RESUMO
The landscape of current cancer immunotherapy is dominated by antibodies targeting PD-1/PD-L1 and CTLA-4 that have transformed cancer therapy, yet their efficacy is limited by primary and acquired resistance. The blockade of additional immune checkpoints, especially TIGIT and LAG-3, has been extensively explored, but so far only a LAG-3 antibody has been approved for combination with nivolumab to treat unresectable or metastatic melanoma. Here we report the development of a PDL1 × TIGIT bi-specific antibody (bsAb) GB265, a PDL1 × LAG3 bsAb GB266, and a PDL1 × TIGIT × LAG3 tri-specific antibody (tsAb) GB266T, all with intact Fc function. In in vitro cell-based assays, these antibodies promote greater T cell expansion and tumor cell killing than benchmark antibodies and antibody combinations in an Fc-dependent manner, likely by facilitating T cell interactions (bridging) with cancer cells and monocytes, in addition to blocking immune checkpoints. In animal models, GB265 and GB266T antibodies outperformed benchmarks in tumor suppression. This study demonstrates the potential of a new generation of multispecific checkpoint inhibitors to overcome resistance to current monospecific checkpoint antibodies or their combinations for the treatment of human cancers.
Assuntos
Melanoma , Neoplasias , Animais , Humanos , Neoplasias/terapia , Nivolumabe , Receptores Imunológicos , Imunoterapia , Linfócitos TRESUMO
SARS-CoV-2 and its variants have persisted in this ongoing COVID-19 pandemic. While the vaccines have greatly reduced the COVID-19 cases, hospitalizations, and death, about half of the world remain unvaccinated due to various reasons. Furthermore, the duration of the immunity gained from COVID-19 vaccination is still unclear. Therefore, there is a need for innovative prophylactic and treatment measures. In response to this need, we previously reported on the successful computer-aided development of potent VHH-based multispecific antibodies that were characterized in vitro. Here, we evaluated in vivo efficacy and safety of the lead trispecific VHH-Fc, ABS-VIR-001. Importantly, our data showed that ABS-VIR-001 treatment prevented SARS-CoV-2 infection and death when provided as an intranasal prophylaxis in a humanized ACE-2 mouse model. In addition, ABS-VIR-001 post-exposure treatment was shown to greatly reduce viral loads by as much as 50-fold. A detailed panel of metabolic and cellular parameters demonstrated that ABS-VIR-001 treatment was overall comparable to the PBS treatment, indicating a favorable safety profile. Notably, our inhibition studies show that ABS-VIR-001 continued to demonstrate unwavering efficacy against SARS-CoV-2 mutants, associated with key variants including Delta and Omicron, owing to its multiple epitope design. Lastly, we rigorously tested and confirmed the excellent thermostability of ABS-VIR-001 when heated to 45 °C for up to 4 weeks. Taken together, our study suggests that ABS-VIR-001 is an efficacious and durable prophylaxis and post-exposure treatment for COVID-19 with promising safety and manufacturability features for global distribution.
Assuntos
Tratamento Farmacológico da COVID-19 , SARS-CoV-2/fisiologia , Anticorpos de Domínio Único/uso terapêutico , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Reações Antígeno-Anticorpo/efeitos dos fármacos , Biomarcadores/metabolismo , COVID-19/virologia , Estabilidade de Medicamentos , Humanos , Hospedeiro Imunocomprometido , Camundongos , Camundongos Transgênicos , SARS-CoV-2/isolamento & purificação , Anticorpos de Domínio Único/imunologia , Anticorpos de Domínio Único/farmacologia , Glicoproteína da Espícula de Coronavírus/imunologia , Carga ViralRESUMO
Despite mounting evidence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) engagement with immune cells, most express little, if any, of the canonical receptor of SARS-CoV-2, angiotensin-converting enzyme 2 (ACE2). Here, using a myeloid cell receptor-focused ectopic expression screen, we identified several C-type lectins (DC-SIGN, L-SIGN, LSECtin, ASGR1, and CLEC10A) and Tweety family member 2 (TTYH2) as glycan-dependent binding partners of the SARS-CoV-2 spike. Except for TTYH2, these molecules primarily interacted with spike via regions outside of the receptor-binding domain. Single-cell RNA sequencing analysis of pulmonary cells from individuals with coronavirus disease 2019 (COVID-19) indicated predominant expression of these molecules on myeloid cells. Although these receptors do not support active replication of SARS-CoV-2, their engagement with the virus induced robust proinflammatory responses in myeloid cells that correlated with COVID-19 severity. We also generated a bispecific anti-spike nanobody that not only blocked ACE2-mediated infection but also the myeloid receptor-mediated proinflammatory responses. Our findings suggest that SARS-CoV-2-myeloid receptor interactions promote immune hyperactivation, which represents potential targets for COVID-19 therapy.
