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1.
Proteins ; 85(3): 487-496, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-27701776

RESUMO

The sixth CAPRI edition included new modeling challenges, such as the prediction of protein-peptide complexes, and the modeling of homo-oligomers and domain-domain interactions as part of the first joint CASP-CAPRI experiment. Other non-standard targets included the prediction of interfacial water positions and the modeling of the interactions between proteins and nucleic acids. We have participated in all proposed targets of this CAPRI edition both as predictors and as scorers, with new protocols to efficiently use our docking and scoring scheme pyDock in a large variety of scenarios. In addition, we have participated for the first time in the servers section, with our recently developed webserver, pyDockWeb. Excluding the CASP-CAPRI cases, we submitted acceptable models (or better) for 7 out of the 18 evaluated targets as predictors, 4 out of the 11 targets as scorers, and 6 out of the 18 targets as servers. The overall success rates were below those in past CAPRI editions. This shows the challenging nature of this last edition, with many difficult targets for which no participant submitted a single acceptable model. Interestingly, we submitted acceptable models for 83% of the evaluated protein-peptide targets. As for the 25 cases of the CASP-CAPRI experiment, in which we used a larger variety of modeling techniques (template-based, symmetry restraints, literature information, etc.), we submitted acceptable models for 56% of the targets. In summary, this CAPRI edition showed that pyDock scheme can be efficiently adapted to the increasing variety of problems that the protein interactions field is currently facing. Proteins 2017; 85:487-496. © 2016 Wiley Periodicals, Inc.


Assuntos
Algoritmos , Biologia Computacional/métodos , Simulação de Acoplamento Molecular/métodos , Peptídeos/química , Proteínas/química , Software , Sequência de Aminoácidos , Benchmarking , Sítios de Ligação , Cristalografia por Raios X , Ligação Proteica , Conformação Proteica , Mapeamento de Interação de Proteínas , Multimerização Proteica , Projetos de Pesquisa , Homologia Estrutural de Proteína , Termodinâmica , Água/química
2.
Proc Natl Acad Sci U S A ; 111(34): E3514-23, 2014 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-25114243

RESUMO

A challenge for microbial pathogens is to assure that their translocated effector proteins target only the correct host cell compartment during infection. The Legionella pneumophila effector vacuolar protein sorting inhibitor protein D (VipD) localizes to early endosomal membranes and alters their lipid and protein composition, thereby protecting the pathogen from endosomal fusion. This process requires the phospholipase A1 (PLA1) activity of VipD that is triggered specifically on VipD binding to the host cell GTPase Rab5, a key regulator of endosomes. Here, we present the crystal structure of VipD in complex with constitutively active Rab5 and reveal the molecular mechanism underlying PLA1 activation. An active site-obstructing loop that originates from the C-terminal domain of VipD is repositioned on Rab5 binding, thereby exposing the catalytic pocket within the N-terminal PLA1 domain. Substitution of amino acid residues located within the VipD-Rab5 interface prevented Rab5 binding and PLA1 activation and caused a failure of VipD mutant proteins to target to Rab5-enriched endosomal structures within cells. Experimental and computational analyses confirmed an extended VipD-binding interface on Rab5, explaining why this L. pneumophila effector can compete with cellular ligands for Rab5 binding. Together, our data explain how the catalytic activity of a microbial effector can be precisely linked to its subcellular localization.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Legionella pneumophila/enzimologia , Fosfolipases A1/química , Fosfolipases A1/metabolismo , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab5 de Ligação ao GTP/química , Proteínas rab5 de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Ligação Competitiva , Domínio Catalítico , Cristalografia por Raios X , Endossomos/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Legionella pneumophila/genética , Legionella pneumophila/patogenicidade , Modelos Moleculares , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Mutagênese Sítio-Dirigida , Fosfolipases A1/genética , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Proteínas de Transporte Vesicular/genética , Proteínas rab5 de Ligação ao GTP/genética
3.
Biochim Biophys Acta ; 1847(12): 1549-59, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26407632

