Identification of Kv4.2 protein complex and modifications by tandem affinity purification-mass spectrometry in primary neurons.
Identification of Kv4.2 protein complex and modifications by tandem affinity purification-mass spectrometry in primary neurons.
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鉴定KV4.2原代神经元中的串联亲和纯度质谱法对蛋白质复合物进行了修饰。
DOI：
10.3389/fncel.2022.1070305
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发表时间：
2022
影响因子：
5.3
通讯作者：
Hoffman, Dax A.
中科院分区：
文献类型：
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作者：
Hu, Jia-Hua;Liu, Ying;Hoffman, Dax A.
蛋白质通常形成复合物以实现不同的生理功能。在神经元中，通信依赖于突触，在突触中受体、通道和锚定蛋白形成复合物以精确地控制信号转导、突触整合和动作电位放电。虽然有许多公开的方案来分离细胞系中的蛋白质复合物，但在神经元中的分离还没有很好地建立。在这里，我们介绍了一种方法，结合慢病毒蛋白表达串联亲和纯化，然后质谱（TAP-MS），以确定神经元中的蛋白质复合物。该方案也可用于鉴定突触蛋白的翻译后修饰（PTM）。我们使用A型电压门控性K+通道亚基Kv4.2作为靶蛋白。Kv4.2在海马体中高度表达，在海马体中其通过调节神经元兴奋性和突触可塑性而有助于学习和记忆。我们在Kv4.2的C端标记了钙调素结合肽（CBP）和链霉亲和素结合肽（SBP），并通过慢病毒在神经元中表达。通过两步TAP纯化Kv4.2，并通过MS分析样品。MS鉴定了两种已知的Kv4.2相互作用蛋白[二肽基肽酶样蛋白（DPP）和Kv通道相互作用蛋白（KChIP）]，以及新型突触蛋白，包括谷氨酸受体、钙通道和锚定蛋白。免疫共沉淀和共定位实验验证了Kv4.2与谷氨酸受体的关联。除了蛋白复合物鉴定外，我们还使用TAP-MS鉴定Kv4.2磷酸化位点。几个已知的和未知的磷酸化位点进行了鉴定。这些发现为识别神经元中的蛋白质-蛋白质相互作用和PTM提供了一种新的途径，并阐明了可能参与神经系统疾病病理学的神经元信号传导机制。
Proteins usually form complexes to fulfill variable physiological functions. In neurons, communication relies on synapses where receptors, channels, and anchoring proteins form complexes to precisely control signal transduction, synaptic integration, and action potential firing. Although there are many published protocols to isolate protein complexes in cell lines, isolation in neurons has not been well established. Here we introduce a method that combines lentiviral protein expression with tandem affinity purification followed by mass-spectrometry (TAP-MS) to identify protein complexes in neurons. This protocol can also be used to identify post-translational modifications (PTMs) of synaptic proteins. We used the A-type voltage-gated K+ channel subunit Kv4.2 as the target protein. Kv4.2 is highly expressed in the hippocampus where it contributes to learning and memory through its regulation of neuronal excitability and synaptic plasticity. We tagged Kv4.2 with the calmodulin-binding-peptide (CBP) and streptavidin-binding-peptide (SBP) at its C-terminus and expressed it in neurons via lentivirus. Kv4.2 was purified by two-step TAP and samples were analyzed by MS. MS identified two prominently known Kv4.2 interacting proteins [dipeptidyl peptidase like (DPPs) and Kv channel-interacting proteins (KChIPs)] in addition to novel synaptic proteins including glutamate receptors, a calcium channel, and anchoring proteins. Co-immunoprecipitation and colocalization experiments validated the association of Kv4.2 with glutamate receptors. In addition to protein complex identification, we used TAP-MS to identify Kv4.2 phosphorylation sites. Several known and unknown phosphorylation sites were identified. These findings provide a novel path to identify protein-protein interactions and PTMs in neurons and shed light on mechanisms of neuronal signaling potentially involved in the pathology of neurological diseases.
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影响因子：
56.9
作者：
Bernard, C;Anderson, A;Johnston, D
通讯作者：
Johnston, D
影响因子：
64.8
作者：
Gavin, AC;Bösche, M;Superti-Furga, G
通讯作者：
Superti-Furga, G
影响因子：
5.3
作者：
Hu, Hui-Juan;Alter, Benedict J.;Gereau, Robert W.
通讯作者：
Gereau, Robert W.
影响因子：
9.9
作者：
Fernandez, Esperanza;Collins, Mark O.;Uren, Rachel T.;Kopanitsa, Maksym V.;Komiyama, Noboru H.;Croning, Mike D. R.;Zografos, Lysimachos;Armstrong, J. Douglas;Choudhary, Jyoti S.;Grant, Seth G. N.
通讯作者：
Grant, Seth G. N.
影响因子：
25
作者：
通讯作者：
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