RESUMO
Ubiquitination is a posttranslational modification in eukaryotes that plays a significant role in the infection of intracellular microbial pathogens, such as Legionella pneumophila. While the Legionella-containing vacuole (LCV) is coated with ubiquitin (Ub), it avoids recognition by autophagy adaptors. Here, we report that the Sdc and Sde families of effectors work together to build ubiquitinated species around the LCV. The Sdc effectors catalyze canonical polyubiquitination directly on host targets or on phosphoribosyl-Ub conjugated to host targets by Sde. Remarkably, Ub moieties within poly-Ub chains are either modified with a phosphoribosyl group by PDE domain-containing effectors or covalently attached to other host substrates via Sde-mediated phosphoribosyl-ubiquitination. Furthermore, these modifications prevent the recognition by Ub adaptors and therefore exclude host autophagy adaptors from the LCV. In this work, we shed light on the nature of the poly-ubiquitinated species present at the surface of the LCV and provide a molecular mechanism for the avoidance of autophagy adaptors by the Ub-decorated LCV.
Assuntos
Autofagia , Proteínas de Bactérias , Legionella pneumophila , Poliubiquitina , Ubiquitinação , Vacúolos , Legionella pneumophila/metabolismo , Legionella pneumophila/genética , Humanos , Vacúolos/metabolismo , Vacúolos/microbiologia , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Poliubiquitina/metabolismo , Interações Hospedeiro-Patógeno , Células HEK293 , Ubiquitina/metabolismoRESUMO
DNA-PKcs is a DNA damage sensor kinase with established roles in DNA double-strand break repair via nonhomologous end joining. Recent studies have revealed additional roles of DNA-PKcs in the regulation of transcription, translation, and DNA replication. However, the substrates through which DNA-PKcs regulates these processes remain largely undefined. Here, we utilized quantitative phosphoproteomics to generate a high coverage map of DNA-PKcs signaling in response to ionizing radiation and mapped its interplay with the ATM kinase. Beyond the detection of the canonical S/T-Q phosphorylation motif, we uncovered a noncanonical mode of DNA-PKcs signaling targeting S/T-ψ-D/E motifs. Sequence and structural analyses of the DNA-PKcs substrate recognition pocket revealed unique features compared to closely related phosphatidylinositol 3-kinase-related kinases that may explain its broader substrate preference. These findings expand the repertoire of DNA-PKcs and ATM substrates while establishing a novel preferential phosphorylation motif for DNA-PKcs.
RESUMO
DNA-PKcs is a DNA damage sensor kinase with established roles in DNA double-strand break repair via non-homologous end joining. Recent studies have revealed additional roles of DNA-PKcs in the regulation of transcription, translation and DNA replication. However, the substrates through which DNA-PKcs regulates these processes remain largely undefined. Here we utilized quantitative phosphoproteomics to generate a high coverage map of DNA-PKcs signaling in response to ionizing radiation and mapped its interplay with the ATM kinase. Beyond the detection of the canonical S/T-Q phosphorylation motif, we uncovered a non-canonical mode of DNA-PKcs signaling targeting S/T-ψ-D/E motifs. Cross-species analysis in mouse pre-B and human HCT116 cell lines revealed splicing factors and transcriptional regulators phosphorylated at this novel motif, several of which contain SAP domains. These findings expand the list of DNA-PKcs and ATM substrates and establish a novel preferential phosphorylation motif for DNA-PKcs that connects it to proteins involved in nucleotide processes and interactions.
RESUMO
Ubiquitination is a crucial posttranslational modification in eukaryotes that plays a significant role in the infection of intracellular microbial pathogens, such as Legionella pneumophila, the bacterium responsible for Legionnaires' disease. While the Legionella-containing vacuole (LCV) is coated with ubiquitin (Ub), it avoids recognition by autophagy adaptors. In this study, we report that the Sdc and Sde families of effectors work together to build ubiquitinated species around the LCV. The Sdc effectors catalyze canonical polyubiquitination directly on host targets or on the phosphoribosyl-Ub (PR-Ub) conjugated to host targets by Sde. Remarkably, the Ub moieties within the poly-Ub chains are either modified with a phosphoribosyl group by Sde and other PDE domain-containing effectors or covalently attached to other host substrates via Sde-mediated PR-ubiquitination. Furthermore, these modifications prevent the recognition by Ub adaptors, such as p62, and therefore exclude host autophagy adaptors from the LCV. Our findings shed light on the nature of the poly-ubiquitinated species present at the surface of the LCV and provide a molecular mechanism for the avoidance of autophagy adaptors by the Ub-decorated LCV.
