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1.
Toxicol Appl Pharmacol ; 483: 116837, 2024 02.
Artigo em Inglês | MEDLINE | ID: mdl-38278496

RESUMO

FLT3L-Fc is a cytokine-Fc fusion agonizing receptor-type tyrosine-protein kinase FLT3 (fms-related tyrosine kinase 3; CD135). FLT3 is expressed on dendritic cells (DCs) as well as myeloid and lymphoid progenitors. Nonclinical pharmacokinetics, pharmacodynamics and safety of FLT3L-Fc were investigated in rats and cynomolgus monkeys. FLT3L-Fc induced robust pharmacodynamic responses, evidenced by marked expansion of peripheral blood cDC1s, cDC2s, and pDCs (up to 301-fold in rats and 378-fold in monkeys), peaking at 8-10 days after the first dose. FLT3L-Fc was well tolerated with no adverse findings at doses up to 10 mg/kg administered intravenously twice three weeks apart. In both species, major clinical pathology findings consisted of expansion of white blood cell (WBC) populations including lymphocytes, monocytes, neutrophils, basophils, and large unstained cells, which were pronounced after the first dose. The WBC findings were associated microscopically with histiocytic and mononuclear cell infiltrates in multiple organs. Tissue immunohistochemistry in monkeys showed that the leukocyte infiltrates consisted of hematopoietic progenitor cells and histiocytes with a reactive morphology and were associated with a slight stimulation of regional T and B cell populations. Additional FLT3L-Fc-associated changes included decreases in red blood cell (RBC) mass, increases in RBC distribution width, variable changes in reticulocytes, and transient alterations in platelet counts (rats only). The RBC and WBC findings were associated microscopically with increased hematopoietic cellularity of the bone marrow in both species and increased splenic megakaryocytic extramedullary hematopoiesis in rats. The totality of nonclinical safety data support the clinical development of FLT3L-Fc.


Assuntos
Proteínas de Membrana , Neoplasias , Ratos , Animais , Células Dendríticas , Células-Tronco Hematopoéticas , Imunoterapia
2.
Nature ; 550(7677): 534-538, 2017 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-29045385

RESUMO

The ubiquitin system regulates essential cellular processes in eukaryotes. Ubiquitin is ligated to substrate proteins as monomers or chains and the topology of ubiquitin modifications regulates substrate interactions with specific proteins. Thus ubiquitination directs a variety of substrate fates including proteasomal degradation. Deubiquitinase enzymes cleave ubiquitin from substrates and are implicated in disease; for example, ubiquitin-specific protease-7 (USP7) regulates stability of the p53 tumour suppressor and other proteins critical for tumour cell survival. However, developing selective deubiquitinase inhibitors has been challenging and no co-crystal structures have been solved with small-molecule inhibitors. Here, using nuclear magnetic resonance-based screening and structure-based design, we describe the development of selective USP7 inhibitors GNE-6640 and GNE-6776. These compounds induce tumour cell death and enhance cytotoxicity with chemotherapeutic agents and targeted compounds, including PIM kinase inhibitors. Structural studies reveal that GNE-6640 and GNE-6776 non-covalently target USP7 12 Å distant from the catalytic cysteine. The compounds attenuate ubiquitin binding and thus inhibit USP7 deubiquitinase activity. GNE-6640 and GNE-6776 interact with acidic residues that mediate hydrogen-bond interactions with the ubiquitin Lys48 side chain, suggesting that USP7 preferentially interacts with and cleaves ubiquitin moieties that have free Lys48 side chains. We investigated this idea by engineering di-ubiquitin chains containing differential proximal and distal isotopic labels and measuring USP7 binding by nuclear magnetic resonance. This preferential binding protracted the depolymerization kinetics of Lys48-linked ubiquitin chains relative to Lys63-linked chains. In summary, engineering compounds that inhibit USP7 activity by attenuating ubiquitin binding suggests opportunities for developing other deubiquitinase inhibitors and may be a strategy more broadly applicable to inhibiting proteins that require ubiquitin binding for full functional activity.


