Biallelic mutations in LSS in autosomal-recessive mutilating palmoplantar keratoderma.

Mutilating palmoplantar keratoderma (PPK) is a heterogeneous genetic disease that poses enormous challenges to clinical diagnosis and genetic counselling. Lanosterol synthase (LSS) gene encodes LSS involved in the biosynthesis pathway of cholesterol. Biallelic mutations in LSS were found to be related to diseases such as cataracts, hypotrichosis and palmoplantar keratoderma-congenital alopecia syndrome. The aim of this study was to investigate the contribution of the LSS mutation to mutilating PPK in a Chinese patient. The clinical and molecular characteristics of the patient were evaluated. A 38-year-old male patient with mutilating PPK was recruited in this study. We identified biallelic variants in the LSS gene (c.683C > T, p.Thr228Ile and c.779G > A, p.Arg260His). Immunoblotting revealed that the Arg260His mutant showed a significantly reduced expression level while Thr228Ile showed an expression level similar to that of the wild type. Thin layer chromatography revealed that mutant Thr228Ile retained partial enzymatic activity and mutant Arg260His did not show any catalytic activity. Our findings show the correlation between LSS mutations and mutilating PPK.

A case of LSS-associated congenital nuclear cataract with hypotrichosis and literature review.

Congenital cataract is the most common cause of lifelong visual loss in children worldwide, which has significant genotypic and phenotypic heterogeneity. The LSS gene encodes lanosterol synthase (LSS), which acts on the cholesterol biosynthesis pathway by converting (S)-2,3-oxidosqualene to lanosterol. The biallelic pathogenic variants in the LSS gene were found in congenital cataract, Alopecia-intellectual disability syndrome, hypotrichosis simplex, and mutilating palmoplantar keratoderma. In this study, we reported the first congenital nuclear cataract combined with hypotrichosis in a 12-year-old boy with biallelic LSS variants (c.1025T>G; p.I342S and c.1531_1532insT; p.L511Ffs*17) by exome sequencing. Reviewing all reported patients with LSS variants indicated that p.W629 might be a hotspot for hypospadias and p.I342S was associated with congenital cataract. Patients with one or two truncation variants tend to have multisystem symptoms compared with those with two missense variants. These findings deepen the understanding of LSS variants and contribute to the genetic counseling of affected families.

Non-syndromic hypotrichosis: A report of two novel variants in the LSS gene.

To date, more than 15 genes have been linked to syndromic and non-syndromic hypotrichosis, among which the LSS gene encoding lanosterol synthase was recently linked to autosomal recessive isolated hypotrichosis. Here we report the case of a 6-year-old girl born to non-consanguineous Iraqi parents and presenting with sparse lanugo hair since birth on the scalp, eyelashes, and eyebrows. Whole exome sequencing followed by Sanger sequencing allowed the detection of two novel compound heterozygous variants in LSS (p.Ile323Thr and p.Gly600Val). Reporting and investigating further cases with LSS variants might help establishing a better genotype-phenotype correlation.

The antiviral enzyme viperin inhibits cholesterol biosynthesis.

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 iNducible) is an endoplasmic reticulum membrane-associated enzyme that exerts broad-ranging antiviral effects, including inhibiting the budding of some enveloped viruses. However, the relationship between viperin expression and the retarded budding of virus particles from lipid rafts on the cell membrane is unclear. Here, we investigated the effect of viperin expression on cholesterol biosynthesis using transiently expressed genes in the human cell line human embryonic kidney 293T (HEK293T). We found that viperin expression reduces cholesterol levels by 20% to 30% in these cells. Following this observation, a proteomic screen of the viperin interactome identified several cholesterol biosynthetic enzymes among the top hits, including lanosterol synthase (LS) and squalene monooxygenase (SM), which are enzymes that catalyze key steps in establishing the sterol carbon skeleton. Coimmunoprecipitation experiments confirmed that viperin, LS, and SM form a complex at the endoplasmic reticulum membrane. While coexpression of viperin was found to significantly inhibit the specific activity of LS in HEK293T cell lysates, coexpression of viperin had no effect on the specific activity of SM, although did reduce SM protein levels by approximately 30%. Despite these inhibitory effects, the coexpression of neither LS nor SM was able to reverse the viperin-induced depletion of cellular cholesterol levels, possibly because viperin is highly expressed in transfected HEK293T cells. Our results establish a link between viperin expression and downregulation of cholesterol biosynthesis that helps explain viperin's antiviral effects against enveloped viruses.

