Huntington’s disease (HD) is caused by mutations that expand a polyglutamine (polyQ) repeat sequence within huntingtin from less than (typically) 25 glutamines to greater than 36. These additional glutamine insertions cause the protein to misfold and aggregate. Recent studies have raised the possibility that the mutant polyQ-expanded monomer can adopt an abnormal compact structure that is more closely linked to pathogenesis than the aggregated forms [1]. However, little is known about this monomeric state and its impact on cell biology. Here we describe our early results in first measuring which proteins in the cell interact with the pathogenic monomer and second, defining the structural features of the pathogenic monomer relative to non-pathogenic forms. We applied immunoprecipitation to pull down binding partners of monomeric huntingtin and compared the differences between a non-pathogenic (25Q) and pathogenic (46Q) variant. Mass spectrometry identified 1,924 protein binding partners of both forms of huntingtin, with 66 significantly different between non-pathogenic and pathogenic polyQ lengths. The two strongest interactors of mutant huntingtin monomers were Fus and Hspa4l. Fus has been linked to huntingtin toxicity and is found primarily in aggregates, while Hsp chaperones have long been a target in modifying protein misfolding [2,3]. We will also investigate the conformation of mutant (36-46Q) vs non-mutant (16-25Q) huntingtin monomers by NMR using two approaches. First we will insert lanthanide probes in the sequence of huntingtin to resolve the polyQ resonances and define the structure and dynamics of these low complexity regions. Second we will examine polyQ resonances on huntingtin expressed directly in mammalian cells, which gives us the opportunity to observe huntingtin in a setting of cellular engagement mechanisms.
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