Nanobodies for Probing CACNA2D2 and CACNA2D3 Function, Expression, and Therapeutics

用于探测 CACNA2D2 和 CACNA2D3 功能、表达和治疗的纳米抗体

• 批准号: 10217683
• 负责人: Henry M. Colecraft
• 金额: $ 16.2万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-15 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10217683
• 关键词: Adult; Affinity; Animal Model; Antibodies; Baculoviruses; Binding; Biological Assay; Biology; Cardiac Myocytes; Cell Separation; Cell surface; Cells; Characteristics; Chronic; Clinical; Data; Disease; Down-Regulation; Epilepsy; Flow Cytometry; Fluorescence; Fluorescence Resonance Energy Transfer; Genes; Glioma; Glutamate Receptor; Human; Immunohistochemistry; In Situ; In Vitro; Individual; Intellectual functioning disability; Knock-out; Libraries; Ligands; Link; Magnetism; Malignant Neoplasms; Mediating; Missense Mutation; Molecular Sieve Chromatography; N-Methylaspartate; Nasopharynx Carcinoma; Neurologic Symptoms; Neurons; Night Blindness; P-Q type voltage-dependent calcium channel; Physiological; Play; Prevention; Production; Property; Protein Family; Proteins; Reagent; Retinal Dystrophy; Role; Synapses; Therapeutic; Thrombospondins; Tissue Fixation; Transfection; Yeasts; disease phenotype; dorsal horn; expectation; experimental study; extracellular; gabapentin; in vivo; loss of function mutation; nanobodies; novel; painful neuropathy; patch clamp; protein function; reconstitution; synaptogenesis; therapeutic target; thrombospondin 2; trafficking; voltage

SUMMARY CACNA2D1 – CACNA2D4 genes encode alpha2delta-1, alpha2delta-2, alpha2delta-3, and alpha2delta-4 proteins which are most well known as auxiliary subunits of high-voltage-activated Ca2+ channels. There are seven different HVA CaV channel types (CaV1.1 – CaV1.4; CaV2.1 – CaV2.3), and in general, alpha2delta-1 – alpha2delta-4 proteins can interact promiscuously with any of the seven pore-forming subunits to enhance trafficking and stabilize channels at the cell surface to increase whole-cell current. Nevertheless, these proteins have broad but not completely overlapping distributions, and loss-of-function mutations in humans, or their individual knockout in animal models, gives rise to distinct disease phenotypes, indicating some unique functional properties. Missense mutations or knockout of alpha2delta-1 and alpha2delta-2 are most prominently linked to neurological symptoms including epilepsy and intellectual disability; loss of alpha2delta-3 is associated with malignancies including gliomas and nasopharyngeal carcinoma; and dysregulation of alpha2delta-4 causes retinal dystrophy and night blindness. The potential of this protein family as therapeutic targets to treat disease was elevated by the serendipitous finding that gabapentinoids which are used clinically to treat neuropathic pain and epilepsy exert their actions via binding alpha2delta-1. It is less clear precisely how gabapentin binding to alpha2delta-1 alleviates neuropathic pain. Proposed candidate mechanisms include chronic down-regulation of CaV2 channel trafficking; disruption of alpha2delta-1 interaction with extracellular thrombospondins; and prevention of alpha2delta-1 mediated trafficking of NMDA type glutamate receptors to the synaptic terminus in the dorsal horn. Thus, beyond their role as auxiliary CaV channel subunits, alpha2delta subunits may also differentially interact with other proteins which may play a role in their distinctive physiological functions. This proposal is primarily focused on CACNA2D2 and CACNA2D3 which are two genes included in the list of IDG-eligible understudied proteins. Ligands that can specifically interact with these proteins and be used to either detect them in situ or manipulate their function are essential for illuminating their specific functional roles as well as realizing their potential as therapeutic targets. Here, we propose to develop nanobodies to alpha2delta-2 and alpha2delta-3 as novel reagents to detect and manipulate the functions of these proteins in vitro, in situ, and in vivo. Our lab has recently developed nanobodies against auxiliary CaV? subunits and used these genetically-encoded molecules to regulate functional expression of CaV1/CaV2 channels with unprecedented levels of precision. The expertise we have gained from these previous studies not only demonstrate overall feasibility, but also provides preliminary data that bolsters our expectations that the experiments proposed here will yield unique reagents to illuminate alpha2delta-2 and alpha2delta-3 biology and therapeutic potential. (1) Isolate nanobodies against alpha2delta-2 and alpha2delta-3 and characterize their binding characteristics. (2) Determine functional impact of alpha2delta-2 and alpha2delta-3 nanobodies on CaV channel functional expression. (3) Determine impact of alpha2delta-2/alpha2delta-3 nanobodies on putative non-CaV channel functions of alpha2delta subunits.