Target identification reveals lanosterol synthase as a vulnerability in glioma.

Diffuse intrinsic pontine glioma (DIPG) remains an incurable childhood brain tumor for which novel therapeutic approaches are desperately needed. Previous studies have shown that the menin inhibitor MI-2 exhibits promising activity in preclinical DIPG and adult glioma models, although the mechanism underlying this activity is unknown. Here, using an integrated approach, we show that MI-2 exerts its antitumor activity in glioma largely independent of its ability to target menin. Instead, we demonstrate that MI-2 activity in glioma is mediated by disruption of cholesterol homeostasis, with suppression of cholesterol synthesis and generation of the endogenous liver X receptor ligand, 24,25-epoxycholesterol, resulting in cholesterol depletion and cell death. Notably, this mechanism is responsible for MI-2 activity in both DIPG and adult glioma cells. Metabolomic and biochemical analyses identify lanosterol synthase as the direct molecular target of MI-2, revealing this metabolic enzyme as a vulnerability in glioma and further implicating cholesterol homeostasis as an attractive pathway to target in this malignancy.

Bi-allelic Mutations in LSS, Encoding Lanosterol Synthase, Cause Autosomal-Recessive Hypotrichosis Simplex.

Hypotrichosis simplex (HS) is a rare form of hereditary alopecia characterized by childhood onset of diffuse and progressive scalp and body hair loss. Although research has identified a number of causal genes, genetic etiology in about 50% of HS cases remains unknown. The present report describes the identification via whole-exome sequencing of five different mutations in the gene LSS in three unrelated families with unexplained, potentially autosomal-recessive HS. Affected individuals showed sparse to absent lanugo-like scalp hair, sparse and brittle eyebrows, and sparse eyelashes and body hair. LSS encodes lanosterol synthase (LSS), which is a key enzyme in the cholesterol biosynthetic pathway. This pathway plays an important role in hair follicle biology. After localizing LSS protein expression in the hair shaft and bulb of the hair follicle, the impact of the mutations on keratinocytes was analyzed using immunoblotting and immunofluorescence. Interestingly, wild-type LSS was localized in the endoplasmic reticulum (ER), whereas mutant LSS proteins were localized in part outside of the ER. A plausible hypothesis is that this mislocalization has potential deleterious implications for hair follicle cells. Immunoblotting revealed no differences in the overall level of wild-type and mutant protein. Analyses of blood cholesterol levels revealed no decrease in cholesterol or cholesterol intermediates, thus supporting the previously proposed hypothesis of an alternative cholesterol pathway. The identification of LSS as causal gene for autosomal-recessive HS highlights the importance of the cholesterol pathway in hair follicle biology and may facilitate novel therapeutic approaches for hair loss disorders in general.

Citrus reticulata peel oil as an antiatherogenic agent: Hypolipogenic effect in hepatic cells, lipid storage decrease in foam cells, and prevention of LDL oxidation.

BACKGROUND AND AIMS: Hypercholesterolemia and oxidative stress are two of the most important risk factors for atherosclerosis. The aim of the present work was to evaluate mandarin (Citrus reticulata) peel oil (MPO) in cholesterol metabolism and lipid synthesis, and its antioxidant capacity. METHODS AND RESULTS: Incubation of hepatic HepG2 cells with MPO (15-60 µL/L) reduced cholesterogenesis and saponifiable lipid synthesis, demonstrated by [14C]acetate radioactivity assays. These effects were associated with a decrease in a post-squalene reaction of the mevalonate pathway. Molecular docking analyses were carried out using three different scoring functions to examine the cholesterol-lowering property of all the components of MPO against lanosterol synthase. Docking simulations proposed that minor components of MPO monoterpenes, like alpha-farnesene and neryl acetate, as well the major component, limonene and its metabolites, could be partly responsible for the inhibitory effects observed in culture assays. MPO also decreased RAW 264.7 foam cell lipid storage and its CD36 expression, and prevented low-density lipoprotein (LDL) lipid peroxidation. CONCLUSION: These results may imply a potential role of MPO in preventing atherosclerosis by a mechanism involving inhibition of lipid synthesis and storage and the decrease of LDL lipid peroxidation.

