scholarly journals Progress in the Discovery of Small-Molecule Inhibitors of Bromodomain–Histone Interactions

2011 ◽  
Vol 16 (10) ◽  
pp. 1170-1185 ◽  
Author(s):  
Chun-wa Chung ◽  
Jason Witherington
Keyword(s):  
Small Molecule ◽  
Protein Interactions ◽  
Transcriptional Activation ◽  
Label Free ◽  
Protein Protein Interactions ◽  
Ligand Selectivity ◽  
Small Molecule Ligands ◽  
Associated Proteins ◽  
In Vitro Systems

Bromodomains are structurally conserved protein modules present in a large number of chromatin-associated proteins and in many nuclear histone acetyltransferases. The bromodomain functions as an acetyl-lysine binding domain and has been shown to be pivotal in regulating protein–protein interactions in chromatin-mediated cellular gene transcription, cell proliferation, and viral transcriptional activation. Structural analyses of these modules in complex with acetyl-lysine peptide ligands provide insights into the molecular basis for recognition and ligand selectivity within this epigenetic reader family. However, there are significant challenges in configuring assays to identify inhibitors of these proteins. This review focuses on the progress made in developing methods to identify peptidic and small-molecule ligands using biophysical label-free and biochemical approaches. The advantage of each technique and the results reported are summarized, highlighting the potential applicably to other reader domains and the caveats in translation from simple in vitro systems to a biological context.

c-Fos-induced activation of a TATA-box-containing promoter involves direct contact with TATA-box-binding protein

1994 ◽  
Vol 14 (9) ◽  
pp. 6021-6029
Author(s):  
R Metz ◽  
A J Bannister ◽  
J A Sutherland ◽  
C Hagemeier ◽  
E C O'Rourke ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Transcriptional Activation ◽  
Binding Protein ◽  
Tata Box ◽  
Binding Motif ◽  
Protein Protein Interactions ◽  
High Concentrations ◽  
Tata Box Binding Protein

Transcriptional activation in eukaryotes involves protein-protein interactions between regulatory transcription factors and components of the basal transcription machinery. Here we show that c-Fos, but not a related protein, Fra-1, can bind the TATA-box-binding protein (TBP) both in vitro and in vivo and that c-Fos can also interact with the transcription factor IID complex. High-affinity binding to TBP requires c-Fos activation modules which cooperate to activate transcription. One of these activation modules contains a TBP-binding motif (TBM) which was identified through its homology to TBP-binding viral activators. This motif is required for transcriptional activation, as well as TBP binding. Domain swap experiments indicate that a domain containing the TBM can confer TBP binding on Fra-1 both in vitro and in vivo. In vivo activation experiments indicate that a GAL4-Fos fusion can activate a promoter bearing a GAL4 site linked to a TATA box but that this activity does not occur at high concentrations of GAL4-Fos. This inhibition (squelching) of c-Fos activity is relieved by the presence of excess TBP, indicating that TBP is a direct functional target of c-Fos. Removing the TBM from c-Fos severely abrogates activation of a promoter containing a TATA box but does not affect activation of a promoter driven only by an initiator element. Collectively, these results suggest that c-Fos is able to activate via two distinct mechanisms, only one of which requires contact with TBP. Since TBP binding is not exhibited by Fra-1, TBP-mediated activation may be one characteristic that discriminates the function of Fos-related proteins.

scholarly journals A Specificity and Targeting Subunit of a Human SWI/SNF Family-Related Chromatin-Remodeling Complex

2000 ◽  
Vol 20 (23) ◽  
pp. 8879-8888 ◽  
Author(s):  
Zuqin Nie ◽  
Yutong Xue ◽  
Dafeng Yang ◽  
Sharleen Zhou ◽  
Bonnie J. Deroo ◽  
...  
Keyword(s):  
Glucocorticoid Receptor ◽  
Chromatin Remodeling ◽  
Protein Interactions ◽  
Transcriptional Activation ◽  
Gene Activation ◽  
Dna Interaction ◽  
Protein Protein Interactions ◽  
Baf Complex ◽  
Chromatin Remodeling Complexes

