A point mutation of human nucleoside diphosphate kinase A found in aggressive neuroblastoma affects protein folding

被引:57
作者
Lascu, I
Schaertl, S
Wang, CQ
Sarger, C
Giartosio, A
Briand, G
Lacombe, ML
Konrad, M
机构
[1] MAX PLANCK INST BIOPHYS CHEM, DEPT MOL GENET, D-37018 GOTTINGEN, GERMANY
[2] UNIV ROMA LA SAPIENZA, DIPARTIMENTO SCI BIOCHIM A ROSSI FANELLI, I-00185 ROME, ITALY
[3] UNIV ROMA LA SAPIENZA, CNR, CTR BIOL MOL, I-00185 ROME, ITALY
[4] UNIV LILLE 2, LAB APPLICAT SPECTROMETRIE MASSE, F-59045 LILLE, FRANCE
[5] UNIV PARIS 06, FAC MED ST ANTOINE, U402 INSERM, F-75571 PARIS, FRANCE
关键词
D O I
10.1074/jbc.272.25.15599
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The point mutation serine 120 to glycine in the human nucleoside diphosphate kinase A has been identified in several aggressive neuroblastomas (Chang, C. L., Zhu, X. X., Thoraval, D. H., Ungar, D., Rawwas, J., Hora, N., Strahler, J. R., Hanash, S. M. & Radany, E. (1994) Nature 370, 335-336). We expressed in bacteria and purified wild-type and S120G mutant nucleoside diphosphate kinase A. The mutant enzyme had enzymatic and structural properties similar to the wild-type enzyme, protein lead whereas its stability to denaturation by heat and urea intermediate. was markedly reduced. More importantly, upon renaturation of the urea-denatured mutant protein, a folding intermediate accumulated, having the characteristics of a molten globule. It had no tertiary structure, as shown by near UV circular dichroism, whereas the secondary structure was substantially recovered. The hydrophobic probe 8-anilino-1-naphthalene sulfonate bound to the intermediate species with an increase in fluorescence intensity and a blue shift. The hydrodynamic size was between that expected for a folded and an unfolded monomer. Finally, electrophoresis in a transverse urea gradient displayed no renaturation curve, and the protein showed the tendency to aggregate at the lowest urea concentrations. The existence of a molten globule folding intermediates resulting from an altered folding in the mutated protein might be related to the aggressiveness of neuroblastomas.
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页码:15599 / 15602
页数:4
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