SUMO-interacting Motif Research Articles

Stepwise phosphorylation and SUMOylation of PIDD1 drive PIDDosome assembly in response to DNA repair failure

SUMOylation regulates numerous cellular stress responses, yet targets in the apoptotic machinery remain elusive. We show that a single, DNA damage-induced monoSUMOylation event controls PIDDosome (PIDD1/RAIDD/caspase-2) formation and apoptotic death in response to unresolved DNA interstrand crosslinks (ICLs). SUMO-1 conjugation occurs on conserved K879 in the PIDD1 death domain (DD); is catalyzed by PIAS1 and countered by SENP3; and is triggered by ATR phosphorylation of neighboring T788 in the PIDD1 DD, which enables PIAS1 docking. Phospho/SUMO-PIDD1 proteins are captured by nucleolar RAIDD monomers via a SUMO-interacting motif (SIM) in the RAIDD DD, thus compartmentalizing nascent PIDDosomes for caspase-2 recruitment. Denying SUMOylation or the SUMO-SIM interaction spares the onset of PIDDosome assembly but blocks its completion, thus eliminating the apoptotic response to ICL repair failure. Conversely, removal of SENP3 forces apoptosis, even in cells with tolerable ICL levels. SUMO-mediated PIDDosome control is also seen in response to DNA breaks but not supernumerary centrosomes. These results illuminate PIDDosome formation in space and time and identify a direct role for SUMOylation in the assembly of a major pro-apoptotic device.

Sumoylation of thymine DNA glycosylase impairs productive binding to substrate sites in DNA.

The base excision repair enzyme thymine DNA glycosylase (TDG) protects against mutations by removing thymine or uracil from guanine mispairs and functions in active DNA demethylation by excising 5-formylcytosine (fC) and 5-carboxylcytosine (caC). Post-translational modification of TDG by SUMO (small ubiquitin-like modifier) reduces its glycosylase activity but the mechanism remains unclear. We investigated this problem using biochemical and biophysical approaches and a TDG construct comprising residues 82-340 (of 410) that includes the SUMOylation site and the motif for non-covalent SUMO binding. Single turnover kinetics experiments were collected at multiple enzyme concentrations ([E]) and the hyperbolic dependence of activity (kobs) on [E] yielded the maximal glycosylase activity (kmax), the enzyme concentration giving half-maximal activity (K0.5), and the catalytic efficiency (kmax/K0.5). Sumoylation of TDG (or TDG82-340) causes large reductions in catalytic efficiency for G·T, G·U, G·fC, and G·caC DNA substrates, due largely to weakened substrate affinity (increased K0.5). 19F NMR experiments show that sumoylation of TDG82-340 reduces productive binding to G·U mispairs and dramatically impairs binding to G·T mispairs. A mutation in the TDG SUMO-interacting motif (SIM), E310Q, shown previously to perturb noncovalent binding of SUMO to unmodified TDG, rescues the glycosylase activity of sumoylated TDG82-340. Similarly, NMR studies show the mutation restores productive binding of sumoylated TDG82-340 to G·U and G·T pairs. Together, the results indicate that intramolecular SUMO-SIM interactions mediate the adverse effect of sumoylation on TDG activity and suggest a model whereby the disruption of SUMO-SIM interactions enables productive binding of sumoylated TDG to substrate sites in DNA.

An Atypical Mechanism of SUMOylation of Neurofibromin SecPH Domain Provides New Insights into SUMOylation Site Selection

Neurofibromin (Nf1) is a giant multidomain protein encoded by the tumour-suppressor gene NF1. NF1 is mutated in a common genetic disease, neurofibromatosis type I (NF1), and in various cancers. The protein has a Ras-GAP (GTPase activating protein) activity but is also connected to diverse signalling pathways through its SecPH domain, which interacts with lipids and different protein partners. We previously showed that Nf1 partially colocalized with the ProMyelocytic Leukemia (PML) protein in PML nuclear bodies, hotspots of SUMOylation, thereby suggesting the potential SUMOylation of Nf1. Here, we demonstrate that the full-length isoform 2 and a SecPH fragment of Nf1 are substrates of the SUMO pathway and identify a well-defined SUMOylation profile of SecPH with two main modified lysines. One of these sites, K1731, is highly conserved and surface-exposed. Despite the presence of an inverted SUMO consensus motif surrounding K1731, and a potential SUMO-interacting motif (SIM) within SecPH, we show that neither of these elements is necessary for K1731 SUMOylation, which is also independent of Ubc9 SUMOylation on K14. A 3D model of an interaction between SecPH and Ubc9 centred on K1731, combined with site-directed mutagenesis, identifies specific structural elements of SecPH required for K1731 SUMOylation, some of which are affected in reported NF1 pathogenic variants. This work provides a new example of SUMOylation dependent on the tertiary rather than primary protein structure surrounding the modified site, expanding our knowledge of mechanisms governing SUMOylation site selection.