RESUMO
The DNA-PKcs kinase mediates the repair of DNA double-strand breaks via classical non-homologous end joining (NHEJ). DNA-PKcs is also recruited to active replication forks, although a role for DNA-PKcs in the control of fork dynamics is unclear. Here, we identify a crucial role for DNA-PKcs in promoting fork reversal, a process that stabilizes stressed replication forks and protects genome integrity. DNA-PKcs promotes fork reversal and slowing in response to several replication stress-inducing agents in a manner independent of its role in NHEJ. Cells lacking DNA-PKcs activity show increased DNA damage during S-phase and cellular sensitivity to replication stress. Notably, prevention of fork slowing and reversal via DNA-PKcs inhibition efficiently restores chemotherapy sensitivity in BRCA2-deficient mammary tumors with acquired PARPi resistance. Together, our data uncover a new key regulator of fork reversal and show how DNA-PKcs signaling can be manipulated to alter fork dynamics and drug resistance in cancer.
Assuntos
Quebras de DNA de Cadeia Dupla , Resistencia a Medicamentos Antineoplásicos , Resistencia a Medicamentos Antineoplásicos/genética , Dano ao DNA , Reparo do DNA por Junção de Extremidades , DNA/genética , Replicação do DNA , Reparo do DNARESUMO
MEDI4276 is a biparatopic tetravalent antibody targeting two nonoverlapping epitopes in subdomains 2 and 4 of the HER2 ecto-domain, with site-specific conjugation to a tubulysin-based microtubule inhibitor payload. MEDI4276 demonstrates enhanced cellular internalization and cytolysis of HER2-positive tumor cells in vitro This was a first-in-human, dose-escalation clinical trial in patients with HER2-positive advanced or metastatic breast cancer or gastric cancer. MEDI4276 doses escalated from 0.05 to 0.9 mg/kg (60- to 90-minute intravenous infusion every 3 weeks). Primary endpoints were safety and tolerability; secondary endpoints included antitumor activity (objective response, progression-free survival, and overall survival), pharmacokinetics, and immunogenicity. Forty-seven patients (median age 59 years; median of seven prior treatment regimens) were treated. The maximum tolerated dose was exceeded at 0.9 mg/kg with two patients experiencing dose-limiting toxicities (DLTs) of grade 3 liver function test (LFT) increases, one of whom also had grade 3 diarrhea, which resolved. Two additional patients reported DLTs of grade 3 LFT increases at lower doses (0.4 and 0.6 mg/kg). The most common (all grade) drug-related adverse events (AEs) were nausea (59.6%), fatigue (44.7%), aspartate aminotransferase (AST) increased (42.6%), and vomiting (38.3%). The most common grade 3/4 drug-related AE was AST increased (21.3%). Five patients had drug-related AEs leading to treatment discontinuation. In the as-treated population, there was one complete response (0.5 mg/kg; breast cancer), and two partial responses (0.6 and 0.75 mg/kg; breast cancer)-all had prior trastuzumab, pertuzumab, and ado-trastuzumab emtansine (T-DM1). MEDI4276 has demonstrable clinical activity but displays intolerable toxicity at doses >0.3 mg/kg.
