scholarly journals ParB of Pseudomonas aeruginosa: Interactions with Its Partner ParA and Its Target parS and Specific Effects on Bacterial Growth

2004 ◽  
Vol 186 (20) ◽  
pp. 6983-6998 ◽  
Author(s):  
Aneta A. Bartosik ◽  
Krzysztof Lasocki ◽  
Jolanta Mierzejewska ◽  
Christopher M. Thomas ◽  
Grazyna Jagura-Burdzy
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Streptomyces Coelicolor ◽  
Consensus Sequence ◽  
Terminal Part ◽  
Dimerization Domain ◽  
Interaction Domain ◽  
C Terminus ◽  
Cell Components

ABSTRACT The par genes of Pseudomonas aeruginosa have been studied to increase the understanding of their mechanism of action and role in the bacterial cell. Key properties of the ParB protein have been identified and are associated with different parts of the protein. The ParB- ParB interaction domain was mapped in vivo and in vitro to the C-terminal 56 amino acids (aa); 7 aa at the C terminus play an important role. The dimerization domain of P. aeruginosa ParB is interchangeable with the dimerization domain of KorB from plasmid RK2 (IncP1 group). The C-terminal part of ParB is also involved in ParB-ParA interactions. Purified ParB binds specifically to DNA containing a putative parS sequence based on the consensus sequence found in the chromosomes of Bacillus subtilis, Pseudomonas putida, and Streptomyces coelicolor. The overproduction of ParB was shown to inhibit the function of genes placed near parS. This “silencing” was dependent on the parS sequence and its orientation. The overproduction of P. aeruginosa ParB or its N-terminal part also causes inhibition of the growth of P. aeruginosa and P. putida but not Escherichia coli cells. Since this inhibitory determinant is located well away from ParB segments required for dimerization or interaction with the ParA counterpart, this result may suggest a role for the N terminus of P. aeruginosa ParB in interactions with host cell components.

scholarly journals L1 Interaction Domains of Papillomavirus L2 Necessary for Viral Genome Encapsidation

2001 ◽  
Vol 75 (9) ◽  
pp. 4332-4342 ◽  
Author(s):  
Martin M. Okun ◽  
Patricia M. Day ◽  
Heather L. Greenstone ◽  
Frank P. Booy ◽  
Douglas R. Lowy ◽  
...  
Keyword(s):  
Viral Genome ◽  
Bovine Papillomavirus ◽  
Interaction Domain ◽  
Binding Domains ◽  
C Terminus ◽  
Interaction Domains ◽  
L1 And L2 ◽  
Genome Encapsidation

ABSTRACT BPHE-1 cells, which harbor 50 to 200 viral episomes, encapsidate viral genome and generate infectious bovine papillomavirus type 1 (BPV1) upon coexpression of capsid proteins L1 and L2 of BPV1, but not coexpression of BPV1 L1 and human papillomavirus type 16 (HPV16) L2. BPV1 L2 bound in vitro via its C-terminal 85 residues to purified L1 capsomers, but not with intact L1 virus-like particles in vitro. However, when the efficiency of BPV1 L1 coimmunoprecipitation with a series of BPV1 L2 deletion mutants was examined in vivo, the results suggested that residues 129 to 246 and 384 to 460 contain independent L1 interaction domains. An L2 mutant lacking the C-terminal L1 interaction domain was impaired for encapsidation of the viral genome. Coexpression of BPV1 L1 and a chimeric L2 protein composed of HPV16 L2 residues 1 to 98 fused to BPV1 L2 residues 99 to 469 generated infectious virions. However, inefficient encapsidation was seen when L1 was coexpressed with either BPV1 L2 with residues 91 to 246 deleted or with BPV1 L2 with residues 1 to 225 replaced with HPV16 L2. Impaired genome encapsidation did not correlate closely with impairment of the L2 proteins either to localize to promyelocytic leukemia oncogenic domains (PODs) or to induce localization of L1 or E2 to PODs. We conclude that the L1-binding domain located near the C terminus of L2 may bind L1 prior to completion of capsid assembly, and that both L1-binding domains of L2 are required for efficient encapsidation of the viral genome.

scholarly journals WBP-2, a WW domain binding protein, interacts with the thyroid-specific transcription factor Pax8

