Structure of Prion Amyloids

朊病毒淀粉样蛋白的结构

基本信息

批准号：
7967191
负责人：
Reed B. WICKNER
金额：
$ 28.49万
依托单位：
NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/7967191
关键词：
Alanine Alzheimer&apos s Disease Amino Acid Sequence Amino Acids Amyloid Amyloid Fibrils Amyloid beta-Protein Biochemistry Biological C-terminal Carbon Cell Nucleus Cells Charge Collaborations Daughter Disease Ethers Event Fiber Filament Frequencies Functional disorder Genes Hydration status Inherited Label Length Location Measures Methods Molds Molecular Structure N Domain N-terminal National Institute of Diabetes and Digestive and Kidney Diseases Non-Insulin-Dependent Diabetes Mellitus Parents Parkinson Disease Phenotype Physiologic pulse Podospora anserina Polymers PrPSc Proteins Prion Diseases Prions Protein Structure Initiative Proteins Radio Recombinants Role Saccharomyces cerevisiae Signal Transduction Solutions Specific qualifier value Structure Time Translations Variant Work X-Ray Crystallography Yeasts amyloid structure base beta pleated sheet fungus human disease insight prevent research study solid state nuclear magnetic resonance termination factor therapy development yeast prion

项目摘要

The non-chromosomal genes URE3, PSI and PIN of Saccharomyces cerevisiae are infectious forms (prions) of the Ure2p, Sup35p and Rnq1p, respectively, while Het-s of Podospora anserina is a prion of the HETs protein (reviewed in 1). The basis of each of these prions is a self-propagating amyloid of the respective protein, as the mammalian infectious protein PrPSc is believed to be an amyloid of the cellular PrP protein. Amyloid is not suitable material for either X-ray crystallography or solution NMR structural studies but can be studied by solid-state NMR (2). We have undertaken solid-state NMR studies of each of the yeast and fungal prion amyloids in collaboration with Dr. Robert Tycko of LCP, NIDDK. We find that each of the yeast infectious prion amyloids, Sup35p, Ure2p and Rnq1p, the basis of the PSI, URE3 and PIN prions, respectively, are parallel in-register beta-sheet structures (3, 5, 6). Sup35p is a subunit of the translation termination factor, consisting of an N-terminal prion domain (N, residues 1-123), a middle highly charged domain (M, residues 124 - 253) and a C-terminal domain (C) that is sufficient for translation termination. We prepared infectious recombinant Sup35NM labeled with Tyr-1-13C, or Leu-1-13C, or Phe-1-13C, and made beta sheet-rich amyloid filaments from each. Beta sheets may be antiparallel, parallel or beta helices. Parallel beta sheets may be either in-register or out of register. An in-register beta sheet has identical residues aligned along the filament. For example, Val31 of the first molecule is 4.7 angstroms (the distance between beta strands in a beta sheet) from Val31 of the second molecule and so on down the fiber. We used a constant-time finite-pulse radio frequency driven recoupling method (4) to measure the distance from each labeled carbonyl carbon to the next nearest labeled nucleus. We found that for Tyr-1-13C and for Leu-1-13C, the decay indicated a distance of about 5 angstroms, demonstrating a parallel beta sheet structure, either in register or out of register by at most one residue (3). Diluting the fully labeled molecules with four parts of unlabeled molecules resulted in substantial decrease in the rate of signal decay, indicating the the interaction being observed was intermolecular, not intramolecular, and again supporting the parallel in-register structure. Ether-precipitated Sup35NM labeled with Leu-1-13C showed a slow decay rate indicating that the results observed were due to the specific amyloid structure, not simply due to aggregation. To confirm that the parallel sheet was indeed in-register, we made Sup35NM labeled with alanine-3-13C, that is, methyl labeled instead of carbonyl carbon labeled as in the other experiments. Because amino acid R groups point in alternate directions with succeeding residues on a beta strand, a parallel beta sheet out of register by a single residue would give a very slow decay rate. In fact the results indicated an in-register structure (3). This was the first structure determined for any prion amyloid. Further work will be needed to determine the details