ABSTRACT
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.
Subject(s)
DNA Repair , Promyelocytic Leukemia Protein , Sumoylation , Telomere Homeostasis , Telomere , Humans , Promyelocytic Leukemia Protein/metabolism , Promyelocytic Leukemia Protein/genetics , Telomere/metabolism , Cell Line, Tumor , SUMO-1 Protein/metabolism , SUMO-1 Protein/genetics , Rad52 DNA Repair and Recombination Protein/metabolism , Rad52 DNA Repair and Recombination Protein/genetics , Cell Line , Small Ubiquitin-Related Modifier Proteins/metabolism , Small Ubiquitin-Related Modifier Proteins/geneticsABSTRACT
Alterations in signaling pathways and modulation of cell metabolism are associated with the pathogenesis of cancers, including hepatocellular carcinoma (HCC). Small ubiquitin-like modifier (SUMO) proteins and NF-κB family play major roles in various cellular processes. The current study aims to determine the expression profile of SUMO and NF-κB genes in HCC tumors and investigate their association with the clinical outcome of HCC. The expression of 5 genes - SUMO1, SUMO2, SUMO3, NF-κB p65, and NF-κB p50 - was quantified in tumor and adjacent non-tumor tissues of 58 HBV-related HCC patients by real-time quantitative PCR and was analyzed for the possible association with clinical parameters of HCC. The expression of SUMO2 was significantly higher in HCC tumor tissues compared to the adjacent non-tumor tissues (Pâ =â .01), while no significant difference in SUMO1, SUMO3, NF-κB p65, and NF-κB p50 expression was observed between HCC tumor and non-tumor tissues (Pâ >â .05). In HCC tissues, a strong correlation was observed between the expression of SUMO2 and NF-κB p50, between SUMO3 and NF-κB p50, between SUMO3 and NF-κB p65 (Spearman rhoâ =â 0.83; 0.82; 0.772 respectively; Pâ <â .001). The expression of SUMO1, SUMO2, SUMO3, NF-κB p65, and NF-κB p50 was decreased in grade 3 compared to grades 1 and 2 in HCC tumors according to the World Health Organization grades system. Our results highlighted that the SUMO2 gene is upregulated in tumor tissues of patients with HCC, and is related to the development of HCC, thus it may be associated with the pathogenesis of HCC.
Subject(s)
Carcinoma, Hepatocellular , Liver Neoplasms , Small Ubiquitin-Related Modifier Proteins , Humans , Carcinoma, Hepatocellular/virology , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/metabolism , Liver Neoplasms/genetics , Liver Neoplasms/virology , Liver Neoplasms/metabolism , Male , Female , Middle Aged , Small Ubiquitin-Related Modifier Proteins/genetics , Small Ubiquitin-Related Modifier Proteins/metabolism , SUMO-1 Protein/genetics , SUMO-1 Protein/metabolism , NF-kappa B/metabolism , Adult , Transcription Factor RelA/metabolism , Transcription Factor RelA/genetics , Hepatitis B virus/genetics , NF-kappa B p50 Subunit/genetics , NF-kappa B p50 Subunit/metabolism , Aged , Gene Expression Regulation, Neoplastic , Ubiquitins/genetics , Ubiquitins/metabolism , Hepatitis B/complications , Hepatitis B/geneticsABSTRACT
The discovery of ferroptosis has unveiled new perspectives for cervical cancer (CC) management. We elucidated the functional mechanism of hypoxia-like conditions in CC cell ferroptosis resistance. CC cells were subjected to normoxia or hypoxia-like conditions, followed by erastin treatment to induce ferroptosis. The assessment of cell viability/ferroptosis resistance was performed by MTT assay/Fe2+, MDA, and glutathione measurement by colorimetry. KDM4A/SUMO1/Ubc9/SENP1 protein levels were determined by Western blot. Interaction and binding sites between KDM4A and SUMO1 were analyzed and predicted by immunofluorescence/co-immunoprecipitation and GPS-SUMO 1.0 software, with the target relationship verified by mutation experiment. SLC7A11/GPX4/H3K9me3 protein levels, and H3K9me3 level in the SLC7A11 gene promoter region were determined by RT-qPCR and Western blot/chromatin immunoprecipitation. H3H9me3/SLC7A11/GPX4 level alterations, and ferroptosis resistance after KDM4A silencing or KDM4A K471 mutation were assessed. Hypoxia-like conditions increased CC cell ferroptosis resistance and KDM4A, SUMO1, and Ubc9 protein levels, while it decreased SENP1 protein level. KDM4A and SUMO1 were co-localized in the nucleus, and hypoxia-like conditions promoted their interaction. Specifically, the K471 locus of KDM4A was the main locus for SUMO1ylation. Hypoxia-like conditions up-regulated SLC7A11 and GPX4 expression levels and decreased H3K9me3 protein level and H3K9me3 abundance in the SLC7A11 promoter region. KDM4A silencing or K471 locus mutation resulted in weakened interaction between KDM4A and SUMO1, elevated H3K9me3 levels, decreased SLC7A11 expression, ultimately, a reduced CC cell ferroptosis resistance. CoCl2-stimulated hypoxia-like conditions enhanced SUMO1 modification of KDM4A at the K471 locus specifically, repressed H3K9me3 levels, and up-regulated SLC7A11/GPX4 to enhance CC cell ferroptosis resistance.