ABSTRACT The SWI/SNF family of chromatin-remodeling complexes facilitates gene activation by assisting transcription machinery to gain access to targets in chromatin. This family includes BAF (also called hSWI/SNF-A) and PBAF (hSWI/SNF-B) from humans and SWI/SNF and Rsc fromSaccharomyces cerevisiae. However, the relationship between the human and yeast complexes is unclear because all human subunits published to date are similar to those of both yeast SWI/SNF and Rsc. Also, the two human complexes have many identical subunits, making it difficult to distinguish their structures or functions. Here we describe the cloning and characterization of BAF250, a subunit present in human BAF but not PBAF. BAF250 contains structural motifs conserved in yeast SWI1 but not in any Rsc components, suggesting that BAF is related to SWI/SNF. BAF250 is also a homolog of the Drosophila melanogaster Osa protein, which has been shown to interact with a SWI/SNF-like complex in flies. BAF250 possesses at least two conserved domains that could be important for its function. First, it has an AT-rich DNA interaction-type DNA-binding domain, which can specifically bind a DNA sequence known to be recognized by a SWI/SNF family-related complex at the β-globin locus. Second, BAF250 stimulates glucocorticoid receptor-dependent transcriptional activation, and the stimulation is sharply reduced when the C-terminal region of BAF250 is deleted. This region of BAF250 is capable of interacting directly with the glucocorticoid receptor in vitro. Our data suggest that BAF250 confers specificity to the human BAF complex and may recruit the complex to its targets through either protein-DNA or protein-protein interactions.

scholarly journals Small molecules that inhibit TNF signalling by stabilising an asymmetric form of the trimer

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
James O’Connell ◽  
John Porter ◽  
Boris Kroeplien ◽  
Tim Norman ◽  
Stephen Rapecki ◽  
...  
Keyword(s):  
Small Molecules ◽  
Small Molecule ◽  
Protein Interactions ◽  
General Mechanism ◽  
Protein Protein Interactions ◽  
Biologic Drugs ◽  
Small Molecule Drugs ◽  
Necrosis Factor

AbstractTumour necrosis factor (TNF) is a cytokine belonging to a family of trimeric proteins; it has been shown to be a key mediator in autoimmune diseases such as rheumatoid arthritis and Crohn’s disease. While TNF is the target of several successful biologic drugs, attempts to design small molecule therapies directed to this cytokine have not led to approved products. Here we report the discovery of potent small molecule inhibitors of TNF that stabilise an asymmetrical form of the soluble TNF trimer, compromising signalling and inhibiting the functions of TNF in vitro and in vivo. This discovery paves the way for a class of small molecule drugs capable of modulating TNF function by stabilising a naturally sampled, receptor-incompetent conformation of TNF. Furthermore, this approach may prove to be a more general mechanism for inhibiting protein–protein interactions.

scholarly journals Label-free methods for optical in vitro characterization of protein–protein interactions

2021 ◽  
Author(s):  
Fabian Soltermann ◽  
Weston B. Struwe ◽  
Philipp Kukura
Keyword(s):  
Protein Interactions ◽  
Label Free ◽  
Protein Protein Interactions ◽  
Cellular Processes ◽  
P Protein ◽  
In Vitro Characterization ◽  
And Function

Protein–protein interactions are involved in the regulation and function of the majority of cellular processes.

scholarly journals Small molecule probes that perturb a protein–protein interface in antithrombin

2014 ◽  
Vol 5 (12) ◽  
pp. 4914-4921 ◽  
Author(s):  
Dongyue Xin ◽  
Andreas Holzenburg ◽  
Kevin Burgess
Keyword(s):  
Small Molecule ◽  
Protein Interactions ◽  
Protein Interface ◽  
Protein Protein Interactions ◽  
Target Proteins ◽  
Small Molecule Probes ◽  
Quaternary Structures

