Purification of polyglutamine aggregates and identification of elongation factor-1alpha and heat shock protein 84 as aggregate-interacting proteins.
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-1alpha (EF-1alpha) 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-1alpha and HSP84 also colocalized with the intracellular aggregates. An assay of the transient expression of EF-1alpha 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-1alpha and HSP84 are involved in the neurodegenerative process of polyglutamine diseases.
Mitsui
Kenichi
K
Laboratories for CAG Repeat Diseases, The Institute of Physical and Chemical Research (RIKEN) Brain Science Institute, Wako-shi, Saitama 351-0198, Japan.
Nakayama
Hiroshi
H
Akagi
Takumi
T
Nekooki
Munenori
M
Ohtawa
Kenji
K
Takio
Koji
K
Hashikawa
Tsutomu
T
Nukina
Nobuyuki
N
D000818:Animals; D001921:Brain / Q000378:metabolism / Q000473:pathology; D002454:Cell Differentiation; D002460:Cell Line; D002470:Cell Survival / Q000235:genetics; D004195:Disease Models, Animal; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins; D018841:HSP90 Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D000071058:Huntingtin Protein; D006816:Huntington Disease / Q000209:etiology / Q000378:metabolism* / Q000473:pathology; D002479:Inclusion Bodies / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D008164:Luminescent Proteins / Q000235:genetics; D046911:Macromolecular Substances; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D016253:Microscopy, Immunoelectron; D009419:Nerve Tissue Proteins / Q000235:genetics; D009474:Neurons / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics; D020648:Peptide Elongation Factor 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010455:Peptides / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D019680:Trinucleotide Repeat Expansion / Q000235:genetics
D018841:HSP90 Heat-Shock Proteins; C086055:HTT protein, human; C506812:Htt protein, mouse; D000071058:Huntingtin Protein; D008164:Luminescent Proteins; D046911:Macromolecular Substances; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D020648:Peptide Elongation Factor 1; D010455:Peptides; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C097188:polyglutamine