p53 domains: structure, oligomerization, and transformation

1994 ◽  
Vol 14 (8) ◽  
pp. 5182-5191
Author(s):  
P Wang ◽  
M Reed ◽  
Y Wang ◽  
G Mayr ◽  
J E Stenger ◽  
...  
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Gel Filtration ◽  
Dominant Negative ◽  
Wild Type ◽  
C Terminus ◽  
Tetramerization Domain ◽  
Wild Type P53 ◽  
Negative Phenotype

Wild-type p53 forms tetramers and multiples of tetramers. Friedman et al. (P. N. Friedman, X. B. Chen, J. Bargonetti, and C. Prives, Proc. Natl. Acad. Sci. USA 90:3319-3323, 1993) have reported that human p53 behaves as a larger molecule during gel filtration than it does during sucrose gradient sedimentation. These differences argue that wild-type p53 has a nonglobular shape. To identify structural and oligomerization domains in p53, we have investigated the physical properties of purified segments of p53. The central, specific DNA-binding domain within murine amino acids 80 to 320 and human amino acids 83 to 323 behaves predominantly as monomers during analysis by sedimentation, gel filtration, and gel electrophoresis. This consistent behavior argues that the central region of p53 is globular in shape. Under appropriate conditions, however, this segment can form transient oligomers without apparent preference for a single oligomeric structure. This region does not enhance transformation by other oncogenes. The biological implications of transient oligomerization by this central segment, therefore, remain to be demonstrated. Like wild-type p53, the C terminus, consisting of murine amino acids 280 to 390 and human amino acids 283 to 393, behaves anomalously during gel filtration and apparently has a nonglobular shape. Within this region, murine amino acids 315 to 350 and human amino acids 323 to 355 are sufficient for assembly of stable tetramers. The finding that murine amino acids 315 to 360 enhance transformation by other oncogenes strongly supports the role of p53 tetramerization in oncogenesis. Amino acids 330 to 390 of murine p53 and amino acids 340 to 393 of human p53, which have been implicated by Sturzbecher et al. in tetramerization (H.-W. Sturzbecher, R. Brain, C. Addison, K. Rudge, M. Remm, M. Grimaldi, E. Keenan, and J. R. Jenkins, Oncogene 7:1513-1523, 1992), do not form stable tetramers under our conditions. Our findings indicate that p53 has at least two autonomous oligomerization domains: a strong tetramerization domain in its C-terminal region and a weaker oligomerization domain in the central DNA binding region of p53. Together, these domains account for the formation of tetramers and multiples of tetramers by wild-type p53. The tetramerization domain is the major determinant of the dominant negative phenotype leading to transformation by mutant p53s.

scholarly journals p53 domains: structure, oligomerization, and transformation.

1994 ◽  
Vol 14 (8) ◽  
pp. 5182-5191 ◽  
Author(s):  
P Wang ◽  
M Reed ◽  
Y Wang ◽  
G Mayr ◽  
J E Stenger ◽  
...  
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Gel Filtration ◽  
Dominant Negative ◽  
Wild Type ◽  
C Terminus ◽  
Tetramerization Domain ◽  
Wild Type P53 ◽  
Negative Phenotype

Wild-type p53 forms tetramers and multiples of tetramers. Friedman et al. (P. N. Friedman, X. B. Chen, J. Bargonetti, and C. Prives, Proc. Natl. Acad. Sci. USA 90:3319-3323, 1993) have reported that human p53 behaves as a larger molecule during gel filtration than it does during sucrose gradient sedimentation. These differences argue that wild-type p53 has a nonglobular shape. To identify structural and oligomerization domains in p53, we have investigated the physical properties of purified segments of p53. The central, specific DNA-binding domain within murine amino acids 80 to 320 and human amino acids 83 to 323 behaves predominantly as monomers during analysis by sedimentation, gel filtration, and gel electrophoresis. This consistent behavior argues that the central region of p53 is globular in shape. Under appropriate conditions, however, this segment can form transient oligomers without apparent preference for a single oligomeric structure. This region does not enhance transformation by other oncogenes. The biological implications of transient oligomerization by this central segment, therefore, remain to be demonstrated. Like wild-type p53, the C terminus, consisting of murine amino acids 280 to 390 and human amino acids 283 to 393, behaves anomalously during gel filtration and apparently has a nonglobular shape. Within this region, murine amino acids 315 to 350 and human amino acids 323 to 355 are sufficient for assembly of stable tetramers. The finding that murine amino acids 315 to 360 enhance transformation by other oncogenes strongly supports the role of p53 tetramerization in oncogenesis. Amino acids 330 to 390 of murine p53 and amino acids 340 to 393 of human p53, which have been implicated by Sturzbecher et al. in tetramerization (H.-W. Sturzbecher, R. Brain, C. Addison, K. Rudge, M. Remm, M. Grimaldi, E. Keenan, and J. R. Jenkins, Oncogene 7:1513-1523, 1992), do not form stable tetramers under our conditions. Our findings indicate that p53 has at least two autonomous oligomerization domains: a strong tetramerization domain in its C-terminal region and a weaker oligomerization domain in the central DNA binding region of p53. Together, these domains account for the formation of tetramers and multiples of tetramers by wild-type p53. The tetramerization domain is the major determinant of the dominant negative phenotype leading to transformation by mutant p53s.