Lanosterol Synthase Genetic Variants, Endogenous Ouabain, and Both Acute and Chronic Kidney Injury.

RATIONALE & OBJECTIVE: Studies of humans and animals have suggested that endogenous ouabain (EO) and related genes are mediators of acute (AKI) and chronic kidney injury. We sought to examine the relationship among EO levels, genetic variants in lanosterol synthase (LSS; an enzyme that catalyzes synthesis of cholesterol, a precursor of EO), and both AKI and chronic kidney injury. STUDY DESIGN: 2 prospective observational cohort studies and a cross-sectional study of kidney tissue. SETTING & PARTICIPANTS: (1) A prospective cohort study of patients undergoing cardiovascular surgery, (2) measurement of EO concentration in kidney tissue removed because of an adjacent tumor, and (3) a prospective cohort study of patients with newly diagnosed essential hypertension. EXPOSURE: Missense variant in LSS (A instead of C allele at rs2254524), which leads to a valine to leucine substitution at amino acid 642. OUTCOMES: Development of postoperative AKI in the cardiovascular surgery cohort, EO concentration in kidney tissue, and estimated glomerular filtration rate (eGFR) reductions in the essential hypertension cohort. ANALYTICAL APPROACH: Logistic regression for analysis of postoperative AKI, analysis of variance for EO concentration in kidney tissue, and generalized linear models for changes in eGFR over time. RESULTS: AKI incidence following cardiovascular surgery was greater among those with the LSS rs2254524 AA genotype (30.7%) than in those with the CC genotype (17.4%; P=0.001). LSS rs2254524 AA kidneys had higher EO concentrations than CC kidneys (2.14±0.29 vs 1.25±0.08ng/g; P<0.001). In the longitudinal study of patients with essential hypertension (median follow-up, 4 years; range, 1-15 years), eGFR decline was greater among the LSS rs2254524 AA genotype group (-4.39±1.18mL/min/1.73m2 per year) than in the AC or CC genotype groups (-1.07±0.55 and -2.00±0.45mL/min/1.73m2 per year respectively; P = 0.03). LIMITATIONS: These associations do not necessarily represent causal relationships; LSS rs2254524 variants may have effects on other steroid hormones. CONCLUSIONS: These findings support the potential value of LSS rs2254524 genotype-based risk stratification to identify patients at high risk for AKI before cardiovascular surgery, as well as predict accelerated eGFR in the setting of hypertension. These findings also suggest that LSS may in part drive EO-mediated kidney damage. EO may represent a new potential therapeutic target for the prevention of AKI and slowing of kidney damage in the setting of hypertension.

Lanosterol Synthase Regulates Human Rhinovirus Replication in Human Bronchial Epithelial Cells.

Human rhinovirus (RV) infections are a significant risk factor for exacerbations of asthma and chronic obstructive pulmonary disease. Thus, approaches to prevent RV infection in such patients would give significant benefit. Through RNA interference library screening, we identified lanosterol synthase (LSS), a component of the cholesterol biosynthetic pathway, as a novel regulator of RV replication in primary normal human bronchial epithelial cells. Selective knock down of LSS mRNA with short interfering RNA inhibited RV2 replication in normal human bronchial epithelial cells. Small molecule inhibitors of LSS mimicked the effect of LSS mRNA knockdown in a concentration-dependent manner. We further demonstrated that the antiviral effect is not dependent on a reduction in total cellular cholesterol but requires a 24-hour preincubation with the LSS inhibitor. The rank order of antiviral potency of the LSS inhibitors used was consistent with LSS inhibition potency; however, all compounds showed remarkably higher potency against RV compared with the LSS enzyme potency. We showed that LSS inhibition led to an induction of 24(S),25 epoxycholesterol, an important regulator of the sterol pathway. We also demonstrated that LSS inhibition led to a profound increase in expression of the innate antiviral defense protein, IFN-ß. We found LSS to be a novel regulator of RV replication and innate antiviral immunity and identified a potential molecular mechanism for this effect, via induction of 24(S),25 epoxycholesterol. Inhibition of LSS could therefore be a novel therapeutic target for prevention of RV-induced exacerbations.

