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.

抽象的。我们的合作基于泰勒实验室的蛋白激酶专业知识和专业知识 Chang Lab以分离合成进化和工程的纳米体（SENS）。我们的目标是隔离感官 这将区分cAMP依赖性蛋白激酶的催化（C）亚基的同工型 （PKA）。这是基于以下意识到，大脑中> 50％的PKA信号传导是由C亚基介导的。 尽管PKA的C亚基是第二个被发现的，但第一个被测序，第一个是 结晶，几乎没有关于C蛋白的了解，它代表了一个不同的蛋白质家族 仅以外显子1编码的N末端序列。C3/4同工型仅在 脑C2在淋巴组织和T细胞中表达。使用视网膜作为“进入大脑的窗户” 泰勒（Taylor）表明，C在这些末端分化的神经元中高度表达，并且它本地化 与C不同，它支持这些同工型的功能性非差额。令人惊讶的发现 那个c，c，rii或rii本地化到线粒体的本地化，这进一步增加了C的重要性和 它与神经退行性疾病的联系。为了验证视网膜成像可以用作的假设 进入大脑中更复杂的信号的窗口，并确定PKA信号是否随着函数的变化而变化 泰勒（Taylor）的AD发作和进展与UCSD的阿尔茨海默氏病研究中心合作 （UCSD ADRC）导演此核心的神经病理学核心和罗伯特·里斯曼（Robert Rissman）现在正在比较广告大脑 来自有轻度和高级广告的人。初步结果，再加上PTAU的成像，标志 高级AD，确认假设并突出具有歧视工具的重要性 在各种C同工型之间。为了实现这一基本下一步，我们转向了新开发的Sens 技术。 Chang Lab使用C纯化的C-亚基蛋白，将荧光标记蛋白 然后选择将区分不同C-亚基同工型的SENS。最初他们会集中精力 基于RNA范围，在C4/C4AB上似乎是与 线粒体和C2在T细胞中表达，具有早期生物标志物的潜力 发作PD。在目标上，泰勒将表达C-亚基同工型以及对应于 每个亚基的N末端尾巴。在Aim II中，Chang将荧光标记这些蛋白质，并通过多个 筛选的综合将隔离选择性高亲和力SENS。这些感官将因其能力而分析 通过蛋白质印迹检测特定的同工型，并在人体和小鼠组织中进行成像。抑制 激酶活性也将进行评估。一个长期目标是结晶sens：c-subunit络合物。 因为这种试剂工具箱可以被临床医生和基本科学家广泛使用，所以这是一个很高的 风险/高回报机会可能会产生很大的影响。我们的工作将在公开访问中迅速发布 所有研究人员将免费提供期刊和SENS。