An Unexpected Enzyme in Vascular Smooth Muscle Cells: Angiotensin II Upregulates Cholesterol-25-Hydroxylase Gene Expression.

Angiotensin II (AngII) is a vasoactive peptide hormone, which, under pathological conditions, contributes to the development of cardiovascular diseases. Oxysterols, including 25-hydroxycholesterol (25-HC), the product of cholesterol-25-hydroxylase (CH25H), also have detrimental effects on vascular health by affecting vascular smooth muscle cells (VSMCs). We investigated AngII-induced gene expression changes in VSMCs to explore whether AngII stimulus and 25-HC production have a connection in the vasculature. RNA-sequencing revealed that Ch25h is significantly upregulated in response to AngII stimulus. The Ch25h mRNA levels were elevated robustly (~50-fold) 1 h after AngII (100 nM) stimulation compared to baseline levels. Using inhibitors, we specified that the AngII-induced Ch25h upregulation is type 1 angiotensin II receptor- and Gq/11 activity-dependent. Furthermore, p38 MAPK has a crucial role in the upregulation of Ch25h. We performed LC-MS/MS to identify 25-HC in the supernatant of AngII-stimulated VSMCs. In the supernatants, 25-HC concentration peaked 4 h after AngII stimulation. Our findings provide insight into the pathways mediating AngII-induced Ch25h upregulation. Our study elucidates a connection between AngII stimulus and 25-HC production in primary rat VSMCs. These results potentially lead to the identification and understanding of new mechanisms in the pathogenesis of vascular impairments.

In Silico Prediction of the Metabolic Resistance of Vitamin D Analogs against CYP3A4 Metabolizing Enzyme.

The microsomal cytochrome P450 3A4 (CYP3A4) and mitochondrial cytochrome P450 24A1 (CYP24A1) hydroxylating enzymes both metabolize vitamin D and its analogs. The three-dimensional (3D) structure of the full-length native human CYP3A4 has been solved, but the respective structure of the main vitamin D hydroxylating CYP24A1 enzyme is unknown. The structures of recombinant CYP24A1 enzymes have been solved; however, from studies of the vitamin D receptor, the use of a truncated protein for docking studies of ligands led to incorrect results. As the structure of the native CYP3A4 protein is known, we performed rigid docking supported by molecular dynamic simulation using CYP3A4 to predict the metabolic conversion of analogs of 1,25-dihydroxyvitamin D2 (1,25D2). This is highly important to the design of novel vitamin D-based drug candidates of reasonable metabolic stability as CYP3A4 metabolizes ca. 50% of the drug substances. The use of the 3D structure data of human CYP3A4 has allowed us to explain the substantial differences in the metabolic conversion of the side-chain geometric analogs of 1,25D2. The calculated free enthalpy of the binding of an analog of 1,25D2 to CYP3A4 agreed with the experimentally observed conversion of the analog by CYP24A1. The metabolic conversion of an analog of 1,25D2 to the main vitamin D hydroxylating enzyme CYP24A1, of unknown 3D structure, can be explained by the binding strength of the analog to the known 3D structure of the CYP3A4 enzyme.

Insight into molecular mechanisms underlying hepatic dysfunction in severe COVID-19 patients using systems biology.