Assuntos
COVID-19/metabolismo , COVID-19/virologia , Interações Hospedeiro-Patógeno , Lectinas Tipo C/metabolismo , Proteínas de Membrana/metabolismo , Células Mieloides/imunologia , Células Mieloides/metabolismo , Proteínas de Neoplasias/metabolismo , SARS-CoV-2/fisiologia , Enzima de Conversão de Angiotensina 2/metabolismo , Sítios de Ligação , COVID-19/genética , Linhagem Celular , Citocinas , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Mediadores da Inflamação/metabolismo , Lectinas Tipo C/química , Proteínas de Membrana/química , Modelos Moleculares , Proteínas de Neoplasias/química , Ligação Proteica , Conformação Proteica , Anticorpos de Domínio Único/imunologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Relação Estrutura-AtividadeRESUMO
SARS-CoV-2 is a newly emergent coronavirus, which has adversely impacted human health and has led to the COVID-19 pandemic. There is an unmet need to develop therapies against SARS-CoV-2 due to its severity and lack of treatment options. A promising approach to combat COVID-19 is through the neutralization of SARS-CoV-2 by therapeutic antibodies. Previously, we described a strategy to rapidly identify and generate llama nanobodies (VHH) from naïve and synthetic humanized VHH phage libraries that specifically bind the S1 SARS-CoV-2 spike protein, and block the interaction with the human ACE2 receptor. In this study we used computer-aided design to construct multi-specific VHH antibodies fused to human IgG1 Fc domains based on the epitope predictions for leading VHHs. The resulting tri-specific VHH-Fc antibodies show more potent S1 binding, S1/ACE2 blocking, and SARS-CoV-2 pseudovirus neutralization than the bi-specific VHH-Fcs or combination of individual monoclonal VHH-Fcs. Furthermore, protein stability analysis of the VHH-Fcs shows favorable developability features, which enable them to be quickly and successfully developed into therapeutics against COVID-19.
Assuntos
Betacoronavirus/metabolismo , Anticorpos de Domínio Único/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Sequência de Aminoácidos , Enzima de Conversão de Angiotensina 2 , Reações Antígeno-Anticorpo , Betacoronavirus/isolamento & purificação , Sítios de Ligação , COVID-19 , Linhagem Celular , Desenho Assistido por Computador , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Epitopos/química , Epitopos/imunologia , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/metabolismo , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Testes de Neutralização , Pandemias , Biblioteca de Peptídeos , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Estabilidade Proteica , SARS-CoV-2 , Anticorpos de Domínio Único/genética , Anticorpos de Domínio Único/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismoRESUMO
Coronaviruses cause severe human viral diseases including SARS, MERS and COVID-19. Most recently SARS-CoV-2 virus (causing COVID-19) has led to a pandemic with no successful therapeutics. The SARS-CoV-2 infection relies on trimeric spike (S) proteins to facilitate virus entry into host cells by binding to ACE2 receptor on host cell membranes. Therefore, blocking this interaction with antibodies are promising agents against SARS-CoV-2. Here we describe using humanized llama antibody VHHs against SARS-CoV-2 that would overcome the limitations associated with polyclonal and monoclonal combination therapies. From two llama VHH libraries, unique humanized VHHs that bind to S protein and block the S/ACE2 interaction were identified. Furthermore, pairwise combination of VHHs showed synergistic blocking. Multi-specific antibodies with enhanced affinity and avidity, and improved S/ACE2 blocking are currently being developed using an in-silico approach that also fuses VHHs to Fc domains. Importantly, our current bi-specific antibody shows potent S/ACE2 blocking (KD - 0.25 nM, IC100 â¼ 36.7 nM, IC95 â¼ 12.2 nM, IC50 â¼ 1 nM) which is significantly better than individual monoclonal VHH-Fcs. Overall, this design would equip the VHH-Fcs multiple mechanisms of actions against SARS-CoV-2. Thus, we aim to contribute to the battle against COVID-19 by developing therapeutic antibodies as well as diagnostics.
