scholarly journals Mapping of a Serine-Rich Domain Essential for the Transcriptional, Antiapoptotic, and Transforming Activities of the v-Rel Oncoprotein

1999 ◽  
Vol 19 (1) ◽  
pp. 307-316 ◽  
Author(s):  
Cailin Chen ◽  
François Agnès ◽  
Céline Gélinas
Keyword(s):  
Biological Activity ◽  
Protein Interactions ◽  
Cell Transformation ◽  
Complete Recovery ◽  
Transformation Model ◽  
Transactivation Domain ◽  
Protein Protein Interactions ◽  
Rich Domain ◽  
C Terminus ◽  
Transforming Activity

ABSTRACT The v-Rel oncoprotein belongs to the Rel/NF-κB family of transcription factors and induces aggressive lymphomas in chickens and transgenic mice. Current models for cell transformation by v-Rel invoke the combined activation of gene expression and the dominant inhibition of transcription mediated by its cellular homologs. Here, we mapped a serine-rich transactivation domain in the C terminus of v-Rel that is necessary for its biological activity. Specific serine-to-alanine substitutions within this region impaired the transcriptional activity of v-Rel, whereas a double mutant abolished its function. In contrast, substitutions with phosphomimetic aspartate residues led to a complete recovery of the transcriptional potential. The transforming activity of v-Rel mutants correlated with their ability to inhibit programmed cell death. The transforming and antiapoptotic activities of v-Rel were abolished by defined Ser-to-Ala mutations and restored by most Ser-to-Asp substitutions. However, one Ser-to-Asp mutant showed wild-type transactivation ability but failed to block apoptosis and to transform cells. These results show that the transactivation function of v-Rel is necessary but not sufficient for cell transformation, adding an important dimension to the transformation model. It is possible that defined protein-protein interactions are also required to block apoptosis and transform cells. Since v-Rel is an acutely oncogenic member of the Rel/NF-κB family, our data raise the possibility that phosphorylation of its serine-rich transactivation domain may regulate its unique biological activity.

Insights into the structure and protein–protein interactions of the pro-apoptotic protein ASPP2

2007 ◽  
Vol 35 (5) ◽  
pp. 966-969 ◽  
Author(s):  
S. Rotem ◽  
C. Katz ◽  
A. Friedler
Keyword(s):  
Intermolecular Interactions ◽  
Protein Interactions ◽  
Intramolecular Interaction ◽  
Protein Protein Interactions ◽  
Sh3 Domains ◽  
Src Homology 3 ◽  
Rich Domain ◽  
C Terminus ◽  
Apoptotic Protein ◽  
Src Homology

ASPP (apoptosis-stimulating protein of p53) 2 is a pro-apoptotic protein that stimulates the p53-mediated apoptotic response. Here, we provide an overview of the structure and protein–protein interactions of ASPP2. The C-terminus of ASPP2 contains Ank (ankyrin) repeats and an SH3 domain (Src homology 3 domain). The Ank–SH3 domains mediate interactions between ASPP2 and numerous proteins involved in apoptosis such as p53 and Bcl-2. The proline-rich domain of ASPP2 is unfolded in its native state, but was not shown to mediate intermolecular interactions. Instead, it makes an intramolecular domain–domain interaction with the Ank–SH3 C-terminal domains of ASPP2. This intramolecular interaction between the unstructured proline-rich domain and the structured Ank–SH3 domains in ASPP2, which is possible due to the unfolded nature of the proline-rich domain, is proposed to have an important role in regulating the intermolecular interactions of ASPP2 with its partner proteins.

scholarly journals The Cac1 subunit of histone chaperone CAF-1 organizes CAF-1-H3/H4 architecture and tetramerizes histones

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Wallace H Liu ◽  
Sarah C Roemer ◽  
Yeyun Zhou ◽  
Zih-Jie Shen ◽  
Briana K Dennehey ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Histone Chaperone ◽  
Protein Protein Interactions ◽  
Rich Domain ◽  
C Terminus ◽  
Assembly Factor ◽  
Hydrogen Deuterium ◽  
And Function ◽  
Minimal Region

