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Liquid–liquid phase separation has emerged as a crucial mechanism driving the formation of membraneless biomolecular condensates, which play important roles in numerous cellular processes. These condensates, found both in the nucleus and cytoplasm, are formed through multivalent, low-affinity interactions between various molecules. P62-containing condensates serve, among other functions, as proteolytic hubs for the ubiquitin–proteasome system. In this study, we investigated the dynamic interplay between nuclear p62 condensates and promyelocytic nuclear bodies (PML-NBs). We show that p62 condensates stabilize PML-NBs under both basal conditions and following exposure to arsenic trioxide which stimulates their degradation. We further show that this effect on the stability of PML-NBs is due to sequestration of their ubiquitin E3 ligase RNF4 in the p62 condensates with subsequent rapid degradation of the ligase. The sequestration of the ligase is made possible by association between the proline-rich domain of the PML protein and the PB1 domain of p62, which results in the formation of a PML-NB shell around the p62 condensates. Importantly, these hybrid structures do not undergo fusion and mixing of their contents which leaves unsolved the mechanism of sequestration of RNF4 in the condensates. These findings suggest an additional possible mechanism of PML-NB as a tumor suppressor which is mediated via interactions between different biomolecular condensates. [ABSTRACT FROM AUTHOR]

Various post-translational modifications (PTMs) collaboratively fine-tune protein activities. SUMO-targeted ubiquitin E3 ligases (STUbLs) emerge as specialized enzymes that recognize SUMO-modified substrates through SUMO-interaction motifs and subsequently ubiquitinate them via the RING domain, thereby bridging the SUMO and ubiquitin signaling pathways. STUbLs participate in a wide array of molecular processes, including cell cycle regulation, DNA repair, replication, and mitosis, operating under both normal conditions and in response to challenges such as genotoxic stress. Their ability to catalyze various types of ubiquitin chains results in diverse proteolytic and non-proteolytic outcomes for target substrates. Importantly, STUbLs are strategically positioned in close proximity to SUMO proteases and deubiquitinases (DUBs), ensuring precise and dynamic control over their target proteins. In this review, we provide insights into the unique properties and indispensable roles of STUbLs, with a particular emphasis on their significance in preserving genome integrity in humans.

DNA replication requires precise regulation achieved through post-translational modifications, including ubiquitination and SUMOylation. These modifications are linked by the SUMO-targeted E3 ubiquitin ligases (STUbLs). Ring finger protein 4 (RNF4), one of only two mammalian STUbLs, participates in double-strand break repair and resolving DNA–protein cross-links. However, its role in DNA replication has been poorly understood. Using CRISPR/Cas9 genetic screens, we discovered an unexpected dependency of RNF4 mutants on ubiquitin specific peptidase 7 (USP7) for survival in TP53-null retinal pigment epithelial cells. TP53−/–/RNF4−/–/USP7−/– triple knockout (TKO) cells displayed defects in DNA replication that cause genomic instability. These defects were exacerbated by the proteasome inhibitor bortezomib, which limited the nuclear ubiquitin pool. A shortage of free ubiquitin suppressed the ataxia telangiectasia and Rad3-related (ATR)-mediated checkpoint response, leading to increased cell death. In conclusion, RNF4 and USP7 work cooperatively to sustain a functional level of nuclear ubiquitin to maintain the integrity of the genome.

Abstract Background Sp1, an important transcription factor, is involved in the progression of various cancers. Our previous studies have indicated that Sp1 levels are increased in the early stage of lung cancer progression but decrease during the late stage, leading to poor prognosis. In addition, estrogen has been shown to be involved in lung cancer progression. According to previous studies, Sp1 can interact with the estrogen receptor (ER) to coregulate gene expression. The role of interaction between Sp1 and ER in lung cancer progression is still unknown and will be clarified in this study. Methods The clinical relevance between Sp1 levels and survival rates in young women with lung cancer was studied by immunohistochemistry. We validated the sex dependence of lung cancer progression in EGFRL858R-induced lung cancer mice. Wound healing assays, chamber assays and sphere formation assays in A549 cells, Taxol-induced drug-resistant A549 (A549-T24) and estradiol (E2)-treated A549 (E2-A549) cells were performed to investigate the roles of Taxol and E2 in lung cancer progression. Luciferase reporter assays, immunoblot and q-PCR were performed to evaluate the interaction between Sp1, microRNAs and CD44. Tail vein-injected xenograft experiments were performed to study lung metastasis. Samples obtained from lung cancer patients were used to study the mRNA level of CD44 by q-PCR and the protein levels of Sp1 and CD44 by immunoblot and immunohistochemistry. Results In this study, we found that Sp1 expression was decreased in premenopausal women with late-stage lung cancer, resulting in a poor prognosis. Tumor formation was more substantial in female EGFRL858R mice than in male mice and ovariectomized female mice, indicating that E2 might be involved in the poor prognosis of lung cancer. We herein report that Sp1 negatively regulates metastasis and cancer stemness in E2-A549 and A549-T24 cells. Furthermore, E2 increases the mRNA and protein levels of RING finger protein 4 (RNF4), which is the E3-ligase of Sp1, and thereby decreases Sp1 levels by promoting Sp1 degradation. Sp1 can be recruited to the promoter of miR-3194-5p, and positively regulate its expression. Furthermore, there was a strong inverse correlation between Sp1 and CD44 levels in clinical lung cancer specimens. Sp1 inhibited CD44 expression by increasing the expression of miR-3194-5p, miR-218-5p, miR-193-5p, miR-182-5p and miR-135-5p, ultimately resulting in lung cancer malignancy. Conclusion Premenopausal women with lung cancer and decreased Sp1 levels have a poor prognosis. E2 increases RNF4 expression to repress Sp1 levels in premenopausal women with lung cancer, thus decreasing the expression of several miRNAs that can target CD44 and ultimately leading to cancer malignancy.

