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）代表了一个持久的公共卫生问题，导致了重大的社会经济伯恩斯前往美国，国内优化与1999年至2017年相关的四倍次数。尽管如此 HUD为基础的神经生物学机制仍然很少理解。所有滥用药物调节多巴胺能信号传导，长期以来一直被认为是多巴胺（DA）信号传导的疾病。然而，针对受体介导的DA信号的药物治疗干预措施并未导致有效治疗。我们的实验室最近确定了一个新的DA信号部分，称为多巴胺基化，从而 DA本身是通过通过跨增材料的底物蛋白的翻译后修饰（PTM）。转谷氨酰胺酶2（TGM2）酶。然后，我感觉到公正地确定多巴胺基化在体内，使用一种新型的化学标记方法耦合到质谱法。我确定了 164在伏伏核（NAC）中多巴胺基化的新型推定突触底物，均为正常的神经功能以及对慢性海洛因自我管理的禁欲的反应。下列的验证了许多推定的底物，我将重点转移到了GCAMKII上，因为：1）这是高度丰富的2）位于自身抑制螺旋（谷氨酰胺[Q] 285）内的单个氨基酸住宅中的多巴胺基因距离关键苏氨酸（T）居住287的氨基酸仅存在两个氨基酸。T287被磷酸化以直接钙调蛋白（CAM）隔离和随后的核易位 - 该站点代表一个令人兴奋的 “测试”案例确定该信号部分在突触可塑性中的重要性3）已更新在戒酒后，在其多巴胺基化状态下，这种效果持续到AD14和4）代表涉及从突触到大脑中核的远程信号的临界底物，最终促进CREB激活。 GCAMKII是一种合成蛋白，证明是学习/记忆所必需的，长期 - 训练（LTP）和兴奋转录耦合。 CREB是重要的转录因子在所有类型的SUD中实施的大脑中，已经观察到激活的动态改变会影响与毒品有关的行为。我还证明了单胺分子可以浸润纹状体和体外皮质神经元，以及用DA的这些神经元的治疗诱导GCAMKII多巴胺基化。还观察到GCAMKII/CAM定位的动态调节以及随后的下游CREB信号传导响应于原发性皮质 - 纹状体神经元培养的DA治疗。因此，我假设 GCAMKIIQ285DOP可能代表大脑中新型的多巴胺能信号传导部分，并且可能在通过对NAC中CREB信号的异常调制来介导海洛因继电器行为。