Markus Veen

Combined overexpression of genes of the ergosterol biosynthetic pathway leads to accumulation of sterols in Saccharomyces cerevisiae

Genes of the post-squalene ergosterol biosynthetic pathway in Saccharomyces cerevisiae have been overexpressed in a systematic approach with the aim to construct yeast strains that produce high amounts of sterols from a squalene-accumulating strain. This strain had previously been deregulated by overexpressing a truncated HMG-CoA reductase (tHMG1) in the main bottleneck of the early ergosterol pathway. The overexpression of the gene ERG1 (squalene epoxidase) induced a significant decrease of the direct substrate squalene, a high increase of lanosterol, and a small increase of later sterols. The overexpression of the ERG11 gene encoding the sterol-14alpha-demethylase resulted in a decrease of lanosterol and an increase of downstream sterols. When these two genes were simultaneously overexpressed, later sterols from zymosterol to ergosterol accumulated and the content of squalene was decreased about three-fold, indicating that these steps had limited the transformation of squalene into sterols. The total sterol content in this strain was three-fold higher than in a wild-type strain.

Mutations causing Greenberg dysplasia but not Pelger anomaly uncouple enzymatic from structural functions of a nuclear membrane protein.

The lamin B receptor (LBR) is an inner nuclear membrane protein with a structural function interacting with chromatin and lamins, and an enzymatic function as a sterol reductase. Heterozygous LBR mutations cause nuclear hyposegmentation in neutrophils (Pelger anomaly), while homozygous mutations cause prenatal death with skeletal defects and abnormal sterol metabolism (Greenberg dysplasia). It has remained unclear whether the lethality in Greenberg dysplasia is due to cholesterol defects or altered nuclear morphology. To answer this question we characterized two LBR missense mutations and showed that they cause Greenberg dysplasia. Both mutations affect residues that are evolutionary conserved among sterol reductases. In contrast to wildtype LBR, both mutations failed to rescue C14 sterol reductase deficient yeast, indicating an enzymatic defect. We found no Pelger anomaly in the carrier parent excluding marked effects on nuclear structure. We studied Lbr in mouse embryos and demonstrate expression in skin and the developing skeletal system consistent with sites of histological changes in Greenberg dysplasia. Unexpectedly we found in disease-relevant cell types not only nuclear but also cytoplasmatic LBR localization. The cytoplasmatic LBR staining co-localized with ER-markers and is thus consistent with the sites of endogeneous sterol synthesis. We conclude that LBR missense mutations can abolish sterol reductase activity, causing lethal Greenberg dysplasia but not Pelger anomaly. The findings separate the metabolic from the structural function and indicate that the sterol reductase activity is essential for human intrauterine development.

Overexpression of genes of the fatty acid biosynthetic pathway leads to accumulation of sterols in Saccharomyces cerevisiae

Saccharomyces cerevisiae strains with deregulated sterol and fatty acid biosynthesis pathways were analysed for sterol and fatty acid content and mRNA profiles, with the aim of identifying interactions between lipid biosynthesis pathways. Acetyl CoA carboxylase ACC1 and fatty acid synthases FAS1/FAS2 were overexpressed in wild‐type and squalene‐overproducing strains. ACC1 overexpression led to decreased fatty acid content in the squalene‐overproducing strain (factor of 0.7), while sterols and squalene were increased (factor of 1.5). In the wild‐type strain, ACC1 overexpression led to increased levels of both fatty acids and squalene/sterols (factors of 4.0 and 1.7, respectively). This parallel activation of the two pathways seems to be due to transcriptional co‐regulation of ACC1 and HMG1. While FAS1 and FAS2 overexpression had no effect in the wild‐type strain, FAS2 overexpression induced significant increase of sterols and squalene (factors of 7.2 and 1.3, respectively) and a concomitant decrease of both saturated and unsaturated fatty acids in the squalene‐overproducing strain (factor of 0.6). The microarray expression profiles showed that genes upregulated in ACC1‐overexpressing strains are FAS1, ERG11, ERG28, ERG5, ERG2 and ERG20, supporting the observed increase of zymosterol and saturated fatty acids. The high ACC1 expression level due to overexpression correlated with increased transcript levels of sphingolipid and sterol biosynthesis genes. The relationship between was shown using the Pathway Studio™ program. Copyright