The histone chaperone Chromatin Assembly Factor 1 (CAF-1) deposits tetrameric (H3/H4)2 histones onto newly-synthesized DNA during DNA replication. To understand the mechanism of the tri-subunit CAF-1 complex in this process, we investigated the protein-protein interactions within the CAF-1-H3/H4 architecture using biophysical and biochemical approaches. Hydrogen/deuterium exchange and chemical cross-linking coupled to mass spectrometry reveal interactions that are essential for CAF-1 function in budding yeast, and importantly indicate that the Cac1 subunit functions as a scaffold within the CAF-1-H3/H4 complex. Cac1 alone not only binds H3/H4 with high affinity, but also promotes histone tetramerization independent of the other subunits. Moreover, we identify a minimal region in the C-terminus of Cac1, including the structured winged helix domain and glutamate/aspartate-rich domain, which is sufficient to induce (H3/H4)2 tetramerization. These findings reveal a key role of Cac1 in histone tetramerization, providing a new model for CAF-1-H3/H4 architecture and function during eukaryotic replication.

scholarly journals E. coli primase and DNA polymerase III holoenzyme are able to bind concurrently to a primed template during DNA replication

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Andrea Bogutzki ◽  
Natalie Naue ◽  
Lidia Litz ◽  
Andreas Pich ◽  
Ute Curth
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Protein Interactions ◽  
Dna Polymerase Iii ◽  
Protein Protein Interactions ◽  
Single Stranded Dna ◽  
E Coli ◽  
Polymerase Iii ◽  
C Terminus ◽  
Pol Iii

Abstract During DNA replication in E. coli, a switch between DnaG primase and DNA polymerase III holoenzyme (pol III) activities has to occur every time when the synthesis of a new Okazaki fragment starts. As both primase and the χ subunit of pol III interact with the highly conserved C-terminus of single-stranded DNA-binding protein (SSB), it had been proposed that the binding of both proteins to SSB is mutually exclusive. Using a replication system containing the origin of replication of the single-stranded DNA phage G4 (G4ori) saturated with SSB, we tested whether DnaG and pol III can bind concurrently to the primed template. We found that the addition of pol III does not lead to a displacement of primase, but to the formation of higher complexes. Even pol III-mediated primer elongation by one or several DNA nucleotides does not result in the dissociation of DnaG. About 10 nucleotides have to be added in order to displace one of the two primase molecules bound to SSB-saturated G4ori. The concurrent binding of primase and pol III is highly plausible, since even the SSB tetramer situated directly next to the 3′-terminus of the primer provides four C-termini for protein-protein interactions.

scholarly journals ArabidopsisImmunophilin-like TWD1 Functionally Interacts with Vacuolar ABC Transporters

2004 ◽  
Vol 15 (7) ◽  
pp. 3393-3405 ◽  
Author(s):  
Markus Geisler ◽  
Marjolaine Girin ◽  
Sabine Brandt ◽  
Vincent Vincenzetti ◽  
Sonia Plaza ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Abc Transporters ◽  
Loss Of Function ◽  
Protein Protein Interactions ◽  
Binding Motifs ◽  
Calmodulin Binding ◽  
C Terminus ◽  
Domain Mapping ◽  
Tetratricopeptide Repeat Domain