Background: Sp1, an important transcription factor, is involved in the progression of various cancers. Our previous studies have indicated that Sp1 levels are increased in the early stage of lung cancer progression but decrease during the late stage, leading to poor prognosis. In addition, estrogen has been shown to be involved in lung cancer progression. According to previous studies, Sp1 can interact with the estrogen receptor (ER) to coregulate gene expression. The role of interaction between Sp1 and ER in lung cancer progression is still unknown and will be clarified in this study. Methods: The clinical relevance between Sp1 levels and survival rates in young women with lung cancer was studied by immunohistochemistry. We validated the sex dependence of lung cancer progression in EGFRL858R-induced lung cancer mice. Wound healing assays, chamber assays and sphere formation assays in A549 cells, Taxol-induced drug-resistant A549 (A549-T24) and estradiol (E2)-treated A549 (E2-A549) cells were performed to investigate the roles of Taxol and E2 in lung cancer progression. Luciferase reporter assays, immunoblot and q-PCR were performed to evaluate the interaction between Sp1, microRNAs and CD44. Tail vein-injected xenograft experiments were performed to study lung metastasis. Samples obtained from lung cancer patients were used to study the mRNA level of CD44 by q-PCR and the protein levels of Sp1 and CD44 by immunoblot and immunohistochemistry. Results: In this study, we found that Sp1 expression was decreased in premenopausal women with late-stage lung cancer, resulting in a poor prognosis. Tumor formation was more substantial in female EGFRL858R mice than in male mice and ovariectomized female mice, indicating that E2 might be involved in the poor prognosis of lung cancer. We herein report that Sp1 negatively regulates metastasis and cancer stemness in E2-A549 and A549-T24 cells. Furthermore, E2 increases the mRNA and protein levels of RING finger protein 4 (RNF4), which is the E3-ligase of Sp1, and thereby decreases Sp1 levels by promoting Sp1 degradation. Sp1 can be recruited to the promoter of miR-3194-5p, and positively regulate its expression. Furthermore, there was a strong inverse correlation between Sp1 and CD44 levels in clinical lung cancer specimens. Sp1 inhibited CD44 expression by increasing the expression of miR-3194-5p, miR-218-5p, miR-193-5p, miR-182-5p and miR-135-5p, ultimately resulting in lung cancer malignancy. Conclusion: Premenopausal women with lung cancer and decreased Sp1 levels have a poor prognosis. E2 increases RNF4 expression to repress Sp1 levels in premenopausal women with lung cancer, thus decreasing the expression of several miRNAs that can target CD44 and ultimately leading to cancer malignancy. [ABSTRACT FROM AUTHOR]

Liquid-liquid phase separation has emerged as a crucial mechanism driving the formation of membraneless biomolecular condensates, which play important roles in numerous cellular processes. These condensates, found both in the nucleus and cytoplasm, are formed through multivalent, low-affinity interactions between various molecules. P62-containing condensates serve, among other functions, as proteolytic hubs for the ubiquitin-proteasome system. In this study, we investigated the dynamic interplay between nuclear p62 condensates and promyelocytic nuclear bodies (PML-NBs). We show that p62 condensates stabilize PML-NBs under both basal conditions and following exposure to arsenic trioxide which stimulates their degradation. We further show that this effect on the stability of PML-NBs is due to sequestration of their ubiquitin E3 ligase RNF4 in the p62 condensates with subsequent rapid degradation of the ligase. The sequestration of the ligase is made possible by association between the proline-rich domain of the PML protein and the PB1 domain of p62, which results in the formation of a PML-NB shell around the p62 condensates. Importantly, these hybrid structures do not undergo fusion and mixing of their contents which leaves unsolved the mechanism of sequestration of RNF4 in the condensates. These findings suggest an additional possible mechanism of PML-NB as a tumor suppressor which is mediated via interactions between different biomolecular condensates., Competing Interests: Competing interests statement:The authors declare no competing interest.