Assuntos
Aminopiridinas/química , Aminopiridinas/farmacologia , Indazóis/química , Indazóis/farmacologia , Fenóis/química , Fenóis/farmacologia , Piridinas/química , Piridinas/farmacologia , Peptidase 7 Específica de Ubiquitina/antagonistas & inibidores , Ubiquitina/metabolismo , Animais , Ligação Competitiva , Linhagem Celular Tumoral , Sinergismo Farmacológico , Feminino , Humanos , Camundongos , Camundongos SCID , Modelos Moleculares , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Neoplasias/patologia , Ligação Proteica , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Especificidade por Substrato , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina/química , Peptidase 7 Específica de Ubiquitina/química , Peptidase 7 Específica de Ubiquitina/deficiência , Peptidase 7 Específica de Ubiquitina/metabolismo
3.
J Biol Chem ; 292(9): 3900-3908, 2017 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-28077575

RESUMO

The antibody Fc region regulates antibody cytotoxic activities and serum half-life. In a therapeutic context, however, the cytotoxic effector function of an antibody is often not desirable and can create safety liabilities by activating native host immune defenses against cells expressing the receptor antigens. Several amino acid changes in the Fc region have been reported to silence or reduce the effector function of antibodies. These earlier studies focused primarily on the interaction of human antibodies with human Fc-γ receptors, and it remains largely unknown how such changes to Fc might translate to the context of a murine antibody. We demonstrate that the commonly used N297G (NG) and D265A, N297G (DANG) variants that are efficacious in attenuating effector function in primates retain potent complement activation capacity in mice, leading to safety liabilities in murine studies. In contrast, we found an L234A, L235A, P329G (LALA-PG) variant that eliminates complement binding and fixation as well as Fc-γ-dependent, antibody-dependent, cell-mediated cytotoxity in both murine IgG2a and human IgG1. These LALA-PG substitutions allow a more accurate translation of results generated with an "effectorless" antibody between mice and primates. Further, we show that both human and murine antibodies containing the LALA-PG variant have typical pharmacokinetics in rodents and retain thermostability, enabling efficient knobs-into-holes bispecific antibody production and a robust path to generating highly effector-attenuated bispecific antibodies for preclinical studies.


Assuntos
Anticorpos Biespecíficos/imunologia , Imunoglobulina G/química , Animais , Formação de Anticorpos , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Complemento C1q/imunologia , Cricetinae , Cristalografia por Raios X , Ensaio de Imunoadsorção Enzimática , Glicosilação , Humanos , Fragmentos Fc das Imunoglobulinas/imunologia , Imunoglobulina G/genética , Camundongos , Conformação Proteica , Receptores de IgG/metabolismo , Temperatura
4.
J Biol Chem ; 291(11): 5986-5996, 2016 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-26797127

RESUMO

FGF21 is a stress-induced hormone with potent anti-obesity, insulin-sensitizing, and hepatoprotective properties. Although proteolytic cleavage of recombinant human FGF21 in preclinical species has been observed previously, the regulation of endogenously produced FGF21 is not well understood. Here we identify fibroblast activation protein (FAP) as the enzyme that cleaves and inactivates human FGF21. A selective chemical inhibitor, immunodepletion, or genetic deletion of Fap stabilized recombinant human FGF21 in serum. In addition, administration of a selective FAP inhibitor acutely increased circulating intact FGF21 levels in cynomolgus monkeys. On the basis of our findings, we propose selective FAP inhibition as a potential therapeutic approach to increase endogenous FGF21 activity for the treatment of obesity, type 2 diabetes, non-alcoholic steatohepatitis, and related metabolic disorders.


