Orthogonal Cross-Seeding: An Approach To Explore Protein Aggregates In Living Cells

Abstract

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.Accumulation of macroscopically observable, abnormal protein deposits is the cytological feature of many age-related neurodegenerative diseases, including Alzheimer's, Parkin-son's, and Huntington's diseases (1-3). Although the characteristic pathological aggregates are primarily made up of the fibrillar aggregated form of the disease-associated protein, multiple aggregate morphologies are accessible to each individual amyloidogenic protein. The heterogeneity of aggregate morphologies may arise from (1) variations in the environment and aggregate growth conditions (4-8), (2) different segments of the primary sequence that govern the formation of the aggregate core (8-13), or (3) complex pathways of formation, including † This project was supported by the SysMO (KOSMOBAC), the DFG (project IG 73/4-1, and the Heisenberg award IG 73/1-1) to Z.I., and by the National Institutes of Health (grant GM027616 and a 2006 NIH Director's Pioneer Award to L.M.G.).

The Relationship between Aggregation and Toxicity of Polyglutamine-Containing Ataxin-3 in the Intracellular Environment of Escherichia coli

“…Indeed, it has been reported that, following IPTG deprivation, protein expression is strongly inhibited in T7-based inducible strains, like the one employed here [45]. Thus, after IPTG removal we monitored growth rates and normalized them with respect to that of the wild type, to allow for the effect of overexpression-related cellular stress.…”

Physicochemical Properties of Cells and Their Effects on Intrinsically Disordered Proteins (IDPs)

“…Both in vitro and in E. coli the aggregation prone P39A CRABP variant seeds early oligomer formation while polyQ tracts with lengths above the pathological threshold seeded the formation of later, detergent resistant aggregates. 888,889 Thus, as observed for polyQs appended to other folded proteins, 890−893 the context of the polyQ sequence is key. Aggregation of polyQ repeats tagged with an FP, (often cerulean fluorescent protein (CFP), GFP, or yellow fluorescent protein (YFP) at the C-terminal end) or complementary GFP fragments has been studied in yeast cells, in mammalian cells and in the transparent nematode C. elegans (for reviews and methods see refs (852, 894, and 895)).…”

Bacterial curli protein promotes the conversion of PAP248-286into the amyloid SEVI: cross-seeding of dissimilar amyloid sequences

“…However, this view has recently been challenged by the observation of efficient cross-polymerization between several non-prion amyloid species (Yagi et al, 2005; Han, Weinreb & Lansbury, 1995; Westermark, Lundmark & Westermark, 2009; Morales et al, 2010; O’Nuallain et al, 2004; Ma & Nussinov, 2012). Seeds formed from amyloid fibers of some non-homologous proteins can either reduce or eliminate the lag-time of amyloid formation of another protein, (Yagi et al, 2005; Han, Weinreb & Lansbury, 1995; Westermark, Lundmark & Westermark, 2009; Morales et al, 2010; O’Nuallain et al, 2004; MacPhee & Dobson, 2000) although the phenomenon is not universal and even single point mutations have been shown to disrupt cross-seeding in some cases (O’Nuallain et al, 2004; Jarrett & Lansbury, 1992; Hinz, Gierasch & Ignatova, 2008; Rajan et al, 2001). Cross-seeding may also be asymmetric, with one protein able to seed another but not be seeded by it (Yu et al, 2012; Siddiqua et al, 2012).…”