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SUMOylated proteins upon ubiquitin E1 inhibition

ABSTRACT: HIS-SUMO2 expressing HeLa and U2OS cells were treated with ubiquitin E1 inhibitor TAK243 with or without the presence of cycloheximide. Subsequently, HIS-SUMO2 conjugates were purified and analyzed by LC-MS/MS

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Hela Cell

SUBMITTER: Román González-Prieto

LAB HEAD: Alfred C.O. Vertegaal

PROVIDER: PXD011852 | Pride | 2019-07-09

REPOSITORIES: Pride

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			Action	DRS
	SamplenumberingShaetal..xlsx	Xlsx
	q103639a.raw	Raw
	q103640a.raw	Raw
	q103641a.raw	Raw
	q103642a.raw	Raw

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Publications

Inhibiting ubiquitination causes an accumulation of SUMOylated newly synthesized nuclear proteins at PML bodies.

Sha Zhe Z Blyszcz Tamara T González-Prieto Román R Vertegaal Alfred C O ACO Goldberg Alfred L AL

The Journal of biological chemistry 20190708 42

Protein ubiquitination and SUMOylation are required for the maintenance of cellular protein homeostasis, and both increase in proteotoxic conditions (<i>e.g.</i> heat shock or proteasome inhibition). However, we found that when ubiquitination was blocked in several human cell lines by inhibiting the ubiquitin-activating enzyme with TAK243, there was an unexpected, large accumulation of proteins modified by SUMO2/3 chains or SUMO1, but not by several other ubiquitin-like proteins. This buildup of ...[more]

PMID: 31285264

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PML Nuclear Bodies are stress-responsive sumoylation/degradation compartments regulating KAP1 silencing function in embryonic stem cells

Project description:PML drives the assembly of PML Nuclear Bodies (NBs), where it recruits many serendipitously-identified proteins, including the SUMO-E2 enzyme UBC9. Interferons, arsenic or oxidative stress, all enhance NB-formation. Here demonstrate that PML is required for efficient basal sumoylation in mouse embryonic stem cells (mESCs) and immediate stress-responsive sumoylation in mouse liver or Acute Promyelocytic Leukemia (APL). Biochemically, PML NBs confer an oxidation-protective environment for the redox-sensitive UBC9 activity, which, together with enzyme/target concentration/immobilization, favors processive SUMO2/3 conjugation. Accordingly, PML-facilitated sumoylation is accompanied by ubiquitination and degradation of some targets. In vivo proteomic analysis of SUMO2 conjugates in APL identified therapy-responsive targets, among which the prominent KAP1 complex of epigenetic regulators. In mESCs, PML is required for SUMO-dependent KAP1 repressive activity and for the resulting silencing of transposable elements and 2-cell-like genes. The ES status is further regulated by a PML-facilitated repressive sumoylation of the Dppa2 zygote genome activation driver. These findings establish the biochemical function of PML NBs in vivo and unveiling an unsuspected epigenetic control of key ESC targets by PML.

2022-09-14 | PXD019609 | Pride

SUMOYLATION AND PROTEOTOSASIS NETWORKS CONVERGE DURING HEAT SHOCK TO MINIMIZE AGGREGATION AND PROMOTE DEGRADATION OF NUCLEAR PROTEINS

Project description:The role of stress-induced increases in SUMO2/3 conjugation during the Heat Shock Response (HSR) has remained enigmatic. We investigated SUMO signal transduction at the proteomic and functional level during the HSR in the context of cells depleted of proteostasis network components via chronic Heat Shock Factor 1 inhibition versus cells with normal pro-teostasis networks. In the recovery phase post-heat shock, SUMO2/3 conjugation remained high for a prolonged time in cells lacking sufficient chaperones. Similar results were obtained upon inhibiting HSP90, indicating that increased chaperone activity during the HSR is critical for the recovery of SUMO2/3 levels post-heat shock. Proteasome inhibition during the re-covery phase likewise prolonged SUMO2/3 conjugation, indicating that stress-induced SU-MO2/3 targets are subsequently degraded by the ubiquitin-proteasome system. Functionally, we show that SUMOylation profoundly enhances solubility of target proteins upon heat shock in vitro. Collectively, our results implicate SUMO2/3 as a rapid response factor protecting the proteome from aggregation upon proteotoxic stress.

