Analysis of the alphaB-crystallin domain responsible for inhibiting tubulin aggregation.
The cytoskeleton has a unique property such that changes of conformation result in polymerization into a filamentous form. alphaB-Crystallin, a small heat shock protein (sHsp), has chaperone activities for various substrates, including proteins constituting the cytoskeleton, such as actin; intermediate filament; and tubulin. However, it is not clear whether the "alpha-crystallin domain" common to sHsps also has chaperone activity for the protein cytoskeleton. To investigate the possibility that the C-terminal alpha-crystallin domain of alpha-crystallin has the aggregation-preventing ability for tubulin, we constructed an N-terminal domain deletion mutant of alphaB-crystallin. We characterized its structural properties and chaperone activities. Far-ultraviolet (UV) circular dichroism measurements showed that secondary structure in the alpha-crystallin domain of the deletion mutant is maintained. Ultracentrifuge analysis of molecular masses indicated that the deletion mutant formed smaller oligomers than did the full-length protein. Chaperone activity assays demonstrated that the N-terminal domain deletion mutant suppressed heat-induced aggregation of tubulin well. Comparison of chaperone activities for 2 other substrates (citrate synthase and alcohol dehydrogenase) showed that it was less effective in the suppression of their aggregation. These results show that alphaB-crystallin recognizes a variety of substrates and especially that alpha-crystallin domain binds free cytoskeletal proteins. We suggest that this feature would be advantageous in its functional role of holding or folding multiple proteins denatured simultaneously under stress conditions.
Ohto-Fujita
Eri
E
Department of Life Sciences, The Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan.
Fujita
Yoshinobu
Y
Atomi
Yoriko
Y
D000426:Alcohol Dehydrogenase / Q000378:metabolism; D000818:Animals; D002021:Buffers; D002950:Citrate (si)-Synthase / Q000378:metabolism; D007700:Kinetics; D050505:Mutant Proteins / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000494:pharmacology*; D011485:Protein Binding / Q000187:drug effects; D020836:Protein Structure, Quaternary / Q000187:drug effects; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D013552:Swine; D014404:Tubulin / Q000378:metabolism*; D038203:alpha-Crystallin B Chain / Q000737:chemistry* / Q000378:metabolism*
D002021:Buffers; D050505:Mutant Proteins; D010446:Peptide Fragments; D014404:Tubulin; D038203:alpha-Crystallin B Chain; D000426:Alcohol Dehydrogenase; D002950:Citrate (si)-Synthase
J Biol Chem. 1999 Nov 19;274(47):33235-43
10559197
Proc Natl Acad Sci U S A. 1992 Nov 1;89(21):10449-53
1438232
J Biol Chem. 2001 May 11;276(19):16059-63
11274139
Cell Stress Chaperones. 2002 Apr;7(2):167-76
12380684
EMBO J. 1994 Feb 15;13(4):945-53
7906647
FEBS Lett. 1995 May 29;365(2-3):133-6
7781765
FEBS Lett. 1995 Aug 7;369(2-3):321-5
7649280
J Biol Chem. 1996 Nov 8;271(45):28558-66
8910485
Eur J Biochem. 1996 Nov 15;242(1):56-66
8954153
J Biol Chem. 1997 Jan 24;272(4):2578-82
8999975
J Biol Chem. 1997 Mar 7;272(10):6220-5
9045637
Cell Struct Funct. 1997 Oct;22(5):539-44
9431459
Methods Enzymol. 1998;290:323-38
9534173
Nature. 1998 Aug 6;394(6693):595-9
9707123
Nat Genet. 1998 Sep;20(1):92-5
9731540
FASEB J. 2005 Jul;19(9):1199-201
15894563
Cell Stress Chaperones. 2006 Summer;11(2):187-97
16817325
Mol Cell Biochem. 2003 Oct;252(1-2):273-8
14577602
J Cell Sci. 2004 Apr 1;117(Pt 9):1719-26
15075233
Biochem Biophys Res Commun. 1983 Jan 27;110(2):463-9
6838530
Biochim Biophys Acta. 1983 Oct 17;748(1):56-67
6615851
FEBS Lett. 1985 Feb 11;181(1):1-6
3972098
Biochem Biophys Res Commun. 1991 Dec 31;181(3):1323-30
1764082
FEBS Lett. 1992 Oct 19;311(2):143-9
1397302
J Biol Chem. 2001 Apr 13;276(15):12024-9
11278766