Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 29
Filtrar
1.
Genes Dev ; 32(5-6): 430-447, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29549180

RESUMO

The p53 tumor suppressor protein is the most well studied as a regulator of transcription in the nucleus, where it exists primarily as a tetramer. However, there are other oligomeric states of p53 that are relevant to its regulation and activities. In unstressed cells, p53 is normally held in check by MDM2 that targets p53 for transcriptional repression, proteasomal degradation, and cytoplasmic localization. Here we discovered a hydrophobic region within the MDM2 N-terminal domain that binds exclusively to the dimeric form of the p53 C-terminal domain in vitro. In cell-based assays, MDM2 exhibits superior binding to, hyperdegradation of, and increased nuclear exclusion of dimeric p53 when compared with tetrameric wild-type p53. Correspondingly, impairing the hydrophobicity of the newly identified N-terminal MDM2 region leads to p53 stabilization. Interestingly, we found that dimeric mutant p53 is partially unfolded and is a target for ubiquitin-independent degradation by the 20S proteasome. Finally, forcing certain tumor-derived mutant forms of p53 into dimer configuration results in hyperdegradation of mutant p53 and inhibition of p53-mediated cancer cell migration. Gaining insight into different oligomeric forms of p53 may provide novel approaches to cancer therapy.


Assuntos
Neoplasias/fisiopatologia , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Linhagem Celular Tumoral , Citoplasma/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Mutação , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Domínios Proteicos , Multimerização Proteica/genética , Proteólise , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/genética
2.
Proc Natl Acad Sci U S A ; 118(44)2021 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-34716260

RESUMO

The p53 tumor suppressor protein, known to be critically important in several processes including cell-cycle arrest and apoptosis, is highly regulated by multiple mechanisms, most certifiably the Murine Double Minute 2-Murine Double Minute X (MDM2-MDMX) heterodimer. The role of MDM2-MDMX in cell-cycle regulation through inhibition of p53 has been well established. Here we report that in cells either lacking p53 or expressing certain tumor-derived mutant forms of p53, loss of endogenous MDM2 or MDMX, or inhibition of E3 ligase activity of the heterocomplex, causes cell-cycle arrest. This arrest is correlated with a reduction in E2F1, E2F3, and p73 levels. Remarkably, direct ablation of endogenous p73 produces a similar effect on the cell cycle and the expression of certain E2F family members at both protein and messenger RNA levels. These data suggest that MDM2 and MDMX, working at least in part as a heterocomplex, may play a p53-independent role in maintaining cell-cycle progression by promoting the activity of E2F family members as well as p73, making them a potential target of interest in cancers lacking wild-type p53.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteína Tumoral p73/metabolismo , Animais , Apoptose , Ciclo Celular/fisiologia , Pontos de Checagem do Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Fator de Transcrição E2F1/metabolismo , Humanos , Proteínas Nucleares/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteína Tumoral p73/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
3.
Proc Natl Acad Sci U S A ; 111(14): 5177-82, 2014 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-24706857

RESUMO

Life requires orchestrated control of cell proliferation, cell maintenance, and cell death. Involved in these decisions are protein complexes that assimilate a variety of inputs that report on the status of the cell and lead to an output response. Among the proteins involved in this response are nutrient-deprivation autophagy factor-1 (NAF-1)- and Bcl-2. NAF-1 is a homodimeric member of the novel Fe-S protein NEET family, which binds two 2Fe-2S clusters. NAF-1 is an important partner for Bcl-2 at the endoplasmic reticulum to functionally antagonize Beclin 1-dependent autophagy [Chang NC, Nguyen M, Germain M, Shore GC (2010) EMBO J 29(3):606-618]. We used an integrated approach involving peptide array, deuterium exchange mass spectrometry (DXMS), and functional studies aided by the power of sufficient constraints from direct coupling analysis (DCA) to determine the dominant docked conformation of the NAF-1-Bcl-2 complex. NAF-1 binds to both the pro- and antiapoptotic regions (BH3 and BH4) of Bcl-2, as demonstrated by a nested protein fragment analysis in a peptide array and DXMS analysis. A combination of the solution studies together with a new application of DCA to the eukaryotic proteins NAF-1 and Bcl-2 provided sufficient constraints at amino acid resolution to predict the interaction surfaces and orientation of the protein-protein interactions involved in the docked structure. The specific integrated approach described in this paper provides the first structural information, to our knowledge, for future targeting of the NAF-1-Bcl-2 complex in the regulation of apoptosis/autophagy in cancer biology.


