scholarly journals A truncated IFN-regulatory factor-8\IFN consensus sequence-binding protein acts as dominant-negative, interferes with endogenous protein-protein interactions and leads to apoptosis of immune cells

2003 ◽  
Vol 15 (7) ◽  
pp. 807-815 ◽  
Author(s):  
S. Hashmueli
Keyword(s):  
Protein Interactions ◽  
Immune Cells ◽  
Binding Protein ◽  
Consensus Sequence ◽  
Dominant Negative ◽  
Regulatory Factor ◽  
Protein Protein Interactions ◽  
Endogenous Protein

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 Regulation of the germinal center gene program by interferon (IFN) regulatory factor 8/IFN consensus sequence-binding protein

2005 ◽  
Vol 203 (1) ◽  
pp. 63-72 ◽  
Author(s):  
Chang Hoon Lee ◽  
Mark Melchers ◽  
Hongsheng Wang ◽  
Ted A. Torrey ◽  
Rebecca Slota ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Transcriptional Activation ◽  
Germinal Center ◽  
Target Genes ◽  
Fluorescent Protein ◽  
Binding Protein ◽  
Consensus Sequence ◽  
B Cell Lymphoma ◽  
Regulatory Factor

Interferon (IFN) consensus sequence-binding protein/IFN regulatory factor 8 (IRF8) is a transcription factor that regulates the differentiation and function of macrophages, granulocytes, and dendritic cells through activation or repression of target genes. Although IRF8 is also expressed in lymphocytes, its roles in B cell and T cell maturation or function are ill defined, and few transcriptional targets are known. Gene expression profiling of human tonsillar B cells and mouse B cell lymphomas showed that IRF8 transcripts were expressed at highest levels in centroblasts, either from secondary lymphoid tissue or transformed cells. In addition, staining for IRF8 was most intense in tonsillar germinal center (GC) dark-zone centroblasts. To discover B cell genes regulated by IRF8, we transfected purified primary tonsillar B cells with enhanced green fluorescent protein–tagged IRF8, generated small interfering RNA knockdowns of IRF8 expression in a mouse B cell lymphoma cell line, and examined the effects of a null mutation of IRF8 on B cells. Each approach identified activation-induced cytidine deaminase (AICDA) and BCL6 as targets of transcriptional activation. Chromatin immunoprecipitation studies demonstrated in vivo occupancy of 5′ sequences of both genes by IRF8 protein. These results suggest previously unappreciated roles for IRF8 in the transcriptional regulation of B cell GC reactions that include direct regulation of AICDA and BCL6.

scholarly journals EndoBind detects endogenous protein-protein interactions in real time

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Anke Bill ◽  
Sheryll Espinola ◽  
Daniel Guthy ◽  
Jacob R. Haling ◽  
Mylene Lanter ◽  
...  
Keyword(s):  
Real Time ◽  
High Throughput ◽  
Protein Interactions ◽  
Continuous Monitoring ◽  
Protein Protein Interactions ◽  
Dimer Formation ◽  
Endogenous Protein ◽  
Extended Duration ◽  
Substrate Addition

AbstractWe present two high-throughput compatible methods to detect the interaction of ectopically expressed (RT-Bind) or endogenously tagged (EndoBind) proteins of interest. Both approaches provide temporal evaluation of dimer formation over an extended duration. Using examples of the Nrf2-KEAP1 and the CRAF-KRAS-G12V interaction, we demonstrate that our method allows for the detection of signal for more than 2 days after substrate addition, allowing for continuous monitoring of endogenous protein-protein interactions in real time.

