Iuchi S, Hoffner G, Verbeke P, Djian P, Green H.
Oligomeric and polymeric aggregates formed by proteins containing expanded polyglutamine.
Proc Natl Acad Sci U S A. 2003 Mar 4;100(5):2409-14.
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The well-documented commonality between protein misfolding or aggregation and a wide range of neurological diseases has resulted in significant efforts directed toward gaining an understanding of molecular mechanisms responsible for this process. In 1994, Max Perutz postulated that the formation of "polar zippers" consisting of antiparallel β-sheets of expanded polyglutamine protein tracts may be responsible for self-aggregation underlying the toxicity associated with Huntington’s disease (HD) and other disorders (Perutz et al., 1994). More recently, elegant studies by the lab of Ron Wetzel at the University of Tennessee (Thakur & Wetzel, 2002), demonstrated requirements for optimum length and conformation of synthetic polyglutamine-containing peptides to nucleate and aggregate. The search for gene products and compounds designed to ameliorate the cellular consequences of protein aggregation has already yielded several important leads, none more promising than that described in the January 23 Nature from the lab of Junying Yuan. These authors report a comprehensive analysis of the numerous cytoprotective effects associated with the azo-dye, Congo red, in the inhibition of polyglutamine oligomerization.
Previous studies (Klunk et al. 1989 and Carter & Chao, 1998) illustrated that Congo red had the capacity to specifically associate with β-amyloid fibrils; it was subsequently shown to inhibit huntingtin fibrillogenesis in a dose-dependent manner (Heiser et al., 2000). In this latest paper, Sanchez et al. utilize a battery of experimental approaches to systematically address the intracellular mechanism by which Congo red reduces polyglutamine aggregation and cytotoxicity. These authors demonstrate that Congo red acts independent of molecular chaperones and the protein synthesis and degradation machinery by directly inhibiting nucleation of expanded polyglutamine aggregates. Moreover, Congo red dissolved preformed aggregates of a Q79 peptide from cell lysates in the absence of other proteins. In contrast, chrysamine G, a structurally similar compound that binds expanded polyglutamine, was incapable of disrupting aggregates or inhibiting polyglutamine-induced cell death.
Through the use of fluorescence resonance energy transfer (FRET), these investigators further showed that Congo red inhibited oligomerization of dual-labeled Q79 fluorescent protein variants and that this inhibition correlated with an increased solubility of Q79 protein in cell lysates. Bence et al., 2001 previously showed that an in vivo consequence of polyglutamine oligomerization was inhibition of the ubiquitin-proteosome system (UPS). The increased solubility of Congo red-treated polyglutamine protein resulted in a selective increase in Q79 degradation, indicating that the dye rendered misfolded proteins more accessible to the UPS-mediated degradation and simultaneously alleviated the cellular stress induced by the presence of polyglutamine aggregates. Importantly, Congo red was also demonstrated to be effective at significantly abrogating multiple features of the disease state when intraperitoneally infused into an HD mouse model at micromolar concentrations. In the absence of any overt toxic effects in these animals, Congo red-treated mice showed a reduction in severe weight loss, hindlimb dyskinesia, and blood glucose levels. Brain slices also showed reductions of polyglutamine immunostaining in the basal ganglia and hippocampus of Congo red-treated mice vs. control animals.
It will be interesting to see what native proteins may coordinately act to enhance cytoplasmic clearance of expanded polyglutamine-repeat containing proteins in the presence of Congo red. Further work aimed at defining gene products that may function to protect cells may expand our understanding of existing cellular mechanisms that might be induced to facilitate or mimic Congo red activity. The exceptional work of Sanchez et al. have set the stage for similar studies and mutational analyses on full-length polyglutamine target proteins. This will likely reveal the specificity of Congo red for these proteins and lead to the rational design of related compounds with potentially therapeutic value.
The accumulation of SCA7 in the knockin model could mean that the expanded polyQ forms oligomers that are invisible under the microscope. Depending on the endogenous level of SCA7, which apparently is very low, a critical
threshold to form visible inclusions may not have been reached until later. It is quite possible that several thousand molecules have to pile up in order to be seen under the microscope. It needs to be tested whether increased polyQ-polyQ interaction is required for the accumulation of SCA7 and if disruption of such interactions might ameliorate neurodegeneration in this model.