Subcellular localization of sterol biosynthesis enzymes

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.

Keywords: 7-Dehydrocholesterol; 8-Dehydrocholesterol; DHCR24; DHCR7; Desmosterol; EBP.

Figure 1.. Schematic drawing of the distal cholesterol biosynthesis pathway, with focus on DHCR7, DHCR24, SC5D and EBP.

Diseases associated with their insufficiency are in red lettering.

Figure 2.. DHCR7, DHCR24 and EBP proteins localize to the endoplasmic reticulum.

Neuro2a cells were transiently transfected with EGFP-DHCR7, EGFPDHCR24 and ptdTomato-EBP fusion constructs. Cells were labeled with anti-PDI (luminal ER protein) antibody 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 all three proteins of interest co-localize with PDI, suggesting ER expression.

Figure 3.. DHCR7 protein localizes to the Golgi apparatus.

Neuro2a cells were transiently transfected with EGFP-DHCR7, EGFP-DHCR24 and ptdTomato-EBP fusion constructs. Cells were labeled with anti-RCAS1 (Golgi resident protein) antibody 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 EGFPDHCR7 localized to the Golgi apparatus while EGFP-DHCR24 and ptdTomato-EBP showed no expression in the Golgi.

Figure 4.. DHCR7, DHCR24 and EBP proteins show comparable expression in the nuclear envelope.

Neuro2a cells were transiently transfected with EGFP-DHCR7, EGFP-DHCR24 and ptdTomato-EBP fusion constructs. Cells were labeled with anti-emerin (localizing to the ER and NE inner and outer membranes) antibody 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 all three recombinant proteins co-localized with emerin in the nuclear envelope, and ER localization for all three proteins was similar to that observed previously with anti-PDI labeling.

Figure 5.. DHCR7, DHCR24, EBP and SC5D cholesterol biosynthesis enzymes show only a partial co-localization.

Neuro2a cells were transiently transfected with EGFP-DHCR7 and EGFP-DHCR24 fusion constructs (rows 1–3) and labeled with anti-EBP and anti-DHCR7. Control Neuro2a cells were double stained with anti-SC5D and anti-EBP antibodies (bottom row). Cells were 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 DHCR7 and DHCR24 showed the most prominent co-localization throughout the cellular compartments, while the more proximal sterol biosynthesis enzyme SC5D in addition to overlap with DHCR7, DHCR24 and EBP within ER is also present in the nucleus.

Figure 6.. DHCR24 expression compensates for the lack of DHCR7 in Dhcr7^−/− newborn mice brains.

Western blot analysis of DHCR7, DHCR24 and SC5D proteins in whole brain tissue samples of Dhcr7^+/+, Dhcr7^–/–, Dhcr7^+/− newborn mice. 40 μg total protein was loaded per lane, 3 biological replicates are shown. Data were normalized to beta-actin. DHCR24 protein expression was greatly increased (2.25-fold, p<0.05) in Dhcr7^–/– (knockout) whole brain tissue lysates when compared to Dhcr7^+/+ (wild type). There was no difference between Dhcr7^+/− (heterozygous) and Dhcr7^+/+ (wild type) samples for DHCR24. SC5D protein expression level did not show any difference in knockout or heterozygous samples in comparison to the wild type samples.