Characterization and functional analysis of chicken promyelocytic leukemia protein

In mammals, promyelocytic leukemia (PML) protein, also named as TRIM19, is the key component of nuclear membrane-less sub structures PML nuclear bodies (PML-NBs) or nuclear domains 10 (ND10). PML-NBs are dynamic foci that consist of numerous permanently or transiently associated proteins. The mammalian PMLs are involved in the regulation of various cellular pathways, including apoptosis, intrinsic and innate antiviral immunity, cell cycle, DNA damage, senescence and etc. Nevertheless, little is known about the role of chicken PML (chPML). In this study, chPML gene was cloned, and its several functions were characterized. We found that chPML was widely expressed in different tissues of chickens, and showed different subcellular distribution pattern in DF-1 cells comparing with LMH and HD11 cells. Like human PML, chPML was identified to be SUMOylated. K463 is one critical SUMOylation site and 240RARRG244 is SUMO interaction motif (SIM) of chPML. Moreover, qPCR showed that chPML could not only up-regulate the expression of host innate immune factor IFN-β and its downstream ISGs, but also antigen presentation-related factors including class II transactivator (CIITA) and MHC II DM beta 2 (DMB2). Notably, over-expression of chIFN-β could promote the expression of endogenous chPML. All these provide novel insights into the function of chPML, and pave the way for further studying the roles of chPML in biological process and anti-infection function.

Sequential post-translational modifications regulate damaged DNA-binding protein DDB2 function.

Nucleotide excision repair (NER) is a major DNA repair system and hereditary defects in this system cause critical genetic diseases (e.g. xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy). Various proteins are involved in the eukaryotic NER system and undergo several post-translational modifications. Damaged DNA-binding protein 2 (DDB2) is a DNA damage recognition factor in the NER pathway. We previously demonstrated that DDB2 was SUMOylated in response to UV irradiation; however, its physiological roles remain unclear. We herein analysed several mutants and showed that the N-terminal tail of DDB2 was the target for SUMOylation; however, this region did not contain a consensus SUMOylation sequence. We found a SUMO-interacting motif (SIM) in the N-terminal tail that facilitated SUMOylation. The ubiquitination of a SUMOylation-deficient DDB2 SIM mutant was decreased, and its retention of chromatin was prolonged. The SIM mutant showed impaired NER, possibly due to a decline in the timely handover of the lesion site to XP complementation group C. These results suggest that the SUMOylation of DDB2 facilitates NER through enhancements in ubiquitination.

SUMO promotes DNA repair protein collaboration to support alternative telomere lengthening in the absence of PML.

The alternative lengthening of telomeres (ALT) pathway maintains telomere length in a significant fraction of cancers that are associated with poor clinical outcomes. A better understanding of ALT mechanisms is therefore necessary for developing new treatment strategies for ALT cancers. SUMO modification of telomere proteins contributes to the formation of ALT telomere-associated PML bodies (APBs), in which telomeres are clustered and DNA repair proteins are enriched to promote homology-directed telomere DNA synthesis in ALT. However, it is still unknown whether-and if so, how-SUMO supports ALT beyond APB formation. Here, we show that SUMO condensates that contain DNA repair proteins enable telomere maintenance in the absence of APBs. In PML knockout ALT cell lines that lack APBs, we found that SUMOylation is required for manifesting ALT features independent of PML and APBs. Chemically induced telomere targeting of SUMO produces condensate formation and ALT features in PML-null cells. This effect requires both SUMOylation and interactions between SUMO and SUMO interaction motifs (SIMs). Mechanistically, SUMO-induced effects are associated with the accumulation of DNA repair proteins, including Rad52, Rad51AP1, RPA, and BLM, at telomeres. Furthermore, Rad52 can undergo phase separation, enrich SUMO at telomeres, and promote telomere DNA synthesis in collaboration with the BLM helicase in a SUMO-dependent manner. Collectively, our findings suggest that SUMO condensate formation promotes collaboration among DNA repair factors to support ALT telomere maintenance without PML. Given the promising effects of SUMOylation inhibitors in cancer treatment, our findings suggest their potential use in perturbing telomere maintenance in ALT cancer cells.