of this structure, but specifying a parallel in-register structure constrains the possibilities very tightly. We have now carried out similar studies on infectious amyloid of the Ure2 prion domain (residues 1-89) with similar results (5). Similarly, we have shown that infectious amyloid of the Rnq1 protein, the basis of the PIN prion of yeast has a parallel in-register beta-sheet structure. Earlier work had shown, surprisingly, that shuffling the prion domain of Ure2p or of Sup35p did not prevent the corresponding proteins from becoming prions (7, 8). We inferred from this fact that the structures must be parallel in-register beta sheets. As mentioned above, this has now been verified for the normal Ure2p and Sup35p prion domains, and we have also shown the same for the shuffled prion domains (9). This confirms that for these prions, it is amino acid composition, not sequence, that determines ability to be a prion. The same amino acid sequence can form biologically distinct, heritable prion variants, which must represent structures differing in some way. We have examined two prion variants of Sup35p, and find that both are parallel in-register beta sheet structures (10). We propose that the differences between variants are in the locations of folds of the beta sheets and in the extent of the beta sheet. Implications: In the parallel in-register beta sheet structure, each residue of the prion domain contacts the same residue in the molecule that was laid down before it and the same residue in the next molecule to join the filament. This provides the possibility of passing on structural variations - such as turns of the sheet or irregularities in the structure - from parent molecules (already in the filament) to daughter molecules newly being laid down. This is the essential "templating" mechanism that must be present for these proteins to act as genes. This can explain the prion "variants", cases where the identical amino acid sequence produces different phenotypes or prion stabilities, doubtless due to differing details of the amyloid structure. This structure also indicates that the sequence of events is not, as often suggested, a primary conformational change of the prion protein, followed by aggregation. It seems evident that the conformational change is coincident with and a consequence of the new molecule joining the filament, and the details of the conformational change are directed by the structure of the end of the filament. 1. Wickner, R. B., H. K. Edskes, et al. (2007). "Prions of fungi: inherited structures and biological roles." Nat. Microbiol. Rev. 5: 611-618. 2. Tycko, R. (2006). Molecular structure of amyloid fibrils: insights from solid-state NMR. Q. Rev. Biophys. 1, 1-55. 3. Shewmaker, F., Wickner, R.B., and Tycko, R. (2006). Amyloid of the -sheet structure.prion domain of Sup35p has an in-register parallel Proc. Natl. Acad. Sci. U. S. A. 103, 19754 - 19759. 4. Balbach, J.J., Petkova, A.T., Oyler, N.A., Antzutkin, O.N., Gordon, D.J., Meredith, S.C., and Tycko, R. (2002). Supramolecular structure in full-length Alzheimer's beta-amyloid fibrils: Evidence for a parallel beta-sheet organization from solid-state nuclear magnetic resonance. Biophys. J. 83, 1205-1216. 5. Baxa, U., R. B. Wickner, A.C. Steven, D. Anderson, D., L. Marekov, W.-M. Yau, R. Tycko, R. (2007) Characterization of beta-sheet structure in Ure2p1-89 yeast prion fibrils by solid state nuclear magnetic resonance. Biochemistry 46: 13149 - 13162. 6. Wickner, R. B., F. Dyda, et al. (2008). "Amyloid of Rnq1p, the basis of the PIN+ prion, has a parallel in-register beta-sheet structure." Proc Natl Acad Sci U S A 105: 2403 - 2408. 7. Ross, E. D. et al. (2004). "Scrambled prion domains form prions and amyloid." Mol Cell Biol 24: 7206-7213. 8. Ross, E. D. et al. (2005). "Primary sequence independence for prion formation." Proc Natl Acad Sci U S A 102: 12825-12830. 9. Shewmaker, F., Ross, E. D., Tycko, R. and Wickner, R. B. (2008) Amyloids of shuffled prion domains that form prions have a parallel beta-sheet structure. Biochemistry 47:4000-4007. 10. Shewmaker F, Kryndushkin D, Chen B, Tycko R & Wickner RB (2009) Two prion variants of Sup35p have in-register beta-sheet structures, independent of hydration. Biochemistry 48: 5074-5082.

