| In vitro AMPylation/Adenylylation of Alpha-synuclein by HYPE/FICD
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Author:
Date:
2020-09-20
[Abstract] One of the major histopathological hallmarks of Parkinson’s disease are Lewy bodies (LBs) –cytoplasmic inclusions, enriched with fibrillar forms of the presynaptic protein alpha-synuclein (α-syn). Progressive deposition of α-syn into LBs is enabled by its propensity to fibrillize into insoluble aggregates. We recently described a marked reduction in α-syn fibrillation in vitro upon posttranslational modification (PTM) by the Fic (Filamentation induced by cAMP) family adenylyltransferase HYPE/FICD (Huntingtin yeast-interacting protein E/FICD). Specifically, HYPE utilizes ATP to covalently decorate key threonine residues in α-syn’s N-terminal and NAC (non-amyloid-β component) regions with AMP (adenosine monophosphate), in a PTM termed AMPylation or adenylylation. Status quo in ...
[摘要] [摘要 ] 帕金森氏病的主要组织病理学标志之一是路易体（LB） –细胞质内含物，富含纤维状形式的突触前蛋白α-突触核蛋白（α-syn）。由于α-syn易于原纤维化成不溶性聚集体，因此可以逐步沉积到LBs中。我们最近描述了Fic（由cAMP诱导的细丝化）家族腺苷酸转移酶HYPE / FICD（与亨廷顿酵母相互作用的蛋白E / FICD）在翻译后修饰（PTM）后体外α-syn纤颤的明显减少。具体而言，HYPE利用ATP在称为AMPylation 或adenylylation 的PTM中，以AMP（单磷酸腺苷）共价修饰α-syn's N末端和NAC（非淀粉样β成分）区域中的关键苏氨酸残基。HYPE底物（例如α-syn）的体外AMPyl化反应现状使用多种ATP类似物，包括放射性标记的α - 32 P-ATP 或α - 33 P-ATP，荧光ATP类似物，生物素化ATP类似物（N6- [6-六甲基] -ATP-生物素），以及基于点击化学的烷基-ATP方法检测基于凝胶的AMPylation 。当前描述HYPE介导AMPylation 的分步方案的文献依赖于α - 33 P-ATP核苷酸，而不是更常见的α - 32 P-ATP。尽管有效，但前一种方法需要长时间且危险的DMSO-PPO（二甲基亚砜- 聚苯基恶唑）沉淀。因此，我们提供了基于α - 33 ...
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| A Low-volume Hydroponic Protocol for Maize and A Sensitive Bud-length Assay for Root-to-Shoot Impacts of The Strigolactone Analog, rac-GR24
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Author:
Date:
2020-08-20
[Abstract] Hydroponic approaches have been a boon for root research by facilitating root-feeding studies as well as secretion analysis. Results have aided our understanding of root-shoot signaling, transport, hormone function and more. However, existing approaches are often restricted to small plants and seedlings by prohibitive cost or availability of experimental substrates. In addition to this, research on the hormone, strigolactone (SL) has been constrained in species like maize by a lack of specific assays for quantifying responses. Here a low-volume hydroponic approach was developed for growing maize plants to the 3-leaf stage (about 2.5 weeks and 12 cm tall at the 2nd leaf collar) using 4 plants with 1 L of aerated media. This protocol also allowed development of an assay for ...
[摘要] [摘要 ] 通过促进根饲研究和分泌分析，水培方法已成为根研究的福音。结果有助于我们理解根射击信号，转运，激素功能等。然而，由于成本过高或实验底物的可获得性，现有方法通常仅限于小型植物和幼苗。除此之外，研究激素，独脚金（SL）已经在玉米等品种由于缺乏量化响应特定试验的限制。这里的低体积水培方法被用于玉米植物生长到3叶阶段（约2.5周和12厘米高的2开发第二LEA f领）使用4种植物和1升充气培养基。该协议还允许开发一种针对SL 类似物rac -GR24 的从根到芽影响的测定方法，该方法对玉米芽上侧芽的生长具有显着的，易于量化的影响。该协议的优势包括经济有效的简便性和精确度，可以量化从根到茎的SL 响应。此外，小体积水培方法可以很容易地用于玉米或其他高粱，C4草（如高粱，甘蔗和桔）的连续或脉冲型根饲研究，根标记实验和/或分泌物采样。
[背景 ] 的用于开发这里介绍的协议初始目的是量化的激素，根对枝条影响独脚金内酯（SL）在玉米中。推定松香内酯在该物种的驯化中起着核心作用，这是由于该激素对植物结构的贡献，以及因此对现代玉米的单茎形态造成的影响（关等人，2012 ）。为了解决这种可能性，需要一种系统，该系统将1 ）允许在这种大草种的初始营养生长期间进行根饲研究，2 ）最小化使用的SL 类似物的数量和成本（rac -GR24），以及3 ...
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| Site-specific DNA Mapping of Protein Binding Orientation Using Azidophenacyl Bromide (APB)
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Author:
Date:
2020-06-20
[Abstract] The orientation of a DNA-binding protein bound on DNA is determinative in directing the assembly of other associated proteins in the complex for enzymatic action. As an example, in a replisome, the orientation of the DNA helicase at the replication fork directs the assembly of the other associated replisome proteins. We have recently determined the orientation of Saccharalobus solfataricus (Sso) Minichromosome maintenance (MCM) helicase at a DNA fork utilizing a site-specific DNA cleavage and mapping assay. Here, we describe a detailed protocol for site-specific DNA footprinting using 4-azidophenacyl bromide (APB). This method provides a straightforward, biochemical method to reveal the DNA binding orientation of SsoMCM helicase and can be applied to other DNA binding ...
[摘要] [摘要 ] 结合在DNA上的DNA结合蛋白的方向决定了复合物中其他相关蛋白的组装，以进行有意的作用。例如，在复制体中，复制叉处的DNA解旋酶的方向指导我们最近通过定点DNA切割和作图分析确定了DNA叉处的Saccharalobus solfataricus （Sso ）微型染色体维持（MCM）解旋酶的方向。 使用4-叠氮苯甲酰溴（APB）进行位点特异性DNA足迹的详细协议。此方法提供了一种直接的生化方法来揭示Sso MCM解旋酶的DNA结合方向，并可应用于其他DNA结合蛋白。
[背景 ] DNA复制是其中基因组双链体链分离成两个模板链中，超前和滞后strands.This功能是通过在生命。如同其他环状六聚体的解旋酶结构域的所有的环状六聚体解旋酶的处理，从理论上讲，这些结构域中的任何一个都可以在易位期间朝向复制叉，并且与已知的3'-5 MCM包含两个结构域； N端结构域（NTD）和C端结构域（CTD）。' 易位directionality.T 他MCM解旋酶负载到DNA起源作为具有面向易位期间解旋酶的每个other.The取向管畸形双六聚体确定两个六聚体是否彼此或旁路彼此活性unwinding.Our最近的一篇论文中离解远表明Saccharolobus solfataricus （Sso MCM ）通过NTD引导DNA解链（Perera和Trakselis ...
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