Acute promyelocytic leukemia (APL) is a hematological malignancy driven by the oncoprotein PML-RARα, which can be treated with arsenic trioxide (As2O3) or/and all-trans retinoic acid. The protein arginine methyltransferase 5 (PRMT5) is involved in tumorigenesis. However, little is known about the biological function and therapeutic potential of PRMT5 in APL. Here, we show that PRMT5 is highly expressed in APL patients. PRMT5 promotes APL by interacting with PML-RARα and suppressing its ubiquitination and degradation. Mechanistically, PRMT5 attenuates the interaction between PML-RARα and its ubiquitin E3 ligase RNF4 by methylating RNF4 at Arg164. Notably, As2O3 treatment triggers the dissociation of PRMT5 from PML nuclear bodies, attenuating RNF4 methylation and promoting RNF4-mediated PML-RARα ubiquitination and degradation. Moreover, knockdown of PRMT5 and pharmacological inhibition of PRMT5 with the specific inhibitor EPZ015666 significantly inhibit APL cells growth. The combination of EPZ015666 with As2O3 shows synergistic effects on As2O3-induced differentiation of bone marrow cells from APL mice, as well as on apoptosis and differentiation of primary APL cells from APL patients. These findings provide mechanistic insight into the function of PRMT5 in APL pathogenesis and demonstrate that inhibition of PRMT5, alone or in combination with As2O3, might be a promising therapeutic strategy against APL. [ABSTRACT FROM AUTHOR]

The post-translational modification of DNA repair and checkpoint proteins by ubiquitin and small ubiquitin-like modifier (SUMO) critically orchestrates the DNA damage response (DDR). The ubiquitin ligase RNF4 integrates signaling by SUMO and ubiquitin, through its selective recognition and ubiquitination of SUMO-modified proteins. Here, we define a key new determinant for target discrimination by RNF4, in addition to interaction with SUMO. We identify a nucleosome-targeting motif within the RNF4 RING domain that can bind DNA and thereby enables RNF4 to selectively ubiquitinate nucleosomal histones. Furthermore, RNF4 nucleosome-targeting is crucially required for the repair of TRF2-depleted dysfunctional telomeres by 53BP1-mediated non-homologous end joining.

Spatial organization of cellular processes in membranous or membrane-less organelles (MLOs, alias molecular condensates) is a key concept for compartmentalizing biochemical pathways. Prime examples of MLOs are the nucleolus, PML nuclear bodies, nuclear splicing speckles or cytosolic stress granules. They all represent distinct sub-cellular structures typically enriched in intrinsically disordered proteins and/or RNA and are formed in a process driven by liquid-liquid phase separation. Several MLOs are critically involved in proteostasis and their formation, disassembly and composition are highly sensitive to proteotoxic insults. Changes in the dynamics of MLOs are a major driver of cell dysfunction and disease. There is growing evidence that post-translational modifications are critically involved in controlling the dynamics and composition of MLOs and recent evidence supports an important role of the ubiquitin-like SUMO system in regulating both the assembly and disassembly of these structures. Here we will review our current understanding of SUMO function in MLO dynamics under both normal and pathological conditions.

The dynamic and reversible post-translational modification of proteins and protein complexes with the ubiquitin-related SUMO modifier regulates a wide variety of nuclear functions, such as transcription, replication and DNA repair. SUMO can be attached as a monomer to its targets, but can also form polymeric SUMO chains. While monoSUMOylation is generally involved in the assembly of protein complexes, multi- or polySUMOylation may have very different consequences. The evolutionary conserved paradigmatic signaling process initiated by multi- or polySUMOylation is the SUMO-targeted Ubiquitin ligase (StUbL) pathway, where the presence of multiple SUMO moieties primes ubiquitylation by the mammalian E3 ubiquitin ligases RNF4 or RNF111, or the yeast Slx5/8 heterodimer. The mammalian SUMO chain-specific isopeptidases SENP6 or SENP7, or yeast Ulp2, counterbalance chain formation thereby limiting StUbL activity. Many facets of SUMO chain signaling are still incompletely understood, mainly because only a limited number of polySUMOylated substrates have been identified. Here we summarize recent work that revealed a highly interconnected network of candidate polySUMO modified proteins functioning in DNA damage response and chromatin organization. Based on these datasets and published work on distinct polySUMO-regulated processes we discuss overarching concepts in SUMO chain function. We propose an evolutionary conserved role of polySUMOylation in orchestrating chromatin dynamics and genome stability networks by balancing chromatin-residency of protein complexes. This concept will be exemplified in processes, such as centromere/kinetochore organization, sister chromatid cohesion, DNA repair and replication.

In a previous study, we characterized Dictyostelium SUMO targeted ubiquitin ligase (StUbL) MIP1 that associates with protein kinase MEK1 and targets SUMOylated MEK1 to ubiquitination (Sobko et al., 2002). These modifications happen in response to activation of MEK1 by the chemoattractant cAMP. Second site genetic suppressor of mek1- null phenotype (SMEK) was also identified in Dictyostelium. MEK1 and SMEK belong to the same linear pathway, in which MEK1 negatively regulates SMEK, which then negatively regulates chemotaxis and aggregation. RNF4 is mammalian homologue of MIP RNF4 interacts with hSMEK2, the human homologue of Dictyostelium SMEK. We propose the existence of an evolutionarily conserved MEK1-SMEK signaling complex that upon MEK1 activation and SUMOylation, recruits ubiqutin ligase MIP1/RNF4, which, in turn, ubiquitinates SMEK and targets this protein for proteasomal degradation. This could be a mechanism for negative regulation of SMEK by MEK1 signaling. [ABSTRACT FROM AUTHOR]

