Recombinant Human SUMO1 Activating Enzyme Subunit 1, His-tagged

• Cat.No.: SAE1-59H
• Product Overview: Recombinant human SAE1 protein, fused to His-T7-tag at N-terminus, was expressed inE.coliand purified by using conventional chromatography techniques.

Specification

• Species: Human
• Source: E.coli
• Tag: His
• Description: SAE1, also known as AOS1, belongs to the ubiquitin-activating E1 family of proteins and plays an important role in the first step of the UBL1 conjugation pathway. Proteins conjugated to Ub are marked for progressive degradation by the 26S Proteasome. SAE1, which is a dimeric enzyme, functions as a UBLI E1 ligase mediating the ATP-dependent activation of UBL1. This protein can bind with UBLE1A and UBLE1B to form a heterodimer which can bind UBL1.
• Sequences: MHHHHHHMAS MTGGQQMGRD LYDDDDKDRW GSMVEKEEAG GGISEEEAAQ YDRQIRLWGL EAQKRLRASR VLLVGLKGLG AEIAKNLILA GVKGLTMLDH EQVTPEDPGA QFLIRTGSVG RNRAEASLER AQNLNPMVDV KVDTEDIEKK PESFFTQFDA VCLTCCSRDV IVKVDQICHK NSIKFFTGDV FGYHGYTFAN LGEHEFVEEK TKVAKVSQGV EDGPDTKRAK LDSSETTMVK KKVVFCPVKE ALEVDWSSEK AKAALKRTTS DYFLLQVLLK FRTDKGRDPS SDTYEEDSEL LLQIRNDVLD SLGISPDLLP EDFVRYCFSE MAPVCAVVGG ILAQEIVKAL SQRDPPHNNF FFFDGMKGNG IVECLGPK.
• Molecular Weight: 42.2 kDa (378aa) confirmed by MALDI-TOF.
• Form: Liquid. In 20 mM Tris-HCl buffer (pH8.0) containing 1mM DTT, 10% glycerol.
• Purity: > 90% by SDS – PAGE.
• Concentration: 1 mg/ml (determined by Bradford assay).
• Storage: Can be stored at +4°C short term (1-2 weeks). For long term storage, aliquot and store at -20°C or -70°C. Avoid repeated freezing and thawing cycles.

Gene Information

• Gene Name: SAE1 SUMO1 activating enzyme subunit 1 [ Homo sapiens ]
• Synonyms:
  SAE1; SUMO1 activating enzyme subunit 1; SUMO-1 activating enzyme E1 N subunit; SUMO-1 activating enzyme subunit 1; SUMO-activating enzyme subunit 1; activator of SUMO1; sentrin/SUMO-activating protein AOS1; ubiquitin-like 1-activating enzyme E1A; ubiquitin-like protein SUMO-1 activating enzyme; AOS1; FLJ3091; HSPC140
• Gene ID: 10055
• mRNA Refseq: NM_001145713
• Protein Refseq: NP_001139185
• MIM: 613294
• UniProt ID: Q9UBE0
• Chromosome Location: 19q13.32
• Pathway: Ubiquitin mediated proteolysis
• Function: ATP-dependent protein binding; contributes_to SUMO activating enzyme activity; enzyme activator activity; ligase activity; protein heterodimerization activity; protein binding; ubiquitin activating enzyme activity

Citation

SUMOylation of E2F1 regulates expression of EZH2

Journal: Cancer research    PubMed ID: 32816857    Data: 2022/1/7

Authors: Li Du, Marwan G. Fakih, Yuan Chen

Article Snippet:In CDH1 promoter luciferase assay, both WT and 4KR EZH2 showed similar dose-dependent suppression of the CDH1 promoter ( ).similar dose-dependent suppression of the CDH1 promoter ( ). ... Taken together, these data suggest that SUMOylation regulates EZH2 function mainly through its expression and not through direct SUMO modification of EZH2. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 6. caption a7 caption a8 EZH2 SUMOylation doesn’t affect its histone methyltranferase activity, protein stability or its suppression of CDH1 promoter. (A) A representative blot of in vitro SUMOylation of purified PRC2 (Creative-Biomart) by incubating with recombinant SUMO E1 (SAE1/SAE2), SUMO E2 enzyme (UBC9), and SUMO1 or SUMO2, and without or with RanBP2 (an E3) at 30oC for 3 h or 16 h. EZH2 was detected by western blot. (B) A representative blot of in vitro histone methytransferase assay with PRC2 after PRC2 in vitro SUMOylation.. SUMOylated PRC2 from in vitro SUMOylation was incubated with or without SENP1 for 30 min, and then histone H3 and S-adenosyl methionine were added for 60 min. Then, western blots were performed to detect H3K27me3 level. (C) Top, schematic diagram indicating the three detected and one predicted SUMOylation sites of EZH2—K20, K307, K419 and K421.SUMOylated PRC2 from in vitro SUMOylation was incubated with..

