Cyclin-dependent kinase 5 (Cdk5) in Physiology and Pathology

生理学和病理学中的细胞周期蛋白依赖性激酶 5 (Cdk5)

• 批准号: 8158243
• 负责人: HARISH C PANT
• 金额: $ 97.87万
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8158243
• 关键词: Cyclin-Dependent Kinase 5; Pathology; Physiology

Cyclin-dependent kinase 5 (Cdk5) is predominantly expressed in the nervous system. Though it is expressed in non- neuronal cell and binds with cyclins but its activity is predominantly found in post mitotic neurons due to its binding to neuron specific molecules P35 and P39. It is involved in neuronal migration, synaptic transmission, and survival. Cdk5,a multifunctional neuronal kinase (targeting proteins from neuronal differentiation to synaptic function), is tightly regulated when complexed with p35, its co-activator. It is one of several kinases that phosphorylate neurofilaments and tau. Its diverse roles stem, in part, from its cross-talk interactions with other kinases in signal transduction networks underlying neuronal cell survival, growth and differentiation. We have shown, for example, that Cdk5 down regulates MAPKs and JNKs and up regulates PI3Ks. These results suggest that Cdk5 normally modulates the intensity of response of other kinases to specific signals underlying neuronal survival. The role of Cdk5 in synaptic transmission is mediated by regulating the cellular functions of presynaptic proteins such as synapsin, Munc18, and dynamin 1. Its multifunctional role at the synapse is complex and probably involves other novel substrates.In our previous studies we have demonstrated neuronal stress modulates Cdk5 activity. Since glucocorticoids are the major end effectors of stress response, play an essential role in the homeostasis of the central nervous system (CNS) and contribute to memory consolidation and emotional control through their intracellular receptors, the glucocorticoid and mineralocorticoid receptors. This raises the possibility that Cdk5 may modulate the behavior of these receptors. Indeed we found, Cdk5 phosphorylates and modulates the transcriptional activity of both glucocorticoid and mineralocorticoid receptor and regulates expression of brain-derived neurotrophic factor. Aldosterone and dexamethasone, respectively, increased and suppressed mRNA/protein expression of brain-derived neurotrophic factor (BDNF) in rat cortical neuronal cells, whereas the endogenous glucocorticoid corticosterone showed a biphasic effect. Cdk5 enhanced the effect of aldosterone and dexamethasone on BDNF expression. Because this neurotrophic factor plays critical roles in neuronal viability, synaptic plasticity, consolidation of memory, and emotional changes, we propose that the activation of CDK5 may modulate the transcriptional activity of the glucocorticoid receptors and regulate the expression of BDNF and thus influence these functions. Normally, Cdk5 activity is tightly regulated but under conditions of neuronal stress it is deregulated leading to hyperactivity, neuronal pathology and cell death. Accordingly, Cdk5 has been implicated in certain neurodegenerative disorders such as Alzheimer's Disease (AD). A model of Cdk5s role in neurodegeneration suggests that a stress-induced influx of calcium ions into neurons activates