Photoactive G-Quadruplex Ligand Identifies Multiple G-Quadruplex-Related Proteins with Extensive Sequence Tolerance in the Cellular Environment

Su, Haomiao; Xu, Jinglei; Chen, Yuqi; Wang, Qi; Lu, Ziang; Chen, Yage; Chen, Kun; Han, Shaoqing; Fang, Zhentian; Wang, Ping; Yuan, Bi-Feng; Zhou, Xiang

doi:10.1021/jacs.0c10792

Cited by 46 publications

(49 citation statements)

References 38 publications

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“…In principle, the approach we describe here should be applicable to a wide range of cell types and cell states, which in turn may help reveal specific differences in G4 interactomes and biology. During the revision of this article, we became aware of an independent study that involved a pyrrolidine derivative of PDS 50 and reported the identification of G4-related proteins in human SV589 and MM231 cells 51 . Although we noted some overlap between the studies (61 shared protein candidates), which somewhat validates the independent approaches, most of the G4-associated proteins identified by our CMPP approach were not found in the independent study.…”

Section: Discussionmentioning

confidence: 99%

Chemical profiling of DNA G-quadruplex-interacting proteins in live cells

et al. 2021

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DNA–protein interactions regulate critical biological processes. Identifying proteins that bind to specific, functional genomic loci is essential to understand the underlying regulatory mechanisms on a molecular level. Here we describe a co-binding-mediated protein profiling (CMPP) strategy to investigate the interactome of DNA G-quadruplexes (G4s) in native chromatin. CMPP involves cell-permeable, functionalized G4-ligand probes that bind endogenous G4s and subsequently crosslink to co-binding G4-interacting proteins in situ. We first showed the robustness of CMPP by proximity labelling of a G4 binding protein in vitro. Employing this approach in live cells, we then identified hundreds of putative G4-interacting proteins from various functional classes. Next, we confirmed a high G4-binding affinity and selectivity for several newly discovered G4 interactors in vitro, and we validated direct G4 interactions for a functionally important candidate in cellular chromatin using an independent approach. Our studies provide a chemical strategy to map protein interactions of specific nucleic acid features in living cells.

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Section: Discussionmentioning

confidence: 99%

Chemical profiling of DNA G-quadruplex-interacting proteins in live cells

et al. 2021

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“…SERBP1 structure and its RNA recognition and binding activity are poorly characterized. SERBP1 was reported to bind preferentially to GC-rich motifs (Kosti et al, 2020), subsequent studies revealed that these motifs could include G-quadruplexes (Su et al, 2021). SERBP1 was identified in the structures of nontranslating 80 S ribosomes, blocking the mRNA entrance channel suggesting that it serves to regulate mRNA translation (Ahn et al, 2015;Brown et al, 2018;Muto et al, 2018).…”

Section: Introductionmentioning

confidence: 99%

Structural Characterization of the RNA-Binding Protein SERBP1 Reveals Intrinsic Disorder and Atypical RNA Binding Modes

Baudin

Moreno-Romero

et al. 2021

Front. Mol. Biosci.

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RNA binding proteins (RBPs) are essential for critical biological processes such as translation regulation and mRNA processing, and misfunctions of these proteins are associated with diseases such as cancer and neurodegeneration. SERBP1 (SERPINE1 mRNA Binding Protein 1) is an RBP that comprises two RG/RGG repeat regions yet lacks other recognizable RNA-binding motifs. It is involved in mRNA maturation, and translational regulation. It was initially identified as a hyaluronic acid binding protein, but recent studies have identified central roles for SERBP1 in brain function and development, especially neurogenesis and synaptogenesis. SERBP1 regulates One-carbon metabolism and epigenetic modification of histones, and increased SERBP1 expression in cancers such as leukemia, ovarian, prostate, liver and glioblastoma is correlated with poor patient outcomes. Despite these important regulatory roles for SERBP1, little is known about its structural and dynamic properties, nor about the molecular mechanisms governing its interaction with mRNA. Here, we define SERBP1 as an intrinsically disordered protein, containing highly conserved elements that were shown to be functionally important. The RNA binding activity of SERBP1 was explored using solution NMR and other biophysical techniques. The outcome of these experiments revealed that SERBP1 preferentially samples compact conformations including a central, stable α-helix and show that SERBP1 recognizes G-rich RNA sequences at the C-terminus involving the RGG box and neighboring residues. Despite the role in RNA recognition, the RGG boxes do not seem to stabilize the central helix and the central helix does not participate in RNA binding. Further, SERBP1 undergoes liquid-liquid phase separation, mediated by salt and RNA, and both RGG boxes are necessary for the efficient formation of condensed phases. Together, these results provide a foundation for understanding the molecular mechanisms of SERBP1 functions in physiological and pathological processes.

