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1.
EMBO Rep ; 20(12): e47964, 2019 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-31680439

RESUMO

RNA-binding proteins (RBPs) participate in all steps of gene expression, underscoring their potential as regulators of RNA homeostasis. We structurally and functionally characterize Mip6, a four-RNA recognition motif (RRM)-containing RBP, as a functional and physical interactor of the export factor Mex67. Mip6-RRM4 directly interacts with the ubiquitin-associated (UBA) domain of Mex67 through a loop containing tryptophan 442. Mip6 shuttles between the nucleus and the cytoplasm in a Mex67-dependent manner and concentrates in cytoplasmic foci under stress. Photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation experiments show preferential binding of Mip6 to mRNAs regulated by the stress-response Msn2/4 transcription factors. Consistent with this binding, MIP6 deletion affects their export and expression levels. Additionally, Mip6 interacts physically and/or functionally with proteins with a role in mRNA metabolism and transcription such as Rrp6, Xrn1, Sgf73, and Rpb1. These results reveal a novel role for Mip6 in the homeostasis of Msn2/4-dependent transcripts through its direct interaction with the Mex67 UBA domain.

2.
Nature ; 568(7753): 557-560, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30971822

RESUMO

The cell cycle is a tightly regulated process that is controlled by the conserved cyclin-dependent kinase (CDK)-cyclin protein complex1. However, control of the G0-to-G1 transition is not completely understood. Here we demonstrate that p38 MAPK gamma (p38γ) acts as a CDK-like kinase and thus cooperates with CDKs, regulating entry into the cell cycle. p38γ shares high sequence homology, inhibition sensitivity and substrate specificity with CDK family members. In mouse hepatocytes, p38γ induces proliferation after partial hepatectomy by promoting the phosphorylation of retinoblastoma tumour suppressor protein at known CDK target residues. Lack of p38γ or treatment with the p38γ inhibitor pirfenidone protects against the chemically induced formation of liver tumours. Furthermore, biopsies of human hepatocellular carcinoma show high expression of p38γ, suggesting that p38γ could be a therapeutic target in the treatment of this disease.


Assuntos
Carcinogênese/patologia , Ciclo Celular , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/patologia , Fígado/enzimologia , Fígado/patologia , Proteína Quinase 12 Ativada por Mitógeno/metabolismo , Idoso , Animais , Carcinogênese/efeitos dos fármacos , Carcinoma Hepatocelular/induzido quimicamente , Carcinoma Hepatocelular/patologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/metabolismo , Feminino , Hepatócitos/citologia , Hepatócitos/patologia , Humanos , Fígado/cirurgia , Neoplasias Hepáticas/induzido quimicamente , Masculino , Camundongos , Pessoa de Meia-Idade , Proteína Quinase 12 Ativada por Mitógeno/antagonistas & inibidores , Fosforilação , Piridonas/farmacologia , Proteína do Retinoblastoma/química , Proteína do Retinoblastoma/metabolismo , Homologia de Sequência , Especificidade por Substrato
3.
J Mol Biol ; 430(17): 2822-2842, 2018 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-29870725

RESUMO

The Escherichia coli homodimeric proteins MnmE and MnmG form a functional complex, MnmEG, that modifies tRNAs using GTP, methylene-tetrahydrofolate, FAD, and glycine or ammonium. MnmE is a tetrahydrofolate- and GTP-binding protein, whereas MnmG is a FAD-binding protein with each protomer composed of the FAD-binding domain, two insertion domains, and the helical C-terminal domain. The detailed mechanism of the MnmEG-mediated reaction remains unclear partially due to incomplete structural information on the free- and substrate-bound forms of the complex. In this study, we show that MnmG can adopt in solution a dimer arrangement (form I) different from that currently considered as the only biologically active (form II). Normal mode analysis indicates that form I can oscillate in a range of open and closed conformations. Using isothermal titration calorimetry and native red electrophoresis, we show that a form-I open conformation, which can be stabilized in vitro by the formation of an interprotomer disulfide bond between the catalytic C277 residues, appears to be involved in the assembly of the MnmEG catalytic center. We also show that residues R196, D253, R436, R554 and E585 are important for the stabilization of form I and the tRNA modification function. We propose that the form I dynamics regulates the alternative access of MnmE and tRNA to the MnmG FAD active site. Finally, we show that the C-terminal region of MnmG contains a sterile alpha motif domain responsible for tRNA-protein and protein-protein interactions.


Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Transferases de Grupo de Um Carbono/química , Transferases de Grupo de Um Carbono/metabolismo , Multimerização Proteica , RNA de Transferência/química , RNA de Transferência/metabolismo , Domínio Catalítico , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas
4.
Chemistry ; 24(8): 1890-1897, 2018 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-29193344

RESUMO

Apoptotic signaling pathways are altered in numerous pathologies such as cancer. In this scenario, caspase-9/PP2Acα interaction constitutes a key target with pharmacological interest to re-establish apoptosis in tumor cells. Very recently, a short peptide (C9h) known to disrupt caspase-9/PP2Acα interaction with subsequent apoptosis induction was described. Here, we prepared two sets of mesoporous silica nanoparticles loaded with safranin O (S2) or with C9h peptide (S4) and functionalized with ϵ-polylysine as capping unit. Aqueous suspensions of both nanoparticles showed negligible cargo release whereas in the presence of pronase, a marked delivery of safranin O or C9h was observed. Confocal microscopy studies carried out with HeLa cells indicated that both materials were internalized and were able to release their entrapped cargos. Besides, a marked decrease in HeLa cell viability (ca. 50 %) was observed when treated with C9h-loaded S4 nanoparticles. Moreover, S4 provides peptide protection from degradation additionally allowing for a dose reduction to observe an apoptotic effect when compared with C9h alone or in combination with a cell-penetrating peptide (i.e., Mut3DPT-C9h). Flow cytometry studies, by means of Annexin V-FITC staining, showed the activation of apoptotic pathways in HeLa as a consequence of S4 internalization, release of C9h peptide and disruption of caspase-9/PP2Acα interaction.


Assuntos
Nanopartículas/química , Peptídeos/química , Polilisina/química , Dióxido de Silício/química , Sequência de Aminoácidos , Apoptose/efeitos dos fármacos , Caspase 9/química , Caspase 9/metabolismo , Dicroísmo Circular , Portadores de Fármacos/síntese química , Portadores de Fármacos/química , Células HeLa , Humanos , Microscopia Confocal , Peptídeos/toxicidade , Fenazinas/química , Fenazinas/toxicidade , Porosidade , Proteína Fosfatase 2/química , Proteína Fosfatase 2/metabolismo
5.
Biomark Res ; 4: 9, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27152197

RESUMO

BACKGROUND: Disruption of alternative splicing in apoptotic factors has been associated to chronic lymphocytic leukemia among other cancers and hematological malignancies. The proapoptotic proteins Caspase-9 and PP2Acα are functionally related in a direct interaction, which constitutes a promising target for cancer therapy. Both proteins present aberrant mRNA splicing variants that are antiapoptotic (Caspase-9b) and catalytically inactive (PP2Acα2), respectively. RESULTS: In this work we have analyzed the relative abundance of the aberrant spliced forms Caspase-9b and PP2Acα2 in several cell lines and chronic lymphocytic leukemia patients and correlated it with several parameters of the disease. Despite 40 % of the patients presented Caspase-9b dysregulation, there was no direct association between alterations in Caspase-9b relative abundance and the parameters analyzed in medical records. More importantly, PP2Acα2 dysregulation was observed in 88 % of CLL patients and was related with advanced stages of the malignancy. CONCLUSIONS: Caspase-9b dysregulation seemed to be associated with the disease, although the differences between healthy donors and CLL patients were not statistically significant. However, PP2Acα2 dysregulation was significantly different between healthy donors and CLL patients and correlated with Binet B and C stages; therefore, we propose the use of PP2Acα2 dysregulation as a potential biomarker for advanced stages of chronic lymphocytic leukemia.