scholarly journals Identification of a minimal transforming domain of p53: negative dominance through abrogation of sequence-specific DNA binding.

1992 ◽  
Vol 12 (12) ◽  
pp. 5581-5592 ◽  
Author(s):  
E Shaulian ◽  
A Zauberman ◽  
D Ginsberg ◽  
M Oren
Keyword(s):  
Dna Binding ◽  
Point Mutation ◽  
P53 Gene ◽  
Dominant Negative ◽  
Mutant P53 ◽  
Wild Type ◽  
Transforming Activity ◽  
Wild Type P53 ◽  
Negative Dominance

Mutations in the p53 gene are most frequent in cancer. Many p53 mutants possess transforming activity in vitro. In cells transformed by such mutants, the mutant protein is oligomerized with endogenous cell p53. To determine the relevance of oligomerization for transformation, miniproteins containing C-terminal portions of p53 were generated. These miniproteins, although carrying no point mutation, transformed at least as efficiently as full-length mutant p53. Transforming activity was coupled with the ability to oligomerize with wild-type p53, as well as with the ability to abrogate sequence-specific DNA binding by coexpressed wild-type p53. These findings suggest that p53-mediated transformation may operate through a dominant negative mechanism, involving the generation of DNA binding-incompetent oligomers.

scholarly journals Chemotaxis of Escherichia coli to Pyrimidines: a New Role for the Signal Transducer Tap

2007 ◽  
Vol 190 (3) ◽  
pp. 972-979 ◽  
Author(s):  
Xianxian Liu ◽  
Rebecca E. Parales
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Growth Conditions ◽  
Wild Type ◽  
E Coli ◽  
C Terminus ◽  
Signaling Domain ◽  
Chemotaxis Proteins ◽  
Periplasmic Domain

ABSTRACT Escherichia coli exhibits chemotactic responses to sugars, amino acids, and dipeptides, and the responses are mediated by methyl-accepting chemotaxis proteins (MCPs). Using capillary assays, we demonstrated that Escherichia coli RP437 is attracted to the pyrimidines thymine and uracil and the response was constitutively expressed under all tested growth conditions. All MCP mutants lacking the MCP Tap protein showed no response to pyrimidines, suggesting that Tap, which is known to mediate dipeptide chemotaxis, is required for pyrimidine chemotaxis. In order to confirm the role of Tap in pyrimidine chemotaxis, we constructed chimeric chemoreceptors (Tapsr and Tsrap), in which the periplasmic and cytoplasmic domains of Tap and Tsr were switched. When Tapsr and Tsrap were individually expressed in an E. coli strain lacking all four native MCPs, Tapsr mediated chemotaxis toward pyrimidines and dipeptides, but Tsrap did not complement the chemotaxis defect. The addition of the C-terminal 19 amino acids from Tsr to the C terminus of Tsrap resulted in a functional chemoreceptor that mediated chemotaxis to serine but not pyrimidines or dipeptides. These results indicate that the periplasmic domain of Tap is responsible for detecting pyrimidines and the Tsr signaling domain confers on Tapsr the ability to mediate efficient chemotaxis. A mutant lacking dipeptide binding protein (DBP) was wild type for pyrimidine taxis, indicating that DBP, which is the primary chemoreceptor for dipeptides, is not responsible for detecting pyrimidines. It is not yet known whether Tap detects pyrimidines directly or via an additional chemoreceptor protein.