酿酒酵母的非染色体基因 URE3、PSI 和 PIN 分别是 Ure2p、Sup35p 和 Rnq1p 的传染性形式（朊病毒），而鹅足孢菌的 Het-s 是 HETs 蛋白的一种朊病毒（已在 1 中综述）。这些朊病毒的基础是各自蛋白质的自我增殖淀粉样蛋白，因为哺乳动物感染性蛋白PrPSc被认为是细胞PrP蛋白的淀粉样蛋白。淀粉样蛋白不适合用于 X 射线晶体学或溶液 NMR 结构研究，但可以通过固态 NMR 进行研究 (2)。我们与 NIDDK LCP 的 Robert Tycko 博士合作，对每种酵母和真菌朊病毒淀粉样蛋白进行了固态核磁共振研究。我们发现，每种酵母感染性朊病毒淀粉样蛋白 Sup35p、Ure2p 和 Rnq1p（分别是 PSI、URE3 和 PIN 朊病毒的基础）是平行的记录内 β 片层结构 (3, 5, 6)。 Sup35p 是翻译终止因子的一个亚基，由 N 端朊病毒结构域（N，残基 1-123）、中间高电荷结构域（M，残基 124 - 253）和 C 端结构域（C）组成，足以终止翻译。我们制备了用 Tyr-1-13C、Leu-1-13C 或 Phe-1-13C 标记的感染性重组 Sup35NM，并从中制备了富含 β 片层的淀粉样蛋白丝。 β折叠可以是反平行、平行或β螺旋。并行测试表可以是注册的，也可以是注册外的。套准内的β片层具有沿细丝排列的相同残基。例如，第一个分子的 Val31 与第二个分子的 Val31 相差 4.7 埃（β 片层中 β 链之间的距离），依此类推，沿着纤维向下。我们使用恒定时间有限脉冲射频驱动重耦合方法 (4) 来测量从每个标记的羰基碳到下一个最近的标记核的距离。我们发现，对于 Tyr-1-13C 和 Leu-1-13C，衰变表明距离约为 5 埃，展示了平行的 β 片层结构，最多有一个残基对齐或不对齐 (3)。用四部分未标记分子稀释完全标记的分子导致信号衰减率显着降低，表明观察到的相互作用是分子间的，而不是分子内的，并且再次支持平行的对准结构。用 Leu-1-13C 标记的醚沉淀 Sup35NM 显示出缓慢的衰减速率，表明观察到的结果是由于特定的淀粉样蛋白结构，而不仅仅是由于聚集。为了确认平行片确实对准，我们制作了用丙氨酸-3-13C标记的Sup35NM，即甲基标记而不是其他实验中标记的羰基碳。由于氨基酸 R 基团与 β 链上的后续残基指向交替方向，因此单个残基未对准的平行 β 片层将给出非常慢的衰减速率。事实上，结果表明存在寄存器内结构 (3)。这是第一个确定的朊病毒淀粉样蛋白的结构。需要进一步的工作来确定该结构的细节，但指定并行寄存器内结构非常严格地限制了可能性。我们现在对 Ure2 朊病毒结构域（残基 1-89）的感染性淀粉样蛋白进行了类似的研究，得到了类似的结果 (5)。类似地，我们已经证明，Rnq1 蛋白的感染性淀粉样蛋白（酵母 PIN 朊病毒的基础）具有平行的记录内 β 片层结构。令人惊讶的是，早期的工作表明，改组 Ure2p 或 Sup35p 的朊病毒结构域并不能阻止相应的蛋白质变成朊病毒 (7, 8)。我们从这个事实推断出这些结构必须是平行的注册β片。如上所述，这一点现已在正常的 Ure2p 和 Sup35p 朊病毒结构域中得到验证，并且我们还对改组的朊病毒结构域进行了同样的验证 (9)。这证实了对于这些朊病毒来说，决定成为朊病毒的能力的是氨基酸组成，而不是序列。相同的氨基酸序列可以形成生物学上不同的、可遗传的朊病毒变体，这些变体必须代表在某些方面不同的结构。