6.
Nucleic Acids Res ; 43(22): 11017-30, 2015 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-26476442

RESUMO

Ribosome biogenesis is one of the most essential pathways in eukaryotes although it is still not fully characterized. Given the importance of this process in proliferating cells, it is obvious that understanding the macromolecular details of the interactions that take place between the assembly factors, ribosomal proteins and nascent pre-rRNAs is essentially required for the development of new non-genotoxic treatments for cancer. Herein, we have studied the association between the WD40-repeat domains of Erb1 and Ytm1 proteins. These are essential factors for the biogenesis of 60S ribosomal subunits in eukaryotes that form a heterotrimeric complex together with the also essential Nop7 protein. We provide the crystal structure of a dimer formed by the C-terminal part of Erb1 and Ytm1 from Chaetomium thermophilum at 2.1 Å resolution. Using a multidisciplinary approach we show that the ß-propeller domains of these proteins interact in a novel manner that leads to a high-affinity binding. We prove that a point mutation within the interface of the complex impairs the interaction between the two proteins and negatively affects growth and ribosome production in yeast. Our study suggests insights into the association of the Erb1-Ytm1 dimer with pre-ribosomal particles.


Assuntos
Proteínas Fúngicas/química , Proteínas Ribossômicas/química , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Chaetomium , Dimerização , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Modelos Moleculares , Mutação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA Ribossômico/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo
7.
Ther Deliv ; 6(10): 1171-94, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26448473

RESUMO

In the era of biomedicines and engineered carrier systems, cell penetrating peptides (CPPs) have been established as a promising tool for therapeutic application. Likewise, other therapeutic peptides, successful in vivo application of CPPs will strongly depend on peptide stability, the bottleneck for this type of biodegradable molecules. In this review, the authors describe the current knowledge of the in vivo degradation for known CPPs and the different strategies available to provide a higher resistance to metabolic degradation while preserving cell penetration efficiency. Peptide stability can be improved by different means, either modifying the structure to make it unrecognizable to proteases, or preventing access of proteolytic enzymes by applying conformation restriction or shielding strategies.


Assuntos
Peptídeos Penetradores de Células/síntese química , Química Farmacêutica/métodos , Sistemas de Liberação de Medicamentos/métodos , Animais , Peptídeos Penetradores de Células/administração & dosagem , Estabilidade de Medicamentos , Humanos , Conformação Molecular
8.
FEBS Lett ; 589(18): 2290-6, 2015 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-26226421

RESUMO

The structural basis of the pH dependency of the dimer-tetramer transition exhibited by Brinda's type II Diocleinae lectins was investigated by equilibrium sedimentation and X-ray crystal structure determination of recombinant wild-type and site-directed single and double mutants of the pH-stable tetrameric Dioclea grandiflora lectin (r-αDGL). Releasing the peripheral site interdimeric contact between R60 and D78 rendered a mutant displaying dimer-tetramer equilibrium in the pH range equivalent to pKa±1 of the γ-COOH. Mutation of both histidines 51 and 131, but not the mutation of each His separately, abolished the formation of the Diocleinae canonical tetramer in the pH range 2.5-8.5. The X-ray structure of the double mutant r-αDGL H51G/H131N suggests that H131 plays a crucial role in networking loop 114-125 residues from all four subunits at the central cavity of the tetrameric lectin, and that H51 maintains the central cavity loops in a proper spatial orientation to make H131-mediated interdimer contacts.