概括 CACNA2D1 - CACNA2D4基因编码Alpha2delta-1，Alpha2delta-2，Alpha2delta-3和Alpha2delta-4蛋白，这些蛋白最著名，它们是高压激活的CA2+通道的辅助亚基。有七种不同的HVA CAV通道类型（CAV1.1 - CAV1.4; CAV2.1 - CAV2.3），通常，Alpha2delta-1 - alpha2delta-4蛋白可以与七个形成的孔子亚基中的任何一个相互交互，以增强运输和稳定在细胞表面上，以增加整个细胞表面。然而，这些蛋白质具有广泛但并不完全重叠的分布，人类或动物模型中的个体敲除功能丧失突变会导致独特的疾病表型，表明某些独特的功能特性。 alpha2delta-1和alpha2delta-2的错义突变或敲除与神经系统症状最为突出的，包括癫痫和智力障碍； α2delta-3的丧失与包括神经胶质瘤和鼻咽癌在内的恶性肿瘤有关。 α2Delta-4的失调会导致视网膜营养不良和夜失明。通过偶然发现，这种蛋白质家族作为治疗疾病的治疗靶标的潜力升高，即在临床上用来治疗神经性疼痛和癫痫的gabapentinoids通过结合α2Delta-1发挥其作用。毫无疑问，加巴喷丁如何与α2DELTA-1结合减轻神经性疼痛。拟议的候选机制包括CAV2通道贩运的慢性下调； α2delta-1与细胞外血小板蛋白的相互作用的破坏；预防α2delta-1介导的NMDA型谷氨酸受体的运输到背角突触末端。除了它们作为辅助CAV通道亚基的作用外，Alpha2delta亚基还可能与其他蛋白质相互作用，这些蛋白可能在其独特的生理功能中起作用。该建议主要集中在CACNA2D2和CACNA2D3上，这是符合IDG资格理解的蛋白质列表中的两个基因。可以特异性与这些蛋白质相互作用并用于检测原位或操纵其功能的配体对于阐明其特定功能作用以及实现其作为治疗靶标的潜力至关重要。在这里，我们建议将纳米构造开发为alpha2delta-2和alpha2delta-3作为新型试剂，以检测和操纵这些蛋白质在体外，原位和体内的功能。我们的实验室最近已经开发了针对辅助CAV的纳米体吗？亚基并使用这些遗传编码的分子来调节具有前所未有的精度水平的CAV1/CAV2通道的功能表达。我们从这些先前的研究中获得的专家不仅证明了总体可行性，而且提供了初步数据，这使我们期望这里提出的实验会产生独特的试剂，以阐明α2DELTA-2和Alpha2delta-3生物学和治疗潜力。 （1）针对alpha2delta-2和alpha2delta-3的分离纳米构造，并表征其结合特性。 （2）确定alpha2delta-2和alpha2delta-3纳米反应对CAV通道功能表达的功能影响。 （3）确定alpha2delta-2/alpha2delta-3纳米化对α2Delta亚基的假定非CAV通道功能的影响。