The effects of KTKEGV repeat motif and intervening ATVA sequence on α-synuclein solubility and assembly.
The effects of KTKEGV repeat motif and intervening ATVA sequence on α-synuclein solubility and assembly.
复制标题
KTKEGV 重复基序和干预 ATVA 序列对 α-突触核蛋白溶解度和组装的影响。
DOI：
10.1111/jnc.15763
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发表时间：
2023
影响因子：
4.7
通讯作者：
Dettmer,Ulf
中科院分区：
文献类型：
--
作者：
Brontesi,Lisa;Imberdis,Thibaut;Ramalingam,Nagendran;Dettmer,Ulf
α-突触核蛋白（αS）是帕金森病的关键蛋白，通常被描述为一种内在无序的蛋白质。与这个概念相一致的是，神经元中可能存在几种依赖于上下文的折叠状态。未折叠的可溶性单体、膜上的螺旋单体和螺旋多聚体（可溶的或膜上的）都已被报道，并且可能彼此处于平衡。我们之前发现，αS可以通过遗传增加αS螺旋的膜包埋一半的疏水性来稳定其膜相关单体形式。膜上αS两亲性螺旋的形成由多达9个具有核心基序KTKEGV的11-氨基酸重复序列控制。然而，该重复序列仅是不完全保守的;例如，它由重复序列#1中的KAKEGV、重复序列#5中的KTKEQV和保守性差的重复序列#6中的AVVTGV组成。在这里，我们探索了将αS核心重复序列完善为9倍KTKEGV（“9 KV”）的效果，并通过连续蛋白提取发现这种工程化突变体在神经细胞的胞质相中积累。完整细胞交联在多聚体位置（30、60、80、100 kDa）捕获了一部分胞质部分。因此，与野生型αS相比，αS 9 KV似乎不太容易占据膜相关单体形式。去除重复序列4和5之间的“ATVA”间插氨基酸序列略微增加了细胞溶质定位，而在所有重复序列1-8之间添加“ATVA”导致αS作为单体被捕获在膜组分中。我们的研究结果有助于对αS动态结构的持续争论，强调野生型αS不太可能在细胞中完全多聚体/单体或完全胞质/膜相关，但蛋白质工程可以产生优先采用某种状态的αS变体。总体而言，KTKEGV重复基序的不完美性质以及重复4和5之间ATVA的存在似乎阻止了αS的强胞质定位，因此在蛋白质动态填充胞质与膜相关状态以及单体与多聚体状态的能力中发挥了重要作用。
Alpha‐synuclein (αS), the key protein in Parkinson's disease, is typically described as an intrinsically disordered protein. Consistent with this notion, several context‐dependent folding states may coexist in neurons. Unfolded soluble monomers, helical monomers at membranes and helical multimers (soluble or at membranes) have all been reported and may be in an equilibrium with each other. We previously found that αS can be stabilized in its membrane‐associated monomeric form by genetically increasing the hydrophobicity of the membrane‐embedded half of the αS helix. αS amphipathic helix formation at membranes is governed by up to nine 11‐amino acid repeats with the core motif KTKEGV. However, this repeat is only imperfectly conserved; for example, it consists of KAKEGV in repeat #1, KTKEQV in repeat #5, and AVVTGV in the poorly conserved repeat #6. Here we explored the effect of perfecting the αS core repeat to nine times KTKEGV (“9KV”) and found by sequential protein extraction that this engineered mutant accumulates in the cytosolic phase of neural cells. Intact‐cell cross‐linking trapped a part of the cytosolic portion at multimeric positions (30, 60, 80, 100 kDa). Thus, compared to wild‐type αS, αS 9KV seems less prone to populating the membrane‐associated monomeric form. Removing the “ATVA” intervening amino‐acid sequence between repeats 4 and 5 slightly increased cytosolic localization while adding “ATVA” in between all repeats 1–8 caused αS to be trapped as a monomer in membrane fractions. Our results contribute to an ongoing debate on the dynamic structure of αS, highlighting that wild‐type αS is unlikely to be fully multimeric/monomeric or fully cytosolic/membrane‐associated in cells, but protein engineering can create αS variants that preferentially adopt a certain state. Overall, the imperfect nature of the KTKEGV repeat motifs and the presence of ATVA in between repeats 4 and 5 seem to prevent a strong cytosolic localization of αS and thus play a major role in the protein's ability to dynamically populate cytosolic vs. membrane‐associated and monomeric vs. multimeric states.