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（蓝精灵）是E3泛素连接酶家族的成员。根据它们的活动，通过促进SMADS上的泛素修饰来调节TGF-beta/bmp信号传导的活动，以调节TGF-beta/bmp信号传导，确定了两个蓝精灵。但是，随后的研究将SMURF底物的曲目扩展到了TGF-BETA/BMP途径之外的蛋白质，例如RhoA，Runx2和Mekk2。为了解决蓝精灵的生理功能，我们产生了缺乏蓝精神或蓝精神的小鼠。我们发现，这两个副E3泛素连接酶在胚胎发生和维持成人生理稳态时具有常见和独特的功能。我们较早的表征SMURF1敲除小鼠的工作揭示了调节成骨细胞功能和骨稳态方面的一种新型机制，这表明靶向SMURF1可能被证明是治疗骨质疏松症中与年龄相关的骨骼损失的有效策略。我们随后在表征SMURF2敲除小鼠的过程中阐明了文献中有关SMURF调节TGF-beta信号传导的矛盾报告，这表明SMURF2确实具有抑制作用，但通过单次泛素化而不是像以前报道的那样促进其降级来减弱SMAD3活性。我们最近使用鼠切开的皮肤模型，最近表明SMURF2通过下调TGF-BETA/SMAD3信号传导来影响皮肤伤口的炎症和胶原蛋白。由于两种蓝精灵的丧失都会导致胚胎致死性，因此我们创建了一个有条件的蓝精灵敲除等位基因，这使我们能够通过使用CRE重组酶的组织特异性表达在所需的组织中完全清除所有蓝精灵活性来研究蓝精灵的生理功能，并获得有条件的SMURF SMURF NULL细胞，并从后期或成人植物中获得有条件的SMURF Null belles embryos。我们对SMURF2基因敲除小鼠的研究还导致了意外发现SMURF2作为原子核中肿瘤抑制剂的先前未识别功能，通常需要通过调节DNA损伤修复和染色质压实来维持基因组稳定性。我们证明了RNF20是控制组蛋白修饰的DNA损伤反应和表观遗传景观的SMURF2核靶标之一。为了进一步了解如何募集SMURF2以调节基因组完整性，我们采用了一种蛋白质组学方法来识别依托泊苷诱导的SMURF2相互作用的蛋白质和Smurf2 - / - MEF中的磷酸化位点，并用Flag-tagged Smurf2重新构成了蛋白质。这导致在ATM共识部位S384上发现了Smurf2的磷酸化。随后，我们表明，Smurf2需要在S384处进行磷酸化以泛素化和降解RNF20，从而揭示了SMURF2 DNA修复活性的关键分子转换。与SMURF2的肿瘤抑制剂功能一致，我们发现细胞核中的Smurf2表达降低，但在乳腺癌细胞的细胞质中增加。 SMURF1表达在乳腺癌细胞的细胞质中也被上调。 Smurf1和Smurf2的过表达促进了裸小鼠模型中的转移，并诱导上皮到中性 - 肾小管转变，迁移和乳腺癌细胞的入侵，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作用的机制，促进了癌细胞迁移和侵袭。