7-Dehydrocholesterol–dependent proteolysis of HMG-CoA reductase suppresses sterol biosynthesis in a mouse model of Smith-Lemli-Opitz/RSH syndrome
Barbara U. Fitzky, Fabian F. Moebius, Hitoshi Asaoka, Heather Waage-Baudet, Liwen Xu, Guorong Xu, Nobuyo Maeda, Kimberly Kluckman, Sylvia Hiller, Hongwei Yu, Ashok K. Batta, Sarah Shefer, Thomas Chen, Gerald Salen, Kathleen Sulik, Robert D. Simoni, Gene C. Ness, Hartmut Glossmann, Shailendra B. Patel, G.S. Tint
Barbara U. Fitzky, Fabian F. Moebius, Hitoshi Asaoka, Heather Waage-Baudet, Liwen Xu, Guorong Xu, Nobuyo Maeda, Kimberly Kluckman, Sylvia Hiller, Hongwei Yu, Ashok K. Batta, Sarah Shefer, Thomas Chen, Gerald Salen, Kathleen Sulik, Robert D. Simoni, Gene C. Ness, Hartmut Glossmann, Shailendra B. Patel, G.S. Tint
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Article

7-Dehydrocholesterol–dependent proteolysis of HMG-CoA reductase suppresses sterol biosynthesis in a mouse model of Smith-Lemli-Opitz/RSH syndrome

Abstract

Smith-Lemli-Opitz/RSH syndrome (SLOS), a relatively common birth-defect mental-retardation syndrome, is caused by mutations in DHCR7, whose product catalyzes an obligate step in cholesterol biosynthesis, the conversion of 7-dehydrocholesterol to cholesterol. A null mutation in the murine Dhcr7 causes an identical biochemical defect to that seen in SLOS, including markedly reduced tissue cholesterol and total sterol levels, and 30- to 40-fold elevated concentrations of 7-dehydrocholesterol. Prenatal lethality was not noted, but newborn homozygotes breathed with difficulty, did not suckle, and died soon after birth with immature lungs, enlarged bladders, and, frequently, cleft palates. Despite reduced sterol concentrations in Dhcr7–/– mice, mRNA levels for 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-controlling enzyme for sterol biosynthesis, the LDL receptor, and SREBP-2 appeared neither elevated nor repressed. In contrast to mRNA, protein levels and activities of HMG-CoA reductase were markedly reduced. Consistent with this finding, 7-dehydrocholesterol accelerates proteolysis of HMG-CoA reductase while sparing other key proteins. These results demonstrate that in mice without Dhcr7 activity, accumulated 7-dehydrocholesterol suppresses sterol biosynthesis posttranslationally. This effect might exacerbate abnormal development in SLOS by increasing the fetal cholesterol deficiency.

Authors

Barbara U. Fitzky, Fabian F. Moebius, Hitoshi Asaoka, Heather Waage-Baudet, Liwen Xu, Guorong Xu, Nobuyo Maeda, Kimberly Kluckman, Sylvia Hiller, Hongwei Yu, Ashok K. Batta, Sarah Shefer, Thomas Chen, Gerald Salen, Kathleen Sulik, Robert D. Simoni, Gene C. Ness, Hartmut Glossmann, Shailendra B. Patel, G.S. Tint

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Figure 1

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(a) Disruption of the Dhcr7 gene. The coding sequence of exon 8 and the ...
(a) Disruption of the Dhcr7 gene. The coding sequence of exon 8 and the 5′ flanking splice acceptor site and part of the 3′ flanking untranslated region (indicated in gray) of the wild-type gene were replaced with a neomycin resistance gene in opposite orientation to Dhcr7 transcription. Restriction sites used for subcloning of short and long Dhcr7 gene segments are shown. The targeting construct was linearized with ClaI (arrow). Primers employed for PCR genotyping are labeled a, b, and neo (see Methods). (b) Tentative topology model of Dhcr7. The induced Dhcr7 mutation is expected to delete one-third of the Dhcr7 protein (the residues shown as filled circles). The box indicates the putative sterol-binding site REF. (c) Total RNA extracted from the livers of Dhcr7–/–, Dhcr7+/–, and wild-type newborn mice was reverse transcribed and amplified using specific primers for the 3′-end of Dhcr7 mRNA and GADPH. (d) Immunoblot analysis of Dhcr7. Upper panel: Loading decreasing amounts of total hepatic protein results in corresponding reductions in Dhcr7 Ab response. Lower panel: Microsomal protein from yeast (WA0) and human tsA-201 cells (tsA) heterologously expressing DHCR7 with an N-terminal myc tag (15) and from livers of newborn wild-type mice demonstrated immunoreactive Dhcr7 protein. Bands corresponding to myc-tagged (myc) and wild-type (WT) DHCR7/Dhcr7 proteins, respectively, are indicated by arrowheads. In contrast, no immunoreactive band corresponding to wild-type Dhcr7 was detected in 40 μg of microsomal protein from either homozygous mice (last two lanes) or mock-transfected yeast (MOCK).