Assuntos
Anticorpos Monoclonais , Antineoplásicos Imunológicos , Neoplasias da Mama , Imunoconjugados , Receptor ErbB-2 , Neoplasias Gástricas , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Anticorpos Monoclonais/química , Antineoplásicos Imunológicos/química , Antineoplásicos Imunológicos/farmacocinética , Antineoplásicos Imunológicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/imunologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Seguimentos , Imunoconjugados/química , Imunoconjugados/farmacocinética , Imunoconjugados/farmacologia , Dose Máxima Tolerável , Prognóstico , Receptor ErbB-2/imunologia , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/imunologia , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Taxa de Sobrevida , Distribuição TecidualRESUMO
Combining PD-L1 blockade with inhibition of oncogenic mitogen-activated protein kinase (MAPK) signaling may result in long-lasting responses in patients with advanced melanoma. This phase 1, open-label, dose-escalation and -expansion study (NCT02027961) investigated safety, tolerability and preliminary efficacy of durvalumab (anti-PD-L1) combined with dabrafenib (BRAF inhibitor) and trametinib (MEK inhibitor) for patients with BRAF-mutated melanoma (cohort A, n = 26), or durvalumab and trametinib given concomitantly (cohort B, n = 20) or sequentially (cohort C, n = 22) for patients with BRAF-wild type melanoma. Adverse events and treatment discontinuation rates were more common than previously reported for these agents given as monotherapy. Objective responses were observed in 69.2% (cohort A), 20.0% (cohort B) and 31.8% (cohort C) of patients, with evidence of improved tumor immune infiltration and durable responses in a subset of patients with available biopsy samples. In conclusion, combined MAPK inhibition and anti-PD-L1 therapy may provide treatment options for patients with advanced melanoma.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Melanoma/tratamento farmacológico , Neoplasias Cutâneas/tratamento farmacológico , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/metabolismo , Estudos de Coortes , Diarreia/induzido quimicamente , Exantema/induzido quimicamente , Feminino , Humanos , Imidazóis/administração & dosagem , Imidazóis/efeitos adversos , Masculino , Melanoma/genética , Melanoma/metabolismo , Pessoa de Meia-Idade , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mutação , Oximas/administração & dosagem , Oximas/efeitos adversos , Intervalo Livre de Progressão , Proteínas Proto-Oncogênicas B-raf/genética , Piridonas/administração & dosagem , Piridonas/efeitos adversos , Pirimidinonas/administração & dosagem , Pirimidinonas/efeitos adversos , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Adulto JovemRESUMO
The maintenance of genomic stability relies on DNA damage sensor kinases that detect DNA lesions and phosphorylate an extensive network of substrates. The Mec1/ATR kinase is one of the primary sensor kinases responsible for orchestrating DNA damage responses. Despite the importance of Mec1/ATR, the current network of its identified substrates remains incomplete due, in part, to limitations in mass spectrometry-based quantitative phosphoproteomics. Phosphoproteomics suffers from lack of redundancy and statistical power for generating high confidence datasets, since information about phosphopeptide identity, site-localization, and quantitation must often be gleaned from a single peptide-spectrum match (PSM). Here we carefully analyzed the isotope label swapping strategy for phosphoproteomics, using data consistency among reciprocal labeling experiments as a central filtering rule for maximizing phosphopeptide identification and quantitation. We demonstrate that the approach allows drastic reduction of false positive quantitations and identifications even from phosphopeptides with a low number of spectral matches. Application of this approach identifies new Mec1/ATR-dependent signaling events, expanding our understanding of the DNA damage signaling network. Overall, the proposed quantitative phosphoproteomic approach should be generally applicable for investigating kinase signaling networks with high confidence and depth.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/fisiologia , Dano ao DNA/genética , Dano ao DNA/fisiologia , Proteômica/métodos , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Instabilidade Genômica/genética , Espectrometria de Massas , Fosfopeptídeos , FosforilaçãoRESUMO
BACKGROUND: For children with cancer in palliative care, pain and worry are common and frequently under-managed, which negatively impacts quality of life (QOL). Massage therapy (MT) can lead to reduced pain in children with chronic illnesses. Children with cancer have experienced lower anxiety after MT. No studies have examined the effects of MT in pediatric oncology patients receiving palliative care. OBJECTIVE: Conduct a MT intervention to determine intervention acceptability and initial effects on ratings of pain, worry reduction, and quality of life. DESIGN: Pre-post single group pilot study. SETTING/SUBJECTS: Eight children with cancer (age 10-17) and one of their parents were recruited from a palliative care service. PROCEDURE/MEASUREMENTS: Baseline (one week prior to intervention): demographics, MT expectations, QOL, and pain measures. Intervention (one month): MT was provided once per week, with children's pain and worry ratings occurring immediately before and after each MT session. Follow Up (4-6 weeks after baseline): QOL, pain, and MT/study acceptability questionnaires. RESULTS: Participants reported significant decreases in pain following two MT sessions, and worry following one session. No significant changes in pain symptoms and QOL were found between baseline and follow up. Participants positively endorsed the study and the MT intervention, and there were no adverse effects reported. CONCLUSIONS: MT may lead to immediate decreases in pain and worry in children with cancer who are receiving palliative care, however the effects may not be sustained long term. Difficulties regarding protocol feasibility including recruitment and study compliance remain important considerations for future work.