DHX9 SUMOylation is required for the suppression of R-loop-associated genome instability

RNA helicase DHX9 is essential for genome stability by resolving aberrant R-loops. However, its regulatory mechanisms remain unclear. Here we show that SUMOylation at lysine 120 (K120) is crucial for DHX9 function. Preventing SUMOylation at K120 leads to R-loop dysregulation, increased DNA damage, and cell death. Cells expressing DHX9 K120R mutant which cannot be SUMOylated are more sensitive to genotoxic agents and this sensitivity is mitigated by RNase H overexpression. Unlike the mutant, wild-type DHX9 interacts with R-loop-associated proteins such as PARP1 and DDX21 via SUMO-interacting motifs. Fusion of SUMO2 to the DHX9 K120R mutant enhances its association with these proteins, reduces R-loop accumulation, and alleviates survival defects of DHX9 K120R. Our findings highlight the critical role of DHX9 SUMOylation in maintaining genome stability by regulating protein interactions necessary for R-loop balance.

Protein SUMOylation promotes cAMP-independent EPAC1 activation

Protein SUMOylation is a prevalent stress-response posttranslational modification crucial for maintaining cellular homeostasis. Herein, we report that protein SUMOylation modulates cellular signaling mediated by cAMP, an ancient and universal stress-response second messenger. We identify K561 as a primary SUMOylation site in exchange protein directly activated by cAMP (EPAC1) via site-specific mapping of SUMOylation using mass spectrometry. Sequence and site-directed mutagenesis analyses reveal that a functional SUMO-interacting motif in EPAC1 is required for the binding of SUMO-conjugating enzyme UBC9, formation of EPAC1 nuclear condensate, and EPAC1 cellular SUMOylation. Heat shock-induced SUMO modification of EPAC1 promotes Rap1/2 activation in a cAMP-independent manner. Structural modeling and molecular dynamics simulation studies demonstrate that SUMO substituent on K561 of EPAC1 promotes Rap1 interaction by increasing the buried surface area between the SUMOylated receptor and its effector. Our studies identify a functional SUMOylation site in EPAC1 and unveil a novel mechanism in which SUMOylation of EPAC1 leads to its autonomous activation. The findings of SUMOylation-mediated activation of EPAC1 not only provide new insights into our understanding of cellular regulation of EPAC1 but also will open up a new field of experimentation concerning the cross-talk between cAMP/EPAC1 signaling and protein SUMOylation, two major cellular stress response pathways, during cellular homeostasis.

Legionella pneumophila exploits the endo-lysosomal network for phagosome biogenesis by co-opting SUMOylated Rab7.

Legionella pneumophila strains harboring wild-type rpsL such as Lp02rpsLWT cannot replicate in mouse bone marrow-derived macrophages (BMDMs) due to induction of extensive lysosome damage and apoptosis. The bacterial factor directly responsible for inducing such cell death and the host factor involved in initiating the signaling cascade that leads to lysosome damage remain unknown. Similarly, host factors that may alleviate cell death induced by these bacterial strains have not yet been investigated. Using a genome-wide CRISPR/Cas9 screening, we identified Hmg20a and Nol9 as host factors important for restricting strain Lp02rpsLWT in BMDMs. Depletion of Hmg20a protects macrophages from infection-induced lysosomal damage and apoptosis, allowing productive bacterial replication. The restriction imposed by Hmg20a was mediated by repressing the expression of several endo-lysosomal proteins, including the small GTPase Rab7. We found that SUMOylated Rab7 is recruited to the bacterial phagosome via SulF, a Dot/Icm effector that harbors a SUMO-interacting motif (SIM). Moreover, overexpression of Rab7 rescues intracellular growth of strain Lp02rpsLWT in BMDMs. Our results establish that L. pneumophila exploits the lysosomal network for the biogenesis of its phagosome in BMDMs.