Small molecule probes for perturbing protein–protein interactions (PPIs) in vitro can be useful if they cause the target proteins to undergo biomedically relevant changes to their tertiary and quaternary structures.

scholarly journals The microtubule-associated histone methyltransferase SET8, facilitated by transcription factor LSF, methylates α-tubulin

2020 ◽  
Vol 295 (14) ◽  
pp. 4748-4759 ◽  
Author(s):  
Hang Gyeong Chin ◽  
Pierre-Olivier Esteve ◽  
Cristian Ruse ◽  
Jiyoung Lee ◽  
Scott E. Schaus ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Protein Interactions ◽  
Posttranslational Modifications ◽  
Organelle Transport ◽  
Histone H4 ◽  
Protein Protein Interactions ◽  
Molecule Inhibitor ◽  
Associated Proteins ◽  
Lysine Methyltransferase

Microtubules are cytoskeletal structures critical for mitosis, cell motility, and protein and organelle transport and are a validated target for anticancer drugs. However, how tubulins are regulated and recruited to support these distinct cellular processes is incompletely understood. Posttranslational modifications of tubulins are proposed to regulate microtubule function and dynamics. Although many of these modifications have been investigated, only one prior study reports tubulin methylation and an enzyme responsible for this methylation. Here we used in vitro radiolabeling, MS, and immunoblotting approaches to monitor protein methylation and immunoprecipitation, immunofluorescence, and pulldown approaches to measure protein–protein interactions. We demonstrate that N-lysine methyltransferase 5A (KMT5A or SET8/PR-Set7), which methylates lysine 20 in histone H4, bound α-tubulin and methylated it at a specific lysine residue, Lys311. Furthermore, late SV40 factor (LSF)/CP2, a known transcription factor, bound both α-tubulin and SET8 and enhanced SET8-mediated α-tubulin methylation in vitro. In addition, we found that the ability of LSF to facilitate this methylation is countered by factor quinolinone inhibitor 1 (FQI1), a specific small-molecule inhibitor of LSF. These findings suggest the general model that microtubule-associated proteins, including transcription factors, recruit or stimulate protein-modifying enzymes to target tubulins. Moreover, our results point to dual functions for SET8 and LSF not only in chromatin regulation but also in cytoskeletal modification.

scholarly journals c-Fos-induced activation of a TATA-box-containing promoter involves direct contact with TATA-box-binding protein.

1994 ◽  
Vol 14 (9) ◽  
pp. 6021-6029 ◽  
Author(s):  
R Metz ◽  
A J Bannister ◽  
J A Sutherland ◽  
C Hagemeier ◽  
E C O'Rourke ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Transcriptional Activation ◽  
Binding Protein ◽  
Tata Box ◽  
Binding Motif ◽  
Protein Protein Interactions ◽  
High Concentrations ◽  
Tata Box Binding Protein

Transcriptional activation in eukaryotes involves protein-protein interactions between regulatory transcription factors and components of the basal transcription machinery. Here we show that c-Fos, but not a related protein, Fra-1, can bind the TATA-box-binding protein (TBP) both in vitro and in vivo and that c-Fos can also interact with the transcription factor IID complex. High-affinity binding to TBP requires c-Fos activation modules which cooperate to activate transcription. One of these activation modules contains a TBP-binding motif (TBM) which was identified through its homology to TBP-binding viral activators. This motif is required for transcriptional activation, as well as TBP binding. Domain swap experiments indicate that a domain containing the TBM can confer TBP binding on Fra-1 both in vitro and in vivo. In vivo activation experiments indicate that a GAL4-Fos fusion can activate a promoter bearing a GAL4 site linked to a TATA box but that this activity does not occur at high concentrations of GAL4-Fos. This inhibition (squelching) of c-Fos activity is relieved by the presence of excess TBP, indicating that TBP is a direct functional target of c-Fos. Removing the TBM from c-Fos severely abrogates activation of a promoter containing a TATA box but does not affect activation of a promoter driven only by an initiator element. Collectively, these results suggest that c-Fos is able to activate via two distinct mechanisms, only one of which requires contact with TBP. Since TBP binding is not exhibited by Fra-1, TBP-mediated activation may be one characteristic that discriminates the function of Fos-related proteins.