BACKGROUND: The coronavirus disease 2019 (COVID-19), a pandemic contributing to more than 105 million cases and more than 2.3 million deaths worldwide, was described to be frequently accompanied by extrapulmonary manifestations, including liver dysfunction. Liver dysfunction and elevated liver enzymes were observed in about 53% of COVID-19 patients. AIM: To gain insight into transcriptional abnormalities in liver tissue of severe COVID-19 patients that may result in liver dysfunction. METHODS: The transcriptome of liver autopsy samples from severe COVID-19 patients against those of non-COVID donors was analyzed. Differentially expressed genes were identified from normalized RNA-seq data and analyzed for the enrichment of functional clusters and pathways. The differentially expressed genes were then compared against the genetic signatures of liver diseases including cirrhosis, fibrosis, non-alcoholic fatty liver disease (NAFLD), and hepatitis A/B/C. Gene expression of some differentially expressed genes was assessed in the blood samples of severe COVID-19 patients with liver dysfunction using qRT-PCR. RESULTS: Analysis of the differential transcriptome of the liver tissue of severe COVID-19 patients revealed a significant upregulation of transcripts implicated in tissue remodeling including G-coupled protein receptors family genes, DNAJB1, IGF2, EGFR, and HDGF. Concordantly, the differential transcriptome of severe COVID-19 liver tissues substantially overlapped with the disease signature of liver diseases characterized with pathological tissue remodeling (liver cirrhosis, Fibrosis, NAFLD, and hepatitis A/B/C). Moreover, we observed a significant suppression of transcripts implicated in metabolic pathways as well as mitochondrial function, including cytochrome P450 family members, ACAD11, CIDEB, GNMT, and GPAM. Consequently, drug and xenobiotics metabolism pathways are significantly suppressed suggesting a decrease in liver detoxification capacity. In correspondence with the RNA-seq data analysis, we observed a significant upregulation of DNAJB1 and HSP90AB1 as well as significant downregulation of CYP39A1 in the blood plasma of severe COVID-19 patients with liver dysfunction. CONCLUSION: Severe COVID-19 patients appear to experience significant transcriptional shift that may ensue tissue remodeling, mitochondrial dysfunction and lower hepatic detoxification resulting in the clinically observed liver dysfunction.

Cholesterol 25-hydroxylase suppresses porcine deltacoronavirus infection by inhibiting viral entry.

Cholesterol 25-hydroxylase (CH25 H) is a key enzyme regulating cholesterol metabolism and also acts as a broad antiviral host restriction factor. Porcine deltacoronavirus (PDCoV) is an emerging swine enteropathogenic coronavirus that can cause vomiting, diarrhea, dehydration and even death in newborn piglets. In this study, we found that PDCoV infection significantly upregulated the expression of CH25H in IPI-FX cells, a cell line of porcine ileum epithelium. Overexpression of CH25H inhibited PDCoV replication, whereas CH25H silencing using RNA interference promoted PDCoV infection. Treatment with 25-hydroxycholesterol (25HC), the catalysate of cholesterol via CH25H, inhibited PDCoV proliferation by impairing viral invasion of IPI-FX cells. Furthermore, a mutant CH25H (CH25H-M) lacking hydroxylase activity also inhibited PDCoV infection to a lesser extent. Taken together, our data suggest that CH25H acts as a host restriction factor to inhibit the proliferation of PDCoV but this inhibitory effect is not completely dependent on its enzymatic activity.

Cholesterol 25-Hydroxylase inhibits SARS-CoV-2 and other coronaviruses by depleting membrane cholesterol.

Coronavirus disease 2019 (COVID-19) is caused by SARS-CoV-2 and has spread across the globe. SARS-CoV-2 is a highly infectious virus with no vaccine or antiviral therapy available to control the pandemic; therefore, it is crucial to understand the mechanisms of viral pathogenesis and the host immune responses to SARS-CoV-2. SARS-CoV-2 is a new member of the betacoronavirus genus like other closely related viruses including SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV). Both SARS-CoV and MERS-CoV have caused serious outbreaks and epidemics in the past eighteen years. Here, we report that one of the interferon-stimulated genes (ISGs), cholesterol 25-hydroxylase (CH25H), is induced by SARS-CoV-2 infection in vitro and in COVID-19-infected patients. CH25H converts cholesterol to 25-hydrocholesterol (25HC) and 25HC shows broad anti-coronavirus activity by blocking membrane fusion. Furthermore, 25HC inhibits USA-WA1/2020 SARS-CoV-2 infection in lung epithelial cells and viral entry in human lung organoids. Mechanistically, 25HC inhibits viral membrane fusion by activating the ER-localized acyl-CoA:cholesterol acyltransferase (ACAT) which leads to the depletion of accessible cholesterol from the plasma membrane. Altogether, our results shed light on a potentially broad antiviral mechanism by 25HC through depleting accessible cholesterol on the plasma membrane to suppress virus-cell fusion. Since 25HC is a natural product with no known toxicity at effective concentrations, it provides a potential therapeutic candidate for COVID-19 and emerging viral diseases in the future.