Metabolism and Biological Activities of 4-Methyl-Sterols.

4,4-Dimethylsterols and 4-methylsterols are sterol biosynthetic intermediates (C4-SBIs) acting as precursors of cholesterol, ergosterol, and phytosterols. Their accumulation caused by genetic lesions or biochemical inhibition causes severe cellular and developmental phenotypes in all organisms. Functional evidence supports their role as meiosis activators or as signaling molecules in mammals or plants. Oxygenated C4-SBIs like 4-carboxysterols act in major biological processes like auxin signaling in plants and immune system development in mammals. It is the purpose of this article to point out important milestones and significant advances in the understanding of the biogenesis and biological activities of C4-SBIs.

The role of sterol-C4-methyl oxidase in epidermal biology.

Deficiency of sterol C4 methyl oxidase, encoded by the SC4MOL gene, has recently been described in four patients from three different families. All of the patients presented with microcephaly, congenital cataracts, and growth delay in infancy. The first patient has suffered since the age of six years from severe, diffuse, psoriasiform dermatitis, sparing only her palms. She is now 20 years old. The second patient is a 5 year old girl who has just started to develop dry skin and hair changes. The third and fourth patients are a pair of affected siblings with a severe skin condition since infancy. Quantitative sterol analysis of plasma and skin scales from all four patients showed marked elevation of 4α-methyl- and 4, 4'-dimethylsterols, consistent with a deficiency in the first step of sterol C4 demethylation in cholesterol biosynthesis. Mutations in the SC4MOL have been identified in all of the patients. SC4MOL deficiency is the first autosomal recessive disorder identified in the sterol demethylation complex. Cellular studies with patient-derived fibroblasts have shown a higher mitotic rate than control cells in cholesterol-depleted medium, with increased de novo cholesterol biosynthesis and accumulation of methylsterols. Immunologic analyses of granulocytes and B cells from patients and obligate carriers in the patients' families indicated dysregulation of immune-related receptors. Inhibition of sterol C4 methyl oxidase in human transformed lymphoblasts induced activation of the cell cycle. Additional studies also demonstrated diminished EGFR signaling and disrupted vesicular trafficking in cells from the affected patients. These findings suggest that methylsterols play an important role in epidermal biology by their influence on cell proliferation, intracellular signaling, vesicular trafficking and immune response. SC4MOL is situated within the psoriasis susceptibility locus PSORS9, and may be a genetic risk factor for common skin conditions. This article is part of a Special Issue entitled The Important Role of Lipids in the Epidermis and their Role in the Formation and Maintenance of the Cutaneous Barrier. Guest Editors: Kenneth R. Feingold and Peter Elias.

Gas chromatography with mass spectrometry analysis of 4,4-dimethylsterols and 4-methylsterols in edible oils after their enrichment by means of solid phase extraction.

4-Methylsterols (4-M-sterols) and 4,4-dimethylsterols (4,4-D-sterols) are a group of underexplored minor sterols that occur in almost all living organisms. Here, we developed a strategy for the determination of the biochemical precursors of the predominant 4-desmethylsterols in edible oils. Due to their low contribution to the sterol content in the samples, a solid phase extraction (SPE) method was developed for the enrichment of 4-M- and 4,4-D-sterols in the hexane extracts of saponified oils. In a two-fold SPE procedure, the bulk of 4,4-D-sterols was collected in one fraction. The residual sample was subjected to a second SPE step which targeted all 4-M-sterols and low shares of 4,4-D-sterols in one fraction and the predominant 4-desmethylsterols in another one. After silylation of the SPE fractions, gas chromatography with mass spectrometry (GC/MS) was used to analyze 4,4-D- and 4-M-sterols. The results were used to define eight subgroups whose characteristic structural features could be linked with the presence of specific m/z values. These m/z values were measured sensitively by GC/MS operated in selected ion monitoring (SIM) mode. Application of the GC/MS method to eighteen edible oils enabled the detection of 55 mostly very low abundant 4-M- and 4,4-D-sterols. Twenty-four of the 4-M- and 4,4-D-sterols could be assigned and the remaining 31 unknown sterols could be traced back to their basic structures.

Evaluation of Antifungal Selective Toxicity Using Candida glabrata ERG25 and Human SC4MOL Knock-In Strains.

