Coactosin-like protein functions as a stabilizing chaperone for 5-lipoxygenase: role of tryptophan 102.
The activity of 5-LO (5-lipoxygenase), which catalyses two initial steps in the biosynthesis of pro-inflammatory LTs (leukotrienes), is strictly regulated. One recently discovered factor, CLP (coactosin-like protein), binds 5-LO and promotes LT formation. In the present paper we report that CLP also stabilizes 5-LO and prevents non-turnover inactivation of the enzyme in vitro. Mutagenesis of tryptophan residues in the 5-LO beta-sandwich showed that 5-LO-Trp102 is essential for binding to CLP, and for CLP to support 5-LO activity. In addition, the stabilizing effect also depended on binding between CLP and 5-LO. After mutations which prevent interaction (5-LO-W102A or CLP-K131A), the protective effect of CLP was absent. A calculated 5-LO-CLP docking model indicates that CLP may bind to additional residues in both domains of 5-LO, thus possibly stabilizing the 5-LO structure. To obtain further support for binding between CLP and 5-LO in a living cell, subcellular localization of CLP and 5-LO in the monocytic cell line Mono Mac 6 was determined. In these cells, 5-LO associates with a nuclear fraction only when differentiated cells are primed with phorbol ester and stimulated with ionophore. The same pattern of redistribution was found for CLP, indicating that the two proteins associate with the nucleus in a co-ordinated fashion. The results of the present study support a role for CLP as a chaperoning scaffold factor, influencing both the stability and the activity of 5-LO.
Esser
Julia
J
Department of Medical Biochemistry and Biophysics, Division of Physiological Chemistry II, Karolinska Institutet, S-171 77 Stockholm, Sweden.
Rakonjac
Marija
M
Hofmann
Bettina
B
Fischer
Lutz
L
Provost
Patrick
P
Schneider
Gisbert
G
Steinhilber
Dieter
D
Samuelsson
Bengt
B
Rådmark
Olof
O
D001094:Arachidonate 5-Lipoxygenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016718:Arachidonic Acid / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D020134:Catalytic Domain; D002460:Cell Line; D002467:Cell Nucleus; D004795:Enzyme Stability; D006801:Humans; D008840:Microfilament Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D009000:Monocytes / Q000166:cytology / Q000378:metabolism; D009154:Mutation; D010713:Phosphatidylcholines / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013820:Thermolysin / Q000378:metabolism; D014364:Tryptophan / Q000235:genetics / Q000378:metabolism* / Q000502:physiology
C118115:COTL1 protein, human; D008840:Microfilament Proteins; D018832:Molecular Chaperones; D010713:Phosphatidylcholines; D016718:Arachidonic Acid; D014364:Tryptophan; D001094:Arachidonate 5-Lipoxygenase; D013820:Thermolysin