48 research outputs found
WWP2 ubiquitylates RNA polymerase II for DNA-PK-dependent transcription arrest and repair at DNA breaks
DNA double-strand breaks (DSBs) at RNA polymerase II (RNAPII) transcribed genes lead to inhibition of transcription. The DNA-dependent protein kinase (DNA-PK) complex plays a pivotal role in transcription inhibition at DSBs by stimulating proteasome-dependent eviction of RNAPII at these lesions. How DNA-PK triggers RNAPII eviction to inhibit transcription at DSBs remains unclear. Here we show that the HECT E3 ubiquitin ligase WWP2 associates with components of the DNA-PK and RNAPII complexes and is recruited to DSBs at RNAPII transcribed genes. In response to DSBs, WWP2 targets the RNAPII subunit RPB1 for K48-linked ubiquitylation, thereby driving DNA-PK- and proteasome-dependent eviction of RNAPII. The lack of WWP2 or expression of nonubiquitylatable RPB1 abrogates the binding of nonhomologous end joining (NHEJ) factors, including DNA-PK and XRCC4/DNA ligase IV, and impairs DSB repair. These findings suggest that WWP2 operates in a DNA-PK-dependent shutoff circuitry for RNAPII clearance that promotes DSB repair by protecting the NHEJ machinery from collision with the transcription machinery
Identification of proximal SUMO-dependent interactors using SUMO-ID
The fast dynamics and reversibility of posttranslational modifications by the ubiquitin family pose significant challenges for research. Here we present SUMO-ID, a technology that merges proximity biotinylation by TurboID and protein-fragment complementation to find SUMO-dependent interactors of proteins of interest. We develop an optimized split-TurboID version and show SUMO interaction-dependent labelling of proteins proximal to PML and RANGAP1. SUMO-dependent interactors of PML are involved in transcription, DNA damage, stress response and SUMO modification and are highly enriched in SUMO Interacting Motifs, but may only represent a subset of the total PML proximal proteome. Likewise, SUMO-ID also allow us to identify interactors of SUMOylated SALL1, a less characterized SUMO substrate. Furthermore, using TP53 as a substrate, we identify SUMO1, SUMO2 and Ubiquitin preferential interactors. Thus, SUMO-ID is a powerful tool that allows to study the consequences of SUMO-dependent interactions, and may further unravel the complexity of the ubiquitin code.Cancer Signaling networks and Molecular Therapeutic
BRCA1/BARD1 ubiquitinates PCNA in unperturbed conditions to promote continuous DNA synthesis
Deficiencies in the BRCA1 tumor suppressor gene are the main cause of hereditary breast and ovarian cancer. BRCA1 is involved in the Homologous Recombination DNA repair pathway and, together with BARD1, forms a heterodimer with ubiquitin E3 activity. The relevance of the BRCA1/BARD1 ubiquitin E3 activity for tumor suppression and DNA repair remains controversial. Here, we observe that the BRCA1/BARD1 ubiquitin E3 activity is not required for Homologous Recombination or resistance to Olaparib. Using TULIP2 methodology, which enables the direct identification of E3-specific ubiquitination substrates, we identify substrates for BRCA1/BARD1. We find that PCNA is ubiquitinated by BRCA1/BARD1 in unperturbed conditions independently of RAD18. PCNA ubiquitination by BRCA1/BARD1 avoids the formation of ssDNA gaps during DNA replication and promotes continuous DNA synthesis. These results provide additional insight about the importance of BRCA1/BARD1 E3 activity in Homologous Recombination.Research and publication of this work was funded by the EMERGIA 2020 program (EMERGIA20_00276) from the ConsejerÃa de EconomÃa, Conocimiento, Empresas y Universidad, Junta de AndalucÃa, Spain to R.G.-P. Research was additionally supported by a Young Investigator Grant from the Dutch Cancer Society (KWF-KIG 11367/2017-2) and Plan Propio de Investigación VI-PP-A.Talento-IV.2 from the University of Sevilla, grants CNS2022-135216 funded by MICIU/AEI/10.13039/501100011033 and by European Union NextGenerationEU/PRTR and PID2021-122361NA-I00 by MICIU/AEI/10.13039/501100011033 and by European Union to R.G.-P. Work in the laboratory of A.C.O.V. has been supported by the European Research Council (ERC; grant 310913) and the Dutch Research Council (NWO; grant 724.016.003). N.G.-R. was supported by the EMERGIA 2021 program (EMERGIA21_00057) from the ConsejerÃa de Universidad, Investigación e Innovación, Andalusian Regional Government- Junta de AndalucÃa to NG-R.Peer reviewe
Human Polycomb 2 Protein Is a SUMO E3 Ligase and Alleviates Substrate-Induced Inhibition of Cystathionine β-Synthase Sumoylation
Human cystathionine β-synthase (CBS) catalyzes the first irreversible
step in the transsulfuration pathway and commits homocysteine to the synthesis
of cysteine. Mutations in CBS are the most common cause of severe hereditary
hyperhomocysteinemia. A yeast two-hybrid approach to screen for proteins that
interact with CBS had previously identified several components of the
sumoylation pathway and resulted in the demonstration that CBS is a substrate
for sumoylation. In this study, we demonstrate that sumoylation of CBS is
enhanced in the presence of human polycomb group protein 2 (hPc2), an
interacting partner that was identified in the initial yeast two-hybrid screen.