Identification of a minimal transforming domain of p53: negative dominance through abrogation of sequence-specific DNA binding

1992 ◽  
Vol 12 (12) ◽  
pp. 5581-5592 ◽  
Author(s):  
E Shaulian ◽  
A Zauberman ◽  
D Ginsberg ◽  
M Oren
Keyword(s):  
Dna Binding ◽  
Point Mutation ◽  
P53 Gene ◽  
Dominant Negative ◽  
Mutant P53 ◽  
Wild Type ◽  
Transforming Activity ◽  
Wild Type P53 ◽  
Negative Dominance

Mutations in the p53 gene are most frequent in cancer. Many p53 mutants possess transforming activity in vitro. In cells transformed by such mutants, the mutant protein is oligomerized with endogenous cell p53. To determine the relevance of oligomerization for transformation, miniproteins containing C-terminal portions of p53 were generated. These miniproteins, although carrying no point mutation, transformed at least as efficiently as full-length mutant p53. Transforming activity was coupled with the ability to oligomerize with wild-type p53, as well as with the ability to abrogate sequence-specific DNA binding by coexpressed wild-type p53. These findings suggest that p53-mediated transformation may operate through a dominant negative mechanism, involving the generation of DNA binding-incompetent oligomers.

Evidence for Mutant p53 Gain-of-Function Effects in Normal Haemopoietic Cells and Myc-Driven Lymphoma

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3589-3589
Author(s):  
Brandon James Aubrey ◽  
Andreas Strasser ◽  
Gemma Kelly ◽  
Lin Tai ◽  
Marco Herold
Keyword(s):  
Cell Death ◽  
Human Cancer ◽  
P53 Protein ◽  
Dominant Negative ◽  
Mutant P53 ◽  
P53 Mutations ◽  
Wild Type ◽  
Over Expression ◽  
Wild Type P53

Abstract Deregulated c-MYC expression and mutations in p53 are among the most common changes detected in human cancer. It is now established that mutant p53 proteins confer a poor prognosis in human cancer through both loss of wild-type p53 activity as well as various proposed gain-of-function properties. The specific role of mutant p53 in MYC-driven tumorigenesis is not known. The Eμ-Myc mouse model carries a c-Myc transgene under the control of the immunoglobulin heavy chain gene enhancer (Eμ), recapitulating the chromosomal translocation underlying human Burkitt Lymphoma (BL). These mice develop aggressive pre-B or B cell lymphomas and ~20% of those tumours exhibit p53 mutations. We have shown that MYC-driven lymphomas are exquisitely dependent on the pro-survival BCL-2 family member MCL-1 such that loss of a single allele of Mcl-1 leads to dramatic tumour regression and prolonged animal survival. Interestingly, we found that this dependency on MCL-1 is reduced, but not completely ablated, by the presence of a p53 mutation. This suggests an important role for mutant p53 in the sustained survival of MYC-driven lymphomas. We are investigating the effects of five different mutant mouse p53 proteins (V170M, I192S, G280, R246Q, R270H) on tumour initiation, sustained growth and chemoresistance in the Eμ-Myc mouse model. We are further examining the effect of p53 mutations on MCL-1 dependence by using a floxed Mcl-1 gene and a tamoxifen-inducible Cre-recombinase in established Eμ-Myc lymphomas. Preliminary data suggest that both loss of wild-type p53 function as well as retroviral over-expression of mutant p53 can compensate for reduced levels of MCL-1 (loss of one Mcl-1 allele). The underlying mechanisms for this are under investigation. The role of mutant p53 in lymphoma cell survival has been further examined in Eμ-Myc lymphoma-derived cell lines. Enforced over-expression of mutant p53 in cell lines containing wild-type p53 impaired induction of apoptosis by Nutlin3A, an inhibitor of Mdm-2 (the major negative regulator of p53). Remarkably, Nutlin-3a-induced apoptosis was impaired although it caused substantial transcriptional induction of the p53 apoptosis effectors, Puma and Noxa. Importantly, different mutant p53 proteins conferred different levels of protection against cell death. The observed protection against cell death may be partly due to dominant-negative effects of mutant p53, however, it does not appear to be robust enough to account for the extent of cell survival. Furthermore, mutant p53 conferred resistance to docetaxol, which is thought to induce cell death through predominantly p53-independent mechanisms. These data suggest that mutant p53 can protect against both p53-dependent and p53-independent cell death processes. Conversely, transcriptional induction of Noxa and Puma implies that “p53-restoration therapy” may remain a feasible treatment strategy even in tumours that bear mutations in p53 and that the role of a dominant-negative effect for some mutant p53 proteins may be less important than previously considered, at least in lymphoma cells. We are also examining the effect of mutant p53 on lymphoma development utilizing a hematopoietic reconstitution model and retroviral over-expression of mutant p53 proteins. The different mutant p53 proteins investigated exhibited distinct effects during tumorigenesis. The R246Q mutant p53 protein markedly accelerated lymphoma development in the context of MYC over-expression. The R246Q mutant p53 protein demonstrated strong selection in p53-deficient (p53-/-) hematopoietic cells during reconstitution indicative of an advantageous activity in emergency hematopoiesis. Overall, these findings provide evidence for a positive oncogenic role of mutant p53 in hematopoietic cells that provides a particularly potent selective advantage in the context of MYC driven lymphoma development. Importantly, different p53 mutations exhibit different functional properties such that different p53 mutations are likely to be associated with distinct risk in human malignant disease. Disclosures No relevant conflicts of interest to declare.