Subject(s)
Amino Acid Transport System y+ , Ferroptosis , Phospholipid Hydroperoxide Glutathione Peroxidase , Sumoylation , Uterine Cervical Neoplasms , Humans , Ferroptosis/drug effects , Uterine Cervical Neoplasms/genetics , Uterine Cervical Neoplasms/pathology , Sumoylation/drug effects , Phospholipid Hydroperoxide Glutathione Peroxidase/metabolism , Phospholipid Hydroperoxide Glutathione Peroxidase/genetics , Female , Amino Acid Transport System y+/genetics , Amino Acid Transport System y+/metabolism , Jumonji Domain-Containing Histone Demethylases/metabolism , Jumonji Domain-Containing Histone Demethylases/genetics , Cell Line, Tumor , Cell Hypoxia , SUMO-1 Protein/metabolism , SUMO-1 Protein/geneticsABSTRACT
Golgin tethers are known to mediate vesicular transport in the secretory pathway, whereas it is relatively unknown whether they may mediate cellular stress response within the cell. Here, we describe a cellular stress response during heat shock stress via SUMOylation of a Golgin tether, Golgin45. We found that Golgin45 is a SUMOylated Golgin via SUMO1 under steady state condition. Upon heat shock stress, the Golgin enters the nucleus by interacting with Importin-ß2 and gets further modified by SUMO3. Importantly, SUMOylated Golgin45 appears to interact with PML and SUMO-deficient Golgin45 mutant functions as a dominant negative for PML-NB formation during heat shock stress, suppressing transcription of lipid metabolism genes. These results indicate that Golgin45 may play a role in heat stress response by transcriptional regulation of lipid metabolism genes in SUMOylation-dependent fashion.
Subject(s)
Heat-Shock Response , Lipid Metabolism , Sumoylation , Ubiquitins , Humans , Lipid Metabolism/genetics , Heat-Shock Response/genetics , Gene Expression Regulation , Promyelocytic Leukemia Protein/metabolism , Promyelocytic Leukemia Protein/genetics , HeLa Cells , SUMO-1 Protein/metabolism , SUMO-1 Protein/genetics , Small Ubiquitin-Related Modifier Proteins/metabolism , Small Ubiquitin-Related Modifier Proteins/genetics , HEK293 Cells , Transcription, Genetic , beta Karyopherins/metabolism , beta Karyopherins/geneticsABSTRACT
Heterologous production of proteins in Escherichia coli has raised several challenges including soluble production of target proteins, high levels of expression and purification. Fusion tags can serve as the important tools to overcome these challenges. SUMO (small ubiquitin-related modifier) is one of these tags whose fusion to native protein sequence can enhance its solubility and stability. In current research, a simple, efficient and cost-effective method is being discussed for the construction of pET28a-SUMO vector. In order to improve the stability and activity of lysophospholipase from Pyrococcus abyssi (Pa-LPL), a 6xHis-SUMO tag was fused to N-terminal of Pa-LPL by using pET28a-SUMO vector. Recombinant SUMO-fused enzyme (6 H-S-PaLPL) works optimally at 35 °C and pH 6.5 with remarkable thermostability at 35-95 °C. Thermo-inactivation kinetics of 6 H-S-PaLPL were also studied at 35-95 °C with first order rate constant (kIN) of 5.58 × 10- 2 h-1 and half-life of 12 ± 0 h at 95 °C. Km and Vmax for the hydrolysis of 4-nitrophenyl butyrate were calculated to be 2 ± 0.015 mM and 3882 ± 22.368 U/mg, respectively. 2.4-fold increase in Vmax of Pa-LPL was observed after fusion of 6xHis-SUMO tag to its N-terminal. It is the first report on the utilization of SUMO fusion tag to enhance the overall stability and activity of Pa-LPL. Fusion of 6xHis-SUMO tag not only aided in the purification process but also played a crucial role in increasing the thermostability and activity of the enzyme. SUMO-fused enzyme, thus generated, can serve as an important candidate for degumming of vegetable oils at industrial scale.
