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: 10133108
• 负责人: Abhishek Seth
• 金额: $ 34.33万
• 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 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10133108
• 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：尽管其患病率很高，但常见的泌尿生殖系统（GU）先天性缺陷的分子基础 是很难理解的。我们已经鉴定并验证了KCTD 13/16p11.2位点的基因损伤， 导致下尿路异常，特别是隐睾和尿道下裂。KCTD13 编码BCR（BTB-CUL 3-RBX 1）E3泛素-蛋白质连接酶复合物的底物特异性衔接子， 通过RHOA的泛素化和降解调节肌动蛋白细胞骨架和细胞迁移。特别是，RHOA 与支持细胞-生殖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 发展和分化。我们发现KCTD 13在GU束中强烈表达，最近已经发现KCTD 13在GU束中表达。 结果显示，与正常对照相比，基因拷贝数变异在患者中非常常见， 有先天缺陷此外，我们获得了体外和体内的证据表明，KCTD 13的损失影响AR水平， 在睾丸和阴茎中。我们还观察到，单倍不足和Kctd 13缺陷小鼠具有显著更高的 隐睾症的发生率和睾丸、精囊和阴茎大小的减小， 生精缺陷，导致严重的生育能力低下。此外，Kctd 13敲除小鼠显示， 雄性化因子SOX 9水平和伴随的雌性化因子RHOA的上调。虽然我们的 收集的数据表明，KCTD 13在男性GU发育中起着关键作用，这种分子如何 对AR和/或SOX 9轴的影响仍然是一个主要的知识空白。我们假设基因剂量 KCTD 13的变化改变了雄性化轴的信号传导，导致异常的GU束 发育、性腺形成缺陷、男性化不足和生育力低下。根据我们的推理，我们 将测试KCTD 13介导下GU道发育的两种合理的独立机制， 分化首先，我们假设和测试KCTD 13基因剂量的变化会影响GU道的发育 通过影响AR降解、亚细胞定位和下游基因靶点（AIM 1）。二是 假设并验证KCTD 13通过调节SOX 9的表达直接影响SOX 9的表达， SOX 9泛素化或间接通过调节RHOA降解，这反过来又导致睾丸缺陷， 阴茎发育和分化（AIM 2）。最后，我们鉴定并表征了KCTD 13- CUL 3途径，使得它们可用于产生用于GU的诊断试剂盒。 性发育障碍（AIM 3）。完成本建议中的研究将提前 我们对常见GU出生缺陷的分子机制的理解。