Synthetic E2-Ub-nucleosome conjugates for studying nucleosome ubiquitination

Ai, Huasong; Tong, Zebin; Deng, Zhiheng; Tian, Jiakun; Zhang, Liying; Sun, Maoshen; Du, Yunxiang; Shen, Qiang; Liang, Lujun; Zheng, Qin; Li, Jiabin; Pan, Man; Liu, Lei

doi:10.1016/j.chempr.2023.01.012

Cited by 33 publications

(32 citation statements)

References 53 publications

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“…Taken together, our results establish a conceptually new family of chemical tools for time-resolved studies on protein ubiquitination in live cells and further exemplify the utility of chemically synthesized customized probes that are more readily synthesizable and are of different structural designs for the study of Ub modifications. − It should be mentioned that although the current photocaging group, Nbg, requires 365 nm of UV light and may cause cytotoxicity in some experiments, our probe can be easily extended to use more benign photosensitive groups that would meet the requirements for specific biological conditions.…”

Section: Discussionmentioning

confidence: 62%

Cell-Permeable Stimuli-Responsive Ubiquitin Probe for Time-Resolved Monitoring of Substrate Ubiquitination in Live Cells

Liang,

Wang,

Hua

et al. 2023

JACS Au

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Dynamic monitoring of intracellular ubiquitin (Ub) conjugates is instrumental to understanding the Ub regulatory machinery. Although many biochemical approaches have been developed to characterize protein ubiquitination, chemical tools capable of temporal resolution probing of ubiquitination events remain to be developed. Here, we report the development of the first cell-permeable and stimuli-responsive Ub probe and its application for the temporal resolution profiling of ubiquitinated substrates in live cells. The probe carrying the photolabile group N-(2-nitrobenzyl)-Gly (Nbg) on the amide bond between Ub Gly75 and Gly76 is readily prepared through chemical synthesis and can be delivered to live cells by conjugation via a disulfide bond with the cyclic cell-penetrating peptide cR10D (i.e., 4-((4-(dimethylamino)phenyl)-azo)-benzoic acid-modified cyclic deca-arginine). Both in vitro and in vivo experiments showed that Ub-modifying enzymes (E1, E2s, and E3s) could not install the Ub probe onto substrate proteins prior to removal of the nitrobenzyl group, which was easily accomplished via photoirradiation. The utility and practicality of this probe were exemplified by the timeresolved biochemical and proteomic investigation of ubiquitination events in live cells during a H 2 O 2 -mediated oxidative stress response. This work shows a conceptually new family of chemical Ub tools for the time-resolved studies on dynamic protein ubiquitination in different biological processes and highlights the utility of modern chemical protein synthesis in obtaining customdesigned tools for biological studies.

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Section: Discussionmentioning

confidence: 62%

Cell-Permeable Stimuli-Responsive Ubiquitin Probe for Time-Resolved Monitoring of Substrate Ubiquitination in Live Cells

Liang,

Wang,

Hua

et al. 2023

JACS Au

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“…The procedure of nucleosome reconstitution was consistent with previous work 38,39 , except for minor modifications. Generally, histone octamer and nucleosomal DNA were mixed at a stoichiometric ratio of 1:1 under high salt conditions (approximately 2 M), then the mixture was dialyzed against histone refolding buffer at 4 using a dialysis tube (D-Tube TM Dialyzer Medi or Maxi, MWCO 6-8 kDa, Merck).…”

Section: Nucleosome Reconstitutionmentioning

confidence: 80%

“…The assembly of histone octamer was based on the previous report with minor modifications 38,39 . Briefly, histone H2A (or H2AUb), H2B, H3 and H4 (15 μM each) were weighted and dissolved in histone unfolding buffer (6 M Gn·HCl, 20 mM Tris, 1 mM DTT, pH 7.5), and dialyzed against histone refolding buffer (2 M NaCl, 10 mM Tris, 1 mM EDTA, pH 7.5) at 4 □.…”

Section: Methodsmentioning

confidence: 99%

Synovial 1 Sarcoma X Breakpoint Protein Uses a Cryptic Groove to Selectively Recognize H2AK119Ub Nucleosomes

Tong

et al. 2023

Preprint

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The cancer-specific fusion oncoprotein SS18-SSX1 disturbs chromatin accessibility by hijacking the chromatin-remodeling BAF complex from the promoters or enhancers to the H2AK119Ub-marked polycomb-repressed chromatin regions. Relocation of the BAF complex was achieved through selective recognition of the H2AK119Ub nucleosomes by the synovial sarcoma X breakpoint 1 protein SSX1, which neither has a ubiquitin (Ub)-binding domain nor binds to free Ub. To elucidate the mechanism by which SSX1 selectively recognizes H2AK119Ub nucleosomes, here we report the cryo-EM structure of SSX1 bound to H2AK119Ub nucleosomes at a resolution of 3.1 Angstrom. The structure reveals that the SSX1 N-terminal basic region (residues W164, R167, L168, R169) is anchored to the H2A/H2B acidic patch, while the aromatic amino acid (residue Y177) in the middle of SSX1 interacts with the hydrophobic pocket formed by the H2A α2 and α3 helixes. Ub recognition by SSX1 is unique and depends on a cryptic basic groove formed by H3 (residues R49, R52, K56) and the Ub motif (residues R42, R72, R74) on the H2AK119 site. Our results describe a novel mode of site-specific ubiquitinated nucleosome recognition that underlies the specific hijacking of the BAF complex to polycomb regions by SS18-SSX1 in synovial sarcoma.

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“…Its combination with other site-specic conjugation chemistries may further facilitate the installation of more complex ubiquitin units onto other protein substrates. 47,48…”

Section: Discussionmentioning

confidence: 99%

The expedient, CAET-assisted synthesis of dual-monoubiquitinated histone H3 enables evaluation of its interaction with DNMT1

Tong

Gong

et al. 2023

Chem. Sci.

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Synthetic E2-Ub-nucleosome conjugates for studying nucleosome ubiquitination

Cited by 33 publications

References 53 publications

Cell-Permeable Stimuli-Responsive Ubiquitin Probe for Time-Resolved Monitoring of Substrate Ubiquitination in Live Cells

Cell-Permeable Stimuli-Responsive Ubiquitin Probe for Time-Resolved Monitoring of Substrate Ubiquitination in Live Cells

Synovial 1 Sarcoma X Breakpoint Protein Uses a Cryptic Groove to Selectively Recognize H2AK119Ub Nucleosomes

The expedient, CAET-assisted synthesis of dual-monoubiquitinated histone H3 enables evaluation of its interaction with DNMT1

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