Oxidized and phosphorylated synthetic peptides corresponding to the second and third tubulin-binding repeats of the tau protein reveal structural features of paired helical filament assembly.
Oxidized and phosphorylated synthetic peptides corresponding to the second and third tubulin-binding repeats of the tau protein reveal structural features of paired helical filament assembly.
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对应于 tau 蛋白第二个和第三个微管蛋白结合重复序列的氧化和磷酸化合成肽揭示了成对螺旋丝组装的结构特征。
DOI：
10.1111/j.1399-3011.1997.tb01178.x
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
OtvosJr,L
中科院分区：
文献类型：
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作者：
Hoffmann,R;Dawson,NF;Wade,JD;OtvosJr,L
正常大脑的微管相关蛋白τ通过其18个氨基酸重复单元附着在微管蛋白上。然而，在阿尔茨海默病的成对螺旋细丝（PHF）中，τ以异常过度磷酸化的形式寡聚化（PHF‐τ）。τ在重复单元2和3中包含两个半胱氨酸残基，但在PHF‐τ中只存在R3‐R3同型二聚体。在R3半胱氨酸下游两个氨基酸的丝氨酸残基是蛋白激酶C的主要磷酸受体位点。在本文报道的工作中，我们使用与R2， R3和磷酸化R3对应的合成肽来确定τ重复肽与β -微管蛋白C端结构域对应的肽片段的结合，并研究同源和异源二聚体形成的动力学。此外，我们研究了区分正常τ和PHF‐τ的肽的两个主要生化特性：构象和代谢稳定性。所有R2和R3肽都与微管蛋白肽特异性结合，无论磷酸化或二聚化状态如何。在微管蛋白肽存在的情况下，τ重复肽的反向构象不受影响。磷酸化使单聚肽和二聚肽的旋转结构略微松动，但并不单独影响肽的血清稳定性或肽形成二聚体的能力。分离的R2和R3单元形成同型二聚体的速率大致相同。然而，当两种肽混合时，R2‐R3异源二聚体优先于同型二聚体形成。二聚体在人血清中通常比单体更稳定。我们对合成肽片段τ的研究结果表明，重复单元的氧化或磷酸化都不能产生PHF‐τ中发现的扩展结构。此外，Ser324的磷酸化似乎不会调节τ的寡聚化动力学，并且在一般的生物化学术语中，不会影响附近二硫桥的形成。与全蛋白水平的研究一致，多肽的同型二聚体的形成（τ的自关联模型）不是首选的。然而，如果二聚体形成，它们的清除速度要比单体慢得多，这解释了受影响大脑中PHF‐τ显著的蛋白酶抗性。©Munksgaard 1997。
The microtubule‐associated protein τ of normal brains is attached to tubulin through its 18‐amino‐acid repeat units. In the paired helical filaments (PHF) of Alzheimer's disease, however, τ is oligomerized in an abnormally hyperphosphorylated form (PHF‐τ). τ contains two cysteine residues in repeat units 2 and 3, but only the R3‐R3 homodimer is present in PHF‐τ. A serine residue two amino acids downstream of the R3 cysteine is a major phosphate acceptor site for protein kinase C. In the work reported here, we used synthetic peptides corresponding to R2, R3 and phosphorylated R3 to determine the binding of the τ repeat peptides to a peptide fragment corresponding to the C‐terminal domain of ß‐tubulin and to study the kinetics of homo‐ and heterodimer formation. Additionally, we studied two major biochemical properties of the peptides that distinguish between normal τ and PHF‐τ: conformation and metabolic stability. All R2 and R3 peptides bound specifically to the tubulin peptide regardless of the state of phosphorylation or dimerization. The reverse‐turn conformation of the τ repeat peptides in the presence of the tubulin peptide remained unaffected. Phosphorylation slightly loosened the turn structure of the monomeric and dimeric peptides, and did not univocally affect the serum stability of the peptides or the ability of the peptides to form dimers. The isolated R2 and R3 units formed homodimers approximately in the same rate. When the two peptides were mixed, however, the R2‐R3 heterodimer was formed preferentially over the homodimers. The dimers were generally more stable in human serum than the monomers. Our results with the synthetic peptide fragments of τ indicate that neither oxidation nor phosphorylation of the repeat units is able to generate extended structure such as that found in PHF‐τ. Additionally, phosphorylation of Ser324 does not appear to modulate the kinetics of oligomerization of τ, and in general biochemistry terms, does not affect disulfide bridge formation nearby. In agreement with studies at the full‐protein level, the formation of homodimers of the peptides, a model of the self‐association of τ, is not preferred. If the dimers are formed, however, their clearance is considerably slower than that of the monomers, explaining the remarkable protease resistance of PHF‐τ in the affected brains. © Munksgaard 1997.