Assuntos
Ansiedade/terapia , Dor do Câncer/terapia , Massagem , Manejo da Dor/métodos , Cuidados Paliativos , Qualidade de Vida , Adolescente , Criança , Feminino , Humanos , Masculino , Medição da Dor , Projetos PilotoRESUMO
BACKGROUND: The safety, efficacy, pharmacokinetics, and pharmacodynamics of the anti-programmed cell death-1 antibody MEDI0680 were evaluated in a phase I, multicenter, dose-escalation study in advanced solid malignancies. METHODS: MEDI0680 was administered intravenously once every 2 weeks (Q2W) or once every 3 weeks at 0.1, 0.5, 2.5, 10 or 20 mg/kg. Two cohorts received 20 mg/kg once a week for 2 or 4 weeks, then 20 mg/kg Q2W. All were treated for 12 months or until progression. The primary endpoint was safety. Secondary endpoints were efficacy and pharmacokinetics. Exploratory endpoints included pharmacodynamics. RESULTS: Fifty-eight patients were treated. Median age was 62.5 years and 81% were male. Most had kidney cancer (n = 36) or melanoma (n = 9). There were no dose-limiting toxicities. Treatment-related adverse events occurred in 83% and were grade ≥ 3 in 21%. Objective clinical responses occurred in 8/58 patients (14%): 5 with kidney cancer, including 1 with a complete response, and 3 with melanoma. The relationship between dose and serum levels was predictable and linear, with apparent receptor saturation at 10 mg/kg Q2W and all 20 mg/kg cohorts. CONCLUSIONS: MEDI0680 induced peripheral T-cell proliferation and increased plasma IFNγ and associated chemokines regardless of clinical response. CD8+ T-cell tumor infiltration and tumoral gene expression of IFNG, CD8A, CXCL9, and granzyme K (GZMK) were also increased following MEDI0680 administration. TRIAL REGISTRATION: NCT02013804 ; date of registration December 12, 2013.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antineoplásicos Imunológicos/uso terapêutico , Melanoma/tratamento farmacológico , Anticorpos Monoclonais/farmacologia , Antineoplásicos Imunológicos/farmacologia , Progressão da Doença , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Resultado do TratamentoRESUMO
This is a pivotal, multicenter, open-label study of moxetumomab pasudotox, a recombinant CD22-targeting immunotoxin, in hairy cell leukemia (HCL), a rare B cell malignancy with high CD22 expression. The study enrolled patients with relapsed/refractory HCL who had ≥2 prior systemic therapies, including ≥1 purine nucleoside analog. Patients received moxetumomab pasudotox 40 µg/kg intravenously on days 1, 3, and 5 every 28 days for ≤6 cycles. Blinded independent central review determined disease response and minimal residual disease (MRD) status. Among 80 patients (79% males; median age, 60.0 years), durable complete response (CR) rate was 30%, CR rate was 41%, and objective response rate (CR and partial response) was 75%; 64 patients (80%) achieved hematologic remission. Among complete responders, 27 (85%) achieved MRD negativity by immunohistochemistry. The most frequent adverse events (AEs) were peripheral edema (39%), nausea (35%), fatigue (34%), and headache (33%). Treatment-related serious AEs of hemolytic uremic syndrome (7.5%) and capillary leak syndrome (5%) were reversible and generally manageable with supportive care and treatment discontinuation (6 patients; 7.5%). Moxetumomab pasudotox treatment achieved a high rate of independently assessed durable response and MRD eradication in heavily pretreated patients with HCL, with acceptable tolerability.