RESUMO

In the Phaeodactylum tricornutum alga, as in most diatoms, cytochrome c6 is the only electron donor to photosystem I, and thus they lack plastocyanin as an alternative electron carrier. We have investigated, by using laser-flash absorption spectroscopy, the electron transfer to Phaeodactylum photosystem I from plastocyanins from cyanobacteria, green algae and plants, as compared with its own cytochrome c6. Diatom photosystem I is able to effectively react with eukaryotic acidic plastocyanins, although with less efficiency than with Phaeodactylum cytochrome c6. This efficiency, however, increases in some green alga plastocyanin mutants mimicking the electrostatics of the interaction site on the diatom cytochrome. In addition, the structure of the transient electron transfer complex between cytochrome c6 and photosystem I from Phaeodactylum has been analyzed by computational docking and compared to that of green lineage and mixed systems. Taking together, the results explain why the Phaeodactylum system shows a lower efficiency than the green systems, both in the formation of the properly arranged [cytochrome c6-photosystem I] complex and in the electron transfer itself.


Assuntos
Citocromos c6/metabolismo , Complexo de Proteína do Fotossistema I/metabolismo , Plastocianina/metabolismo , Estramenópilas/metabolismo , Citocromos c6/química , Cinética , Simulação de Acoplamento Molecular , Fotossíntese , Complexo de Proteína do Fotossistema I/química , Plastocianina/química , Ligação Proteica , Estramenópilas/fisiologia
4.
PLoS Pathog ; 9(5): e1003382, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23696742

RESUMO

The covalent attachment of adenosine monophosphate (AMP) to proteins, a process called AMPylation (adenylylation), has recently emerged as a novel theme in microbial pathogenesis. Although several AMPylating enzymes have been characterized, the only known virulence protein with de-AMPylation activity is SidD from the human pathogen Legionella pneumophila. SidD de-AMPylates mammalian Rab1, a small GTPase involved in secretory vesicle transport, thereby targeting the host protein for inactivation. The molecular mechanisms underlying Rab1 recognition and de-AMPylation by SidD are unclear. Here, we report the crystal structure of the catalytic region of SidD at 1.6 Å resolution. The structure reveals a phosphatase-like fold with additional structural elements not present in generic PP2C-type phosphatases. The catalytic pocket contains a binuclear metal-binding site characteristic of hydrolytic metalloenzymes, with strong dependency on magnesium ions. Subsequent docking and molecular dynamics simulations between SidD and Rab1 revealed the interface contacts and the energetic contribution of key residues to the interaction. In conjunction with an extensive structure-based mutational analysis, we provide in vivo and in vitro evidence for a remarkable adaptation of SidD to its host cell target Rab1 which explains how this effector confers specificity to the reaction it catalyses.


Assuntos
Monofosfato de Adenosina/química , Proteínas de Bactérias/química , Legionella pneumophila/enzimologia , Simulação de Acoplamento Molecular , Proteínas rab1 de Ligação ao GTP/química , Monofosfato de Adenosina/genética , Monofosfato de Adenosina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Humanos , Legionella pneumophila/genética , Fosfoproteínas Fosfatases/química , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Proteína Fosfatase 2C , Estrutura Quaternária de Proteína , Relação Estrutura-Atividade , Proteínas rab1 de Ligação ao GTP/genética , Proteínas rab1 de Ligação ao GTP/metabolismo
5.
Proteins ; 82(4): 620-32, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24155158

