The Isolated C-Terminal Domain of Ring1B Is a Dimer Made of Stable, Well-Structured Monomers

Czypionka, Anna; Paños, Olga Ruiz de los; Mateu, Mauricio G.; Barrera, Francisco N.; Hurtado-Gómez, Estefanía; Gómez, Javier; Vidal, Miguel; Neira, José L.

doi:10.1021/bi701343q

Cited by 50 publications

(103 citation statements)

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“…Fluorescence spectra were collected on a Cary Varian spectrofluorimeter (Agilent) interfaced with a Peltier, at 25°C. Experiments were carried out at pH 6.8 (20 mM phosphate buffer), where C-RING1B has a wellfolded conformation (17). The samples were prepared the day before and left overnight at 5°C.…”

Section: Methodsmentioning

confidence: 99%

“…C-RING1B is a dimeric protein (its self-association constant is ∼200 μM) (17). Except for the NMR titration experiments, the rest of the experiments were carried out under conditions where the dominant C-RING1B species was the monomer.…”

Section: Structural Determinants Of the C-ring1b/nupr1 Interaction Inmentioning

confidence: 99%

“…S3). We did not determine K d from changes in the denaturation midpoints due to the irreversibility of C-RING1B thermal denaturations (17) (with all NUPR1 species we observed precipitation). Thus, we conclude that we could detect binding by far-UV CD of wild-type NUPR1 and C-RING1B, leading to changes in the secondary structure of, at least, one of the proteins.…”

mentioning

confidence: 99%

“…We have described the conformational properties of the C-terminal domain of RING1B (C-RING1B), encompassing the residues 227-334 (17), and shown that it is a dimer of well-formed monomers. The X-ray structure of the monomers of C-RING1B resembles that of a ubiquitin module, and it serves to interact with CBX proteins (18).…”

mentioning

confidence: 99%

“…We carried out the experiments at pH 7.0, where C-RING1B was stable (17). At this pH, the most protected residues from exchange of wild-type NUPR1 that could be unambiguously assigned were (Fig.…”

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confidence: 99%

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Intrinsically disordered chromatin protein NUPR1 binds to the C-terminal region of Polycomb RING1B

Santofimia‐Castaño

Rizzuti

Pey

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Intrinsically disordered proteins (IDPs) are ubiquitous in eukaryotes, and they are often associated with diseases in humans. The protein NUPR1 is a multifunctional IDP involved in chromatin remodeling and in the development and progression of pancreatic cancer; however, the details of such functions are unknown. Polycomb proteins are involved in specific transcriptional cascades and gene silencing. One of the proteins of the Polycomb complex is the Ring finger protein 1 (RING1). RING1 is related to aggressive tumor features in multiple cancer types. In this work we characterized the interaction between NUPR1 and the paralogue RING1B in vitro, in silico, and in cellulo. The interaction occurred through the C-terminal region of RING1B (C-RING1B), with an affinity in the low micromolar range (∼10 μM). The binding region of NUPR1, mapped by NMR, was a hydrophobic polypeptide patch at the 30s region of its sequence, as pinpointed by computational results and site-directed mutagenesis at Ala33. The association between C-RING1B and wild-type NUPR1 also occurred in cellulo as tested by protein ligation assays; this interaction is inhibited by trifluoperazine, a drug known to hamper binding of wild-type NUPR1 with other proteins. Furthermore, the Thr68Gln and Ala33Gln/Thr68Gln mutants had a reduction in the binding toward C-RING1B as shown by in vitro, in silico, and in cellulo studies. This is an example of a well-folded partner of NUPR1, because its other interacting proteins are also unfolded. We hypothesize that NUPR1 plays an active role in chromatin remodeling and carcinogenesis, together with Polycomb proteins.G ene expression control occurs through the combined activities of transcription factors and chromatin regulators, considered as effectors of epigenetic mechanisms. Posttranslational modifications of nucleosomal histones, DNA methylation, local compaction, and long-range interactions determine a variety of chromatin structures that can affect the transcriptional output.A group of chromatin regulators are the Polycomb Repressive complexes (PRCs) (1, 2). These Polycomb group (PcG) proteins are encoded by genes initially discovered over 60 years ago as repressors of the Hox genes in Drosophila (3). The PcG proteins form two main silencing heteromeric complexes called PRC1 and PRC2; both are highly conserved in eukaryotes and either in isolation or synergistically can silence genes (4, 5). The catalytic functions of both complexes include ubiquitin-ligase (H3K119ub1) and methyltransferase activity (H3K27Me3), respectively. In mammals, the PRC2 core is formed by, at least, four heteromeric units, one of which is EZH2 (or its paralog, EZH1), the methyltransferase that catalyzes the trimethylation of histone H3 at Lys27 (5). This modification can act as an anchoring site of PRC1 complexes containing chromobox (CBX) proteins. These CBX proteins recruit PRC1 complex onto PRC2-enriched chromatin, facilitating the monoubiquitylation. The PRC1-dependent monoubiquitylation at Lys119 of histone H2A correlates with transc...

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Section: Methodsmentioning

confidence: 99%

Section: Structural Determinants Of the C-ring1b/nupr1 Interaction Inmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Intrinsically disordered chromatin protein NUPR1 binds to the C-terminal region of Polycomb RING1B

Santofimia‐Castaño

Rizzuti

Pey

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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A survey of the year 2007 literature on applications of isothermal titration calorimetry

Bjelić

Jelesarov

2008

J of Molecular Recognition

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Characterization of Reversible Associations by Sedimentation Velocity with UltraScan

Demeler

Brookes

Wang

et al. 2010

Macromolecular Bioscience

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We compare here the utility of sedimentation velocity (SV) to sedimentation equilibrium (SE) analysis for the characterization of reversible systems. Genetic algorithm optimization in UltraScan is used to optimize the model and to obtain solution properties of all components present in the system. We apply our method to synthetic and experimental data, and suggest limits for the accessible kinetic range. We conclude that equilibrium constants obtained from SV and SE analysis are equivalent, but that SV experiments provide better confidence for the K(d), can better account for the presence of contaminants and provide additional information including rate constants and shape parameters.

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The Isolated C-Terminal Domain of Ring1B Is a Dimer Made of Stable, Well-Structured Monomers

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Cited by 50 publications

References 71 publications

Intrinsically disordered chromatin protein NUPR1 binds to the C-terminal region of Polycomb RING1B

Intrinsically disordered chromatin protein NUPR1 binds to the C-terminal region of Polycomb RING1B

A survey of the year 2007 literature on applications of isothermal titration calorimetry

Characterization of Reversible Associations by Sedimentation Velocity with UltraScan

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