Assessing POR and CYB5R1 oxidoreductase-mediated oxidative rupture of PUFA in liposomes

Bo Yan; Youwei Ai; Zhiyuan Zhang; Xiaodong Wang

doi:10.1016/j.xpro.2021.100360

Assessing POR and CYB5R1 oxidoreductase-mediated oxidative rupture of PUFA in liposomes

STAR Protoc. 2021 Mar 2;2(1):100360. doi: 10.1016/j.xpro.2021.100360. eCollection 2021 Mar 19.

Authors

Bo Yan^{1

2}, Youwei Ai^{1

2}, Zhiyuan Zhang^{1

2}, Xiaodong Wang^{1

2}

Affiliations

¹ National Institute of Biological Sciences, 7 Science Park Road, Zhongguancun Life Science Park, Beijing 102206, China.
² Tsinghua Institute of Multidisciplinary Biomedical Research, Tsinghua University, Beijing 100871, China.

Abstract

Lipid peroxidation of polyunsaturated fatty acid (PUFA) phospholipids induces necrotic cell death through compromised cell membrane integrity during ferroptosis. We established assays to investigate oxidoreductase-mediated oxidative rupture, specifically via NADPH-cytochrome P450 reductase (POR) and NADH-cytochrome b5 reductase (CYB5R1), of PUFA phospholipids in artificially generated protein-free liposomes. Liposome breakage was detected via Tb³⁺ liposome release and electron microscopy liposome morphology imaging. This protocol was also applied to other oxidoreductases with analogous functions and investigation of ferroptotic membrane damage in cell-free systems. For complete details on the use and execution of this protocol, please refer to Yan et al. (2020).

Keywords: Cell membrane; Metabolism.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Cattle
Cytochrome P-450 Enzyme System / chemistry*
Cytochrome-B(5) Reductase / chemistry*
Fatty Acids, Unsaturated / chemistry*
Humans
Liposomes
Oxidation-Reduction

Substances

Fatty Acids, Unsaturated
Liposomes
POR protein, human
Cytochrome P-450 Enzyme System
CYB5R1 protein, human
Cytochrome-B(5) Reductase