HMGCR and the peroxisome
Description: The mevalonate pathway produces sterols, especially cholesterol modulating the fluidity of eukaryotic membrane structures, dolichol required for glycoprotein synthesis, haem A and ubiquinone participating in the electron transport, isopentyladenine present in some transfer RNA, certain isoprenoids as farnesyl involved in the posttranslational modification of proteins and vitamin D and many more. To ensure a constant production of the multiple isoprenoid compounds at all stages of growth, this pathway is tightly controlled by feedback systems that operate via sterols and nonsterols through multiple mechanisms, including inhibition or induction of transcription, as well as on translational and posttranslational levels. Currently it is highly contradictory whether the peroxiosmes play a role in the mevalonate pathway. We have formulated a hypothesis fitting most of the available results. This proposal is focused on the 3-hydroxy-3-methylglutaryl CoA reductase (HMGCR) catalyzing a rate-controlling step in the mevalonate pathway. HMGCR is known as an integral membrane protein of endoplasmic reticulum. Our previous studies indicate that a HMGCR activity exists that is insensitive to lovastatin and was hypothesised to be localized in the peroxisomes. We hypothesize that the soluble peroxisomal, lovastatine resistant isoform of HMGCR results form the endoplasmic reticulum-localized HMGCR during the complex sterol-triggered degradation process. The soluble cytoplasmatic HMGCR is than recognized by the cytoplasmatic receptor pex7 at the newly generated N-terminal putative peroxisomal targeting signal 2 and get targeted into the peroxisome by the import machinery. It has already been described by the group of Brown and Goldste that the soluble catalytic part of the HMGCR is fully functional. Thus we hypothesise that a peroxisomal soluble HMGCR contribute to mevalonate synthesis under conditions of high cholesterol levels adding an additional level of complexity for HMGCR regulation. Using density gradient centrifugation sub cellular fractionation will be preformed form mouse liver and kidney homogenates, and form cell clultures under different conditions, and the soluble HMGCR will be investigated by enzymatic and western blot analysis in different sub cellular compartments. We will characterize the peroxisomal targeting signal by subcloning, in vitro mutagenesis and by testing the interaction with the pex7 receptor using the Yeast Two Hybrid system. We further overexpress the soluble HMGCR in the cytoplasm and in the peroxiosmes in order to investigate the influences on de novo biosynthesis of cholesterol and farnesyl. This proposal will finally prove whether a peroxisomal HMGCR exists under certain conditions and whether a peroxisomal HMGCR can contribute to mevalonate pathway.
Funder: Austrian Science Fund
Amount: €149,500
Funding type: grant
Funder scheme: Principal Investigator Projects
Start date: 2009-08-01
End date: 2013-03-31
Investigator: Johannes Berger
Institution: Medizinische Universität Wien
Subfield: Molecular Biology
Domain: Life Sciences
Funded outputs count: 3
Funder award ID: P21950
OpenAlex ID: G1719061908