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

CaMKII 鉴定为海洛因自我给药后多巴胺酰化的新型底物

基本信息

批准号：
10462196
负责人：
Andrew F Stewart
金额：
$ 4.52万
依托单位：
ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-01-19 至 2024-01-18
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10462196
关键词：
Abstinence Acute Affect Alanine Amino Acids Back Behavior Behavior Control Binding Brain Calcium Calmodulin Cell Culture Techniques Cell Nucleus Cells Cessation of life ChIP-seq Chemicals Chronic Co-Immunoprecipitations Complex Corpus striatum structure Coupled Coupling Cues Custom Cyclic AMP Response Element Data Disease Dopamine Enzymes Epigenetic Process Event Functional disorder Gene Expression Profile Genetic Transcription Glutamine Heroin Histone H3 Immunoprecipitation In Vitro Intervention Laboratories Learning Link Long-Term Potentiation Mass Spectrum Analysis Mediating Memory Modeling Mutate Neurons Neurophysiology - biologic function Nuclear Nuclear Translocation Nucleus Accumbens Opioid Pharmaceutical Preparations Phosphorylation Phosphotransferases Play Post-Translational Protein Processing Proteins Public Health Rattus Recombinants Regulation Relapse Research Rewards Role Saline Self Administration Signal Transduction Site Source Substance Use Disorder Substance abuse problem Sucrose Synapses Synaptic plasticity Testing Threonine Tissues United States Validation Ventral Tegmental Area Viral Virus Western Blotting adeno-associated viral vector behavioral outcome calmodulin-dependent protein kinase II drug of abuse efficacious treatment experimental study heroin use in vivo knock-down mesolimbic system monoamine mutant neurobiological mechanism new therapeutic target novel postsynaptic receptor response socioeconomics transcription factor transcriptome sequencing transglutaminase 2

项目摘要

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.

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 1999 年至 2017 年间，国内鸦片相关死亡人数翻了两番，这给美国带来了负担。 HUD 背后的神经生物学机制仍然知之甚少，所有滥用药物都会调节。多巴胺能信号传导，长期以来一直被认为是多巴胺 (DA) 信号传导障碍。针对受体介导的 DA 信号传导的药物治疗干预尚未产生有效的效果我们的实验室最近发现了一种新的 DA 信号传导部分，称为多巴胺化。 DA 本身通过转酰胺基作用作为底物蛋白的翻译后修饰 (PTM) 然后，我寻求公正地鉴定转谷氨酰胺酶 2 (TGM2) 的其他突触底物。我发现了体内多巴胺酰化，利用一种新颖的化学标记方法与质谱联用。伏核 (NAc) 中 164 个新推定的多巴胺酰化突触底物，均在正常的神经功能以及对长期自我服用海洛因戒断的反应。在验证了许多假定的底物后，我将注意力转向了 gCaMKII，因为：1) 它非常丰富 2) 它是在位于其自身抑制螺旋（谷氨酰胺 [Q]285）内的单个氨基酸残基上进行多巴胺酰化，该位点距离关键的苏氨酸 (T) 残基 287 仅存在两个氨基酸。T287 被磷酸化以直接钙调蛋白 (CaM) 隔离和随后的核转位——因此，这个位点代表了一个令人兴奋的用于确定该信号部分在突触后可塑性中的重要性的“测试”案例 3) 它被上调戒除海洛因 SA 后处于多巴胺化状态，这种作用持续到 AD14 和 4) 代表最终参与介导从突触到大脑细胞核的长距离信号的关键底物促进 CREB 激活，gCaMKII 是一种突触蛋白，被证明是学习/记忆所必需的，晚期长时程增强（LTP）和激发转录耦合是一个重要的转录因子。在涉及所有类型 SUD 的大脑中，已观察到激活的动态变化会影响我还证明单胺分子可以渗透纹状体和体外皮质神经元，以及用 DA 处理这些神经元会诱导 gCaMKII 多巴胺酰化。还观察到 gCaMKII/CaM 定位和后续下游 CREB 信号传导的动态调节因此，我在原代皮质纹状体神经元培养中对 DA 治疗做出了反应。 gCaMKIIQ285dop 可能代表大脑中一种新型的多巴胺能信号传导部分，并且可能在通过 NAc 中 CREB 信号的异常调节来介导海洛因复吸行为。