Towards Elucidation of the Role of Ubiquitination in the Pathogenesis of Parkinson's Disease with Semisynthetic Ubiquitinated α-Synuclein
Towards Elucidation of the Role of Ubiquitination in the Pathogenesis of Parkinson's Disease with Semisynthetic Ubiquitinated α-Synuclein
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DOI：
10.1002/anie.201005546
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发表时间：
2011-01-01
影响因子：
16.6
通讯作者：
Lashuel, Hilal A.
中科院分区：
文献类型：
--
作者：
Hejjaoui, Mirva;Haj-Yahya, Mahmood;Lashuel, Hilal A.
赖氨酸残基的泛素化已成为调节多种细胞过程的重要机制，如细胞信号传导、DNA修复和26 S蛋白酶体的蛋白质降解。[1，2]直到最近，旨在破译泛素化对蛋白质功能的影响的研究主要依赖于生物化学方法在体外重建泛素化的蛋白质。因此，该领域的进展在很大程度上依赖于对目的蛋白高度特异性的E2-E3酶机制的发现。[3]缺乏一个有效的和通用的方法，位点特异性掺入的泛素（Ub）或聚泛素链阻碍了努力阐明的分子和结构基础的影响，泛素化的蛋白质功能的调节。一些研究小组最近报道了促进位点特异性肽和蛋白质泛素化的优雅的化学方法。这些方法中的几种是基于通过非天然异肽键连接泛素以产生肽或重组蛋白的酶促稳定的Ub缀合物。[4-9]通过形成天然异肽键的肽和蛋白质的位点特异性泛素化现在是可能的，这是Muir和同事，Liu和同事以及我们研究小组的研究结果。[10 - 13]我们最近开发了一种高效和化学选择性的方法来促进肽和蛋白质的位点特异性泛素化。[12]在我们的方法中，δ-巯基赖氨酸残基用于介导与泛素硫酯的硫代转酯化，然后进行S-N酰基转移以在泛素和赖氨酸衍生物之间形成异肽键。然后通过脱硫除去δ-巯基赖氨酸的巯基柄，以在泛素和赖氨酸之间提供所需的天然异肽连接，而不改变靶肽/蛋白质的天然序列。为了便于在固相肽合成（SPPS）和顺序连接中使用这个独特的残基，我们还合成了具有不同保护基团的δ巯基赖氨酸。[14]在此，我们描述了一种基于使用这些（先进的）合成工具的有效策略，用于半合成和表征在Lys6处单泛素化的α-突触核蛋白（T7-Ub α-syn（K6））。α-突触核蛋白（α-synuclein，α-syn）是一种天然未折叠的140个氨基酸的突触前蛋白，与帕金森病（Parkinson's disease，PD）及相关的神经退行性疾病（统称为"突触核蛋白病"）的发病机制有关。[15 - 17] PD的病理特征是多巴胺能神经元的丢失和细胞内包涵体的存在，称为路易体（LB），主要由α-syn组成。[15]已证明几种翻译后修饰（包括磷酸化、C末端截短和泛素化）与PD病理学密切相关，并在从PD患者脑中分离的LB中的α-syn中鉴定。[18 - 21]了解这些修饰在调节α-syn聚集、LB形成和毒性中的作用，对于了解α-syn的生物学和阐明其在PD发病机制中的作用至关重要，并可能导致识别治疗该疾病的新治疗靶点。在路易体中发现的大多数α-syn种类在多个赖氨酸残基处是单或双泛素化的。[18α-syn在单个或多个赖氨酸残基上的定向位点特异性泛素化是不可能的。例如，α-syn与泛素连接酶的共表达主要导致多个位点的单泛素化和双泛素化。[22，23...
The ubiquitination of lysine residues has emerged as an important mechanism for the regulation of a variety of cellular processes, such as cell signaling, DNA repair, and protein degradation by the 26S proteasome.[1, 2] Until recently, studies aimed at deciphering the effect of ubiquitination on protein function have relied primarily on biochemical approaches to reconstitute the ubiquitinated protein in vitro. As a result, progress in the field has been very much dependent on the discovery of the E2–E3 enzymatic machinery that is highly specific to the protein of interest.[3] The lack of an effective and general method for the site-specific incorporation of ubiquitin (Ub) or polyubiquitin chains has hampered efforts to elucidate the molecular and structural basis underlying the effects of ubiquitination on the regulation of protein function. Several research groups have recently reported elegant chemical methods that facilitate site-specific peptide and protein ubiquitination. Several of these methods are based on the attachment of ubiquitin through non-native isopeptide bonds to generate enzymatically stable Ub conjugates of peptides or recombinant proteins.[4–9] The site-specific ubiquitination of peptides and proteins through the formation of a native isopeptide bond is now possible as a result of studies by Muir and co-workers, Liu and co-workers, and our research group.[10–13] We recently developed a highly efficient and chemoselective method to facilitate the site-specific ubiquitination of peptides and proteins.[12] In our approach, a δ-mercaptolysine residue is used to mediate transthioesterification with an ubiquitin thioester, followed by S–N acyl transfer to form an isopeptide bond between ubiquitin and the lysine derivative. The thiol handle of δ-mercaptolysine is then removed by desulfurization to furnish the desired native isopeptide linkage between ubiquitin and lysine without altering the native sequence of the target peptides/proteins. To facilitate the use of this unique residue in solid-phase peptide synthesis (SPPS) and sequential ligation, we also synthesized δmercaptolysine with different protecting groups.[14] Herein, we describe an efficient strategy based on the use of these (advanced) synthetic tools for the semisynthesis and characterization of α-synuclein monoubiquitinated at Lys6 (T7-Ubα-syn (K6)). α-Synuclein (α-syn) is a natively unfolded 140 amino acid presynaptic protein that is implicated in the pathogenesis of Parkinson s disease (PD) and related neurodegenerative diseases that are collectively termed “synucleinopathies”.[15–17] The pathology of PD is characterized by the loss of dopaminergic neurons and the presence of intracellular inclusions, known as Lewy bodies (LBs), which are composed primarily of α-syn.[15] Several posttranslational modifications, including phosphorylation, C-terminal truncations, and ubiquitination, have been shown to be closely associated with PD pathology and were identified in α-syn within LBs isolated from PD patients brains.[18–21] Understanding of the role of these modifications in the regulation of α-syn aggregation, LB formation, and toxicity is crucial to the understanding of the biology of α-syn and the elucidation of its role in the pathogenesis of PD, and may lead to the identification of novel therapeutic targets for the treatment of the disease. The majority of α-syn species found in Lewy bodies are mono-or diubiquitinated at multiple lysine residues.[18, 19, 21] The directed site-specific ubiquitination of α-syn at a single or multiple lysine residues has not been possible. For example, the coexpression of α-syn with ubiquitin ligases results predominantly in mono-and diubiquitination at multiple sites.[22, 23 …