Yeast Sterol C₈−C₇ Isomerase:  Identification and Characterization of a High-Affinity Binding Site for Enzyme Inhibitors

Abstract

The yeast gene ERG2 encodes a sterol C8-C7 isomerase and is essential for ergosterol synthesis and cell proliferation. Its striking homology with the so-called sigma1 receptor of guinea pig brain, a polyvalent steroid and drug binding protein, suggested that the yeast sterol C8-C7 isomerase (ERG2) carries a similar high affinity drug binding domain. Indeed the sigma ligands [3H]haloperidol (Kd = 0.3 nM) and [3H]ifenprodil (Kd = 1.4 nM) bound to a single population of sites in ERG2 wild type yeast microsomes (Bmax values of 77 and 61 pmol/mg of protein, respectively), whereas binding activity was absent in strains carrying ERG2 gene mutations or disruptions. [3H]Ifenprodil binding was inhibited by sterol isomerase inhibitors such as fenpropimorph (Ki = 0.05 nM), tridemorph (Ki = 0.09 nM), MDL28,815 (Ki = 0.44 nM), triparanol (Ki = 1.5 nM), and AY-9944 (Ki = 5.8 nM). [3H]Haloperidol specifically photoaffinity-labeled a protein with an apparent molecular weight of 27400, in agreement with the molecular mass of the sterol C8-C7 isomerase (24900 Da). 9E10 c-myc antibodies specifically immunoprecipitated the c-myc tagged protein after [3H]haloperidol photolabeling, unequivocally proving that the drug binding site is localized on the ERG2 gene product. Haloperidol, trifluperidol, and ifenprodil inhibited the growth of Saccharomyces cerevisiae and reduced the ergosterol content of cells grown in their presence. Our results demonstrate that the yeast sterol C8-C7 isomerase has a polyvalent high-affinity drug binding site similar to mammalian sigma receptors and that in yeast sigma ligands inhibit sterol biosynthesis.

Antiproliferative effects of SR31747A in animal cell lines are mediated by inhibition of cholesterol biosynthesis at the sterol isomerase step

“…However, the IC 50 value deduced from the in vitro assay is much higher. This result is not surprising in the light of the previous results obtained by others [21] and by ourselves [6] with the yeast sterol isomerase. Indeed, a similar discrepancy has already been observed with various inhibitors of the yeast sterol isomerase, including SR31747A.…”

“…Inhibition of cholesterol biosynthesis, whether at the early HMG CoA reductase step (by lovastatin or compactin [24]), or at final steps [by 24(R,S),25-iminolanosterol [25], BM 17.766 [26]], has been shown to induce growth inhibition of tumour cells. Although we did not show that SR31747A inhibits liver sterol isomerase in vivo, such an inhibition can be expected as liver sterol isomerase is pharmacologically indistinguishable from the yeast and mammalian enzymes [21]. Furthermore, it is worth noting that tamoxifen is another M1 sterol isomerase inhibitor, which competitively inhibits SR31747A binding to this enzyme [27].…”

Molecular cloning and expression of the human Δ7-sterol reductase

“…This isomerase is a putative target of drugs effective in animal models of stroke (13). Surprisingly, the mammalian genome contains another protein without homology to the human sterol ⌬8-⌬7 isomerase that shows significant pharmacological and structural similarities with the sterol ⌬8-⌬7 isomerase of Saccharomyces cerevisiae (14)(15)(16)(17). We now report the cloning of the ultimate enzyme of mammalian sterol biosynthesis, the ⌬7-sterol reductase.…”

Activation of the molecular chaperone, sigma 1 receptor, preserves cone function in a murine model of inherited retinal degeneration

“…Sigma 1 receptor (Sig-1R), a promising target for the treatment of neurodegenerative disease because of its multifaceted roles in cellular survival (6)(7)(8), is a unique membrane protein with no homology to other mammalian proteins (9,10). N,N-dimethyltryptamine is the only endogenous Sig1R ligand identified (11).…”