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.

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 The loop region of the helix-loop-helix protein Id1 is critical for its dominant negative activity.

1993 ◽  
Vol 13 (12) ◽  
pp. 7874-7880 ◽  
Author(s):  
S Pesce ◽  
R Benezra
Keyword(s):  
Amino Acids ◽  
Protein Interactions ◽  
Dominant Negative ◽  
Site Directed Mutagenesis ◽  
Protein Protein Interactions ◽  
Helix Loop Helix ◽  
Loop Region ◽  
Dominant Negative Activity

Id1, a helix-loop-helix (HLH) protein which lacks a DNA binding domain, has been shown to negatively regulate other members of the HLH family by direct protein-protein interactions, both in vitro and in vivo. In this study, we report the results of site-directed mutagenesis experiments aimed at defining the regions of Id1 which are important for its activity. We have found that the HLH domain of Id1 is necessary and nearly sufficient for its activity. In addition, we show that two amino acid residues at the amino terminus of the Id1 loop are critical for its activity, perhaps by specifying the correct dimerization partners. In this regard, replacing the first four amino acids of the loops of the basic HLH proteins E12 and E47 with the corresponding amino acids of Id1 confers Id1 dimerization specificity. These studies point to the loop region as an important structural and functional element of the Id subfamily of HLH proteins.

scholarly journals Differential Regulation of Hand1 Homodimer and Hand1-E12 Heterodimer Activity by the Cofactor FHL2

2004 ◽  
Vol 24 (22) ◽  
pp. 9835-9847 ◽  
Author(s):  
Alison A. Hill ◽  
Paul R. Riley
Keyword(s):  
Protein Interactions ◽  
Differential Regulation ◽  
Protein Protein Interactions ◽  
Helix Loop Helix ◽  
Developing Heart ◽  
Unknown Protein ◽  
Class B ◽  
Bhlh Proteins

ABSTRACT The basic helix-loop-helix (bHLH) factor Hand1 plays an essential role in cardiac morphogenesis, and yet its precise function remains unknown. Protein-protein interactions involving Hand1 provide a means of determining how Hand1-induced gene expression in the developing heart might be regulated. Hand1 is known to form either heterodimers with near-ubiquitous E-factors and other lineage-restricted class B bHLH proteins or homodimers with itself in vitro. To date, there have been no reported Hand1 protein interactions involving non-bHLH proteins. Heterodimer-versus-homodimer choice is mediated by the phosphorylation status of Hand1; however, little is known about the in vivo function of these dimers or, importantly, how they are regulated. In an effort to understand how Hand1 activity in the heart might be regulated postdimerization, we have investigated tertiary Hand1-protein interactions with non-bHLH factors. We describe a novel interaction of Hand1 with the LIM domain protein FHL2, a known transcriptional coactivator and corepressor expressed in the developing cardiovascular system. FHL2 interacts with Hand1 via the bHLH domain and is able to repress Hand1/E12 heterodimer-induced transcription but has no effect on Hand1/Hand1 homodimer activity. This effect of FHL2 is not mediated either at the level of dimerization or via an effect of Hand1/E12 DNA binding. In summary, our data describe a novel differential regulation of Hand1 heterodimers versus homodimers by association of the cofactor FHL2 and provide insight into the potential for a tertiary level of control of Hand1 activity in the developing heart.

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.

The loop region of the helix-loop-helix protein Id1 is critical for its dominant negative activity

1993 ◽  
Vol 13 (12) ◽  
pp. 7874-7880
Author(s):  
S Pesce ◽  
R Benezra
Keyword(s):  
Amino Acids ◽  
Protein Interactions ◽  
Dominant Negative ◽  
Site Directed Mutagenesis ◽  
Protein Protein Interactions ◽  
Helix Loop Helix ◽  
Loop Region ◽  
Dominant Negative Activity

Id1, a helix-loop-helix (HLH) protein which lacks a DNA binding domain, has been shown to negatively regulate other members of the HLH family by direct protein-protein interactions, both in vitro and in vivo. In this study, we report the results of site-directed mutagenesis experiments aimed at defining the regions of Id1 which are important for its activity. We have found that the HLH domain of Id1 is necessary and nearly sufficient for its activity. In addition, we show that two amino acid residues at the amino terminus of the Id1 loop are critical for its activity, perhaps by specifying the correct dimerization partners. In this regard, replacing the first four amino acids of the loops of the basic HLH proteins E12 and E47 with the corresponding amino acids of Id1 confers Id1 dimerization specificity. These studies point to the loop region as an important structural and functional element of the Id subfamily of HLH proteins.

