scholarly journals Impact of the N-Terminal Domain of STAT3 in STAT3-Dependent Transcriptional Activity

2015 ◽  
Vol 35 (19) ◽  
pp. 3284-3300 ◽  
Author(s):  
Tiancen Hu ◽  
Jennifer E. Yeh ◽  
Luca Pinello ◽  
Jaison Jacob ◽  
Srinivas Chakravarthy ◽  
...  
Keyword(s):  
Transcriptional Regulation ◽  
Dna Binding ◽  
Protein Interactions ◽  
Target Genes ◽  
Nuclear Translocation ◽  
Biological Effects ◽  
Null Mouse ◽  
Protein Protein Interactions ◽  
Terminal Domain

The transcription factor STAT3 is constitutively active in many cancers, where it mediates important biological effects, including cell proliferation, differentiation, survival, and angiogenesis. The N-terminal domain (NTD) of STAT3 performs multiple functions, such as cooperative DNA binding, nuclear translocation, and protein-protein interactions. However, it is unclear which subsets of STAT3 target genes depend on the NTD for transcriptional regulation. To identify such genes, we compared gene expression inSTAT3-null mouse embryonic fibroblasts (MEFs) stably expressing wild-type STAT3 or STAT3 from which NTD was deleted. NTD deletion reduced the cytokine-induced expression of specific STAT3 target genes by decreasing STAT3 binding to their regulatory regions. To better understand the potential mechanisms of this effect, we determined the crystal structure of the STAT3 NTD and identified a dimer interface responsible for cooperative DNA bindingin vitro. We also observed an Ni2+-mediated oligomer with an as yet unknown biological function. Mutations on both dimer and Ni2+-mediated interfaces affected the cytokine induction of STAT3 target genes. These studies shed light on the role of the NTD in transcriptional regulation by STAT3 and provide a structural template with which to design STAT3 NTD inhibitors with potential therapeutic value.

scholarly journals Ways into Understanding HIF Inhibition

Cancers ◽  
2021 ◽  
Vol 13 (1) ◽  
pp. 159
Author(s):  
Tina Schönberger ◽  
Joachim Fandrey ◽  
Katrin Prost-Fingerle
Keyword(s):  
Protein Interactions ◽  
Target Genes ◽  
Protein Protein Interactions ◽  
Low Oxygen ◽  
Drug Candidates ◽  
Open Questions ◽  
Wide Range ◽  
Hif Pathway

Hypoxia is a key characteristic of tumor tissue. Cancer cells adapt to low oxygen by activating hypoxia-inducible factors (HIFs), ensuring their survival and continued growth despite this hostile environment. Therefore, the inhibition of HIFs and their target genes is a promising and emerging field of cancer research. Several drug candidates target protein–protein interactions or transcription mechanisms of the HIF pathway in order to interfere with activation of this pathway, which is deregulated in a wide range of solid and liquid cancers. Although some inhibitors are already in clinical trials, open questions remain with respect to their modes of action. New imaging technologies using luminescent and fluorescent methods or nanobodies to complement widely used approaches such as chromatin immunoprecipitation may help to answer some of these questions. In this review, we aim to summarize current inhibitor classes targeting the HIF pathway and to provide an overview of in vitro and in vivo techniques that could improve the understanding of inhibitor mechanisms. Unravelling the distinct principles regarding how inhibitors work is an indispensable step for efficient clinical applications and safety of anticancer compounds.

Topoisomerase II forms multimers in vitro: effects of metals, beta-glycerophosphate, and phosphorylation of its C-terminal domain

1994 ◽  
Vol 14 (10) ◽  
pp. 6962-6974
Author(s):  
Y S Vassetzky ◽  
Q Dang ◽  
P Benedetti ◽  
S M Gasser
Keyword(s):  
Chromosome Segregation ◽  
Protein Interactions ◽  
Topoisomerase Ii ◽  
Dna Topoisomerase Ii ◽  
Protein Protein Interactions ◽  
Dna Topoisomerase ◽  
Terminal Domain ◽  
Terminal Amino ◽  
In Vitro Effects

We present a novel assay for the study of protein-protein interactions involving DNA topoisomerase II. Under various conditions of incubation we observe that topoisomerase II forms complexes at least tetrameric in size, which can be sedimented by centrifugation through glycerol. The multimers are enzymatically active and can be visualized by electron microscopy. Dephosphorylation of topoisomerase II inhibits its multimerization, which can be restored at least partially by rephosphorylation of multiple sites within its 200 C-terminal amino acids by casein kinase II. Truncation of topoisomerase II just upstream of the major phosphoacceptor sites reduces its aggregation, rendering the truncated enzyme insensitive to either kinase treatments or phosphatase treatments. This is consistent with a model in which interactions involving the phosphorylated C-terminal domain of topoisomerase II aid either in chromosome segregation or in chromosome condensation.

scholarly journals Analysis of the SWI4/SWI6 protein complex, which directs G1/S-specific transcription in Saccharomyces cerevisiae.