Assuntos
Antineoplásicos/uso terapêutico , Toxinas Bacterianas/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Exotoxinas/uso terapêutico , Leucemia de Células Pilosas/tratamento farmacológico , Recidiva Local de Neoplasia/tratamento farmacológico , Terapia de Salvação , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Seguimentos , Humanos , Leucemia de Células Pilosas/patologia , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/patologia , Prognóstico , Indução de Remissão , Taxa de SobrevidaRESUMO
Marfan syndrome (MFS) is a systemic disorder of connective tissue caused by mutations in fibrillin-1. Cardiac dysfunction in MFS has not been characterized halting the development of therapies of cardiac complication in MFS. We aimed to study the age-dependent cardiac remodeling in the mouse model of MFS FbnC1039G+/- mouse [Marfan heterozygous (HT) mouse] and its association with valvular regurgitation. Marfan HT mice of 2-4 mo demonstrated a mild hypertrophic cardiac remodeling with predominant decline of diastolic function and increased transforming growth factor-ß canonical (p-SMAD2/3) and noncanonical (p-ERK1/2 and p-p38 MAPK) signaling and upregulation of hypertrophic markers natriuretic peptides atrium natriuretic peptide and brain natriuretic peptide. Among older HT mice (6-14 mo), cardiac remodeling was associated with two distinct phenotypes, manifesting either dilated or constricted left ventricular chamber. Dilatation of left ventricular chamber was accompanied by biochemical evidence of greater mechanical stress, including elevated ERK1/2 and p38 MAPK phosphorylation and higher brain natriuretic peptide expression. The aortic valve regurgitation was registered in 20% of the constricted group and 60% of the dilated group, whereas mitral insufficiency was observed in 40% of the constricted group and 100% of the dilated group. Cardiac dysfunction was not associated with the increase of interstitial fibrosis and nonmyocyte proliferation. In the mouse model fibrillin-1, haploinsufficiency results in the early onset of nonfibrotic hypertrophic cardiac remodeling and dysfunction, independently from valvular abnormalities. MFS heart is vulnerable to stress-induced cardiac dilatation in the face of valvular regurgitation, and stress-activated MAPK signals represent a potential target for cardiac management in MFS.
Assuntos
Síndrome de Marfan/patologia , Miocárdio/patologia , Animais , Cardiomegalia/diagnóstico por imagem , Cardiomegalia/patologia , Fibrilina-1 , Fibrilinas , Fibrose/patologia , Hemodinâmica , Sistema de Sinalização das MAP Quinases , Masculino , Síndrome de Marfan/diagnóstico por imagem , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/metabolismo , Insuficiência da Valva Mitral/etiologia , Insuficiência da Valva Mitral/patologia , Fenótipo , Proteína Smad2/genética , Proteína Smad2/metabolismo , Proteína Smad3/genética , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Ultrassonografia , Função Ventricular Esquerda , Proteínas Quinases p38 Ativadas por Mitógeno/biossíntese , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
A recurring theme of a host of gerontologic studies conducted in either experimental animals or in humans is related to documenting the functional decline with age. We hypothesize that elevated circulating levels of a powerful antiangiogenic peptide, endostatin, represent one of the potent systemic causes for multiorgan microvascular rarefaction and functional decline due to fibrosis. It is possible that during the life span of an organism there is an accumulation of dormant transformed cells producing antiangiogenic substances (endostatin) that maintain the dormancy of such scattered malignant cells. The proof of this postulate cannot be obtained by physically documenting these scattered cells, and it rests exclusively on the detection of sequelae of shifted pro- and antiangiogenic balance toward the latter. Here we compared circulating levels of endostatin in young and aging mice of two different strains and showed that endostatin levels are elevated in the latter. Renal expression of endostatin increased ~5.6-fold in aging animals. This was associated with microvascular rarefaction and progressive tubulointerstitial fibrosis. In parallel, the levels of sirtuins 1 and 3 were significantly suppressed in aging mice in conjunction with the expression of markers of senescence. Treating young mice with endostatin for 28 days showed delayed recovery of circulation after femoral artery ligation and reduced patency of renal microvasculature but no fibrosis. In conclusion, the findings are consistent with the hypothesis on elevation of endostatin levels and parallel microvascular rarefaction and induction of renal fibrosis in aging mice.
Assuntos
Envelhecimento/sangue , Envelhecimento/patologia , Endostatinas/sangue , Pleiotropia Genética , Rim/irrigação sanguínea , Rim/patologia , Animais , Endostatinas/genética , Fibrose , Camundongos , Microvasos/fisiologia , Modelos Animais , Neovascularização Fisiológica/fisiologia , Fluxo Sanguíneo Regional/fisiologia , Sirtuína 1/sangue , Sirtuína 3/sangueRESUMO
We evaluated the therapeutic efficacy and mechanisms of action of both mouse and human B7-H4 Immunoglobulin fusion proteins (mB7-H4Ig; hB7-H4Ig) in treating EAE. The present data show that mB7-H4Ig both directly and indirectly (via increasing Treg function) inhibited CD4⺠T-cell proliferation and differentiation in both Th1- and Th17-cell promoting conditions while inducing production of IL-10. B7-H4Ig treatment effectively ameliorated progression of both relapsing (R-EAE) and chronic EAE correlating with decreased numbers of activated CD4⺠T-cells within the CNS and spleen, and a concurrent increase in number and function of Tregs. The functional requirement for Treg activation in treating EAE was demonstrated by a loss of therapeutic efficacy of hB7-H4Ig in R-EAE following inactivation of Treg function either by anti-CD25 treatment or blockade of IL-10. Significant to the eventual translation of this treatment into clinical practice, hB7-H4Ig similarly inhibited the in vitro differentiation of naïve human CD4⺠T-cells in both Th1- and Th17-promoting conditions, while promoting the production of IL-10. B7-H4Ig thus regulates pro-inflammatory T-cell responses by a unique dual mechanism of action and demonstrates significant promise as a therapeutic for autoimmune diseases, including MS.