Assuntos
Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ribonucleoproteínas/metabolismo , Sequência de Aminoácidos , Humanos , Espectrometria de Massas , Modelos Moleculares , Dados de Sequência Molecular , Oligopeptídeos/química , Ligação Proteica
4.
Proc Natl Acad Sci U S A ; 109(50): 20467-72, 2012 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-23169665

RESUMO

Impairment of ribosomal biogenesis can activate the p53 protein independently of DNA damage. The ability of ribosomal proteins L5, L11, L23, L26, or S7 to bind Mdm2 and inhibit its ubiquitin ligase activity has been suggested as a critical step in p53 activation under these conditions. Here, we report that L5 and L11 are particularly important for this response. Whereas several other newly synthesized ribosomal proteins are degraded by proteasomes upon inhibition of Pol I activity by actinomycin D, L5 and L11 accumulate in the ribosome-free fraction where they bind to Mdm2. This selective accumulation of free L5 and L11 is due to their mutual protection from proteasomal degradation. Furthermore, the endogenous, newly synthesized L5 and L11 continue to be imported into nucleoli even after nucleolar disruption and colocalize with Mdm2, p53, and promyelocytic leukemia protein. This suggests that the disrupted nucleoli may provide a platform for L5- and L11-dependent p53 activation, implying a role for the nucleolus in p53 activation by ribosomal biogenesis stress. These findings may have important implications with respect to understanding the pathogenesis of diseases caused by impaired ribosome biogenesis.


Assuntos
Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Sequência de Bases , Linhagem Celular Tumoral , Nucléolo Celular/metabolismo , Dactinomicina/farmacologia , Humanos , Camundongos , Modelos Biológicos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteína da Leucemia Promielocítica , Complexo de Endopeptidases do Proteassoma/metabolismo , Estabilidade Proteica , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Proteínas Ribossômicas/antagonistas & inibidores , Proteínas Ribossômicas/genética , Estresse Fisiológico , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Regulação para Cima
5.
J Microbiol Methods ; 222: 106959, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38782300

RESUMO

Salmonella enterica serovar Infantis (S. infantis) is an important emerging pathogen, associated with poultry and poultry products and related to an increasing number of human infections in many countries. A concerning trend among S. infantis isolates is the presence of plasmid-mediated multidrug resistance. In many instances, the genes responsible for this resistance are carried on a megaplasmid known as the plasmid of emerging S. infantis (pESI) or pESI like plasmids. Plasmids can be remarkably stable due to the presence of multiple replicons and post-segregational killing systems (PSKs), which contribute to their maintenance within bacterial populations. To enhance our understanding of S. infantis and its multidrug resistance determinants toward the development of new vaccination strategies, we have devised a new method for targeted plasmid curing. This approach effectively overcomes plasmid addiction by leveraging the temporal overproduction of specific antitoxins coupled with the deletion of the partition region. By employing this strategy, we successfully generated a plasmid-free strain from a field isolate derived from S. infantis 119,944. This method provides valuable tools for studying S. infantis and its plasmid-borne multidrug resistance mechanisms and can be easily adopted for plasmid curing from other related bacteria.


