Related Experiment Videos
Differential localization of 5- and 15-lipoxygenases to the nuclear envelope in RAW macrophages
P Christmas1, J W Fox, S R Ursino
1Arthritis Unit, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Charlestown, Massachusetts 02129, USA.
Abstract:
Leukotriene formation is initiated in myeloid cells by an increase in intracellular calcium and translocation of 5-lipoxygenase from the cytoplasm to the nuclear envelope where it can utilize arachidonic acid. Monocyte- macrophages and eosinophils also express 15-lipoxygenase, which converts arachidonic acid to 15(S)-hydroxyeicosatetraenoic acid. Enhanced green fluorescent 5-lipoxygenase (5-LO) and 15-lipoxygenase (15-LO) fusion proteins were expressed in the cytoplasm of RAW 264.7 macrophages. Only 5-lipoxygenase translocated to the nuclear envelope after cell stimulation, suggesting that differential subcellular compartmentalization can regulate the generation of leukotrienes versus 15(S)-hydroxyeicosatetraenoic acid in cells that possess both lipoxygenases. A series of truncation mutants of 5-LO were created to identify putative targeting domains; none of these mutants localized to the nuclear envelope. The lack of targeting of 15-LO was then exploited to search for specific targeting motifs in 5-LO, by creating 5-LO/15-LO chimeric molecules. The only chimera that could sustain nuclear envelope translocation was one which involved replacement of the N-terminal 237 amino acids with the corresponding segment of 15-LO. Significantly, no discrete targeting domain could be identified in 5-LO, suggesting that sequences throughout the molecule are required for nuclear envelope localization.
Insights
Leukotriene production is regulated by the movement of 5-lipoxygenase (5-LO) to the nuclear envelope. Researchers found that sequences throughout the 5-LO molecule, not a specific domain, are crucial for this nuclear envelope localization.
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Biology
Background:
- Leukotriene synthesis is initiated by 5-lipoxygenase (5-LO) translocation to the nuclear envelope in myeloid cells.
- Both 5-LO and 15-lipoxygenase (15-LO) are present in macrophages, producing different eicosanoids.
- Subcellular localization of lipoxygenases may control the balance of eicosanoid production.
Purpose of the Study:
- To investigate the subcellular localization of 5-LO and 15-LO in macrophages.
- To identify the molecular determinants responsible for 5-LO nuclear envelope targeting.
- To understand how differential localization regulates eicosanoid synthesis.
Main Methods:
- Expression of enhanced green fluorescent protein-tagged 5-LO and 15-LO fusion proteins in RAW 264.7 macrophages.
- Cell stimulation to induce lipoxygenase translocation.
- Creation and analysis of 5-LO truncation mutants and 5-LO/15-LO chimeric molecules.
Main Results:
- Only 5-LO, not 15-LO, translocated to the nuclear envelope upon stimulation.
- Truncation mutants of 5-LO failed to localize to the nuclear envelope.
- A chimeric molecule with the N-terminal 237 amino acids of 15-LO fused to 5-LO retained nuclear envelope translocation capability.
- No discrete targeting domain was identified within 5-LO for nuclear envelope localization.
Conclusions:
- Differential subcellular localization of 5-LO and 15-LO regulates the production of distinct eicosanoids.
- Nuclear envelope targeting of 5-LO requires sequences distributed throughout the protein, rather than a specific domain.
- Understanding lipoxygenase localization is key to controlling inflammatory mediator synthesis.