Exoenzyme S binds its cofactor 14-3-3 through a non-phosphorylated motif

2002 ◽  
Vol 30 (4) ◽  
pp. 401-405 ◽  
Author(s):  
B. Hallberg
Keyword(s):  
Specific Interaction ◽  
Phage Display Library ◽  
Bacterial Toxin ◽  
Inositol Polyphosphate ◽  
Checkpoint Control ◽  
Exoenzyme S ◽  
Infected Cells ◽  
Adp Ribosyltransferase

14-3-3 proteins belong to a family of conserved molecules, which play a regulatory role and participate in signal transduction and checkpoint control pathways. 14-3-3 proteins bind phosphoserine-phosphorylated ligands, such as the Raf-1 kinase and Bad, through recognition of the phosphorylated consensus motif, RSXpSXP (where pS is phosphoserine). Recently, a phosphorylation-independent interaction has been reported to occur between 14-3-3 and a small number of proteins, for example the 43 kDa inositol polyphosphate 5-phosphatase, glycoprotein Ib, p75NTR-associated cell-death executor (NADE) and the bacterial ADP-ribosyltransferase toxin exoenzyme S (ExoS). It has been suggested that specific residues of 14-3-3 proteins are required for activation of the bacterial toxin ExoS. An unphosphorylated peptide derived from a phage display library, known as the R18 peptide, and a synthetic peptide derived from ExoS inhibit the interaction between ExoS and 14-3-3. In this report we identify the amino acid sequence on ExoS which is responsible for its specific interaction with 14-3-3, both in vitro and in vivo. In addition, we believe that this interaction is critical for the ADP-ribosylation of an endogenous target, Ras, by ExoS both in vitro and in vivo. Loss of the 14-3-3-binding site on ExoS results in an ExoS molecule that is unable to efficiently inactivate Ras and shows a reduced capacity to change the morphology of infected cells, together with reduced killing activity.

scholarly journals 14-3-3 proteins are required for the inhibition of Ras by exoenzyme S

2000 ◽  
Vol 349 (3) ◽  
pp. 697-701 ◽  
Author(s):  
Maria Lena HENRIKSSON ◽  
Ulrika TROLLÉR ◽  
Bengt HALLBERG
Keyword(s):  
Signal Transduction ◽  
Pseudomonas Aeruginosa ◽  
Binding Site ◽  
Phage Display Library ◽  
Checkpoint Control ◽  
Exoenzyme S ◽  
Interaction Site ◽  
Killing Activity

14-3-3 proteins play a regulatory role and participate in both signal transduction and checkpoint control pathways. 14-3-3 proteins bind phosphoserine ligands, such as Raf-1 kinase and Bad, by recognizing the phosphorylated consensus motif, Arg-Ser-Xaa-pSer-Xaa-Pro (where ‘Xaa’ represents ‘any residue’, and ‘pSer’ is ‘phosphoserine’) . However, 14-3-3 proteins must bind unphosphorylated ligands, such as glycoprotein Ibα and Pseudomonas aeruginosa exoenzyme S (ExoS), since it has been suggested that specific residues of 14-3-3 proteins are required for activation of ExoS. Furthermore, an unphosphorylated peptide derived from a phage display library inhibited the binding of both ExoS and Raf-1 to 14-3-3, and bound within the same conserved amphipathic groove on the surface of 14-3-3 as the Raf-derived phosphopeptide (pS-Raf-259). In the present study we identify the interaction site on ExoS for 14-3-3, and show that ExoS and 14-3-3 do indeed interact in vivo. In addition, we show that this interaction is critical for the ADP-ribosylation of Ras by ExoS, both in vitro and in vivo. Loss of the 14-3-3 binding site on ExoS results in an ExoS molecule that is unable to efficiently inactivate Ras, and displays reduced killing activity.

scholarly journals Modification of Ras in Eukaryotic Cells by Pseudomonas aeruginosa Exoenzyme S

1998 ◽  
Vol 66 (6) ◽  
pp. 2607-2613 ◽  
Author(s):  
Eileen M. McGuffie ◽  
Dara W. Frank ◽  
Timothy S. Vincent ◽  
Joan C. Olson
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Host Cells ◽  
Eukaryotic Cells ◽  
Exoenzyme S ◽  
Ras Family ◽  
Isogenic Mutant Strain ◽  
Related Proteins ◽  
Adp Ribosyltransferase

