Abstract
Cholesterol synthesis is a complex, coordinated process involving a series of enzymes. As of today, our understanding of subcellular localization of cholesterol biosynthesis enzymes is far from complete. Considering the complexity and intricacies of this pathway and the importance of functions of DHCR7, DHCR24 and EBP enzymes for human health, we undertook a study to determine their subcellular localization and co-localization. Using expression constructs and antibody staining in cell cultures and transgenic mice, we found that all three enzymes are expressed in ER and nuclear envelope. However, their co-localization was considerably different across the cellular compartments. Furthermore, we observed that in the absence of DHCR7 protein, DHCR24 shows a compensatory upregulation in a Dhcr7−/− transgenic mouse model. The overall findings suggest that the sterol biosynthesis enzymes might not always work in a same functional complex, but that they potentially have different, multifunctional roles that go beyond the sterol biosynthesis pathway. Furthermore, the newly uncovered compensatory mechanism between DHCR7 and DHCR24 could be of importance for designing medications that would improve cholesterol production in patients with desmosterolosis and Smith–Lemli–Opitz syndrome.
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Acknowledgements
This work was supported by The National Institutes of Health, NIMH MH110636 (KM) and MN067234 (KM). Katalin Koczok was the Rosztoczy Foundation scholar.
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10735_2018_9807_MOESM1_ESM.pptx
Supplementary Figure 1. Co-localization of fluorescently tagged recombinant proteins and antibody staining. In this validation study, Neuro2a cells were transiently transfected with EGFP-DHCR7, EGFP-DHCR24 and ptdTomato-EBP fusion constructs. Cells were labeled with anti-DHCR7, anti-DHCR24 and anti-EBP antibodies, respectively, in order to verify antibody specificity and analyzed by confocal microscopy. Nuclei were visualized by Hoechst dye. Original images were collected with a 60× oil objective. Calibration bar, 5 µm. Note that the recombinant proteins and antibody staining fully co-localize. (PPTX 2331 KB)
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Koczok, K., Gurumurthy, C.B., Balogh, I. et al. Subcellular localization of sterol biosynthesis enzymes. J Mol Hist 50, 63–73 (2019). https://doi.org/10.1007/s10735-018-9807-y
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DOI: https://doi.org/10.1007/s10735-018-9807-y