Physiological and pathological functions of E3 ubiquitin ligases Smurfs

Smad泛素调节因子(SNURF)是E3泛素连接酶Hect家族的成员。根据它们通过促进Smads上的泛素修饰来调节转化生长因子-β/骨形态发生蛋白信号的活性，确定了两个蓝精灵，即SMurf1和SMurf2；然而，随后的研究扩大了SMurf底物的研究范围，将RhoA、Runx2和MEKK2等蛋白质包括在转化生长因子-β/骨形态发生蛋白途径之外。为了解决蓝精灵的生理功能，我们培育了缺乏蓝精灵1或蓝精灵2的小鼠。我们发现这两个相似的E3泛素连接酶在胚胎发育和维持成人生理平衡方面既有共同的功能，也有独特的功能。我们早期对S-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，提示靶向S-1可能被证明是治疗骨质疏松中年龄相关性骨丢失的有效策略。我们随后对SMurf2基因敲除小鼠的研究澄清了文献中关于SMurf调控转化生长因子-β信号的相互矛盾的报告，表明SMurf2确实具有抑制作用，但它是通过单一泛素化来减弱Smad3的活性，而不是像之前报道的那样促进其降解。利用小鼠皮肤伤口模型，我们最近发现SMurf2通过下调转化生长因子-β/Smad3信号影响皮肤伤口的炎症和胶原蛋白的处理。由于这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SMurf2基因敲除等位基因，它使我们能够通过组织特异性表达cre重组酶完全去除所需组织中的所有蓝精灵活性来研究蓝精灵的生理功能，并从晚期胚胎或成人组织中获得条件性SMurf零细胞。我们对SMurf2基因敲除小鼠的研究还意外地发现了SMurf2作为核内肿瘤抑制因子的一个以前未被认识的功能，通常需要通过调节DNA损伤修复和染色质压缩来维持基因组的稳定性。我们证明了RNF20是控制DNA损伤反应和组蛋白修饰表观遗传格局的SMurf2核靶点之一。为了进一步了解SMurf2是如何被招募到DNA损伤焦点来调节基因组完整性的，我们采用了蛋白质组学的方法来鉴定Etoposide诱导的SMurf2相互作用蛋白和与Flag标记的SMurf2重组的SMurf2-/-MEF中的磷酸化位点。这导致在ATM共识位点S384发现了S-MURF2的磷酸化。我们随后发现，SMurf2需要S384的磷酸化才能泛素化并降解RNF20，从而揭示了SMurf2 DNA修复活性的关键分子开关。与SMurf2的肿瘤抑制功能一致，我们发现SMurf2在乳腺癌细胞的细胞核中表达减少，但在细胞质中的表达增加。S-1的表达也在乳腺癌细胞的细胞质中上调。SMurf1和SMurf2的过表达促进了裸鼠模型的转移，并诱导了乳腺癌细胞向上皮间充质的转化、迁移和侵袭，提示SMurfs在乳腺癌的发展中发挥着重要作用。我们目前正在研究S-1和S-2促进癌细胞迁移和侵袭的作用机制。

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.