RNF4, a poly‐SUMO‐specific E3 ubiquitin ligase, is associated with protein degradation, DNA damage repair and tumour progression. However, the effect of RNF4 in cardiomyocytes remains to be explored. Here, we identified the alteration of RNF4 from ischaemic hearts and oxidative stress‐induced apoptotic cardiomyocytes. Upon myocardial infarction (MI) or H2O2/ATO treatment, RNF4 increased rapidly and then decreased gradually. PML SUMOylation and PML nuclear body (PML‐NB) formation first enhanced and then degraded upon oxidative stress. Reactive oxygen species (ROS) inhibitor was able to attenuate the elevation of RNF4 expression and PML SUMOylation. PML overexpression and RNF4 knockdown by small interfering RNA (siRNA) enhanced PML SUMOylation, promoted p53 recruitment and activation and exacerbated H2O2/ATO‐induced cardiomyocyte apoptosis which could be partially reversed by knockdown of p53. In vivo, knockdown of endogenous RNF4 via in vivo adeno‐associated virus infection deteriorated post‐MI structure remodelling including more extensive interstitial fibrosis and severely fractured and disordered structure. Furthermore, knockdown of RNF4 worsened ischaemia‐induced cardiac dysfunction of MI models. Our results reveal a novel myocardial apoptosis regulation model that is composed of RNF4, PML and p53. The modulation of these proteins may provide a new approach to tackling cardiac ischaemia. [ABSTRACT FROM AUTHOR]

Eukaryotic genome stability is maintained by a complex and diverse set of molecular processes. One class of enzymes that promotes proper DNA repair, replication and cell cycle progression comprises small ubiquitin-like modifier (SUMO)-targeted E3 ligases, or STUbLs. Previously, we reported a role for the budding yeast STUbL synthetically lethal with sgs1 (Slx) 5/8 in preventing G 2 /M-phase arrest in a minichromosome maintenance protein 10 (Mcm10)-deficient model of replication stress. Here, we extend these studies to human cells, examining the requirement for the human STUbL RING finger protein 4 (RNF4) in MCM10 mutant cancer cells. We find that MCM10 and RNF4 independently promote origin firing but regulate DNA synthesis epistatically and, unlike in yeast, the negative genetic interaction between RNF4 and MCM10 causes cells to accumulate in G 1 -phase. When MCM10 is deficient, RNF4 prevents excessive DNA under-replication at hard-to-replicate regions that results in large DNA copy number alterations and severely reduced viability. Overall, our findings highlight that STUbLs participate in species-specific mechanisms to maintain genome stability, and that human RNF4 is required for origin activation in the presence of chronic replication stress. [Display omitted] • It is unknown if human MCM10 and RNF4 genetically interact like in budding yeast. • MCM10 / RNF4 genetic interaction causes severe replicative stress in HCT116 cells. • MCM10 and RNF4 separately promote origin firing but together control DNA synthesis. • RNF4 loss exacerbates large chromosomal changes driven by MCM10 deficiency. • STUbLs promote eukaryotic genome stability via organism specific mechanisms. [ABSTRACT FROM AUTHOR]

Perturbation of protein homeostasis and aggregation of misfolded proteins is a major cause of many human diseases. A hallmark of the neurodegenerative disease spinocerebellar ataxia type 7 (SCA7) is the intranuclear accumulation of mutant, misfolded ataxin-7 (polyQ-ATXN7). Here, we show that endogenous ATXN7 is modified by SUMO proteins, thus also suggesting a physiological role for this modification under conditions of proteotoxic stress caused by the accumulation of polyQ-ATXN7. Co-immunoprecipitation experiments, immunofluorescence microscopy and proximity ligation assays confirmed the colocalization and interaction of polyQ-ATXN7 with SUMO2 in cells. Moreover, upon inhibition of the proteasome, both endogenous SUMO2/3 and the RNF4 ubiquitin ligase surround large polyQ-ATXN7 intranuclear inclusions. Overexpression of RNF4 and/or SUMO2 significantly decreased levels of polyQ-ATXN7 and, upon proteasomal inhibition, led to a marked increase in the polyubiquitination of polyQ-ATXN7. This provides a mechanism for the clearance of polyQ-ATXN7 from affected cells that involves the recruitment of RNF4 by SUMO2/3-modified polyQ-ATXN7, thus leading to its ubiquitination and proteasomal degradation. In a SCA7 knock-in mouse model, we similarly observed colocalization of SUMO2/3 with polyQ-ATXN7 inclusions in the cerebellum and retina. Furthermore, we detected accumulation of SUMO2/3 high-molecular-mass species in the cerebellum of SCA7 knock-in mice, compared with their wild-type littermates, and changes in SUMO-related transcripts. Immunohistochemical analysis showed the accumulation of SUMO proteins and RNF4 in the cerebellum of SCA7 patients. Taken together, our results show that the SUMO pathway contributes to the clearance of aggregated ATXN7 and suggest that its deregulation might be associated with SCA7 disease progression.

