Biochemical Analysis of Protein SUMOylation

Alontaga, Aileen Y.; Bobkova, Ekaterina V.; Chen, Yuan

doi:10.1002/0471142727.mb1029s99

Cited by 9 publications

(14 citation statements)

References 39 publications

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“…In our study, the immunoblotting was performed in reducing condition to prevent formation of non-specific protein aggregates, but without affecting true protein modifications. Additionally, the inclusion of 1 mM DTT to lysis buffer or β-mercaptoethanol to sample buffer, should not affect SUMO or ubiquitin mediated modifications under reducing condition, as previously reported 54 55 . Moreover, we were unable to ascertain phosphorylation status of FoxM1 in vivo, because availability of phosphorylated site-specific FoxM1 antibodies is currently limited.…”

Section: Discussionsupporting

confidence: 54%

Control of regional decidualization in implantation: Role of FoxM1 downstream of Hoxa10 and cyclin D3

Gao

Bian

et al. 2015

Sci Rep

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Appropriate regulation of regional uterine stromal cell decidualization in implantation, at the mesometrial triangle and secondary decidual zone (SDZ) locations, is critical for successful pregnancy, although the regulatory mechanisms remain poorly understood. In this regard, the available animal models that would specifically allow mechanistic analysis of site-specific decidualization are strikingly limited. Our study found that heightened expression of FoxM1, a Forkhead box transcription factor, is regulated during decidualization, and its conditional deletion in mice reveals failure of implantation with regional decidualization defects such as a much smaller mesometrial decidua with enlarged SDZ. Analysis of cell cycle progression during decidualization both in vivo and in vitro demonstrates that the loss of FoxM1 elicits diploid cell deficiency with enhanced arrests prior to mitosis and concomitant upregulation of polyploidy. We further showed that Hoxa10 and cyclin D3, two decidual markers, control transcriptional regulation and intra-nuclear protein translocation of FoxM1 in polyploid cells, respectively. Overall, we suggest that proper regional decidualization and polyploidy development requires FoxM1 signaling downstream of Hoxa10 and cyclin D3.

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Section: Discussionsupporting

confidence: 54%

Control of regional decidualization in implantation: Role of FoxM1 downstream of Hoxa10 and cyclin D3

Gao

Bian

et al. 2015

Sci Rep

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“…Alternatively, RNF4 could interact directly with Ubc13-Uev1A to stimulate the synthesis of K63-linked polyubiquitin chains on SUMO. Consistent with this latter model, in vitro studies indicate that RNF4 and the yeast homolog, the Sxl5/Slx8 heterodimer, can function with multiple E2 enzymes, including Ubc13 [33]. Importantly, depletion of RNF4 from cells also reduces the accumulation of K63-linked polyubiquitin chains at sites of DNA damage [25].…”

Section: Discussionmentioning

confidence: 95%

RNF4-Dependent Hybrid SUMO-Ubiquitin Chains Are Signals for RAP80 and Thereby Mediate the Recruitment of BRCA1 to Sites of DNA Damage

et al. 2012

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The DNA repair function of the breast cancer susceptibility protein BRCA1 depends in part on its interaction with RAP80, which targets BRCA1 to DNA double strand breaks (DSBs) through recognition of K63-linked polyubiquitin chains. The localization of BRCA1 to DSBs also requires sumoylation. Here, we demonstrated that, in addition to having ubiquitin-interacting motifs, RAP80 also contains a SUMO-interacting motif (SIM) that is critical for recruitment to DSBs. In combination with the ubiquitin-binding activity of RAP80, this SIM enabled RAP80 to bind with nanomolar affinity to hybrid chains consisting of ubiquitin conjugated to SUMO. Furthermore, RNF4, a SUMO-targeted ubiquitin E3 ligase that synthesizes hybrid SUMO-ubiquitin chains, localized to DSBs and was critical for the recruitment of RAP80 and BRCA1 to sites of DNA damage. Our findings, therefore, connect ubiquitin-dependent and SUMO-dependent DSB recognition, revealing that RNF4 synthesized hybrid SUMO-ubiquitin chains are recognized by RAP80 to promote BRCA1 recruitment and DNA repair.

