Site-specific phosphorylation of Huntingtin exon 1 recombinant proteins enabled by the discovery of novel kinases

Abstract

Posttranslational modifications (PTMs) within the first 17 amino acids (Nt17) of exon1 of the Huntingtin protein (Httex1) play important roles in modulating its cellular properties and functions in health and disease. In particular, phosphorylation of threonine and serine residues (T3, S13, and/or S16) has been shown to inhibit Htt aggregation in vitro and inclusion formation in cellular and animal models of Huntington’s disease (HD). In this manuscript, we describe a new and simple methodology for producing milligram quantities of highly pure wild type or mutant Httex1 proteins that are site-specifically phosphorylated at T3 or at both S13 and S16. This advance was enabled by 1) the discovery and validation of novel kinases that efficiently phosphorylate Httex1 at S13 and S16 (TBK1), at T3 (GCK) or T3 and S13 (TNIK and HGK); and, 2) the development of an efficient methodology for producing recombinant native Httex1 proteins using a SUMO-fusion expression and purification strategy. As proof of concept, we demonstrate how this method can be applied to produce Httex1 proteins that are both site- specifically phosphorylated and fluorescently labeled or isotopically labeled. Together, these advances should increase access to these valuable tools and expand the range of methods and experimental approaches that can be used to elucidate the mechanisms by which phosphorylation influences Httex1 structure, aggregation, interactome and function(s) in health and disease.

Investigating crosstalk among PTMs provides novel insight into the structural basis underlying the differential effects of Nt17 PTMs on mutant Httex1 aggregation

“…We took advantage of recent advances from our lab that allow for the generation of microgram quantities of highly pure mutant Httex1 fused to the SUMO protein [48], which enhances the solubility of mutant Httex1 (Figure 1-B). The desired PTMs (oxidation or phosphorylation) are then introduced, using enzymatic or chemical approaches, into Nt17 of the fusion protein, followed by removal of the SUMO protein and purification of the modified mutant Httex1 by RP-HPLC[49].…”

“…To produce mHttex1 oxidized at M8 and phosphorylated at T3 (mHttex1-oxM8/pT3), we first generated SUMO-mHttex1-oxM8 as described above. After overnight dialysis to remove the excess of H 2 O 2 , the fusion protein was co-incubated overnight with GCK kinase, a kinase that we recently reported to efficiently phosphorylate Httex1 specifically at T3[49]. The extent of phosphorylation was monitored by ESI/MS.…”

“…To produce mutant Httex1 that is both oxidized at M8 and acetylated at lysine 6, we employed a semisynthetic protein strategy (Figure 1-C) that we previously used to introduce single or multiple PTMs within Nt17 of mHttex1 [49]. mHttex1-AcK6/oxM8 was produced using native chemical ligation between the Htt A10C-90 43Q and Ac-2-9-Nbz AcK6/oxM8 peptides (characterization by ESI-MS and UPLC is shown in Figure S2-B), followed by desulfurization to convert the cysteine to the native Alanine 10 (Figure S2-C).…”

The Nt17 domain and its helical conformation regulate the aggregation, cellular properties and neurotoxicity of mutant huntingtin exon 1

“…Therefore, the proteins used in the cellular studies were produced by E. coli and purified as described previously [41]. The phosphorylated proteins were produced using a chemoenzymatic approach recently developed by our group-based kinase-mediated sitespecific phosphorylation of Httex1 proteins [132]. The expression, purification and characterization methods of all the proteins used in this manuscript have been extensively detailed in our previous studies [41,85,94,[132][133][134].…”

A new chemoenzymatic semisynthetic approach provides novel insight into the role of phosphorylation beyond exon1 of Huntingtin and reveals N-terminal fragment length-dependent distinct mechanisms of aggregation

“…Our laboratory has developed several semisynthetic and chemoenzymatic strategies for the introduction of single or multiple PTMs in the Nt17 domain of Httex1. 17,22,18,23,24 Notably, prior to the work described here, there were no methods that made this possible in domains beyond Httex1, for example, in longer N-terminal Htt fragments. Thus, precluding systematic studies to elucidate the role of PTMs, such as T107, S116, and S120, in regulating Htt structure and aggregation and/or potential crosstalk between these phosphorylation sites and neighboring PTMs (such proteolytic cleavage site between C105-A114 13 ).…”