(A) EZH2 mRNA levels were determined using real-time qPCR in HCT116 cells (left panel) and HCC1937 cells (right panel). Doxycycline (Dox, 5μg/ml) was added to induce SAE2 knockdown (+Dox) in HCT116 cells and HCC1937 cells for 3 days. Knockdown for 3 days was necessary to decrease enzyme level due to the slow turnover rate of the SUMO E1. (B) EZH2 mRNA levels decreased in HT29 and MB-MDA-231 cell lines by stable expression of shSAE2 in comparison to the matched control cell line (shCtrl). (C) Western blot shows SAE2, EZH2, UBC9, EZH2, H3K27me3 (tri-methylation at residue lysine 27 of histone H3) levels and SUMO-2,3 modifications in HCT116 and HT29 stable cell lines with SAE2 knockdown by shRNA. GAPDH and Histone H3 are used as loading controls. (D) Transient knockdown of SAE2 or UBC9 by siRNA transfection reduced EZH2 and H3K27me3 levels and SUMO-2,3 modifications in HCT116 and HT29 cells as determined by western blot. (E) Western blot for EZH2 and global SUMOylation (SUMO1 and SUMO2,3) levels in HCT116 cells treated with 0, 0.2, or 1.0 μM ML-792 SUMO E1 inhibitor for 24 h. Estimated variation is indicated as SD, p values were derived using a two-tailed Student’s t-test. * p < 0.05, ** p < 0.01.

(A) Nude mice were xenografted with HCT116 cells transduced with Dox-inducible SAE2 shRNA by subcutaneous injection. Recipient animals were treated with (+Dox) or without (-Dox) Dox and tumor weight was measured after 16 days. (B) NSG mice were xenografted with HT29 cells transduced with SAE2-targeting shRNA lentivirus (shSAE2) or control shRNA (shCtrl) and tumor weight was measured after 35 days. (C) Two different colorectal PDX tissues, PDX356531 and PDX354313, were cultured and transduced with lentivirus containing SAE2-targeting shRNA (shSAE2) or control shRNA (shCtrl). Cell viability was determined after 72 h. SAE2 and EZH2 levels and SUMO2,3 modifications in tumor tissue from HCT116 xenografts (D) or HT29 xenografts (E) were determined using western blot (top) and quantified using Image J (bottom). (F) PDX356531 and PDX354313 cells with SAE2-targeting shRNA lentivirus (shSAE2) or control shRNA (shCtrl) were harvested after 72 h, then SAE2 and EZH2 levels and SUMO-2,3 modifications were determined using western blot (top) and quantified (bottom). GAPDH was used as a loading control. Estimated variation is indicated as SD, p values were derived using a two-tailed Student’s t-test. * p < 0.05, ** p < 0.01, *** p < 0.001.

(A) A representative blot of in vitro SUMOylation of purified PRC2 (Creative-Biomart) by incubating with recombinant SUMO E1 (SAE1/SAE2), SUMO E2 enzyme (UBC9), and SUMO1 or SUMO2, and without or with RanBP2 (an E3) at 30oC for 3 h or 16 h. EZH2 was detected by western blot. (B) A representative blot of in vitro histone methytransferase assay with PRC2 after PRC2 in vitro SUMOylation. SUMOylated PRC2 from in vitro SUMOylation was incubated with or without SENP1 for 30 min, and then histone H3 and S-adenosyl methionine were added for 60 min. Then, western blots were performed to detect H3K27me3 level. (C) Top, schematic diagram indicating the three detected and one predicted SUMOylation sites of EZH2—K20, K307, K419 and K421. 293T cells were transfected with plasmids expressing HA-tagged WT or mutant EZH2 with 4 SUMOylation sites mutated to arginine (4KR) along with an UBC9-expressing vector. Then, the expression of both WT and mutant EZH2 were detected by western blots with an anti-HA antibody. Cell lysates were immunoprecipitated with an anti-HA antibody under denaturing condition followed by immunoblotting with an anti-HA antibody and SUMO1 antibody. (D) SUMOylation did not affect EZH2 protein stability. Representative western blot of EZH2 in HCT116 cells transfected with HA-tagged EZH2 WT or 4KR mutant expression plasmid for 2 days, followed by treatment with 100 μg ml?1 CHX for indicated time. GAPDH was used as a loading control. (E) SUMOylation doesn’t affect EZH2 function in suppressing CDH1 promoter. HCT116 cells were transfected with empty vector (Ctrl), EZH2 WT or 4KR mutant together with CDH1 promoter reporter plasmid and renilla plasmid. EZH2 WT or 4KR plasmids were transfect with different doses (100 ng/well “+” and 200 ng/well “++”). CDH1 promoter activity was measured 48 h post transfection and normalized to renilla. Estimated variation is indicated as SD, p values were derived using a two-tailed Student’s t-test or ANOVA. ns, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.