Synthetically-evolved and engineered Nanobodies selective for Cb isoforms of PKA

对 PKA Cb 亚型具有选择性的合成进化和工程纳米抗体

• 批准号: 10525796
• 负责人: GEOFFREY A CHANG
• 金额: $ 43.45万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-15 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10525796
• 关键词: Affinity; Alzheimer' s Disease; Alzheimer' s disease brain; Animals; Antigens; Bacteria; Binding; Biological Assay; Brain; Catalytic Domain; Cells; Collaborations; Communities; Complex; Coupled; Crystallization; Cyclic AMP-Dependent Protein Kinases; Engineering; Exons; Family; Goals; Human; Image; In Vitro; Incentives; Individual; Inflammation; Journals; Kinetics; Knowledge; Label; Laboratories; Late Onset Alzheimer Disease; Length; Link; Lymphoid Tissue; Maps; Mediating; Metabolic Diseases; Methods; Mitochondria; Molecular Evolution; Mus; N-terminal; Neurodegenerative Disorders; Neurons; Peptide Fragments; Peptides; Phosphotransferases; Protein Analysis; Protein Family; Protein Isoforms; Protein Kinase; Protein Subunits; Proteins; Publishing; RNA; RNA Splicing; Reagent; Research; Research Personnel; Retina; Role; Sampling; Scientist; Screening procedure; Signal Transduction; Specificity; Structure; T-Lymphocyte; Tail; Technology; Tissue Model; Tissues; Variant; Western Blotting; Work; alpha helix; base; diagnostic tool; early detection biomarkers; early onset; extracellular; high risk; human imaging; human tissue; mitochondrial dysfunction; mouse model; nanobodies; neuropathology; protein complex; rapid technique; retinal imaging; retinal neuron; screening; tool; virtual

ABSTRACT. Our collaboration builds on the protein kinase expertise of the Taylor lab and the expertise of the Chang lab to isolate Synthetically-evolved and engineered Nanobodies (SENs). Our goal is to isolate SENs that will discriminate between the isoforms of the catalytic (C) subunits of cAMP-dependent protein kinase (PKA). This builds on the realization that >50% of PKA signaling in the brain is mediated by C subunits. Although the C subunit of PKA was the second to be discovered, the first to be sequenced, and the first to be crystallized, virtually nothing is known about the C proteins, which represent a family of proteins that differ only in their N-terminal sequence encoded for by Exon 1. The C3/4 isoforms are expressed exclusively in brain while C2 is expressed in lymphoid tissues and T cells. Using the retina as a “Window into the Brain” Taylor showed that C is highly expressed in these terminally differentiated neurons and that it localizes differently from C, which supports the functional non-redundancy of these isoforms. The surprising discovery that C, but not C, RII, or RII, localizes to mitochondria adds further credence to the importance of C and its link to neurodegenerative diseases. To validate the hypothesis that imaging of the retina could serve as a window into the more complex signaling in brain and to determine if PKA signaling changes as a function of the onset and progression of AD, Taylor, in collaboration with the Alzheimer Disease Research Center at UCSD (UCSD ADRC) Neuropathology Core and Robert Rissman who directs this core is now comparing AD brains from individuals with mild and advanced AD. The preliminary results, coupled with imaging of pTau, a hallmark of advanced AD, confirm the hypothesis and highlight the importance of having tools that will discriminate between the various C isoforms. To achieve this essential next step we turned to the newly developed SENS technology. Using C-subunit proteins, purified in the Taylor lab, the Chang lab will fluorescently tag the proteins and then select for SENs that will discriminate between the different C-subunit isoforms. Initially they will focus on C4/C4ab as these, based on RNA scope, appeared to be the best candidates for association with mitochondria, and C2 which is expressed in T cells and has the potential to serve as a Biomarker for early onset PD. In Aim I Taylor will express C-subunit isoforms as well as peptide fragments that correspond to the N-terminal tails of each subunit. In Aim II Chang will fluorescently tag these proteins and through multiple rounds of screening will isolate selective high affinity SENs. These SENs will be analyzed for their ability to detect specific isoforms by Western blots and by imaging in both human and mouse tissues. Inhibition of kinase activity will also be assessed. A long-term goal will be to crystallize SENs: C-subunit complexes. Because this toolbox of reagents can be widely used by both clinicians and basic scientists, this is a high risk/high return opportunity that can have a high impact. Our work will be published quickly in open access journals and the SENS will be freely available to all researchers.

摘要。我们的合作建立在泰勒实验室的蛋白激酶专业知识和 Chang实验室分离合成进化和工程纳米抗体（SENs）。我们的目标是分离SENs 其将区分cAMP依赖性蛋白激酶的催化（C）亚基的同种型 （PKA）.这是基于以下认识：大脑中>50%的PKA信号传导是由C β亚基介导的。 虽然PKA的C β亚基是第二个被发现的，第一个被测序的，也是第一个被发现的。 结晶，几乎没有什么是已知的C蛋白，这代表了一个家庭的蛋白质， 仅在由外显子1编码的N端序列中。C β 3/4同种型仅在 而C β 2在淋巴组织和T细胞中表达。将视网膜作为“大脑的窗口” Taylor表明，C β在这些终末分化的神经元中高度表达， 与C β不同，C β支持这些同种型的功能非冗余。令人惊讶的发现 线粒体定位于C β，而不是C β、RII β或RII β，这进一步证实了C β的重要性， 与神经退行性疾病的联系为了验证视网膜成像可以作为一种 打开大脑中更复杂的信号传导的窗口，并确定PKA信号传导是否作为大脑中 泰勒与加州大学圣地亚哥分校阿尔茨海默病研究中心合作， (UCSD ADRC）神经病理学核心和指导该核心的罗伯特·里斯曼现在正在比较AD大脑 从轻度和晚期AD患者身上。初步结果，加上pTau的成像，一个标志性的 先进的AD，证实了假设，并强调了工具，将区分的重要性 不同亚型之间的联系为了实现这一重要的下一步，我们转向了新开发的SENS 技术.使用泰勒实验室纯化的C亚基蛋白质，Chang实验室将荧光标记蛋白质 然后选择能够区分不同C亚基同种型的SEN。首先，他们将专注于 基于RNA范围，这些似乎是与C144/C144 ab相关的最佳候选者。 线粒体，以及在T细胞中表达并有可能作为早期免疫缺陷的生物标志物的C β 2。 发病PD。在Aim I中，Taylor将表达C亚基同种型以及对应于C亚基的肽片段。 每个亚基的N末端尾部。在Aim II中，Chang将荧光标记这些蛋白质，并通过多个 几轮筛选将分离选择性高亲和力的SENs。这些SENs将被分析它们的能力， 通过蛋白质印迹和成像在人和小鼠组织中检测特异性同种型。抑制 还将评估激酶活性。一个长期的目标将是结晶SENs：C-亚基复合物。 因为这种试剂工具箱可以被临床医生和基础科学家广泛使用，这是一个高 风险/高回报的机会，可以有很大的影响。我们的工作将很快以开放获取的方式发表 期刊和SENS将免费提供给所有研究人员。