CRCNS: Modeling Activation of CaMKII in Spines

描述（由申请人提供）：本提案的直接目的是建立一个准确的动态模型，用于在Ca 2+通过NMDA受体流入突触后棘期间，信号蛋白Ca 2 +/钙调蛋白（CaM）依赖性蛋白激酶II（CaMKII）的激活和自磷酸化。这项工作将分三个阶段进行。首先，研究人员将验证由Ca 2+和CaM激活单个单体催化亚基的模型，并通过将模型与实验进行比较来改进其动力学参数。在Mathematica中实现确定性模型;将针对CaMKII单体亚基的酶活性的实验室测定在亚基、CaM和Ca 2+的广泛浓度范围内测试模型的输出。浓度将模拟体内和体外条件。在第二阶段，研究人员将根据第一阶段验证的模型构建CaMKII十二聚体全酶激活模型。他们将模拟全酶内亚基二聚体的协同激活，以及其亚基自磷酸化的三种不同途径。该模型将在程序MCell中构建，该程序支持蛋白质相互作用和酶激活的空间正确随机模型，在生物学上逼真的几何形状内。该模型将采用一种新的基于规则的算法来指定全酶中亚基的位置和行为。它将在充分混合的体积中构建，以通过与在宽范围的亚基、CaM和Ca 2+浓度下使用全酶的实验室实验进行比较来进行测试。比较将用于优化全酶模型中的四个新参数，并选择全酶内自磷酸化进展的最准确模型。在第三阶段，研究人员将把CaMKII全酶的优化模型引入到一个更大的MCell模型中，该模型通过NMDA受体将Ca 2+流入脊柱，并通过泵和交换器将其去除。在MCell中使用该模型的模拟将用于测试关于支配棘中CaMKII激活的参数的假设。 该建议的智力价值在于其在中枢神经系统学习机制研究中的实用性。脊柱中的调节机制通过调节LTP和LTD等活性依赖性变化来控制突触强度。我们对脊柱中的调节酶了解很多，并且我们对酶网络进行了假设，这些酶网络调节控制突触可塑性的细胞过程，包括谷氨酸受体的插入和移除以及脊柱肌动蛋白细胞骨架形状的变化。然而，在目前的分析阶段，对突变动物的定性研究，或特定酶的过表达和敲低是该领域的主导范式，它们不足以将我们的知识带到下一个层次，即建立这些参与者中每一个的行动时机，以及每一个变得重要的调控网络中的精确条件和位置。为了达到这种理解水平，我们需要更好的量化模型和方法。CaMK Ⅱ是LTP诱导过程中由NMDA受体介导的Ca 2+激活的起始酶之一。一个经过充分验证的定量模型，其激活功能强大的MCell程序将提供一个起点和一个例子，为建设动态模型的连续步骤，在脊柱调节途径。 更广泛的影响包括促进将计算技术引入细胞神经生物学研究的教育目标。一名女博士后研究员将接受测试计算模型的实验技术和MCell使用方面的培训。本科生（包括少数民族学生）将通过加州理工学院和索尔克的暑期研究项目参与这项工作。所有模型都将提供给社区下载。CaMKII全酶的模型将是在MCell中模拟胞质多蛋白复合物内相互作用的第一个例子。这样做的语法将被公布，并在NSF赞助的MCell定期研讨会上教授。这个建议具有医学意义。使用CaMKII的脊柱信号通路的缺陷与工作记忆缺陷有关，类似于精神分裂症和相关思维障碍的基础。定量了解突触活动过程中CaMKII激活的调控因素及其在控制突触可塑性中的作用，将有助于开发临床上有用的药理学药物，这些药物靶向突触功能障碍的特定方面，副作用较少。

DESCRIPTION (provided by applicant): The immediate objective of this proposal is to build an accurate dynamic model of activation and autophosphorylation of the signaling protein Ca2+/calmodulin (CaM)-dependent protein kinase II (CaMKII) during influx of Ca2+ into the postsynaptic spine through NMDA receptors. The work will proceed in three stages. First, investigators will validate a model of activation of individual monomeric catalytic subunits by Ca2+ and CaM and refine its kinetic parameters by comparing the model to experiments. The deterministic model is implemented in Mathematica; the output of the model will be tested against bench assays of the enzymatic activity of monomeric subunits of CaMKII under a wide range of concentrations of the subunits, CaM, and Ca2+. The concentrations will mimic both in vivo and in vitro conditions. In the second stage, investigators will construct a model of activation of the dodecameric holoenzyme of CaMKII, based on the model validated in the first stage. They will model cooperative activation of subunit dimers within the holoenzyme, and three different paths of autophosphorylation of its subunits. The models will be constructed in the program MCell, which supports spatially correct stochastic models of protein interactions and enzymatic activation, within biologically realistic geometries. This model will employ a new rule-based algorithm to specify the locations and behavior of subunits in holoenzymes. It will be constructed in a well-mixed volume to enable testing by comparison to bench experiments with holoenzymes under a wide range of concentrations of subunits, CaM, and Ca2+. The comparisons will be used to optimize four new parameters in the holoenzyme model, and to choose the most accurate model for progression of autophosphorylation within the holoenzyme. In the third stage, investigators will introduce optimized models of the CaMKII holoenzyme into a larger MCell model of Ca2+ influx into spines through NMDA receptors and its removal by pumps and exchangers. Simulations in MCell with this model will be used to test hypotheses about parameters governing activation of CaMKII in spines. The intellectual merit of the proposal lies in its utility in the study of mechanisms of learning in the central nervous system. The regulatory machinery in a spine controls synaptic strength by regulating activity-dependent changes such as LTP and LTD. We know much about the regulatory enzymes in a spine and we have hypotheses about enzymatic networks that regulate the cellular processes controlling synaptic plasticity, including insertion and removal of glutamate receptors and changes in the shape of the spine actin cytoskeleton. However, at the present stage of analysis, qualitative studies with mutant animals, or over-expression and knock-down of particular enzymes are the dominant paradigm in the field and they are not adequate to bring our knowledge to the next level, which is to establish the timing of the action of each of these players, and the precise conditions and position in the regulatory network at which each one becomes important. To reach that level of understanding, we need better quantitative models and methods. CaMKII is one of the the initial enzymes activated by Ca2+ coming through NMDA receptors during induction of LTP. A well-validated quantitative model of its activation in the powerful MCell program will provide a starting point and an example for the construction of dynamic models of successive steps in spine regulatory pathways. The broader impacts include the educational goal of fostering introduction of computational techniques into cellular neurobiological research. A female postdoctoral fellow will be trained in experimental techniques to test computational models, and in the use of MCell. Undergraduate students (including minority students) will be involved in the work through summer research programs at Caltech and Salk. All models will be made available to the community for download. The models of CaMKII holoenzymes will be a first example of simulation in MCell of interactions within a cytosolic multiprotein complex. The syntax for doing this will be published, and taught in the regular workshops on MCell sponsored by NSF. The proposal has medical significance. Deficiencies in spine signaling pathways that use CaMKII are associated with working memory deficits similar to those that underlie schizophrenia and related thought disorders. A quantitative understanding of the factors governing activation of CaMKII during synaptic activity, and its role in controlling synaptic plasticity will facilitate development of clinically useful pharmacological agents that target specific aspects of synaptic dysfunction with fewer undesirable side effects.