A crosslinked and ribosylated actin trimer does not interact productively with myosin

2019 ◽  
Vol 97 (2) ◽  
pp. 140-147 ◽  
Author(s):  
Navneet Sidhu ◽  
John F. Dawson
Keyword(s):  
Binding Site ◽  
Protein Interactions ◽  
Binding Proteins ◽  
Binding Protein ◽  
Dnase I ◽  
Protein Protein Interactions ◽  
Adp Ribosylation ◽  
Myosin Binding ◽  
Pointed End

A purified F-actin-derived actin trimer that interacts with end-binding proteins did not activate or bind the side-binding protein myosin under rigor conditions. Remodeling of the actin trimer by the binding of gelsolin did not rescue myosin binding, nor did the use of different means of inhibiting the polymerization of the trimer. Our results demonstrate that ADP-ribosylation on all actin subunits of an F-actin-derived trimer inhibits myosin binding and that the binding of DNase-I to the pointed end subunits of a crosslinked trimer also remodels the myosin binding site. Taken together, this work highlights the need for a careful balance between modification of actin subunits and maintaining protein–protein interactions to produce a physiologically relevant short F-actin complex.

scholarly journals Bcl-2 and FKBP12 bind to IP3 and ryanodine receptors at overlapping sites: the complexity of protein–protein interactions for channel regulation

2015 ◽  
Vol 43 (3) ◽  
pp. 396-404 ◽  
Author(s):  
Tim Vervliet ◽  
Jan B. Parys ◽  
Geert Bultynck
Keyword(s):  
Protein Interactions ◽  
Hippocampal Neurons ◽  
Binding Protein ◽  
Ryanodine Receptors ◽  
B Cell Lymphoma ◽  
Experimental Models ◽  
Amino Acid Sequences ◽  
Protein Protein Interactions ◽  
Fk506 Binding Protein ◽  
Fk506 Binding Proteins

The 12- and 12.6-kDa FK506-binding proteins, FKBP12 (12-kDa FK506-binding protein) and FKBP12.6 (12.6-kDa FK506-binding protein), have been implicated in the binding to and the regulation of ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (IP3Rs), both tetrameric intracellular Ca2+-release channels. Whereas the amino acid sequences responsible for FKBP12 binding to RyRs are conserved in IP3Rs, FKBP12 binding to IP3Rs has been questioned and could not be observed in various experimental models. Nevertheless, conservation of these residues in the different IP3R isoforms and during evolution suggested that they could harbour an important regulatory site critical for IP3R-channel function. Recently, it has become clear that in IP3Rs, this site was targeted by B-cell lymphoma 2 (Bcl-2) via its Bcl-2 homology (BH)4 domain, thereby dampening IP3R-mediated Ca2+ flux and preventing pro-apoptotic Ca2+ signalling. Furthermore, vice versa, the presence of the corresponding site in RyRs implied that Bcl-2 proteins could associate with and regulate RyR channels. Recently, the existence of endogenous RyR–Bcl-2 complexes has been identified in primary hippocampal neurons. Like for IP3Rs, binding of Bcl-2 to RyRs also involved its BH4 domain and suppressed RyR-mediated Ca2+ release. We therefore propose that the originally identified FKBP12-binding site in IP3Rs is a region critical for controlling IP3R-mediated Ca2+ flux by recruiting Bcl-2 rather than FKBP12. Although we hypothesize that anti-apoptotic Bcl-2 proteins, but not FKBP12, are the main physiological inhibitors of IP3Rs, we cannot exclude that Bcl-2 could help engaging FKBP12 (or other FKBP isoforms) to the IP3R, potentially via calcineurin.

scholarly journals Use of periplasmic target protein capture for phage display engineering of tight-binding protein–protein interactions

2011 ◽  
Vol 24 (11) ◽  
pp. 819-828 ◽  
Author(s):  
Bartlomiej G. Fryszczyn ◽  
Nicholas G. Brown ◽  
Wanzhi Huang ◽  
Miriam A. Balderas ◽  
Timothy Palzkill
Keyword(s):  
Phage Display ◽  
Protein Interactions ◽  
Binding Protein ◽  
Tight Binding ◽  
Target Protein ◽  
Protein Protein Interactions ◽  
Protein Capture

scholarly journals Interferon consensus sequence-binding protein, a member of the interferon regulatory factor family, suppresses interferon-induced gene transcription.

