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.

SMURF2-mediated ubiquitin signaling plays an essential role in the regulation of PARP1 PARylating activity, molecular interactions, and functions in mammalian cells.

Poly(ADP-ribose) polymerase 1 (PARP1) is a key molecular stress sensor and response mediator implicated in multiple cellular functions in health and diseases. Despite its importance and intrinsic involvement in pivotal molecular and cellular processes, including DNA repair, transcription regulation, chromatin organization, and cell death, the regulatory mechanisms of PARP1 are poorly understood. In this study, we show that SMURF2, a HECT-type E3 ubiquitin ligase and suggested tumor suppressor, physically interacts with PARP1 in different cellular settings, directly ubiquitinates it in vitro and stimulates its PARylation activity in cells, the phenomenon that required SMURF2 E3 ubiquitin ligase function. Intriguingly, in the cellular environment SMURF2 was found to regulate the dynamic exchange of ubiquitin moieties on PARP1, mostly decreasing its monoubiquitination. Through the set of systematic mass spectrometry analyses conducted on SMURF2-modified cells, we identified on PARP1 18 lysine residues (out of 126 present in PARP1) as sites which ubiquitination was considerably affected by SMURF2. Subsequent site-directed mutagenesis coupled with in cellula ubiquitination and PARylation assays unveiled K222 as a critical site enabling a cross talk between SMURF2-modulated monoubiquitination of PARP1 and its activity, and pointed to K498, S507, and a KTR triad (K498/K521/K524) as the main auto-PARylation sites affected by SMURF2. The results also uncovered that SMURF2 controls PARP1 interactome, influencing its functions and expression in a context-dependent manner. Taken together, these findings suggest that SMURF2-mediated ubiquitin signaling plays an essential role in PARP1 regulation, beyond the regulation of its protein expression.

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.

Molecular functions of NEDD4 E3 ubiquitin ligases in cancer.

The initiation, progression and cure of cancer rely heavily on altered gene expression and posttranslational functions. Protein ubiquitination is a major mechanism for posttranslational reorganization of the genome. This evolutionary conserved cascade, through regulation of protein stability, distribution, and function, governs nearly every biological process in the cell. E3 ubiquitin ligases are pivotal components of the ubiquitination pathway. Genetic alterations, abnormal expression, and dysfunctions of E3s have been implicated in the pathogenesis of a wide spectrum of human malignancies. In this review, we summarize and discuss recent discoveries on the roles of NEDD4 E3s in cancer. Over the past decade, members of this family have increasingly surfaced as fundamental components and critical regulators of molecular pathways central to the pathogenesis and cure of the disease.

Functional analysis of protein ubiquitination.

Alterations in the protein ubiquitination can lead to the development of serious pathological conditions and diseases and, therefore, are under extensive investigation. Here we detail the revised/updated version of two approaches for analyzing the functional activities of the ubiquitin transferring system and target protein ubiquitination. These approaches permit the analysis of protein ubiquitination within the cellular environment as well as in a tube when the purified components are used. The updates introduced in the protocols allow both to increase the sensitivity of the assays and to reduce the false positives often experienced in the analyses.

The Emerging Role of E3 Ubiquitin Ligase SMURF2 in the Regulation of Transcriptional Co-Repressor KAP1 in Untransformed and Cancer Cells and Tissues.

KAP1 is an essential nuclear factor acting as a scaffold for protein complexes repressing transcription. KAP1 plays fundamental role in normal and cancer cell biology, affecting cell proliferation, DNA damage response, genome integrity maintenance, migration and invasion, as well as anti-viral and immune response. Despite the foregoing, the mechanisms regulating KAP1 cellular abundance are poorly understood. In this study, we identified the E3 ubiquitin ligase SMURF2 as an important regulator of KAP1. We show that SMURF2 directly interacts with KAP1 and ubiquitinates it in vitro and in the cellular environment in a catalytically-dependent manner. Interestingly, while in the examined untransformed cells, SMURF2 mostly exerted a negative impact on KAP1 expression, a phenomenon that was also monitored in certain Smurf2-ablated mouse tissues, in tumor cells SMURF2 stabilized KAP1. This stabilization relied on the unaltered E3 ubiquitin ligase function of SMURF2. Further investigations showed that SMURF2 regulates KAP1 post-translationally, interfering with its proteasomal degradation. The conducted immunohistochemical studies showed that the reciprocal relationship between the expression of SMURF2 and KAP1 also exists in human normal and breast cancer tissues and suggested that this relationship may be disrupted by the carcinogenic process. Finally, through stratifying KAP1 interactome in cells expressing either SMURF2 wild-type or its E3 ligase-dead form, we demonstrate that SMURF2 has a profound impact on KAP1 protein-protein interactions and the associated functions, adding an additional layer in the SMURF2-mediated regulation of KAP1. Cumulatively, these findings uncover SMURF2 as a novel regulator of KAP1, governing its protein expression, interactions, and functions.

Smurfs in Protein Homeostasis, Signaling, and Cancer.

Protein ubiquitination is an evolutionary conserved highly-orchestrated enzymatic cascade essential for normal cellular functions and homeostasis maintenance. This pathway relies on a defined set of cellular enzymes, among them, substrate-specific E3 ubiquitin ligases (E3s). These ligases are the most critical players, as they define the spatiotemporal nature of ubiquitination and confer specificity to this cascade. Smurf1 and Smurf2 (Smurfs) are the C2-WW-HECT-domain E3 ubiquitin ligases, which recently emerged as important determinants of pivotal cellular processes. These processes include cell proliferation and differentiation, chromatin organization and dynamics, DNA damage response and genomic integrity maintenance, gene expression, cell stemness, migration, and invasion. All these processes are intimately connected and profoundly altered in cancer. Initially, Smurf proteins were identified as negative regulators of the bone morphogenetic protein (BMP) and the transforming growth factor beta (TGF-ß) signaling pathways. However, recent studies have extended the scope of Smurfs' biological functions beyond the BMP/TGF-ß signaling regulation. Here, we provide a critical literature overview and updates on the regulatory roles of Smurfs in molecular and cell biology, with an emphasis on cancer. We also highlight the studies demonstrating the impact of Smurf proteins on tumor cell sensitivity to anticancer therapies. Further in-depth analyses of Smurfs' biological functions and influences on molecular pathways could provide novel therapeutic targets and paradigms for cancer diagnosis and treatment.

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.

Smurf2-Mediated Stabilization of DNA Topoisomerase IIα Controls Genomic Integrity.

DNA topoisomerase IIα (Topo IIα) ensures genomic integrity and unaltered chromosome inheritance and serves as a major target of several anticancer drugs. Topo IIα function is well understood, but how its expression is regulated remains unclear. Here, we identify the E3 ubiquitin ligase Smurf2 as a physiologic regulator of Topo IIα levels. Smurf2 physically interacted with Topo IIα and modified its ubiquitination status to protect Topo IIα from the proteasomal degradation in dose- and catalytically dependent manners. Smurf2-depleted cells exhibited a reduced ability to resolve DNA catenanes and pathological chromatin bridges formed during mitosis, a trait of Topo IIα-deficient cells and a hallmark of chromosome instability. Introducing Topo IIα into Smurf2-depleted cells rescued this phenomenon. Smurf2 was a determinant of Topo IIα protein levels in normal and cancer cells and tissues, and its levels affected cell sensitivity to the Topo II-targeting drug etoposide. Our results identified Smurf2 as an essential regulator of Topo IIα, providing novel insights into its control and into the suggested tumor-suppressor functions of Smurf2. Cancer Res; 77(16); 4217-27. ©2017 AACR.