Assuntos
Mutação , Lectinas de Plantas/química , Lectinas de Plantas/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Cristalografia por Raios X , Modelos Moleculares , Lectinas de Plantas/genética , Estrutura Quaternária de Proteína , Proteínas Recombinantes/genética , Ultracentrifugação
9.
Acta Crystallogr F Struct Biol Commun ; 71(Pt 6): 684-7, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26057796

RESUMO

Acidic leucine-rich nuclear phosphoprotein 32A (PP32A) is a tumour suppressor whose expression is altered in many cancers. It is an apoptotic enhancer that stimulates apoptosome-mediated caspase activation and also forms part of a complex involved in caspase-independent apoptosis (the SET complex). Crystals of a fragment of human PP32A corresponding to the leucine-rich repeat domain, a widespread motif suitable for protein-protein interactions, have been obtained. The structure has been refined to 1.56 Šresolution. This domain was previously solved at 2.4 and 2.69 Šresolution (PDB entries 2je0 and 2je1, respectively). The new high-resolution structure shows some differences from previous models: there is a small displacement in the turn connecting the first α-helix (α1) to the first ß-strand (ß1), which slightly changes the position of α1 in the structure. The shift in the turn is observed in the context of a new crystal packing unrelated to those of previous structures.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/química , Leucina/química , Sequência de Aminoácidos , Clonagem Molecular , Cristalização , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA , Proteínas Recombinantes/química , Proteínas Recombinantes/genética
10.
PLoS One ; 10(4): e0123463, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25880847

RESUMO

Erb1 (Eukaryotic Ribosome Biogenesis 1) protein is essential for the maturation of the ribosomal 60S subunit. Functional studies in yeast and mammalian cells showed that altogether with Nop7 and Ytm1 it forms a stable subcomplex called PeBoW that is crucial for a correct rRNA processing. The exact function of the protein within the process remains unknown. The N-terminal region of the protein includes a well conserved region shown to be involved in PeBoW complex formation whereas the carboxy-terminal half was predicted to contain seven WD40 repeats. This first structural report on Erb1 from yeast describes the architecture of a seven-bladed ß-propeller domain that revealed a characteristic extra motif formed by two α-helices and a ß-strand that insert within the second WD repeat. We performed analysis of molecular surface and crystal packing, together with multiple sequence alignment and comparison of the structure with other ß-propellers, in order to identify areas that are more likely to mediate protein-protein interactions. The abundance of many positively charged residues on the surface of the domain led us to investigate whether the propeller of Erb1 might be involved in RNA binding. Three independent assays confirmed that the protein interacted in vitro with polyuridilic acid (polyU), thus suggesting a possible role of the domain in rRNA rearrangement during ribosome biogenesis.


Assuntos
RNA/metabolismo , Proteínas Ribossômicas/química , Proteínas Ribossômicas/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Dicroísmo Circular , Cristalografia por Raios X , Evolução Molecular , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Poli U/química , Poli U/metabolismo , Conformação Proteica , Estrutura Terciária de Proteína , RNA/química , Proteínas Ribossômicas/genética , Proteínas de Saccharomyces cerevisiae/genética
11.
Ther Deliv ; 6(2): 139-47, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25690083

RESUMO

AIM: Before starting preclinical studies, we have analyzed the integrity in serum of DPT-C9h, a promising therapeutic peptide, and performed modifications in order to improve its stability. MATERIALS & METHODS: Mutant peptides exchanging arginine 8 for either lysine, asparagine or alanine were synthesized and compared with the parental peptide. RESULTS: All mutants clearly improved peptide stability while keeping their functional activity. PK studies showed an enhanced stability, being Mut3DPT-C9h the most promising candidate. Biodistribution studies demonstrate that the modified peptide is able to reach the targeted tumor and accumulate there at higher concentration than the parental peptide. DISCUSSION: Small modifications in the peptide sequence result in improvements allowing the selection of better candidates for preclinical studies.


