Increased resistance to oxysterol cytotoxicity in fibroblasts transfected with a lysosomally targeted Chromobacterium oxidase

Abstract

7-Ketocholesterol (7KC) is a cytotoxic oxysterol that plays a role in many age-related degenerative diseases. 7KC formation and accumulation often occurs in the lysosome, which hinders enzymatic transformations that reduce its toxicity and increase the sensitivity to lysosomal membrane permeabilization. We assayed the potential to mitigate 7KC cytotoxicity and enhance cell viability by overexpressing 7KC-active enzymes in human fibroblasts. One of the enzymes tested, a cholesterol oxidase engineered for lysosomal targeting, significantly increased cell viability in the short term upon treatment with up to 50 µM 7KC relative to controls. These results suggest targeting the lysosome for optimal treatment of oxysterol-mediated cytotoxicity, and support the use of introducing novel catalytic function into the lysosome for therapeutic and research applications.

Introducing inducible fluorescent split cholesterol oxidase to mammalian cells

“…Oxysterols accumulate in lysosomes and cause membrane permeabilization and provoke cell death in a concentration-dependent manner. Overexpression of lysosome-targeted COase was found to decrease oxysterol-induced apoptosis, although the exact mechanism of protection is unknown (18). Our observations show that cytoplasmically expressed COase results in cellular cholesterol reduction and plasma membrane cholesterol biosensor translocation.…”

“…It has an isoelectric point of 8.6 and possesses high positive electrostatic surface potential that increases its affinity toward negatively charged membranes. Additionally, its activity in mammalian cells is reported (18), providing an additional justification for choosing this enzyme. The best splitting strategy generates separate fragments containing isolated domains that are correctly folded and functional by themselves, as in the classical two-hybrid system.…”

“…Interestingly, expression of the lysosome-targeted COase alleviated oxysterol cytotoxicity (18). Oxysterols accumulate in lysosomes and cause membrane permeabilization and provoke cell death in a concentration-dependent manner.…”

“…An additional cytotoxic effect is caused by peroxide that increases the intracellular reactive oxygen species level (17). Despite that, a lysosome-targeted COase has been reported to attenuate the cytotoxicity of 7-ketocholesterol in human fibroblasts (18). Thus, COase activity influences multiple cellular processes.…”

Taking a lipidation-dependent path toward endolysosomes

“…However, overexpression of these constructs is often accompanied by unwanted effects, including the enlarged lysosomes that accumulate at perinuclear regions upon transfection with Rab7 fluorescent constructs, 6 or the dilation and fusion of early endosomes observed upon overexpression of GFP-Rab5. 7 Fluorescent constructs of other endolysosomal constituents such as Lamp1, which have been extensively validated as endolysosomal markers, 9,11 can also display various biological effects when overexpressed in cells, 8 and give rise to enlargement, aggregation and/or rounding of these compartments 9 (see below).…”