Assuntos
Farmacorresistência Bacteriana Múltipla , Plasmídeos , Aves Domésticas , Salmonella enterica , Plasmídeos/genética , Animais , Salmonella enterica/genética , Salmonella enterica/isolamento & purificação , Aves Domésticas/microbiologia , Farmacorresistência Bacteriana Múltipla/genética , Sorogrupo , Salmonelose Animal/microbiologia , Doenças das Aves Domésticas/microbiologia
6.
Mol Cell Biol ; 44(1): 27-42, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38270135

RESUMO

The p63 transcription factor, a member of the p53 family, plays an oncogenic role in squamous cell carcinomas, while in breast cancers its expression is often repressed. In the canonical conserved Hippo pathway, known to play a complex role in regulating growth of cancer cells, protein kinases MST1/2 and LATS1/2 act sequentially to phosphorylate and inhibit the YAP/TAZ transcription factors. We found that in MCF10A mammary epithelial cells as well as in squamous and breast cancer cell lines, expression of ΔNp63 RNA and protein is strongly repressed by inhibition of the Hippo pathway protein kinases. While MST1/2 and LATS1 are required for p63 expression, the next step of the pathway, namely phosphorylation and degradation of the YAP/TAZ transcriptional activators is not required for p63 repression. This suggests that regulation of p63 expression occurs by a noncanonical version of the Hippo pathway. We identified similarly regulated genes, suggesting the broader importance of this pathway. Interestingly, lowering p63 expression lead to increased YAP protein levels, indicating crosstalk of the YAP/TAZ-independent and -dependent branches of the Hippo pathway. These results, which reveal the intersection of the Hippo and p63 pathways, may prove useful for the control of their activities in cancer cells.


Assuntos
Via de Sinalização Hippo , Transdução de Sinais , Humanos , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Sinalização YAP , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo
8.
J Biol Chem ; 287(18): 15016-23, 2012 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-22416135

RESUMO

The molecular basis of the interaction between mitochondrial carrier homologue 2 (MTCH2) and truncated BID (tBID) was characterized. These proteins participate in the apoptotic pathway, and the interaction between them may serve as a target for anticancer lead compounds. In response to apoptotic signals, MTCH2 recruits tBID to the mitochondria, where it activates apoptosis. A combination of peptide arrays screening with biochemical and biophysical techniques was used to characterize the mechanism of the interaction between tBID and MTCH2 at the structural and molecular levels. The regions that mediate the interaction between the proteins were identified. The two specific binding sites between the proteins were determined to be tBID residues 59-73 that bind MTCH2 residues 140-161, and tBID residues 111-125 that bind MTCH2 residues 240-290. Peptides derived from tBID residues 111-125 and 59-73 induced cell death in osteosarcoma cells. These peptides may serve as lead compounds for anticancer drugs that act by targeting the tBID-MTCH2 interaction.


Assuntos
Apoptose , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Antineoplásicos/farmacologia , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/antagonistas & inibidores , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/genética , Linhagem Celular Tumoral , Humanos , Mitocôndrias/genética , Proteínas de Transporte da Membrana Mitocondrial/genética , Osteossarcoma/tratamento farmacológico , Osteossarcoma/genética , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Peptídeos/metabolismo , Peptídeos/farmacologia , Análise Serial de Proteínas
9.
bioRxiv ; 2023 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-36824867

RESUMO

The p63 transcription factor, a member of the p53 family, plays an oncogenic role in squamous cancers, while in breast cancers its expression is often repressed. In the canonical conserved Hippo pathway, known to play a complex role in regulating growth of cancer cells, the protein kinases MST1/2 and LATS1/2 act sequentially to phosphorylate and inhibit the YAP/TAZ transcription factors. We found that in the MCF10A mammary epithelial cell line as well as in squamous and breast cancer cell lines, expression of ΔNp63 RNA and protein is strongly repressed by inhibition of the Hippo pathway protein kinases in a manner that is independent of p53. While MST1/2 and LATS1 are required for p63 expression, the next step of the pathway, namely phosphorylation and degradation of the YAP/TAZ transcriptional activators is not required for repression of p63. This suggests that regulation of p63 expression occurs by a non-canonical version of the Hippo pathway. We additionally identified additional genes that were similarly regulated suggesting the broader importance of this pathway. Interestingly, we observed that experimentally lowering p63 expression leads to increased YAP protein levels, thereby constituting a feedback loop. These results, which reveal the intersection of the Hippo and p63 pathways, may prove useful for the control of their activities in cancer cells. One Sentence Summary: Regulation of p63 expression occurs by a non-canonical version of the Hippo pathway in mammary epithelial, breast carcinoma and head and neck squamous carcinoma cells.