Assuntos
Encefalomielite Autoimune Experimental/tratamento farmacológico , Imunoglobulinas/farmacologia , Interleucina-10/imunologia , Linfócitos T/efeitos dos fármacos , Inibidor 1 da Ativação de Células T com Domínio V-Set/farmacologia , Animais , Doenças Autoimunes/tratamento farmacológico , Doenças Autoimunes/imunologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Encefalomielite Autoimune Experimental/imunologia , Feminino , Humanos , Imunoglobulinas/imunologia , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Camundongos , Linfócitos T/imunologia , Inibidor 1 da Ativação de Células T com Domínio V-Set/imunologiaRESUMO
The fibroblast growth factor (FGF) pathway promotes tumor growth and angiogenesis in many solid tumors. Although there has long been interest in FGF pathway inhibitors, development has been complicated: An effective FGF inhibitor must block the activity of multiple mitogenic FGF ligands but must spare the metabolic hormone FGFs (FGF-19, FGF-21, and FGF-23) to avoid unacceptable toxicity. To achieve these design requirements, we engineered a soluble FGF receptor 1 Fc fusion protein, FP-1039. FP-1039 binds tightly to all of the mitogenic FGF ligands, inhibits FGF-stimulated cell proliferation in vitro, blocks FGF- and vascular endothelial growth factor (VEGF)-induced angiogenesis in vivo, and inhibits in vivo growth of a broad range of tumor types. FP-1039 antitumor response is positively correlated with RNA levels of FGF2, FGF18, FGFR1c, FGFR3c, and ETV4; models with genetic aberrations in the FGF pathway, including FGFR1-amplified lung cancer and FGFR2-mutated endometrial cancer, are particularly sensitive to FP-1039-mediated tumor inhibition. FP-1039 does not appreciably bind the hormonal FGFs, because these ligands require a cell surface co-receptor, klotho or ß-klotho, for high-affinity binding and signaling. Serum calcium and phosphate levels, which are regulated by FGF-23, are not altered by administration of FP-1039. By selectively blocking nonhormonal FGFs, FP-1039 treatment confers antitumor efficacy without the toxicities associated with other FGF pathway inhibitors.
Assuntos
Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Imunoglobulina G/uso terapêutico , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Proteínas de Fusão Oncogênica/uso terapêutico , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/uso terapêutico , Cálcio/sangue , Fator de Crescimento de Fibroblastos 23 , Fatores de Crescimento de Fibroblastos/metabolismo , Humanos , Fosfatos/sangue , Proteínas Recombinantes de FusãoRESUMO
There is no proven therapy or prevention for vascular Ehlers-Danlos syndrome (vEDS), a genetic disorder associated with the mutation of procollagen type III and characterized by increased fragility of vascular and hollow organ walls. Heterozygous COL3A1-deficient (HT) mice recapitulate a mild presentation of one of the variants of vEDS: haploinsufficiency for collagen III. Adult HT mice are characterized by increased metalloproteinase (MMP) activity, reduced collagen content in the arterial walls, and spontaneous development of various severity lesions in aorta. We hypothesized that chronic treatment with a MMP inhibitor would increase collagen content and prevent the development of spontaneous aortic lesions. HT mice were treated since weaning with the broad-spectrum MMP inhibitor doxycycline added to food. At the age of 9 months MMP-9 expression was twice as high in the tunica media of aorta in untreated HT mice, whereas total collagen content was 30% lower (p < 0.01) and the cumulative score of aortic lesions was eight times higher than in wild-type (WT) mice (p < 0.01). After 9 months of doxycycline treatment, MMP-9 activity, collagen content, and lesions in the aortas of HT mice were at the level of those of WT mice (p > 0.05). In the mouse model of collagen III haploinsufficiency treatment with broad-spectrum MMP inhibitor that was started early in life normalized increased MMP activity, reduced aortic collagen content in adults, and prevented the development of spontaneous aortic lesions. Our findings provide experimental justification for the clinical evaluation of the benefit of doxycycline at least in the haploinsufficient variety of vEDS.