Assuntos
Antagonistas de Receptores de Angiotensina/imunologia , Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Camelídeos Americanos/imunologia , Peptidil Dipeptidase A/imunologia , Enzima de Conversão de Angiotensina 2 , Animais , Anticorpos Biespecíficos/imunologia , Humanos , Peptidil Dipeptidase A/metabolismo , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/imunologiaRESUMO
The advantages of site-specific over stochastic bioconjugation technologies include homogeneity of product, minimal perturbation of protein structure/function, and - increasingly - the ability to perform structure activity relationship studies at the conjugate level. When selecting the optimal location for site-specific payload placement, many researchers turn to in silico modeling of protein structure to identify regions predicted to offer solvent-exposed conjugatable sites while conserving protein function. Here, using the aldehyde tag as our site-specific technology platform and human IgG1 antibody as our target protein, we demonstrate the power of taking an unbiased scanning approach instead. Scanning insertion of the human formylglycine generating enzyme (FGE) recognition sequence, LCTPSR, at each of the 436 positions in the light and heavy chain antibody constant regions followed by co-expression with FGE yielded a library of antibodies bearing an aldehyde functional group ready for conjugation. Each of the variants was expressed, purified, and conjugated to a cytotoxic payload using the Hydrazinyl Iso-Pictet-Spengler ligation to generate an antibody-drug conjugate (ADC), which was analyzed in terms of conjugatability (assessed by drug-to-antibody ratio, DAR) and percent aggregate. We searched for insertion sites that could generate manufacturable ADCs, defined as those variants yielding reasonable antibody titers, DARs of ≥ 1.3, and ≥ 95% monomeric species. Through this process, we discovered 58 tag insertion sites that met these metrics, including 14 sites in the light chain, a location that had proved refractory to the placement of manufacturable tag sites using in silico modeling/rational approaches.
Assuntos
Aldeídos/imunologia , Imunoconjugados/imunologia , Regiões Constantes de Imunoglobulina/imunologia , Imunoglobulina G/imunologia , Aldeídos/química , Sequência de Aminoácidos , Sítios de Ligação , Simulação por Computador , Composição de Medicamentos/métodos , Glicina/análogos & derivados , Glicina/química , Glicina/genética , Glicina/imunologia , Humanos , Imunoconjugados/química , Imunoconjugados/genética , Regiões Constantes de Imunoglobulina/química , Regiões Constantes de Imunoglobulina/genética , Imunoglobulina G/química , Imunoglobulina G/genética , Biblioteca de Peptídeos , Ligação ProteicaRESUMO
Hematologically derived tumors make up â¼10% of all newly diagnosed cancer cases in the United States. Of these, the non-Hodgkin lymphoma (NHL) designation describes a diverse group of cancers that collectively rank among the top 10 most commonly diagnosed cancers worldwide. Although long-term survival trends are improving, there remains a significant unmet clinical need for treatments to help patients with relapsed or refractory disease, one cause of which is drug efflux through upregulation of xenobiotic pumps, such as MDR1. CD22 is a clinically validated target for the treatment of NHL, but no anti-CD22 agents have yet been approved for this indication. Recent approval of an anti-CD22 antibody-drug conjugate (ADC) for the treatment of relapsed/refractory ALL supports the rationale for targeting this protein. An opportunity exists for a next-generation anti-CD22 antibody-drug conjugate (ADC) to address unmet medical needs in the relapsed/refractory NHL population. We describe a site-specifically conjugated antibody-drug conjugate, made using aldehyde tag technology, targeted against CD22 and bearing a noncleavable maytansine payload that is resistant to MDR1-mediated efflux. The construct was efficacious against CD22+ NHL xenografts and could be repeatedly dosed in cynomolgus monkeys at 60 mg/kg with no observed significantly adverse effects. Exposure to total ADC at these doses (as assessed by AUC0-inf) indicated that the exposure needed to achieve efficacy was below tolerable limits. Together, the data suggest that this drug has the potential to be used effectively in patients with CD22+ tumors that have developed MDR1-related resistance to prior therapies. Mol Cancer Ther; 17(1); 161-8. ©2017 AACR.