RESUMO

We report the first assessment of blind predictions of water positions at protein-protein interfaces, performed as part of the critical assessment of predicted interactions (CAPRI) community-wide experiment. Groups submitting docking predictions for the complex of the DNase domain of colicin E2 and Im2 immunity protein (CAPRI Target 47), were invited to predict the positions of interfacial water molecules using the method of their choice. The predictions-20 groups submitted a total of 195 models-were assessed by measuring the recall fraction of water-mediated protein contacts. Of the 176 high- or medium-quality docking models-a very good docking performance per se-only 44% had a recall fraction above 0.3, and a mere 6% above 0.5. The actual water positions were in general predicted to an accuracy level no better than 1.5 Å, and even in good models about half of the contacts represented false positives. This notwithstanding, three hotspot interface water positions were quite well predicted, and so was one of the water positions that is believed to stabilize the loop that confers specificity in these complexes. Overall the best interface water predictions was achieved by groups that also produced high-quality docking models, indicating that accurate modelling of the protein portion is a determinant factor. The use of established molecular mechanics force fields, coupled to sampling and optimization procedures also seemed to confer an advantage. Insights gained from this analysis should help improve the prediction of protein-water interactions and their role in stabilizing protein complexes.


Assuntos
Colicinas/química , Mapeamento de Interação de Proteínas , Água/química , Algoritmos , Biologia Computacional , Modelos Moleculares , Simulação de Acoplamento Molecular , Conformação Proteica
6.
Proteins ; 81(12): 2192-200, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23934865

RESUMO

In addition to protein-protein docking, this CAPRI edition included new challenges, like protein-water and protein-sugar interactions, or the prediction of binding affinities and ΔΔG changes upon mutation. Regarding the standard protein-protein docking cases, our approach, mostly based on the pyDock scheme, submitted correct models as predictors and as scorers for 67% and 57% of the evaluated targets, respectively. In this edition, available information on known interface residues hardly made any difference for our predictions. In one of the targets, the inclusion of available experimental small-angle X-ray scattering (SAXS) data using our pyDockSAXS approach slightly improved the predictions. In addition to the standard protein-protein docking assessment, new challenges were proposed. One of the new problems was predicting the position of the interface water molecules, for which we submitted models with 20% and 43% of the water-mediated native contacts predicted as predictors and scorers, respectively. Another new problem was the prediction of protein-carbohydrate binding, where our submitted model was very close to being acceptable. A set of targets were related to the prediction of binding affinities, in which our pyDock scheme was able to discriminate between natural and designed complexes with area under the curve = 83%. It was also proposed to estimate the effect of point mutations on binding affinity. Our approach, based on machine learning methods, showed high rates of correctly classified mutations for all cases. The overall results were highly rewarding, and show that the field is ready to move forward and face new interesting challenges in interactomics.


Assuntos
Carboidratos/química , Simulação de Acoplamento Molecular , Proteínas/química , Água/química , Biologia Computacional , Mutação , Ligação Proteica , Conformação Proteica , Espalhamento a Baixo Ângulo , Software , Difração de Raios X
7.
Proteins ; 81(11): 1980-7, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23843247

RESUMO

Community-wide blind prediction experiments such as CAPRI and CASP provide an objective measure of the current state of predictive methodology. Here we describe a community-wide assessment of methods to predict the effects of mutations on protein-protein interactions. Twenty-two groups predicted the effects of comprehensive saturation mutagenesis for two designed influenza hemagglutinin binders and the results were compared with experimental yeast display enrichment data obtained using deep sequencing. The most successful methods explicitly considered the effects of mutation on monomer stability in addition to binding affinity, carried out explicit side-chain sampling and backbone relaxation, evaluated packing, electrostatic, and solvation effects, and correctly identified around a third of the beneficial mutations. Much room for improvement remains for even the best techniques, and large-scale fitness landscapes should continue to provide an excellent test bed for continued evaluation of both existing and new prediction methodologies.