Background/Aims: Promyelocytic leukemia (PML) protein is a tumor suppressor that fuses with retinoic acid receptor-α (PML-RARα) to contribute to the initiation of acute promyelocytic leukemia (APL). Arsenic trioxide (ATO) upregulates expression of TGF-β1, promoting collagen synthesis in osteoblasts, and ATO binds directly to PML to induce oligomerization, sumoylation, and ubiquitination. However, how ATO upregulates TGF-β1 expression is uncertain. Thus, we suggested that PML sumoylation is responsible for regulation of TGF-β1 protein expression. Methods: Kunming mice were treated with ATO, and osteoblasts were counted under scanning electron microscopy. Masson's staining was used to quantify collagen content. hFOB1.19 cells were transfected with siRNA against UBC9 or RNF4, and then treated with ATO or FBS. TGF-β1, PML expression, and sumoylation were quantified with Western blot, and collagen quantified via immunocytochemistry. Results: ATO enhanced osteoblast accumulation, collagen synthesis, and PML-NB formation in vivo. Knocking down UBC9 in hFOB1.19 cells inhibited ATO- and FBS-induced PML sumoylation, TGF-β1 expression, and collagen synthesis. Conversely, knocking down RNF4 enhanced ATO- and FBS-induced PML sumoylation, TGF-β1 expression, and collagen synthesis. Conclusion: These data suggest that PML sumoylation is required for ATO-induced collagen synthesis in osteoblasts.

ABSTRACT SUMOylation is recently found to function as a targeting signal for the degradation of substrates through the ubiquitin-proteasome system. RNF4 is the most studied human SUMO-targeted ubiquitin E3 ligase. However, the relationship between SUMO proteases, SENPs, and RNF4 remains obscure. There are limited examples of the SENP regulation of SUMO2/3-targeted proteolysis mediated by RNF4. The present study investigated the role of SENP3 in the global protein turnover related to SUMO2/3-targeted ubiquitination and focused in particular on the SENP3 regulation of the stability of Sp1. Our data demonstrated that SENP3 impaired the global ubiquitination profile and promoted the accumulation of many proteins. Sp1, a cancer-associated transcription factor, was among these proteins. SENP3 increased the level of Sp1 protein via antagonizing the SUMO2/3-targeted ubiquitination and the consequent proteasome-dependent degradation that was mediated by RNF4. De-conjugation of SUMO2/3 by SENP3 attenuated the interaction of Sp1 with RNF4. In gastric cancer cell lines and specimens derived from patients and nude mice, the level of Sp1 was generally increased in parallel to the level of SENP3. These results provided a new explanation for the enrichment of the Sp1 protein in various cancers, and revealed a regulation of SUMO2/3 conjugated proteins whose levels may be tightly controlled by SENP3 and RNF4.

Various post-translational modifications (PTMs) collaboratively fine-tune protein activities. SUMO-targeted ubiquitin E3 ligases (STUbLs) emerge as specialized enzymes that recognize SUMO-modified substrates through SUMO-interaction motifs and subsequently ubiquitinate them via the RING domain, thereby bridging the SUMO and ubiquitin signaling pathways. STUbLs participate in a wide array of molecular processes, including cell cycle regulation, DNA repair, replication, and mitosis, operating under both normal conditions and in response to challenges such as genotoxic stress. Their ability to catalyze various types of ubiquitin chains results in diverse proteolytic and non-proteolytic outcomes for target substrates. Importantly, STUbLs are strategically positioned in close proximity to SUMO proteases and deubiquitinases (DUBs), ensuring precise and dynamic control over their target proteins. In this review, we provide insights into the unique properties and indispensable roles of STUbLs, with a particular emphasis on their significance in preserving genome integrity in humans., Competing Interests: The authors declare no competing interests., (© 2023 The Authors.)

Human adenovirus (HAdV) E1B-55K is a multifunctional regulator of productive viral replication and oncogenic transformation in nonpermissive mammalian cells. These functions depend on E1B-55K's posttranslational modification with the SUMO protein and its binding to HAdV E4orf6. Both early viral proteins recruit specific host factors to form an E3 ubiquitin ligase complex that targets antiviral host substrates for proteasomal degradation. Recently, we reported that the PML-NBassociated factor Daxx represses efficient HAdV productive infection and is proteasomally degraded via a SUMO-E1B-55K-dependent, E4orf6-independent pathway, the details of which remained to be established. RNF4, a cellular SUMO-targeted ubiquitin ligase (STUbL), induces ubiquitinylation of specific SUMOylated proteins and plays an essential role during DNA repair. Here, we show that E1B-55K recruits RNF4 to the insoluble nuclear matrix fraction of the infected cell to support RNF4/Daxx association, promoting Daxx PTM and thus inhibiting this antiviral factor. Removing RNF4 from infected cells using RNA interference resulted in blocking the proper establishment of viral replication centers and significantly diminished viral gene expression. These results provide a model for how HAdV antagonize the antiviral host responses by exploiting the functional capacity of cellular STUbLs. Thus, RNF4 and its STUbL function represent a positive factor during lytic infection and a novel candidate for future therapeutic antiviral intervention strategies. IMPORTANCE Daxx is a PML-NB-associated transcription factor that was recently shown to repress efficient HAdV productive infection. To counteract this antiviral measurement during infection, Daxx is degraded via a novel pathway including viral E1B-55K and host proteasomes. This virus-mediated degradation is independent of the classical HAdV E3 ubiquitin ligase complex, which is essential during viral infection to target other host antiviral substrates. To maintain a productive viral life cycle, HAdV E1B-55K early viral protein inhibits the chromatin-remodeling factor Daxx in a SUMO-dependent manner. In addition, viral E1B-55K protein recruits the STUbL RNF4 and sequesters it into the insoluble fraction of the infected cell. E1B-55K promotes complex formation between RNF4- and E1B-55K-targeted Daxx protein, supporting Daxx posttranslational modification prior to functional inhibition. Hence, RNF4 represents a novel host factor that is beneficial for HAdV gene expression by supporting Daxx counteraction. In this regard, RNF4 and other STUbL proteins might represent novel targets for therapeutic intervention. [ABSTRACT FROM AUTHOR]