Previously, the immunophilin-like protein TWD1 from Arabidopsis has been demonstrated to interact with the ABC transporters AtPGP1 and its closest homologue, AtPGP19. Physiological and biochemical investigation of pgp1/pgp19 and of twd1 plants suggested a regulatory role of TWD1 on AtPGP1/AtPGP19 transport activities. To further understand the dramatic pleiotropic phenotype that is caused by loss-of-function mutation of the TWD1 gene, we were interested in other TWD1 interacting proteins. AtMRP1, a multidrug resistance-associated (MRP/ABCC)-like ABC transporter, has been isolated in a yeast two-hybrid screen. We demonstrate molecular interaction between TWD1 and ABC transporters AtMRP1 and its closest homologue, AtMRP2. Unlike AtPGP1, AtMRP1 binds to the C-terminal tetratricopeptide repeat domain of TWD1, which is well known to mediate protein-protein interactions. Domain mapping proved that TWD1 binds to a motif of AtMRP1 that resembles calmodulin-binding motifs; and calmodulin binding to the C-terminus of MRP1 was verified. By membrane fractionation and GFP-tagging, we localized AtMRP1 to the central vacuolar membrane and the TWD1-AtMRP1 complex was verified in vivo by coimmunoprecipitation. We were able to demonstrate that TWD1 binds to isolated vacuoles and has a significant impact on the uptake of metolachlor-GS and estradiol-β-glucuronide, well-known substrates of vacuolar transporters AtMRP1 and AtMRP2.

scholarly journals Aspartic Acid Isomerization Characterized by High Definition Mass Spectrometry Significantly Alters the Bioactivity of a Novel Toxin from Poecilotheria

Toxins ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 207 ◽  
Author(s):  
Stephen R. Johnson ◽  
Hillary G. Rikli
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
Ion Mobility ◽  
Protein Protein Interactions ◽  
High Definition ◽  
Vast Number ◽  
Post Translational Modifications ◽  
C Terminus ◽  
Acid Isomerization

Research in toxinology has created a pharmacological paradox. With an estimated 220,000 venomous animals worldwide, the study of peptidyl toxins provides a vast number of effector molecules. However, due to the complexity of the protein-protein interactions, there are fewer than ten venom-derived molecules on the market. Structural characterization and identification of post-translational modifications are essential to develop biological lead structures into pharmaceuticals. Utilizing advancements in mass spectrometry, we have created a high definition approach that fuses conventional high-resolution MS-MS with ion mobility spectrometry (HDMSE) to elucidate these primary structure characteristics. We investigated venom from ten species of “tiger” spider (Genus: Poecilotheria) and discovered they contain isobaric conformers originating from non-enzymatic Asp isomerization. One conformer pair conserved in five of ten species examined, denominated PcaTX-1a and PcaTX-1b, was found to be a 36-residue peptide with a cysteine knot, an amidated C-terminus, and isoAsp33Asp substitution. Although the isomerization of Asp has been implicated in many pathologies, this is the first characterization of Asp isomerization in a toxin and demonstrates the isomerized product’s diminished physiological effects. This study establishes the value of a HDMSE approach to toxin screening and characterization.

scholarly journals Divisome under Construction: Distinct Domains of the Small Membrane Protein FtsB Are Necessary for Interaction with Multiple Cell Division Proteins

2009 ◽  
Vol 191 (8) ◽  
pp. 2815-2825 ◽  
Author(s):  
Mark D. Gonzalez ◽  
Jon Beckwith
Keyword(s):  
Cell Division ◽  
Membrane Proteins ◽  
Protein Interactions ◽  
Cell Envelope ◽  
Coiled Coil ◽  
Main Function ◽  
Protein Protein Interactions ◽  
Molecular Scaffold ◽  
C Terminus ◽  
Under Construction

ABSTRACT Cell division in bacteria requires the coordinated action of a set of proteins, the divisome, for proper constriction of the cell envelope. Multiple protein-protein interactions are required for assembly of a stable divisome. Within the Escherichia coli divisome is a conserved subcomplex of inner membrane proteins, the FtsB/FtsL/FtsQ complex, which is necessary for linking the upstream division proteins, which are predominantly cytoplasmic, with the downstream division proteins, which are predominantly periplasmic. FtsB and FtsL are small bitopic membrane proteins with predicted coiled-coil motifs, which themselves form a stable subcomplex that can recruit downstream division proteins independently of FtsQ; however, the details of how FtsB and FtsL interact together and with other proteins remain to be characterized. Despite the small size of FtsB, we identified separate interaction domains of FtsB that are required for interaction with FtsL and FtsQ. The N-terminal half of FtsB is necessary for interaction with FtsL and sufficient, when in complex with FtsL, for recruitment of downstream division proteins, while a portion of the FtsB C terminus is necessary for interaction with FtsQ. These properties of FtsB support the proposal that its main function is as part of a molecular scaffold to allow for proper formation of the divisome.