Subject(s)
Enzyme Stability , Escherichia coli , Pyrococcus abyssi , Recombinant Fusion Proteins , Temperature , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Recombinant Fusion Proteins/chemistry , Escherichia coli/genetics , Escherichia coli/metabolism , Hydrogen-Ion Concentration , Kinetics , Pyrococcus abyssi/genetics , Pyrococcus abyssi/enzymology , Small Ubiquitin-Related Modifier Proteins/metabolism , Small Ubiquitin-Related Modifier Proteins/genetics , Genetic Vectors/metabolism , SUMO-1 Protein/genetics , SUMO-1 Protein/metabolism , SUMO-1 Protein/chemistry , Cloning, Molecular , SolubilityABSTRACT
The mechanistic target of rapamycin (mTOR) signaling is influenced by multiple regulatory proteins and post-translational modifications; however, underlying mechanisms remain unclear. Here, we report a novel role of small ubiquitin-like modifier (SUMO) in mTOR complex assembly and activity. By investigating the SUMOylation status of core mTOR components, we observed that the regulatory subunit, GßL (G protein ß-subunit-like protein, also known as mLST8), is modified by SUMO1, 2, and 3 isoforms. Using mutagenesis and mass spectrometry, we identified that GßL is SUMOylated at lysine sites K86, K215, K245, K261, and K305. We found that SUMO depletion reduces mTOR-Raptor (regulatory protein associated with mTOR) and mTOR-Rictor (rapamycin-insensitive companion of mTOR) complex formation and diminishes nutrient-induced mTOR signaling. Reconstitution with WT GßL but not SUMOylation-defective KR mutant GßL promotes mTOR signaling in GßL-depleted cells. Taken together, we report for the very first time that SUMO modifies GßL, influences the assembly of mTOR protein complexes, and regulates mTOR activity.
Subject(s)
Signal Transduction , Sumoylation , TOR Serine-Threonine Kinases , Humans , TOR Serine-Threonine Kinases/metabolism , TOR Serine-Threonine Kinases/genetics , HEK293 Cells , SUMO-1 Protein/metabolism , SUMO-1 Protein/genetics , Small Ubiquitin-Related Modifier Proteins/metabolism , Small Ubiquitin-Related Modifier Proteins/genetics , mTOR Associated Protein, LST8 Homolog/metabolism , mTOR Associated Protein, LST8 Homolog/genetics , Ubiquitins/metabolism , Ubiquitins/genetics , Lysine/metabolismABSTRACT
Head and neck squamous cell carcinoma (HNSCC) is currently one of the most common malignancies with a poor prognosis worldwide. Meanwhile, small ubiquitinlike modifier (SUMO) specific peptidase 1 (SENP1) was associated with ferroptosis. However, the specific functions and underlying mechanisms of action of SENP1 in ferroptosis and tumor progression of HNSCC remain to be established. The findings of the present study implicated a novel ferroptosis pathway in the initiation and progression of HNSCC, providing new functional targets to guide future therapy. In the present study, The Cancer Genome Atlas database was employed to establish a gene model related to ferroptosis and verified SENP1 as a key gene via transcriptome sequencing. Expression of SENP1 in HNSCC tissue and CAL27 cells was detected based on reverse transcriptionquantitative PCR and western blot analysis. Proliferation and migration abilities of cells were determined using Cell Counting Kit8, wound healing and Transwell experiments. Expression levels of iron, glutathione (GSH) and lipid peroxidation endproduct malondialdehyde (MDA) under conditions of silencing of SENP1 with shRNA lentivirus were assayed. Additionally, the relationship between SENP1 and longchain acylcoenzyme A synthase 4 (ACSL4) was validated with the aid of immunoblotting and coimmunoprecipitation (coIP). Finally, the influence of shSENP1 on the expression of key ferroptosis proteins, glutathione peroxidase 4 (GPX4) and solute carrier family 7 member 11, was evaluated via western blotting. It was revealed that SENP1 was significantly overexpressed in HNSCC and associated with low patient survival. Silencing of SENP1 led to significant suppression of cell proliferation, migration and invasion, increase in the contents of iron ions and MDA and decline in GSH levels in HNSCC cells, thereby enhancing ferroptosis and inhibiting disease progression. Conversely, overexpression of SENP1 suppressed ferroptosis and promoted progression of HNSCC. CoIP and western blot analyses revealed a SUMOylation link between SENP1 and ACSL4. SENP1 reduced the stability of ACSL4 protein through deSUMOylation, leading to inhibition of ferroptosis. SENP1 silencing further inhibited the expression of the key iron death protein, GPX4, to regulate ferroptosis. Taken together, SENP1 deficiency promoted ferroptosis and inhibited tumor progression through reduction of SUMOylation of ACSL4 in HNSCC. The collective results of the present study supported the utility of SENP1 as an effective predictive biomarker for targeted treatment of HNSCC.