Abstract
Cholesterol is an essential component of membranes, which is acquired by cells via receptor-mediated endocytosis of lipoproteins or via de novo synthesis. In specialized cells, anabolic enzymes metabolize cholesterol, generating steroid hormones or bile acids. However, surplus cholesterol cannot be catabolized due to the lack of enzymes capable of degrading the cholestane ring. The inability to degrade cholesterol becomes evident in the development and progression of cardiovascular disease, where the accumulation of cholesterol/cholesteryl-esters in macrophages can elicit a maladaptive immune response leading to the development and progression of atherosclerosis. The discovery of cholesterol catabolic pathways in Actinomycetes led us to the hypothesis that if enzymes enabling cholesterol catabolism could be genetically engineered and introduced into human cells, the atherosclerotic process may be prevented or reversed. Comparison of bacterial enzymes that degrade cholesterol to obtain carbon and generate energy with the action of human enzymes revealed that humans lack a 3-ketosteroid Δ1-dehydrogenase (Δ1-KstD), which catalyzes the C-1 and C-2 desaturation of ring A. Here we describe the construction, heterologous expression, and actions of a synthetic humanized Δ1-KstD expressed in Hep3B and U-937 cells, providing proof that one of three key enzymes required for cholesterol ring opening can be functionally expressed in human cells.
Highlights
Www.nature.com/scientificreports sufficient to maintain normal cholesterol homeostasis
Reverse-phase high-performance liquid chromatography (RP-HPLC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) based methods were used to measure the ability of Δ1-ketosteroid dehydrogenases (KstD) to introduce a double bond between C-1 and C-2 (Figs S2–S4)
Our idea to genetically engineer human cells to enable cholesterol catabolism was founded upon a series of unexpected observations made by others studying chronic tuberculosis[48,49,50]
Summary
Www.nature.com/scientificreports sufficient to maintain normal cholesterol homeostasis. When hepatic cholesterol levels are insufficient to meet this metabolic need, the expression of LDL-receptors is induced This allows the liver to “recycle” cholesterol from the blood via the endocytosis of circulating LDLs. Drugs, collectively referred to as statins, exploit this biological process. Both Mycobacteria and Rhodococci have similar enzymes that catalyze cholestane B-ring opening (Fig. 1) These observations lead us to the provocative hypothesis that if we could enable controlled cholesterol catabolism in human cells, surplus cholesterol could be degraded. After migrating to inflamed atherosclerotic lesions and differentiating into macrophages, the cholesterol-catabolizing macrophages may be able to prevent or even reverse the atherosclerotic process To test this hypothesis, here we describe the generation of three synthetic humanized enzymes predicted to enable cholestane B-ring opening [cholesterol-3-OH-dehydrogenase (CholD), 3-ketosteroid Δ1-dehydrogenase (Δ1-KstD), and 3-ketosteroid-9α-hydroxylase (Kst-9αH)]. This represents an integral step needed for cholestane A-ring aromatization and B-ring opening for which humans have no known ortholog
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