1993 ◽  
Vol 13 (2) ◽  
pp. 1069-1077 ◽  
Author(s):  
J Sidorova ◽  
L Breeden
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Binding ◽  
Protein Interactions ◽  
Protein Protein Interactions ◽  
C Terminus ◽  
Specific Complex ◽  
Dependent Cell ◽  
The Stability

SWI4 and SWI6 play a crucial role in START-specific transcription in Saccharomyces cerevisiae. SWI4 and SWI6 form a specific complex on the SCB (SWI4/6-dependent cell cycle box) sequences which have been found in the promoters of HO and G1 cyclin genes. Overproduction of SWI4 eliminates the SWI6 dependency of HO transcription in vivo and results in a new SWI6-independent, SCB-specific complex in vitro, which is heterogeneous and reacts with SWI4 antibodies. The C terminus of SWI4 is not required for SWI6-independent binding of SWI4 to SCB sequences, but it is necessary and sufficient for association with SWI6. Both SWI4 and SWI6 contain two copies of a 33-amino-acid TPLH repeat, which has been implicated in protein-protein interactions in other proteins. These repeats are not required for the SWI4-SWI6 association. Alanine substitutions in both TPLH repeats of SWI6 reduce its activity but do not affect the stability of the protein or its association with SWI4. However, these mutations reduce the ability of the SWI4/6 complex to bind DNA. Deletion of the lucine zipper motif in SWI6 also allows SWI4/6 complex formation, but it eliminates the DNA-binding ability of the SWI4/6 complex. This indicates that the integrity of two different regions of SWI6 is required for DNA binding by the SWI4/6 complex. From these data, we propose that the sequence-specific DNA-binding domain resides in SWI4 but that SWI6 controls the accessibility of this domain in the SWI4/6 complex.

scholarly journals Myogenic Basic Helix-Loop-Helix Proteins and Sp1 Interact as Components of a Multiprotein Transcriptional Complex Required for Activity of the Human Cardiac α-Actin Promoter

1999 ◽  
Vol 19 (4) ◽  
pp. 2577-2584 ◽  
Author(s):  
Elzbieta Biesiada ◽  
Yasuo Hamamori ◽  
Larry Kedes ◽  
Vittorio Sartorelli
Keyword(s):  
Dna Binding ◽  
Protein Interactions ◽  
Serum Response Factor ◽  
Multiprotein Complex ◽  
Protein Protein Interactions ◽  
Basic Helix Loop Helix ◽  
Helix Loop Helix ◽  
Transcriptional Complex

ABSTRACT Activation of the human cardiac α-actin (HCA) promoter in skeletal muscle cells requires the integrity of DNA binding sites for the serum response factor (SRF), Sp1, and the myogenic basic helix-loop-helix (bHLH) family. In this study we report that activation of the HCA correlates with formation of a muscle-specific multiprotein complex on the promoter. We provide evidence that proteins eluted from the multiprotein complex specifically react with antibodies directed against myogenin, Sp1, and SRF and that the complex can be assembled in vitro by using the HCA promoter and purified MyoD, E12, SRF, and Sp1. In vitro and in vivo assays revealed a direct association of Sp1 and myogenin-MyoD mediated by the DNA-binding domain of Sp1 and the HLH motif of myogenin. The results obtained in this study indicate that protein-protein interactions and the cooperative DNA binding of transcriptional activators are critical steps in the formation of a transcriptionally productive multiprotein complex on the HCA promoter and suggest that the same mechanisms might be utilized to regulate the transcription of muscle-specific and other genes.

