Researchers at Michael Mancini’s lab at Baylor College of Medicine, Houston, Texas, studied inclusion bodies formed by the polyQ protein ataxin1. By measuring fluorescence recovery after photobleaching (see related news item) of green fluorescent protein (GFP)-ataxin1 chimeras in transfected cells, the authors determined how quickly free cytoplasmic protein could exchange with protein in polyQ aggregates. They found that aggregates comprise both fast- and slow-exchanging components with the speed of the exchange depending on the length of the polyQ expansion. Those proteins with 84 glutamines, for example, exchanged much more slowly than those with only two glutamines, such that 87 percent of inclusions with 84Q chimeras but only 19 percent of 2Q chimeras were classified as slow-exchanging. The authors found that the expansion length was also a predictor of the size of inclusions. 2Q, 30Q, and 84Q expansions result in large inclusions in 4.4, 12.3, and 18.2 percent of infected cells, respectively.

Stenoien, et al., also examined mobility in inclusions containing ubiquitin, CREB-binding protein (CBP), and the LMP2 subunit of the proteasome core, proteins that have been shown previously to associate with polyQ inclusions. Low and high amounts of ubiquitin correlated with fast- and slow-exchange of ataxin-84Q, respectively, whereas the effect of LMP2 on ataxin mobility was just the opposite. These results suggest that the ubiquitin-proteasome degradation pathway has a significant influence on the dynamics of these aggregates. Interestingly, LMP2 and CBP mobility was faster than ataxin 1, even in aggregates where the latter exchanged very slowly with its cytoplasmic form, indicating that at least some proteins are not irreversibly trapped in these inclusions.

This was also the conclusion reached by Soojin Kim and colleagues working under the direction of Richard Morimoto at Northwestern University, Evanston, Illinois. Using similar techniques they studied the interaction between polyQ proteins, the TATA binding protein (containing a 37-glutamine stretch) and the chaperone Hsp70, which has been shown previously to attenuate the toxicity associated with polyQ expansions. They found that only 17 percent of the TATA binding protein found in aggregates was readily diffusible, while in contrast over 70 percent of Hsp70 associated with aggregates was mobile. In fact, the rate of diffusion of Hsp70 was comparable to that observed between the chaperone and unfolded protein substrates in the nucleolus. This fact, coupled with the observation that Hsp70 lacking its substrate binding domain diffused even faster, suggests some active role for the chaperone, perhaps in inhibiting aggregate growth.

One caveat to these and similar studies, is that not all aggregates and inclusion bodies may behave similarly. Previous work by Chai et al, for example, has shown that aggregates of ataxin 1 and ataxin 3 behave quite differently, the latter tightly sequestering CBP.

“These are elegant papers,” says Henry Paulson, University of Iowa. “They extend the idea that these inclusions are complex. They do have aggregated protein, that seems very clear, but they also have proteins that associate in a dynamic way.” But there are differences. “We found that ataxin3 behaved like it was stuck whereas ataxin 1 had a lot of dynamic behavior,” recalls Paulson, “and Mancini’s group really shows that ataxin1 protein is a dynamic protein. It has become very clear of the last couple of years that researchers must investigate these polyglutamine domains within the context of the protein. Some of the differences that exist in disease clinically, may relate to differences in the actual proteins in which polyglutamine occurs.”-Tom Fagan

Reference:
Stenoien DL, Mielke M, Mancini MA. Intranuclear ataxin1 inclusions contain both fast- and slow-exchanging components. Nat Cell Biol. 2002 Oct ;4(10):806-10. (Abstract)

Kim S, Nollen EAA, Kitagawa K, Bindokas VP, Morimoto RI. Polyglutamine protein aggregates are dynamic. Nat Cell Biol. 2002 Oct ;4(10):826-31. (Abstract)