The Sites Of Prolyl Isomerase PIN1 Interaction And Cis/trans Isomerization Catalysis On Huntingtin-N Terminal

Huntington’s disease(HD) is a genetically inherited autosomal dominant neurodegenerative disorder, the mutation for HD is an abnormal expansion of a CAG repeat in the IT15 gene which encodes for huntingtin. The nmmber of CAG expansions exceeding 35 repeats that caused abnormal polyglutamine(Poly-Q) expansion in HD. Mutant huntingtin(m Htt) may activate a variety of proteolytic enzymes, these proteolytic enzyme will cut m Htt into different length segments, m Htt toxic fragments. The m Htt toxic fragments misfold in the neurons, form dense aggregates and cause strong cytotoxicity.The peptidyl prolyl cis/trans isomerase NIMA-interacting 1(PIN1) is a proline-directed phosphorylation enzyme, parvulin subfamily of PPIases. Peptidyl-prolyl reserved ammonia acyl bond contain proline, so it can form the cis or trans transformation proteins. Many substrates in the peptide protein function key switch control corresponding cis/trans transformation, and the transformation of this structure under normal circumstances is very slow. PIN1 is a kind of cis/trans isomerase, specificity identification of phosphorylation of serine/threonine- proline motifs(p S/T-P). PIN1 play a role in proline directional phosphorylation related signaling pathways. PIN1 is associated with a variety of pathogenic proteins in neurodegenerative diseases, such as Tau protein of Alzheimer’s disease, α-synuclein of Parkinson’s disease.Our previous research had found that PIN1 could interact with segments of m Htt toxicity, inhibit m Htt misfolding and degradation, and inhibiting m Htt cytotoxicity.But their specific binding sites and combination mechanism were not clear. We also found that the formation of aggregates rate increased significantly after m Htt 62 site glutamate mutate to alanine. 62E63 P motifs are similarity with PIN1 specificity identification of p S/T-P motifs. So it may be becatalytic site of PIN1. We analyzed the interaction sites of PIN1 and amino terminus of huntingtin by Co-Immunoprecipitation technique to, and tested the catalytic sites of PIN1 in huntingtin-N terminal(Htt-Nt) by nuclear magnetic resonance(NMR) methods.1. Huntington’s protein amino terminal 1-17 amino acids(N17Htt) and Proline-rich region(PRRHtt) is interact with PIN1. In order to find the interact with PIN1 sites in the Htt-Nt, we constructed PIN1 with HA plasmid(p HA-PIN1) and different dominant with green fluorescent protein(GFP) plasmid, amino terminal 1-17 amino acids(p N17Htt-GFP), poly glutamine region(p Poly Q-GFP), and Proline-rich region(PRRHtt-GFP). The p N17Htt-GFP, p Poly Q-GFP and PRRHtt-GFP separately with p HA-PIN1 cotransfection N2 a cells, and then analyzing by Co-Immunoprecipitation. We found that: PIN1 combined with N17 Htt and PRRHtt, and later was stronger. So the N17 Htt and PRRHtt was domains interaction with PIN1.2. Huntington’s protein amino terminal 1-17 amino acids on the 13 th and 16 th sites are the key combining site to the PIN1. The Htt-Nt could interact with PIN1, s inhibit m Htt misfolding, and reduce aggregates after 13 th and 16 th serines phosphorylation. In order to define the N17 Htt interact with PIN1 key sites, we found that N17Htt12 th to 16 th motifs is Ep SLKp S, it electric charge is negative, negative, hydrophobic, zero charge and negative. We also found that corresponding motifs in PIN1, HIILR, it electric charge is positive, hydrophobic, hydrophobic, hydrophobic and positive. We constructed point mutants of N17 Htt 12th to 16 th motifs, separately with p HA-PIN1 cotransfection N2 a cells, and than analyzing by Co-Immunoprecipitation. The experimental results found that: 13 th and 16 th sites of two serine when single point mutations, the interaction strength decreased significantly, and double point mutations, the interaction disappeared. So amino terminal 1-17 amino acids on the 13 th and 16 th sites is the key combining site to the PIN1.3.62E63 P in the PRRHtt is the cis/trans isomeric sites by PIN1. Previously we found that a point mutation from 62 Glu to Ala(Htt-E62 A, AEAP) in PRRHtt, Htt160Q-E62 A could increase aggregates, and the amount of Htt160Q-E62 A could not be effectively reduced by PIN1, so we suspected that the AEEP sequences in the PRRHtt region may be the catalytic site of Htt-Nt cis/trans isomerization by PIN1. In order to prove this hypothesis, we use short peptide synthesis GPAVAEEPLHRP and purified PIN1 to for NMR experiment. The experimental results were that PIN1 and the short peptide had direct interaction, and there was a cis/trans isomeric sites by PIN1. Further attribution experimental showed that 62E63 P in the PRRHtt was the cis/trans isomeric sites by PIN1Conclusions. This study use Co-Immunoprecipitation and NMR to analyze the motif of Prolyl Isomerase PIN1 interaction and Cis/trans isomerization catalysis on Huntingtin-N terminal. We demonstrated that huntingtin amino terminal 1-17 amino acids and Proline-rich region is interact with PIN1, Huntington’s protein amino terminal 1-17 amino acids on the 13 th and 16 th sites is the key combining site to the PIN1, 62E63 P in the PRRHtt was the cis/trans isomeric sites by PIN1.From this results, The Htt is one of the PIN1 substrate, 62E63 P was cis/trans isomeric sites by PIN1 is nonspecific identification. This research supplied new experimental evidences to reveal abnormal aggregation of m Htt, and provided a scientific reference for finding more effective gene therapy of HD.