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：
9814720
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 2023-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9814720
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 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 Subfamily lentivirinae Suspensions Testing Thyroid Function Tests Thyroid Gland Thyroid Hormones Thyrotropin Receptor Time Ultrasonography Viral base cell growth clinically relevant epidemiology study genetic approach genetic regulatory protein inhibitor/antagonist 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

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，在最后几个年。因果因素仍然不明确。 TSH是参与甲状腺的甲状腺的主要调节剂生长，分化和甲状腺激素分泌。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。因此，完全了解 TSHR下游涉及的机制可能会为治疗提供潜在的新目标。 TSH通过规范的TSHR-GS-TMAC单位行动以综合营地，第二个Messenger中介许多激素的作用。 TMAC是大型跨膜蛋白家族的成员，蛋白质依赖性腺苷酸环化酶活性负责转导膜受体信号。可溶 Adenylyl Cyclase（SAC）是腺苷酸环化酶家族的进化保守成员，是一个相对较高的新发现的活性不是由G蛋白调节的，而是被碳酸氢盐和钙激活。这该建议的前提是基于新型SAC/Cyclase相关蛋白1的鉴定（CAP1）/RAP1B复合物。我们将在四个集成的特定目标中解决其参与TSH生物学的参与。在AIM＃1中，我们将利用基于FRET的营地传感器评估药理学和遗传学（SHRNA/CRISPR-CAS9）干扰SAC活性会影响TSH介导的营地水平的方法；初步数据表明SAC参与调节PKA-PDE4单元的TSH介导的CAMP动力学。在AIM＃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。药理（SAC抑制剂）和遗传（SH-SAC/SHCAP1）干预抑制了TSH介导的增殖。为了评估CAP1相关SAC的特定作用，我们将使用SHRNA/CRISPR-CAS9 在细胞中下调CAP1的方法，我们将在感染慢病毒后进行救援测定法编码SH-或CAS9的全长WT和突变体E171R-CAP1，缺乏SAC结合。在AIM＃3中，我们将解决核囊池在TSH介导的G1/S进展中的参与。我们将使用新开发的光遗传学工具，该工具融合了融合到蓝色的蓝光活化环化酶（BPAC）发射纳米酸酯酶（NLUC）。将BPAC-NLUC结构瞄准不同的隔间将使我们能够直接测试核中产生的cAMP是否能够在SAC下调时挽救增殖。我们将通过小鼠的甲状腺特异性缺失来补充这些细胞研究，并通过超声引导的甲状腺特异性SAC和BPAC-NLUC病毒悬浮液的注射。在AIM＃4中，我们将利用质谱方法来识别使用新的CAP1相关蛋白新开发的试剂和优化的CAP1-PULL DOWN分析的协议。我们的长期目标是提供新型SAC-CAP1-RAP1复合物及其其基础的机械细节参与TSH生物学。