National Institutes of Health/National Institute on Aging (NIH/NIA)
R01 AG029430
米国
Howard Hughes Medical Institute (HHMI)
米国
引用
ジャーナル: Elife / 年: 2017 タイトル: Atomic structures of fibrillar segments of hIAPP suggest tightly mated β-sheets are important for cytotoxicity. 著者: Pascal Krotee / Jose A Rodriguez / Michael R Sawaya / Duilio Cascio / Francis E Reyes / Dan Shi / Johan Hattne / Brent L Nannenga / Marie E Oskarsson / Stephan Philipp / Sarah Griner / Lin ...著者: Pascal Krotee / Jose A Rodriguez / Michael R Sawaya / Duilio Cascio / Francis E Reyes / Dan Shi / Johan Hattne / Brent L Nannenga / Marie E Oskarsson / Stephan Philipp / Sarah Griner / Lin Jiang / Charles G Glabe / Gunilla T Westermark / Tamir Gonen / David S Eisenberg / 要旨: hIAPP fibrils are associated with Type-II Diabetes, but the link of hIAPP structure to islet cell death remains elusive. Here we observe that hIAPP fibrils are cytotoxic to cultured pancreatic β- ...hIAPP fibrils are associated with Type-II Diabetes, but the link of hIAPP structure to islet cell death remains elusive. Here we observe that hIAPP fibrils are cytotoxic to cultured pancreatic β-cells, leading us to determine the structure and cytotoxicity of protein segments composing the amyloid spine of hIAPP. Using the cryoEM method MicroED, we discover that one segment, 19-29 S20G, forms pairs of β-sheets mated by a dry interface that share structural features with and are similarly cytotoxic to full-length hIAPP fibrils. In contrast, a second segment, 15-25 WT, forms non-toxic labile β-sheets. These segments possess different structures and cytotoxic effects, however, both can seed full-length hIAPP, and cause hIAPP to take on the cytotoxic and structural features of that segment. These results suggest that protein segment structures represent polymorphs of their parent protein and that segment 19-29 S20G may serve as a model for the toxic spine of hIAPP.
装置: Hanging Drop Vapor Diffusion Tray / Atmosphere: Air, sealed chamber 詳細: 20 mg/mL Lyophilized peptide in ice-cold water. Crystals were grown using the hanging drop vapor diffusion method at 4 degrees C in 0.35 M sodium thiocyanate and 35% MPD. 温度: 277 K / Time: 7 DAY
緩衝液
pH: 7
緩衝液成分
ID
濃度
名称
式
Buffer-ID
1
0.35M
sodiumthiocyanate
NaSCN
1
2
35 %
MPD
1
試料
濃度: 20 mg/ml / 包埋: NO / シャドウイング: NO / 染色: NO / 凍結: YES
温度: 277 K / 手法: 蒸気拡散法, ハンギングドロップ法 詳細: 2 uL 20 mg/mL FLVHSSNNFGA + 1 uL 35% MPD, 0.35 M sodium thiocyanate against a reservoir of 35% MPD, 0.35 M sodium thiocyanate. Crystals appeared within several hours.
凍結剤: NITROGEN 試料ホルダーモデル: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER 最高温度: 100 K / 最低温度: 100 K
撮影
平均露光時間: 2 sec. / 電子線照射量: 0.01 e/Å2 フィルム・検出器のモデル: TVIPS TEMCAM-F416 (4k x 4k) Num. of diffraction images: 879 / 撮影したグリッド数: 2 詳細: The detector was operated in rolling shutter mode with 2x2 pixel binning.