Huntington's disease (HD) is caused by the expansion mutation above a length threshold of a polyglutamine (polyQ) stretch in the huntingtin (Htt) protein. Mutant Htt (mHtt) pathogenicity is proposed to rely on its malfunction and propensity to misfold and aggregate. Htt has scaffolding properties and has been reported to interact with hundreds of partners. Many interactors show apparent increased or decreased affinity (dysinteraction) for mHtt, which may account for selective malfunctions and striatal degeneration in HD. These dysinteractions are proposed to result from mutant polyQ conformational changes that remain elusive. To date, dysinteractions have only been studied using semi-quantitative techniques with their outcome potentially influenced by the presence of mHtt aggregates. Therefore, the molecular mechanism underlying these dysinteractions remains to be determined. Here, we have used purified proteins devoid of aggregates to quantify the interaction of normal and mHtt with two partners: SH3GL3, reported to have increased binding to mHtt, and the 2B4 antibody, a model partner. Using surface plasmon resonance and pull-down techniques, we show that in the absence of aggregation polyQ length has no effect on Htt interactions. We demonstrate that the presence of aggregates affects the spatial distribution and solubility of Htt partners and strongly influences the outcome of pull-down experiments. Our results show that expanded polyQ per se does not alter Htt interactions and suggest that aggregated mHtt form molecular platforms that influence the Htt interacting network. Modulating mHtt aggregation could thus have beneficial effects on specific cellular pathways deregulated in HD.
Background Alterations in cellular calcium homeostasis have been recognised as one of several pathogenic mechanisms in Huntington's disease (HD). The discovery of a direct interaction between the C terminal extremity (C-ter) of inositol-3-phosphate receptor (IP3R) and huntingtin (htt) associated protein 1 (HAP1) has shed light on the function of HAP1 in calcium homeostasis regulation. The C-ter of IP3R consists of a small domain in continuation of the transmembrane region; it has important regulatory functions on the IP3R. Aim We wondered whether this regulatory domain is present in other membrane proteins, which might therefore interact with HAP1 and Htt, and could display altered functions in the presence of mutant htt. We searched for Cter-IP3R homologous sequences and discovered that the C-ter domain of another transmembrane channel—the Ryanodine Receptor (RYR), which is also a key regulator of calcium homeostasis—displays a striking sequence homology with C-ter-IP3R. Methods/techniques GST-pull down, Co-IP, immunocytochemistry, FLIM-FRET. Results In GST-pull down experiments, a specific interaction of HAP1 with C-ter-RYR and C-ter-IP3R, respectively, was observed. Mutant and wt Htt was pulled down in co-immunoprecipitation experiments using four different antibodies against RYR. Transiently expressed GFP tagged C-ter-RYR together with mRFP labelled N-ter-htt colocalised in N2a cells whereas expression of C-ter-RYR with mRFP alone did not. Immunocytochemistry of primary neurons showed a colocalisation of endogenous htt and RYR. To further assess the interaction of htt and RYR in primary neurons we performed FLIM-FRET based assays in a 96 well format using a high resolution fluorescent plate reader. Close proximity of RYR and htt was detected between the two proteins, as indicated by a significant shortening in lifetime of the donor fluorochrome compared with the negative control in which the acceptor fluorophore was not present. Significant lifetime shortening of the donor fluorochrome was observed independently of RYR or htt used as donor molecule. In summary, these results clearly suggest an interaction of RYR with htt and HAP1. Further work is in progress to clarify the effect of mutant htt on the channel activity of the ryanodine receptor.
Background Huntington's disease (HD) is due to an expansion above a polyQ length threshold in the huntingtin (Htt) protein. This expansion confers a toxic gain of function to the Htt which relies on its malfunction and propensity to misfold and aggregate. A first model proposed to explain this polyQ length dependent toxicity threshold stated that it is triggered by a toxic conformational change that occurs only above a certain polyQ length. However, we and others showed that normal and expanded polyQ display strikingly similar structural properties, supporting another model in which polyQ tracts are inherently toxic sequences whose deleterious effect gradually increases with polyQ length and aggregation kinetics. In this model, polyQ toxicity manifests when cellular factors that prevent proteotoxicity are overwhelmed. Aims Hundreds of proteins have been found to interact with Htt and many of them showed increased or decreased affinity with mutant Htt. If small and expanded polyQ share similar structural properties, what are the molecular mechanisms underlying these dysinteractions? The question has therapeutic implication, as the specificity of neuronal vulnerability in HD is thought to be due, in part, to mutant Htt dysinteractions with protein partners. Methods Up to now, the polyQ length influence on Htt interactions was exclusively studied using semiquantitative techniques prone to be biased by polyQ aggregation (Y2H, IP and pull downs). We report the first precise quantification of the polyQ length influence on Htt/partners affinity, using a dedicated biophysical technique (surface plasmon resonance), in conditions where the absence of aggregation is strictly controlled. Results In conditions where the absence of aggregates is strictly controlled, we observe that polyQ length variations do not affect the interactions between soluble Htt exon-1 and protein partners. However, we provide evidence that the presence of aggregated polyQ species can dramatically influence the behaviour of Htt partners. Conclusions We thus propose that aggregated polyQ species influence Htt's and its partners' behaviours and result in aberrant interactions. Our work suggests that modulating mutant Htt aggregation could have positive effects on specific cellular pathways deregulated in HD.
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