10.
Vaccine ; 41(33): 4918-4925, 2023 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-37400285

RESUMO

The most common source of foodborne Salmonella infection in humans is poultry eggs and meat, such that prevention of human infection is mostly achieved by vaccination of farm animals. While inactivated and attenuated vaccines are available, both present drawbacks. This study aimed to develop a novel vaccination strategy, which combines the effectiveness of live-attenuated and safety of inactivated vaccines by construction of inducible self-destructing bacteria utilizing toxin-antitoxin (TA) systems. Hok-Sok and CeaB-CeiB toxin-antitoxin systems were coupled with three induction systems aimed for activating cell killing upon lack of arabinose, anaerobic conditions or low concentration of metallic di-cations. The constructs were transformed into a pathogenic Salmonella enterica serovar Enteritidis strain and bacteria elimination was evaluated in vitro under specific activating conditions and in vivo following administration to chickens. Four constructs induced bacterial killing under the specified conditions, both in growth media and within macrophages. Cloacal swabs of all chicks orally administered transformed bacteria had no detectable levels of bacteria within 9 days of inoculation. By day ten, no bacteria were identified in the spleen and liver of most birds. Antibody immune response was raised toward TA carrying Salmonella which resembled response toward the wildtype bacteria. The constructs described in this study led to self-destruction of virulent Salmonella enteritidis both in vitro and in inoculated animals within a period which is sufficient for the induction of a protective immune response. This system may serve as a safe and effective live vaccine platform against Salmonella as well as other pathogenic bacteria.


Assuntos
Antitoxinas , Doenças das Aves Domésticas , Salmonelose Animal , Vacinas contra Salmonella , Toxinas Biológicas , Animais , Humanos , Galinhas , Salmonella enteritidis , Vacinação/veterinária , Vacinas Atenuadas
11.
Front Immunol ; 14: 1311658, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-38152397

RESUMO

Background: Immune checkpoint therapies have led to significant breakthroughs in cancer patient treatment in recent years. However, their efficiency is variable, and resistance to immunotherapies is common. VISTA is an immune-suppressive checkpoint inhibitor of T cell response belonging to the B7 family and a promising novel therapeutic target. VISTA is expressed in the immuno-suppressive tumor microenvironment, primarily by myeloid lineage cells, and its genetic knockout or antibody blockade restores an efficient antitumor immune response. Methods: Fully human monoclonal antibodies directed against VISTA were produced after immunizing humanized Trianni mice and single B cell sequencing. Anti-VISTA antibodies were evaluated for specificity, cross-reactivity, monocyte and T cell activation, Fc-effector functions, and antitumor efficacy using in vitro and in vivo models to select the KVA12123 antibody lead candidate. The pharmacokinetics and safety profiles of KVA12123 were evaluated in cynomolgus monkeys. Results: Here, we report the development of a clinical candidate anti-VISTA monoclonal antibody, KVA12123. KVA12123 showed high affinity binding to VISTA through a unique epitope distinct from other clinical-stage anti-VISTA monoclonal antibodies. This clinical candidate demonstrated high specificity against VISTA with no cross-reactivity detected against other members of the B7 family. KVA12123 blocked VISTA binding to its binding partners. KVA12123 induced T cell activation and demonstrated NK-mediated monocyte activation. KVA12123 treatment mediated strong single-agent antitumor activity in several syngeneic tumor models and showed enhanced efficacy in combination with anti-PD-1 treatment. This clinical candidate was engineered to improve its pharmacokinetic characteristics and reduce Fc-effector functions. It was well-tolerated in preclinical toxicology studies in cynomolgus monkeys, where hematology, clinical chemistry evaluations, and clinical observations revealed no indicators of toxicity. No cytokines associated with cytokine release syndrome were elevated. Conclusion: These results establish that KVA12123 is a promising drug candidate with a distinct but complementary mechanism of action of the first generation of immune checkpoint inhibitors. This antibody is currently evaluated alone and in combination with pembrolizumab in a Phase 1/2 open-label clinical trial in patients with advanced solid tumors.