When the substrates for CBS, homocysteine and serine for cystathionine
generation and homocysteine and cysteine for H2S generation, are
added to the sumoylation mixture, they inhibit the sumoylation reaction, but
only in the absence of hPc2. Similarly, the product of the CBS reaction,
cystathionine, inhibits sumoylation in the absence of hPc2. Sumoylation in turn
decreases CBS activity by ∼28% in the absence of hPc2 and by
70% in its presence. Based on these results, we conclude that hPc2
serves as a SUMO E3 ligase for CBS, increasing the efficiency of sumoylation. We
also demonstrate that γ-cystathionase, the second enzyme in the
transsulfuration pathway is a substrate for sumoylation under in vitro
conditions. We speculate that the role of this modification may be for nuclear
localization of the cysteine-generating pathway under conditions where nuclear
glutathione demand is high
Uncovering Ubiquitin and Ubiquitin-like Signaling Networks
Microscopic imaging and technolog
Interaction for Immersive Analytics
International audienceIn this chapter, we briefly review the development of natural user interfaces and discuss their role in providing human-computer interaction that is immersive in various ways. Then we examine some opportunities for how these technologies might be used to better support data analysis tasks. Specifically, we review and suggest some interaction design guidelines for immersive analytics. We also review some hardware setups for data visualization that are already archetypal. Finally, we look at some emerging system designs that suggest future directions
Ubiquitin-specific Protease 11 (USP11) Deubiquitinates Hybrid Small Ubiquitin-like Modifier (SUMO)-Ubiquitin Chains to Counteract RING Finger Protein 4 (RNF4)
Ring finger protein 4 (RNF4) is a SUMO-targeted ubiquitin E3 ligase with a pivotal function in the DNA damage response (DDR). SUMO interaction motifs (SIMs) in the N-terminal part of RNF4 tightly bind to SUMO polymers, and RNF4 can ubiquitinate these polymers in vitro. Using a proteomic approach, we identified the deubiquitinating enzyme ubiquitin-specific protease 11 (USP11), a known DDR-component, as a functional interactor of RNF4. USP11 can deubiquitinate hybrid SUMO-ubiquitin chains to counteract RNF4. SUMO-enriched nuclear bodies are stabilized by USP11, which functions downstream of RNF4 as a counterbalancing factor. In response to DNA damage induced by methyl methanesulfonate, USP11 could counteract RNF4 to inhibit the dissolution of nuclear bodies. Thus, we provide novel insight into cross-talk between ubiquitin and SUMO and uncover USP11 and RNF4 as a balanced SUMO-targeted ubiquitin ligase/protease pair with a role in the DDR
SENP6 regulates localization and nuclear condensation of DNA damage response proteins by group deSUMOylation
Abstract The SUMO protease SENP6 maintains genomic stability, but mechanistic understanding of this process remains limited. We find that SENP6 deconjugates SUMO2/3 polymers on a group of DNA damage response proteins, including BRCA1-BARD1, 53BP1, BLM and ERCC1-XPF. SENP6 maintains these proteins in a hypo-SUMOylated state under unstressed conditions and counteracts their polySUMOylation after hydroxyurea-induced stress. Co-depletion of RNF4 leads to a further increase in SUMOylation of BRCA1, BARD1 and BLM, suggesting that SENP6 antagonizes targeting of these proteins by RNF4. Functionally, depletion of SENP6 results in uncoordinated recruitment and persistence of SUMO2/3 at UVA laser and ionizing radiation induced DNA damage sites. Additionally, SUMO2/3 and DNA damage response proteins accumulate in nuclear bodies, in a PML-independent manner driven by multivalent SUMO-SIM interactions. These data illustrate coordinated regulation of SUMOylated DNA damage response proteins by SENP6, governing their timely localization at DNA damage sites and nuclear condensation state
Sumoylation of poly(ADP-ribose) polymerase 1 inhibits its acetylation and restrains transcriptional coactivator function
Poly(ADP-ribose) polymerase 1 (PARP1)
is a chromatin-associated nuclear protein and functions
as a molecular stress sensor. At the cellular level,
PARP1 has been implicated in a wide range of processes,
such as maintenance of genome stability, cell
death, and transcription. PARP1 functions as a transcriptional
coactivator of nuclear factor B (NF-B)
and hypoxia inducible factor 1 (HIF1). In proteomic
studies, PARP1 was found to be modified by small
ubiquitin-like modifiers (SUMOs). Here, we characterize
PARP1 as a substrate for modification by SUMO1
and SUMO3, both in vitro and in vivo. PARP1 is
sumoylated at the single lysine residue K486 within its
automodification domain. Interestingly, modification
of PARP1 with SUMO does not affect its ADP-ribosylation
activity but completely abrogates p300-mediated
acetylation of PARP1, revealing an intriguing crosstalk
of sumoylation and acetylation on PARP1. Genetic
complementation of PARP1-depleted cells with wildtype
and sumoylation-deficient PARP1 revealed that
SUMO modification of PARP1 restrains its transcriptional
coactivator function and subsequently reduces
gene expression of distinct PARP1-regulated target
genes. Messner, S., Schuermann, D., Altmeyer, M.,
Kassner, I., Schmidt, D., Scha¨r, P., Mu¨ller, S., and
Hottiger, M. O. Sumoylation of poly(ADP-ribose) polymerase
1 inhibits its acetylation and restrains transcriptional
coactivator function