RNF4, a SUMO-targeted ubiquitin ligase, stabilizes a selected group of oncoproteins. It potentiates oncoprotein activity and serves as a positive feedback agonist of Wnt and Notch pathways. RNF4 is essential for cancer cell survival and its levels are elevated in human cancers, correlating with poor outcome in a subset of cancer patients.

Respiratory syncytial virus (RSV) is the most important cause of viral acute respiratory tract infections and hospitalizations in children, for which no vaccine or specific treatments are available. RSV causes airway mucosa inflammation and cellular oxidative damage by triggering production of reactive oxygen species and by inhibiting at the same time expression of antioxidant enzymes, via degradation of the transcription factor NF-E2-related factor 2 (NRF2). RSV infection induces NRF2 deacetylation, ubiquitination, and degradation through a proteasome-dependent pathway. Although degradation via KEAP1 is the most common mechanism, silencing KEAP1 expression did not rescue NRF2 levels during RSV infection. We found that RSV-induced NRF2 degradation occurs in an SUMO-specific E3 ubiquitin ligase - RING finger protein 4 (RNF4)-dependent manner. NRF2 is progressively SUMOylated in RSV infection and either blocking SUMOylation or silencing RNF4 expression rescued both NRF2 nuclear levels and transcriptional activity. RNF4 associates with promyelocytic leukemia – nuclear bodies (PML-NBs). RSV infection induces the expression of PML and PML-NBs formation in an interferon (INF)-dependent manner and also induces NRF2 – PMN-NBs association. Inhibition of PML-NB formation by blocking IFN pathway or silencing PML expression resulted in a significant reduction of RSV-associated NRF2 degradation and increased antioxidant enzyme expression, identifying the RNF4-PML pathway as a key regulator of antioxidant defenses in the course of viral infection. [ABSTRACT FROM AUTHOR]

SLX4, disabled in the Fanconi anemia group P, is a scaffolding protein that coordinates the action of structure-specific endonucleases and other proteins involved in the replication-coupled repair of DNA interstrand cross-links. Here, we show that SLX4 dimerization and SUMO-SIM interactions drive the assembly of SLX4 membraneless compartments in the nucleus called condensates. Super-resolution microscopy reveals that SLX4 forms chromatin-bound clusters of nanocondensates. We report that SLX4 compartmentalizes the SUMO-RNF4 signaling pathway. SENP6 and RNF4 regulate the assembly and disassembly of SLX4 condensates, respectively. SLX4 condensation per se triggers the selective modification of proteins by SUMO and ubiquitin. Specifically, SLX4 condensation induces ubiquitylation and chromatin extraction of topoisomerase 1 DNA-protein cross-links. SLX4 condensation also induces the nucleolytic degradation of newly replicated DNA. We propose that the compartmentalization of proteins by SLX4 through site-specific interactions ensures the spatiotemporal control of protein modifications and nucleolytic reactions during DNA repair. [Display omitted] • SLX4 assembles μm-sized globular clusters of nanocondensates on chromatin • Site-specific interactions yield SLX4 condensates that are regulated by SENP6 and RNF4 • SLX4 compartmentalizes and activates the SUMO/RNF4 pathway • SLX4 condensation triggers TOP1-DPCs chromatin extraction and nascent DNA degradation In this study, Alghoul et al. show that the Fanconi anemia protein SLX4 forms globular clusters of condensates on chromatin that compartmentalize DNA damage response proteins. Compartmentalization of the SUMO/RNF4 pathway by SLX4 triggers the extraction of topoisomerase 1-DNA-protein cross-links from chromatin and the degradation of nascent DNA. [ABSTRACT FROM AUTHOR]