Analysis of the SWI4/SWI6 protein complex, which directs G1/S-specific transcription in Saccharomyces cerevisiae

1993 ◽  
Vol 13 (2) ◽  
pp. 1069-1077
Author(s):  
J Sidorova ◽  
L Breeden
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Binding ◽  
Protein Interactions ◽  
Protein Protein Interactions ◽  
C Terminus ◽  
Specific Complex ◽  
Dependent Cell ◽  
The Stability

SWI4 and SWI6 play a crucial role in START-specific transcription in Saccharomyces cerevisiae. SWI4 and SWI6 form a specific complex on the SCB (SWI4/6-dependent cell cycle box) sequences which have been found in the promoters of HO and G1 cyclin genes. Overproduction of SWI4 eliminates the SWI6 dependency of HO transcription in vivo and results in a new SWI6-independent, SCB-specific complex in vitro, which is heterogeneous and reacts with SWI4 antibodies. The C terminus of SWI4 is not required for SWI6-independent binding of SWI4 to SCB sequences, but it is necessary and sufficient for association with SWI6. Both SWI4 and SWI6 contain two copies of a 33-amino-acid TPLH repeat, which has been implicated in protein-protein interactions in other proteins. These repeats are not required for the SWI4-SWI6 association. Alanine substitutions in both TPLH repeats of SWI6 reduce its activity but do not affect the stability of the protein or its association with SWI4. However, these mutations reduce the ability of the SWI4/6 complex to bind DNA. Deletion of the lucine zipper motif in SWI6 also allows SWI4/6 complex formation, but it eliminates the DNA-binding ability of the SWI4/6 complex. This indicates that the integrity of two different regions of SWI6 is required for DNA binding by the SWI4/6 complex. From these data, we propose that the sequence-specific DNA-binding domain resides in SWI4 but that SWI6 controls the accessibility of this domain in the SWI4/6 complex.

ftsZ mutations affecting cell division frequency, placement and morphology in Bacillus subtilis

Microbiology ◽  
2005 ◽  
Vol 151 (6) ◽  
pp. 2053-2064 ◽  
Author(s):  
Andrea Feucht ◽  
Jeffery Errington
Keyword(s):  
Bacillus Subtilis ◽  
Protein Interactions ◽  
Temperature Sensitive ◽  
Protein Protein Interactions ◽  
Terminal Domain ◽  
Simple Strategy ◽  
Division Site ◽  
C Terminus ◽  
Division Frequency

A key event in cytokinesis in bacteria is the assembly of the essential division protein FtsZ into ring-like structures at the nascent division site. FtsZ is the prokaryotic homologue of tubulin, and is found in nearly all bacteria. In vitro, FtsZ polymerizes in the presence of GTP to form higher-ordered polymers. FtsZ consists of two domains, with the GTP-binding site located in the N-terminal domain. The less-conserved C-terminal domain contains residues important for GTP hydrolysis, but its overall function is still unclear. This paper reports the development of a simple strategy to generate mutations in the essential division gene ftsZ. Nine novel and viable ftsZ mutants of Bacillus subtilis are described. Eight of the mutations would affect the C-terminus of FtsZ. The collection of mutants exhibits a range of morphological phenotypes, ranging from normal to highly filamentous cells; some produce minicells, or divide in a twisted configuration; one mutation has a temperature-sensitive effect specifically impairing sporulation. The sites of the amino acid changes generated by the mutations could be informative about FtsZ function and its protein–protein interactions.

scholarly journals Novel trans-Acting Bacillus subtilis glnA Mutations That Derepress glnRA Expression

2009 ◽  
Vol 191 (8) ◽  
pp. 2485-2492 ◽  
Author(s):  
Susan H. Fisher ◽  
Lewis V. Wray
Keyword(s):  
Bacillus Subtilis ◽  
Transcription Factors ◽  
Dna Binding ◽  
Glutamine Synthetase ◽  
Protein Interactions ◽  
Feedback Inhibition ◽  
Wild Type ◽  
Protein Protein Interactions ◽  
Enzymatic Assays

ABSTRACT Bacillus subtilis contains two nitrogen transcription factors, GlnR and TnrA. The activities of GlnR and TnrA are regulated by direct protein-protein interactions with the feedback-inhibited form of glutamine synthetase (GS). To look for other factors involved in regulating GlnR activity, we isolated mutants with constitutive glnRA expression (GlnC). The twenty-seven GlnC mutants isolated in this mutant screen all contained mutations tightly linked to the glnRA operon which encodes GlnR (glnR) and GS (glnA). Four GlnC mutants contained mutations in the glnR gene that most likely impair the ability of GlnR to bind DNA. Three other GlnC mutants contained novel glnA mutations (S55F, V173I, and L174F). GlnR regulation was completely relieved in the three glnA mutants, while only modest defects in TnrA regulation were observed. In vitro enzymatic assays showed that the purified S55F mutant enzyme was catalytically defective while the V173I and L174F enzymes were highly resistant to feedback inhibition. The V173I and L174F GS proteins were found to require higher glutamine concentrations than the wild-type GS to regulate the DNA-binding activities of GlnR and TnrA in vitro. These results are consistent with a model where feedback-inhibited GS is the only cellular factor involved in regulating the activity of GlnR in B. subtilis.

