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

基本信息

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
项目类别：
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&apos s Disease Alzheimer&apos 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

项目摘要

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 (SEN) 我们的目标是分离 SEN。将区分 cAMP 依赖性蛋白激酶催化 (C) 亚基的亚型 (PKA)。这是基于这样的认识：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、第一个被测序、第一个被结晶后，我们对 C 蛋白几乎一无所知，它代表了一个不同的蛋白质家族仅在由外显子 1 编码的 N 端序列中。C3/4 同工型仅在大脑，而 C2 在淋巴组织和 T 细胞中表达，利用视网膜作为“进入大脑的窗口”。 Taylor 表明 C 在这些终末分化神经元中高度表达，并且它定位于与 C 不同，C 支持这些亚型的功能非冗余性，这是令人惊讶的发现。 C（而非 C、RII 或 RII）定位于线粒体，这进一步证明了 C 的重要性，并且验证视网膜成像可以作为神经退行性疾病的联系。观察大脑中更复杂的信号传导的窗口，并确定 PKA 信号传导是否随 AD 的发病和进展，Taylor 与加州大学圣地亚哥分校阿尔茨海默病研究中心合作（加州大学圣地亚哥分校 ADRC）神经病理学核心和指导该核心的 Robert Rissman 现在正在比较 AD 大脑来自患有轻度和晚期 AD 的个体的初步结果，加上 pTau 的成像，这是一个标志。高级 AD 的研究，证实假设并强调拥有能够区分的工具的重要性为了实现这一重要的下一步，我们转向了新开发的 SENS。 Chang 实验室使用在 Taylor 实验室纯化的 C 亚基蛋白质，对蛋白质进行荧光标记。然后选择能够区分不同 C 亚基同工型的 SEN。最初，它们将重点关注。在 C4/C4ab 上，因为基于 RNA 范围，这些似乎是与线粒体和 C2，它在 T 细胞中表达，有可能作为早期的生物标志物在 Aim I Taylor 中，将表达 C 亚基亚型以及对应于的肽片段。 In Aim II Chang 会通过多个亚基的 N 末端尾部对这些蛋白质进行荧光标记。多轮筛选将选择性地分离出高亲和力 SEN，并对这些 SEN 的能力进行分析。通过蛋白质印迹和人类和小鼠组织中的成像来检测特定亚型。激酶活性也将被评估，长期目标是结晶 SEN：C 亚基复合物。因为这个试剂工具箱可以被封建科学家和基础科学家广泛使用，所以这是一个很高的标准。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。