Lanosterol synthase deficiency promotes tumor progression by orchestrating PDL1-dependent tumor immunosuppressive microenvironment.

Lipid metabolic reprogramming is closely related to tumor progression with the mechanism not fully elucidated. Here, we report the immune-regulated role of lanosterol synthase (LSS), an essential enzyme in cholesterol synthesis. Database analysis and clinical sample experiments suggest that LSS was lowly expressed in colon and breast cancer tissues, which indicates poor prognosis. The biological activity of tumor cell lines and tumor progression in NOD scid gamma (NSG) mice were not affected after LSS knockdown, whereas LSS deficiency obviously aggravated tumor burden in fully immunized mice. Flow cytometry analysis showed that LSS knockdown significantly promoted the formation of tumor immunosuppressive microenvironment, characterized by the increase in M2 macrophages and polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs), as well as the decrease in anti-tumoral T lymphocytes. With the inhibition of myeloid infiltration or loss function of T lymphocytes, the propulsive effect of LSS knockdown on tumor progression disappeared. Mechanistically, LSS knockdown increased programmed death ligand 1 (PDL1) protein stability by 2,3-oxidosqualene (OS) binding to PDL1 protein. Anti-PDL1 therapy abolished LSS deficiency-induced immunosuppressive microenvironment and cancer progression. In conclusion, our results show that LSS deficiency promotes tumor progression by establishing an OS-PDL1 axis-dependent immunosuppressive microenvironment, indicative of LSS or OS as a potential hallmark of response to immune checkpoint blockade.

ATR promotes mTORC1 activation via de novo cholesterol synthesis in p16-low cancer cells.

DNA damage and cellular metabolism are intricately linked with bidirectional feedback. Two of the main effectors of the DNA damage response and control of cellular metabolism are ATR and mTORC1, respectively. Prior work has placed ATR upstream of mTORC1 during replication stress, yet the direct mechanism for how mTORC1 is activated in this context remain unclear. We previously published that p16-low cells have mTORC1 hyperactivation, which in part promotes their proliferation. Using this model, we found that ATR, but not ATM, is upstream of mTORC1 activation via de novo cholesterol synthesis and is associated with increased lanosterol synthase (LSS). Indeed, p16-low cells showed increased cholesterol abundance. Additionally, knockdown of either ATR or LSS decreased mTORC1 activity. Decreased mTORC1 activity due to ATR knockdown was rescued by cholesterol supplementation. Finally, using both LSS inhibitors and multiple FDA-approved de novo cholesterol synthesis inhibitors, we found that the de novo cholesterol biosynthesis pathway is a metabolic vulnerability of p16-low cells. Together, our data provide new evidence coupling the DNA damage response and cholesterol metabolism and demonstrate the feasibility of using FDA-approved cholesterol-lowering drugs in tumors with loss of p16.

Lanosterol synthase loss of function decreases the malignant phenotypes of HepG2 cells by deactivating the Src/MAPK signaling pathway.

Cholesterol is critical for tumor cells to maintain their membrane components, cell morphology and activity functions. The inhibition of the cholesterol pathway may be an efficient strategy with which to limit tumor growth and the metastatic process. In the present study, lanosterol synthase (LSS) was knocked down by transfecting LSS short hairpin RNA into HepG2 cells, and cell growth, apoptosis and migratory potential were then detected by Cell Counting Kit-8 cell proliferation assay, flow cytometric analysis and wound healing assay, respectively. In addition, proteins associated with the regulation of the aforementioned cell biological behaviors were analyzed by western blot analysis. The activity of the Src/MAPK signaling pathway was measured by western blotting to elucidate the possible signal transduction mechanisms. LSS knockdown in the HepG2 liver cancer cell line inhibited cell proliferation, with cell cycle arrest at the S phase; it also decreased cell migratory ability and increased apoptosis. The expression proteins involved in the regulation of cell cycle, cell apoptosis and migration was altered by LSS knockdown in HepG2 cells. Furthermore, a decreased Src/MAPK activity was observed in the HepG2 cells subjected to LSS knockdown. LSS loss of function decreased the malignant phenotypes of HepG2 cells by deactivating the Src/MAPK signaling pathway and regulating expression of genes involved in cell cycle regulation, cell apoptosis and migration.