GPS-SUMO 2.0: an updated online service for the prediction of SUMOylation sites and SUMO-interacting motifs.

Small ubiquitin-like modifiers (SUMOs) are tiny but important protein regulators involved in orchestrating a broad spectrum of biological processes, either by covalently modifying protein substrates or by noncovalently interacting with other proteins. Here, we report an updated server, GPS-SUMO 2.0, for the prediction of SUMOylation sites and SUMO-interacting motifs (SIMs). For predictor training, we adopted three machine learning algorithms, penalized logistic regression (PLR), a deep neural network (DNN), and a transformer, and used 52404 nonredundant SUMOylation sites in 8262 proteins and 163 SIMs in 102 proteins. To further increase the accuracy of predicting SUMOylation sites, a pretraining model was first constructed using 145545 protein lysine modification sites, followed by transfer learning to fine-tune the model. GPS-SUMO 2.0 exhibited greater accuracy in predicting SUMOylation sites than did other existing tools. For users, one or multiple protein sequences or identifiers can be input, and the prediction results are shown in a tabular list. In addition to the basic statistics, we integrated knowledge from 35 public resources to annotate SUMOylation sites or SIMs. The GPS-SUMO 2.0 server is freely available at https://sumo.biocuckoo.cn/. We believe that GPS-SUMO 2.0 can serve as a useful tool for further analysis of SUMOylation and SUMO interactions.

Concerted SUMO-targeted ubiquitin ligase activities of TOPORS and RNF4 are essential for stress management and cell proliferation

Protein SUMOylation provides a principal driving force for cellular stress responses, including DNA–protein crosslink (DPC) repair and arsenic-induced PML body degradation. In this study, using genome-scale screens, we identified the human E3 ligase TOPORS as a key effector of SUMO-dependent DPC resolution. We demonstrate that TOPORS promotes DPC repair by functioning as a SUMO-targeted ubiquitin ligase (STUbL), combining ubiquitin ligase activity through its RING domain with poly-SUMO binding via SUMO-interacting motifs, analogous to the STUbL RNF4. Mechanistically, TOPORS is a SUMO1-selective STUbL that complements RNF4 in generating complex ubiquitin landscapes on SUMOylated targets, including DPCs and PML, stimulating efficient p97/VCP unfoldase recruitment and proteasomal degradation. Combined loss of TOPORS and RNF4 is synthetic lethal even in unstressed cells, involving defective clearance of SUMOylated proteins from chromatin accompanied by cell cycle arrest and apoptosis. Our findings establish TOPORS as a STUbL whose parallel action with RNF4 defines a general mechanistic principle in crucial cellular processes governed by direct SUMO–ubiquitin crosstalk.

Cold-induced FOXO1 nuclear transport aids cold survival and tissue storage

Cold-induced injuries severely limit opportunities and outcomes of hypothermic therapies and organ preservation, calling for better understanding of cold adaptation. Here, by surveying cold-altered chromatin accessibility and integrated CUT&Tag/RNA-seq analyses in human stem cells, we reveal forkhead box O1 (FOXO1) as a key transcription factor for autonomous cold adaptation. Accordingly, we find a nonconventional, temperature-sensitive FOXO1 transport mechanism involving the nuclear pore complex protein RANBP2, SUMO-modification of transporter proteins Importin-7 and Exportin-1, and a SUMO-interacting motif on FOXO1. Our conclusions are supported by cold survival experiments with human cell models and zebrafish larvae. Promoting FOXO1 nuclear entry by the Exportin-1 inhibitor KPT-330 enhances cold tolerance in pre-diabetic obese mice, and greatly prolongs the shelf-life of human and mouse pancreatic tissues and islets. Transplantation of mouse islets cold-stored for 14 days reestablishes normoglycemia in diabetic mice. Our findings uncover a regulatory network and potential therapeutic targets to boost spontaneous cold adaptation.

Chigno/CG11180 and SUMO are Chinmo-interacting proteins with a role in Drosophila testes somatic support cells.

