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The Journal of Neuroscience, November 1, 2002, 22(21):9267-9277
Purification of Polyglutamine Aggregates and Identification of
Elongation Factor-1 and Heat Shock Protein 84 as
Aggregate-Interacting Proteins
Kenichi
Mitsui1,
Hiroshi
Nakayama4,
Takumi
Akagi2,
Munenori
Nekooki1,
Kenji
Ohtawa3,
Koji
Takio4,
Tsutomu
Hashikawa2, and
Nobuyuki
Nukina1
Laboratories for 1 CAG Repeat Diseases and
2 Neural Architecture and 3 Advanced Technology
Development Center, The Institute of Physical and Chemical Research
(RIKEN) Brain Science Institute, and 4 Division of
Biomolecular Characterization, RIKEN, Wako-shi, Saitama 351-0198, Japan
Aggregates of green fluorescent protein (GFP)-fused truncated
N-terminal huntingtin containing abnormally long polyglutamine tracts
(150 repeats of glutamine residue) were purified from an ecdysone-inducible mutant neuro2A cell line (HD150Q-28) by using a
fluorescence-activated cell sorter. To analyze the
aggregate-interacting proteins, we subjected the purified
aggregates to SDS-PAGE; prominent protein bands in the gel were
digested with Achromobactor lysyl endopeptidase,
followed by a HPLC-mass spectrometry (MS) analysis. The
resulting data of tandem MS analysis revealed that, in addition to
ubiquitin and widely reported chaperone proteins such as heat shock
cognate 70 (HSC70), human DNA J-1 (HDJ-1), and HDJ-2, the translational
elongation factor-1 (EF-1) and heat shock protein 84 (HSP84)
also were recognized as aggregate-interacting proteins. Sequestration of these proteins to aggregates was confirmed further by
several immunochemical methods. We confirmed that, in addition to the
other known proteins, EF-1 and HSP84 also colocalized with the
intracellular aggregates. An assay of the transient expression of
EF-1 and HSP84 in HD150Q-28 cells revealed that both proteins improved cell viability. Moreover, the rate of aggregate formation decreased in both transfectants. Our study suggests that both EF-1
and HSP84 are involved in the neurodegenerative process of
polyglutamine diseases.
Key words:
huntingtin; polyglutamine aggregate; GFP; cell sorter; mass spectrometry; HSP84; EF-1
Copyright © 2002 Society for Neuroscience 0270-6474/02/22219267-11$05.00/0
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