Quantitative proteomics analysis of inborn errors of cholesterol synthesis: identification of altered metabolic pathways in DHCR7 and SC5D deficiency

Abstract

Smith-Lemli-Opitz syndrome (SLOS) and lathosterolosis are malformation syndromes with cognitive deficits caused by mutations of 7-dehydrocholesterol reductase (DHCR7) and lathosterol 5-desaturase (SC5D), respectively. DHCR7 encodes the last enzyme in the Kandutsch-Russel cholesterol biosynthetic pathway, and impaired DHCR7 activity leads to a deficiency of cholesterol and an accumulation of 7-dehydrocholesterol. SC5D catalyzes the synthesis of 7-dehydrocholesterol from lathosterol. Impaired SC5D activity leads to a similar deficiency of cholesterol but an accumulation of lathosterol. Although the genetic and biochemical causes underlying both syndromes are known, the pathophysiological processes leading to the developmental defects remain unclear. To study the pathophysiological mechanisms underlying SLOS and lathosterolosis neurological symptoms, we performed quantitative proteomics analysis of SLOS and lathosterolosis mouse brain tissue and identified multiple biological pathways affected in Dhcr7(Delta3-5/Delta3-5) and Sc5d(-/-) E18.5 embryos. These include alterations in mevalonate metabolism, apoptosis, glycolysis, oxidative stress, protein biosynthesis, intracellular trafficking, and cytoskeleton. Comparison of proteome alterations in both Dhcr7(Delta3-5/Delta3-5) and Sc5d(-/-) brain tissues helps elucidate whether perturbed protein expression was due to decreased cholesterol or a toxic effect of sterol precursors. Validation of the proteomics results confirmed increased expression of isoprenoid and cholesterol synthetic enzymes. This alteration of isoprenoid synthesis may underlie the altered posttranslational modification of Rab7, a small GTPase that is functionally dependent on prenylation with geranylgeranyl, that we identified and validated in this study. These data suggested that although cholesterol synthesis is impaired in both Dhcr7(Delta3-5/Delta3-5) and Sc5d(-/-) embryonic brain tissues the synthesis of nonsterol isoprenoids may be increased and thus contribute to SLOS and lathosterolosis pathology. This proteomics study has provided insight into the pathophysiological mechanisms of SLOS and lathosterolosis, and understanding these pathophysiological changes will help guide clinical therapy for SLOS and lathosterolosis.

Fig. 1.

Representative 2-DE maps of SLOS and lathosterolosis mouse brain proteins. A, SLOS and lathosterolosis are inborn errors of cholesterol synthesis. SLOS is caused by mutations in the DHCR7 gene. DHCR7 catalyzes the final step in cholesterol synthesis. Lathosterolosis is caused by mutations of the SC5D gene. Cholesterol levels are decreased in both SLOS and lathosterolosis, but the accumulating precursor sterol differs. In SLOS, 7DHC accumulates, whereas in lathosterolosis, the accumulating sterol is lathosterol. B, representative 2-DE maps of control (Dhcr7^+/+ and Sc5d^+/+), Dhcr7^{Δ3–5/Δ3–5}, and Sc5d^−/− mouse brain proteins. Eighty micrograms of the pooled protein sample from Dhcr7^+/+, Dhcr7^{Δ3–5/Δ3–5}, Sc5d^+/+, and Sc5d^−/− embryonic mouse brain tissues were separated on a pH 3–10 nonlinear IPG strip followed by electrophoretic separation on a 12% SDS-polyacrylamide gel. Acidic pH is to the left, and increased molecular mass is at the top. Compared with Dhcr7^+/+ mouse brains, the protein spots with significantly decreased or increased expression in Dhcr7^{Δ3–5/Δ3–5} mouse brains are marked in Dhcr7^+/+ and Dhcr7^{Δ3–5/Δ3–5} mouse brain 2-DE maps, respectively. Compared with Sc5d^+/+ mouse brains, the protein spots with significantly decreased or increased expression in Sc5d^−/− mouse brains are marked in Sc5d^+/+ and Sc5d^−/− mouse brain 2-DE maps, respectively. Supplemental Table 2 provides detailed information on the differentially expressed protein spots.

Fig. 2.

