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: 10641191
• 负责人: Abhishek Seth
• 金额: $ 13.66万
• 依托单位: NEMOURS CHILDREN'S HOSPITAL, ORLANDO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-02 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10641191
• 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：尽管发病率很高，但常见的泌尿生殖系统(GU)先天缺陷的分子基础 人们对此知之甚少。我们已经确定并验证了KCTD13/16p11.2基因座的基因损伤为 引起下尿路异常，特别是隐睾症和尿道下裂。KCTD13 编码BCR(BTB-CUL3-RBX1)E3泛素-蛋白连接酶复合体的底物特异性接头，该接头 通过泛素化和RHOA的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 与维持支持-生殖细胞连接以促进配子发生和性腺有关 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 发展与差异化。我们发现KCTD13在胃肠道中有很强的表达，并且最近 研究表明，与正常对照组相比，基因拷贝数变异在患者中非常常见 有古氏先天缺陷。此外，我们在体外和体内获得了KCTD13缺失影响AR水平的证据 在睾丸和阴茎中。我们还观察到单倍体不足和KCTD13缺陷的小鼠显著更高 隐睾症的发生率和睾丸、精囊和阴茎大小的减少与 生精缺陷，导致严重的不育症。此外，KCTD13基因缺失的小鼠显示出显著的减少 男性化因子SOX9水平和伴随的女性化因子RHOA的上调。而我们的 集体数据表明KCTD13在男性GU发育中起着关键作用，该分子如何 对AR和/或SOX9轴的冲击仍然是知识的一个主要缺口。我们假设基因剂量 KCTD13基因的改变改变了雄化轴的信号传递，导致胃束异常 发育、性腺形成缺陷、性功能低下和不育。根据我们的推理，我们 将测试两种看似独立的机制，KCTD13通过这两种机制介导下消化道发育和 差异化。首先，我们假设并检验了KCTD13基因剂量的变化对胃肠道发育的影响。 通过影响AR降解、亚细胞定位和下游基因靶点(AIM 1)。第二，我们 KCTD13通过调控直接影响男性化因子SOX9表达的假设与检验 Sox9泛素化或间接调节RHOA降解，进而导致睾丸和 阴茎发育和分化(AIM 2)。最后，我们鉴定和表征了KCTD13-的突变。 CUL3途径在GU发育中是关键的，因此它们可以用来产生一种诊断试剂盒 性发育障碍患者(AIM3)。这项建议中的研究工作将提前完成 我们对常见GU出生缺陷的分子机制的理解。