2019-03-25 | PXD010231 | Pride

Unbiased in vivo exploration of nuclear bodies-enhanced sumoylation reveals that PML orchestrates embryonic stem cell fate (II)

Project description:Membrane-less organelles are condensates formed by phase separation whose functions often remain enigmatic. Upon oxidative stress, PML scaffolds Nuclear Bodies (NBs) to regulate senescence or metabolic adaptation, but their role in pluripotency remains elusive. Here we establish that PML is required for basal SUMO2/3 conjugation in mESCs and oxidative stress-driven sumoylation in mESCs or in vivo. PML NBs create an oxidationprotective environment for UBC9-driven SUMO2/3 conjugation of PML partners, often followed by their poly-ubiquitination and degradation. Differential in vivo proteomics identified several members of the KAP1 complex as PML NB-dependent SUMO2-targets. The latter drives functional activation of this key epigenetic repressor. Accordingly, Pml-/- mESCs reexpress transposable elements and display features of totipotent-like cells, a process further enforced by PML-controlled SUMO2-conjugation of DPPA2. Finally, PML is required for adaptive stress responses in mESCs. Collectively, PML orchestrates mESC fate through SUMO2-conjugation of key transcriptional or epigenetic regulators, raising new mechanistic hypotheses about PML roles in normal or cancer stem cells.

2023-03-06 | PXD032240 | Pride

Unbiased in vivo exploration of nuclear bodies-enhanced sumoylation reveals that PML orchestrates embryonic stem cell fate

2022-09-14 | PXD028865 | Pride

Changes to SUMO-modified proteome and total cellular proteome during EBV ractivation in AGS gastric carcinoma cells

Project description:To monitor changes to cellular and viral SUMO-modified proteins during EBV reactivation. SUMO1 and SUMO2 conjugates were studied using KGG mutant forms of SUMO and GG-K specific antibody enrichment of SUMO modified peptides. Time-points of 12h and 24h post-reactivation were monitored. Total cellular proteome was also analysed to study changes to the total protein levels of proteins under the same conditions.

2023-06-19 | PXD034631 | Pride

Identification of SUMO targets required to maintain human stem cells in the pluripotent state. - Identification of SUMO modified proteins from hiPS cells

Project description:To determine the role of SUMO modification in the maintenance of pluripotent stem cells we used ML792, a potent and selective inhibitor of SUMO Activating Enzyme. Treatment of human induced pluripotent stem cells with ML792 initiated changes associated with loss of pluripotency such as reduced expression of key pluripotency markers. To identify putative effector proteins and establish sites of SUMO modification, cells were engineered to stably express either SUMO1 or SUMO2 with TGG to KGG mutations that facilitate GlyGly-K peptide immunoprecipitation and identification. A total of 976 SUMO sites were identified in 427 proteins. STRING enrichment created 3 networks of proteins with functions in regulation of gene expression, ribosome biogenesis and RNA splicing, although the latter two categories represented only 5% of the total GGK peptide intensity. The remainder have roles in transcription and chromatin structure regulation. Many of the most heavily SUMOylated proteins form a network of zinc-finger transcription factors centred on TRIM28 and associated with silencing of retroviral elements. At the level of whole proteins there was only limited evidence for SUMO paralogue-specific modification, although at the site level there appears to be a preference for SUMO2 modification over SUMO1 in acidic domains. We show that SUMO is involved in the maintenance of the pluripotent state in hiPSCs and identify many chromatin-associated proteins as bona fide SUMO substrates in human induced pluripotent stem cells.

2021-11-03 | PXD028050 | Pride

Transcriptional targets of EphB2 and Ephrin-A4 signaling in epidermal keratinocytes

Project description:Human epidermal keratinocytes were treated with 25 ng.ml EphB2 or EFNA4, both as-Fc conjugates (Sigma). Human epidermal keratinocytes are treated with 25 ng/ml EphB2 or EFNA4 Fc conjugates in a 48hr time course.

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SUMO proteomics in Caenorhabditis elegans

Project description:Comprehensive list of SUMO targets from the nematode Caenorhabditis elegans. SUMO conjugates isolated from transgenic worms carrying 8His and GFP tagged SUMO. The constructs rescues the lethal knock-out of a single SUMO gene, smo-1. SUMO conjugates where isolated from heat shock, arsenite exposure, and UV treated SUMO-GFP worms as well as from control non treated animals. In parallel identical purification procedure was performed with non-transgenic worms and proteins identified with this control where excluded.

2018-01-24 | PXD006644 | Pride

Targets of Ephrin-A signaling in epidermal keratinocytes

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