Assuntos
Anticorpos Monoclonais , Inibidores de Checkpoint Imunológico , Neoplasias , Animais , Humanos , Camundongos , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Macaca fascicularis , Neoplasias/terapia , Linfócitos T , Microambiente Tumoral
12.
Cancer Discov ; 13(5): 1250-1273, 2023 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-37067901

RESUMO

Cancer-relevant mutations in the oligomerization domain (OD) of the p53 tumor suppressor protein, unlike those in the DNA binding domain, have not been well elucidated. Here, we characterized the germline OD mutant p53(A347D), which occurs in cancer-prone Li-Fraumeni syndrome (LFS) patients. Unlike wild-type p53, mutant p53(A347D) cannot form tetramers and exists as a hyperstable dimeric protein. Further, p53(A347D) cannot bind or transactivate the majority of canonical p53 target genes. Isogenic cell lines harboring either p53(A347D) or no p53 yield comparable tumorigenic properties, yet p53(A347D) displays remarkable neomorphic activities. Cells bearing p53(A347D) possess a distinct transcriptional profile and undergo metabolic reprogramming. Further, p53(A347D) induces striking mitochondrial network aberration and associates with mitochondria to drive apoptotic cell death upon topoisomerase II inhibition in the absence of transcription. Thus, dimer-forming p53 demonstrates both loss-of-function (LOF) and gain-of-function (GOF) properties compared with the wild-type form of the protein. SIGNIFICANCE: A mutant p53 (A347D), which can only form dimers, is associated with increased cancer susceptibility in LFS individuals. We found that this mutant wields a double-edged sword, driving tumorigenesis through LOF while gaining enhanced apoptogenic activity as a new GOF, thereby yielding a potential vulnerability to select therapeutic approaches. See related commentary by Stieg et al., p. 1046. See related article by Gencel-Augusto et al., p. 1230. This article is highlighted in the In This Issue feature, p. 1027.


Assuntos
Síndrome de Li-Fraumeni , Humanos , Síndrome de Li-Fraumeni/genética , Síndrome de Li-Fraumeni/metabolismo , Síndrome de Li-Fraumeni/patologia , Ativação Transcricional , Proteína Supressora de Tumor p53/metabolismo , Apoptose/genética , Mitocôndrias/metabolismo
13.
Chem Soc Rev ; 40(5): 2131-45, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21243154

RESUMO

Screening of arrays and libraries of compounds is well-established as a high-throughput method for detecting and analyzing interactions in both biological and chemical systems. Arrays and libraries can be composed from various types of molecules, ranging from small organic compounds to DNA, proteins and peptides. The applications of libraries for detecting and characterizing biological interactions are wide and diverse, including for example epitope mapping, carbohydrate arrays, enzyme binding and protein-protein interactions. Here, we will focus on the use of peptide arrays to study protein-protein interactions. Characterization of protein-protein interactions is crucial for understanding cell functionality. Using peptides, it is possible to map the precise binding sites in such complexes. Peptide array libraries usually contain partly overlapping peptides derived from the sequence of one protein from the complex of interest. The peptides are attached to a solid support using various techniques such as SPOT-synthesis and photolithography. Then, the array is incubated with the partner protein from the complex of interest. Finally, the detection of the protein-bound peptides is carried out by using immunodetection assays. Peptide array screening is semi-quantitative, and quantitative studies with selected peptides in solution are required to validate and complement the screening results. These studies can improve our fundamental understanding of cellular processes by characterizing amino acid patterns of protein-protein interactions, which may even develop into prediction algorithms. The binding peptides can then serve as a basis for the design of drugs that inhibit or activate the target protein-protein interactions. In the current review, we will introduce the recent work on this subject performed in our and in other laboratories. We will discuss the applications, advantages and disadvantages of using peptide arrays as a tool to study protein-protein interactions.