scholarly journals Differentiation-Dependent Interactions between RUNX-1 and FLI-1 during Megakaryocyte Development

2009 ◽  
Vol 29 (15) ◽  
pp. 4103-4115 ◽  
Author(s):  
Hui Huang ◽  
Ming Yu ◽  
Thomas E. Akie ◽  
Tyler B. Moran ◽  
Andrew J. Woo ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Protein Interactions ◽  
Transcriptional Activation ◽  
Fetal Liver ◽  
Gel Filtration ◽  
Cell Types ◽  
Protein Protein Interactions ◽  
Megakaryocyte Differentiation

ABSTRACT The transcription factor RUNX-1 plays a key role in megakaryocyte differentiation and is mutated in cases of myelodysplastic syndrome and leukemia. In this study, we purified RUNX-1-containing multiprotein complexes from phorbol ester-induced L8057 murine megakaryoblastic cells and identified the ets transcription factor FLI-1 as a novel in vivo-associated factor. The interaction occurs via direct protein-protein interactions and results in synergistic transcriptional activation of the c-mpl promoter. Interestingly, the interaction fails to occur in uninduced cells. Gel filtration chromatography confirms the differentiation-dependent binding and shows that it correlates with the assembly of a complex also containing the key megakaryocyte transcription factors GATA-1 and Friend of GATA-1 (FOG-1). Phosphorylation analysis of FLI-1 with uninduced versus induced L8057 cells suggests the loss of phosphorylation at serine 10 in the induced state. Substitution of Ser10 with the phosphorylation mimic aspartic acid selectively impairs RUNX-1 binding, abrogates transcriptional synergy with RUNX-1, and dominantly inhibits primary fetal liver megakaryocyte differentiation in vitro. Conversely, substitution with alanine, which blocks phosphorylation, augments differentiation of primary megakaryocytes. We propose that dephosphorylation of FLI-1 is a key event in the transcriptional regulation of megakaryocyte maturation. These findings have implications for other cell types where interactions between runx and ets family proteins occur.

scholarly journals Phosphorylation of Enabled by the DrosophilaAbelson Tyrosine Kinase Regulates the In Vivo Function and Protein-Protein Interactions of Enabled

1998 ◽  
Vol 18 (1) ◽  
pp. 152-160 ◽  
Author(s):  
Allen R. Comer ◽  
Shawn M. Ahern-Djamali ◽  
Jyh-Lyh Juang ◽  
P. David Jackson ◽  
F. M. Hoffmann
Keyword(s):  
Tyrosine Kinase ◽  
Protein Interactions ◽  
Phosphorylation Sites ◽  
Protein Protein Interactions ◽  
Tyrosine Residues ◽  
Abelson Tyrosine Kinase ◽  
Genetic Suppressors ◽  
Associated Proteins

ABSTRACT Drosophila Enabled (Ena) is a member of a family of cytoskeleton-associated proteins including mammalian vasodilator-stimulated phosphoprotein and murine Enabled that regulate actin cytoskeleton assembly. Mutations in Drosophila enawere discovered as dominant genetic suppressors of mutations in the Abelson tyrosine kinase (Abl), suggesting that Ena and Abl function in the same pathway or process. We have identified six tyrosine residues on Ena that are phosphorylated by Abl in vitro and in vivo. Mutation of these phosphorylation sites to phenylalanine partially impaired the ability of Ena to restore viability to ena mutant animals, indicating that phosphorylation is required for optimal Ena function. Phosphorylation of Ena by Abl inhibited the binding of Ena to SH3 domains in vitro, suggesting that one effect of Ena phosphorylation may be to modulate its association with other proteins.

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