In-cell aggregation of a polyglutamine-containing chimera is a multistep process initiated by the flanking sequence.
Toxicity in amyloid diseases is intimately linked to the nature of aggregates, with early oligomeric species believed to be more cytotoxic than later fibrillar aggregates. Yet mechanistic understanding of how aggregating species evolve with time is currently lacking. We have explored the aggregation process of a chimera composed of a globular protein (cellular retinoic acid-binding protein, CRABP) and huntingtin exon 1 with polyglutamine tracts either above (Q53) or below (Q20) the pathological threshold using Escherichia coli cells as a model intracellular environment. Previously we showed that fusion of the huntingtin exon 1 sequence with >40Q led to structural perturbation and decreased stability of CRABP (Ignatova, Z., and Gierasch, L. M. (2006) J. Biol. Chem. 281, 12959-12967). Here we report that the Q53 chimera aggregates in cells via a multistep process: early stage aggregates are spherical and detergent-soluble, characteristics of prefibrillar aggregates, and appear to be dominated structurally by CRABP, in that they can promote aggregation of a CRABP variant but not oligoglutamine aggregation, and the CRABP domain is relatively sequestered based on its protection from proteolysis. Late stage aggregates appear to be dominated by polyGln; they are fibrillar, detergent-resistant, capable of seeding aggregation of oligoglutamine but not the CRABP variant, and show relative protection of the polyglutamine-exon1 domain from proteolysis. These results point to an evolution of the dominant sequences in intracellular aggregates and may provide molecular insight into origins of toxic prefibrillar aggregates.
Ignatova
Zoya
Z
Max Planck Institute of Biochemistry, Am Klopferspitz 18, Martinsried 82152, Germany. ignatova@biochem.mpg.de
Thakur
Ashwani K
AK
Wetzel
Ronald
R
Gierasch
Lila M
LM
D000682:Amyloid / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000686:Amyloidosis / Q000235:genetics / Q000378:metabolism; D000818:Animals; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics; D006801:Humans; D000071058:Huntingtin Protein; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018168:Receptors, Retinoic Acid / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D000682:Amyloid; C086055:HTT protein, human; D000071058:Huntingtin Protein; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D010455:Peptides; D018168:Receptors, Retinoic Acid; D011993:Recombinant Fusion Proteins; C097188:polyglutamine