权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Role of soluble adenylyl cyclase in TSH biology

可溶性腺苷酸环化酶在 TSH 生物学中的作用

基本信息

批准号：
10372085
负责人：
DANIEL L ALTSCHULER
金额：
$ 39.13万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-07-01 至 2024-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10372085
关键词：
Actins Address Adenylate Cyclase Affinity Animal Model Benign Bicarbonates Binding Biological Assay Biology CREB1 gene CRISPR/Cas technology Calcium Catalytic Domain Cell Nucleus Cell Proliferation Cells Code Complement Complex Concentration Camps Critical Pathways Cyclic AMP Cyclic AMP-Dependent Protein Kinases Cytoskeleton Data Diffusion Down-Regulation Endocrine Endocrine System Diseases Engineering Family Fluorescence Resonance Energy Transfer GTP-Binding Protein alpha Subunits, Gs GTP-Binding Proteins Genetic Goals Growth Hormone secretion Hormones Infection Injections Integral Membrane Protein Intervention Length Lentivirus Light Link Malignant Neoplasms Malignant neoplasm of thyroid Mass Spectrum Analysis Mediating Membrane Methods Mus Nuclear Pathway interactions Pharmacology Phosphorylation Positioning Attribute Proteins Proteolysis Protocols documentation Reagent Receptor Signaling Recording of previous events Reporting Research Resistance Role Scaffolding Protein Second Messenger Systems Signal Pathway Signal Transduction Suspensions Testing Thyroid Function Tests Thyroid Gland Thyroid Hormones Thyrotropin Receptor Time Viral base cell growth clinically relevant epidemiology study genetic approach genetic regulatory protein inhibitor knock-down member mutant nanoluciferase new therapeutic target novel optogenetics radixin protein scaffold sensor small hairpin RNA synergism therapeutic target tool transcription factor tumor progression ultrasound

项目摘要

Thyroid cancer is the most common endocrine malignancy, with an alarmingly steady increase in the last few years. Causal factors remain ill-defined. TSH is the major regulator of the thyroid gland involved in thyroid growth, differentiation, and thyroid hormone secretion. Evidence from animal models and new epidemiological studies suggest that TSH signaling pathways are critical for tumor progression. Thus, fully understanding the mechanisms involved downstream of the TSHR might provide potential new targets for therapy. TSH acts via the canonical TSHR-Gs-tmAC unit to synthesize cAMP, a second messenger mediating the action of numerous hormones. tmACs are members of a large family of transmembrane proteins with a G- protein-dependent adenylyl cyclase activity responsible for transducing membrane receptor signaling. Soluble adenylyl cyclase (sAC), an evolutionarily conserved member of the adenylyl cyclase family, is a relatively newly discovered activity not modulated by G-proteins, but instead activated by bicarbonate and calcium. The premise of this proposal is based on the identification of a novel sAC/Cyclase-Associated Protein 1 (CAP1)/Rap1b complex. We will address its involvement in TSH biology in four integrated specific aims. In Aim #1 we will utilize FRET-based cAMP sensors to assess whether pharmacological and genetic (shRNA/CRISPR-Cas9) methods that interfere with sAC activity impact TSH-mediated cAMP levels; preliminary data indicate sAC is involved in TSH-mediated cAMP dynamics regulating a PKA-PDE4 unit. In Aim#2 we will assess the involvement of the sAC-CAP1 complex in TSH-mediated G1/S progression. Pharmacological (sAC inhibitors) and genetic (sh-sAC/shCAP1) intervention inhibited TSH-mediated proliferation. To assess a specific role for CAP1-associated sAC, we will use shRNA/CRISPR-Cas9 approaches to downregulate CAP1 in cells and we will perform rescue assays upon infection with lentivirus coding for sh- or Cas9-resistant full length WT and mutant E171R-CAP1, deficient in sAC binding. In Aim #3 we will address the involvement of a nuclear sAC pool in TSH-mediated G1/S progression. We will use a newly developed optogenetic tool incorporating a blue-light activated cyclase (bPAC) fused to a blue- emitting nanoluciferase (nLuc). Targeting the bPAC-nLuc construct to distinct compartments will allow us to directly test whether cAMP generated in the nucleus is able to rescue proliferation upon sAC downregulation. We will complement these cell studies by thyroid-specific deletion of sAC in mice and rescue mediated by ultrasound-guided thyroid injection of compartment-specific sAC and bPAC-nLuc viral suspensions. In Aim #4 we will exploit mass spectrometry approaches to identify new CAP1-associated proteins utilizing newly developed reagents and optimized protocols for CAP1-pull down assays. Our long-term goal is to provide mechanistic details underlying the novel sAC-CAP1-Rap1 complex and its involvement in TSH biology.