Probing the 14-3-3 Isoform-Specificity Profile of Interactions Stabilized by Fusicoccin A

2020 ◽  
Author(s):  
James Frederich ◽  
Ananya Sengupta ◽  
Josue Liriano ◽  
Ewa A. Bienkiewicz ◽  
Brian G. Miller
Keyword(s):  
Protein Interactions ◽  
Neurological Diseases ◽  
Adapter Proteins ◽  
Protein Protein Interactions ◽  
Specific Expression ◽  
Individual Client ◽  
Isoform Specificity ◽  
Fusicoccin A

Fusicoccin A (FC) is a fungal phytotoxin that stabilizes protein–protein interactions (PPIs) between 14-3-3 adapter proteins and their phosphoprotein interaction partners. In recent years, FC has emerged as an important chemical probe of human 14-3-3 PPIs implicated in cancer and neurological diseases. These previous studies have established the structural requirements for FC-induced stabilization of 14-3-3·client phosphoprotein complexes; however, the effect of different 14-3-3 isoforms on FC activity has not been systematically explored. This is a relevant question for the continued development of FC variants because there are seven distinct isoforms of 14-3-3 in humans. Despite their remarkable sequence and structural similarities, a growing body of experimental evidence supports both tissue-specific expression of 14-3-3 isoforms and isoform-specific functions <i>in vivo</i>. Herein, we report the isoform-specificity profile of FC <i>in vitro</i>using recombinant human 14-3-3 isoforms and a focused library of fluorescein-labeled hexaphosphopeptides mimicking the C-terminal 14-3-3 recognition domains of client phosphoproteins targeted by FC in cell culture. Our results reveal modest isoform preferences for individual client phospholigands and demonstrate that FC differentially stabilizes PPIs involving 14-3-3s. Together, these data provide strong motivation for the development of non-natural FC variants with enhanced selectivity for individual 14-3-3 isoforms.

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.

scholarly journals EEF1A2 interacts with HSP90AB1 to promote lung adenocarcinoma metastasis via enhancing TGF-β/SMAD signalling

2021 ◽  
Author(s):  
Liqing Jia ◽  
Xiaolu Ge ◽  
Chao Du ◽  
Linna Chen ◽  
Yanhong Zhou ◽  
...  
Keyword(s):  
Lung Adenocarcinoma ◽  
Protein Interactions ◽  
Elongation Factor ◽  
Protein Translation ◽  
Protein Protein Interactions ◽  
Promising Target ◽  
Tgfβ Receptor ◽  
Mesenchymal Transformation

Abstract Background Eukaryotic protein translation elongation factor 1α2 (EEF1A2) is an oncogene that promotes the progression of breast and pancreatic cancer. In this study, we aimed to elucidate the oncogenic function of EEF1A2 in the metastasis of lung adenocarcinoma (LUAD). Methods Immunohistochemistry and western blot were used to study EEF1A2 expression levels in LUAD tissues and cells, respectively. The role of EEF1A2 in LUAD progression were investigated in vitro and in vivo. We identified potential EEF1A2-binding proteins by liquid chromatography-electrospray mass spectrometry (LC-MS)/MS. Protein–protein interactions were determined by immunofluorescence and co-immunoprecipitation (Co-IP). Results In this study, we report that EEF1A2 mediates the epithelial–mesenchymal transformation (EMT), to promote the metastasis of LUAD cells in vitro and in vivo. Moreover, EEF1A2 interacts with HSP90AB1 to increase TGFβ Receptor (TβR)-I, and TβRII expression, followed by enhanced SMAD3 and pSMAD3 expression and nuclear localisation, which promotes the EMT of LUAD cells. Overexpression of EEF1A2 in cancer tissues is associated with poor prognosis and short survival of patients with LUAD. Conclusions These findings underscore the molecular functions of EEF1A2 in LUAD metastasis and indicate that EEF1A2 represents a promising target in the treatment of aggressive LUAD.

Differential use of SCL/TAL-1 DNA-binding domain in developmental hematopoiesis

Blood ◽  
2008 ◽  
Vol 112 (4) ◽  
pp. 1056-1067 ◽  
Author(s):  
Mira T. Kassouf ◽  
Hedia Chagraoui ◽  
Paresh Vyas ◽  
Catherine Porcher
Keyword(s):  
Dna Binding ◽  
Molecular Mechanisms ◽  
Binding Activity ◽  
Hematopoietic Stem ◽  
Helix Loop Helix ◽  
Experimental Systems ◽  
Dna Binding Activity ◽  
Independent Functions

Abstract Dissecting the molecular mechanisms used by developmental regulators is essential to understand tissue specification/differentiation. SCL/TAL-1 is a basic helix-loop-helix transcription factor absolutely critical for hematopoietic stem/progenitor cell specification and lineage maturation. Using in vitro and forced expression experimental systems, we previously suggested that SCL might have DNA-binding–independent functions. Here, to assess the requirements for SCL DNA-binding activity in vivo, we examined hematopoietic development in mice carrying a germline DNA-binding mutation. Remarkably, in contrast to complete absence of hematopoiesis and early lethality in scl-null embryos, specification of hematopoietic cells occurred in homozygous mutant embryos, indicating that direct DNA binding is dispensable for this process. Lethality was forestalled to later in development, although some mice survived to adulthood. Anemia was documented throughout development and in adulthood. Cellular and molecular studies showed requirements for SCL direct DNA binding in red cell maturation and indicated that scl expression is positively autoregulated in terminally differentiating erythroid cells. Thus, different mechanisms of SCL's action predominate depending on the developmental/cellular context: indirect DNA binding activities and/or sequestration of other nuclear regulators are sufficient in specification processes, whereas direct DNA binding functions with transcriptional autoregulation are critically required in terminal maturation processes.

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.

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