The E3 ubiquitin ligase SMURF2 stabilizes RNA editase ADAR1p110 and promotes its adenosine-to-inosine (A-to-I) editing function.

Epitranscriptomic changes in RNA catalyzed by the RNA-editing enzyme ADAR1 play an essential role in the regulation of diverse molecular and cellular processes, both under physiological conditions and in disease states, including cancer. Yet, despite a growing body of evidence pointing to ADAR1 as a potential therapeutic target, the mechanisms regulating its cellular abundance and activity, particularly of its constitutively expressed and ubiquitous form, ADAR1p110, are poorly understood. Here, we report the HECT-type E3 ubiquitin ligase SMURF2 as a pivotal regulator of ADAR1p110. We show that SMURF2, which is primarily known to promote the ubiquitin-mediated degradation of its protein substrates, protects ADAR1p110 from proteolysis and promotes its A-to-I editase activity in human and mouse cells and tissues. ADAR1p110's interactome analysis performed in human cells also showed a positive influence of SMURF2 on the stability and function of ADAR1p110. Mechanistically, we found that SMURF2 directly binds, ubiquitinates and stabilizes ADAR1p110 in an E3 ubiquitin ligase-dependent manner, through ADAR1p110 ubiquitination at lysine-744 (K744). Mutation of this residue to arginine (K744R), which is also associated with several human disorders, including dyschromatosis symmetrica hereditaria (DSH) and some types of cancer, abolished SMURF2-mediated protection of ADAR1p110 from both proteasomal and lysosomal degradation and inactivated ADAR1p110-mediated RNA editing. Our findings reveal a novel mechanism underlying the regulation of ADAR1 in mammalian cells and suggest SMURF2 as a key cellular factor influencing the protein abundance, interactions and functions of ADAR1p110.

Development and characterisation of SMURF2-targeting modifiers.

The C2-WW-HECT-domain E3 ubiquitin ligase SMURF2 emerges as an important regulator of diverse cellular processes. To date, SMURF2-specific modulators were not developed. Here, we generated and investigated a set of SMURF2-targeting synthetic peptides and peptidomimetics designed to stimulate SMURF2's autoubiquitination and turnover via a disruption of the inhibitory intramolecular interaction between its C2 and HECT domains. The results revealed the effects of these molecules both in vitro and in cellulo at the nanomolar concentration range. Moreover, the data showed that targeting of SMURF2 with either these modifiers or SMURF2-specific shRNAs could accelerate cell growth in a cell-context-dependent manner. Intriguingly, a concomitant cell treatment with a selected SMURF2-targeting compound and the DNA-damaging drug etoposide markedly increased the cytotoxicity produced by this drug in growing cells. Altogether, these findings demonstrate that SMURF2 can be druggable through its self-destructive autoubiquitination, and inactivation of SMURF2 might be used to affect cell sensitivity to certain anticancer drugs.

Generation of SMURF2 knockout human cells using the CRISPR/Cas9 system.

The HECT domain E3 ubiquitin ligase SMURF2 regulates stability of several key protein targets involved in tumorigenesis, cell proliferation, migration, differentiation, and senescence. While altered levels and aberrant cellular distribution of SMURF2 were reported in different types of cancer, its role in tumorigenesis is far from understood. To elucidate the role of SMURF2 in cancer, appropriate human cancer cell models are needed. Here, we describe approaches that can be used to generate human normal and cancer cell strains knocked-out for SMURF2 using the clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) gene-editing technology.

SMURF2 prevents detrimental changes to chromatin, protecting human dermal fibroblasts from chromosomal instability and tumorigenesis.

