Formic acid dissolves aggregates of an N-terminal huntingtin fragment containing an expanded polyglutamine tract: applying to quantification of protein components of the aggregates.
Huntington's disease (HD) is caused by an expansion of the CAG repeat that encodes polyglutamine in huntingtin. Transient expression of an N-terminal huntingtin fragment containing an expanded polyglutamine tract induced formation of protein aggregates in cultured cells. The turnover of protein components in such aggregates has been difficult to study because of their insolubility in aqueous solutions. Here we describe a method of solubilizing the aggregates and quantifying their protein components. Insoluble pellets were collected from COS7 cells expressing an N-terminal huntingtin fragment containing an expanded polyglutamine tract and subjected to treatment with various detergent, acid, and alkaline reagents. Treatment with 100% formic acid at 37 degrees C for 30 min induced essentially complete dissociation of the aggregates to monomer. We used this solubilization technique to quantify huntingtin fusion protein in the aggregates formed in transient expression experiments. The frequency of aggregate formation increased when the proteasome inhibitor beta-lactone was added to culture media. However, the total amount of accumulated huntingtin fusion protein did not differ between cells cultured with or without beta-lactone. These results suggest that other protein components which are degraded by the proteasome, in addition to huntingtin, might be related to the dynamics of polyglutamine protein aggregates.
Hazeki
N
N
CREST, Tokyo, Japan. nhazeki-tky@umin.ac.jp
Tukamoto
T
T
Goto
J
J
Kanazawa
I
I
D000818:Animals; D019556:COS Cells; D002424:Caustics / Q000494:pharmacology; D003546:Cysteine Endopeptidases; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005091:Exons; D005561:Formates / Q000494:pharmacology*; D049452:Green Fluorescent Proteins; D019791:Guanidine / Q000494:pharmacology; D006490:Hemostatics / Q000494:pharmacology; D006851:Hydrochloric Acid / Q000494:pharmacology; D015151:Immunoblotting; D007783:Lactones / Q000494:pharmacology; D008164:Luminescent Proteins / Q000378:metabolism; D008567:Membranes, Artificial; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors; D009419:Nerve Tissue Proteins / Q000737:chemistry*; D009687:Nuclear Proteins / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry*; D046988:Proteasome Endopeptidase Complex; D011485:Protein Binding / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012972:Sodium Hydroxide / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection; D019680:Trinucleotide Repeat Expansion; D014508:Urea / Q000494:pharmacology
D002424:Caustics; D005561:Formates; D006490:Hemostatics; D007783:Lactones; D008164:Luminescent Proteins; D008567:Membranes, Artificial; D009097:Multienzyme Complexes; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; C030544:formic acid; D049452:Green Fluorescent Proteins; C097188:polyglutamine; D012972:Sodium Hydroxide; D014508:Urea; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D019791:Guanidine; D006851:Hydrochloric Acid