A Stable Chemical SUMO1–Ubc9 Conjugate Specifically Binds as a Thioester Mimic to the RanBP2–E3 Ligase Complex

Sommer, Stefanie; Ritterhoff, Tobias; Melchior, Frauke; Mootz, Henning D.

doi:10.1002/cbic.201500011

Cited by 6 publications

(6 citation statements)

References 167 publications

(158 reference statements)

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“…Notably, R54 is located outside of the known class II and class III interaction interfaces on opposite surfaces of the protein modifier and thus not expected to report on the corresponding sets of SUMO binding proteins. Bode and co-workers incorporated photo-cross-linking amino acids into synthetically prepared versions of Ubc9 and demonstrated cross-linking with the E3 ligase RanBP2, which is known to form ternary complexes with SUMO-E2 and a substrate protein. − …”

Section: Introductioncontrasting

confidence: 86%

Identification of SUMO Binding Proteins Enriched after Covalent Photo-Cross-Linking

Brüninghoff¹,

Aust²,

Taupitz³

et al. 2020

ACS Chem. Biol.

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Post-translational modification with the small ubiquitinlike modifier (SUMO) affects thousands of proteins in the human proteome and is implicated in numerous cellular processes. The main outcome of SUMO conjugation is a rewiring of protein−protein interactions through recognition of the modifier's surface by SUMO binding proteins. The SUMO-interacting motif (SIM) mediates binding to a groove on SUMO; however, the low affinity of this interaction and the poor conservation of SIM sequences complicates the isolation and identification of SIM proteins. To address these challenges, we have designed and biochemically characterized monomeric and multimeric SUMO-2 probes with a genetically encoded photo-cross-linker positioned next to the SIM binding groove. Following photoinduced covalent capture, even weak SUMO binders are not washed away during the enrichment procedure, and very stringent washing conditions can be applied to remove nonspecifically binding proteins. A total of 329 proteins were isolated from nuclear HeLa cell extracts and identified using mass spectrometry. We found the molecular design of our probes was corroborated by the presence of many established SUMO interacting proteins and the high percentage (>90%) of hits containing a potential SIM sequence, as predicted by bioinformatic analyses. Notably, 266 of the 329 proteins have not been previously reported as SUMO binders using traditional noncovalent enrichment procedures. We confirmed SUMO binding with purified proteins and mapped the position of the covalent cross-links for selected cases. We postulate a new SIM in MRE11, involved in DNA repair. The identified SUMO binding candidates will help to reveal the complex SUMO-mediated protein network.

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Section: Introductioncontrasting

confidence: 86%

Identification of SUMO Binding Proteins Enriched after Covalent Photo-Cross-Linking

Brüninghoff¹,

Aust²,

Taupitz³

et al. 2020

ACS Chem. Biol.

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“…We foresee that UbDha may be used to expedite generation of crystal structures of E1, E2 or E3 enzymes and their complexes 27 . In addition, we hypothesize that the stability of our E2-UbDha adducts immobilized on affinity beads could enable proteomic profiling of cognate RING E3 enzymes, which cannot themselves be directly trapped in a mechanism-dependent manner 43 .…”

Section: Discussionmentioning

confidence: 99%

A cascading activity-based probe sequentially targets E1–E2–E3 ubiquitin enzymes

et al. 2016

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Post-translational modifications of proteins with ubiquitin (Ub) and ubiquitin-like (Ubl) modifiers, orchestrated by a cascade of specialized E1, E2 and E3 enzymes, control a staggering breadth of cellular processes. To monitor catalysis along these complex reaction pathways, we developed a cascading activity-based probe, UbDha. Akin to the native Ub, upon ATP-dependent activation by the E1, UbDha can travel downstream to the E2 (and subsequently E3) enzymes through sequential trans-thioesterifications. Unlike the native Ub, at each step along the cascade UbDha has the option to react irreversibly with active site cysteine residues of target enzymes, thus enabling their detection. We show that our cascading probe ‘hops’ and ‘traps’ catalytically active ubiquitin-modifying enzymes (but not their substrates) by a mechanism diversifiable to Ubls. Our founder methodology, amenable to structural studies, proteome-wide profiling and monitoring of enzymatic activities in living cells, presents novel and versatile tools to interrogate the Ub/Ubl cascades.

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“…SUMOs are a class of important ubiquitin-like proteins. There are four SUMO family members that have been identified in humans (SUMO1-4), by contrast, the SUMO-conjugating enzyme E2 has only one form (UBC9), and RNA interference knockdown of UBC9 expression can effectively block SUMOylation of intracellular proteins (30). SUMOs have both free and conjugate forms that maintain the balance of cellular SUMOylation.…”

Section: Discussionmentioning

confidence: 99%

Moderate hypothermia induces protein SUMOylation in bone marrow stromal cells and enhances their tolerance to hypoxia

Ren

Liu

et al. 2017

Molecular Medicine Reports

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Acute cerebral infarction can progress rapidly, and there are limited specific and effective treatments. Small ubiquitin‑like modifiers (SUMOs) provide an important post‑translational modification of proteins. Following cerebral infarction, multiple proteins can combine with SUMOs to protect nerve cells. Furthermore, moderate hypothermia (core body temperature, 33‑34˚C) can increase the level of SUMOylation on multiple proteins. In the present study, it was examined whether moderate hypothermia increases the survival rate of bone marrow stromal stem cells (BMSCs) implanted in the cerebral ischemic penumbra via SUMOylation of multiple proteins. Firstly, BMSCs were exposed to oxygen‑glucose deprivation (OGD) under moderate hypothermic (33˚C) conditions. Subsequently, adult rats with middle cerebral artery occlusion were treated with a combination of BMSCs and moderate hypothermia (32‑34˚C). The results demonstrated that hypothermia promoted the combination of multiple proteins with SUMOs in BMSCs, and induced transport of SUMOs from the cytoplasm to the nucleus. Moderate hypothermia additionally reduced damage to BMSCs following OGD and improved BMSC survival following transplantation into the penumbra. These data suggest that moderate hypothermia may protect against BMSC injury via rapid SUMOylation of intracellular proteins. Thus, BMSC transplantation combined with moderate hypothermia may be a potential therapeutic strategy to treat cerebral infarction.

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A Stable Chemical SUMO1–Ubc9 Conjugate Specifically Binds as a Thioester Mimic to the RanBP2–E3 Ligase Complex

Cited by 6 publications

References 167 publications

Identification of SUMO Binding Proteins Enriched after Covalent Photo-Cross-Linking

Identification of SUMO Binding Proteins Enriched after Covalent Photo-Cross-Linking

A cascading activity-based probe sequentially targets E1–E2–E3 ubiquitin enzymes

Moderate hypothermia induces protein SUMOylation in bone marrow stromal cells and enhances their tolerance to hypoxia

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