Stem cells are critical for replenishment of cells lost to death, damage or differentiation. Drosophila testes are a key model system for elucidating mechanisms regulating stem cell maintenance and differentiation. An intriguing gene identified through such studies is the transcription factor, chronologically inappropriate morphogenesis (Chinmo). Chinmo is a downstream effector of the Jak-STAT signaling pathway that acts in testis somatic stem cells to ensure maintenance of male stem cell fate and sexual identity. Defects in these processes can lead to infertility and the formation of germ cell tumors. While Chinmo's effect on testis stem cell behavior has been investigated in detail, there is still much to be learned about its structure, function, and interactions with other proteins. Using a two-hybrid screen, we find that Chinmo interacts with itself, the small ubiquitin-like modifier SUMO, the novel protein CG11180, and four other proteins (CG4318, Ova (ovaries absent), Taf3 (TBP-associated factor 3), and CG18269). Since both Chinmo and CG11180 contain sumoylation sites and SUMO-interacting motifs (SIMs), we analyzed their interaction in more detail. Using site-directed mutagenesis of a unique SIM in CG11180, we demonstrate that Chinmo's interaction with CG11180 is SUMO-dependent. Furthermore, to assess the functional relevance of both SUMO and CG11180, we performed RNAi-mediated knockdown of both proteins in somatic cells of the Drosophila testis. Using this approach, we find that CG11180 and SUMO are required in somatic cells of adult testes, and that reduction of either protein causes formation of germ cell tumors. Overall, our work suggests that SUMO may be involved in the interaction of Chinmo and CG11180 and that these genes are required in somatic cells of the adult Drosophila testis. Consistent with the CG11180 knockdown phenotype in male testes, and to underscore its connection to Chinmo, we propose the name Chigno (Childless Gambino) for CG11180.

HSV-1 ICP0 dimer domain adopts a novel β-barrel fold.

Infected cell protein 0 (ICP0) is an immediate-early regulatory protein of herpes simplex virus 1 (HSV-1) that possesses E3 ubiquitin ligase activity. ICP0 transactivates viral genes, in part, through its C-terminal dimer domain (residues 555-767). Deletion of this dimer domain results in reduced viral gene expression, lytic infection, and reactivation from latency. Since ICP0's dimer domain is associated with its transactivation activity and efficient viral replication, we wanted to determine the structure of this specific domain. The C-terminus of ICP0 was purified from bacteria and analyzed by X-ray crystallography to solve its structure. Each subunit or monomer in the ICP0 dimer is composed of nine β-strands and two α-helices. Interestingly, two adjacent β-strands from one monomer "reach" into the adjacent subunit during dimer formation, generating two β-barrel-like structures. Additionally, crystallographic analyses indicate a tetramer structure is formed from two β-strands of each dimer, creating a "stacking" of the β-barrels. The structural protein database searches indicate the fold or structure adopted by the ICP0 dimer is novel. The dimer is held together by an extensive network of hydrogen bonds. Computational analyses reveal that ICP0 can either form a dimer or bind to SUMO1 via its C-terminal SUMO-interacting motifs but not both. Understanding the structure of the dimer domain will provide insights into the activities of ICP0 and, ultimately, the HSV-1 life cycle.

Evolution of a novel regulatory mechanism of hypoxia inducible factor in hypoxia-tolerant electric fishes

Hypoxia is a significant source of metabolic stress that activates many cellular pathways involved in cellular differentiation, proliferation, and cell death. Hypoxia is also a major component in many human diseases and a known driver of many cancers. Despite the challenges posed by hypoxia, there are animals that display impressive capacity to withstand lethal levels of hypoxia for prolonged periods of time and thus offer a gateway to a more comprehensive understanding of the hypoxic response in vertebrates. The weakly electric fish genus Brachyhypopomus inhabits some of the most challenging aquatic ecosystems in the world, with some species experiencing seasonal anoxia, thus providing a unique system to study the cellular and molecular mechanisms of hypoxia tolerance. In this study, we use closely related species of Brachyhypopomus that display a range of hypoxia tolerances to probe for the underlying molecular mechanisms via hypoxia inducible factors (HIFs)-transcription factors known to coordinate the cellular response to hypoxia in vertebrates. We find that HIF1⍺ from hypoxia tolerant Brachyhypopomus species displays higher transactivation in response to hypoxia than that of intolerant species, when overexpressed in live cells. Moreover, we identified two SUMO-interacting motifs near the oxygen-dependent degradation and transactivation domains of the HIF1⍺ protein that appear to boost transactivation of HIF1, regardless of the genetic background. Together with computational analyses of selection, this shows that evolution of HIF1⍺ are likely to underlie adaptations to hypoxia tolerance in Brachyhypopomus electric fishes, with changes in two SUMO-interacting motifs facilitating the mechanism of this tolerance.