calpain, a Ca++- activated protease, which cleaves p35 into p25 and a p10 fragment. p25, in turn, forms a more stable Cdk5/p25 hyperactive complex, that hyperphosphorylates tau and other neuronal cytoskeletal proteins, and induces cell death. Indeed, increased levels of p25 and Cdk5 activity have been reported in AD brains. That p25 may be toxic comes from studies of cortical neurons treated with Abeta-amyloid peptide,a key marker of AD pathology, where p35 is converted to p25 accompanied by hyper-activated Cdk5, tau and neurofilament hyperphosphorylation and apoptosis. Expression of the Cdk5/p25 complex seems to be primarily responsible for the tau and neurofilament pathology and suggests that a therapeutic approach directed specifically at this target might prove successful. For most of these studies, however, the focus has been amon various laboratories around world on aminothiazol compounds resembling roscovitine, a kinase inhibitor that ccompetes with the ATP binding site in Cdk5 and other kinases. These drugs do not act specifically on Cdk5/p25 but also inhibit Cdk5/p35 and other kinases essential for normal development and function. This could be responsible for serious secondary side effects and thereby compromise any therapeutic value. Our approach to this problem, however, is based on earlier studies where we identified a peptide of 125 amino acid (aa) residues of p35 (CIP) that inhibited Cdk5/p25 activity and rescued cortical neurons from induced apoptosis without affecting Cdk5/p35 activity. This approach might prove to be a more effective way to suppress deregulated Cdk5/p25 hyperactivity inducing neurodegenerative pathology. For a therapy to be effective, however, it must be small enough molecule to pass the blood-brain barrier; a large peptide of 125 residues may be problematic, to say the least. For that reason, we set out to produce a smaller peptide of CIP with equivalent specificity. Based on an analysis of Cdk5/p25 crystal structure and molecular dynamics, several smaller peptides were generated and tested. We identified a novel 24 amino acid peptide, called p5, that more effectively inhibited Cdk5/p25 activity in cortical neurons than CIP without affecting endogenous Cdk5/p35, nor other Cdks. The small size and specificity of p5 inhibition make it an excellent candidate for therapeutic trials in animal models of AD and other neurodegenerative disorders associated with Cdk5 deregulation. This may provide a possible new and novel therapeutic route for intervention to prevent or reduce the neurodegenerative pathology induced by Cdk5 deregulation. The Cdks ligand binding mechanisms are not understood , although a large numbers of molecules including various inhibitors and activators have been used to study their effects on its activity. In a recent study we have used crystal structure of the Cdk5/p25 complex to understand the possible molecular mechanisms of the ligand binding, specificity, and regulation of the kinase using comparative molecular dynamics simulations under physiological conditions. This study provides new insight on the mechanisms that modulate such processes, which may be exploited to control pathological activation by p25. The structural changes observed