Hsp70 Promotes SUMO of HIF-1α and Promotes Lung Cancer Invasion and Metastasis

Objective. This study aims to investigate the effect of heat shock protein-70 (Hsp70) on epithelial-mesenchymal transition (EMT) of lung cancer cells under heat stimulation and to explore its possible molecular mechanism. Methods. qRT-PCR and immunohistochemistry assay were used to detect the expression of Hsp70 in lung cancer tissues and adjacent tissues. EdU assay was used to detect the cell activity. The effect of Hsp70 on the migration and invasion of A549 and NCI-H446 cells was detected by the wound-healing assay and Transwell assay. A tumor transplantation animal model was established to detect the effect of overexpression of Hsp70 on proliferation and metastasis of lung cancer cells. Western blot assay was used to detect the effect of thermal stimulation and overexpression of Hsp70 on SUMO modification of HIF-1α. Results. The wound-healing rate of A549 and NCI-H446 cells under Hsp70 stimulation was significantly higher than blank control group. At the same time, the number of cells passing through the membrane increased significantly. Hypodermic tumor transplantation in nude mice proved that knockout Hsp70 can inhibit proliferation and metastasis of lung cancer cells. Thermal stimulation upregulated the expression of Hsp70 and promoted SUMO modification of HIF-1α, ultimately promoting the proliferation and metastasis of lung cancer. Inhibition of Hsp70 reverses the effect of thermal stimulation on lung cancer by reducing the SUMO modification of HIF-1α. Conclusion. Thermal stimulation can promote EMT in A549 and NCI-H446 cells and promote cell migration and invasion in vitro and in vivo by upregulation of Hsp70. This process is associated with the promotion of SUMO modification of HIF-1α.

Identification of proximal SUMO-dependent interactors using SUMO-ID

The fast dynamics and reversibility of posttranslational modifications by the ubiquitin family pose significant challenges for research. Here we present SUMO-ID, a technology that merges proximity biotinylation by TurboID and protein-fragment complementation to find SUMO-dependent interactors of proteins of interest. We develop an optimized split-TurboID version and show SUMO interaction-dependent labelling of proteins proximal to PML and RANGAP1. SUMO-dependent interactors of PML are involved in transcription, DNA damage, stress response and SUMO modification and are highly enriched in SUMO Interacting Motifs, but may only represent a subset of the total PML proximal proteome. Likewise, SUMO-ID also allow us to identify interactors of SUMOylated SALL1, a less characterized SUMO substrate. Furthermore, using TP53 as a substrate, we identify SUMO1, SUMO2 and Ubiquitin preferential interactors. Thus, SUMO-ID is a powerful tool that allows to study the consequences of SUMO-dependent interactions, and may further unravel the complexity of the ubiquitin code.

Knockdown of ubiquitin-like modifier-activating enzyme 2 promotes apoptosis of clear cell renal cell carcinoma cells

Small ubiquitin-related modifier (SUMO) proteins are involved in the development of tumors. Ubiquitin-like modifier-activating enzyme 2 (UBA2) is an important member of the SUMO modification system; however, its role in clear cell renal cell carcinoma (ccRCC) is unclear. Therefore, we investigated the expression and function of UBA2 in ccRCC. Both mRNA and protein expression levels of UBA2 were found to be higher in ccRCC than in normal renal tissues and significantly related to the tumor size, Fuhrman grade, and tumor stage. UBA2 knockdown inhibited ccRCC cell growth, promoted apoptosis in vitro and in vivo, and decreased the abundance of a p53 mutant, c-Myc, and key enzymes of the SUMO modification system. Meanwhile, overexpression of UBA2 had the opposite effects. Overexpression of the p53 mutant or c-Myc alleviated the effects of UBA2 knockdown on ccRCC cell proliferation and apoptosis. In conclusion, targeting UBA2 may have a therapeutic potential against ccRCC.

