细胞内
细胞生物学
相互作用体
蛋白质组学
化学
铁硫簇
功能(生物学)
生物
泛素连接酶
生物化学
泛素
基因
酶
作者
Vijaya Pandey,Adarsh K. Mayank,James A. Wohlschlegel
出处
期刊:Methods in molecular biology
日期:2023-01-01
卷期号:: 155-166
标识
DOI:10.1007/978-1-0716-3080-8_10
摘要
Multicellular organisms have evolved elaborate strategies to sense and adapt to changes in intracellular oxygen. The canonical cellular pathway responsible for oxygen sensing consists of the von Hippel-Lindau (pVHL) tumor suppressor protein, prolyl hydroxylases (PHD), and hypoxia-inducible factors (HIFs), which together regulate expression of downstream genes involved in oxygen homeostasis. In recent years, it has become increasingly clear that oxygen regulatory mechanisms are intertwined with cellular iron-sensing pathways. Key members of these networks such as prolyl-hydroxylases, E3 ubiquitin ligase adaptor protein FBXL5, iron regulatory proteins (IRPs), and Fe-S cluster proteins require both iron and oxygen for their optimal function and/or are tightly regulated by intracellular concentrations of these molecules. Monitoring how protein interactomes are remodeled as a function of intracellular oxygen and iron levels gives insights into the nature and dynamics of these pathways. We have recently described an oxygen-sensitive interaction between FBXL5 and the cytoplasmic Fe-S cluster targeting complex (CIA targeting complex) with implications in the FBXL5-dependent regulation of IRPs. Based on this work, we present a protocol describing the induction and maintenance of hypoxia in mammalian cell cultures and a mass-spectrometry-based proteomics approach aimed at interrogating changes in interactome of key proteins as a function of intracellular oxygen and iron levels. These methods are widely applicable to understanding the dynamics of iron and oxygen signaling.
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