Selective and brain-penetrant lanosterol synthase inhibitors target glioma stem-like cells by inducing 24(S),25-epoxycholesterol production.

Glioblastoma (GBM) is an aggressive adult brain cancer with few treatment options due in part to the challenges of identifying brain-penetrant drugs. Here, we investigated the mechanism of MM0299, a tetracyclic dicarboximide with anti-glioblastoma activity. MM0299 inhibits lanosterol synthase (LSS) and diverts sterol flux away from cholesterol into a "shunt" pathway that culminates in 24(S),25-epoxycholesterol (EPC). EPC synthesis following MM0299 treatment is both necessary and sufficient to block the growth of mouse and human glioma stem-like cells by depleting cellular cholesterol. MM0299 exhibits superior selectivity for LSS over other sterol biosynthetic enzymes. Critical for its application in the brain, we report an MM0299 derivative that is orally bioavailable, brain-penetrant, and induces the production of EPC in orthotopic GBM tumors but not normal mouse brain. These studies have implications for the development of an LSS inhibitor to treat GBM or other neurologic indications.

Expression of oxysterols in human lenses: Implications of the sterol pathway in age-related cataracts.

Lanosterol, an oxysterol molecule, has been proposed to help maintain lens transparency by inhibiting the formation of protein aggregates. This sterol is produced by the enzyme lanosterol synthase and is part of a metabolic pathway that forms cholesterol as a final step. Abnormalities in lanosterol synthase are responsible for congenital cataracts. The αA-crystallin protein, which acts as a molecular chaperone to lanosterol synthase, has been reported to have anti-protein aggregation, anti-inflammatory and anti-apoptotic properties. In this work, we evaluated the correlation of lanosterol synthase and αA-crystallin in human cataractous lenses with the grade of opacity, as well as the expression of lanosterol synthase, farnesyl DPP, geranyl synthase and squalene epoxidase genes. Lanosterol synthase and αA-crystallin were overexpressed in cataractous lenses as well as farnesyl-DP synthase, squalene epoxidase, lanosterol synthase and geranyl synthase genes in cataratous lenses in comparison with normal lenses. Our data confirm that lanosterol synthase and the sterol pathway are upregulated in cataractous lenses. This argues for a functional role of the oxysterol pathway and its products as an important mediator in the pathogenesis of human cataracts.

Expression Analysis of Lanosterol Synthase Gene in Dynamic Accumulation of Triterpenoids in Sanghuangporus baumii.

BACKGROUND: Sanghuangporus baumii is a traditional Chinese medicine with anti- cancer, anti-tumor, and anti-inflammatory effects. Triterpenoids are one of the main medicinal ingredients found in S. baumii. However, the dynamic changes of triterpenoids content and its molecular regulation mechanism are still unclear. OBJECTIVE: Some studies have shown that Lanosterol synthase ( LS) is a key enzyme involved in the mevalonate pathway (MVA pathway) to produce lanosterol, which is a precursor for synthesizing S. baumii triterpenoids. Therefore, the study of LS gene and expression characteristics can provide clues for the further study of triterpenoids synthesis. METHODS: The PCR, RACE PCR, RT-PCR, homologous recombination and prokaryotic expression technology were used to research the gene characteristic and dynamic changes of LS transcription level. RESULTS: The S. baumii LS sequence included a 5'-untranslated region (129 bp), a 3'-untranslated region (87 bp), and an open reading frame (2,229 bp) encoding 734 amino acids. The S. baumii LS protein was expressed in E. coli BL21 (DE3). The transcription start site of the S. baumii LS promoter sequence ranged from 1 740 bp to 1790 bp. The LS promoter contained 12 CAAT-boxes, 5 ABREs, 6 G-Boxes, 6 CGTCA-motifs, and so on. The LS transcription levels were the highest on day 11 in mycelia (1.6-fold), and the triterpenoids content also gradually increased. The transcription levels began to decrease on day 13, but the triterpenoids content still increased. CONCLUSION: The S. baumii LS was cloned and characterized to help to understand the mechanism of triterpenoids synthesis. In addition, we studied the relationship between LS transcription level and triterpenoid dynamic accumulation, and we found that they had a certain correlation.