Assuntos
Imunoconjugados/farmacologia , Maitansina/administração & dosagem , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/imunologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/biossíntese , Animais , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Macaca fascicularis , Masculino , Camundongos , Ratos , Ratos Sprague-DawleyRESUMO
Phage display antibody libraries have a proven track record for the discovery of therapeutic human antibodies, increasing the demand for large and diverse phage antibody libraries for the discovery of new therapeutics. We have constructed naïve antibody phage display libraries in both Fab and scFv formats, with each library having more than 250 billion clones that encompass the human antibody repertoire. These libraries show high fidelity in open reading frame and expression percentages, and their V-gene family distribution, VH-CDR3 length and amino acid usage mirror the natural diversity of human antibodies. Both the Fab and scFv libraries show robust sequence diversity in target-specific binders and differential V-gene usage for each target tested, supporting the use of libraries that utilize multiple display formats and V-gene utilization to maximize antibody-binding diversity. For each of the targets, clones with picomolar affinities were identified from at least one of the libraries and for the two targets assessed for activity, functional antibodies were identified from both libraries.
Assuntos
Técnicas de Visualização da Superfície Celular , Fragmentos Fab das Imunoglobulinas/imunologia , Biblioteca de Peptídeos , Receptor de Insulina/imunologia , Receptor TIE-2/imunologia , Anticorpos de Cadeia Única/imunologia , Sequência de Aminoácidos , Animais , Afinidade de Anticorpos , Especificidade de Anticorpos , Células CHO , Cricetinae , Cricetulus , Humanos , Fragmentos Fab das Imunoglobulinas/biossíntese , Fragmentos Fab das Imunoglobulinas/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fases de Leitura Aberta , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor de Insulina/genética , Receptor TIE-2/genética , Anticorpos de Cadeia Única/biossíntese , Anticorpos de Cadeia Única/genética , TransfecçãoRESUMO
The sequence and conformational effects on the gas-phase acidities of peptides have been studied by using two pairs of isomeric cysteine-polyglycine peptides, CysGly(3,4)NH(2) and Gly(3,4)CysNH(2). The extended Cooks kinetic method was employed to determine the gas-phase acidities using a triple quadrupole mass spectrometer with an electrospray ionization source. The ion activation was achieved via collision-induced dissociation experiments. The deprotonation enthalpies (Delta(acid)H) were determined to be 323.9 +/- 2.5 kcal/mol (CysGly(3)NH(2)), 319.2 +/- 2.3 kcal/mol (CysGly(4)NH(2)), 333.8 +/- 2.1 kcal/mol (Gly(3)CysNH(2)), and 321.9 +/- 2.8 kcal/mol (Gly(4)CysNH(2)), respectively. The corresponding deprotonation entropies (Delta(acid)S) of the peptides were estimated. The gas-phase acidities (Delta(acid)G) were derived to be 318.4 +/- 2.5 kcal/mol (CysGly(3)NH(2)), 314.9 +/- 2.3 kcal/mol (CysGly(4)NH(2)), 327.5 +/- 2.1 kcal/mol (Gly(3)CysNH(2)), and 317.4 +/- 2.8 kcal/mol (Gly(4)CysNH(2)), respectively. Conformations and energetic information of the neutral and anionic peptides were calculated through simulated annealing (Tripos), geometry optimization (AM1), and single point energy calculations (B3LYP/6-31+G(d)), respectively. Both neutral and deprotonated peptides adopt many possible conformations of similar energies. All neutral peptides are mainly random coils. The two C-cysteine anionic peptides, Gly(3,4)(Cys-H)(-)NH(2), are also random coils. The two N-cysteine anionic peptides, (Cys-H)(-)Gly(3,4)NH(2), may exist in both random coils and stretched helices. The two N-cysteine peptides, CysGly(3)NH(2) and CysGly(4)NH(2), are significantly more acidic than the corresponding C-terminal cysteine ones, Gly(3)CysNH(2) and Gly(4)CysNH(2). The stronger acidities of the former may come from the greater stability of the thiolate anion resulting from the interaction with the helix-macrodipole, in addition to the hydrogen bonding interactions.
