Post-Translational Modifications (PTMs), Identified on Endogenous Huntingtin, Cluster within Proteolytic Domains between HEAT Repeats

Abstract

Post-translational modifications (PTMs) of proteins regulate various cellular processes. PTMs of polyglutamine-expanded huntingtin (Htt) protein, which causes Huntington’s disease (HD), are likely modulators of HD pathogenesis. Previous studies have identified and characterized several PTMs on exogenously expressed Htt fragments, but none of them were designed to systematically characterize PTMs on the endogenous full-length Htt protein. We found that full-length endogenous Htt, which was immunoprecipitated from HD knock-in mouse and human post-mortem brain, is suitable for detection of PTMs by mass spectrometry. Using label-free and mass tag labeling-based approaches, we identified near 40 PTMs, of which half are novel (data are available via ProteomeXchange with identifier PXD005753). Most PTMs were located in clusters within predicted unstructured domains rather than within the predicted α-helical structured HEAT repeats. Using quantitative mass spectrometry, we detected significant differences in the stoichiometry of several PTMs between HD and WT mouse brain. The mass-spectrometry identification and quantitation were verified using phospho-specific antibodies for selected PTMs. To further validate our findings, we introduced individual PTM alterations within full-length Htt and identified several PTMs that can modulate its subcellular localization in striatal cells. These findings will be instrumental in further assembling the Htt PTM framework and highlight several PTMs as potential therapeutic targets for HD.

Design and characterisation of mutant and wild-type huntingtin proteins produced from a toolkit of scalable eukaryotic expression systems

“…Our constructs permit production of HTT in complex with the HTT binding protein HAP40, as well as in its apo form and we have characterised these HTT protein samples. This includes mapping post-translational modifications (PTMs) of the proteins derived from both insect and mammalian cells, revealing similar modification motifs to those previously reported in the literature (17)(18)(19). Both apo and HTT-HAP40 complex samples are folded, as judged by reasonable thermal melting transitions of protein samples in solution.…”

“…Monomethylation of some lysine and arginine residues was also detected ( Table 2). Sequence analysis of HTT using CIDER (32) and IUPred (33) in conjunction with analysis of the recently published near-atomic resolution cryo-electron microscopy structure of HTT in complex with HAP40 (23) (Figure 5), reveals that most of the phosphorylation sites are within disordered regions of the protein structure as described previously (18). Whilst some of these previously unreported modifications may be artefacts of the Sf9 expression system, they appear to have minimal effect on global huntingtin function, as seen by the ability of this sample to form a complex with HAP40.…”

“…Interestingly, we also observed acetylation (K826 and K2932), monomethylation (R2781 and H2786) and dimethylation (R2053) of our samples, none of which have been previously described in the literature. Acetylation of HTT at other sites has been previously described and methylation motifs are observed in MS data of post-mortem human brain tissue samples of HTT (18), indicating that HTT protein methylation is a physiological modification.…”

“…PTM of HTT is well described for protein derived from various mammalian cell systems and in some detail for HTT extracted from post-mortem brain tissue (17)(18)(19) . However, it is not known if these PTMs are conserved in HTT expressed in Sf9 insect cells.…”

“…In vitro or animal models S421 9 (9) Reported (16,18,26,(54)(55)(56)(57)(58)(59)(60)(61)(61)(62)(63)(64)(65)(66)(67)(68)(69)(70)(71)(72)(73)(74)(75)(76)(77)(78)(79) Yes Yes S421 / S434…”