On the difficulties of characterizing weak protein interactions that are critical for neurotransmitter release.
On the difficulties of characterizing weak protein interactions that are critical for neurotransmitter release.
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DOI：
10.1002/2211-5463.13473
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发表时间：
2022-11
期刊：
影响因子：
2.6
通讯作者：
Jaczynska, Klaudia
中科院分区：
文献类型：
--
作者：
Rizo, Josep;David, Guillaume;Fealey, Michael E.;Jaczynska, Klaudia
神经递质释放的机制已被广泛研究，表明囊泡融合是由Synaxin-1、SNAP-25和Synaptobrevin形成的SNARE复合体介导的。该复合体被N-乙基马来酰亚胺敏感因子(NSF)和SNAP分解以回收SNARE，而Munc18-1和Munc13S以NSF-SNAP抗性的方式组织SNARE复合体组装。突触素-1作为钙离子感受器，在与SNARs和复合蛋白的紧密相互作用中触发胞吐。在这里，我们回顾了与研究这些步骤背后的蛋白质相互作用相关的技术方面，这一过程受到阻碍，因为释放机制组装在两个膜之间，并且是高度动态的。此外，难以描述的弱相互作用在神经递质释放中发挥关键作用，例如通过降低在这一高度调控的过程中需要克服的能量障碍。我们说明了结构生物学在揭示神经递质释放的潜在机制方面发挥的关键作用，但也讨论了考虑所用技术的局限性的重要性，包括从我们实验室和其他实验室的研究中学到的教训。我们特别强调：(A)一些蛋白质序列的混杂，包括在没有天然靶标的情况下可以介导与蛋白质无关的相互作用的膜结合区；(B)需要确保在晶体结构中观察到的弱相互作用具有生物学意义；以及(C)等温滴定量热法分析弱相互作用的局限性。最后，我们强调，即使是需要重新解释的研究，也往往有助于推动这一领域的发展，因为它提高了我们对该系统的理解，并提供了可检验的假设。已描述的突触素-1-SNARE相互作用的种类说明了确定控制导致钙离子触发的突触小泡融合的不同步骤的蛋白质相互作用所涉及的挑战。
The mechanism of neurotransmitter release has been extensively characterized, showing that vesicle fusion is mediated by the SNARE complex formed by syntaxin‐1, SNAP‐25 and synaptobrevin. This complex is disassembled by N‐ethylmaleimide sensitive factor (NSF) and SNAPs to recycle the SNAREs, whereas Munc18‐1 and Munc13s organize SNARE complex assembly in an NSF‐SNAP‐resistant manner. Synaptotagmin‐1 acts as the Ca2+ sensor that triggers exocytosis in a tight interplay with the SNAREs and complexins. Here, we review technical aspects associated with investigation of protein interactions underlying these steps, which is hindered because the release machinery is assembled between two membranes and is highly dynamic. Moreover, weak interactions, which are difficult to characterize, play key roles in neurotransmitter release, for instance by lowering energy barriers that need to be overcome in this highly regulated process. We illustrate the crucial role that structural biology has played in uncovering mechanisms underlying neurotransmitter release, but also discuss the importance of considering the limitations of the techniques used, including lessons learned from research in our lab and others. In particular, we emphasize: (a) the promiscuity of some protein sequences, including membrane‐binding regions that can mediate irrelevant interactions with proteins in the absence of their native targets; (b) the need to ensure that weak interactions observed in crystal structures are biologically relevant; and (c) the limitations of isothermal titration calorimetry to analyze weak interactions. Finally, we stress that even studies that required re‐interpretation often helped to move the field forward by improving our understanding of the system and providing testable hypotheses. The variety of synaptotagmin‐1‐SNARE interactions that have been described illustrates the challenges involved in determining the protein interactions that control the different steps leading to Ca2+‐triggered synaptic vesicle fusion.
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