Orthogonal cross-seeding: an approach to explore protein aggregates in living cells.
Protein aggregation is associated with the pathology of many diseases, especially neurodegenerative diseases. A variety of structurally polymorphic aggregates or preaggregates including amyloid fibrils is accessible to any aggregating protein. Preaggregates are now believed to be the toxic culprits in pathologies rather than mature aggregates. Although clearly valuable, understanding the mechanism of formation and the structural characteristics of these prefibrillar species is currently lacking. We report here a simple new approach to map the nature of the aggregate core of transient aggregated species directly in the cell. The method is conceptually based on the highly discriminating ability of aggregates to recruit new monomeric species with equivalent molecular structure. Different soluble segments comprising parts of an amyloidogenic protein were transiently pulse-expressed in a tightly controlled, time-dependent manner along with the parent aggregating full-length protein, and their recruitment into the insoluble aggregate was monitored immunochemically. We used this approach to determine the nature of the aggregate core of the metastable aggregate species formed during the course of aggregation of a chimera containing a long polyglutamine repeat tract in a bacterial host. Strikingly, we found that different segments of the full-length protein dominated the aggregate core at different times during the course of aggregation. In its simplicity, the approach is also potentially amenable to screen also for compounds that can reshape the aggregate core and induce the formation of alternative nonamyloidogenic species.
Hinz
Justyna
J
Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany.
Gierasch
Lila M
LM
Ignatova
Zoya
Z
D000818:Animals; D002470:Cell Survival; D005091:Exons / Q000235:genetics; D051379:Mice; D011487:Protein Conformation; D018168:Receptors, Retinoic Acid / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D050486:Serotonin Plasma Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors
D018168:Receptors, Retinoic Acid; D050486:Serotonin Plasma Membrane Transport Proteins; C082927:retinoic acid binding protein I, cellular