2004 ◽  
Vol 377 (3) ◽  
pp. 553-560 ◽  
Author(s):  
Roberto NITSCH ◽  
Tina DI PALMA ◽  
Anna MASCIA ◽  
Mariastella ZANNINI
Keyword(s):  
Protein Interaction ◽  
Transcriptional Activation ◽  
Binding Protein ◽  
Interaction Domain ◽  
Ww Domain ◽  
Transcriptional Activation Domain ◽  
Culture Cells ◽  
C Terminus

The Pax gene family encodes transcription factors that are essential in organogenesis and in the differentiation of various organs in higher eukaryotes. Pax proteins have a DNA binding domain at the N-terminus, and a transcriptional activation domain at the C-terminus. How these domains interact with the transcriptional machinery of the cell is still unclear. In the present paper, we describe the identification by means of immunological screening of the WW domain binding protein WBP-2 as a biochemical interactor of Pax8 (a WW domain is a protein-interaction domain containing two conserved tryptophan residues). Pax8 is required for the morphogenesis of the thyroid gland and for the maintenance of the thyroid differentiated cellular phenotype. WBP-2 was identified originally as a WW domain binding protein, and its function is still unknown. WBP-2 binds to Pax8 in vitro in pulldown assays, and in vivo in tissue culture cells in co-immunoprecipitation assays. Interestingly, Pax8 does not contain a WW domain. Our results point to the identification of a new protein-interacting domain that is present in the C-terminal portion of Pax8 and that is required for protein–protein interaction with WBP-2. Our results demonstrate that WBP-2 is not a transcriptional co-activator of Pax8, but rather behaves as an adaptor molecule, as suggested in other studies.

scholarly journals Interaction of the Streptomyces Wbl protein WhiD with the principal sigma factor σHrdB depends on the WhiD [4Fe-4S] cluster

2020 ◽  
Vol 295 (28) ◽  
pp. 9752-9765
Author(s):  
Melissa Y. Y. Stewart ◽  
Matthew J. Bush ◽  
Jason C. Crack ◽  
Mark J. Buttner ◽  
Nick E. Le Brun
Keyword(s):  
Sigma Factor ◽  
Streptomyces Coelicolor ◽  
Tight Binding ◽  
Gel Filtration ◽  
Binding Interaction ◽  
C Terminus ◽  
Highly Sensitive ◽  
Late Stages

The bacterial protein WhiD belongs to the Wbl family of iron-sulfur [Fe-S] proteins present only in the actinomycetes. In Streptomyces coelicolor, it is required for the late stages of sporulation, but precisely how it functions is unknown. Here, we report results from in vitro and in vivo experiments with WhiD from Streptomyces venezuelae (SvWhiD), which differs from S. coelicolor WhiD (ScWhiD) only at the C terminus. We observed that, like ScWhiD and other Wbl proteins, SvWhiD binds a [4Fe-4S] cluster that is moderately sensitive to O2 and highly sensitive to nitric oxide (NO). However, although all previous studies have reported that Wbl proteins are monomers, we found that SvWhiD exists in a monomer-dimer equilibrium associated with its unusual C-terminal extension. Several Wbl proteins of Mycobacterium tuberculosis are known to interact with its principal sigma factor SigA. Using bacterial two-hybrid, gel filtration, and MS analyses, we demonstrate that SvWhiD interacts with domain 4 of the principal sigma factor of Streptomyces, σHrdB (σHrdB4). Using MS, we determined the dissociation constant (Kd) for the SvWhiD-σHrdB4 complex as ∼0.7 μm, consistent with a relatively tight binding interaction. We found that complex formation was cluster dependent and that a reaction with NO, which was complete at 8–10 NO molecules per cluster, resulted in dissociation into the separate proteins. The SvWhiD [4Fe-4S] cluster was significantly less sensitive to reaction with O2 and NO when SvWhiD was bound to σHrdB4, consistent with protection of the cluster in the complex.

scholarly journals Self-Association and Mapping of the Interaction Domain of Hepatitis E Virus ORF3 Protein

2001 ◽  
Vol 75 (5) ◽  
pp. 2493-2498 ◽  
Author(s):  
Shweta Tyagi ◽  
Shahid Jameel ◽  
Sunil K. Lal
Keyword(s):  
Hepatitis E Virus ◽  
Phosphorylation Site ◽  
Hepatitis E ◽  
Mitogen Activated Protein Kinase ◽  
Dimerization Domain ◽  
Interaction Domain ◽  
Orf3 Protein ◽  
Self Association

