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: 10702494
• 负责人: YING E Zhang
• 金额: $ 94.15万
• 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/10702494
• 关键词: Address; Adult; Affect; Age-Related Bone Loss; Alleles; Attenuated; Breast Cancer Cell; Cell Cycle Progression; Cell Nucleus; Cell physiology; Chromatin; Collagen; Consensus; Cutaneous; Cytoplasm; DNA Damage; DNA Repair; Embryo; Embryonic Development; Enterobacteria phage P1 Cre recombinase; Enzymes; Epigenetic Process; Epithelial; Etoposide; Excision; Family; Genome Stability; Homeostasis; Inflammation; Inflammatory Response; Knock-out; Knockout Mice; Literature; Malignant Neoplasms; Modeling; Modification; Molecular; Monoubiquitination; Mus; Mutation; Neoplasm Metastasis; Nuclear; Nude Mice; Null Lymphocytes; Osteoblasts; Osteoporosis; Pathologic; Pathway interactions; Phosphorylation; Phosphorylation Site; Physiological; Play; Proteasome Inhibitor; Proteins; Proteomics; Regulation; Reporting; Research; Role; Signal Transduction; Site; Surgical incisions; System; Therapeutic Uses; Tissues; Transforming Growth Factor beta; Tumor Suppressor Proteins; Ubiquitin; Ubiquitination; Work; base; bone; breast cancer progression; cancer cell; cell motility; genome integrity; histone modification; member; migration; mouse model; multicatalytic endopeptidase complex; novel; overexpression; protein degradation; reconstitution; recruit; response; skin wound; success; tumor; tumor progression; 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 subsequent 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. Using a murine incisional cutaneous model, we recently showed that Smurf2 affects inflammation and collagen processing in cutaneous wounds by down-regulating TGF-beta/Smad3 signaling. 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. Our study of Smurf2 knockout mice also led to an unexpected discovery of a previously unrecognized function of Smurf2 as a tumor suppressor in the nucleus that is normally required to maintain genomic stability through regulation of DNA damage repair and chromatin compaction. We demonstrated that RNF20 is one of the Smurf2 nuclear targets in controlling DNA damage response and epigenetic landscape of histone modifications. To further understand how Smurf2 is recruited to DNA damage foci to regulate genomic integrity, we undertook a proteomic approach to identify Etoposide-induced Smurf2 interacting proteins and phosphorylation sites in Smurf2-/- MEFs reconstituted with Flag-tagged Smurf2. This led to the discovery of phosphorylation of Smurf2 at S384, an ATM consensus site. We subsequently showed that phosphorylation at S384 is required for Smurf2 to ubiquitinate and degrade RNF20, thus revealing a crucial molecular switch for the DNA repair activity of Smurf2. 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 cancer cell migration and invasion.

Smad泛素调节因子（Smurfs）是E3的HECT家族的成员 泛素连接酶。两个蓝精灵，蓝精灵1和蓝精灵2，根据他们的活动， 通过促进Smads上的泛素修饰来调节TGF-β/BMP信号传导;然而， 随后的研究扩大了Smurf底物的库， 如RhoA、Runx2和MEKK 2在TGF-β/BMP途径之外。以应对所述生理 为了研究蓝精灵的功能，我们已经产生了缺乏Smurf1或Smurf2的小鼠。我们发现 这两种旁系同源的E3泛素连接酶具有共同和独特的功能， 胚胎发生和维持成年生理稳态。我们早期的工作 描述Smurf1基因敲除小鼠揭示了一种调节成骨细胞的新机制 功能和骨稳态，这表明靶向Smurf1可能被证明是一种有效的治疗方法。 治疗骨质疏松症中年龄相关性骨丢失的策略。我们随后的工作 描述Smurf2敲除小鼠澄清了文献中关于 通过显示Smurf2确实具有抑制作用， 但它是通过单泛素化而不是促进Smad3的活性， 如前所述，其降解。使用小鼠切口皮肤模型，我们最近 显示Smurf2通过以下方式影响皮肤伤口中的炎症和胶原蛋白加工： 下调TGF-β/Smad3信号传导。因为失去两个蓝精灵会导致 致命性，我们已经创造了一个条件Smurf2敲除等位基因，这使我们能够 研究蓝精灵的生理功能，通过完全删除所有蓝精灵活动， 利用组织特异性表达的Cre重组酶， 来自晚期胚胎或成体组织的蓝精灵零细胞。我们对Smurf2基因敲除小鼠的研究 这也导致了一个意外的发现，即以前未被认识到的Smurf2作为肿瘤的功能。 细胞核中的抑制子，通常需要通过 调节DNA损伤修复和染色质致密化。我们证明了RNF20是 Smurf2核靶点在控制DNA损伤反应和表观遗传景观中的作用 组蛋白修饰为了进一步了解Smurf 2如何被招募到DNA损伤灶， 调节基因组的完整性，我们进行了蛋白质组学的方法来确定依托泊苷诱导的 Smurf2-/-MEFs中Smurf2相互作用蛋白和磷酸化位点 蓝精灵2号这导致了Smurf2在S384处磷酸化的发现， 共识网站我们随后发现，Smurf2需要S384的磷酸化， 泛素化并降解RNF20，从而揭示了DNA修复的关键分子开关 蓝精灵2号的活动。与Smurf2的肿瘤抑制功能一致，我们发现， Smurf2在乳腺癌细胞核中表达减少，但在细胞质中表达增加 细胞Smurf1表达在乳腺癌细胞的细胞质中也上调。 Smurf1和Smurf2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， 这表明蓝精灵在乳腺癌进展中起着重要作用。我们目前正在 研究Smurf1和Smurf2促进癌细胞增殖的机制 移民和入侵。