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
Huntingtons disease is a neurodegenerative disorder caused by the expansion of a polyglutamine (poly Q) repeat (>36Q) in the N-terminal domain of the huntingtin protein (Htt), which renders the protein or fragments thereof more prone to aggregate and form inclusions. Although several Htt N-terminal fragments of different lengths have been identified within Htt inclusions, most studies on the mechanisms, sequence, and structural determinants of Htt aggregation have focused on the Htt exon1 (Httex1). Herein, we investigated the aggregation properties of mutant N-terminal Htt fragments of various lengths (Htt171, Htt140, and Htt104) in comparison to mutant Httex1. We also present a new chemoenzymatic semisynthetic strategy that enables site-specific phosphorylation of Htt beyond Httex1. These advances yielded novel insights into how PTMs and structured domains beyond Httex1 influence aggregation mechanisms, kinetics, and fibril morphology of longer N-terminal Htt fragments. We demonstrate that phosphorylation at T107 significantly slowed its aggregation, whereases phosphorylation at T107 and S116 accelerated the aggregation of Htt171, underscoring the importance of crosstalk between different PTMs. We demonstrate that mutant Htt171 proteins aggregate via a different mechanism and form oligomers and fibrillar aggregates with morphological properties that are distinct from that of mutant Httex1. These observations suggest that different N-terminal fragments could have distinct mechanisms of aggregation and that a single polyQ-targeting anti-aggregation strategy may not effectively inhibit the aggregation of all N-terminal Htt fragments. Finally, our results underscore the importance of further studies to investigate the aggregation mechanisms of Htt fragments and how the various fragments interact with each other and influence Htt toxicity, pathology formation, and disease progression. Table of content O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=136 SRC="FIGDIR/small/436743v1_ufig1.gif" ALT="Figure 1"> View larger version (60K): org.highwire.dtl.DTLVardef@113aebborg.highwire.dtl.DTLVardef@1349236org.highwire.dtl.DTLVardef@6b273borg.highwire.dtl.DTLVardef@dcad5f_HPS_FORMAT_FIGEXP M_FIG C_FIG