Structural study of polyglutamine and molecular mechanism of toll-like receptor signaling



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Huntington?s disease (HD) is caused by the expansion of a CAG repeats encoding polyglutamine (polyQ) in the first exon of Huntingtin (Htt) gene. In HD patients, polyQ contains 36-183 glutamine residues, whereas normal individuals have a polyQ of only 8-35 residues. To elucidate this threshold phenomenon of polyQ aggregation, fluorescence proteins CFP and YFP were attached to both ends of polyQ of different lengths. FRET (fluorescence resonance energy transfer) was conducted to characterize the conformation of polyQ in the pre-aggregation state. Our FRET data show that both the normal and expanded polyQ tracts reveal the same extended structure in low concentration. Longer polyQ has multiple cooperative binding sites with higher avidity. PolyQ tracts form aggregates when proteins exceed a critical concentration. The antibody MW1 Fv fragment binds to polyQ, breaks apart polyQ oligomer and stabilizes it in a more extended conformation. The addition of polyproline to the C-terminus inhibits polyQ aggregation by inducing PPII-like Helix structure. To understand how the flanking sequence affects the polyQ structure, the structure of Q10P10 peptide in complex with MW1 Fv was determined by protein crystallography and compared with Q10/Fv crystal structure. Q10P10 peptide bound to Fv has a similar extended structure as Q10 peptide when a polyproline tract adopts PPII helical structure sticking out of the complex. Toll-like receptors are transmembrane receptors on different kinds of leukocytes. They can recognize the structural conserved molecular motifs derived from microbes. On the upstream of the TLR signal pathway, TLRs recruit the adaptor protein-MyD88 through TIR/TIR domain interaction, and MyD88 recruits the downstream kinases IRAK4 and IRAK1 through death domain/death domain interaction. Pellino1, a newly identified E3 ubiquitin ligase, is also involved in TLR signaling by adding polyubiquitin chain to IRAK1 in conjugation with Ubc13/Uev1a E2 complex. TIR/TIR and DD/DD binding motifs were studied with techniques including mutagenesis, analytical gel filtration, NMR spectroscopy and crystallography. We identified a MyD88DD (E52QR62S) double-mutant that attenuates protein aggregation without interrupting the binding with IRAK4. This double mutant is a good candidate for structure determination by NMR spectroscopy. Our ubiquitination assay showed Pellino1 catalyzes polyubiquitination in the presence of Ubc13/Uev1a in vitro. Needle cluster-shaped crystals of Pellino1/Ubc13/ Uev1a protein complex were obtained by ?hanging drop? method of vapor diffusion. Once the crystallization conditions are optimized, we will be able to collect X-ray diffraction data for this E2/E3 complex.