Assuntos
Bases de Dados de Proteínas , Mapeamento de Interação de Proteínas , Algoritmos , Mutação , Ligação Proteica
8.
Nat Commun ; 14(1): 3318, 2023 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-37308482

RESUMO

p38α is a versatile protein kinase that can control numerous processes and plays important roles in the cellular responses to stress. Dysregulation of p38α signaling has been linked to several diseases including inflammation, immune disorders and cancer, suggesting that targeting p38α could be therapeutically beneficial. Over the last two decades, numerous p38α inhibitors have been developed, which showed promising effects in pre-clinical studies but results from clinical trials have been disappointing, fueling the interest in the generation of alternative mechanisms of p38α modulation. Here, we report the in silico identification of compounds that we refer to as non-canonical p38α inhibitors (NC-p38i). By combining biochemical and structural analyses, we show that NC-p38i efficiently inhibit p38α autophosphorylation but weakly affect the activity of the canonical pathway. Our results demonstrate how the structural plasticity of p38α can be leveraged to develop therapeutic opportunities targeting a subset of the functions regulated by this pathway.


Assuntos
Inflamação , Transdução de Sinais , Humanos , Fosforilação
9.
Sci Rep ; 12(1): 15810, 2022 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-36138080

RESUMO

Oncogenic RAS proteins are important for driving tumour formation, and for maintenance of the transformed phenotype, and thus their relevance as a cancer therapeutic target is undeniable. We focused here on obtaining peptidomimetics, which have good pharmacological properties, to block Ras-effector interaction. Computational analysis was used to identify hot spots of RAS relevant for these interactions and to screen a library of peptidomimetics. Nine compounds were synthesized and assayed for their activity as RAS inhibitors in cultured cells. Most of them induced a reduction in ERK and AKT activation by EGF, a marker of RAS activity. The most potent inhibitor disrupted Raf and PI3K interaction with oncogenic KRAS, corroborating its mechanism of action as an inhibitor of protein-protein interactions, and thus validating our computational methodology. Most interestingly, improvement of one of the compounds allowed us to obtain a peptidomimetic that decreased the survival of pancreatic cancer cell lines harbouring oncogenic KRAS.


Assuntos
Neoplasias Pancreáticas , Peptidomiméticos , Linhagem Celular Tumoral , Fator de Crescimento Epidérmico/metabolismo , Humanos , Neoplasias Pancreáticas/metabolismo , Peptidomiméticos/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transdução de Sinais/genética
10.
Biochim Biophys Acta Biomembr ; 1861(7): 1302-1316, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31077676

RESUMO

Aggregated LDL is the first ligand reported to interact with the cluster II CR9 domain of low-density lipoprotein receptor-related protein 1 (LRP1). In particular, the C-terminal half of domain CR9, comprising the region Gly1127-Cys1140 exclusively recognizes aggregated LDL and it is crucial for aggregated LDL binding. Our aim was to study the effect of the sequence Gly1127-Cys1140 (named peptide LP3 and its retro-enantio version, named peptide DP3) on the structural characteristics of sphingomyelinase- (SMase) and phospholipase 2 (PLA2)-modified LDL particles. Turbidimetry, gel filtration chromatography (GFC) and transmission electronic microscopy (TEM) analysis showed that LP3 and DP3 peptides strongly inhibited SMase- and PLA2-induced LDL aggregation. Nondenaturing polyacrylamide gradient gel electrophoresis (GGE), agarose gel electrophoresis and high-performance thin-layer chromatography (HPTLC) indicated that LP3 and DP3 prevented SMase-induced alterations in LDL particle size, electric charge and phospholipid content, respectively, but not those induced by PLA2. Western blot analysis showed that LP3 and DP3 counteracted changes in ApoB-100 conformation induced by the two enzymes. LDL proteomics (LDL trypsin digestion followed by mass spectroscopy) and computational modeling methods evidenced that peptides preserve ApoB-100 conformation due to their electrostatic interactions with a basic region of ApoB-100. These results demonstrate that LRP1-derived peptides are protective against LDL aggregation, even in conditions of extreme lipolysis, through their capacity to bind to ApoB-100 regions critical for ApoB-100 conformational preservation. These results suggests that these LRP1(CR9) derived peptides could be promising tools to prevent LDL aggregation induced by the main proteolytic enzymes acting in the arterial intima.