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“…Since there are currently no such high throughput assays that utilize single purified enzymes in the SUMO pathway (Alontaga et al, 2012; Carlson et al, 2009), we turned to a reconstituted biochemical cascade using recombinant SUMO-1, SUMO-activating enzyme 1/2 (SAE-1/2, the SUMO E1 enzyme) and UBC9 (the SUMO E2 enzyme) proteins with a peptide substrate. Importantly, this approach enables the assay to simultaneously probe for inhibitors of both the E1 and E2 enzymes.…”

Section: Resultsmentioning

confidence: 99%

“…Our initial screening assay involved a reconstituted sumoylation cascade, including recombinant SAE 1/2, UBC9, and SUMO-1. In order to evaluate whether 2-D08 inhibited individual steps in the cascade, we utilized a fluorescently labeled SUMO-1 (referred to as FL-SUMO-1) (Alontaga et al, 2012), rather than a fluorescent substrate. Straightforward gel shift experiments clearly indicated the activation and transfer of SUMO-1 between the enzymes.…”

Section: Resultsmentioning

confidence: 99%

“…There are few biochemical assays available to identify and evaluate small molecule inhibitors in these pathways, and the kinetic study of sumoylation remains nontrivial (Alontaga et al, 2012). In the specific case of sumoylation there have only been two reports of small molecule inhibitors to date, both of which appear to affect the E1 SUMO activating enzyme (SAE) (Fukuda et al, 2009a; Fukuda et al, 2009b).…”

Section: Introductionmentioning

confidence: 99%

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An Electrophoretic Mobility Shift Assay Identifies a Mechanistically Unique Inhibitor of Protein Sumoylation

Kim

Nagy

Keyser

et al. 2013

Chemistry & Biology

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SUMMARY The dynamic, posttranslational modification of proteins with a SUMO tag has been recognized as an important cellular regulatory mechanism relevant to a number of cancers as well as normal embryonic development. As part of a program aimed towards the identification of inhibitors of SUMO conjugating enzymes, we have developed a microfluidic electrophoretic mobility shift assay to monitor sumoylation events in real time. We disclose herein the use of this assay to discover the first cell permeable compound capable of blocking the transfer of SUMO-1 from the E2 enzyme UBC9 to the substrate. We screened a small collection of compounds and identified an oxygenated flavonoid derivative that inhibits sumoylation in vitro. Next, we carried out an in-depth mechanistic analysis that ruled out many common false positive mechanisms such as aggregation or alkylation. Furthermore, we report that this flavonoid inhibits a single step in the sumoylation cascade: the transfer of SUMO from the E2 enzyme (UBC9) thioester conjugate to the substrate. In addition to being the first example of a compound with this unique mechanism of action, this inhibitor has a discreet structure-activity relationship uncharacteristic of a promiscuous inhibitor. Cell-based studies showed that the flavonoid inhibits the sumoylation of topoisomerase-I in response to camptothecin treatment in two different breast cancer cell lines, while isomeric analogs are inactive. Importantly, this compound blocks sumoylation while not affecting ubiquitylation in cells. This work identifies a novel point of entry for pharmacological inhibition of the sumoylation cascade, and will serve as the basis for continued study of additional pharmacophores that modulate SUMO-conjugating enzymes such as UBC9.

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Biochemical Analysis of Protein SUMOylation

Cited by 9 publications

References 39 publications

Control of regional decidualization in implantation: Role of FoxM1 downstream of Hoxa10 and cyclin D3

Control of regional decidualization in implantation: Role of FoxM1 downstream of Hoxa10 and cyclin D3

RNF4-Dependent Hybrid SUMO-Ubiquitin Chains Are Signals for RAP80 and Thereby Mediate the Recruitment of BRCA1 to Sites of DNA Damage

An Electrophoretic Mobility Shift Assay Identifies a Mechanistically Unique Inhibitor of Protein Sumoylation

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