Assuntos
Aorta Torácica/efeitos dos fármacos , Modelos Animais de Doenças , Doxiciclina/administração & dosagem , Síndrome de Ehlers-Danlos/tratamento farmacológico , Síndrome de Ehlers-Danlos/patologia , Inibidores de Metaloproteinases de Matriz/administração & dosagem , Animais , Aorta Torácica/enzimologia , Aorta Torácica/patologia , Síndrome de Ehlers-Danlos/enzimologia , Camundongos , Camundongos Knockout , Resultado do TratamentoRESUMO
Induction of a pluripotent state in somatic cells through nuclear reprogramming has ushered in a new era of regenerative medicine. Heterogeneity and varied differentiation potentials among induced pluripotent stem cell (iPSC) lines are, however, complicating factors that limit their usefulness for disease modeling, drug discovery, and patient therapies. Thus, there is an urgent need to develop nonmutagenic rapid throughput methods capable of distinguishing among putative iPSC lines of variable quality. To address this issue, we have applied a highly specific chemoproteomic targeting strategy for de novo discovery of cell surface N-glycoproteins to increase the knowledge-base of surface exposed proteins and accessible epitopes of pluripotent stem cells. We report the identification of 500 cell surface proteins on four embryonic stem cell and iPSCs lines and demonstrate the biological significance of this resource on mouse fibroblasts containing an oct4-GFP expression cassette that is active in reprogrammed cells. These results together with immunophenotyping, cell sorting, and functional analyses demonstrate that these newly identified surface marker panels are useful for isolating iPSCs from heterogeneous reprogrammed cultures and for isolating functionally distinct stem cell subpopulations.
Assuntos
Separação Celular/métodos , Glicoproteínas/análise , Imunofenotipagem/métodos , Proteínas de Membrana/análise , Células-Tronco Pluripotentes/metabolismo , Proteômica/métodos , Animais , Células Cultivadas , Receptor gp130 de Citocina/análise , Embrião de Mamíferos/citologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/transplante , Fibroblastos/citologia , Fibroblastos/metabolismo , Citometria de Fluxo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/transplante , Espectrometria de Massas , Camundongos , Camundongos da Linhagem 129 , Camundongos Transgênicos , Microscopia Confocal , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Células-Tronco Pluripotentes/citologia , Teratoma/metabolismo , Teratoma/patologiaRESUMO
Our goal is to develop accurate electrostatic models that can be implemented in current computational protein design protocols. To this end, we improve upon a previously reported pairwise decomposable, finite difference Poisson-Boltzmann (FDPB) model for protein design (Marshall et al., Protein Sci 2005, 14, 1293). The improvement involves placing generic sidechains at positions with unknown amino acid identity and explicitly capturing two-body perturbations to the dielectric environment. We compare the original and improved FDPB methods to standard FDPB calculations in which the dielectric environment is completely determined by protein atoms. The generic sidechain approach yields a two to threefold increase in accuracy per residue or residue pair over the original pairwise FDPB implementation, with no additional computational cost. Distance dependent dielectric and solvent-exclusion models were also compared with standard FDPB energies. The accuracy of the new pairwise FDPB method is shown to be superior to these models, even after reparameterization of the solvent-exclusion model.
Assuntos
Biologia Computacional/métodos , Simulação por Computador , Modelos Químicos , Proteínas/química , Aminoácidos/química , Desenho Assistido por Computador , Distribuição de Poisson , Reprodutibilidade dos Testes , Eletricidade Estática , TermodinâmicaRESUMO
Electrostatic interactions are important for both protein stability and function, including binding and catalysis. As protein design moves into these areas, an accurate description of electrostatic energy becomes necessary. Here, we show that a simple distance-dependent Coulombic function parameterized by a comparison to Poisson-Boltzmann calculations is able to capture some of these electrostatic interactions. Specifically, all three helix N-capping interactions in the engrailed homeodomain fold are recovered using the newly parameterized model. The stability of this designed protein is similar to a protein forced by sequence restriction to have beneficial electrostatic interactions.