Assuntos
Lipoproteínas LDL/química , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Peptídeos/metabolismo , Proteínas de Artrópodes/sangue , Humanos , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/química , Oligopeptídeos/sangue , Fosfolipases A2/metabolismo , Fosfolipídeos/química , Ligação Proteica , Esfingomielina Fosfodiesterase/química , Eletricidade Estática
11.
Adv Protein Chem Struct Biol ; 110: 203-249, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29412997

RESUMO

A huge amount of genetic information is available thanks to the recent advances in sequencing technologies and the larger computational capabilities, but the interpretation of such genetic data at phenotypic level remains elusive. One of the reasons is that proteins are not acting alone, but are specifically interacting with other proteins and biomolecules, forming intricate interaction networks that are essential for the majority of cell processes and pathological conditions. Thus, characterizing such interaction networks is an important step in understanding how information flows from gene to phenotype. Indeed, structural characterization of protein-protein interactions at atomic resolution has many applications in biomedicine, from diagnosis and vaccine design, to drug discovery. However, despite the advances of experimental structural determination, the number of interactions for which there is available structural data is still very small. In this context, a complementary approach is computational modeling of protein interactions by docking, which is usually composed of two major phases: (i) sampling of the possible binding modes between the interacting molecules and (ii) scoring for the identification of the correct orientations. In addition, prediction of interface and hot-spot residues is very useful in order to guide and interpret mutagenesis experiments, as well as to understand functional and mechanistic aspects of the interaction. Computational docking is already being applied to specific biomedical problems within the context of personalized medicine, for instance, helping to interpret pathological mutations involved in protein-protein interactions, or providing modeled structural data for drug discovery targeting protein-protein interactions.


Assuntos
Pesquisa Biomédica , Biologia Computacional , Simulação de Acoplamento Molecular , Proteínas , Descoberta de Drogas , Humanos , Modelos Moleculares , Ligação Proteica , Proteínas/química , Proteínas/metabolismo
12.
Sci Rep ; 8(1): 11830, 2018 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-30087384

RESUMO

MAPK/ERK kinase (MEK) 1/2 are central signaling proteins that serve as specificity determinants of the MAPK/ERK cascade. More than twenty activating mutations have been reported for MEK1/2, and many of them are known to cause diseases such as cancers, arteriovenous malformation and RASopathies. Changes in their intrinsic activity do not seem to correlate with the severity of the diseases. Here we studied four MEK1/2 mutations using biochemical and molecular dynamic methods. Although the studied mutants elevated the activating phosphorylation of MEK they had no effect on the stimulated ERK1/2 phosphorylation. Studying the regulatory mechanism that may explain this lack of effect, we found that one type of mutation affects MEK stability and two types of mutations demonstrate a reduced sensitivity to PP2A. Together, our results indicate that some MEK mutations exert their function not only by their elevated intrinsic activity, but also by modulation of regulatory elements such as protein stability or dephosphorylation.


Assuntos
Regulação Enzimológica da Expressão Gênica , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 2/genética , Peptídeo Hidrolases/genética , Monoéster Fosfórico Hidrolases/genética , Animais , Células COS , Chlorocebus aethiops , Células HeLa , Humanos , MAP Quinase Quinase 1/química , MAP Quinase Quinase 1/metabolismo , Sistema de Sinalização das MAP Quinases/genética , Mutação , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Peptídeo Hidrolases/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação , Estabilidade Proteica
13.
Methods Mol Biol ; 1529: 139-159, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-27914049

RESUMO

An important aspect of protein functionality is the formation of specific complexes with other proteins, which are involved in the majority of biological processes. The functional characterization of such interactions at molecular level is necessary, not only to understand biological and pathological phenomena but also to design improved, or even new interfaces, or to develop new therapeutic approaches. X-ray crystallography and NMR spectroscopy have increased the number of 3D protein complex structures deposited in the Protein Data Bank (PDB). However, one of the more challenging objectives in biological research is to functionally characterize protein interactions and thus identify residues that significantly contribute to the binding. Considering that the experimental characterization of protein interfaces remains expensive, time-consuming, and labor-intensive, computational approaches represent a significant breakthrough in proteomics, assisting or even replacing experimental efforts. Thanks to the technological advances in computing and data processing, these techniques now cover a vast range of protocols, from the estimation of the evolutionary conservation of amino acid positions in a protein, to the energetic contribution of each residue to the binding affinity. In this chapter, we review several existing computational protocols to model the phylogenetic, structural, and energetic properties of residues within protein-protein interfaces.