ABSTRACT Genetic and functional data suggest that Pseudomonas aeruginosa exoenzyme S (ExoS), an ADP-ribosyltransferase, is translocated into eukaryotic cells by a bacterial type III secretory mechanism activated by contact between bacteria and host cells. Although purified ExoS is not toxic to eukaryotic cells, ExoS-producing bacteria cause reduced proliferation and viability, possibly mediated by bacterially translocated ExoS. To investigate the activity of translocated ExoS, we examined in vivo modification of Ras, a preferred in vitro substrate. The ExoS-producing strain P. aeruginosa388 and an isogenic mutant strain, 388ΔexoS, which fails to produce ExoS, were cocultured with HT29 colon carcinoma cells. Ras was found to be ADP-ribosylated during coculture with 388 but not with 388ΔexoS, and Ras modification by 388 corresponded with reduction in HT29 cell DNA synthesis. Active translocation by bacteria was found to be required, since exogenous ExoS, alone or in the presence of 388ΔexoS, was unable to modify intracellular Ras. Other ExoS-producing strains caused modification of Ras, indicating that this is not a strain-specific event. ADP-ribosylation of Rap1, an additional Ras family substrate for ExoS in vitro, was not detectable in vivo under conditions sufficient for Ras modification, suggesting possible ExoS substrate preference among Ras-related proteins. These results confirm that intracellular Ras is modified by bacterially translocated ExoS and that the inhibition of target cell proliferation correlates with the efficiency of Ras modification.

Correlation between in vitro and in vivo Data of Radiolabeled Peptide for Tumor Targeting

2019 ◽  
Vol 19 (12) ◽  
pp. 950-960
Author(s):  
Soghra Farzipour ◽  
Seyed Jalal Hosseinimehr
Keyword(s):  
Tumor Targeting ◽  
Single Photon ◽  
Specific Binding ◽  
Specific Interaction ◽  
Photon Emission ◽  
Emission Computed Tomography ◽  
Mixed Effect ◽  
Radiolabeled Peptides

Tumor-targeting peptides have been generally developed for the overexpression of tumor specific receptors in cancer cells. The use of specific radiolabeled peptide allows tumor visualization by single photon emission computed tomography (SPECT) and positron emission tomography (PET) tools. The high affinity and specific binding of radiolabeled peptide are focusing on tumoral receptors. The character of the peptide itself, in particular, its complex molecular structure and behaviors influence on its specific interaction with receptors which are overexpressed in tumor. This review summarizes various strategies which are applied for the expansion of radiolabeled peptides for tumor targeting based on in vitro and in vivo specific tumor data and then their data were compared to find any correlation between these experiments. With a careful look at previous studies, it can be found that in vitro unblock-block ratio was unable to correlate the tumor to muscle ratio and the success of radiolabeled peptide for in vivo tumor targeting. The introduction of modifiers’ approaches, nature of peptides, and type of chelators and co-ligands have mixed effect on the in vitro and in vivo specificity of radiolabeled peptides.

scholarly journals ADP-ribosylation of integrin α7 modulates the binding of integrin α7β1 to laminin

2004 ◽  
Vol 385 (1) ◽  
pp. 309-317 ◽  
Author(s):  
Zhefeng ZHAO ◽  
Joanna GRUSZCZYNSKA-BIEGALA ◽  
Anna ZOLKIEWSKA
Keyword(s):  
C2c12 Myotubes ◽  
Post Translational Modification ◽  
Integrin Β1 ◽  
Adp Ribosylation ◽  
In Vitro Binding ◽  
Vitro Binding ◽  
C2c12 Skeletal Muscle Cells ◽  
Adp Ribosyltransferase

The extracellular domain of integrin α7 is ADP-ribosylated by an arginine-specific ecto-ADP-ribosyltransferase after adding exogenous NAD+ to intact C2C12 skeletal muscle cells. The effect of ADP-ribosylation on the structure or function of integrin α7β1 has not been explored. In the present study, we show that ADP-ribosylation of integrin α7 takes place exclusively in differentiated myotubes and that this post-translational modification modulates the affinity of α7β1 dimer for its ligand, laminin. ADP-ribosylation in the 37-kDa ‘stalk’ region of α7 that takes place at micromolar NAD+ concentrations increases the binding of the α7β1 dimer to laminin. Increased in vitro binding of integrin α7β1 to laminin after ADP-ribosylation of the 37-kDa fragment of α7 requires the presence of Mn2+ and it is not observed in the presence of Mg2+. In contrast, ADP-ribosylation of the 63-kDa N-terminal region comprising the ligand-binding site of α7 that occurs at approx. 100 μM NAD+ inhibits the binding of integrin α7β1 to laminin. Furthermore, incubation of C2C12 myotubes with NAD+ increases the expression of an epitope on integrin β1 subunit recognized by monoclonal antibody 9EG7. We discuss our results based on the current models of integrin activation. We also hypothesize that ADP-ribosylation may represent a mechanism of regulation of integrin α7β1 function in myofibres in vivo when the continuity of the membrane is compromised and NAD+ is available as a substrate for ecto-ADP-ribosylation.

