Nucleation Inhibition of Huntingtin Protein (htt) by Polyproline PPII Helices: A Potential Interaction with the N-Terminal α-Helical Region of Htt

Abstract

Huntington's disease (HD) is a genetic neurodegenerative disorder characterized by the formation of amyloid fibrils of the huntingtin protein (htt). The seventeen-residue N-terminal region of htt (Nt 17 ) has been implicated in formation of early-phase oligomeric species, which may be neurotoxic. Because tertiary interactions with a downstream (C-terminal) polyproline (polyP) region of htt may disrupt oligomer formation which are precursors to fibrillar species, the effect of co-incubation of a region of htt with a 10-residue polyP peptide on oligomerization and fibrilization has been examined by atomic force microscopy (AFM). From multiple, time-course experiments, morphological changes in oligomeric species are observed for the protein/peptide mixture compared with the protein alone. Additionally, an overall decrease in fibril formation is observed for the heterogeneous mixture. To consider potential sites of interaction between the Nt 17 region and polyP, mixtures containing Nt 17 and polyP peptides have been examined by ion mobility spectrometry (IMS) and gas-phase hydrogen deuterium exchange (HDX) coupled with mass spectrometry (MS). These data combined with molecular dynamics simulations (MDS) suggest that the C-terminal region of Nt 17 may be a primary point of contact. One interpretation of the results is that polyP may possibly regulate Nt 17 by inducing a random coil region in the Cterminal portion of Nt 17 , thus, decreasing the propensity to form the reactive amphipathic α-helix. A separate interpretation is that residues important for helix-helix interactions are blocked by polyP association.

Membrane Interactions Accelerate the Self-Aggregation of Huntingtin Exon 1 Fragments in a Polyglutamine Length-Dependent Manner

“…Therefore, quantitative studies of the speed of aggregation were performed with constructs carrying a limiting number of glutamines (see also [83]). It should be noted that in their natural context the solubility of the full-length protein or its exon 1 domain is increased by the polyproline flanking sequence following the polyQ domain [31,32,40,48]. Therefore, as an alternative, exon 1 fragments were investigated where the carboxy-terminal proline-rich domain slows down the aggregation process, probably through interactions with polyQ and htt17 (e.g., [26,42,84]).…”

“…Recent structural investigations reveal high conformational plasticity of htt17 and the subsequent polyQ domain where htt17 association [26], its interactions with the membrane [43][44][45][46][47] or with other polypeptide domains are associated with random coil-helix structural transitions [48,49]. A recent NMR study has shown that in the solution, the htt17 sequence associates in a dimer of dimers preaggregation state [50,51].…”

Membrane interactions accelerate the self-aggregation of huntingtin exon 1 fragments in a polyglutamine length-dependent manner

“…Therefore, quantitative studies of the speed of aggregation were performed with constructs carrying a limiting number of glutamines (see also (73)). It should be noted that in their natural context the solubility of the full-length protein or its exon 1 domain is increased by the polyproline flanking sequence following the polyQ domain (28,29,37,44). Furthermore, in the cellular environment, interactions with other proteins, chaperones and proteases, as well as other domains of the full-length protein assure that healthy cells are protected from huntingtin aggregation (74,75).…”

“…Once the interactions with the membrane are established the aggregation process is enhanced by high peptide density (i.e. P/L ratio) and by extended polyQ chains (Tables 2 and 3) but slowed down by the presence of the polyproline stretch that follows the poly-Q domain (44,83). Thereby these and other biophysical observations provide a rationale for a number of biochemical and cell biological experiments where huntingtin has been shown associated with membranes of intracellular organelles (6,10,13,(16)(17)(18)(19)(20)25) or where the membrane anchoring domain htt17 has been demonstrated to promote the development of the disease (6,13,26,30,(36)(37)(38).…”

“…Recent structural investigations reveal a highly dynamic behaviour of htt17 and the subsequent polyQ domain where htt17 association (23), its interactions with the membrane (39)(40)(41)(42)(43) or with other polypeptide domains are associated with random coilhelix structural transitions (44,45). Interestingly, the htt17 and the polyQ domains mutually influence each other and their conformational properties are coupled (46,47).…”