Investigating Crosstalk Among PTMs Provides Novel Insight Into the Structural Basis Underlying the Differential Effects of Nt17 PTMs on Mutant Httex1 Aggregation

Abstract

Post-translational modifications (PTMs) within the first 17 amino acids (Nt17) of the Huntingtin protein (Htt) have been shown to inhibit the aggregation and attenuate the toxicity of mutant Htt proteins in vitro and in various models of Huntington’s disease. Here, we expand on these studies by investigating the effect of methionine eight oxidation (oxM8) and its crosstalk with lysine 6 acetylation (AcK6) or threonine 3 phosphorylation (pT3) on the aggregation of mutant Httex1 (mHttex1). We show that M8 oxidation delays but does not inhibit the aggregation and has no effect on the final morphologies of mHttex1aggregates. The presence of both oxM8 and AcK6 resulted in dramatic inhibition of Httex1 fibrillization. Circular dichroism spectroscopy and molecular dynamics simulation studies show that PTMs that lower the mHttex1 aggregation rate (oxM8, AcK6/oxM8, pT3, pT3/oxM8, and pS13) result in increased population of a short N-terminal helix (first eight residues) in Nt17 or decreased abundance of other helical forms, including long helix and short C-terminal helix. PTMs that did not alter the aggregation rate (AcK6) of mHttex1 exhibit a similar distribution of helical conformation as the unmodified peptides. These results show that the relative abundance of N- vs. C-terminal helical conformations and long helices, rather than the overall helicity of Nt17, better explains the effect of different Nt17 PTMs on mHttex1; thus, explaining the lack of correlation between the effect of PTMs on the overall helicity of Nt17 and mHttex1 aggregation in vitro. Taken together, our results provide novel structural insight into the differential effects of single PTMs and crosstalk between different PTMs in regulating mHttex1 aggregation.

CREB1K292 and HINFPK330 as Putative Common Therapeutic Targets in Alzheimer’s and Parkinson’s Disease

“…For instance, Nguyen et al, 2016, concluded that glutamine triggers acetylation-dependent degradation of glutamine synthetase, whereas Son et al, 2020, demonstrated that leucine regulates autophagy through acetylation of the mTORC1. , Moreover, the role of methionine involvement in lysine acetylation is not studied so far in AD and PD, but yet at the same time demonstrated the potential relationship between methionine and lysine acetylation in other neurological defects. For instance, Chiki et al, 2021, concluded that the presence of oxidation of methionine at position 8 and acetylation at K6 resulted in the dramatic inhibition of Httex1 fibrilization . Thus, these studies correlate with our results and suggest that glutamic acid (E), leucine (L), methionine (M), valine (V), and glutamine (Q) could be critical amino acid residues in acetylation and HDAC binding.…”

“…For instance, Chiki et al, 2021, concluded that the presence of oxidation of methionine at position 8 and acetylation at K6 resulted in the dramatic inhibition of Httex1 fibrilization. 106 Thus, these studies correlate with our results and suggest that glutamic acid (E), leucine (L), methionine (M), valine (V), and glutamine (Q) could be critical amino acid residues in acetylation and HDAC binding. Further, structural information of CREB reveals that it consists of 11 exons and three isoforms that are produced through alternative splicing.…”

Huntingtin exon 1 deletion does not alter the subcellular distribution of huntingtin and gene transcription in mice

“…N-terminal HTT encoded by exon 1 has been found to be important for membrane anchoring, protein interactions, and subcellular distribution (Michalek et al, 2013 ; Tao et al, 2019 ; Riguet et al, 2021 ). In the exon 1 domain of HTT, the first 1–17 amino acids can modulate mutant HTT oligomerization, toxicity, and subcellular localization (Cornett et al, 2005 ; Gu et al, 2009 ; Arndt et al, 2019 ; Chiki et al, 2021 ; Vieweg et al, 2021 ). However, our findings showed that removing exon 1 of mouse Htt did not significantly alter its subcellular distribution.…”

“…N17 amino acids in exon 1 are well conserved across species and are thought to be important for HTT's function as well as the toxicity of mutant HTT. In support of this, N17 amino acids can markedly modify the toxicity of mutant HTT and its nuclear localization (Cornett et al, 2005;Gu et al, 2009;Arndt et al, 2019;Chiki et al, 2021;Vieweg et al, 2021).…”

Deep Learning-Assisted Single-Molecule Detection of Protein Post-translational Modifications with a Biological Nanopore

“…10 Therefore, a method to accurately identify protein PTMs is essential as increasing evidence suggests the PTM code is combinatorial in nature and involves complex interplay and cross-talk among PTMs. 11,12 PTMs have been implicated in a wide variety of diseases, such as characterizing the changes in protein PTM types and levels is highly relevant for early diagnosis and monitoring the progression of diseases, as well as for evaluating the efficacy of emerging therapies. 4,13 For example, several neurodegenerative diseases including Alzheimer's disease (AD) and Parkinson's disease (PD) are caused by the accumulation of misfolded and aggregated proteins in the brain regions that are affected by the disease (e.g., amyloid-β in amyloid plaques, Tau in neurofibrillary tangles, and α-synuclein in Lewy bodies and Lewy neurites).…”