Central and carboxy-terminal regions of human p53 protein are essential for interaction and complex formation with PARP-1

2003 ◽  
Vol 89 (2) ◽  
pp. 220-232 ◽  
Author(s):  
J�zefa W?sierska-G?dek ◽  
Jacek Wojciechowski ◽  
Gerald Schmid
Keyword(s):  
Complex Formation ◽  
P53 Protein ◽  
Carboxy Terminal ◽  
Parp 1

scholarly journals Advantage of a baculovirus expression system for protein-protein interaction studies. Involvement of posttranslational phosphorylation in the interaction between wt p53 protein and poly(ADP-ribose) polymerase-1.

2005 ◽  
Vol 52 (3) ◽  
pp. 713-719 ◽  
Author(s):  
Gerald Schmid ◽  
Jacek Wojciechowski ◽  
Józefa Wesierska-Gadek
Keyword(s):  
Complex Formation ◽  
Protein Interaction ◽  
P53 Protein ◽  
Full Length ◽  
Functional Domain ◽  
Terminal Part ◽  
Protein Protein Interaction ◽  
Amino Terminal ◽  
Carboxy Terminal ◽  
Parp 1

We recently observed an interaction between poly(ADP-ribose) polymerase-1 (PARP-1) and the tumor suppressor p53 protein. However, more extensive studies on both proteins, especially those on characterization of their domains involved in the interaction were difficult due to very low expression levels of p53 in mammalian cells. Therefore, we generated recombinant proteins for such studies. To clarify which domains of human PARP-1 and of human wild-type (wt) p53 were involved in this protein-protein interaction, we generated baculoviral constructs encoding full length or distinct functional domains of both proteins. Full length PARP-1 was simultaneously coexpressed in insect cells with full length wt p53 protein or its distinct truncated fragments and vice versa. Reciprocal immunoprecipitation of Sf9 cell lysates revealed that the central and carboxy-terminal fragments of p53 each were sufficient to confer binding to PARP-1, whereas the amino-terminal part harbouring the transactivation functional domain was dispensable. On the other hand, the amino-terminal and central fragments of PARP-1 were both necessary for complex formation with p53 protein. Since the most important features of p53 protein are regulated by phosphorylation, we addressed the question whether its phosphorylation is essential for the binding between the two proteins. Baculovirally expressed wt p53 was post-translationally modified. At least six distinct p53 isomers were resolved by immunoblotting following two-dimensional separation of baculovirally expressed wt p53 protein. Using specific phospho-serine antibodies, we identified phosphorylation of baculovirally expressed p53 protein at five distinct sites. To define the role of p53 phosphorylation, pull-down assays using untreated and dephosphorylated p53 protein were performed. Dephosphorylated p53 failed to bind PARP-1, indicating that complex formation between the two proteins was regulated by phosphorylation of p53. The marked phosphorylation of p53 at Ser392 observed in unstressed cells suggests that the phosphorylated carboxy-terminal part of p53 undergoes complex formation with PARP-1 resulting in masking of the NES and thereby preventing its export.

Phosphorylation regulates the interaction and complex formation between wt p53 protein and PARP-1

2003 ◽  
Vol 89 (6) ◽  
pp. 1260-1248 ◽  
Author(s):  
J�zefa W?sierska-G?dek ◽  
Jacek Wojciechowski ◽  
Gerald Schmid
Keyword(s):  
Complex Formation ◽  
P53 Protein ◽  
Parp 1

scholarly journals Rab GDI: a solubilizing and recycling factor for rab9 protein.

1993 ◽  
Vol 4 (4) ◽  
pp. 425-434 ◽  
Author(s):  
T Soldati ◽  
M A Riederer ◽  
S R Pfeffer
Keyword(s):  
Complex Formation ◽  
Intracellular Transport ◽  
Rab Proteins ◽  
Rab Protein ◽  
Transport Vesicles ◽  
Late Endosomes ◽  
Geranylgeranyl Transferase ◽  
Carboxy Terminal ◽  
Golgi Network

Rab proteins are thought to function in the processes by which transport vesicles identify and/or fuse with their respective target membranes. The bulk of these proteins are membrane associated, but a measurable fraction can be found in the cytosol. The cytosolic forms of rab3A, rab11, and Sec4 occur as equimolar complexes with a class of proteins termed "GDIs," or "GDP dissociation inhibitors." We show here that the cytosolic form of rab9, a protein required for transport between late endosomes and the trans Golgi network, also occurs as a complex with a GDI-like protein, with an apparent mass of approximately 80 kD. Complex formation could be reconstituted in vitro using recombinant rab9 protein, cytosol, ATP, and geranylgeranyl diphosphate, and was shown to require an intact rab9 carboxy terminus, as well as rab9 geranylgeranylation. Monoprenylation was sufficient for complex formation because a mutant rab9 protein bearing the carboxy terminal sequence, CLLL, was prenylated in vitro by geranylgeranyl transferase I and was efficiently incorporated into 80-kD complexes. Purified, prenylated rab9 could also assemble into 80-kD complexes by addition of purified, rab3A GDI. Finally, rab3A-GDI had the capacity to solubilize rab9GDP, but not rab9GTP, from cytoplasmic membranes. These findings support the proposal that GDI proteins serve to recycle rab proteins from their target membranes after completion of a rab protein-mediated, catalytic cycle. Thus GDI proteins have the potential to regulate the availability of specific intracellular transport factors.