ABSTRACT Hepatitis E virus (HEV) is a major human pathogen in the developing world. In the absence of an in vitro culture system, very little information on the basic biology of the virus exists. A small protein (∼13.5 kDa) of unknown function, pORF3, is encoded by the third open reading frame of HEV. The N-terminal region of pORF3 is associated with the cytoskeleton using one of its hydrophobic domains. The C-terminal half of pORF3 is rich in proline residues and contains a putativesrc homology 3 (SH3) binding domain and a mitogen-activated protein kinase phosphorylation site. In this study, we demonstrate that pORF3 can homodimerize in vivo, using the yeast two-hybrid system. We have isolated a 43-amino-acid interaction domain of pORF3 which is capable of self-association in vivo and in vitro. The overlap of the dimerization domain with the SH3 binding and phosphorylation domains suggests that pORF3 may have a dimerization-dependent regulatory role to play in the signal transduction pathway.

scholarly journals The transcription factor DNR from Pseudomonas aeruginosa specifically requires nitric oxide and haem for the activation of a target promoter in Escherichia coli

Microbiology ◽  
2009 ◽  
Vol 155 (9) ◽  
pp. 2838-2844 ◽  
Author(s):  
Nicoletta Castiglione ◽  
Serena Rinaldo ◽  
Giorgio Giardina ◽  
Francesca Cutruzzolà
Keyword(s):  
Nitric Oxide ◽  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Consensus Sequence ◽  
Signal Molecules ◽  
Reporter System ◽  
Nucleotide Substitutions ◽  
E Coli

Pseudomonas aeruginosa is a well-known pathogen in chronic respiratory diseases such as cystic fibrosis. Infectivity of P. aeruginosa is related to the ability to grow under oxygen-limited conditions using the anaerobic metabolism of denitrification, in which nitrate is reduced to dinitrogen via nitric oxide (NO). Denitrification is activated by a cascade of redox-sensitive transcription factors, among which is the DNR regulator, sensitive to nitrogen oxides. To gain further insight into the mechanism of NO-sensing by DNR, we have developed an Escherichia coli-based reporter system to investigate different aspects of DNR activity. In E. coli DNR responds to NO, as shown by its ability to transactivate the P. aeruginosa norCB promoter. The direct binding of DNR to the target DNA is required, since mutations in the helix–turn–helix domain of DNR and specific nucleotide substitutions in the consensus sequence of the norCB promoter abolish the transcriptional activity. Using an E. coli strain deficient in haem biosynthesis, we have also confirmed that haem is required in vivo for the NO-dependent DNR activity, in agreement with the property of DNR to bind haem in vitro. Finally, we have shown, we believe for the first time, that DNR is able to discriminate in vivo between different diatomic signal molecules, NO and CO, both ligands of the reduced haem iron in vitro, suggesting that DNR responds specifically to NO.

Single-Stranded DNA Binding Proteins (SSBPs) Interact with LDB1 Homodimers to Facilitate DNA Looping.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1469-1469
Author(s):  
Ying Cai ◽  
Lalitha Nagarajan ◽  
Stephen J. Brandt
Keyword(s):  
Dna Binding ◽  
Luciferase Activity ◽  
Conflicts Of Interest ◽  
Dna Probes ◽  
Dominant Negative ◽  
Cross Linking ◽  
Dimerization Domain ◽  
Interaction Domain