Studies of two temperature-sensitive mutants of Mengo virus

1979 ◽  
Vol 57 (6) ◽  
pp. 902-913 ◽  
Author(s):  
Patrick W. K. Lee ◽  
John S. Colter
Keyword(s):  
Rna Synthesis ◽  
Viral Rna ◽  
Temperature Sensitive ◽  
Supporting Evidence ◽  
Structural Polypeptide ◽  
Infected Cells ◽  
Mengo Virus ◽  
In Vitro Stability

Studies of the synthesis of viral ribonucleates and polypeptides in cells infected with two RNA−ts mutants of Mengo virus (ts 135 and ts 520) have shown that when ts 135 infected cells are shifted from the permissive (33 °C) to the nonpermissive (39 °C) temperature: (i) the synthesis of all three species of viral RNA (single stranded, replicative form, and replicative intermediate) is inhibited to about the same extent, and (ii) the posttranslational cleavage of structural polypeptide precursors A and B is partially blocked. Investigations of the in vivo and in vitro stability of the viral RNA replicase suggest that the RNA− phentotype reflects a temperature-sensitive defect in the enzyme. The second defect does not appear to result from the inhibition of viral RNA synthesis at 39 °C, since normal cleavage of polypeptides A and B occurs in wt Mengo-infected cells in which viral RNA synthesis is blocked by cordycepin, and at the nonpermissive temperature in ts 520 infected cells. Considered in toto, the evidence suggests that ts 135 is a double mutant.Subviral (53 S) particles have been shown to accumulate in ts 520 (but not ts 135) infected cells when cultures are shifted from 33 to 39 °C. This observation provides supporting evidence for the proposal that this recently discovered particle is an intermediate in the assembly pathway of Mengo virions.

scholarly journals Analysis of the Interaction of the Adenovirus L1 52/55-Kilodalton and IVa2 Proteins with the Packaging Sequence In Vivo and In Vitro

2005 ◽  
Vol 79 (4) ◽  
pp. 2366-2374 ◽  
Author(s):  
Pilar Perez-Romero ◽  
Ryan E. Tyler ◽  
Johanna R. Abend ◽  
Monica Dus ◽  
Michael J. Imperiale
Keyword(s):  
Viral Protein ◽  
Direct Interaction ◽  
Dna Interaction ◽  
The Other ◽  
Infected Cells ◽  
In Vitro Interaction ◽  
Packaging Sequence

ABSTRACT We previously showed that the adenovirus IVa2 and L1 52/55-kDa proteins interact in infected cells and the IVa2 protein is part of two virus-specific complexes (x and y) formed in vitro with repeated elements of the packaging sequence called the A1-A2 repeats. Here we demonstrate that both the IVa2 and L1 52/55-kDa proteins bind in vivo to the packaging sequence and that each protein-DNA interaction is independent of the other. There is a strong and direct interaction of the IVa2 protein with DNA in vitro. This interaction is observed when probes containing the A1-A2 or A4-A5 repeats are used, but it is not found by using an A5-A6 probe. Furthermore, we show that complex x is likely a heterodimer of IVa2 and an unknown viral protein, while complex y is a monomer or multimer of IVa2. No in vitro interaction of purified L1 52/55-kDa protein with the packaging sequence was found, suggesting that the L1 52/55-kDa protein-DNA interaction may be mediated by an intermediate protein. Results support roles for both the L1 52/55-kDa and IVa2 proteins in DNA encapsidation.

scholarly journals RanBP2 and SENP3 Function in a Mitotic SUMO2/3 Conjugation-Deconjugation Cycle on Borealin

2009 ◽  
Vol 20 (1) ◽  
pp. 410-418 ◽  
Author(s):  
Ulf R. Klein ◽  
Markus Haindl ◽  
Erich A. Nigg ◽  
Stefan Muller
Keyword(s):  
Mammalian Cells ◽  
Spindle Assembly Checkpoint ◽  
Critical Role ◽  
Specific Interaction ◽  
Regulatory Function ◽  
Mitotic Progression ◽  
Chromosome Congression ◽  
Chromosomal Passenger

