Development of novel methods and tools to decipher the huntingtin post-translation modifications code

Huntington's disease (HD) is a fatal genetic neurodegenerative disorder caused by a CAG repeat expansion in the Huntingtin gene of more than 36 repeats. This repeat is translated into a polyglutamine (polyQ) stretch within the first exon-encoded region of the Huntingtin protein (Httex1). The aggregation of expanded Huntingtin protein (Htt) in the striatum and cortex has been implicated in neuronal dysfunction and death. Recent studies have shown that mutations that mimic post-translational modifications (PTMs) in the first 17 N-terminal amino acids (Nt17) of the protein dramatically influence the aggregation, subcellular localization, and toxicity of Httex1 and full-length Htt proteins. These findings suggest that these modifications may serve as reversible molecular switches for regulating Htt function in health and disease. However, assessing the effect of authentic PTMs and their cross-talk has not been possible due to the lack of knowledge about the enzymes responsible for these modifications. Moreover, the high aggregation propensity of Httex1 with increased polyQ content hampered the development of an efficient method to produce post-translationally modified proteins. By consequence, previous biophysical studies were mostly carried out using tagged Httex1 protein, or model peptides bearing PTM mimetics mutations which do not fully capture the chemical properties of the bona fide PTMs. Therefore, we developed a semisynthetic methodology that permits site-specific modification of the mutant Httex1 (at single or multiple residues). We used this strategy to investigate the effect of acetylation (at K6, K9, or K15) and phosphorylation (at T3), as well as the cross-talk between these modifications on the structure and aggregation of wild-type and mutant Httex1. Our results demonstrated that T3 phosphorylation (pT3) significantly inhibited the aggregation of mutant Httex1 (43Q), whereas only partial inhibition of aggregation was achieved by the phosphomimetic mutation (T3D). Acetylation of single ly-sine residues, K6, K9, or K15 (AcK6, AcK9, or AcK15), did not affect the aggregation of Httex1. Interestingly, AcK6, but not at AcK9 or K15, reversed the inhibitory effect of pT3. Next, we discovered novel kinases GCK and TBK1 that phosphorylate efficiently and specifically T3 and S13/S16, respectively. We exploited these kinases to produce homogenously phosphorylated recombinant Httex1 at T3 or both S13 and S16. We generated, using this method, suitable for structural and cellular applications. Finally, we combined chemical, semisynthetic and enzymatic methods to produce mutant Httex1 proteins bearing oxidized Methionine at position 8 (oxM8) alone or in combination with other Nt17 PTMs (pT3 or AcK6). We ob-served that oxM8 delayed the aggregation of mutant Httex1 and decreased the helical content of the Nt17 independently of the presence of other PTMs. Together, these findings provide novel insight into the role of Nt17 PTMs in regulating the aggregation of Httex1 and suggest that the aggregation and possibly the function (s) of Httex1 are controlled by complex regulatory mechanisms involving cross-talk between different PTMs. The tools presented in this thesis will open new windows of opportunity to decipher the role of Htt PTMs code in the regulation of Htt function in health and disease.