Recombinant Reconstitution of Sumoylation Reactions In Vitro

Flotho, Annette; Werner, Andreas; Winter, Tobias; Frank, Andrea; Ehret, Heidi; Melchior, Frauke

doi:10.1007/978-1-61779-474-2_5

Cited by 21 publications

(17 citation statements)

References 16 publications

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“…GST-RanGAP fragment 418–587 and GST-PML fragment 485–495 were purchased from Biomol International (Farmingdale, NY). MmUbc9 WT and K49R variant proteins were expressed and purified from the pET28a backbone as described earlier 63 . In vitro reactions were composed of 0.5 µM substrate (RanGAP, PML and E2–25K), 12.5 µM His 6 -SUMO3 Q92R, 65 nM SAE1/SAE2 and 0.5 µM wild type or K49R Ubc9 in activity buffer (20 mM HEPES (pH 7.8), 50 mM NaCl and 1 mM DTT).…”

Section: Methodsmentioning

confidence: 99%

Quantitative SUMO proteomics reveals the modulation of several PML nuclear body associated proteins and an anti-senescence function of UBC9

McManus

Bourdeau

Acevedo

et al. 2018

Sci Rep

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Several regulators of SUMOylation have been previously linked to senescence but most targets of this modification in senescent cells remain unidentified. Using a two-step purification of a modified SUMO3, we profiled the SUMO proteome of senescent cells in a site-specific manner. We identified 25 SUMO sites on 23 proteins that were significantly regulated during senescence. Of note, most of these proteins were PML nuclear body (PML-NB) associated, which correlates with the increased number and size of PML-NBs observed in senescent cells. Interestingly, the sole SUMO E2 enzyme, UBC9, was more SUMOylated during senescence on its Lys-49. Functional studies of a UBC9 mutant at Lys-49 showed a decreased association to PML-NBs and the loss of UBC9’s ability to delay senescence. We thus propose both pro- and anti-senescence functions of protein SUMOylation.

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Section: Methodsmentioning

confidence: 99%

Quantitative SUMO proteomics reveals the modulation of several PML nuclear body associated proteins and an anti-senescence function of UBC9

McManus

Bourdeau

Acevedo

et al. 2018

Sci Rep

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“…Recombinant protein components of the SUMO conjugation machinery were produced in E. coli using pET28a-Aos1 (SAE1), pET28b-Uba2 (SAE2), pET23a-Ubc9, pET-11a-SUMO-1, and pET11-hRanGAP1, and were purified as previously described (25). …”

Section: Methodsmentioning

confidence: 99%

“…Protein levels were confirmed by Coomassie Brilliant Blue staining. Pre-acetylated proteins were incorporated into in vitro SUMOylation assays with RanGAP1 substrate based on prior optimization studies; SAE1/SAE2 (140 ng), Ubc9 (220 ng), SUMO-1 (2 μg), RanGAP1 (2 μg, unacetylated) (27). Reaction buffer consisted of 40 mM HEPES pH 7.3, 220 mM KOAc, 4 mM Mg(OAc) 2 , 4 mM DTT, and protease/phosphatase inhibitor cocktail (Thermo Fisher).…”

Section: Methodsmentioning

confidence: 99%

Class I HDAC inhibition stimulates cardiac protein SUMOylation through a post-translational mechanism

Blakeslee

Wysoczynski

Fritz

et al. 2014

Cellular Signalling

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Lysine residues are subject to a multitude of reversible post-translational modifications, including acetylation and SUMOylation. In the heart, enhancement of lysine acetylation or SUMOylation using histone deacetylase (HDAC) inhibitors or SUMO-1 gene transfer, respectively, has been shown to be cardioprotective. Here, we addressed whether there is crosstalk between lysine acetylation and SUMOylation in the heart. Treatment of cardiomyocytes and cardiac fibroblasts with pharmacological inhibitors of HDAC catalytic activity robustly increased conjugation of SUMO-1, but not SUMO-2/3, to several high molecular weight proteins in both cell types. Use of a battery of selective HDAC inhibitors and short hairpin RNAs demonstrated that HDAC2, which is a class I HDAC, is the primary HDAC isoform that controls cardiac protein SUMOylation. HDAC inhibitors stimulated protein SUMOylation in the absence of de novo gene transcription or protein synthesis, revealing a post-translational mechanism of HDAC inhibitor action. HDAC inhibition did not suppress the activity of de-SUMOylating enzymes, suggesting that increased protein SUMOylation in HDAC inhibitor-treated cells is due to stimulation of SUMO-1 conjugation rather than blockade of SUMO-1 cleavage. Consistent with this, multiple components of the SUMO conjugation machinery were capable of being acetylated in vitro. These findings reveal a novel role for reversible lysine acetylation in the control of SUMOylation in the heart, and suggest that cardioprotective actions of HDAC inhibitors are in part due to stimulation of protein SUMO-1-ylation in myocytes and fibroblasts.

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“…Enrichment of native endogenous SUMOylated proteins by cell fractionation is generally limited by the low abundance of such proteins and the action of efficient SUMO proteases [14]. In vitro modification of enriched target proteins with recombinant SUMOylation enzymes is a more promising approach, typically yielding mixtures of modified and unmodified target proteins contaminated with the SUMOylation enzymes, hence requiring subsequent purification steps [15], [16]. Also, co-expression of SUMO targets with SUMO1 and SUMO-activating and -conjugating enzymes of different origin (human, mouse, Xenopus laevis ) in E.coli was shown to produce SUMO-modified protein [17]–[19], but in our experience, the yields of specifically modified proteins were often poor, impeding efficient purification and subsequent biochemical analysis.…”

Section: Introductionmentioning

confidence: 99%

Versatile Recombinant SUMOylation System for the Production of SUMO-Modified Protein

2014

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Posttranslational modification by small ubiquitin-like modifiers (SUMO) is being associated with a growing number of regulatory functions in diverse cellular processes. The biochemical investigation into the underlying molecular mechanisms, however, has been lagging behind due to the difficulty to generate sufficient amounts of recombinant SUMOylated proteins. Here, we present two newly designed two-component vector systems for the expression and purification of SUMO-modified target proteins in Escherichia coli. One system consists of a vector for SUMO conjugation, expressing human SUMO-activating (SAE1/SAE2) and conjugating (Ubc9) enzymes together with His6-tagged SUMO1, 2 or 3, that can be combined with commonly used expression constructs for any gene of interest. To facilitate SUMOylation of targets normally requiring a SUMO-E3 ligase for efficient modification, a second system is designed to express the target protein as a fusion with the human SUMO-conjugating enzyme Ubc9, thus compensating the absence of a potential SUMO ligase. We demonstrate the proficiency of these systems by SUMOylation of two DNA repair proteins, the thymine DNA glycosylase (TDG) and XRCC1, and describe purification schemes for SUMOylated proteins in native and active form. This SUMO toolbox facilitates “in-cell” and “in-extract” production and purification of recombinant SUMO-modified target proteins for functional and structural analysis.

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Recombinant Reconstitution of Sumoylation Reactions In Vitro

Cited by 21 publications

References 16 publications

Quantitative SUMO proteomics reveals the modulation of several PML nuclear body associated proteins and an anti-senescence function of UBC9

Quantitative SUMO proteomics reveals the modulation of several PML nuclear body associated proteins and an anti-senescence function of UBC9

Class I HDAC inhibition stimulates cardiac protein SUMOylation through a post-translational mechanism

Versatile Recombinant SUMOylation System for the Production of SUMO-Modified Protein

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