The Role of E3-Ubiquitin Ligase Complex in Genitourinary Tract Development and Function.

E3-泛素连接酶复合物在泌尿生殖道发育和功能中的作用。

• 批准号: 10349531
• 负责人: Abhishek Seth
• 金额: $ 20.66万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-25 至 2022-07-01
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10349531
• 关键词: 16p11.2; Actins; Affect; Androgen Receptor; Birth; CUL3 gene; Cell Line; Complex; Congenital Abnormality; Copy Number Polymorphism; Critical Pathways; Cryptorchidism; Cytoskeleton; Data; Defect; Development; Diagnosis; Diagnostic Reagent Kits; Disease; Essential Genes; Exhibits; Feminization; Gametogenesis; Gene Dosage; Gene Targeting; Genes; Genetic; Genital; Genitalia; Genitourinary system; Germ Cells; Goals; High Prevalence; Human; Hypospadias; In Vitro; Incidence; Individual; Intercellular Junctions; Knockout Mice; Knowledge; Lead; Lesion; Link; Maintenance; Male Genital Organs; Malignant Neoplasms; Masculine; Mediating; Molecular; Mus; Mutant Strains Mice; Mutation; Neurologic; Newborn Infant; Pathway interactions; Patients; Physiological; Play; Proteins; RBX1 gene; RHOA gene; Receptor Signaling; Recovery; Regulation; Risk; Role; Seminal Vesicles; Sex Behavior; Sex Differentiation; Signal Transduction; Testing; Testis; Tissues; Ubiquitination; Up-Regulation; Urinary tract; Urination; Virilism; cell motility; design; experimental study; gonad development; in vitro Model; in vivo; knock-down; mRNA Expression; male; mouse model; overexpression; penis; receptor expression; sertoli cell; sex development disorder; subfertility; tool; ubiquitin-protein ligase; urogenital tract

Summary: Despite their high prevalence, the molecular basis for common genitourinary (GU) congenital defects is poorly understood. We have identified and validated gene lesions at the KCTD13/16p11.2 locus as responsible for lower GU tract abnormalities, particularly cryptorchidism and hypospadias. KCTD13 encodes a substrate-specific adapter of a BCR (BTB-CUL3-RBX1) E3 ubiquitin-protein ligase complex, which regulates the actin cytoskeleton and cell migration via ubiquitination and degradation of RHOA. Notably, RHOA has been implicated in the maintenance of Sertoli-germ cell junctions to promote gametogenesis and gonads development. E3 ubiquitin ligases also regulate the androgen receptor (AR), which is key to male genitalia development and differentiation. We found that KCTD13 is robustly expressed in the GU tract and have recently shown that in comparison to normal controls, gene copy number variants are remarkably common in patients with GU birth defects. Moreover, we obtained in vitro and in vivo evidence that KCTD13 loss affects AR levels in testis and penis. We also observed that haploinsufficient and Kctd13 deficient mice had significantly higher incidence of cryptorchidism and decreased size of testes, seminal vesicles and penis size in conjunction with spermatogenic defects, causing severe subfertility. Further, Kctd13 null mice revealed a significant decrease in masculinization factor SOX9 levels and concomitant upregulation of the feminization factor RHOA. While our collective data suggest a key role of KCTD13 in male GU development, the mechanisms of how this molecule impinges upon the AR and/or SOX9 axes remain a major gap of knowledge. We hypothesize that gene dosage changes in KCTD13 alter the signaling of the masculinization axes, leading to abnormal GU tract development, defective gonad formation, undervirilization, and subfertility. In line with our reasoning, we will test two plausible independent mechanisms by which KCTD13 mediates lower GU tract development and differentiation. First, we hypothesize and test that gene dosage changes in KCTD13 affect GU tract development by affecting AR degradation, subcellular localization and downstream gene targets (AIM 1). Second, we hypothesize and test that KCTD13 affects the expression of masculinization factor SOX9 directly by modulating SOX9 ubiquitination or indirectly by regulating RHOA degradation, which in turn leads to defects in testis and penile development and differentiation (AIM 2). Finally, we identify and characterize the mutations in KCTD13- CUL3 pathway that are critical in GU development such that they could be used to generate a diagnostic kit for patients with disorders of sexual development (AIM3). Completion of the studies in this proposal will advance our understanding of the molecular mechanisms that underlie common GU birth defects.

摘要：尽管患病率很高，但普通泌尿生殖率（GU）先天性缺陷的分子基础 理解很差。我们已经在KCTD13/16P11.2基因座确定并验证了基因病变是 负责较低的GU道异常，尤其是隐齿术和催眠术。 KCTD13 编码BCR（BTB-CUL3-RBX1）E3泛素蛋白 - 蛋白连接酶复合物的底物特异性适配器，该适配器 通过RhoA的泛素化和降解来调节肌动蛋白细胞骨架和细胞迁移。值得注意的是，Rhoa 与维持Sertoli-Germ细胞连接的维持有关，以促进配子发生和性腺 发展。 E3泛素连接酶还调节雄激素受体（AR），这是男性生殖器的关键 发展与分化。我们发现KCTD13在GU道中强烈表达，最近有 表明与正常对照相比，基因拷贝数变异在患者中非常普遍 与GU先生缺陷。此外，我们获得了体外和体内证据，表明KCTD13损失会影响AR水平 在睾丸和阴茎中。我们还观察到，单倍弹性和KCTD13缺乏小鼠的小鼠明显更高 隐式术和睾丸大小，精确囊泡和阴茎大小的发生率与与之结合 精子缺陷，导致严重的亚生物。此外，KCTD13无效小鼠显示出显着下降 男性化因子SOX9水平和女性化因子RhoA的上调。而我们的 集体数据表明，KCTD13在男性GU发育中的关键作用，该分子的机制如何 撞击AR和/或Sox9轴仍然是知识的主要差距。我们假设基因剂量 KCTD13的变化改变了男性化轴的信号传导，导致异常GU道 发育，性腺形成缺陷，缺乏效率和不良性。与我们的推理一致，我们 将测试KCTD13介导较低的GU道发展和 分化。首先，我们假设并测试KCTD13的基因剂量变化会影响GU道的发展 通过影响AR降解，亚细胞定位和下游基因靶标（AIM 1）。第二，我们 假设并测试KCTD13直接通过调节直接影响男性化因子SOX9的表达 Sox9泛素化或间接调节RhoA降解，这又导致睾丸和 阴茎发展和差异化（AIM 2）。最后，我们确定并表征了KCTD13-中的突变。 CUL3途径在GU开发中至关重要，因此可以用来生成诊断套件 性发育障碍的患者（AIM3）。该提案中的研究完成 我们对基于GU先生缺陷的分子机制的理解。