Assuntos
Peptídeos/química , Análise Serial de Proteínas/métodos , Mapeamento de Interação de Proteínas/métodos , Sítios de Ligação , Simulação por Computador , Biblioteca de Peptídeos , Ligação Proteica , Estrutura Terciária de Proteína
14.
Vaccine ; 40(5): 726-733, 2022 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-34998606

RESUMO

The devastating impact of infectious bronchitis (IB) triggered by the IB virus (IBV), on poultry farms is generally curbed by livestock vaccination with live attenuated or inactivated vaccines. Yet, this approach is challenged by continuously emerging variants and by time limitations of vaccine preparation techniques. This work describes the design and evaluation of an anti-IBV vaccine comprised of E. coli expressing and secreting viral spike 1 subunit (S1) and nucleocapsid N-terminus and C-terminus polypeptides fused to heat-labile enterotoxin B (LTB) (LS1, LNN, LNC, respectively). Following chicken oral vaccination, anti-IBV IgY levels and cellular-mediated immunity as well as protection against virulent IBV challenge, were evaluated 14 days following the booster dose. Oral vaccination induced IgY levels that exceeded those measured following vaccination with each component separately. Following exposure to inactivated IBV, splenocytes isolated from chicks orally vaccinated with LNN or LNC -expressing bacteria, showed a higher percentage of CD8+ cells as compared to splenocytes isolated from chicks vaccinated with wild type or LTB-secreting E. coli and to chicks subcutaneously vaccinated. Significant reduction in viral load and percent of shedders in the vaccinated chicks was evident starting 3 days following challenge with 107.5 EID50/ml virulent IBV. Taken together, orally delivered LTB-fused IBV polypeptide-expressing bacteria induced virus-specific IgY antibody production and was associated with significantly shorter viral shedding on challenge with a live IBV. The proposed vaccine design and delivery route promise an effective and rapidly adaptable means of protecting poultry farms from devastating IB outbreaks.


Assuntos
Infecções por Coronavirus , Gammacoronavirus , Vírus da Bronquite Infecciosa , Doenças das Aves Domésticas , Vacinas Virais , Animais , Anticorpos Antivirais , Galinhas , Infecções por Coronavirus/prevenção & controle , Infecções por Coronavirus/veterinária , Escherichia coli , Doenças das Aves Domésticas/prevenção & controle , Vacinação , Vacinas Atenuadas , Proteínas Virais
15.
Vaccine ; 40(8): 1098-1107, 2022 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-35078662

RESUMO

The rapid spread of the COVID-19 pandemic, with its devastating medical and economic impacts, triggered an unprecedented race toward development of effective vaccines. The commercialized vaccines are parenterally administered, which poses logistic challenges, while adequate protection at the mucosal sites of virus entry is questionable. Furthermore, essentially all vaccine candidates target the viral spike (S) protein, a surface protein that undergoes significant antigenic drift. This work aimed to develop an oral multi-antigen SARS-CoV-2 vaccine comprised of the receptor binding domain (RBD) of the viral S protein, two domains of the viral nucleocapsid protein (N), and heat-labile enterotoxin B (LTB), a potent mucosal adjuvant. The humoral, mucosal and cell-mediated immune responses of both a three-dose vaccination schedule and a heterologous subcutaneous prime and oral booster regimen were assessed in mice and rats, respectively. Mice receiving the oral vaccine compared to control mice showed significantly enhanced post-dose-3 virus-neutralizing antibody, anti-S IgG and IgA production and N-protein-stimulated IFN-γ and IL-2 secretion by T cells. When administered as a booster to rats following parenteral priming with the viral S1 protein, the oral vaccine elicited markedly higher neutralizing antibody titres than did oral placebo booster. A single oral booster following two subcutaneous priming doses elicited serum IgG and mucosal IgA levels similar to those raised by three subcutaneous doses. In conclusion, the oral LTB-adjuvanted multi-epitope SARS-CoV-2 vaccine triggered versatile humoral, cellular and mucosal immune responses, which are likely to provide protection, while also minimizing technical hurdles presently limiting global vaccination, whether by priming or booster programs.


