CtBP represses Dpp signaling as a dimer

Bi, Caili; Meng, Fei; Yang, Lin; Cheng, Lin; Wang, Ping; Chen, Mengmeng; Fang, Ming; Xie, Hao

doi:10.1016/j.bbrc.2017.12.018

Cited by 13 publications

(10 citation statements)

References 31 publications

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“…Previous investigations into the role of assembly in CtBP have investigated the activity of mutants specifically designed to disrupt the CtBP dimer (33)(34)(35)(36). Our results strongly suggest that the CtBP dimer is required for the assembly of a CtBP tetramer formed from the pairing of two CtBP dimers.…”

Section: Assembly Of Tetrameric Ctbpmentioning

confidence: 52%

“…Regulation of gene expression through the oligomerization of transcriptional factors is an important paradigm (27,28). In the case of CtBP, substantial evidence exists that oligomerization is linked with NAD(H) binding (3, 25, 29 -32), and dimerdestabilizing mutants have been found to inhibit transcriptional function (33)(34)(35)(36). Assembly of CtBP has primarily been considered in terms of dimers, as NADH-bound CtBP crystal structures reveal a predominant dimer with extensive interactions between subunit pairs (23,37,38).…”

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confidence: 99%

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Assembly of human C-terminal binding protein (CtBP) into tetramers

Bellesis

Jecrois

Hayes

et al. 2018

Journal of Biological Chemistry

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C-terminal binding protein 1 (CtBP1) and CtBP2 are transcriptional coregulators that repress numerous cellular processes, such as apoptosis, by binding transcription factors and recruiting chromatin-remodeling enzymes to gene promoters. The NAD(H)-linked oligomerization of human CtBP is coupled to its co-transcriptional activity, which is implicated in cancer progression. However, the biologically relevant level of CtBP assembly has not been firmly established; nor has the stereochemical arrangement of the subunits above that of a dimer. Here, multi-angle light scattering (MALS) data established the NAD- and NADH-dependent assembly of CtBP1 and CtBP2 into tetramers. An examination of subunit interactions within CtBP1 and CtBP2 crystal lattices revealed that both share a very similar tetrameric arrangement resulting from assembly of two dimeric pairs, with specific interactions probably being sensitive to NAD(H) binding. Creating a series of mutants of both CtBP1 and CtBP2, we tested the hypothesis that the crystallographically observed interdimer pairing stabilizes the solution tetramer. MALS data confirmed that these mutants disrupt both CtBP1 and CtBP2 tetramers, with the dimer generally remaining intact, providing the first stereochemical models for tetrameric assemblies of CtBP1 and CtBP2. The crystal structure of a subtle destabilizing mutant suggested that small structural perturbations of the hinge region linking the substrate- and NAD-binding domains are sufficient to weaken the CtBP1 tetramer. These results strongly suggest that the tetramer is important in CtBP function, and the series of CtBP mutants reported here can be used to investigate the physiological role of the tetramer.

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Section: Assembly Of Tetrameric Ctbpmentioning

confidence: 52%

mentioning

confidence: 99%

Assembly of human C-terminal binding protein (CtBP) into tetramers

Bellesis

Jecrois

Hayes

et al. 2018

Journal of Biological Chemistry

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“…It has long been established that binding of NAD (H) induces oligomerization and transcriptional activation of CtBP [25,26]. However, there are conflicting hypotheses about the level of oligomerization [27][28][29][30][31][32][33] and the relative affinity of binding NADH vs NAD + [33][34][35]. These issues are key to understanding the basis for transcriptional activation of CtBP and also for guiding the design of inhibitors to disrupt transcriptional activity in cancer.…”

Section: Discussionmentioning

confidence: 99%

“…Although NAD(H) binding to CtBP has been linked to oligomerization and transcriptional activity for nearly two decades [25,26], the level of assembly and the relative affinity of NADH vs NAD + are key issues without consensus at present. Numerous reports have suggested that NAD(H) triggers activation through assembly of CtBP monomers into dimers [27][28][29][30], but others have shown evidence that NAD(H) triggers assembly of dimers into tetramers [31][32][33]. Additionally, published data of the affinity of NADH and NAD + binding to CtBP have led to conflicting conclusions [33][34][35].…”