in the kinase are stabilized by a network of interactions involving highly conserved residues within the cyclin-dependent kinase (cdk) family. Collective motions of the proteins (cdk5, p25, and inhibitor,CIP) and their complexes are identified by principal component analysis, revealing two conformational states of the activation loop upon p25 complexation, which are absent in the uncomplexed kinase and not apparent from the crystal. Simulations of the uncomplexed inhibitor CIP show structural rearrangements and increased flexibility of the interfacial loop containing the critical residue E240, which becomes fully hydrated and available for interactions with one of several positively charged residues in the kinase. These changes provide a rationale for the observed high affinity and enhanced inhibitory action of CIP when compared to either p25 or the physiological activators of cdk5, p35.

细胞周期蛋白依赖性激酶 5 (Cdk5) 主要在神经系统中表达。虽然它在非神经元细胞中表达并与细胞周期蛋白结合，但由于它与神经元特异性分子 P35 和 P39 结合，其活性主要在有丝分裂后神经元中发现。它参与神经元迁移、突触传递和存活。 Cdk5 是一种多功能神经元激酶（针对从神经元分化到突触功能的蛋白质），当与其共激活剂 p35 复合时受到严格调节。它是磷酸化神经丝和 tau 蛋白的几种激酶之一。其多样化的作用部分源于其与信号转导网络中其他激酶的串扰相互作用，这些信号转导网络是神经元细胞存活、生长和分化的基础。例如，我们已经证明，Cdk5 下调 MAPK 和 JNK，上调 PI3K。这些结果表明，Cdk5 通常调节其他激酶对神经元存活的特定信号的反应强度。 Cdk5 在突触传递中的作用是通过调节突触前蛋白（如突触蛋白、Munc18 和 dynamin 1）的细胞功能来介导的。它在突触中的多功能作用很复杂，可能涉及其他新底物。在我们之前的研究中，我们已经证明神经元应激调节 Cdk5 活性。由于糖皮质激素是应激反应的主要末端效应器，在中枢神经系统 (CNS) 的稳态中发挥着重要作用，并通过其细胞内受体、糖皮质激素和盐皮质激素受体有助于记忆巩固和情绪控制。这提出了 Cdk5 调节这些受体行为的可能性。事实上，我们发现，Cdk5 磷酸化并调节糖皮质激素和盐皮质激素受体的转录活性，并调节脑源性神经营养因子的表达。醛固酮和地塞米松分别增加和抑制大鼠皮质神经元细胞中脑源性神经营养因子（BDNF）的mRNA/蛋白表达，而内源性糖皮质激素皮质酮则表现出双相效应。 Cdk5 增强醛固酮和地塞米松对 BDNF 表达的影响。由于这种神经营养因子在神经元活力、突触可塑性、记忆巩固和情绪变化中发挥着关键作用，因此我们认为 CDK5 的激活可能调节糖皮质激素受体的转录活性并调节 BDNF 的表达，从而影响这些功能。 正常情况下，Cdk5 活性受到严格调节，但在神经元应激条件下，它会失调，导致多动、神经元病理和细胞死亡。因此，Cdk5 与某些神经退行性疾病有关，例如阿尔茨海默病 (AD)。 Cdk5s 在神经退行性变中的作用模型表明，应激诱导的钙离子流入神经元会激活钙蛋白酶（一种 Ca++ 激活的蛋白酶），它将 p35 裂解为 p25 和 p10 片段。 p25 反过来形成更稳定的 Cdk5/p25 过度活跃复合物，使 tau 蛋白和其他神经元细胞骨架蛋白过度磷酸化，并诱导细胞死亡。事实上，据报道，AD 大脑中 p25 和 Cdk5 活性水平升高。 p25 可能具有毒性，这一研究来自对用 Aβ-淀粉样肽（AD 病理学的关键标志物）处理的皮质神经元的研究，其中 p35 转化为 p25，并伴有过度激活的 Cdk5、tau 和神经丝过度磷酸化和细胞凋亡。 Cdk5/p25 复合物的表达似乎主要负责 tau 蛋白和神经丝病理学，并表明专门针对该靶点的治疗方法可能会成功。然而，对于大多数这些研究，世界各地的各个实验室都将重点放在类似于 roscovitine 的氨基噻唑化合物上，roscovitine 是一种激酶抑制剂，与 Cdk5 和其他激酶中的 ATP 结合位点竞争。这些药物并不专门作用于 Cdk5/p25，但也会抑制 Cdk5/p35 和其他对正常发育和功能至关重要的激酶。这可能会导致严重的继发副作用，从而损害任何治疗价值。 然而，我们解决这个问题的方法是基于早期的研究，其中我们鉴定了 p35 (CIP) 的 125 个氨基酸 (aa) 残基的肽，它抑制 Cdk5/p25 活性，并在不影响 Cdk5/p35 活性的情况下拯救皮层神经元免于诱导细胞凋亡。这种方法可能被证明是抑制失调的 Cdk5/p25 过度活跃引起的神经退行性病理的更有效方法。然而，要使治疗有效，它的分子必须足够小，才能通过血脑屏障；至少可以说，125 个残基的大肽可能会产生问题。因此，我们开始生产具有同等特异性的更小的 CIP 肽。基于对 Cdk5/p25 晶体结构和分子动力学的分析，生成并测试了几种较小的肽。 我们发现了一种新的 24 个氨基酸肽，称为 p5，它比 CIP 更有效地抑制皮质神经元中的 Cdk5/p25 活性，而不影响内源性 Cdk5/p35 或其他 Cdks。 p5 抑制的小尺寸和特异性使其成为 AD 和与 Cdk5 失调相关的其他神经退行性疾病动物模型中治疗试验的绝佳候选者。这可能提供一种可能的新的干预治疗途径，以预防或减少 Cdk5 失调引起的神经退行性病理。 尽管包括各种抑制剂和激活剂在内的大量分子已被用来研究它们对其活性的影响，但 Cdks 配体结合机制尚不清楚。在最近的一项研究中，我们利用生理条件下的比较分子动力学模拟，利用 Cdk5/p25 复合物的晶体结构来了解配体结合、特异性和激酶调节的可能分子机制。这项研究为调节此类过程的机制提供了新的见解，可用于控制 p25 的病理激活。在激酶中观察到的结构变化通过涉及细胞周期蛋白依赖性激酶 (cdk) 家族中高度保守残基的相互作用网络来稳定。通过主成分分析鉴定了蛋白质（cdk5、p25 和抑制剂 CIP）及其复合物的集体运动，揭示了 p25 复合时激活环的两种构象状态，这在未复合的激酶中不存在，并且在晶体中不明显。对未复合的抑制剂 CIP 的模拟显示，含有关键残基 E240 的界面环发生了结构重排并提高了灵活性，E240 变得完全水合，可与激酶中几个带正电的残基之一相互作用。与 p25 或 cdk5 的生理激活剂、p35 相比，这些变化为观察到的 CIP 的高亲和力和增强的抑制作用提供了理论依据。