scholarly journals Ligand-dependent recruitment of the Arnt coregulator determines DNA recognition by the dioxin receptor

1993 ◽  
Vol 13 (4) ◽  
pp. 2504-2514
Author(s):  
M Whitelaw ◽  
I Pongratz ◽  
A Wilhelmsson ◽  
J A Gustafsson ◽  
L Poellinger
Keyword(s):  
Dna Binding ◽  
Target Genes ◽  
Nuclear Translocation ◽  
Mutational Analysis ◽  
Hepatoma Cell ◽  
Hepatoma Cell Line ◽  
Receptor System ◽  
Ligand Free ◽  
Dioxin Receptor

The intracellular basic region/helix-loop-helix (bHLH) dioxin receptor mediates signal transduction by dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin) and functions as a ligand-activated DNA binding protein directly interacting with target genes by binding to dioxin response elements. Here we show that the partially purified, ligand-bound receptor alone could not bind target DNA. In contrast, DNA binding by the receptor could be induced by addition of a cytosolic auxiliary activity which functionally and biochemically corresponded to the bHLH factor Arnt. While Arnt exhibited no detectable affinity for the dioxin response element in the absence of the dioxin receptor, it strongly promoted the DNA binding function of the ligand-activated but not the ligand-free receptor forms. Arnt also functionally reconstituted in vitro the DNA binding activity of a mutant, nuclear translocation-deficient dioxin receptor phenotype in cytosolic extracts from a dioxin-resistant hepatoma cell line. Importantly, coimmunoprecipitation experiments showed that Arnt physically interacted in solution with the ligand-activated dioxin receptor but failed to heterodimerize with the ligand-free, hsp90-associated receptor form. Mutational analysis suggested that the functional interaction between these two factors occurred via the bHLH motif of Arnt. These data suggest that dioxin receptor activity is governed by a complex pattern of combinatorial regulation involving repression by hsp90 and then by ligand-dependent recruitment of the positive coregulator Arnt. The dioxin receptor system also provides the first example of signal-controlled dimerization of bHLH factors.

scholarly journals SUMOylation of the Corepressor N-CoR Modulates Its Capacity to Repress Transcription

2006 ◽  
Vol 17 (4) ◽  
pp. 1643-1651 ◽  
Author(s):  
Jens Tiefenbach ◽  
Natalia Novac ◽  
Miryam Ducasse ◽  
Maresa Eck ◽  
Frauke Melchior ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Posttranslational Modifications ◽  
Transcriptional Repression ◽  
Histone Deacetylases ◽  
Hormone Receptors ◽  
Target Genes ◽  
Protein Protein Interactions ◽  
Lysine Residues

In the absence of ligands the corepressor N-CoR mediates transcriptional repression by some nuclear hormone receptors. Several protein–protein interactions of N-CoR are known, of which mainly complex formation with histone deacetylases (HDACs) leads to the repression of target genes. On the other hand, the role of posttranslational modifications in corepressor function is not well established. Here, we show that N-CoR is modified by Sumo-1. We found SUMO-E2–conjugating enzyme Ubc9 and SUMO-E3 ligase Pias1 as novel N-CoR interaction partners. The SANT1 domain of N-CoR was found to mediate this interaction. We show that K152, K1117, and K1330 of N-CoR can be conjugated to SUMO and that mutation of all sites is necessary to fully block SUMOylation in vitro. Because these lysine residues are located within repression domains I and III, respectively, we investigated a possible correlation between the functions of the repression domains and SUMOylation. Coexpression of Ubc9 protein resulted in enhanced N-CoR–dependent transcriptional repression. Studies using SUMOylation-deficient N-CoR RDI mutants suggest that SUMO modification contributes to repression by N-CoR. Mutation of K152 to R in RD1, for example, not only significantly reduced repression of a reporter gene, but also abolished the effect of Ubc9 on transcriptional repression.

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