E3 ubiquitin ligases (E3s) play essential roles in the maintenance of tissue homeostasis under normal and stress conditions, as well as in disease states, particularly in cancer. However, the role of E3s in the initiation of human tumors is poorly understood. Previously, we reported that genetic ablation of the HECT-type E3 ubiquitin ligase Smurf2 induces carcinogenesis in mice; but whether and how these findings are pertinent to the inception of human cancer remain unknown. Here we show that SMURF2 is essential to protect human dermal fibroblasts (HDFs) from malignant transformation, and its depletion converts HDFs into tumorigenic entity. This phenomenon was associated with the radical changes in chromatin structural and epigenetic landscape, dysregulated gene expression and cell-cycle control, mesenchymal-to-epithelial transition and impaired DNA damage response. Furthermore, we show that SMURF2-mediated tumor suppression is interlinked with SMURF2's ability to regulate the expression of two central chromatin modifiers-an E3 ubiquitin ligase RNF20 and histone methyltransferase EZH2. Silencing these factors significantly reduced the growth and transformation capabilities of SMURF2-depleted cells. Finally, we demonstrate that SMURF2-compromised HDFs are highly tumorigenic in nude mice. These findings suggest the critical role that SMURF2 plays in preventing malignant alterations, chromosomal instability and cancer.

Altered Expression and Localization of Tumor Suppressive E3 Ubiquitin Ligase SMURF2 in Human Prostate and Breast Cancer.

SMURF2, an E3 ubiquitin ligase and suggested tumor suppressor, operates in normal cells to prevent genomic instability and carcinogenesis. However, the mechanisms underlying SMURF2 inactivation in human malignancies remain elusive, as SMURF2 is rarely found mutated or deleted in cancers. We hypothesized that SMURF2 might have a distinct molecular biodistribution in cancer versus normal cells and tissues. The expression and localization of SMURF2 were analyzed in 666 human normal and cancer tissues, with primary focus on prostate and breast tumors. These investigations were accompanied by SMURF2 gene expression analyses, subcellular fractionation and biochemical studies, including SMURF2's interactome analysis. We found that while in normal cells and tissues SMURF2 has a predominantly nuclear localization, in prostate and aggressive breast carcinomas SMURF2 shows a significantly increased cytoplasmic sequestration, associated with the disease progression. Mechanistic studies showed that the nuclear export machinery was not involved in cytoplasmic accumulation of SMURF2, while uncovered that its stability is markedly increased in the cytoplasmic compartment. Subsequent interactome analyses pointed to 14-3-3s as SMURF2 interactors, which could potentially affect its localization. These findings link the distorted expression of SMURF2 to human carcinogenesis and suggest the alterations in SMURF2 localization as a potential mechanism obliterating its tumor suppressor activities.

Smurf2 regulates stability and the autophagic-lysosomal turnover of lamin A and its disease-associated form progerin.

A-lamins, encoded by the LMNA gene, are major structural components of the nuclear lamina coordinating essential cellular processes. Mutations in the LMNA gene and/or alterations in its expression levels have been linked to a distinct subset of human disorders, collectively known as laminopathies, and to cancer. Mechanisms regulating A-lamins are mostly obscure. Here, we identified E3 ubiquitin ligase Smurf2 as a physiological regulator of lamin A and its disease-associated mutant form progerin (LAΔ50), whose expression underlies the development of Hutchinson-Gilford progeria syndrome (HGPS), a devastating premature aging syndrome. We show that Smurf2 directly binds, ubiquitinates, and negatively regulates the expression of lamin A and progerin in Smurf2 dose- and E3 ligase-dependent manners. Overexpression of catalytically active Smurf2 promotes the autophagic-lysosomal breakdown of lamin A and progerin, whereas Smurf2 depletion increases lamin A levels. Remarkably, acute overexpression of Smurf2 in progeria fibroblasts was able to significantly reduce the nuclear deformability. Furthermore, we demonstrate that the reciprocal relationship between Smurf2 and A-lamins is preserved in different types of mouse and human normal and cancer tissues. These findings establish Smurf2 as an essential regulator of lamin A and progerin and lay a foundation for evaluating the efficiency of progerin clearance by Smurf2 in HGPS, and targeting of the Smurf2-lamin A axis in age-related diseases such as cancer.