Assuntos
Biologia Computacional/métodos , Modelos Moleculares , Engenharia de Proteínas , Proteínas , Aminoácidos/química , Sítios de Ligação , Simulação por Computador , Bases de Dados de Proteínas , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Mutação , Ligação Proteica , Conformação Proteica , Engenharia de Proteínas/métodos , Mapeamento de Interação de Proteínas/métodos , Proteínas/química , Proteínas/genética , Proteínas/metabolismo , Software , Navegador
14.
J Chem Theory Comput ; 12(7): 3236-49, 2016 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-27294484

RESUMO

To understand cellular processes at the molecular level we need to improve our knowledge of protein-protein interactions, from a structural, mechanistic, and energetic point of view. Current theoretical studies and computational docking simulations show that protein dynamics plays a key role in protein association and support the need for including protein flexibility in modeling protein interactions. Assuming the conformational selection binding mechanism, in which the unbound state can sample bound conformers, one possible strategy to include flexibility in docking predictions would be the use of conformational ensembles originated from unbound protein structures. Here we present an exhaustive computational study about the use of precomputed unbound ensembles in the context of protein docking, performed on a set of 124 cases of the Protein-Protein Docking Benchmark 3.0. Conformational ensembles were generated by conformational optimization and refinement with MODELLER and by short molecular dynamics trajectories with AMBER. We identified those conformers providing optimal binding and investigated the role of protein conformational heterogeneity in protein-protein recognition. Our results show that a restricted conformational refinement can generate conformers with better binding properties and improve docking encounters in medium-flexible cases. For more flexible cases, a more extended conformational sampling based on Normal Mode Analysis was proven helpful. We found that successful conformers provide better energetic complementarity to the docking partners, which is compatible with recent views of binding association. In addition to the mechanistic considerations, these findings could be exploited for practical docking predictions of improved efficiency.


Assuntos
Ligação Proteica/fisiologia , Proteínas/química , Humanos , Simulação de Dinâmica Molecular , Conformação Proteica
15.
Mol Oncol ; 8(5): 1026-42, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24816189

RESUMO

The tumor suppressor p53 regulates the expression of genes involved in cell cycle progression, senescence and apoptosis. Here, we investigated the effect of single point mutations in the oligomerization domain (OD) on tetramerization, transcription, ubiquitylation and stability of p53. As predicted by docking and molecular dynamics simulations, p53 OD mutants show functional defects on transcription, Mdm2-dependent ubiquitylation and 26S proteasome-mediated degradation. However, mutants unable to form tetramers are well degraded by the 20S proteasome. Unexpectedly, despite the lower structural stability compared to WT p53, p53 OD mutants form heterotetramers with WT p53 when expressed transiently or stably in cells wild type or null for p53. In consequence, p53 OD mutants interfere with the capacity of WT p53 tetramers to be properly ubiquitylated and result in changes of p53-dependent protein expression patterns, including the pro-apoptotic proteins Bax and PUMA under basal and adriamycin-induced conditions. Importantly, the patient derived p53 OD mutant L330R (OD1) showed the more severe changes in p53-dependent gene expression. Thus, in addition to the well-known effects on p53 stability, ubiquitylation defects promote changes in p53-dependent gene expression with implications on some of its functions.


Assuntos
Mutação Puntual , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Ativação Transcricional , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Ubiquitinação , Linhagem Celular Tumoral , Humanos , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Complexo de Endopeptidases do Proteassoma/metabolismo , Multimerização Proteica , Proteólise , Proteína Supressora de Tumor p53/química
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