Interactions in vivo between the proteins of infectious bursal disease virus: capsid protein VP3 interacts with the RNA-dependent RNA polymerase, VP1

Microbiology ◽  
2000 ◽  
Vol 81 (1) ◽  
pp. 209-218 ◽  
Author(s):  
Mirriam G. J. Tacken ◽  
Peter J. M. Rottier ◽  
Arno L. J. Gielkens ◽  
Ben P. H. Peeters
Keyword(s):  
Disease Virus ◽  
Protein Interactions ◽  
Infectious Bursal Disease Virus ◽  
Viral Proteins ◽  
Infectious Bursal Disease ◽  
Transactivation Domain ◽  
Protein Protein Interactions ◽  
Bursal Disease ◽  
Infected Cells

Little is known about the intermolecular interactions between the viral proteins of infectious bursal disease virus (IBDV). By using the yeast two-hybrid system, which allows the detection of protein–protein interactions in vivo, all possible interactions were tested by fusing the viral proteins to the LexA DNA-binding domain and the B42 transactivation domain. A heterologous interaction between VP1 and VP3, and homologous interactions of pVP2, VP3, VP5 and possibly VP1, were found by co-expression of the fusion proteins in Saccharomyces cerevisiae. The presence of the VP1–VP3 complex in IBDV-infected cells was confirmed by co-immunoprecipitation studies. Kinetic analyses showed that the complex of VP1 and VP3 is formed in the cytoplasm and eventually is released into the cell-culture medium, indicating that VP1–VP3 complexes are present in mature virions. In IBDV-infected cells, VP1 was present in two forms of 90 and 95 kDa. Whereas VP3 initially interacted with both the 90 and 95 kDa proteins, later it interacted exclusively with the 95 kDa protein both in infected cells and in the culture supernatant. These results suggest that the VP1–VP3 complex is involved in replication and packaging of the IBDV genome.

scholarly journals Mutations in the v-Rel Transactivation Domain Indicate Altered Phosphorylation and Identify a Subset of NF-κB-Regulated Cell Death Inhibitors Important for v-Rel Transforming Activity

2003 ◽  
Vol 23 (5) ◽  
pp. 1520-1533 ◽  
Author(s):  
Béatrice Rayet ◽  
Yongjun Fan ◽  
Céline Gélinas
Keyword(s):  
Transcriptional Activity ◽  
Lymphoid Cells ◽  
Transactivation Domain ◽  
Constitutive Activation ◽  
Rich Domain ◽  
Endogenous Expression ◽  
Regulated Cell Death ◽  
Transforming Activity ◽  
Selection For

ABSTRACT Consistent with the constitutive activation of Rel/NF-κB in human hematopoietic tumors, the v-Rel oncoprotein induces aggressive leukemia/lymphomas in animal models. v-Rel is thus a valuable tool to characterize the role of Rel/NF-κB in cancer and the mechanisms involved. Prior studies by our group identified a serine-rich domain in v-Rel that was required for biological activity. Here, we investigated the molecular basis for the transformation defect of specific serine mutants. We show that the transforming efficiency of these mutants in primary lymphoid cells is correlated with their ability to mediate κB site-dependent transactivation and with specific changes in phosphorylation profiles. Interestingly, coexpression of the death antagonists Bcl-xL and Bcl-2 significantly increased their oncogenicity, whereas other NF-κB-regulated death inhibitors showed little or no effect. The fact that a subset of apoptosis inhibitors could rescue v-Rel transactivation mutants suggests that their reduced transcriptional activity may critically affect expression of defined death antagonists essential for oncogenesis. Consistent with this hypothesis, we observed selection for high endogenous expression of Bcl-2-related death antagonists in cells transformed by weakly transforming v-Rel mutants. These results emphasize the need for Rel/NF-κB to efficiently activate expression of a subset of antiapoptotic genes from the Bcl-2 family to manifest its oncogenic phenotype.