Assuntos
Cisteína/química , Gases/química , Mapeamento de Peptídeos/métodos , Peptídeos/química , Sequência de Aminoácidos , Isomerismo , Dados de Sequência Molecular , Transição de Fase , Conformação ProteicaRESUMO
Early cellular events associated with tumorigenesis often include loss of cell cycle checkpoints or alteration in growth signaling pathways. Identification of novel genes involved in cellular proliferation may lead to new classes of cancer therapeutics. By screening a tetracycline-inducible cDNA library in A549 cells for genes that interfere with proliferation, we have identified a fragment of UHRF1 (ubiquitin-like protein containing PHD and RING domains 1), a nuclear RING finger protein, that acts as a dominant negative effector of cell growth. Reduction of UHRF1 levels using an UHRF1-specific shRNA decreased growth rates in several tumor cell lines. In addition, treatment of A549 cells with agents that activated different cell cycle checkpoints resulted in down-regulation of UHRF1. The primary sequence of UHRF1 contains a PHD and a RING motif, both of which are structural hallmarks of ubiquitin E3 ligases. We have confirmed using an in vitro autoubiquitination assay that UHRF1 displays RING-dependent E3 ligase activity. Overexpression of a GFP-fused UHRF1 RING mutant that lacks ligase activity sensitizes cells to treatment with various chemotherapeutics. Taken together, our results suggest a general requirement for UHRF1 in tumor cell proliferation and implicate the RING domain of UHRF1 as a functional determinant of growth regulation.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Divisão Celular/fisiologia , Neoplasias/enzimologia , Sítios de Ligação , Proteínas Estimuladoras de Ligação a CCAAT/química , Proteínas Estimuladoras de Ligação a CCAAT/genética , Linhagem Celular Tumoral , Clonagem Molecular , Células HeLa , Humanos , Cinética , Oligonucleotídeos Antissenso , Proteínas Recombinantes/metabolismo , Retroviridae/genética , Transcrição Gênica , Ubiquitina-Proteína LigasesRESUMO
TRAC-1 (T cell RING (really interesting new gene) protein identified in activation screen) is a novel E3 ubiquitin ligase identified from a retroviral vector-based T cell surface activation marker screen. The C-terminal truncated TRAC-1 specifically inhibited anti-TCR-mediated CD69 up-regulation in Jurkat cells, a human T leukemic cell line. In this study, we show that TRAC-1 is a RING finger ubiquitin E3 ligase with highest expression in lymphoid tissues. Point mutations that disrupt the Zn(2+)-chelating ability of its amino-terminal RING finger domain abolished TRAC-1's ligase activity and the dominant inhibitory effect of C-terminal truncated TRAC-1 on TCR stimulation. The results of in vitro biochemical studies indicate that TRAC-1 can stimulate the formation of both K48- and K63-linked polyubiquitin chains and therefore could potentially activate both degradative and regulatory ubiquitin-dependent pathways. Antisense oligonucleotides to TRAC-1 specifically reduced TRAC-1 mRNA levels in Jurkat and primary T cells and inhibited their activation in response to TCR cross-linking. Collectively, these results indicate that the E3 ubiquitin ligase TRAC-1 functions as a positive regulator of T cell activation.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Ativação Linfocitária/imunologia , Proteínas Nucleares/fisiologia , Linfócitos T/enzimologia , Linfócitos T/imunologia , Ubiquitina-Proteína Ligases/fisiologia , Regulação para Cima/imunologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Sequência de Bases , Catálise , Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/isolamento & purificação , Linhagem Celular , Linhagem Celular Tumoral , Proteínas de Ligação a DNA , Humanos , Células Jurkat , Ativação Linfocitária/genética , Tecido Linfoide/citologia , Tecido Linfoide/enzimologia , Tecido Linfoide/imunologia , Dados de Sequência Molecular , Proteínas Nucleares/biossíntese , Proteínas Nucleares/genética , Proteínas Nucleares/isolamento & purificação , Correpressor 2 de Receptor Nuclear , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Poliubiquitina/metabolismo , Receptores de Antígenos de Linfócitos T/fisiologia , Proteínas Repressoras , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Linfócitos T/metabolismo , Fatores de Transcrição/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/biossíntese , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/isolamento & purificação , Regulação para Cima/genéticaRESUMO