我们检查了 Sup35p 的两个朊病毒变体，发现两者都是平行的注册内 β 片层结构 (10)。我们认为变体之间的差异在于β折叠的折叠位置和β折叠的范围。影响：在平行对准β折叠结构中，朊病毒结构域的每个残基接触在其之前的分子中的相同残基以及下一个分子中的相同残基以连接细丝。这提供了将结构变化（例如片材的转动或结构的不规则性）从母体分子（已经在细丝中）传递到新铺设的子体分子的可能性。这是这些蛋白质作为基因必须存在的基本“模板”机制。这可以解释朊病毒“变体”，即相同的氨基酸序列产生不同的表型或朊病毒稳定性，这无疑是由于淀粉样蛋白结构的不同细节造成的。这种结构还表明，事件的顺序并不像人们经常认为的那样，是朊病毒蛋白的主要构象变化，然后是聚集。似乎很明显，构象变化与新分子加入细丝一致，并且是新分子加入细丝的结果，并且构象变化的细节由细丝末端的结构决定。 1. Wickner, R. B., H. K. Edskes 等人。（2007）。 “真菌朊病毒：遗传结构和生物学作用。”纳特。微生物。修订版 5：611-618。 2.Tycko, R. (2006)。淀粉样原纤维的分子结构：来自固态核磁共振的见解。 Q.Rev.Biophys。 1、1-55。 3. Shewmaker, F.、Wickner, R.B. 和 Tycko, R. (2006)。 -sheet 结构的淀粉样蛋白。Sup35p 的朊病毒结构域有一个登记内并行过程。国家。阿卡德。科学。美国 103, 19754 - 19759。 4. Balbach, J.J.、Petkova, A.T.、Oyler, N.A.、Anzutkin, O.N.、Gordon, D.J.、Meredith, S.C. 和 Tycko, R. (2002)。全长阿尔茨海默病β-淀粉样蛋白原纤维中的超分子结构：来自固态核磁共振的平行β-折叠组织的证据。生物物理学。 J.83, 1205-1216。 5. Baxa, U.、R. B. Wickner、A.C. Steven、D. Anderson, D.、L. Marekov, W.-M.丘，R. 泰科，R. (2007) 通过固态核磁共振表征 Ure2p1-89 酵母朊病毒原纤维中的 β-折叠结构。生物化学 46：13149 - 13162。 6. Wickner, R. B., F. Dyda 等人。（2008）。 “Rnq1p 的淀粉样蛋白是 PIN+ 朊病毒的基础，具有平行的登记内 β-折叠结构。”美国国家科学院院刊 105：2403 - 2408。 7.罗斯，E.D.等人。（2004）。 “乱序的朊病毒结构域形成朊病毒和淀粉样蛋白。”分子细胞生物学 24：7206-7213。 8.罗斯，E.D.等人。（2005）。 “朊病毒形成的初级序列独立性。”美国国家科学院院刊 102：12825-12830。 9. Shewmaker, F.、Ross, E. D.、Tycko, R. 和 Wickner, R. B. (2008) 形成朊病毒的改组朊病毒结构域的淀粉样蛋白具有平行的 β 片层结构。生物化学 47：4000-4007。 10. Shewmaker F、Kryndushkin D、Chen B、Tycko R 和 Wickner RB (2009) Sup35p 的两种朊病毒变体具有注册内 β-折叠结构，与水合作用无关。生物化学 48：5074-5082。