Identification of CaMKII as a novel substrate of dopaminylation following heroin self administration

海洛因使用障碍（HUD）是一个持久的公共卫生问题，导致重大的社会经济问题。 这给美国带来了沉重的负担，从1999年到2017年，国内与鸦片有关的死亡人数翻了两番。尽管如此 HUD的神经生物学机制仍然知之甚少。所有滥用药物都能调节 多巴胺能信号传导障碍，长期以来被认为是多巴胺（DA）信号传导障碍。然而，在这方面， 靶向受体介导的DA信号传导的药物干预没有导致有效的 治疗。我们的实验室最近发现了一种新的DA信号部分，称为多巴胺化， DA本身作为底物蛋白的翻译后修饰（PTM），通过转酰胺作用， 转氨酶2（TGM 2）酶。然后，我试图无偏见地确定额外的突触基质， 多巴胺在体内，利用一种新的化学标记的方法耦合到质谱。我鉴定 164个新的推定突触底物多巴胺在伏核（NAc），无论是在上下文中， 正常的神经功能和对慢性海洛因自我给药戒断的反应。以下 为了验证一些假定的底物，我将重点转向gCaMKII，因为：1）它是高度丰富的2）它是 多巴胺在一个单一的氨基酸残基，位于其自身抑制螺旋（谷氨酰胺[Q]285），一个网站 其存在于距离关键的苏氨酸（T）残基287仅两个氨基酸处。T287被磷酸化以指导 钙调素（CaM）螯合和随后的核转位-因此，这个网站，代表了一个令人兴奋的 用于确定该信号传导部分在突触后可塑性中的重要性的“测试”情况3）它被上调 在海洛因SA戒断后其多巴胺化状态下，持续至AD 14的作用，4）代表 一种重要的底物，参与介导从突触到脑细胞核的长距离信号， 促进CREB活化。gCaMKII是一种被证明是学习/记忆所必需的突触蛋白， 晚期-长时程增强（LTP）和激发转录偶联。CREB是一种重要的转录因子 在涉及所有类型SUD的脑中，已观察到激活的动态变化影响 与毒品有关的行为。我还证明了单胺分子可以渗透到纹状体， 体外皮质神经元，以及用DA处理这些神经元诱导gCaMK II多巴胺化。我 还观察到gCaMKII/CaM定位和随后的下游CREB信号传导的动态调节 在原代皮层-纹状体神经元培养中对DA处理的响应。因此，我假设， gCaMKIIQ 285 dop可能代表脑中一种新的多巴胺能信号传导部分，并可能在脑内多巴胺能神经元的表达中发挥直接作用。 通过NAc中CREB信号传导的异常调节介导海洛因复吸行为。

Heroin use disorder (HUD) represents an enduring public health issue resulting in significant socioeconomic burdens to the United States, with domestic opiate-related deaths quadrupling from 1999 to 2017. Despite this the neurobiological mechanisms underlying HUD remain poorly understood. All drugs of abuse modulate dopaminergic signaling and have long been thought of as disorders of dopamine (DA) signaling. However, pharmacotherapeutic interventions targeting receptor mediated DA-signaling have not resulted in efficacious treatments. Our laboratory recently identified a novel signaling moiety for DA, termed dopaminylation, whereby DA itself acts as a post-translational modification (PTM) on substrate proteins via transamidation by the Transglutaminase 2 (TGM2) enzyme. I sought, then, to unbiasedly identify additional synaptic substrates of dopaminylation in vivo, utilizing a novel chemical tagging approach coupled to mass spectrometry. I identified 164 novel putative synaptic substrates of dopaminylation in Nucleus Accumbens (NAc), both in the context of normal neural function and in response to abstinence from chronic heroin self-administration. Following validation of a number of putative substrates, I turned my focus to gCaMKII as: 1) it is highly abundant 2) it is dopaminylated at a single amino acid residue, located within it’s autoinhibitory helix (glutamine [Q]285), a site that exists only two amino acids away from the critical threonine (T) residue 287. T287 is phosphorylated to direct Calmodulin (CaM) sequestration and subsequent nuclear translocation – thus, this site, represents an exciting ‘test’ case for establishing the importance of this signaling moiety in post synaptic plasticity 3) it is upregulated in its dopaminylation state following abstinence from heroin SA, an effect that persists to AD14 and 4)represents a critical substrate involved in mediating long range signals from the synapse to the nucleus in brain, ultimately promoting CREB activation. gCaMKII is a synaptic protein demonstrated to be necessary for learning/memory, late-long-term-potentiation (LTP) and excitation transcription coupling. CREB is an important transcription factor in brain implicated in all types of SUD, where dynamic alterations in activation have been observed to affect drug-related behaviors. I have additionally demonstrated that monoamine molecules can infiltrate striatal and cortical neurons in vitro, as well as that treatment of these neurons with DA induces gCaMKII dopaminylation. I also observed the dynamic regulation of gCaMKII/CaM localization and subsequent downstream CREB signaling in response to DA treatment in primary cortical-striatal neuronal culture. I therefore hypothesize that gCaMKIIQ285dop may represent a novel dopaminergic signaling moiety in brain, and may play a direct role in mediating heroin relapse behaviors via aberrant modulation of CREB signaling in NAc.