Assuntos
Neoplasias da Mama/patologia , Peptídeos Penetradores de Células/química , Proteólise , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Peptídeos Penetradores de Células/farmacocinética , Peptídeos Penetradores de Células/farmacologia , Feminino , Humanos , Camundongos Nus , Mutação , Análise de Sequência de Proteína , Distribuição Tecidual , Ensaios Antitumorais Modelo de Xenoenxerto
12.
J Mol Biol ; 426(3): 674-90, 2014 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-24239949

RESUMO

TAX1BP1 is a novel ubiquitin-binding adaptor protein involved in the negative regulation of the NF-kappaB transcription factor, which is a key player in inflammatory responses, immunity and tumorigenesis. TAX1BP1 recruits A20 to the ubiquitinated signaling proteins TRAF6 and RIP1, leading to their A20-mediated deubiquitination and the disruption of IL-1-induced and TNF-induced NF-kappaB signaling, respectively. The two zinc fingers localized at its C-terminus function as novel ubiquitin-binding domains (UBZ, ubiquitin-binding zinc finger). Here we present for the first time both the solution and crystal structures of two classical UBZ domains in tandem within the human TAX1BP1. The relative orientation of the two domains is slightly different in the X-ray structure with respect to the NMR structure, indicating some degree of conformational flexibility, which is rationalized by NMR relaxation data. The observed degree of flexibility and stability between the two UBZ domains might have consequences on the recognition mechanism of interacting partners.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Ubiquitina/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Cristalografia por Raios X , Humanos , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Espalhamento a Baixo Ângulo , Homologia de Sequência de Aminoácidos , Transdução de Sinais
14.
PLoS One ; 8(9): e73018, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24039852

RESUMO

SH3 domains constitute a new type of ubiquitin-binding domains. We previously showed that the third SH3 domain (SH3-C) of CD2AP binds ubiquitin in an alternative orientation. We have determined the structure of the complex between first CD2AP SH3 domain and ubiquitin and performed a structural and mutational analysis to decipher the determinants of the SH3-C binding mode to ubiquitin. We found that the Phe-to-Tyr mutation in CD2AP and in the homologous CIN85 SH3-C domain does not abrogate ubiquitin binding, in contrast to previous hypothesis and our findings for the first two CD2AP SH3 domains. The similar alternative binding mode of the SH3-C domains of these related adaptor proteins is characterised by a higher affinity to C-terminal extended ubiquitin molecules. We conclude that CD2AP/CIN85 SH3-C domain interaction with ubiquitin constitutes a new ubiquitin-binding mode involved in a different cellular function and thus changes the previously established mechanism of EGF-dependent CD2AP/CIN85 mono-ubiquitination.


Assuntos
Ubiquitina/química , Domínios de Homologia de src , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Modelos Moleculares , Mutação , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Ubiquitina/metabolismo
15.
FEBS J ; 280(15): 3647-57, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23711155

RESUMO

Human Drg1, a guanine nucleotide binding protein conserved in archaea and eukaryotes, is regulated by Lerepo4. Together they form a complex which interacts with translating ribosomes. Here we have purified and characterized the GTPase activity of Drg1 and three variants, a shortened mutant depleted of the TGS domain, a phosphomimicking mutant and a construct with the two combined mutations. Our data reveal that potassium strongly stimulates the GTPase activity, without changing the monomeric status of Drg1 and that this activity is notably reduced in the mutants. The nature of Lerepo4 association has also been investigated. Dissecting the role of the different domains revealed that Dfrp domain is the sole responsible for the Drg1 increase in thermal stability and the four fold stimulation over its catalytic activity. Lerepo4 action leaves Drg1 affinity for nucleotides unaffected, feasibly favoring a switch I reorientation, mainly via the TGS domain. Drg1 displayed a high temperature optimum of activity at 42°C, suggesting the ability of being active under possible heat stress conditions.