Sumoylation of SAP130 regulates its interaction with FAF1 as well as its protein stability and transcriptional repressor function

BackgroundFas-associated factor 1 (FAF1) is a multidomain protein that interacts with diverse partners to affect numerous cellular processes. Previously, we discovered two Small Ubiquitin-like Modifier (SUMO)-interacting motifs (SIMs) within FAF1 that are crucial for transcriptional modulation of mineralocorticoid receptor. Recently, we identified Sin3A-associated protein 130 (SAP130), a putative sumoylated protein, as a candidate FAF1 interaction partner by yeast two-hybrid screening. However, it remained unclear whether SAP130 sumoylation might occur and functionally interact with FAF1.ResultsIn this study, we first show that SAP130 can be modified by SUMO1 at Lys residues 794, 878 and 932 both in vitro and in vivo. Mutation of these three SUMO-accepting Lys residues to Ala had no impact on SAP130 association with Sin3A or its nuclear localization, but the mutations abrogated the association of SAP130 with the FAF1. The mutations also potentiated SAP130 trans-repression activity and attenuated SAP130-mediated promotion of cell growth. Additionally, SUMO1-modified SAP130 was less stable than unmodified SAP130. Transient transfection experiments further revealed that FAF1 mitigated the trans-repression and cell proliferation-promoting functions of SAP130, and promoted SAP130 degradation by enhancing its polyubiquitination in a sumoylation-dependent manner.ConclusionsTogether, these results demonstrate that sumoylation of SAP130 regulates its biological functions and that FAF1 plays a crucial role in controlling the SUMO-dependent regulation of transcriptional activity and protein stability of SAP130.

TRIM28-mediated nucleocapsid protein SUMOylation enhances SARS-CoV-2 virulence

Viruses, as opportunistic intracellular parasites, hijack the cellular machinery of host cells to support their survival and propagation. Numerous viral proteins are subjected to host-mediated post-translational modifications. Here, we demonstrate that the SARS-CoV-2 nucleocapsid protein (SARS2-NP) is SUMOylated on the lysine 65 residue, which efficiently mediates SARS2-NP’s ability in homo-oligomerization, RNA association, liquid-liquid phase separation (LLPS). Thereby the innate antiviral immune response is suppressed robustly. These roles can be achieved through intermolecular association between SUMO conjugation and a newly identified SUMO-interacting motif in SARS2-NP. Importantly, the widespread SARS2-NP R203K mutation gains a novel site of SUMOylation which further increases SARS2-NP’s LLPS and immunosuppression. Notably, the SUMO E3 ligase TRIM28 is responsible for catalyzing SARS2-NP SUMOylation. An interfering peptide targeting the TRIM28 and SARS2-NP interaction was screened out to block SARS2-NP SUMOylation and LLPS, and consequently inhibit SARS-CoV-2 replication and rescue innate antiviral immunity. Collectively, these data support SARS2-NP SUMOylation is critical for SARS-CoV-2 virulence, and therefore provide a strategy to antagonize SARS-CoV-2.

Understanding β-strand mediated protein-protein interactions: tuning binding behaviour of intrinsically disordered sequences by backbone modification.

A significant challenge in chemical biology is to understand and modulate protein-protein interactions (PPIs). Given that many PPIs involve a folded protein domain and a peptide sequence that is intrinsically disordered in isolation, peptides represent powerful tools to understand PPIs. Using the interaction between small ubiquitin-like modifier (SUMO) and SUMO-interacting motifs (SIMs), here we show that N-methylation of the peptide backbone can effectively restrict accessible peptide conformations, predisposing them for protein recognition. Backbone N-methylation in appropriate locations results in faster target binding, and thus higher affinity, as shown by relaxation-based NMR experiments and computational analysis. We show that such higher affinities occur as a consequence of an increase in the energy of the unbound state, and a reduction in the entropic contribution to the binding and activation energies. Thus, backbone N-methylation may represent a useful modification within the peptidomimetic toolbox to probe β-strand mediated interactions.