Functional annotation of differentially expressed proteins that were identified in Dhcr7^{Δ3–5/Δ3–5} and Sc5d^−/− embryonic brain tissues. A, functional annotation of 36 differentially expressed (r > 1.5, p < 0.05) proteins in Dhcr7^{Δ3–5/Δ3–5} embryonic brain tissue. B, functional annotation of 30 differentially expressed (r > 1.5, p < 0.05) proteins in Sc5d^−/− embryonic brain tissue.

Fig. 3.

Activation of mevalonate metabolism pathway in Dhcr7^{Δ3–5/Δ3–5} and Sc5d^−/− embryonic brain tissues. A, representative regions from the 2-DE gels demonstrating differential expression of protein spots 17 and 49, which were subsequently identified as HMGCS1 and IDI1, respectively. B, Western blot analysis of pooled protein samples (20 μg) with anti-HMGCS1 and anti-GGPS validated altered expression of these two proteins. C, Western blot analysis of the pooled protein samples (20 μg) with SREBP-2 and SREBP-1 antibodies showed increased activation of SREBP-2 but no change in SREBP1. D, Western blot analysis of HMGCS1, GGPS, c-SREBP-2, and c-SREBP-1 from five independent Dhcr7^+/+ or Dhcr7^{Δ3–5/Δ3–5} brain tissue protein samples (20 μg) confirmed increased protein expression for HMGCS1, GGPS, and c-SREBP2 (mean ± S.D., n = 5, *, p < 0.05; **, p < 0.01). E, Western blot analysis of HMGCS1, GGPS, c-SREBP-2, and c-SREBP-1 from five independent Sc5d^+/+ or Sc5d^−/− brain tissue protein samples (20 μg) confirmed increased protein expression for HMGCS1, GGPS, and c-SREBP2 (mean ± S.D.; n = 5; *, p < 0.05). Band intensity was normalized to that of actin. Error bars indicate the S.D. calculated from five individual samples.

Fig. 4.

Differential expression of Rab7 and Rab5 in Dhcr7^{Δ3–5/Δ3–5} and Sc5d^−/− embryonic brain tissues. A, representative 2-DE gel sections of protein spots 50 and 51 identified as Rab7. B, Western blot analysis of pooled protein samples (20 μg) with anti-Rab7 and anti-Rab5 antibodies. C, increased expression of both Rab7 and Rab5 was confirmed in five independent Dhcr7^+/+ or Dhcr7^{Δ3–5/Δ3–5} brain tissue samples (mean ± S.D.; n = 5; *, p < 0.05; **, p < 0.01). D, increased expression of both Rab7 and Rab5 was confirmed in five independent Sc5d^+/+ or Sc5d^−/− brain tissue samples (mean ± S.D.; n = 5; *, p < 0.05). Band intensity on Western blots was quantified and normalized to that of actin. Error bars indicate the S.D. calculated from five individual samples.

Fig. 5.

Activation of caspase-3 in Dhcr7^{Δ3–5/Δ3–5} and Sc5d^−/− embryonic brain tissues. A, representative regions from the 2-DE gels demonstrating differential expression of protein spot 39, which was subsequently identified as caspase-3 precursor. B, Western blot analysis of pooled protein samples (20 μg) with anti-caspase-3 showed increased cleaved caspase-3 in Dhcr7^{Δ3–5/Δ3–5} and Sc5d^−/− brain tissues. C, Western blot analysis of caspase-3 from five independent Dhcr7^+/+ or Dhcr7^{Δ3–5/Δ3–5} brain tissue protein samples (20 μg) confirmed increased cleaved caspase-3 (mean ± S.D.; n = 5; *, p < 0.05). D, Western blot analysis of caspase-3 from five independent Sc5d^+/+ or Sc5d^−/− brain tissue protein samples (20 μg) confirmed increased cleaved caspase-3 (mean ± S.D.; n = 5; **, p < 0.01). Band intensity was normalized to that of actin. Error bars indicate the S.D. calculated from five individual samples. E, Western blot analysis showed that cleaved caspase-3 increased both in DSF and in DRM of Dhcr7^{Δ3–5/Δ3–5} brain tissue. Transferrin receptor and clathrin heavy chain were used as marker proteins for DSF and DRM, respectively.