Abstract Abstract 1469 Poster Board I-492 The LIM domain binding protein LDB1 is an essential cofactor of LIM-homeodomain (LIM-HD) and LIM-only (LMO) proteins in hematopoiesis and other developmental programs. We have shown that LDB1 and its LIM-HD and LMO interaction partners are protected from ubiquitylation by a small family of SSBPs. Recently, we demonstrated that these SSBPs bind to LDB1 homodimers to promote formation of a ternary complex containing two molecules of LDB1 and SSBP. This was dependent on an intact LDB1 dimerization domain (DD) and produced a shift, directly or indirectly, in the equilibrium between LDB1 monomer and dimer. In the present study, we introduced a 24-glycine linker between two full-length LDB1 peptide-coding sequences and expressed this forced or tethered LDB1 dimer (TD-LDB1) in vitro and in vivo. First, both TD-LDB1, introduced into cells by transfection, and endogenous LDB1 were found to have the same turnover rate, indicating that protection from ubiquitylation was independent of dimerization status. Second, TD-LDB1 was fused to the DNA binding domain of GAL4 (GAL4DBD) and in a second construct to the activation domain of herpesvirus VP16 (VP16AD) and these constructs were expressed in cells with a GAL4 reporter plasmid. Co-expression of GAL4-LDB1 and VP16-LDB1 significantly increased reporter luciferase activity as a result of dimerization, while co-expression of GAL4-TD-LDB1 with VP16-LDB1 did not, ruling out formation of LDB1 trimers. Likewise, co-expression of GAL4-TD-LDB1 with VP16-TD-LDB1 did not significantly affect luciferase activity, indicating that LDB1 also cannot form protein tetramers. In contrast, TD-LDB1 was able to bind SSBP2 and SSBP3 in both chemical cross-linking and mammalian two-hybrid assays, consistent with the SSBP interacting with preformed LDB1 dimers. Finally, the complete 200-amino acid DD of LDB1, reported as necessary and sufficient for protein dimerization, was confirmed in cross-linking analysis to act as a dominant negative inhibitor of LDB1 dimerization. When the DD was introduced into Lhx2-, Ldb1-, and Ssbp3-expressing cells, application of a modified electrophoretic mobility assay that can detect linking of two DNA probes in solution revealed that the DD reduced formation of a ‘looped’ complex containing two DNA probes and led to the appearance of a new complex containing Lhx2, Ssbp3, and Ldb1, apparently in a monomeric form. In summary, this work elucidates a novel function of SSBPs in enhancing LDB1 dimerization and, ultimately, long-range communication between cis regulatory regions in genes. In addition, it suggests that SSBPs bind dimeric LDB1 and induce an allosteric change in the adjacent SSBP interaction domain rather than vice versa. Finally, these results lead to the prediction that an SSBP- and LDB1-containing complex could promote looping between promoter-proximal and promoter-distal LIM-HD binding elements. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Oligomerization Mediated by a Helix-Loop-Helix-Like Domain of Baculovirus IE1 Is Required for Early Promoter Transactivation

2001 ◽  
Vol 75 (13) ◽  
pp. 6042-6051 ◽  
Author(s):  
Victoria A. Olson ◽  
Justin A. Wetter ◽  
Paul D. Friesen
Keyword(s):  
Homologous Region ◽  
Dimerization Domain ◽  
Helix Loop Helix ◽  
C Terminus ◽  
Viral Promoters ◽  
Primary Determinant ◽  
Plasmid Transfection ◽  
Enhancer Sequences

ABSTRACT IE1 is a principal transcriptional regulator of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). Transactivation by IE1 is stimulated when early viral promoters are cis linked to homologous-region (hr) enhancer sequences of AcMNPV. This transcriptional enhancement is correlated with the binding of IE1 as a dimer to the 28-bp palindromic repeats comprising the hr enhancer. To define the role of homophilic interactions in IE1 transactivation, we have mapped the IE1 domains required for oligomerization. We report here that IE1 oligomerizes by a mechanism independent of enhancer binding, as demonstrated by in vitro pull-down assays using fusions of IE1 (582 residues) to the C terminus of glutathioneS-transferase. In vivo oligomerization of IE1 was verified by immunoprecipitation of IE1 complexes from extracts of plasmid-transfected SF21 cells. Analyses of a series of site-directed IE1 insertion mutations indicated that a helix-loop-helix (HLH)-like domain extending from residue 543 to residue 568 is the primary determinant of oligomerization. Replacement of residues within the hydrophobic face of the putative dimerization domain disrupted IE1 homophilic interactions and caused loss of IE1 transactivation of hr-dependent promoters in plasmid transfection assays. Thus, oligomerization is required for IE1 transcriptional stimulation. HLH mutations also reduced IE1 stability and abrogated transactivation of non-hr-dependent promoters. These data support a model wherein IE1 oligomerizes prior to DNA binding to facilitate proper interaction with the symmetrical recognition sites within the hr enhancer and thereby promote the transcription of early viral genes.

