HMGCR and the peroxisome

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.

Many different organelles are responsible for the appropriate function of each cell type within the human body. The mitochondria, for example, are among other functions responsible for energy production or the lysosomes for the degradation and recycling of metabolites. The peroxisomes, another essential organelle, are responsible for the synthesis (e.g. plasmalogens) or the degradation (e.g. very long-chain fatty acids) of different lipids. It addition, it has been suggested that the peroxisomes play a role in cholesterol metabolism. Within this project, we have focused on the rate limiting enzyme of the cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR). Competitive inhibition of HMGCR is the target for statins, the main cholesterol lowering drugs. A cholesterol-dependent negative feedback loop promotes degradation of HMGCR localized at the membrane of the endoplasmic reticulum. This degradation leads to soluble but functional truncated HMGCR cleavage products, which are normally further degraded in the proteasome. By using fluorescence microscopy and density gradient centrifugation, we could demonstrate that in cell lines representing phagocytosing cells (monocyte and microglia), a truncated degradation product of HMGCR localizes to the peroxisome. We demonstrated that the transport of this truncated degradation product into peroxisomes is an active process. We identified a peroxisomal targeting signal 2 near the N-terminus of this truncated HMGCR and demonstrated a functional interaction with the receptor PEX7, which mediates the trafficking to the peroxisome. Furthermore, we could demonstrate that this truncated HMGCR is enzymatically active in the cytosol but inactive when targeted to peroxisomes. Thus, the peroxisomal import of the truncated HMGCR contributes to the clearance of HMGCR activity in the cytosol under high cholesterol conditions in monocytes. The peroxisomal import of the HMGCR degradation products in monocytes adds an additional level of complexity to the mechanisms regulating the HMGCR activity under different metabolic conditions in different cell types.