27 Sep 2004

In the September 24 issue of Molecular Cell, Erich Wanker and colleagues demonstrate the power of combining cDNA and yeast two-hybrid (Y2H) screens in pursuit of protein accomplices of disease-suspect molecules, in this case huntingtin (htt). They found numerous potential new protein-protein interactions (PPIs) in a huntingtin-centered network, and zeroed in on the protein GIT1, which may just be an active player in the formation of htt-rich aggregates found in Huntington disease.

By combining library and Y2H screens, Wanker and first author Heike Goehler of the Max Delbruek Center for Molecular Medicine in Berlin, along with colleagues at several other institutions, were able to create a protein-protein interaction (PPI) network with htt at the hub. The researchers focused their search on proteins known to participate in cellular processes where htt has been spotted, among them clathrin-mediated endocytosis, apoptosis, vesicle transport, cell signaling, morphogenesis, and transcriptional regulation. In the htt PPI network, Goehler and colleagues identified 186 Y2H interactions between 35 bait and 51 prey proteins, including 165 new potential interactions.

There were 19 direct htt partners in the PPI, and the researchers found that only one—GIT1, a G-protein-coupled receptor kinase-interacting protein—was able to enhance htt aggregation when cotransfected into cells with an aggregation-prone N-terminal htt fragment. They also found that a C-terminal GIT1 fragment promoted the localization of the htt fragment to vesicles near the nucleus; the C-terminal fragment of the protein is known to promote the formation of intracellular vesicles. The same was true in an assay with both full-length proteins, but with lower rates of vesicle formation and htt recruitment into the vesicles.

After showing that RNA interference against endogenous GIT1 prevents the formation of htt aggregates in vitro, the researchers began to look for evidence of a physiological association between the two proteins. They found GIT1 and htt co-precipitating in transfected cells, but more importantly, they detected the two together in extracts from human cortex. Finally, they detected GIT1 in htt aggregates from human HD brain. In particular, they found a large proportion of the protein was missing its N-terminus, indicating that GIT1 is N-terminally cleaved in HD, but not normal, brain.

So what are GIT1 and htt up to? The authors note that GIT1 is thought to be an integrator of signaling pathways controlling vesicle trafficking, adhesion, and cytoskeletal organization. Given their findings that GIT1 seems to induce htt to enter perinuclear vesicles, "this suggests that GIT1-mediated concentration of mutant htt in membranous compartments may trigger polar zipper formation (Perutz, 1999) and subsequent accumulation of insoluble protein aggregates," write the authors.

Another mystery to be solved is the overabundance of N-terminally cleaved GIT1 in HD brain. Does this indicate that GIT1 is a risk factor for HD, or does the mutant htt induce abnormal proteolysis of GIT1? Either way, the evidence shows that the shortened GIT1 can have various deleterious effects inside cells.—Hakon Heimer

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References

Paper Citations

1. Perutz MF. Glutamine repeats and neurodegenerative diseases: molecular aspects. Trends Biochem Sci. 1999 Feb;24(2):58-63. PubMed.

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