The role of the COP9 signalosome in distal nephron remodeling

COP9信号体在远端肾单位重塑中的作用

• 批准号: 10723624
• 负责人: Ryan J Cornelius
• 金额: $ 23.1万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10723624
• 关键词: Atrophic; Biological; Blood Vessels; COPS5 gene; Carrier Proteins; Catalytic Domain; Cell Maintenance; Cell Nucleus; Cell physiology; Cells; Clinical; Cullin Proteins; Data; Dietary Potassium; Disease; Distal; Distal convoluted renal tubule structure; Diuretics; Familial disease; Family; Fluorescence; Functional disorder; Genes; Genetic Transcription; Hyperplasia; Hypertrophy; Impairment; Kidney; Knock-out; Lead; Ligase; Light; Lysine; Mediating; Modeling; Molecular; Mus; Mutation; Nephrons; Nuclear; Pathway interactions; Pharmaceutical Preparations; Phosphotransferases; Physiological; Population; Potassium; Potassium Deficiency; Proteins; Reporter; Research; Resistance; Resolution; Role; SLC11A2 gene; SLC12A3 gene; Sodium; Sorting; Specificity; Stimulus; Syndrome; Techniques; Technology; Testing; Transcript; Ubiquitin; Work; blood pressure regulation; career; career development; cell dedifferentiation; cell type; clinically significant; cullin-3; design; dietary salt; experimental study; familial hyperkalemic hypertension; human disease; kidney cortex; mouse model; novel; protein degradation; response; salt intake; single nucleus RNA-sequencing; transcriptome; transcriptome sequencing; transcriptomics; ubiquitin ligase; ubiquitin-protein ligase

Abstract DCT remodeling has been associated with human diseases, changes in dietary salt intake, and drug administration. The disease familial hyperkalemic hypertension (FHHt) and diuretic resistance cause hypertrophy and hyperplasia; whereas, Gitelman and EAST syndromes lead to DCT atrophy. Yet despite its clinical significance, the cellular and molecular basis for this plasticity is unclear. The DCT comprises two subsegments, the early DCT1 the late DCT2. Knepper and colleagues, using single cell transcriptomics, recently identified a rare proliferative cell population within the DCT1, which they suggested may be responsible for the DCT's unique plasticity. Preliminary data from our lab showed that dietary potassium deficiency increased the percent of these proliferative cells in the DCT and caused DCT hypertrophy, especially along the DCT1. Furthermore, this work showed the proliferative DCT cell type to have lower levels of transport protein transcripts and higher levels of proliferative transcripts, suggesting that DCT1 cells dedifferentiate into a more proliferative state. Cullin-RING-ligases (CRLs) are a family of E3 ubiquitin ligases that mediate regulated degradation of proteins and are involved in many cellular functions important for cell maintenance and elimination of unwanted proteins. CRLs were recently discovered to regulate blood pressure via proteasomal degradation of with-no- lysine kinases (WNKs). Cullin 3 (CUL3) is the critical component of CRLs, which add ubiquitin moieties targeting proteins for proteasomal degradation. Mutations in CUL3 cause the disease FHHt. All CRLs are regulated by the COP9 signalosome (CSN), which interacts with the CRL and turns off ubiquitin ligase activity. Disease- causing CUL3 mutations inhibit the ability of CUL3 to interact with the CSN and therefore leave CUL3 hyperactivated. To investigate the role of impaired CSN-CUL3 interaction in human disease, we inactivated the CSN by deleting Jab1 (the key CSN catalytic subunit) along the entire nephron. Despite the fact that both CUL3 and the CSN are expressed all along the nephron, these mice (KS-Jab1-/-) showed remodeling only along the distal nephron, with shortening of the DCT and a large reduction in DCT1-specific proteins. Here, we plan to test our hypothesis that CSN dysfunction causes dedifferentiation of DCT1 cells leading to DCT remodeling using recent technological advances and new mouse models that allow us to study DCT remodeling at the protein and transcript level in unprecedented detail. We have utilized fluorescence-activated nucleus sorting (FANS) of DCT- specific INTACT (Isolation of Nuclei Tagged in specific Cell Types) reporter mice, which have inducible nuclear GFP expression to greatly enrich for DCT cells. We pair this with single nucleus RNA sequencing (snRNA-seq) that generates remarkably granular data about DCT cell populations. The proposed experiments with Jab1-/- mice will shine a light on the plasticity of the DCT, helping to uncover the mechanisms for DCT remodeling caused by CSN dysfunction. This will reveal valuable information that could have clinical and biological importance in advancing therapies in cases where the DCT remodels.

抽象的 DCT 重塑与人类疾病、膳食盐摄入量的变化以及药物相关 行政。家族性高钾性高血压（FHHt）和利尿剂抵抗引起的疾病 肥大和增生；而Gitelman和EAST综合征则导致DCT萎缩。然而尽管其 尽管具有临床意义，但这种可塑性的细胞和分子基础尚不清楚。 DCT 包括两个 子段，早期 DCT1 和晚期 DCT2。 Knepper 和同事最近使用单细胞转录组学 在 DCT1 中发现了一种罕见的增殖细胞群，他们认为这可能是造成 DCT独特的可塑性。我们实验室的初步数据表明，饮食中缺钾会增加 DCT 中这些增殖细胞的百分比并导致 DCT 肥大，尤其是沿 DCT1。 此外，这项工作表明增殖性 DCT 细胞类型的转运蛋白转录物水平较低 和更高水平的增殖转录本，表明 DCT1 细胞去分化为更具增殖性的细胞 状态。 Cullin-RING-连接酶 (CRL) 是 E3 泛素连接酶家族，可介导 蛋白质并参与许多细胞功能，对于细胞维持和消除不需要的细胞很重要 蛋白质。最近发现 CRL 通过蛋白酶体降解来调节血压 赖氨酸激酶（WNK）。 Cullin 3 (CUL3) 是 CRL 的关键组成部分，它添加了靶向的泛素部分 用于蛋白酶体降解的蛋白质。 CUL3 突变会导致 FHHt 疾病。所有 CRL 均受监管 COP9 信号体 (CSN)，它与 ​​CRL 相互作用并关闭泛素连接酶活性。疾病- 导致 CUL3 突变抑制 CUL3 与 CSN 相互作用的能力，从而使 CUL3 消失 过度活跃。为了研究受损的 CSN-CUL3 相互作用在人类疾病中的作用，我们灭活了 CSN 通过沿整个肾单位删除 Jab1（关键的 CSN 催化亚基）来实现。尽管事实上 CUL3 并且 CSN 沿着肾单位表达，这些小鼠 (KS-Jab1-/-) 仅沿着肾单位表现出重塑 远端肾单位，DCT 缩短，DCT1 特异性蛋白大量减少。在这里，我们计划测试一下 我们的假设是 CSN 功能障碍导致 DCT1 细胞去分化，从而导致 DCT 重塑 最近的技术进步和新的小鼠模型使我们能够研究蛋白质和 转录水平前所未有的详细。我们利用 DCT 的荧光激活核分选 (FANS) 特定的 INTACT（特定细胞类型中标记的细胞核的分离）报告小鼠，具有可诱导的核 GFP 表达极大丰富了 DCT 细胞。我们将其与单核 RNA 测序 (snRNA-seq) 配对 生成有关 DCT 细胞群的非常精细的数据。拟议的 Jab1-/- 实验 小鼠将揭示 DCT 的可塑性，有助于揭示 DCT 重塑的机制 CSN功能障碍所致。这将揭示可能具有临床和生物学意义的有价值的信息 在 DCT 重塑的情况下推进治疗的重要性。