scholarly journals Tetramerization domain of human butyrylcholinesterase is at the C-terminus

1997 ◽  
Vol 327 (3) ◽  
pp. 747-757 ◽  
Author(s):  
M. Renee BLONG ◽  
Elliott BEDOWS ◽  
Oksana LOCKRIDGE
Keyword(s):  
Amino Acids ◽  
Cho Cells ◽  
Chinese Hamster ◽  
Disulphide Bond ◽  
Wild Type ◽  
Flag Epitope ◽  
Bche Activity ◽  
C Terminus ◽  
Tetramerization Domain ◽  
Inactive Protein

Butyrylcholinesterase (BChE) in human serum consists predominantly of tetramers. Recombinant BChE, however, expressed in Chinese hamster ovary (CHO) cells, consists of approx. 55% dimers, 10-30% tetramers and 15-40% monomers. To determine the origin of the monomer species we added the FLAG epitope (epitope tag, amino acid sequence DYKDDDDK) to the C-terminus of the enzyme, and expressed BChE-FLAG in CHO cells. We found that secreted, active monomers had lost their FLAG epitope, suggesting that the monomers were made by proteolysis of dimers or tetramers at the C-terminus. To estimate the number of amino acids that could be deleted from the C-terminus without losing BChE activity, we expressed deletion mutants. We found that deletion of up to 50 amino acids from the C-terminus yielded active monomers, but that deletion of 51 amino acids destroyed BChE activity and caused the inactive protein to remain within the cell. Deletion of eight or more amino acids from the N-terminus also resulted in inactive protein that remained inside the cell. Monomeric BChE had wild-type Km and kcat values (8 μM and 24000 min-1 for butyrylthiocholine) and showed substrate activation. The Cys-571→Ala mutant, though incapable of forming the interchain disulphide bond, had nearly the same amount of tetrameric BChE as recombinant wild-type BChE. These results support the conclusion that the tetramerization domain of BChE is at the C-terminus, within the terminal 50 amino acids, and that the interchain disulphide bond is not essential for tetramerization. Molecular modelling suggested that the tetramerization domain was a four-helix bundle, stabilized by interactions of seven conserved aromatic amino acids.

scholarly journals Interaction of p53 with its consensus DNA-binding site.

1995 ◽  
Vol 15 (4) ◽  
pp. 2157-2165 ◽  
Author(s):  
Y Wang ◽  
J F Schwedes ◽  
D Parks ◽  
K Mann ◽  
P Tegtmeyer
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Consensus Sequence ◽  
Specific Interaction ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Wild Type ◽  
Virtual Absence ◽  
Wild Type P53

We have analyzed the specific interaction of murine p53 with the consensus DNA-binding sequence 5'-AGACATGCCT-AGACATGCCT-3'. We used segments of p53 lacking the C-terminal, nonspecific DNA-binding domain because the presence of an autonomous nonspecific DNA-binding domain in wild-type p53 would complicate analysis of site-specific DNA binding. p53 amino acids 1 to 360 bind the consensus sequence as tetramers, and DNA binding promotes tetramer-tetramer interactions. p53 amino acids 80 to 290, lacking both the nonspecific DNA-binding and tetramerization domains, consistently bind consensus DNA as four monomers and only as four monomers. The virtual absence of stable binding by fewer than four monomers, even at low concentrations of p53, argues that binding by amino acids 80 to 290 is strongly cooperative. Because p53 tetramers and monomers do not simultaneously bind a single DNA consensus sequence, we conclude that a single tetramer of wild-type p53 engages the recognition sequences of the entire DNA consensus site. We further show that consensus DNA consists of two functional half-sites. Insertions, deletions, or rearrangements within the half-sites reduce DNA binding dramatically. In contrast, two half-sites separated by insertions bind p53 relatively efficiently. Insertions that place half-sites on opposite faces of the DNA helix reduce DNA binding more than insertions that place half-sites on the same face of the helix. Transcription studies, in vivo, strongly confirm the rotational specificity of the p53 interaction with consensus DNA. The ability of single p53 tetramers to bind separated DNA half-sites argues that p53 has a flexible tetramerization region.