DNA replication requires precise regulation achieved through post-translational modifications, including ubiquitination and SUMOylation. These modifications are linked by the SUMO-targeted E3 ubiquitin ligases (STUbLs). Ring finger protein 4 (RNF4), one of only two mammalian STUbLs, participates in double-strand break repair and resolving DNA-protein cross-links. However, its role in DNA replication has been poorly understood. Using CRISPR/Cas9 genetic screens, we discovered an unexpected dependency of RNF4 mutants on ubiquitin specific peptidase 7 ( USP7) for survival in TP53 -null retinal pigment epithelial cells. TP53 -/- /RNF4 -/- /USP7 -/- triple knockout (TKO) cells displayed defects in DNA replication that cause genomic instability. These defects were exacerbated by the proteasome inhibitor bortezomib, which limited the nuclear ubiquitin pool. A shortage of free ubiquitin suppressed the ataxia telangiectasia and Rad3-related (ATR)-mediated checkpoint response, leading to increased cell death. In conclusion, RNF4 and USP7 work cooperatively to sustain a functional level of nuclear ubiquitin to maintain the integrity of the genome.

The s mall u biquitin related mo difier SUMO regulates protein functions to maintain cell homeostasis. SUMO attachment is executed by the hierarchical action of E1, E2 and E3 enzymes of which E3 ligases ensure substrate specificity. We recently identified the ZNF451 family as novel class of SUMO2/3 specific E3 ligases and characterized their function in SUMO chain formation. The founding member, ZNF451 isoform1 (ZNF451-1) partially resides in PML bodies, nuclear structures organized by the promyelocytic leukemia gene product PML. As PML and diverse PML components are well known SUMO substrates the question arises whether ZNF451-1 is involved in their sumoylation. Here, we show that ZNF451-1 indeed functions as SUMO2/3 specific E3 ligase for PML and selected PML components in vitro . Mutational analysis indicates that substrate sumoylation employs an identical biochemical mechanism as we described for SUMO chain formation. In vivo , ZNF451-1 RNAi depletion leads to PML stabilization and an increased number of PML bodies. By contrast, PML degradation upon arsenic trioxide treatment is not ZNF451-1 dependent. Our data suggest a regulatory role of ZNF451-1 in fine-tuning physiological PML levels in a RNF4 cooperative manner in the mouse neuroblastoma N2a cell-line. [ABSTRACT FROM AUTHOR]

SUMOylation is recently found to function as a targeting signal for the degradation of substrates through the ubiquitin-proteasome system. RNF4 is the most studied human SUMO-targeted ubiquitin E3 ligase. However, the relationship between SUMO proteases, SENPs, and RNF4 remains obscure. There are limited examples of the SENP regulation of SUMO2/3-targeted proteolysis mediated by RNF4. The present study investigated the role of SENP3 in the global protein turnover related to SUMO2/3-targeted ubiquitination and focused in particular on the SENP3 regulation of the stability of Sp1. Our data demonstrated that SENP3 impaired the global ubiquitination profile and promoted the accumulation of many proteins. Sp1, a cancer-associated transcription factor, was among these proteins. SENP3 increased the level of Sp1 protein via antagonizing the SUMO2/3-targeted ubiquitination and the consequent proteasome-dependent degradation that was mediated by RNF4. De-conjugation of SUMO2/3 by SENP3 attenuated the interaction of Sp1 with RNF4. In gastric cancer cell lines and specimens derived from patients and nude mice, the level of Sp1 was generally increased in parallel to the level of SENP3. These results provided a new explanation for the enrichment of the Sp1 protein in various cancers, and revealed a regulation of SUMO2/3 conjugated proteins whose levels may be tightly controlled by SENP3 and RNF4. [ABSTRACT FROM AUTHOR]

Among the hallmarks of cholangiocarcinoma (CCA) progression and unresponsiveness to therapy is impaired ubiquitin-dependent degradation of nuclear tumor suppressor protein. In the previous stage, our research group found that as a key tumor suppressor, nuclear dysfunction of p27kip1 is closely related to chemotherapy resistance of CCA, but the specific mechanism is unclear. It was recently shown that p27kip1-driven tumors were strongly dependent on the SUMO pathway. RNF4, as the SUMO-targeted ubiquitin ligase (STUbL), identifies SUMOylated proteins as a substrate through sumo-interacting motifs (SIM) and causes its degradation via the ubiquitin proteasome pathway. Here we described that the expression of RNF4 was upregulated in CCA tissues and related to malignant features. Silencing RNF4 arrested human CCA cells at the G1 phase, which was associated with the upregulation of p27kip1 and the downregulation of its downstream cycle-related proteins. Silencing RNF4 inhibited cell proliferation and migration, increased cell apoptosis, and sensitized CCA cells to treatment of chemotherapeutic drugs in vitro. Immunofluorescence showed that p27kip1 and RNF4 were mainly co-located in the nucleus. Immunoprecipitation and Western blot showed that p27kip1 was a target protein for SUMOylation and high expression of RNF4 decreased the levels of nuclear p27kip1, enhanced the levels of ubiquitinated and SUMOylated p27kip1, indicating that RNF4 could regulate cell cycle progression via recognizing SUMOylated p27kip1 and facilitating its ubiquitination degradation. These data indicate that RNF4-mediated ubiquitination degradation of SUMOylated proteins is a novel regulatory mechanism of p27kip1 dysfunction and CCA tumorigenesis, which provides a potential option for therapeutic intervention of CCA. • Silenced RNF4 inhibits CCA cell proliferation, migration and increases cell apoptosis. • RNF4 accelerRNF4 accelerates the progression of CCA via facilitaing p27kip1 ubiquitination. • RNF4 knockdown increases chemosensitivity of CCA cells. [ABSTRACT FROM AUTHOR]