A Tale of Usurpation and Subversion: SUMO-Dependent Integrity of Promyelocytic Leukemia Nuclear Bodies at the Crossroad of Infection and Immunity

Promyelocytic leukemia nuclear bodies (PML NBs) are multi-protein assemblies representing distinct sub-nuclear structures. As phase-separated molecular condensates, PML NBs exhibit liquid droplet-like consistency. A key organizer of the assembly and dynamics of PML NBs is the ubiquitin-like SUMO modification system. SUMO is covalently attached to PML and other core components of PML NBs thereby exhibiting a glue-like function by providing multivalent interactions with proteins containing SUMO interacting motifs (SIMs). PML NBs serve as the catalytic center for nuclear SUMOylation and SUMO-SIM interactions are essential for protein assembly within these structures. Importantly, however, formation of SUMO chains on PML and other PML NB-associated proteins triggers ubiquitylation and proteasomal degradation which coincide with disruption of these nuclear condensates. To date, a plethora of nuclear activities such as transcriptional and post-transcriptional regulation of gene expression, apoptosis, senescence, cell cycle control, DNA damage response, and DNA replication have been associated with PML NBs. Not surprisingly, therefore, SUMO-dependent PML NB integrity has been implicated in regulating many physiological processes including tumor suppression, metabolism, drug-resistance, development, cellular stemness, and anti-pathogen immune response. The interplay between PML NBs and viral infection is multifaceted. As a part of the cellular antiviral defense strategy, PML NB components are crucial restriction factors for many viruses and a mutual positive correlation has been found to exist between PML NBs and the interferon response. Viruses, in turn, have developed counterstrategies for disarming PML NB associated immune defense measures. On the other end of the spectrum, certain viruses are known to usurp specific PML NB components for successful replication and disruption of these sub-nuclear foci has recently been linked to the stimulation rather than curtailment of antiviral gene repertoire. Importantly, the ability of invading virions to manipulate the host SUMO modification machinery is essential for this interplay between PML NB integrity and viruses. Moreover, compelling evidence is emerging in favor of bacterial pathogens to negotiate with the SUMO system thereby modulating PML NB-directed intrinsic and innate immunity. In the current context, we will present an updated account of the dynamic intricacies between cellular PML NBs as the nuclear SUMO modification hotspots and immune regulatory mechanisms in response to viral and bacterial pathogens.

SUMO Modification of OsFKBP20-1b Is Integral to Proper Pre-mRNA Splicing upon Heat Stress in Rice

OsFKBP20-1b, a plant-specific cyclophilin protein, has been implicated to regulate pre-mRNA splicing under stress conditions in rice. Here, we demonstrated that OsFKBP20-1b is SUMOylated in a reconstituted SUMOylation system in E.coli and in planta, and that the SUMOylation-coupled regulation was associated with enhanced protein stability using a less SUMOylated OsFKBP20-1b mutant (5KR_OsFKBP20-1b). Furthermore, OsFKBP20-1b directly interacted with OsSUMO1 and OsSUMO2 in the nucleus and cytoplasm, whereas the less SUMOylated 5KR_OsFKBP20-1b mutant had an impaired interaction with OsSUMO1 and 2 in the cytoplasm but not in the nucleus. Under heat stress, the abundance of an OsFKBP20-1b-GFP fusion protein was substantially increased in the nuclear speckles and cytoplasmic foci, whereas the heat-responsiveness was remarkably diminished in the presence of the less SUMOylated 5KR_OsFKBP20-1b-GFP mutant. The accumulation of endogenous SUMOylated OsFKBP20-1b was enhanced by heat stress in planta. Moreover, 5KR_OsFKBP20-1b was not sufficiently associated with the U snRNAs in the nucleus as a spliceosome component. A protoplast transfection assay indicated that the low SUMOylation level of 5KR_OsFKBP20-1b led to inaccurate alternative splicing and transcription under heat stress. Thus, our results suggest that OsFKBP20-1b is post-translationally regulated by SUMOylation, and the modification is crucial for proper RNA processing in response to heat stress in rice.

TRIM28 SUMOylates and stabilizes NLRP3 to facilitate inflammasome activation

The cellular NLRP3 protein level is crucial for assembly and activation of the NLRP3 inflammasome. Various posttranslational modifications (PTMs), including phosphorylation and ubiquitination, control NLRP3 protein degradation and inflammasome activation; however, the function of small ubiquitin-like modifier (SUMO) modification (called SUMOylation) in controlling NLRP3 stability and subsequent inflammasome activation is unclear. Here, we show that the E3 SUMO ligase tripartite motif-containing protein 28 (TRIM28) is an enhancer of NLRP3 inflammasome activation by facilitating NLRP3 expression. TRIM28 binds NLRP3, promotes SUMO1, SUMO2 and SUMO3 modification of NLRP3, and thereby inhibits NLRP3 ubiquitination and proteasomal degradation. Concordantly, Trim28 deficiency attenuates NLRP3 inflammasome activation both in vitro and in vivo. These data identify a mechanism by which SUMOylation controls the cellular NLRP3 level and inflammasome activation, and reveal correlations and interactions of NLRP3 SUMOylation and ubiquitination during inflammasome activation.