甲状腺癌是最常见的内分泌恶性肿瘤，近几年呈惊人的稳定增长趋势年。因果因素仍然不明确。 TSH是甲状腺的主要调节因子，参与甲状腺生长、分化和甲状腺激素分泌。来自动物模型和新流行病学的证据研究表明 TSH 信号通路对于肿瘤进展至关重要。从而充分认识 TSHR 下游涉及的机制可能为治疗提供潜在的新靶点。 TSH 通过典型的 TSHR-Gs-tmAC 单元发挥作用，合成 cAMP，这是介导多种激素的作用。 tmAC 是具有 G- 的跨膜蛋白大家族的成员蛋白质依赖性腺苷酸环化酶活性负责转导膜受体信号传导。可溶腺苷酸环化酶（sAC）是腺苷酸环化酶家族中进化上保守的成员，是一种相对新发现的活性不受 G 蛋白调节，而是由碳酸氢盐和钙激活。这该提案的前提是基于新型 sAC/环化酶相关蛋白 1 的鉴定 (CAP1)/Rap1b 复合体。我们将通过四个综合具体目标来解决其对 TSH 生物学的参与。在目标#1中，我们将利用基于 FRET 的 cAMP 传感器来评估药理学和遗传是否 (shRNA/CRISPR-Cas9) 干扰 sAC 活性的方法会影响 TSH 介导的 cAMP 水平；初步数据表明 sAC 参与 TSH 介导的 cAMP 动力学调节 PKA-PDE4 单元。在目标#2 中，我们将评估 sAC-CAP1 复合物在 TSH 介导的 G1/S 进展中的参与情况。药理学（sAC 抑制剂）和遗传（sh-sAC/shCAP1）干预抑制 TSH 介导增殖。为了评估 CAP1 相关 sAC 的特定作用，我们将使用 shRNA/CRISPR-Cas9 下调细胞中 CAP1 的方法，我们将在慢病毒感染后进行救援试验编码 sh 或 Cas9 抗性全长 WT 和突变体 E171R-CAP1，缺乏 sAC 结合。在目标 3 中，我们将解决核 sAC 库在 TSH 介导的 G1/S 进展中的参与问题。我们将使用新开发的光遗传学工具，将蓝光激活环化酶（bPAC）与蓝光融合发射纳米荧光素酶（nLuc）。将 bPAC-nLuc 构建体靶向不同的区室将使我们能够直接测试细胞核中产生的cAMP是否能够在sAC下调时挽救增殖。我们将通过小鼠甲状腺特异性删除 sAC 并通过以下方法介导的救援来补充这些细胞研究：超声引导下甲状腺注射区室特异性 sAC 和 bPAC-nLuc 病毒悬浮液。在目标 #4 中，我们将利用质谱方法来鉴定新的 CAP1 相关蛋白新开发的试剂和优化的 CAP1-pull down 检测方案。我们的长期目标是提供新型 sAC-CAP1-Rap1 复合物及其背后的机制细节参与 TSH 生物学。

项目成果

期刊论文数量（4）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Soluble cyclase-mediated nuclear cAMP synthesis is sufficient for cell proliferation.

可溶性环化酶介导的核 cAMP 合成足以促进细胞增殖。

DOI：
10.1073/pnas.2208749120
发表时间：
2023-01-24
期刊：
Proceedings of the National Academy of Sciences of the United States of America
影响因子：
11.1
作者：
通讯作者：

From membrane to nucleus: A three-wave hypothesis of cAMP signaling.

DOI：
10.1016/j.jbc.2023.105497
发表时间：
2024-01
期刊：
JOURNAL OF BIOLOGICAL CHEMISTRY
影响因子：
4.8
作者：
Pizzoni, Alejandro;Zhang, Xuefeng;Altschuler, Daniel L
通讯作者：
Altschuler, Daniel L

CAP1 binds and activates adenylyl cyclase in mammalian cells.

CAP1 结合并激活哺乳动物细胞中的腺苷酸环化酶。

DOI：
10.1073/pnas.2024576118
发表时间：
2021
期刊：
Proceedings of the National Academy of Sciences of the United States of America
影响因子：
11.1
作者：
Zhang,Xuefeng;Pizzoni,Alejandro;Hong,Kyoungja;Naim,Nyla;Qi,Chao;Korkhov,Volodymyr;Altschuler,DanielL
通讯作者：
Altschuler,DanielL

Dual Activation of cAMP Production Through Photostimulation or Chemical Stimulation.