Assuntos
Fatores de Crescimento de Fibroblastos/metabolismo , Gelatinases/metabolismo , Proteínas de Membrana/metabolismo , Serina Endopeptidases/metabolismo , Sequência de Aminoácidos , Animais , Endopeptidases , Fatores de Crescimento de Fibroblastos/química , Gelatinases/genética , Deleção de Genes , Células HEK293 , Humanos , Macaca fascicularis , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Proteólise , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Serina Endopeptidases/genética
5.
J Biol Chem ; 288(16): 11470-81, 2013 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-23476017

RESUMO

H5 is a constitutively expressed, phosphorylated vaccinia virus protein that has been implicated in viral DNA replication, post-replicative gene expression, and virus assembly. For the purpose of understanding the role of H5 in vaccinia biology, we have characterized its biochemical and biophysical properties. Previously, we have demonstrated that H5 is associated with an endoribonucleolytic activity. In this study, we have shown that this cleavage results in a 3'-OH end suitable for polyadenylation of the nascent transcript, corroborating a role for H5 in vaccinia transcription termination. Furthermore, we have shown that H5 is intrinsically disordered, with an elongated rod-shaped structure that preferentially binds double-stranded nucleic acids in a sequence nonspecific manner. The dynamic phosphorylation status of H5 influences this structure and has implications for the role of H5 in multiple processes during virus replication.


Assuntos
Endorribonucleases/metabolismo , Terminação da Transcrição Genética/fisiologia , Vaccinia virus/fisiologia , Proteínas Virais/metabolismo , Replicação Viral/fisiologia , Replicação do DNA/fisiologia , DNA Viral/biossíntese , DNA Viral/genética , Endorribonucleases/genética , Células HeLa , Humanos , Fosforilação/fisiologia , Estrutura Terciária de Proteína , Vacínia/genética , Vacínia/metabolismo , Proteínas Virais/genética
6.
Pharmaceutics ; 16(5)2024 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-38794321

RESUMO

FLT3L-Fc is a half-life extended, effectorless Fc-fusion of the native human FLT3-ligand. In cynomolgus monkeys, treatment with FLT3L-Fc leads to a complex pharmacokinetic/pharmacodynamic (PK/PD) relationship, with observed nonlinear PK and expansion of different immune cell types across different dose levels. A minimal physiologically based PK/PD model with expansion-enhanced target-mediated drug disposition (TMDD) was developed to integrate the molecule's mechanism of action, as well as the complex preclinical and clinical PK/PD data, to support the preclinical-to-clinical translation of FLT3L-Fc. In addition to the preclinical PK data of FLT3L-Fc in cynomolgus monkeys, clinical PK and PD data from other FLT3-agonist molecules (GS-3583 and CDX-301) were used to inform the model and project the expansion profiles of conventional DC1s (cDC1s) and total DCs in peripheral blood. This work constitutes an essential part of our model-informed drug development (MIDD) strategy for clinical development of FLT3L-Fc by projecting PK/PD in healthy volunteers, determining the first-in-human (FIH) dose, and informing the efficacious dose in clinical settings. Model-generated results were incorporated in regulatory filings to support the rationale for the FIH dose selection.

7.
Sci Immunol ; 6(59)2021 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-33963061

RESUMO

Repair of the intestinal epithelium is tightly regulated to maintain homeostasis. The response after epithelial damage needs to be local and proportional to the insult. How different types of damage are coupled to repair remains incompletely understood. We report that after distinct types of intestinal epithelial damage, IL-1R1 signaling in GREM1+ mesenchymal cells increases production of R-spondin 3 (RSPO3), a Wnt agonist required for intestinal stem cell self-renewal. In parallel, IL-1R1 signaling regulates IL-22 production by innate lymphoid cells and promotes epithelial hyperplasia and regeneration. Although the regulation of both RSPO3 and IL-22 is critical for epithelial recovery from Citrobacter rodentium infection, IL-1R1-dependent RSPO3 production by GREM1+ mesenchymal cells alone is sufficient and required for recovery after dextran sulfate sodium-induced colitis. These data demonstrate how IL-1R1-dependent signaling orchestrates distinct repair programs tailored to the type of injury sustained that are required to restore intestinal epithelial barrier function.