Characterization of mutant p53-hsp72/73 protein-protein complexes by transient expression in monkey COS cells

1988 ◽  
Vol 8 (9) ◽  
pp. 3740-3747
Author(s):  
H W Stürzbecher ◽  
C Addison ◽  
J R Jenkins
Keyword(s):  
Complex Formation ◽  
Protein Complexes ◽  
P53 Protein ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Mutant P53 ◽  
Cos Cells

Several mutant, but not wild-type, p53 proteins form complexes with hsp72/73 heat shock-related proteins in simian virus 40-transformed monkey COS cells. We carried out a detailed biochemical and structural mapping analysis of p53 and report here that p53-hsp72/73 complex formation showed considerable structural specificity. Such complexes were remarkably stable, but unlike analogous complexes formed between p53 and simian virus 40 T antigen, they did not form in in vitro association assays. p53-hsp72/73 complex formation in vivo appears to be dependent on aspects of mutant p53 protein conformation. However, absence of the conformation-sensitive epitope recognized by monoclonal antibody PAb 246 was not reliably diagnostic of such complexes, nor was p53-hsp72173 binding reliably diagnostic of oncogenic activation.

scholarly journals Carboxy-terminal domain of AID required for its mRNA complex formation in vivo

2009 ◽  
Vol 106 (8) ◽  
pp. 2747-2751 ◽  
Author(s):  
Taichiro Nonaka ◽  
Tomomitsu Doi ◽  
Takae Toyoshima ◽  
Masamichi Muramatsu ◽  
Tasuku Honjo ◽  
...  
Keyword(s):  
Complex Formation ◽  
Terminal Domain ◽  
Carboxy Terminal Domain ◽  
Carboxy Terminal

scholarly journals Physical Requirements and Functional Consequences of Complex Formation between the Cytomegalovirus IE1 Protein and Human STAT2

2009 ◽  
Vol 83 (24) ◽  
pp. 12854-12870 ◽  
Author(s):  
Steffen Krauss ◽  
Julia Kaps ◽  
Nathalie Czech ◽  
Christina Paulus ◽  
Michael Nevels
Keyword(s):  
Amino Acids ◽  
Complex Formation ◽  
Low Complexity ◽  
Murine Cytomegalovirus ◽  
Interferon Response ◽  
Protein Accumulation ◽  
Type I ◽  
Functional Consequences ◽  
Carboxy Terminal ◽  
Interferon Responses

ABSTRACT Our previous work has shown that efficient evasion from type I interferon responses by human cytomegalovirus (hCMV) requires expression of the 72-kDa immediate-early 1 (IE1) protein. It has been suggested that IE1 inhibits interferon signaling through intranuclear sequestration of the signal transducer and activator of transcription 2 (STAT2) protein. Here we show that physical association and subnuclear colocalization of IE1 and STAT2 depend on short acidic and serine/proline-rich low-complexity motifs in the carboxy-terminal region of the 491-amino-acid viral polypeptide. These motifs compose an essential core (amino acids 373 to 420) and an adjacent ancillary site (amino acids 421 to 445) for STAT2 interaction that are predicted to form part of a natively unstructured domain. The presence of presumably “disordered” carboxy-terminal domains enriched in low-complexity motifs is evolutionarily highly conserved across all examined mammalian IE1 orthologs, and the murine cytomegalovirus IE1 protein appears to interact with STAT2 just like the human counterpart. A recombinant hCMV specifically mutated in the IE1 core STAT2 binding site displays hypersensitivity to alpha interferon, delayed early viral protein accumulation, and attenuated growth in fibroblasts. However, replication of this mutant virus is specifically restored by knockdown of STAT2 expression. Interestingly, complex formation with STAT2 proved to be entirely separable from disruption of nuclear domain 10 (ND10), another key activity of IE1. Finally, our results demonstrate that IE1 counteracts the antiviral interferon response and promotes viral replication by at least two distinct mechanisms, one depending on sequestration of STAT2 and the other one likely involving ND10 interaction.