1993 ◽  
Vol 13 (1) ◽  
pp. 588-599 ◽  
Author(s):  
N Nelson ◽  
M S Marks ◽  
P H Driggers ◽  
K Ozato
Keyword(s):  
Major Histocompatibility Complex ◽  
Retinoic Acid ◽  
Major Histocompatibility Complex Class ◽  
Binding Protein ◽  
Consensus Sequence ◽  
Interferon Regulatory Factor ◽  
Chloramphenicol Acetyltransferase ◽  
Complex Class ◽  
Regulatory Factor ◽  
Histocompatibility Complex

We previously isolated a cDNA clone encoding interferon consensus sequence-binding protein (ICSBP), a member of the interferon regulatory factor (IRF) family, that binds to the interferon (IFN)-stimulated response element (ISRE) of many IFN-regulated genes. In this investigation, we studied the functional role of ICSBP by transient cotransfection of ICSBP cDNA with IFN-responsive reporter genes into the human embryonal carcinoma cell line N-Tera2. These cells were shown not to express ICSBP or IRF-2, thus allowing functional analysis of transfected cDNAs. Cotransfection of ICSBP into cells treated with retinoic acid or any of the IFNs (alpha, beta, or gamma) repressed expression of a chloramphenicol acetyltransferase reporter driven by the major histocompatibility complex class I gene promoter. Similarly, ICSBP repressed expression of chloramphenicol acetyltransferase reporters driven by the ISREs of the 2'-5' oligoadenylate synthetase, guanylate-binding protein, and ISG-15 genes in IFN-treated cells. The repression was dependent on the presence of the ISRE in the reporter. Deletion analysis showed that the putative N-terminal DNA binding domain of ICSBP by itself is capable of mediating the repression. Using the same cotransfection conditions as for ICSBP, a similar repression of these reporters was observed with IRF-2. Finally, ICSBP repressed the IRF-1-mediated induction of major histocompatibility complex class I and IFN-beta reporters in the absence of IFN or retinoic acid. Taken together, these results suggest that ICSBP is a negative regulatory factor capable of repressing transcription of target genes induced by IFN, retinoic acid, or IRF-1.

PKCε activation induces dichotomous cardiac phenotypes and modulates PKCε-RACK interactions and RACK expression

2001 ◽  
Vol 280 (3) ◽  
pp. H946-H955 ◽  
Author(s):  
Jason M. Pass ◽  
Yuting Zheng ◽  
William B. Wead ◽  
Jun Zhang ◽  
Richard C. X. Li ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Protein Expression ◽  
Protein Interactions ◽  
Dominant Negative ◽  
The Other ◽  
Protein Protein Interactions ◽  
Kinase C ◽  
Transgenic Lines ◽  
Pkc Activation

Receptors for activated C kinase (RACKs) have been shown to facilitate activation of protein kinase C (PKC). However, it is unknown whether PKC activation modulates RACK protein expression and PKC-RACK interactions. This issue was studied in two PKCε transgenic lines exhibiting dichotomous cardiac phenotypes: one exhibits increased resistance to myocardial ischemia (cardioprotected phenotype) induced by a modest increase in PKCε activity (228 ± 23% of control), whereas the other exhibits cardiac hypertrophy and failure (hypertrophied phenotype) induced by a marked increase in PKCε activity (452 ± 28% of control). Our data demonstrate that activation of PKC modulates the expression of RACK isotypes and PKC-RACK interactions in a PKCε activity- and dosage-dependent fashion. We found that, in mice displaying the cardioprotected phenotype, activation of PKCε enhanced RACK2 expression (178 ± 13% of control) and particulate PKCε-RACK2 protein-protein interactions (178 ± 18% of control). In contrast, in mice displaying the hypertrophied phenotype, there was not only an increase in RACK2 expression (330 ± 33% of control) and particulate PKCε-RACK2 interactions (154 ± 14% of control) but also in RACK1 protein expression (174 ± 10% of control). Most notably, PKCε-RACK1 interactions were identified in this line. With the use of transgenic mice expressing a dominant negative PKCε, we found that the changes in RACK expression as well as the attending cardiac phenotypes were dependent on PKCε activity. Our observations demonstrate that RACK expression is dynamically regulated by PKCε and suggest that differential patterns of PKCε-RACK interactions may be important determinants of PKCε-dependent cardiac phenotypes.

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