Molecular docking studies of Chenopodium album Linn with Lanosterol synthase enzyme

Cardiovascular diseases (CVD) are the major cause of death among people across the globe. Hypercholesterolemia is one of the major contributing factors for CVD. Molecules that bind with Lanosterol synthase enzyme, can be potential drug targets. Statin group of compounds like Simvastatin, cerivastatin, Atorvastatin etc., used for treating hypercholesterolemia have side effects and hence there is a growing demand for plant derived flavonoids. This work focusses on studying the compounds quercetin-3-O-(2??,6??-di-O-?-l-rhamnopyranosyl)-?-d-glucopyranoside, kaempferol-3-O-(2??,6??-di-O-?-l-rhamnopyranosyl)-?-d-glucopyranoside, rutin; quercetin-3-O-?-d-glucopyranoside (Iso quercetin); and kaempferol-3-O-?-d-glucopyranoside (Astragalin) present in Chenopodium album Linn to inhibit Lanosterol synthase. Bioactivity score, drug likeness character was assessed in silico. Based on bioactivity spectrum, it is observed that the molecules are biologically active and the probability of these compounds to be biologically active is ranging from 0.784 to 0.992, suggesting that these compounds are effective for treating hypercholesterolemia. In the molecular docking studies, the compounds binding affinity score was in agreement that the molecules have the potential to be used as an alternative to the statin group of compounds in treating cholesterol.

Viperin inhibits cholesterol biosynthesis and interacts with enzymes in the cholesterol biosynthetic pathway

Many enveloped viruses bud from cholesterol-rich lipid rafts on the cell membrane. Depleting cellular cholesterol impedes this process and results in viral particles with reduced viability. Viperin (virus inhibitory protein endoplasmic reticulum-associated, interferon-induced) is an ER membrane-associated enzyme that when expressed in response to viral infections exerts broad-ranging antiviral effects, including inhibiting the budding of some enveloped viruses. Here we have investigated the effect of viperin expression on cholesterol biosynthesis. We found that viperin expression reduces cholesterol levels by 20 – 30 % in HEK293T cells. A proteomic screen of the viperin interactome identified several cholesterol biosynthetic enzymes among the top hits. The two most highly enriched proteins were lanosterol synthase and squalene monooxygenase, enzymes that catalyze key steps establishing the sterol carbon skeleton. Co-immunoprecipitation experiments established that viperin, lanosterol synthase and squalene monooxygenase form a complex at the ER membrane. Co-expression of viperin was found to significantly inhibit the specific activity of lanosterol synthase in HEK293T cell lysates. Co-expression of viperin had no effect on the specific activity of squalene monooxygenase, but reduced its expression levels in the cells by approximately 30 %. Despite these inhibitory effects, co-expression of either LS or SM failed to reverse the viperin-induced depletion of cellular cholesterol levels in HEK293T cells. Our results establish a clear link between the down-regulation of cholesterol biosynthesis and viperin, although at this point the effect cannot be unambiguously attributed interactions between viperin and a specific biosynthetic enzyme.

Identification and Characterization of A Lanosterol synthase Gene from Sanghuangporus Baumii

Lanosterol synthase (LS) is a key enzyme involved in the mevalonate pathway (MVA pathway) to produce lanosterol, which is a precursor for synthesizing Sanghuangporus baumii triterpenoids. To research the characteristics and construction of LS, LS ORF and promoter were cloned from S. baumii. A 2,445 bp S. baumii LS sequence was obtained by rapid amplification of cDNA ends (RACE) technology and recombinant PCR. S. baumii LS sequence includes a 5’-untranslated region (129 bp), a 3’-untranslated region (87 bp), and an open reading frame (2,229 bp) encoding a 734 amino acids. The molecular weight of LS is 84.99 kDa, and transcription start site of S. baumii LS promoter sequence ranged from 1 740 bp to 1790 bp. LS promoter contained 12 CAAT-boxes, 5 ABREs, 6 G-Boxes, 6 CGTCA-motifs, and so on. The S. baumii LS protein was expressed in E. coli BL21 (DE3) (84.99 kDa + 21.15 kDa tag protein). The transcription level of S. baumii LS was the highest on day 11 in mycelia (1.6-fold).

Identification of differential gene expression pattern in lens epithelial cells derived from cataractous and non-cataractous lenses of Shumiya cataract rat

The Shumiya cataract rat (SCR) is a model for hereditary cataract. Two-third of these rats develop lens opacity within 10-11-weeks. Onset of cataract is attributed to the synergetic effect of lanosterol synthase (Lss) and farnesyl-diphosphate farnesyltransferase 1 (Fdft1) mutant alleles that lead to cholesterol deficiency in the lenses, which in turn adversely affects lens biology including the growth and differentiation of lens epithelial cells (LECs). Nevertheless, the molecular events and changes in gene expression associated with the onset of lens opacity in SCR is poorly understood. In the present study, a microarray-based approach was employed to analyze comparative gene expression changes in LECs isolated from the pre-cataractous and cataractous stages of lenses of 5-, 8- and 10-week-old SCRs. The changes in gene expression observed in microarray results in the LECs were further validated using real-time PCR and western blot analyses. Lens opacity was not observed in 5-week-old rats. However, histological analysis revealed mild dysplasia in the anterior suture and poorly differentiated fiber cells at the bow region. Expression of approximately 100 genes, including major intrinsic protein of lens fiber (MIP and Aquaporin0), deoxyribonuclease II beta (Dnase2b), heat shock protein B1 (Hspb1), and crystallin γ (γCry) B, C, and F were found to be significantly downregulated (0.07-0.5 fold) in rat LECs derived from cataract lenses compared to that in noncataractous lenses (control). Thus, our study aimed to identify the gene expression patterns during cataract formation in SCRs, which may be responsible for cataractogenesis in SCR. We proposed that mutation in lanosterol synthase was responsible for the downregulation of genes associated with lens fiber differentiation, which in turn leads to the formation of cataract in these rats. Our findings may have wider implications in understanding the effect of cholesterol deficiency and the role of cholesterol-lowering therapeutics on cataractogenesis.

The cholesterol synthesis enzyme lanosterol 14α-demethylase is post-translationally regulated by the E3 ubiquitin ligase MARCH6

Cholesterol synthesis is a tightly controlled pathway, with over 20 enzymes involved. Each of these enzymes can be distinctly regulated, helping to fine-tune the production of cholesterol and its functional intermediates. Several enzymes are degraded in response to increased sterol levels, whilst others remain stable. We hypothesised that an enzyme at a key branch point in the pathway, lanosterol 14α-demethylase (LDM) may be post-translationally regulated. Here, we show that the preceding enzyme, lanosterol synthase is stable, whilst LDM is rapidly degraded. Surprisingly, this degradation is not triggered by sterols. However, the E3 ubiquitin ligase membrane-associated ring-CH-type finger 6 (MARCH6), known to control earlier rate-limiting steps in cholesterol synthesis, also control levels of LDM and the terminal cholesterol synthesis enzyme, 24-dehydrocholesterol reductase. Our work highlights MARCH6 as the first example of an E3 ubiquitin ligase that targets multiple steps in a biochemical pathway and indicates new facets in the control of cholesterol synthesis.