Orientation of the Carboxy-terminal Regions of Fibrin γ Chain Dimers Determined from the Crosslinked Products Formed in Mixtures of Fibrin, Fragment D, and Factor XIIIa

1995 ◽  
Vol 74 (04) ◽  
pp. 1113-1119 ◽  
Author(s):  
Kevin R Siebenlist ◽  
David A Meh ◽  
Joseph S Wall ◽  
James F Hainfeld ◽  
Michael W Mosesson
Keyword(s):  
Acetic Acid ◽  
Complex Formation ◽  
Factor Xiiia ◽  
Electron Microscopic ◽  
Fiber Growth ◽  
Sds Page ◽  
Isopeptide Bonds ◽  
Covalently Linked ◽  
Oriented Parallel ◽  
Carboxy Terminal

SummaryThere are two schools of thought regarding the orientation of the intermolecular ∈-amino-(γ-glutamyl) lysine isopeptide bonds formed between γ chains in the D domains of assembled fibrin fibers. Some investigators believe that these bonds are oriented parallel to the direction of fiber growth (longitudinally) at the contacting ends of fibrin D domains (‘DD-long’), whereas others believe that these bonds are oriented across the two-stranded fibril, between D domains in opposing strands (‘DD-transverse’). To distinguish between these two possibilities, the structure of crosslinked products formed in mixtures of fibrin, plasmic fragment D, and factor XIIIa were analyzed, based upon this rationale: Complex formation between D fragments and a fibrin template depends upon the non-covalent ‘D:E’ interaction between each fibrin E domain and two D fragments (‘D:fibrin:D’). If carboxy-terminal γ chains in the D:fibrin:D complex become aligned in a DD-long configuration, only crosslinked fragment D dimers (‘D-D’) will result and the fibrin ‘template’ will not become crosslinked to the associated D fragments. If instead, γ chain crosslinks form transversely between the D fragments and fibrin, covalently linked D-fibrin complexes will result.SDS-PAGE of factor XIIIa crosslinked mixtures of fibrin and fragment D demonstrated products of a size and subunit composition indicating D-fibrin and D-fibrin-D formation. Small amounts of D dimers were also formed at the same levels as were formed in mixtures of fragment D and factor XIIIa alone. Electron microscopic images of D-fibrin-D complexes prepared under physiological buffer conditions demonstrated that the D fragments were associated with the central E domain of the fibrin molecule, but that they could be dissociated from this non-covalent association in 2% acetic acid. These findings indicate that γ chain crosslinks occur transversely in D:fibrin:D complexes and permit the extrapolated conclusion that γ chain crosslinks are also positioned transversely in an assembled fibrin polymer.

Mapping of the Coronavirus Membrane Protein Domains Involved in Interaction with the Spike Protein

1999 ◽  
Vol 73 (9) ◽  
pp. 7441-7452 ◽  
Author(s):  
Cornelis A. M. de Haan ◽  
M. Smeets ◽  
F. Vernooij ◽  
H. Vennema ◽  
P. J. M. Rottier
Keyword(s):  
Complex Formation ◽  
Virus Particle ◽  
Transmembrane Domains ◽  
Viral Envelope ◽  
M Protein ◽  
Particle Assembly ◽  
S Protein ◽  
Amino Terminal ◽  
Heterotypic Interactions ◽  
Carboxy Terminal

ABSTRACT The coronavirus membrane (M) protein is the key player in virion assembly. One of its functions is to mediate the incorporation of the spikes into the viral envelope. Heterotypic interactions between M and the spike (S) protein can be demonstrated by coimmunoprecipitation and by immunofluorescence colocalization, after coexpression of their genes in eukaryotic cells. Using these assays in a mutagenetic approach, we have mapped the domains in the M protein that are involved in complex formation between M and S. It appeared that the 25-residue luminally exposed amino-terminal domain of the M protein is not important for M-S interaction. A 15-residue deletion, the insertion of a His tag, and replacement of the ectodomain by that of another coronavirus M protein did not affect the ability of the M protein to associate with the S protein. However, complex formation was sensitive to changes in the transmembrane domains of this triple-spanning protein. Deletion of either the first two or the last two transmembrane domains, known not to affect the topology of the protein, led to a considerable decrease in complex formation, but association was not completely abrogated. Various effects of changes in the part of the M protein that is located at the cytoplasmic face of the membrane were observed. Deletions of the extreme carboxy-terminal tail appeared not to interfere with M-S complex formation. However, deletions in the amphipathic domain severely affected M-S interaction. Interestingly, changes in the amino-terminal and extreme carboxy-terminal domains of M, which did not disrupt the interaction with S, are known to be fatal to the ability of the protein to engage in virus particle formation (C. A. M. de Haan, L. Kuo, P. S. Masters, H. Vennema, and P. J. M. Rottier, J. Virol. 72:6838–6850, 1998). Apparently, the structural requirements of the M protein for virus particle assembly differ from the requirements for the formation of M-S complexes.

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