The androgen receptor transactivation domain: the interplay between protein conformation and protein–protein interactions

2003 ◽  
Vol 31 (5) ◽  
pp. 1042-1046 ◽  
Author(s):  
J. Reid ◽  
R. Betney ◽  
K. Watt ◽  
I.J. McEwan
Keyword(s):  
Androgen Receptor ◽  
Protein Interactions ◽  
Transcriptional Activation ◽  
Specific Protein ◽  
Transactivation Domain ◽  
Protein Protein Interactions ◽  
Induced Folding ◽  
Large N ◽  
Tryptophan Residues ◽  
Receptor Transactivation

The AR (androgen receptor) belongs to the nuclear receptor superfamily and directly regulates patterns of gene expression in response to the steroids testosterone and dihydrotestosterone. Sequences within the large N-terminal domain of the receptor have been shown to be important for transactivation and protein–protein interactions; however, little is known about the structure and folding of this region. Folding of the AR transactivation domain was observed in the presence of the helix-stabilizing solvent trifluorethanol and the natural osmolyte TMAO (trimethylamine N-oxide). TMAO resulted in the movement of two tryptophan residues to a less solvent-exposed environment and the formation of a protease-resistant conformation. Critically, binding to a target protein, the RAP74 subunit of the general transcription factor TFIIF, resulted in a similar resistance to protease digestion, consistent with induced folding of the receptor transactivation domain. Our current hypothesis is that the folding of the transactivation domain in response to specific protein–protein interactions creates a platform for subsequent interactions, resulting in the formation of a competent transcriptional activation complex.

scholarly journals Positive and Negative Regulation of Poly(A) Nuclease

2004 ◽  
Vol 24 (12) ◽  
pp. 5521-5533 ◽  
Author(s):  
David A. Mangus ◽  
Matthew C. Evans ◽  
Nathan S. Agrin ◽  
Mandy Smith ◽  
Preetam Gongidi ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Protein Interactions ◽  
Binding Pocket ◽  
Negative Regulation ◽  
Single Amino Acid ◽  
Carboxy Terminus ◽  
Protein Protein Interactions ◽  
C Terminus

ABSTRACT PAN, a yeast poly(A) nuclease, plays an important nuclear role in the posttranscriptional maturation of mRNA poly(A) tails. The activity of this enzyme is dependent on its Pan2p and Pan3p subunits, as well as the presence of poly(A)-binding protein (Pab1p). We have identified and characterized the associated network of factors controlling the maturation of mRNA poly(A) tails in yeast and defined its relevant protein-protein interactions. Pan3p, a positive regulator of PAN activity, interacts with Pab1p, thus providing substrate specificity for this nuclease. Pab1p also regulates poly(A) tail trimming by interacting with Pbp1p, a factor that appears to negatively regulate PAN. Pan3p and Pbp1p both interact with themselves and with the C terminus of Pab1p. However, the domains required for Pan3p and Pbp1p binding on Pab1p are distinct. Single amino acid changes that disrupt Pan3p interaction with Pab1p have been identified and define a binding pocket in helices 2 and 3 of Pab1p's carboxy terminus. The importance of these amino acids for Pab1p-Pan3p interaction, and poly(A) tail regulation, is underscored by experiments demonstrating that strains harboring substitutions in these residues accumulate mRNAs with long poly(A) tails in vivo.

Sign in / Sign up

Export Citation Format

Share Document