Aims: The invasive intramyocardial injection of mesenchymal stromal cells (MSCs) allows for limited repeat injections and shows poor therapeutic efficacy against ischemic heart failure. Intravenous injection is an alternative method because this route allows for repeated, noninvasive, and easy delivery. However, the lack of targeting of MSCs hinders the ability of these cells to accumulate in the ischemic area after intravenous injections. We investigated whether and how the overexpression of colony-stimulating factor 2 receptor beta subunit (CSF2RB) may regulate the cardiac homing of MSCs and their cardioprotective effects against ischemic heart failure. Methods and Results: Adult mice were subjected to myocardial ischemia/reperfusion (MI/R) or sham operations. We observed significantly higher CSF2 protein expression and secretion by the ischemic heart from 1 day to 2 weeks after MI/R. Mouse adipose tissue-derived MSCs (ADSCs) were infected with adenovirus harboring CSF2RB or control adenovirus. Enhanced green fluorescent protein (EGFP)-labeled ADSCs were intravenously injected into MI/R mice every three days for a total of 7 times. Compared with ADSCs infected with control adenovirus, intravenously delivered ADSCs overexpressing CSF2RB exhibited markedly increased cardiac homing. Histological analysis revealed that CSF2RB overexpression significantly enhanced the ADSC-mediated proangiogenic, antiapoptotic, and antifibrotic effects. More importantly, ADSCs overexpressing CSF2RB significantly increased the left ventricular ejection fraction and cardiac contractility/relaxation in MI/R mice. In vitro experiments demonstrated that CSF2RB overexpression increases the migratory capacity and reduces the hypoxia/reoxygenation-induced apoptosis of ADSCs. We identified STAT5 phosphorylation as the key mechanism underlying the effects of CSF2RB on promoting ADSC migration and inhibiting ADSC apoptosis. RNA sequencing followed by cause-effect analysis revealed that CSF2RB overexpression increases the expression of the ubiquitin ligase RNF4. Coimmunoprecipitation and coimmunostaining experiments showed that RNF4 binds to phosphorylated STAT5. RNF4 knockdown reduced STAT5 phosphorylation as well as the antiapoptotic and promigratory actions of ADSCs overexpressing CSF2RB. Conclusions: We demonstrate for the first time that CSF2RB overexpression optimizes the efficacy of intravenously delivered MSCs in the treatment of ischemic heart injury by increasing the response of the MSCs to a CSF2 gradient and CSF2RB-dependent STAT5/RNF4 activation., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Background/Aims: Promyelocytic leukemia (PML) protein is a tumor suppressor that fuses with retinoic acid receptor-α (PML-RARα) to contribute to the initiation of acute promyelocytic leukemia (APL). Arsenic trioxide (ATO) upregulates expression of TGF-β1, promoting collagen synthesis in osteoblasts, and ATO binds directly to PML to induce oligomerization, sumoylation, and ubiquitination. However, how ATO upregulates TGF-β1 expression is uncertain. Thus, we suggested that PML sumoylation is responsible for regulation of TGF-β1 protein expression. Methods: Kunming mice were treated with ATO, and osteoblasts were counted under scanning electron microscopy. Masson's staining was used to quantify collagen content. hFOB1.19 cells were transfected with siRNA against UBC9 or RNF4, and then treated with ATO or FBS. TGF-β1, PML expression, and sumoylation were quantified with Western blot, and collagen quantified via immunocytochemistry. Results: ATO enhanced osteoblast accumulation, collagen synthesis, and PML-NB formation in vivo. Knocking down UBC9 in hFOB1.19 cells inhibited ATO- and FBS-induced PML sumoylation, TGF-β1 expression, and collagen synthesis. Conversely, knocking down RNF4 enhanced ATO- and FBS-induced PML sumoylation, TGF-β1 expression, and collagen synthesis. Conclusion: These data suggest that PML sumoylation is required for ATO-induced collagen synthesis in osteoblasts. © 2015 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Most of NF-κB (nuclear factor kappa B) signaling molecules have various types of post-translational modifications. In this study, we focused on ubiquitination and designed a siRNA library including most ubiquitin-binding domains. With this library, we identified several candidate regulators of canonical NF-κB pathway, including RNF4. Overexpression of RNF4 impaired NF-κB activation in a dose-dependent manner, whereas RNF4 knockdown potentiated NF-κB activation. We showed that RNF4 interacts with the TAK1–TAB2–TAB3 complex, but not TAB1. Further, we found that RNF4 specifically down-regulated TAB2 through a lysosomal pathway, and knockdown of RNF4 impaired endogenous TAB2 degradation. Therefore, our findings will provide new insights into the negative regulation of NF-κB signaling. [ABSTRACT FROM AUTHOR]