Assuntos
Anticorpos Neutralizantes , COVID-19 , Animais , Anticorpos Antivirais , Vacinas contra COVID-19 , Humanos , Imunidade Celular , Imunoglobulina A , Imunoglobulina G , Camundongos , Pandemias , Ratos , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Vacinação
16.
Proc Natl Acad Sci U S A ; 105(34): 12277-82, 2008 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-18719108

RESUMO

We have characterized the molecular basis of the interaction between ASPP2 and Bcl-2, which are key proteins in the apoptotic pathway. The C-terminal ankyrin repeats and SH3 domain of ASPP2 (ASPP2(Ank-SH3)) mediate its interactions with the antiapoptotic protein Bcl-2. We used biophysical and computational methods to identify the interaction sites of Bcl-2 and its homologues with ASPP2. Using peptide array screening, we found that ASPP2(Ank-SH3) binds two homologous sites in all three Bcl proteins tested: (i) the conserved BH4 motif, and (ii) a binding site for proapoptotic regulators. Quantitative binding studies revealed that binding of ASPP2(Ank-SH3) to the Bcl-2 family members is selective at two levels: (i) interaction with Bcl-2-derived peptides is the tightest compared to peptides from the other family members, and (ii) within Bcl-2, binding of ASPP2(Ank-SH3) to the BH4 domain is tightest. Sequence alignment of the ASPP2-binding peptides combined with binding studies of mutated peptides revealed that two nonconserved positions where only Bcl-2 contains positively charged residues account for its tighter binding. The experimental binding results served as a basis for docking analysis, by which we modeled the complexes of ASPP2(Ank-SH3) with the full-length Bcl proteins. Using peptide arrays and quantitative binding studies, we found that Bcl-2 binds three loops in ASPP2(Ank-SH3) with similar affinity, in agreement with our predicted model. Based on our results, we propose a mechanism in which ASPP2 induces apoptosis by inhibiting functional sites of the antiapoptotic Bcl-2 proteins.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Transporte/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Sítios de Ligação , Simulação por Computador , Humanos , Modelos Moleculares , Mutação , Ligação Proteica
17.
Mol Cancer Res ; 19(9): 1522-1533, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34045312

RESUMO

p53 mutations that result in loss of transcriptional activity are commonly found in numerous types of cancer. While the majority of these are missense mutations that map within the central DNA-binding domain, truncations and/or frameshift mutations can also occur due to various nucleotide substitutions, insertions, or deletions. These changes result in mRNAs containing premature stop codons that are translated into a diverse group of C-terminally truncated proteins. Here we characterized three p53 frameshift mutant proteins expressed from the endogenous TP53 locus in U2OS osteosarcoma and HCT116 colorectal cancer cell lines. These mutants retain intact DNA-binding domains but display altered oligomerization properties. Despite their abnormally high expression levels, they are mostly transcriptionally inactive and unable to initiate a stimuli-induced transcriptional program characteristic of wild-type p53. However, one of these variant p53 proteins, I332fs*14, which resembles naturally expressed TAp53 isoforms ß and γ, retains some residual antiproliferative activity and can induce cellular senescence in HCT116 cells. Cells expressing this mutant also display decreased motility in migration assays. Hence, this p53 variant exhibits a combination of loss-of-gain and gain-of-function characteristics, distinguishing it from both wild type p53 and p53 loss. IMPLICATIONS: p53 frameshift mutants display a mixture of residual antiproliferative and neomorphic functions that may be differentially exploited for targeted therapy.


Assuntos
Biomarcadores Tumorais/genética , Neoplasias Colorretais/patologia , Mutação da Fase de Leitura , Regulação Neoplásica da Expressão Gênica , Mutação com Perda de Função , Osteossarcoma/patologia , Proteína Supressora de Tumor p53/genética , Apoptose , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Ciclo Celular , Movimento Celular , Proliferação de Células , Neoplasias Colorretais/genética , Humanos , Osteossarcoma/genética , Células Tumorais Cultivadas
18.
Proteins ; 77(3): 602-11, 2009 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-19507243

