A Novel Fluorescent Sensor Protein for Visualization of Redox States in the Cytoplasm and in Peroxisomes

Abstract

Reactive oxygen species are generated within peroxisomes during peroxisomal metabolism. However, due to technological difficulties, the intraperoxisomal redox state remain elusive, and the effect of peroxisome deficiency on the intracellular redox state is controversial. A newly developed, genetically encoded fluorescence resonance energy transfer (FRET) probe, Redoxfluor, senses the physiological redox state via its internal disulfide bonds, resulting in a change in the conformation of the protein leading to a FRET response. We made use of Redoxfluor to measure the redox states at the subcellular level in yeast and Chinese hamster ovary (CHO) cells. In wild-type peroxisomes harboring an intact fatty acid ␤-oxidation system, the redox state within the peroxisomes was more reductive than that in the cytosol, despite the fact that reactive oxygen species were generated within the peroxisomes. Interestingly, we observed that the redox state of the cytosol of cell mutants for peroxisome assembly, regarded as models for a neurological metabolic disorder, was more reductive than that of the wild-type cells in yeast and CHO cells. Furthermore, Redoxfluor was utilized to develop an efficient system for the screening of drugs that moderate the abnormal cytosolic redox state in the mutant CHO cell lines for peroxisome assembly without affecting the redox state of normal cells.

Redox-regulated Cargo Binding and Release by the Peroxisomal Targeting Signal Receptor, Pex5

“…The redox state of the peroxisome lumen is more reducing than in the cytosol (21). Consistent with this observation, we show that the oligomeric states of Pex5 and their differential ability to bind cargo are under redox regulation.…”

Red fluorescent proteins (RFPs) and RFP-based biosensors for neuronal imaging applications

“…These "active" FP constructs are interchangeably referred to as biosensors, sensors, indicators, or reporters. Examples of such FP-based biosensors include ones for intracellular pH, [56][57][58][59] concentration of various ions, 60 second messengers such as ATP, 61 redox potential, 62 membrane voltage, 63 reactive oxygen species, 64 and various enzyme activities. 65 The utility of these biosensors can be extended by combining them with specific promoters and/or targeting signals for specific organelles, cells, or tissues, for either in vitro or in vivo applications.…”

Oxidative Shielding or Oxidative Stress?

“…1). These include: lysosomes (Gille and Nohl, 2000), smooth ER, rough ER (Hwang et al, 1992), the Golgi (Navas et al, 2010), the cytosol (Go and Jones, 2008), peroxisomes (Yano et al, 2010), the nucleus (Go and Jones, 2011), and the mitochondrial matrix (Go and Jones, 2008). These compartments are not in equilibrium because the proteins that maintain the redox in each compartment, and the membranes that separate them, are not diffusible (Jones, 2010).…”