Hochauflösende Proteinmarkierung für in vivo und in vitro Untersuchungen der Mechanismen der Proteinaggregation
Final Report Abstract
A fascinating problem in biology is to understand the principles of integration of various processes in the cell. The studies in the test tube have provided so far solid baseline knowledge; however the highly crowded cellular environment and the plethora of interaction partners in the cell introduce another level of complexity to these processes. In this project we have developed and established several approaches that allowed us to study non-invasively and directly in the cell the highly heterogeneous, misfolding and aggregation processes. Using a conformational-sensitive labeling assay based on the FIAsH-labeling protocol that reports on misfolding and aggregation we were able to quantitatively measure the cellular aggregation rates of mutants of a model protein with various aggregation propensity. This data set which to our knowledge is the first quantitative in vivo set served as a base-line experimental set to develop a model to predict the aggregation propensity of any protein in the cell environment directly from the primary amino acid sequence. Subsequently, we dedicated a substantial effort to elucidate the aggregation pathway of polyglutamine-containing (polyQ) proteins that are implicated in the neurodegenerative diseases, i.e., Huntington's, dentatorubral and pallldoluyslan atrophy, and several spinocerebellar ataxias. Formation of macroscopically discernible inclusions composed of fibrillar aggregates is a major pathological hallmark of these diseases, however their role in the disease pathology is controversial and still a subject of intense debate. Transiently existing, intermediate aggregate species are rather suggested to be the main culprits exerting the toxic effect; their transient nature hinders their isolation and structural characterization. We developed an orthogonal cross-seeding assay allowing mapping the aggregate core directly in the cell. Using this approach combined with several ex vivo characterization of the cell aggregates we could determine three various species in the aggregation pathway of polyQ-containing model protein (that is a chimera of a stably folded protein and exon1 of Huntingtin with various length of the polyQ stretch): 1) Thee aggregation starts from a monomeric nucleus which is alternatively folded monomer as revealed by intramolecular FRET measurements. 2) In the first phase of aggregation small, oligomeric detergent-labile species with more amorphous phenotype are formed. Interestingly, the polyQ stretch Is not participating in the aggregate core. 3) The late species have fibrillar structure and are largely detergent-resistant with clearly defined polyQ aggregate core. Furthermore, we tested whether the naturally evolved mechanism, the osmocompensatory response, can combat both amorphous and structured aggregation. Protection against osmotic stress involves uptake of small organic osmolytes that stabilize the own protein bulk against denaturation and subsequent aggregation under stress conditions. Clearly, we saw a difference in the function of various osmotic substances with effect ranging from favoring the aggregation (by glycine-betaine) to solubilizing amorphous aggregates (proline). Intriguingly, in the case of the structured polyQ-aggregation, proline inhibited the formation of fibrillar species and introduced offpathway amorphous aggregate lacking the polyQ-core. The latter are benign for the cell viability.
Publications
- (2007) Effect of osmolytes on protein folding and aggregation in cells. Meth. Enzymol. 428, 355-372
Ignatova, Z. and Gierasch, L.M.
- (2007) In vivo aggregation of polyglutamine tracts is a multistep process with early involvement of flanking sequences. J. Biol. Chem.282, 36736-36743
Ignatova, Z., Thakur, A.K., Wetzel, R.B., and Gierasch, L.M.
- (2008) A fluorescent window into protein folding and aggregation in cells. Meth. Cell. Biol. 89, 59-70
Ignatova, Z. and Gierasch, L.M.
- (2008) Cross-seeding in vivo: an approach for direct determination of mechanism of aggregation in living cells. Biochem. 47, 4196-4200
Hinz, J., Gierasch, L.M., and Ignatova, Z.
- (2009) A method for direct measurement of protein stability in vivo. Meth. Mol. Biol. 490, 165-178
Ignatova, Z. and Gierasch, L.M.
- (2009) Isolation of homogeneous ribosome-nascent complexes from coupled cell-free transcription-translation system. Nature Protocols, online
Zhang, G. and Ignatova, Z.
- (2009) Transient ribosomal attenuation coordinates protein synthesis and co-translational folding. Nature Struct. Mol. Biol. 16, 274-280
Zhang, G., Hubalewska, M., and Ignatova, Z.