RESUMO

We used computational methods to study the interaction between two key proteins in apoptosis regulation: the transcription factor NF-kappa-B (NFkappaB) and the proapoptotic protein ASPP2. The C-terminus of ASPP2 contains ankyrin repeats and SH3 domains (ASPP2(ANK-SH3)) that mediate interactions with numerous apoptosis-related proteins, including the p65 subunit of NFkappaB (NFkappaB(p65)). Using peptide-based methods, we have recently identified the interaction sites between NFkappaB(p65) and ASPP2(ANK-SH3) (Rotem et al., J Biol Chem 283, 18990-18999). Here we conducted a computational study of protein docking and molecular dynamics to obtain a structural model of the complex between the full length proteins and propose a mechanism for the interaction. We found that ASPP2(ANK-SH3) binds two sites in NFkappaB(p65), at residues 236-253 and 293-313 that contain the nuclear localization signal (NLS). These sites also mediate the binding of NFkappaB to its natural inhibitor IkappaB, which also contains ankyrin repeats. Alignment of the ankyrin repeats of ASPP2(ANK-SH3) and IkappaB revealed that both proteins share highly similar interfaces at their binding sites to NFkappaB. Protein docking of ASPP2(ANK-SH3) and NFkappaB(p65), as well as molecular dynamics simulations of the proteins, provided structural models of the complex that are energetically similar to the NFkappaB-IkappaB determined structure. Our results show that ASPP2(ANK-SH3) binds NFkappaB(p65) in a similar manner to its natural inhibitor IkappaB, suggesting a possible novel role for ASPP2 as an NFkappaB inhibitor.


Assuntos
Proteínas Reguladoras de Apoptose/química , Quinase I-kappa B/química , NF-kappa B/química , Algoritmos , Apoptose , Sítios de Ligação , Biologia Computacional/métodos , Simulação por Computador , Citoplasma/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , NF-kappa B/metabolismo , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Eletricidade Estática , Proteína Supressora de Tumor p53/química
19.
J Bacteriol ; 190(21): 7117-22, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18776015

RESUMO

In Escherichia coli, FtsH (HflB) is a membrane-bound, ATP-dependent metalloendoprotease belonging to the AAA family (ATPases associated with diverse cellular activities). FtsH has a limited spectrum of known substrates, including the transcriptional activator sigma32. FtsH is the only known E. coli protease that is essential, as it regulates the concentration of LpxC, which carries out the first committed step in the synthesis of lipid A. Here we identify a new FtsH substrate--3-deoxy-D-manno-octulosonate (KDO) transferase--which carries out the attachment of two KDO residues to the lipid A precursor (lipid IVA) to form the minimal essential structure of the lipopolysaccharide (LPS) (KDO2-lipid A). Thus, FtsH regulates the concentration of the lipid moiety of LPS (lipid A) as well as the sugar moiety (KDO-based core oligosaccharides), ensuring a balanced synthesis of LPS.


Assuntos
Proteases Dependentes de ATP/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Lipopolissacarídeos/biossíntese , Proteases Dependentes de ATP/genética , Proteases Dependentes de ATP/fisiologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/fisiologia , Lipídeo A/genética , Lipídeo A/metabolismo , Lipopolissacarídeos/química , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Açúcares Ácidos/química , Açúcares Ácidos/metabolismo , Transferases/genética , Transferases/metabolismo
20.
Biotechnol Adv ; 36(3): 818-838, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29305895

RESUMO

The negative impact of the massive use of synthetic pesticides on the environment and on human health has stimulated the search for environment-friendly practices for controlling plant diseases and pests. Among them, biocontrol, which relies on using beneficial organisms or their products (bioactive molecules and/or hydrolytic enzymes), holds the greatest promise and is considered a pillar of integrated pest management. Chitinases are particularly attractive to this purpose since they have fungicidal, insecticidal, and nematicidal activities. Here, current knowledge on the biopesticidal action of microbial and viral chitinases is reviewed, together with a critical analysis of their future development as biopesticides.


Assuntos
Quitinases/farmacologia , Controle Biológico de Vetores/métodos , Praguicidas/farmacologia , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Agentes de Controle Biológico/farmacologia , Quitinases/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fungicidas Industriais/farmacologia , Inseticidas/farmacologia , Metagenômica/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Vertebrados , Proteínas Virais/genética , Proteínas Virais/metabolismo , Proteínas Virais/farmacologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA