent proteases Lon and ClpXP; (2) to identify endogenous mitochondrial substrates of these proteases and chaperones. We hypothesize that mitochondrial quality control proteases and chaperones function to- (a) prevent the toxic accumulation of abnormal proteins, (b) facilitate the degradation of non-native polypeptides and (c) reactivate the functional state of proteins once they are disaggregated. Our research strategy is to develop and optimize biochemical and cell culture assays to study chaperone-mediated protein disaggregation, reactivation/refolding and degradation of reporter substrates. For example, a purified protein system will be employed to study Mortalin- and Tid1- dependent disaggregation and reactivation of insoluble and enzymatically inactive glucose-6-phosphate dehydrogenase (aggG6PDH); whereas denatured luciferase (unfoldedLuc) will be used to examine chaperone-assisted refolding. In addition, the interplay between Mortalin, Tid1 and mitochondrial proteases in protein degradation of an aggregation-prone mutant of ornithine transcarbamylase (aggOTC) will be carried out in cell culture experiments using knockdown cell lines for Tid1, Lon or ClpP, as well as with purified chaperones, proteases and aggOTC. Lastly, a proteomics approach will be undertaken to identify endogenous mitochondrial substrates of Tid1, Lon and ClpP. Mitochondrial proteins that accumulate or aggregate upon depletion of Lon, ClpP or Tid1 are likely to be substrates of these quality control proteins. Electron microscopy data show the accumulation of inclusion bodies within mitochondria of Lon-depleted cells. We predict a similar phenotype in ClpP- or Tid1- depleted mitochondria. Taken together, the results obtained from this project will provide the knowledge and experimental assays needed to elucidate further and exploit the protein quality control function of these mitochondrial chaperones and proteases in the management or treatment of protein-misfolding diseases.
StatusFinished
Effective start/end date8/15/117/31/14

Funding

  • National Institute on Aging: $153,158.00
  • National Institute on Aging: $133,646.00
  • National Institute on Aging: $40,012.00

ASJC

  • Clinical Neurology
  • Cell Biology

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