Development of a Synthetic 3-ketosteroid Δ1-dehydrogenase for the Generation of a Novel Catabolic Pathway Enabling Cholesterol Degradation in Human Cells

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 Δ 1 -dehydrogenase (Δ 1 -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 Δ 1 -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.

Recent developments in the enzymatic modifications of steroid scaffolds

“…103 In recent years, various KstDs with high activities have been discovered and applied. [104][105][106][107][108][109][110][111][112] For instance, Guo and coworkers used Nocardioides for the Δ 1,2 -dehydrogenation of 71 to produce 72, which is an important intermediate for the synthesis of fluocinolone acetonide (Fig. 13B).…”

Healthspan Extension through Innovative Genetic Medicines

“… 38 A therapy based on work from the University of South Alabama is introducing a gene in macrophages ex vivo that can metabolize oxidized cholesterol. 39 Efforts to treat epidermolysis bullosa have had dramatic results from two groups using either transgenic ex vivo keratinocytes or intradermal injection of collagen VII. 40 , 41 Using their genetic medicine platform, the authors are developing multiple gene replacement therapies to treat sarcopenia, mitochondrial diseases, collagen defects, and skin disease.…”