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confidence: 99%

NADH/NAD⁺ binding and linked tetrameric assembly of the oncogenic transcription factors CtBP1 and CtBP2

et al. 2022

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The activation of oncogenic C‐terminal binding Protein (CtBP) transcriptional activity is coupled with NAD(H) binding and homo‐oligomeric assembly, although the level of CtBP assembly and nucleotide binding affinity continues to be debated. Here, we apply biophysical techniques to address these fundamental issues for CtBP1 and CtBP2. Our ultracentrifugation results unambiguously demonstrate that CtBP assembles into tetramers in the presence of saturating NAD+ or NADH with tetramer to dimer dissociation constants about 100 nm. Isothermal titration calorimetry measurements of NAD(H) binding to CtBP show dissociation constants between 30 and 500 nm, depending on the nucleotide and paralog. Given cellular levels of NAD+, CtBP is likely to be fully saturated with NAD under physiological concentrations suggesting that CtBP is unable to act as a sensor for NADH levels.

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“…Although previous studies have proposed dimeric CtBP as the relevant oligomeric state (Nardini et al, 2003;Thio et al, 2004;Nardini et al, 2009;Bi et al, 2018;Mani-Telang et al, 2007;Dcona et al, 2019), our studies with multi-angle light scattering and sitedirected mutagenesis have shown that the primary effect of NADH binding is to promote the assembly of two CtBP dimers into tetramers (Bellesis et al, 2018). This was further supported by the observation that CtBP1 and CtBP2 exhibit similar tetrameric assemblies within distinct crystal lattices used for structure determination (Hilbert et al, 2014), resulting in a tetramer model for CtBP.…”

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confidence: 96%

CryoEM Structure of CtBP2 Confirms Tetrameric Architecture

Jecrois

Dcona

Deng

et al. 2020

Preprint

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C-terminal binding proteins 1 and 2 (CtBP1 and CtBP2) are transcriptional regulators that activate or repress many genes involved in cellular development, apoptosis and metastasis. CtBP proteins are activated under hypoxic conditions where NAD(H) levels tend to be higher. NADH-dependent activation of CtBP2 has direct implication in multiple types of cancers and poor patient prognosis. Previous studies have proposed dimeric CtBP as the relevant oligomeric state, however our studies with multi-angle light scattering have shown that the primary effect of NADH binding is to promote the assembly of two CtBP dimers into tetramers. Here, we present the cryoEM structures of two different constructs of CtBP2 corroborating that the native state of CtBP2 in the presence of NADH is indeed tetrameric. The physiological relevance of tetrameric CtBP2 was tested in HCT116; CtBP2 -/-cells transfected with tetramer destabilizing mutants.Mutants that inhibit tetramer formation show a decrease in expression of the CtBP transcriptional target TIAM1 and exhibit a decrease in the ability to promote cell migration. Together with our cryoEM studies, these results highlight the tetramer as the functional oligomeric form of CtBP2.

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CtBP represses Dpp signaling as a dimer

Cited by 13 publications

References 31 publications

Assembly of human C-terminal binding protein (CtBP) into tetramers

Assembly of human C-terminal binding protein (CtBP) into tetramers

NADH/NAD⁺ binding and linked tetrameric assembly of the oncogenic transcription factors CtBP1 and CtBP2

CryoEM Structure of CtBP2 Confirms Tetrameric Architecture

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CtBP represses Dpp signaling as a dimer

Cited by 13 publications

References 31 publications

Assembly of human C-terminal binding protein (CtBP) into tetramers

Assembly of human C-terminal binding protein (CtBP) into tetramers

NADH/NAD+ binding and linked tetrameric assembly of the oncogenic transcription factors CtBP1 and CtBP2

CryoEM Structure of CtBP2 Confirms Tetrameric Architecture

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NADH/NAD⁺ binding and linked tetrameric assembly of the oncogenic transcription factors CtBP1 and CtBP2