scholarly journals An MDM2 inhibitor achieves synergistic cytotoxic effects with adenoviruses lacking E1B55kDa gene on mesothelioma with the wild-type p53 through augmenting NFI expression

2021 ◽  
Vol 12 (7) ◽  
Author(s):  
Thao Thi Thanh Nguyen ◽  
Masato Shingyoji ◽  
Michiko Hanazono ◽  
Boya Zhong ◽  
Takao Morinaga ◽  
...  
Keyword(s):  
Wild Type ◽  
Inhibitory Effects ◽  
Cytotoxic Effects ◽  
P53 Expression ◽  
Nuclear Factor I ◽  
Infected Cells ◽  
Wild Type P53 ◽  
Mdm2 Inhibitor ◽  
P16 Expression

AbstractA majority of mesothelioma specimens were defective of p14 and p16 expression due to deletion of the INK4A/ARF region, and the p53 pathway was consequently inactivated by elevated MDM2 functions which facilitated p53 degradaton. We investigated a role of p53 elevation by MDM2 inhibitors, nutlin-3a and RG7112, in cytotoxicity of replication-competent adenoviruses (Ad) lacking the p53-binding E1B55kDa gene (Ad-delE1B). We found that a growth inhibition by p53-activating Ad-delE1B was irrelevant to p53 expression in the infected cells, but combination of Ad-delE1B and the MDM2 inhibitor produced synergistic inhibitory effects on mesothelioma with the wild-type but not mutated p53 genotype. The combination augmented p53 phosphorylation, activated apoptotic but not autophagic pathway, and enhanced DNA damage signals through ATM-Chk2 phosphorylation. The MDM2 inhibitors facilitated production of the Ad progenies through augmented expression of nuclear factor I (NFI), one of the transcriptional factors involved in Ad replications. Knocking down of p53 with siRNA did not increase the progeny production or the NFI expression. We also demonstrated anti-tumor effects by the combination of Ad-delE1B and the MDM2 inhibitors in an orthotopic animal model. These data collectively indicated that upregulation of wild-type p53 expression contributed to cytotoxicity by E1B55kDa-defective replicative Ad through NFI induction and suggested that replication-competent Ad together with augmented p53 levels was a therapeutic strategy for p53 wild-type mesothelioma.

scholarly journals A CACNA1A variant associated with trigeminal neuralgia alters the gating of Cav2.1 channels

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Eder Gambeta ◽  
Maria A. Gandini ◽  
Ivana A. Souza ◽  
Laurent Ferron ◽  
Gerald W. Zamponi
Keyword(s):  
Trigeminal Neuralgia ◽  
Missense Mutation ◽  
Neurotransmitter Release ◽  
Trigeminal System ◽  
Wild Type ◽  
Calcium Dependent ◽  
Whole Cell Patch Clamp ◽  
C Terminus ◽  
Dependent Inactivation

AbstractA novel missense mutation in the CACNA1A gene that encodes the pore forming α1 subunit of the CaV2.1 voltage-gated calcium channel was identified in a patient with trigeminal neuralgia. This mutation leads to a substitution of proline 2455 by histidine (P2455H) in the distal C-terminus region of the channel. Due to the well characterized role of this channel in neurotransmitter release, our aim was to characterize the biophysical properties of the P2455H variant in heterologously expressed CaV2.1 channels. Whole-cell patch clamp recordings of wild type and mutant CaV2.1 channels expressed in tsA-201 cells reveal that the mutation mediates a depolarizing shift in the voltage-dependence of activation and inactivation. Moreover, the P2455H mutant strongly reduced calcium-dependent inactivation of the channel that is consistent with an overall gain of function. Hence, the P2455H CaV2.1 missense mutation alters the gating properties of the channel, suggesting that associated changes in CaV2.1-dependent synaptic communication in the trigeminal system may contribute to the development of trigeminal neuralgia.

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