Misshapen/NIKs-related kinase (MINK) is a member of the germinal center family of kinases that are homologous to the yeast sterile 20 (Ste20) kinases and regulate a wide variety of cellular processes, including cell morphology, cytoskeletal rearrangement, and survival. Here, we present the cloning and functional characterization of a novel human Misshapen/NIKs-related kinase beta (hMINK beta) that encodes a polypeptide of 1312 amino acids. hMINK beta is ubiquitously expressed in most tissues with at least five alternatively spliced isoforms. Similar to Nck interacting kinase (NIK) and Traf2 and Nck-interacting kinase (TNIK), hMINK beta moderately activates c-Jun N-terminal kinase (JNK) and associates with Nck via the intermediate domain in the yeast two-hybrid system and in a glutathione S-transferase (GST) pull-down assay. Interestingly, overexpression of the kinase domain deleted and kinase-inactive mutants of hMINK beta in human fibrosarcoma HT1080 cells enhanced cell spreading, actin stress fiber formation, and adhesion to extracellular matrix, as well as decreased cell motility and cell invasion. Furthermore, these mutants also promoted cell-cell adhesion in human breast carcinoma MCF7 cells, evidenced with cell growth in clusters and increased membrane localization of beta-catenin, a multifunctional protein involved in E-cadherin-mediated cell adhesion. Finally, hMINK beta protein was found to colocalize with the Golgi apparatus, implicating that hMINK beta might exert its functions, at least in part, through the modulation of intracellular protein transport. Taken together, these results suggest that hMINK beta plays an important role in cytoskeleton reorganization, cell adhesion, and cell motility.
Assuntos
Clonagem Molecular , Expressão Gênica , Proteínas Serina-Treonina Quinases/análise , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Processamento Alternativo , Animais , Sítios de Ligação , Northern Blotting , Neoplasias da Mama , Adesão Celular , Divisão Celular , Membrana Celular/química , Movimento Celular , Proteínas do Citoesqueleto/análise , Citoesqueleto/fisiologia , Citoesqueleto/ultraestrutura , Ativação Enzimática , Matriz Extracelular , Fibrossarcoma , Imunofluorescência , Deleção de Genes , Glutationa Transferase , Complexo de Golgi/química , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , MAP Quinase Quinase 4 , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Dados de Sequência Molecular , Mutagênese , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Especificidade de Órgãos , Mutação Puntual , Proteínas Serina-Treonina Quinases/genética , Homologia de Sequência , Transativadores/análise , Transfecção , Células Tumorais Cultivadas , Técnicas do Sistema de Duplo-Híbrido , beta CateninaRESUMO
Unrepaired DNA double-strand breaks can lead to apoptosis or tumorigenesis. In mammals double-strand breaks are repaired mainly by nonhomologous end-joining mediated by the DNA-PK complex. The core protein of this complex, DNA-PKcs, is a DNA-dependent serine/threonine kinase that phosphorylates protein targets as well as itself. Although the (auto)phosphorylation activity has been shown to be essential for repair of both random double-strand breaks and induced breaks at the immunoglobulin locus, the corresponding phosphatase has been elusive. In fact, to date, none of the putative phosphatases in DNA double-strand break repair has been identified. Here we show that protein phosphatase 5 interacts with DNA-PKcs and dephosphorylates with surprising specificity at least two functional sites. Cells with either hypo- or hyperphosphorylation of DNA-PKcs at these sites show increased radiation sensitivity.