PTEN-mediated dephosphorylation of 53BP1 confers cellular resistance to DNA damage in cancer cells.

Homologous recombination (HR) repair for DNA double-strand breaks (DSBs) is critical for maintaining genome stability and conferring the resistance of tumor cells to chemotherapy. Nuclear PTEN which contains both phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase and protein phosphatase plays a key role in HR repair, but the underlying mechanism remains largely elusive. We find that SUMOylated PTEN promotes HR repair but represses nonhomologous end joining (NHEJ) repair by directly dephosphorylating TP53-binding protein 1 (53BP1). During DNA damage responses (DDR), tumor suppressor ARF (p14ARF) was phosphorylated and then interacted efficiently with PTEN, thus promoting PTEN SUMOylation as an atypical SUMO E3 ligase. Interestingly, SUMOylated PTEN was subsequently recruited to the chromatin at DSB sites. This was because SUMO1 that was conjugated to PTEN was recognized and bound by the SUMO-interacting motif (SIM) of breast cancer type 1 susceptibility protein (BRCA1), which has been located to the core of 53BP1 foci on chromatin during S/G2 stage. Furthermore, these chromatin-loaded PTEN directly and specifically dephosphorylated phosphothreonine-543 (pT543) of 53BP1, resulting in the dissociation of the 53BP1 complex, which facilitated DNA end resection and ongoing HR repair. SUMOylation-site-mutated PTENK254R mice also showed decreased DNA damage repair in vivo. Blocking the PTEN SUMOylation pathway with either a SUMOylation inhibitor or a p14ARF(2-13) peptide sensitized tumor cells to chemotherapy. Our study therefore provides a new mechanistic understanding of PTEN in HR repair and clinical intervention of chemoresistant tumors.

SUMOylation-triggered ALIX activation modulates extracellular vesicles circTLCD4-RWDD3 to promote lymphatic metastasis of non-small cell lung cancer

Lymph node (LN) metastasis is one of the predominant metastatic routes of non-small cell lung cancer (NSCLC) and is considered as a leading cause for the unsatisfactory prognosis of patients. Although lymphangiogenesis is well-recognized as a crucial process in mediating LN metastasis, the regulatory mechanism involving lymphangiogenesis and LN metastasis in NSCLC remains unclear. In this study, we employed high-throughput sequencing to identify a novel circular RNA (circRNA), circTLCD4-RWDD3, which was significantly upregulated in extracellular vesicles (EVs) from LN metastatic NSCLC and was positively associated with deteriorated OS and DFS of patients with NSCLC from multicenter clinical cohort. Downregulating the expression of EV-packaged circTLCD4-RWDD3 inhibited lymphangiogenesis and LN metastasis of NSCLC both in vitro and in vivo. Mechanically, circTLCD4-RWDD3 physically interacted with hnRNPA2B1 and mediated the SUMO2 modification at K108 residue of hnRNPA2B1 by upregulating UBC9. Subsequently, circTLCD4-RWDD3-induced SUMOylated hnRNPA2B1 was recognized by the SUMO interaction motif (SIM) of ALIX and activated ALIX to recruit ESCRT-III, thereby facilitating the sorting of circTLCD4-RWDD3 into NSCLC cell-derived EVs. Moreover, EV-packaged circTLCD4-RWDD3 was internalized by lymphatic endothelial cells to activate the transcription of PROX1, resulting in the lymphangiogenesis and LN metastasis of NSCLC. Importantly, blocking EV-mediated transmission of circTLCD4-RWDD3 via mutating SIM in ALIX or K108 residue of hnRNPA2B1 inhibited the lymphangiogenesis and LN metastasis of NSCLC in vivo. Our findings reveal a precise mechanism underlying SUMOylated hnRNPA2B1-induced EV packaging of circTLCD4-RWDD3 in facilitating LN metastasis of NSCLC, suggesting that EV-packaged circTLCD4-RWDD3 could be a potential therapeutic target against LN metastatic NSCLC.