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“…An approach to probe protein-G4 interactions is through the use of G4 ligands, and this was recently used to identify G4-binding proteins in cells. 57,58 A common G4 ligand used for such studies is pyridostatin (PDS; Figure 8A), which shows high selectivity for G4 folds over other DNA or RNA structures. 59 The same approach outlined herein was followed to study the impact of the PDS ligand added at 5 μ M for 30 min before following APE1 cleavage activity or binding as described above in buffered solutions with 50 mM K + ions and 10 mM Mg 2+ ions.…”

Section: Impact Of Ape1 Binding To the Vegf G4s In The Presence Of A G4 Ligandmentioning

confidence: 99%

“…These data demonstrate that G4 ligands block APE1 binding to parallel-stranded G4s; additionally, this may explain why, in the intracellular studies to identify G4-binding proteins, APE1 was not found in one study and not a major G4 binding partner in the second study. 57,58 The Sugimoto laboratory has reported the properties of DNA and its interactions with proteins can change with the addition of the cosolvent polyethylene glycol 200 (PEG-200) to the buffer; they propose PEG-200 functions to mimic the molecular crowding that occurs as a result of the high concentration of proteins in the cellular milieu, or that could also occur via water exclusion. 60,61 Studies were conducted following their guidelines with 20% PEG-200 added to the 50 mM Mg 2+ -containing buffer, and activity and binding assays were conducted.…”

Section: Impact Of Ape1 Binding To the Vegf G4s In The Presence Of A G4 Ligandmentioning

confidence: 99%

Binding of AP endonuclease-1 to G-quadruplex DNA depends on the N-terminal domain, Mg²⁺and ionic strength

Fleming

Manage

Burrows

2021

Preprint

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The base excision repair enzyme apurinic/apyrimidinic endonuclease-1 (APE1) is also engaged in transcriptional regulation. APE1 can function in both pathways when the protein binds to a promoter G-quadruplex (G4) bearing an abasic site (modeled with tetrahydrofuran, F) that leads to enzymatic stalling on the non-canonical fold to recruit activating transcription factors. Biochemical and biophysical studies to address the binding and catalytic activity of APE1 with the vascular endothelial growth factor (VEGF) promoter G4 are lacking, and the present work provides insight on this topic. Herein, the native APE1 was used for cleavage assays, and the catalytically inactive mutant D210A was used for binding assays with double-stranded DNA (dsDNA) versus the native G4 or the G4 with F at various positions, revealing dependencies of the interaction on the cation concentrations K+ and Mg2+ and the N-terminal domain of the protein. Assays in 0, 1, or 10 mM Mg2+ found dsDNA and G4 substrates required the cation for both binding and catalysis, in which G4 binding increased with [Mg2+]. Studies with 50 versus physiological 140 mM K+ ions present showed that F-containing dsDNA was bound and cleaved by APE1; whereas, the G4s with F were poorly cleaved in low salt and not cleaved at all at higher salt while the binding remained robust. Using delta 33 or delta 61 N-terminal truncated APE1 proteins, the binding and cleavage of dsDNA with F was minimally impacted; in contrast, the G4s required the N-terminus for binding and catalysis. With this knowledge, we found APE1 could remodel the F-containing VEGF promoter dsDNA to G4 folds in solution. Lastly, the addition of the G4 ligand pyridostatin inhibited APE1 binding and cleavage of F-containing G4s but not dsDNA. The biological and medicinal chemistry implications of the results are discussed.

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Photoactive G-Quadruplex Ligand Identifies Multiple G-Quadruplex-Related Proteins with Extensive Sequence Tolerance in the Cellular Environment

Cited by 46 publications

References 38 publications

Chemical profiling of DNA G-quadruplex-interacting proteins in live cells

Chemical profiling of DNA G-quadruplex-interacting proteins in live cells

Structural Characterization of the RNA-Binding Protein SERBP1 Reveals Intrinsic Disorder and Atypical RNA Binding Modes

Binding of AP endonuclease-1 to G-quadruplex DNA depends on the N-terminal domain, Mg²⁺and ionic strength

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Photoactive G-Quadruplex Ligand Identifies Multiple G-Quadruplex-Related Proteins with Extensive Sequence Tolerance in the Cellular Environment

Cited by 46 publications

References 38 publications

Chemical profiling of DNA G-quadruplex-interacting proteins in live cells

Chemical profiling of DNA G-quadruplex-interacting proteins in live cells

Structural Characterization of the RNA-Binding Protein SERBP1 Reveals Intrinsic Disorder and Atypical RNA Binding Modes

Binding of AP endonuclease-1 to G-quadruplex DNA depends on the N-terminal domain, Mg2+and ionic strength

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Binding of AP endonuclease-1 to G-quadruplex DNA depends on the N-terminal domain, Mg²⁺and ionic strength