Cleavage of Syndecan-1 Promotes the Proliferation of the Basal-Like Breast Cancer Cell Line BT-549 Via Akt SUMOylation

Basal-like breast cancer is characterized by an aggressive clinical outcome and presence of metastasis, for which effective therapies are unavailable. We have previously shown that chondroitin 4-O-sulfotransferase-1 (C4ST-1) controls the invasive properties of the basal-like breast cancer cell line BT-549 by inducing matrix metalloproteinase (MMP) expression through the N-cadherin/β-catenin pathway. Here we report that C4ST-1 controls the proliferation of BT-549 cells via the MMP-dependent cleavage of syndecan-1. Syndecan-1 is a membrane-bound proteoglycan associated with an aggressive phenotype and poor prognosis in breast cancer. In addition, the cleavage of syndecan-1 at a specific juxtamembrane cleavage site is implicated in the pathophysiological response in breast cancer. Knockout of C4ST-1 remarkably suppressed both the cleavage of syndecan-1 and proliferation of BT-549 cells. Kinases (AKT1, ERK1/2, PI3K, and STAT3) comprising cancer proliferative pathways are phosphorylated in C4ST-1 knockout cells at a level similar to that in parental BT-549 cells, whereas levels of phosphorylated S6 kinase and SUMOylated AKT (hyperactivated AKT observed in breast cancer) decreased in C4ST-1 knockout cells. An MMP inhibitor, GM6001, suppressed the small ubiquitin-like modifier (SUMO) modification of AKT, suggesting that cleavage of syndecan-1 by MMPs is involved in the SUMO modification of AKT. Forced expression of the cytoplasmic domain of syndecan-1, which is generated by MMP-dependent cleavage, increased the SUMO modification of AKT and global protein SUMOylation. Furthermore, syndecan-1 C-terminal domain-expressing BT-549 cells were more proliferative and sensitive to a potent SUMOylation inhibitor, tannic acid, compared with BT-549 cells transfected with an empty expression vector. These findings assign new functions to the C-terminal fragment of syndecan-1 generated by MMP-dependent proteolysis, thereby broadening our understanding of their physiological importance and implying that the therapeutic inhibition of syndecan-1 cleavage could affect the progression of basal-like breast cancer.

Identification of proximal SUMO-dependent interactors using SUMO-ID

ABSTRACTThe fast dynamics and reversibility of posttranslational modifications by the ubiquitin family pose significant challenges for research. Here we present SUMO-ID, a technology that merges proximity biotinylation by TurboID and protein-fragment complementation to find SUMO-dependent interactors of proteins of interest. We developed an optimized split-TurboID version and show SUMO interaction-dependent labelling of proteins proximal to PML and RANGAP1. SUMO-dependent interactors of PML are involved in transcription, DNA damage, stress response and SUMO modification and are highly enriched in SUMO Interacting Motifs, but may only represent a subset of the total PML proximal proteome. Likewise, SUMO-ID also allowed us to identify novel interactors of SUMOylated SALL1, a less characterized SUMO substrate. Thus, SUMO-ID is a powerful tool that allows to study the consequences of SUMO-dependent interactions, and may further unravel the complexity of the ubiquitin code.

SUMO maintains the chromatin environment of human induced pluripotent stem cells

Pluripotent stem cells represent a powerful system to identify the mechanisms governing cell fate decisions during early mammalian development. Covalent attachment of the Small Ubiquitin Like Modifier (SUMO) to proteins has emerged as an important factor in stem cell maintenance. Here we show that SUMO is required to maintain stem cells in their pluripotent state and identify many chromatin-associated proteins as bona fide SUMO substrates in human induced pluripotent stem cells (hiPSCs). Loss of SUMO increases chromatin accessibility and expression of long non-coding RNAs and human endogenous retroviral elements, indicating a role for the SUMO modification of SETDB1 and a large TRIM28 centric network of zinc finger proteins in silencing of these elements. While most protein coding genes are unaffected, the Preferentially Expressed Antigen of Melanoma (PRAME) gene locus becomes more accessible and transcription is dramatically increased after inhibition of SUMO modification. When PRAME is silent, a peak of SUMO over the transcriptional start site overlaps with ChIP-seq peaks for cohesin, RNA pol II, CTCF and ZNF143, with the latter two heavily modified by SUMO. These associations suggest that silencing of the PRAME gene is maintained by the influence of SUMO on higher order chromatin structure. Our data indicate that SUMO modification plays an important role in hiPSCs by repressing genes that disrupt pluripotency networks or drive differentiation.