The ubiquitin-like SUMO system controls cellular key functions, and several lines of evidence point to a critical role of SUMO for mitotic progression. However, in mammalian cells mitotic substrates of sumoylation and the regulatory components involved are not well defined. Here, we identify Borealin, a component of the chromosomal passenger complex (CPC), as a mitotic target of SUMO. The CPC, which additionally comprises INCENP, Survivin, and Aurora B, regulates key mitotic events, including chromosome congression, the spindle assembly checkpoint, and cytokinesis. We show that Borealin is preferentially modified by SUMO2/3 and demonstrate that the modification is dynamically regulated during mitotic progression, peaking in early mitosis. Intriguingly, the SUMO ligase RanBP2 interacts with the CPC, stimulates SUMO modification of Borealin in vitro, and is required for its modification in vivo. Moreover, the SUMO isopeptidase SENP3 is a specific interaction partner of Borealin and catalyzes the removal of SUMO2/3 from Borealin. These data thus delineate a mitotic SUMO2/3 conjugation–deconjugation cycle of Borealin and further assign a regulatory function of RanBP2 and SENP3 in the mitotic SUMO pathway.

Participation of the upstream region of the fibroin gene in the formation of transcription complex in vitro

1986 ◽  
Vol 6 (11) ◽  
pp. 3928-3933
Author(s):  
M Tsuda ◽  
S Hirose ◽  
Y Suzuki
Keyword(s):  
Complex Formation ◽  
Specific Interaction ◽  
Inhibitory Effect ◽  
Silk Gland ◽  
Upstream Region ◽  
Fibroin Gene ◽  
Transcription Complexes ◽  
Posterior Silk Gland

The addition of exogenous histones has an inhibitory effect on fibroin gene transcription in posterior silk gland extracts. The histones probably disturb a process in complex formation, because when transcription complexes were constructed by preincubation of the templates with the extracts, the inhibitory effect of histones was greatly reduced. Transcription of a fibroin gene construct, pFb5' delta-238, having the upstream region beyond the TATA box was relatively less inhibited than that of pFb5' delta-44 lacking the upstream region. This tendency toward differential inhibition was observed in the silk gland extracts but not in a HeLa cell extract and persisted even after complex formation in the silk gland extracts, suggesting a specific interaction of the upstream region with some factors in the extracts. The complexes formed on pFb5' delta-44 are probably more susceptible to the inhibitory effect of histones. On the basis of these results we propose a participation of the upstream region of the fibroin gene in the formation of stable transcription complexes at the promoter through an interaction with specific factors in the silk gland. Since the transcription-enhancing effect via the upstream region is augmented at a high histone/DNA ratio, it may mimic the in vivo situation in which the fibroin gene can be transcribed in the posterior silk gland even in the presence of excess suppressive materials.

Detection of Circulating Immune Complexes in the Sera of Rabbits with Experimental Syphilis: Possible Role in Immunoregulation

1980 ◽  
Vol 29 (2) ◽  
pp. 575-582
Author(s):  
Robert E. Baughn ◽  
Kenneth S. K. Tung ◽  
Daniel M. Musher
Keyword(s):  
Immune Complexes ◽  
Proteolytic Enzymes ◽  
Suppressor Cell ◽  
Suppressive Effect ◽  
Circulating Immune Complexes ◽  
Time Period ◽  
Infected Cells ◽  
Disseminated Disease

The in vivo and in vitro immunoglobulin G plaque-forming cell responses to sheep erythrocytes (SRBC) are nearly obliterated during disseminated syphilitic infection (3 to 8 weeks post-intravenous injection) in rabbits. Splenic and lymph node cells obtained from infected rabbits during this time period were capable of suppressing the normal in vitro responses of uninfected, SRBC-primed cells. Cell-free washings of cells from infected animals were also suppressive. This finding coupled with the fact that treatment of infected cells with proteolytic enzymes abrogated the suppressive effect constitute arguments against involvement of a specific suppressor cell population. The incidence of elevated levels of circulating immune complexes in the sera of rabbits with disseminated disease was also significantly different from that of uninfected controls or infected rabbits before the onset or after the regression of lesions. When added to cultures of lymphocytes from uninfected, SRBC-sensitized rabbits, sera containing complexes caused dose-related suppression of the in vitro immunoglobulin responses. Unlike immune complexes, no correlation was found between the presence of mucopolysaccharide materials and the stage of infection or the ability of serum to suppress the immunoglobulin responses to SRBC.

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