Assuntos
Proteínas de Transporte/química , Proteínas de Ligação ao GTP/química , Potássio/química , Estabilidade Enzimática , Guanosina Difosfato/química , Guanosina Trifosfato/química , Humanos , Concentração de Íons de Hidrogênio , Cinética , Fosforilação , Multimerização Proteica , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA
16.
PLoS One ; 8(4): e60816, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23637769

RESUMO

PURPOSE: PP2A is a serine/threonine phosphatase critical to physiological processes, including apoptosis. Cell penetrating peptides are molecules that can translocate into cells without causing membrane damage. Our goal was to develop cell-penetrating fusion peptides specifically designed to disrupt the caspase-9/PP2A interaction and evaluate their therapeutic potential in vitro and in vivo. EXPERIMENTAL DESIGN: We generated a peptide containing a penetrating sequence associated to the interaction motif between human caspase-9 and PP2A (DPT-C9h), in order to target their association. Using tumour cell lines, primary human cells and primary human breast cancer (BC) xenografts, we investigated the capacity of DPT-C9h to provoke apoptosis in vitro and inhibition of tumour growth (TGI) in vivo. DPT-C9h was intraperitoneally administered at doses from 1 to 25 mg/kg/day for 5 weeks. Relative Tumour Volume (RTV) was calculated. RESULTS: We demonstrated that DPT-C9h specifically target caspase-9/PP2A interaction in vitro and in vivo and induced caspase-9-dependent apoptosis in cancer cell lines. DPT-C9h also induced significant TGI in BC xenografts models. The mouse-specific peptide DPT-C9 also induced TGI in lung (K-Ras model) and breast cancer (PyMT) models. DPT-C9h has a specific effect on transformed B cells isolated from chronic lymphocytic leukemia patients without any effect on primary healthy cells. Finally, neither toxicity nor immunogenic responses were observed. CONCLUSION: Using the cell-penetrating peptides blocking caspase-9/PP2A interactions, we have demonstrated that DPT-C9h had a strong therapeutic effect in vitro and in vivo in mouse models of tumour progression.


Assuntos
Antineoplásicos/farmacologia , Caspase 9/metabolismo , Peptídeos Penetradores de Células/farmacologia , Desenho de Drogas , Terapia de Alvo Molecular , Proteína Fosfatase 2/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Sítios de Ligação , Neoplasias da Mama/tratamento farmacológico , Caspase 9/química , Linhagem Celular Tumoral , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/uso terapêutico , Citocromos c/metabolismo , Feminino , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Dados de Sequência Molecular , Ligação Proteica/efeitos dos fármacos , Proteína Fosfatase 2/química , Especificidade da Espécie , Ensaios Antitumorais Modelo de Xenoenxerto
17.
FEBS J ; 280(14): 3399-415, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23663663

RESUMO

The CD2AP (CD2-associated protein) and CIN85 (Cbl-interacting protein of 85 kDa) adaptor proteins each employ three Src homology 3 (SH3) domains to cluster protein partners and ensure efficient signal transduction and down-regulation of tyrosine kinase receptors. Using NMR, isothermal titration calorimetry and small-angle X-ray scattering methods, we have characterized several binding modes of the N-terminal SH3 domain (SH3A) of CD2AP and CIN85 with two natural atypical proline-rich regions in CD2 (cluster of differentiation 2) and Cbl-b (Casitas B-lineage lymphoma), and compared these data with previous studies and published crystal structures. Our experiments show that the CD2AP-SH3A domain forms a type II dimer with CD2 and both type I and type II dimeric complexes with Cbl-b. Like CD2AP, the CIN85-SH3A domain forms a type II complex with CD2, but a trimeric complex with Cbl-b, whereby the type I and II interactions take place at the same time. Together, these results explain how multiple interactions among similar SH3 domains and ligands produce a high degree of diversity in tyrosine kinase, cell adhesion or T-cell signaling pathways.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Antígenos CD2/química , Proteínas do Citoesqueleto/química , Proteínas Proto-Oncogênicas c-cbl/química , Sequência de Aminoácidos , Humanos , Ligações de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Prolina , Ligação Proteica , Estrutura Secundária de Proteína , Espalhamento a Baixo Ângulo , Termodinâmica , Titulometria , Difração de Raios X , Domínios de Homologia de src
18.
J Mol Biol ; 425(12): 2147-63, 2013 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-23500495

