Physiological and pathological functions of E3 ubiquitin ligases Smurfs

E3泛素连接酶Smurfs的生理和病理功能

• 批准号: 9153805
• 负责人: YING E Zhang
• 金额: $ 76.07万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9153805
• 关键词: Address; Adult; Age-Related Bone Loss; Alleles; Attenuated; Biological; Biological Assay; Breast Cancer Cell; Cell Culture Techniques; Cell Cycle Progression; Cell Nucleus; Cell physiology; Cells; Cytoplasm; Cytoplasmic Granules; DNA Repair; DNA biosynthesis; Embryo; Embryonic Development; Enzymes; Epigenetic Process; Epithelial; Erinaceidae; Excision; Family; Genome Stability; Homeostasis; In Vitro; Inflammatory Response; Knock-out; Knockout Mice; Literature; Malignant Neoplasms; Modification; Mono-S; Mus; Mutation; Neoplasm Metastasis; Neurons; Nude Mice; Null Lymphocytes; Osteoblasts; Osteoporosis; Pathway interactions; Physiological; Play; Process; Proteasome Inhibitor; Proteins; Regulation; Reporting; Research; Role; Signal Transduction; Staging; Substrate Specificity; System; Therapeutic Uses; Tissues; Transforming Growth Factor beta; Tumor Suppressor Proteins; Ubiquitin; Ubiquitin-Activating Enzymes; Ubiquitination; Work; base; bone; cell motility; chromatin remodeling; gamma-Glutamyl Hydrolase; histone modification; in vivo; malignant breast neoplasm; member; migration; mouse model; multicatalytic endopeptidase complex; novel; overexpression; protein degradation; recombinase; smoothened signaling pathway; sonic hedgehog receptor; success; tumor; tumor progression; tumorigenesis; ubiquitin-protein ligase

Smad ubiquitin regulatory factors (Smurfs) are members of the HECT family of E3 ubiquitin ligases. Two Smurfs, Smurf1 and Smurf2, were identified based on their activities to modulate TGF-beta/BMP signaling by promoting ubiquitin modification on Smads; however, subsequent studies have expanded the repertoire of Smurf substrates to include proteins such as RhoA, Runx2 and MEKK2 outside the TGF-beta/BMP pathway. To address the physiological function of Smurfs, we have generated mice lacking either Smurf1 or Smurf2. We found that these two paralogous E3 ubiquitin ligases have both common and unique functions during embryogenesis and in maintaining adult physiological homeostasis. Our earlier work on characterizing Smurf1 knockout mice revealed a novel mechanism in regulating osteoblast function and bone homeostasis, suggesting that targeting Smurf1 may prove to be an effective strategy for treating age-related bone losses in osteoporosis. Our more recent work on characterizing Smurf2 knockout mice clarified contradictory reports in the literature about Smurf regulation of TGF-beta signaling by showing that Smurf2 indeed has an inhibitory role, but it does so by attenuating Smad3 activity through mono-ubiquitination rather than promoting its degradation as previously reported. Because loss of both Smurfs leads to embryonic lethality, we have created a conditional Smurf2 knockout allele, which enabled us to investigate physiological functions of Smurfs by completely removal of all Smurf activities in desired tissues using tissue-specific expression of cre recombinase and obtain conditional Smurf null cells from late stages embryos or adult tissues. Using Smurf double knockout cells, we identified Smurfs as the E3 ligases that control endocytic turnover of Sonic hedgehog (Shh) receptor Patched1 via ubiquitin modification. We demonstrated that this regulation is crucial for the activation of Shh signaling both in cell culture assays and in sustaining the Shh-dependent proliferation of cerebellar granule neuron precursorsOur study of Smurf2 knockout mice also led to an unexpected discovery of a previously unrecognized function of Smurf2 as a tumor suppressor that normally maintains genomic stability by controlling epigenetic landscape of histone modifications. We found that Smurf2 interacts with a group of proteins that regulate DNA replication, chromatin remodeling and histone modification. We are currently validating these findings using in vitro and in vivo approaches and to investigate the biological relevance of these interactions in genomic stability and tumorigenesis. Consistent with the tumor suppresser function of Smurf2, we found that Smurf2 expression is decreased in the nucleus but increased in the cytoplasm of breast cancer cells. Smurf1 expression is also upregulated in the cytoplasm of the breast cancer cells. Overexpression of Smurf1 and Smurf2 promotes metastasis in nude mouse models and induces epithelial-to-mesenmchymal transition, migration, and invasion of breast cancer cells, suggesting that Smurfs play important roles in breast cancer progression. We are currently studying the mechanism underpinning the role of Smurf1 and Smurf2 promoting breast cancer cell migration and invasion.

Smad泛素调节因子（Smurfs）是E3泛素连接酶的HECT家族的成员。两个Smurf，Smurf 1和Smurf 2，基于它们通过促进Smads上的泛素修饰来调节TGF-β/BMP信号传导的活性而被鉴定;然而，随后的研究已经扩展了Smurf底物的库，以包括TGF-β/BMP通路之外的蛋白质，如RhoA，Runx 2和MEKK 2。为了解决蓝精灵的生理功能，我们已经产生了缺乏Smurf 1或Smurf 2的小鼠。我们发现，这两个旁系同源的E3泛素连接酶在胚胎发生和维持成人生理稳态的共同和独特的功能。我们早期对Smurf 1基因敲除小鼠的研究揭示了一种调节成骨细胞功能和骨稳态的新机制，这表明靶向Smurf 1可能被证明是治疗骨质疏松症中与年龄相关的骨丢失的有效策略。我们最近对Smurf 2基因敲除小鼠的研究澄清了文献中关于Smurf调节TGF-β信号传导的矛盾报道，表明Smurf 2确实具有抑制作用，但它是通过单泛素化减弱Smad 3活性而不是促进其降解来实现的。由于两个蓝精灵的缺失会导致胚胎死亡，我们创造了一个条件性Smurf 2敲除等位基因，这使我们能够通过使用cre重组酶的组织特异性表达完全去除所需组织中的所有蓝精灵活动来研究蓝精灵的生理功能，并获得条件性蓝精灵无效细胞来自晚期胚胎或成人组织。使用Smurf双敲除细胞，我们确定Smurfs作为E3连接酶，其通过泛素修饰控制Sonic hedgehog（Shh）受体Patched 1的内吞周转。我们证明了这种调节对于Shh信号的激活至关重要，无论是在细胞培养试验中还是在维持小脑颗粒神经元细胞的Shh依赖性增殖中。我们对Smurf 2敲除小鼠的研究还意外地发现了Smurf 2作为肿瘤抑制因子的先前未被认识的功能，该功能通常通过控制组蛋白修饰的表观遗传景观来维持基因组的稳定性。我们发现Smurf 2与一组调节DNA复制、染色质重塑和组蛋白修饰的蛋白质相互作用。我们目前正在验证这些发现，在体外和体内的方法，并调查这些相互作用在基因组稳定性和肿瘤发生的生物学相关性。与Smurf 2的肿瘤抑制功能一致，我们发现Smurf 2在乳腺癌细胞的细胞核中表达减少，但在细胞质中表达增加。Smurf 1表达在乳腺癌细胞的细胞质中也上调。Smurf 1和Smurf 2的过表达促进裸鼠模型中的转移，并诱导乳腺癌细胞的上皮-间充质转化、迁移和侵袭，表明Smurfs在乳腺癌进展中起重要作用。我们目前正在研究Smurf 1和Smurf 2促进乳腺癌细胞迁移和侵袭的机制。