scholarly journals Regulation of Plant Phototropic Growth by NPH3/RPT2-like Substrate Phosphorylation and 14-3-3 Binding

2021 ◽  
Author(s):  
Stuart Sullivan ◽  
Thomas Waksman ◽  
Louise Henderson ◽  
Dimitra Paliogianni ◽  
Melanie Lütkemeyer ◽  
...  
Keyword(s):  
Consensus Sequence ◽  
Phosphorylation Site ◽  
Common Mechanism ◽  
Binding Motif ◽  
Growth Responses ◽  
Directional Growth ◽  
Agc Kinases ◽  
C Terminus

Polarity underlies all plant physiology and directional growth responses such as phototropism. Yet, our understanding of how plant tropic responses are established is far from complete. The plasma-membrane associated BTB-containing protein, NON-PHOTOTROPIC HYPOCOTYL 3 (NPH3) is a key determinant of phototropic growth which is regulated by AGC kinases known as the phototropins (phots). However, the mechanism by which phots initiate phototropic signalling via NPH3, and other NPH3/RPT2-like (NRL) members, has remained unresolved. Here we demonstrate that NPH3 is directly phosphorylated by phot1 both in vitro and in vivo. Light-dependent phosphorylation within a conserved consensus sequence (RxS) located at the extreme C-terminus of NPH3 is necessary to promote its functionality for phototropism and petiole positioning in Arabidopsis. Phosphorylation of this region by phot1 also triggers 14-3-3 binding combined with changes in NPH3 phosphorylation and localisation status. Seedlings expressing mutants of NPH3 that are unable to bind or constitutively bind 14-3-3s show compromised functionality that is consistent with a model where signalling outputs arising from a gradient in NPH3 RxS phosphorylation/localisation across the stem are a major contributor to phototropic responsiveness. Our current findings provide further evidence that 14-3-3 proteins are instrumental components regulating auxin-dependent growth and show for the first time that NRL proteins are direct phosphorylation targets for plant AGC kinases. Moreover, the C-terminal phosphorylation site/14-3-3-binding motif of NPH3 is conserved in several members of the NRL family, suggesting a common mechanism of regulation.

scholarly journals Unique Properties of the Large Antigen of Hepatitis Delta Virus

1999 ◽  
Vol 73 (9) ◽  
pp. 7147-7152 ◽  
Author(s):  
Gloria Moraleda ◽  
Steven Seeholzer ◽  
Vadim Bichko ◽  
Roland Dunbrack ◽  
James Otto ◽  
...  
Keyword(s):  
Hepatitis Delta Virus ◽  
Surface Proteins ◽  
Translation System ◽  
Dimerization Domain ◽  
Particle Assembly ◽  
Hepatitis Delta ◽  
C Terminus ◽  
Delta Virus

ABSTRACT The large form of the hepatitis delta virus (HDV) protein (L) can be isoprenylated near its C terminus, and this modification is considered essential for particle assembly. Using gel electrophoresis, we separated L into two species of similar mobilities. The slower species could be labeled by the incorporation of [14C]mevalonolactone and is interpreted to be isoprenylated L (Li). In serum particles, infected liver, transfected cells, and assembled particles, 25 to 85% of L was isoprenylated. Isoprenylation was also demonstrated by 14C incorporation in vitro with a rabbit reticulocyte coupled transcription-translation system. However, the species obtained migrated even slower than that detected by labeling in vivo. Next, in studies of HDV particle assembly in the presence of the surface proteins of human hepatitis B virus, we observed the following. (i) Relative to L, Li was preferentially assembled into virus-like particles. (ii) Li could coassemble the unmodified L and the small delta protein, S. (iii) In contrast, a form of L with a deletion in the dimerization domain was both isoprenylated and assembled, but it could not support the coassembly of S. Finally, to test the expectation that the isoprenylation of L would increase its hydrophobicity, we applied a phase separation strategy based on micelle formation with the nonionic detergent Triton X-114. We showed the following. (i) The unique C-terminal 19 amino acids present on L relative to S caused a significant increase in the hydrophobicity. (ii) This increase was independent of isoprenylation. (iii) In contrast, other, artificial modifications at either the N or C terminus of S did not increase the hydrophobicity. (iv) The increased hydrophobicity was not sufficient for particle assembly; nevertheless, we speculate that it might facilitate virion assembly.

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