Engineering Critical Amino Acid Residues of Lanosterol Synthase to Improve the Production of Triterpenoids in Saccharomyces cerevisiae.

Triterpenoids are a subgroup of terpenoids and have wide applications in the food, cosmetics, and pharmaceutical industries. The heterologous production of various triterpenoids in Saccharomyces cerevisiae, as well as other microbes, has been successfully implemented as these production hosts not only produce the precursor of triterpenoids 2,3-oxidosqualene by the mevalonate pathway but also allow simple expression of plant membrane-anchored enzymes. Nevertheless, 2,3-oxidosqualene is natively converted to lanosterol catalyzed by the endogenous lanosterol synthase (Erg7p), causing low production of recombinant triterpenoids. While simple deletion of ERG7 was not effective, in this study, the critical amino acid residues of Erg7p were engineered to lower this critical enzyme activity. The engineered S. cerevisiae indeed accumulated 2,3-oxidosqualene up to 180 mg/L. Engineering triterpenoid synthesis into the ERG7-modified strain resulted in 7.3- and 3-fold increases in the titers of dammarane-type and lupane-type triterpenoids, respectively. This study presents an efficient inducer-free strategy for lowering Erg7p activity, thereby providing 2,3-oxidosqualene for the enhanced production of various triterpenoids.

Two cases of severe congenital hypotrichosis caused by compound heterozygous mutations in the LSS gene.

It has recently been shown that bi-allelic mutations in the lanosterol synthase (LSS) gene, which was originally reported as a causative gene for congenital cataracts, underlie a non-syndromic form of hypotrichosis. Furthermore, it has also been revealed that mutations in the LSS gene can cause syndromic forms of hypotrichosis. To date, however, clear genotype-phenotype correlations have not completely been characterized. In this study, we identified two Japanese patients who had severe congenital hypotrichosis without any other associated findings. Their scalp hairs were extremely short and thin, and were able to be plucked easily. Observation of the plucked hairs showed aberrantly-miniaturized anagen hair follicles. Genetic analysis demonstrated that both patients carried bi-allelic mutations in the LSS gene in a compound heterozygote state. Our findings further underscore the crucial roles of the LSS gene in hair follicle development and hair growth in humans.

Defect of LSS Disrupts Lens Development in Cataractogenesis.

Congenital cataract is one of the leading causes of blindness in children worldwide. About one-third of congenital cataracts are caused by genetic defects. LSS, which encodes lanosterol synthase, is a causal gene for congenital cataracts. LSS is critical in preventing abnormal protein aggregation of various cataract-causing mutant crystallins; however, its roles in lens development remain largely unknown. In our study, we generated a mouse model harboring Lss G589S mutation, which is homologous to cataract-causing G588S mutation in human LSS. LssG589S/G589S mice exhibited neonatal lethality at postal day 0 (P0), whereas these mice showed severe opacity in eye lens. Also, we found that cataract was formed at E17.5 after we examined the opacity of embryonic lens from E13.5 to E18.5. Moreover, disrupted lens differentiation occurred at E14.5 prior to formation of the opacity of eye lens, shown as delayed differentiation of lens secondary fiber and disordered lens fiber organization. In addition, RNA-seq analysis indicated that cholesterol synthesis signaling pathways were significantly downregulated. Overall, our findings provide clear evidence that a mouse model harboring a homozygous Lss G589S mutation can recapitulate human congenital cataract. Our study points out that LSS functions as a critical determinant of lens development, which will contribute to better understanding LSS defects in cataractogenesis and developing therapies for cataracts.