RESUMO

The breast cancer metastasis suppressor 1 (BRMS1) gene suppresses metastasis without affecting the primary tumor growth. Cellular localization of BRMS1 appears to be important for exerting its effects on metastasis inhibition. We recently described a nucleo-cytoplasmic shuttling for BRMS1 and identified a nuclear export signal within the N-terminal coiled coil. The structure of these regions shows an antiparallel coiled coil capable of oligomerizing, which compromises the accessibility to the nuclear export signal consensus residues. We have studied the structural and biophysical features of this region to further understand the contribution of the N-terminal coiled coil to the biological function of BRMS1. We have observed that residues 85 to 98 might be important in defining the oligomerization state of the BRMS1 N-terminal coiled coil. The fragments are mainly disordered in solution, with evidence of residual structure. In addition, we report the presence of a conformational dynamic equilibrium (oligomeric folded species ↔ oligomeric unfolded) in solution in the BRMS1 N-terminal coiled coil that might facilitate the nuclear export of BRMS1 to the cytoplasm.


Assuntos
Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Multimerização Proteica , Cristalografia por Raios X , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação Proteica , Proteínas Repressoras
19.
Nucleic Acids Res ; 40(21): 11100-14, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23002146

RESUMO

Developmentally Regulated GTP-binding (DRG) proteins are highly conserved GTPases that associate with DRG Family Regulatory Proteins (DFRP). The resulting complexes have recently been shown to participate in eukaryotic translation. The structure of the Rbg1 GTPase, a yeast DRG protein, in complex with the C-terminal region of its DFRP partner, Tma46, was solved by X-ray diffraction. These data reveal that DRG proteins are multimodular factors with three additional domains, helix-turn-helix (HTH), S5D2L and TGS, packing against the GTPase platform. Surprisingly, the S5D2L domain is inserted in the middle of the GTPase sequence. In contrast, the region of Tma46 interacting with Rbg1 adopts an extended conformation typical of intrinsically unstructured proteins and contacts the GTPase and TGS domains. Functional analyses demonstrate that the various domains of Rbg1, as well as Tma46, modulate the GTPase activity of Rbg1 and contribute to the function of these proteins in vivo. Dissecting the role of the different domains revealed that the Rbg1 TGS domain is essential for the recruitment of this factor in polysomes, supporting further the implication of these conserved factors in translation.


Assuntos
Proteínas Fúngicas/química , Proteínas de Ligação ao GTP/química , Polirribossomos/metabolismo , Sequência de Aminoácidos , Dimerização , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Deleção de Genes , Guanosina Trifosfato/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Eletricidade Estática
20.
J Mol Biol ; 411(5): 1114-27, 2011 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-21777593

RESUMO

We present here the first structural report derived from breast cancer metastasis suppressor 1 (BRMS1), a member of the metastasis suppressor protein group, which, during recent years, have drawn much attention since they suppress metastasis without affecting the growth of the primary tumor. The relevance of the predicted N-terminal coiled coil on the molecular recognition of some of the BRMS1 partners, on its cellular localization and on the role of BRMS1 biological functions such as transcriptional repression prompted us to characterize its three-dimensional structure by X-ray crystallography. The structure of BRMS1 N-terminal region reveals that residues 51-98 form an antiparallel coiled-coil motif and, also, that it has the capability of homo-oligomerizing in a hexameric conformation by forming a trimer of coiled-coil dimers. We have also performed hydrodynamic experiments that strongly supported the prevalence in solution of this quaternary structure for BRMS1(51-98). This work explores the structural features of BRMS1 N-terminal region to help clarify the role of this area in the context of the full-length protein. Our crystallographic and biophysical results suggest that the biological function of BRMS1 may be affected by its ability to promote molecular clustering through its N-terminal coiled-coil region.


Assuntos
Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Sinais de Exportação Nuclear , Nexinas de Classificação/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X , Humanos , Hidrodinâmica , Dados de Sequência Molecular , Proteínas de Neoplasias/genética , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Proteínas Repressoras , Homologia de Sequência de Aminoácidos , Nexinas de Classificação/química , Ultracentrifugação
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