Assuntos
Proteínas de Ligação a DNA , Proteínas Nucleares/fisiologia , Fosfoproteínas Fosfatases/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Animais , Células CHO , Domínio Catalítico , Cricetinae , Reparo do DNA , Proteína Quinase Ativada por DNA , Células HeLa , Humanos , Fosforilação , Tolerância a RadiaçãoAssuntos
Sinais de Localização Nuclear/metabolismo , Biblioteca de Peptídeos , Peptídeos/metabolismo , Alquil e Aril Transferases/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Divisão Celular , Membrana Celular/química , Membrana Celular/metabolismo , Citoplasma/química , Citoplasma/metabolismo , Avaliação Pré-Clínica de Medicamentos , Farnesiltranstransferase , Técnicas Genéticas , Proteínas de Fluorescência Verde , Humanos , Imuno-Histoquímica , Proteínas Luminescentes , Dados de Sequência Molecular , Mutação , Sinais de Localização Nuclear/química , Sinais de Localização Nuclear/genética , Peptídeos/química , Peptídeos/genética , Fenótipo , Polilisina/química , Polilisina/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Células Tumorais CultivadasRESUMO
BACKGROUND: The activation of T cells, mediated by the T-cell receptor (TCR), activates a battery of specific membrane-associated, cytosolic and nuclear proteins. Identifying the signaling proteins downstream of TCR activation will help us to understand the regulation of immune responses and will contribute to developing therapeutic agents that target immune regulation. RESULTS: In an effort to identify novel signaling molecules specific for T-cell activation we undertook a large-scale dominant effector genetic screen using retroviral technology. We cloned and characterized 33 distinct genes from over 2,800 clones obtained in a screen of 7 x 108 Jurkat T cells on the basis of a reduction in TCR-activation-induced CD69 expression after expressing retrovirally derived cDNA libraries. We identified known signaling molecules such as Lck, ZAP70, Syk, PLC gamma 1 and SHP-1 (PTP1C) as truncation mutants with dominant-negative or constitutively active functions. We also discovered molecules not previously known to have functions in this pathway, including a novel protein with a RING domain (found in a class of ubiquitin ligases; we call this protein TRAC-1), transmembrane molecules (EDG1, IL-10R alpha and integrin alpha2), cytoplasmic enzymes and adaptors (PAK2, A-Raf-1, TCPTP, Grb7, SH2-B and GG2-1), and cytoskeletal molecules (moesin and vimentin). Furthermore, using truncated Lck, PLC gamma 1, EDG1 and PAK2 mutants as examples, we showed that these dominant immune-regulatory molecules interfere with IL-2 production in human primary lymphocytes. CONCLUSIONS: This study identified important signal regulators in T-cell activation. It also demonstrated a highly efficient strategy for discovering many components of signal transduction pathways and validating them in physiological settings.
Assuntos
Ativação Linfocitária/fisiologia , Proteínas/fisiologia , Antígenos CD/fisiologia , Antígenos de Diferenciação de Linfócitos T/fisiologia , Linhagem Celular Tumoral , Regulação da Expressão Gênica , Genes/fisiologia , Humanos , Células Jurkat/química , Células Jurkat/metabolismo , Lectinas Tipo C , Receptores de Antígenos de Linfócitos T/fisiologia , Projetos de Pesquisa , Transdução de Sinais/fisiologia , Linfócitos T/fisiologia , Regulação para Cima/fisiologiaRESUMO
Phosphorylation of mitogen-activated protein kinases (MAPKs) on specific tyrosine and threonine sites by MAP kinase kinases (MAPKKs) is thought to be the sole activation mechanism. Here, we report an unexpected activation mechanism for p38alpha MAPK that does not involve the prototypic kinase cascade. Rather it depends on interaction of p38alpha with TAB1 [transforming growth factor-beta-activated protein kinase 1 (TAK1)-binding protein 1] leading to autophosphorylation and activation of p38alpha. We detected formation of a TRAF6-TAB1-p38alpha complex and showed stimulus-specific TAB1-dependent and TAB1-independent p38alpha activation. These findings suggest that alternative activation pathways contribute to the biological responses of p38alpha to various stimuli.