With only four classes of antifungal drugs available for the treatment of invasive systemic fungal infections, the number of resistant fungi is increasing, highlighting the urgent need for novel antifungal drugs. Ergosterol, an essential component of cell membranes, and its synthetic pathway have been targeted for antifungal drug development. Sterol-C4-methyl monooxygenase (Erg25p), which is a greater essential target than that of existing drugs, represents a promising drug target. However, the development of antifungal drugs must consider potential side effects, emphasizing the importance of evaluating their selective toxicity against fungi. In this study, we knocked in ERG25 of Candida glabrata and its human ortholog, SC4MOL, in ERG25-deleted Saccharomyces cerevisiae. Utilizing these strains, we evaluated 1181-0519, an Erg25p inhibitor, that exhibited selective toxicity against the C. glabrata ERG25 knock-in strain. Furthermore, 1181-0519 demonstrated broad-spectrum antifungal activity against pathogenic Candida species, including Candida auris. The approach of utilizing a gene that is functionally conserved between yeast and humans and subsequently screening for molecular target drugs enables the identification of selective inhibitors for both species.

MAGEA6 positively regulates MSMO1 and promotes the migration and invasion of oesophageal cancer cells.

The melanoma antigen gene family A (MAGEA) family of proteins comprises of cancer-testis antigens that are highly expressed in a number of tumours but are minimally expressed in normal cells. Due to its expression characteristics, this protein family has become a popular target for anti-cancer drugs and immunotherapy research over recent years. Although, elevated expression levels of MAGEA6 has been found in different types of tumours, there remains to be insufficient information on the function of MAGEA6 and its associated gene regulation pathways. The present study used Transwell, Cell Counting Kit-8 and wound healing assays to analyse the effects of MAGEA6 on Eca109 cell invasion, migration and proliferation. The main functions and pathways involved in MAGEA6 were predicted by Illumina Hiseq screening for mutually regulated genes and core genes. Eca109 cell line with a high expression of MAGEA6 was a stable cell line obtained by transfection in the early stage, and this cell line was used in subsequent experiments. Transcriptome sequencing was performed on this cell line and the Eca109 cell line that normally expressed MAGEA6. It was revealed that a high expression of MAGEA6 conferred a significant stimulating effect on cell proliferation whilst also significantly increasing cell invasion and migration. Transcriptomic analysis identified 14 differentially expressed genes and 13 core regulatory genes closely associated with MAGEA6 expression regulation, such as methylsterol monooxygenase 1 (MSMO1). The present study suggest that MAGEA6 positively regulated MSMO1 expression, which may serve an oncogenic role in cells through this regulatory effect. Overall, this provided a novel route of investigation for an in-depth study of the regulatory function of MAGEA6.

Mitochondrial gene polymorphisms alter hepatic cellular energy metabolism and aggravate diet-induced non-alcoholic steatohepatitis.

OBJECTIVE: Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease and is associated with an enhanced risk for liver and cardiovascular diseases and mortality. NAFLD can progress from simple hepatic steatosis to non-alcoholic steatohepatitis (NASH). However, the mechanisms predisposing to this progression remain undefined. Notably, hepatic mitochondrial dysfunction is a common finding in patients with NASH. Due to a lack of appropriate experimental animal models, it has not been evaluated whether this mitochondrial dysfunction plays a causative role for the development of NASH. METHODS: To determine the effect of a well-defined mitochondrial dysfunction on liver physiology at baseline and during dietary challenge, C57BL/6J-mt(FVB/N) mice were employed. This conplastic inbred strain has been previously reported to exhibit decreased mitochondrial respiration likely linked to a non-synonymous gene variation (nt7778 G/T) of the mitochondrial ATP synthase protein 8 (mt-ATP8). RESULTS: At baseline conditions, C57BL/6J-mt(FVB/N) mice displayed hepatic mitochondrial dysfunction characterized by decreased ATP production and increased formation of reactive oxygen species (ROS). Moreover, genes affecting lipid metabolism were differentially expressed, hepatic triglyceride and cholesterol levels were changed in these animals, and various acyl-carnitines were altered, pointing towards an impaired mitochondrial carnitine shuttle. However, over a period of twelve months, no spontaneous hepatic steatosis or inflammation was observed. On the other hand, upon dietary challenge with either a methionine and choline deficient diet or a western-style diet, C57BL/6J-mt(FVB/N) mice developed aggravated steatohepatitis as characterized by lipid accumulation, ballooning of hepatocytes and infiltration of immune cells. CONCLUSIONS: We observed distinct metabolic alterations in mice with a mitochondrial polymorphism associated hepatic mitochondrial dysfunction. However, a second hit, such as dietary stress, was required to cause hepatic steatosis and inflammation. This study suggests a causative role of hepatic mitochondrial dysfunction in the development of experimental NASH.