Assuntos
Citrobacter rodentium , Infecções por Enterobacteriaceae/imunologia , Mucosa Intestinal/fisiologia , Receptores Tipo I de Interleucina-1/imunologia , Animais , Células Cultivadas , Técnicas de Cocultura , Colite/induzido quimicamente , Colite/imunologia , Colite/patologia , Colo/efeitos dos fármacos , Colo/imunologia , Colo/patologia , Sulfato de Dextrana , Células Epiteliais , Fibroblastos , Interleucinas/imunologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Camundongos Transgênicos , Organoides , Receptores Tipo I de Interleucina-1/genética , Regeneração , Transdução de Sinais , Trombospondinas/imunologia , Interleucina 22
8.
Cancer Discov ; 10(2): 232-253, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31699795

RESUMO

With only a fraction of patients responding to cancer immunotherapy, a better understanding of the entire tumor microenvironment is needed. Using single-cell transcriptomics, we chart the fibroblastic landscape during pancreatic ductal adenocarcinoma (PDAC) progression in animal models. We identify a population of carcinoma-associated fibroblasts (CAF) that are programmed by TGFß and express the leucine-rich repeat containing 15 (LRRC15) protein. These LRRC15+ CAFs surround tumor islets and are absent from normal pancreatic tissue. The presence of LRRC15+ CAFs in human patients was confirmed in >80,000 single cells from 22 patients with PDAC as well as by using IHC on samples from 70 patients. Furthermore, immunotherapy clinical trials comprising more than 600 patients across six cancer types revealed elevated levels of the LRRC15+ CAF signature correlated with poor response to anti-PD-L1 therapy. This work has important implications for targeting nonimmune elements of the tumor microenvironment to boost responses of patients with cancer to immune checkpoint blockade therapy. SIGNIFICANCE: This study describes the single-cell landscape of CAFs in pancreatic cancer during in vivo tumor evolution. A TGFß-driven, LRRC15+ CAF lineage is associated with poor outcome in immunotherapy trial data comprising multiple solid-tumor entities and represents a target for combinatorial therapy.This article is highlighted in the In This Issue feature, p. 161.


Assuntos
Fibroblastos Associados a Câncer/imunologia , Carcinoma Ductal Pancreático/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/genética , Inibidores de Checkpoint Imunológico/farmacologia , Proteínas de Membrana/metabolismo , Miofibroblastos/imunologia , Neoplasias Pancreáticas/tratamento farmacológico , Animais , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/imunologia , Fibroblastos Associados a Câncer/efeitos dos fármacos , Fibroblastos Associados a Câncer/metabolismo , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/imunologia , Linhagem Celular Tumoral , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Ensaios Clínicos como Assunto , Biologia Computacional , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/imunologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/imunologia , Humanos , Inibidores de Checkpoint Imunológico/uso terapêutico , Camundongos , Miofibroblastos/efeitos dos fármacos , Miofibroblastos/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/imunologia , RNA-Seq , Análise de Célula Única , Fator de Crescimento Transformador beta/metabolismo , Resultado do Tratamento , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/genética , Microambiente Tumoral/imunologia
9.
Alzheimers Res Ther ; 11(1): 97, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31787113

RESUMO

BACKGROUND: Accumulation of amyloid ß (Aß) in the brain is proposed as a cause of Alzheimer's disease (AD), with Aß oligomers hypothesized to be the primary mediators of neurotoxicity. Crenezumab is a humanized immunoglobulin G4 monoclonal antibody that has been shown to bind to synthetic monomeric and aggregated Aß in vitro; however, less is known about the binding characteristic in vivo. In this study, we evaluated the binding patterns of crenezumab to synthetic and native forms of Aß both in vitro and in vivo. METHODS: Crenezumab was used to immunoprecipitate Aß from synthetic Aß preparations or brain homogenates from a PS2APP mouse model of AD to determine the forms of Aß that crenezumab interacts with. Following systemic dosing in PS2APP or nontransgenic control mice, immunohistochemistry was used to localize crenezumab and assess its relative distribution in the brain, compared with amyloid plaques and markers of neuritic dystrophies (BACE1; LAMP1). Pharmacodynamic correlations were performed to investigate the relationship between peripheral and central target engagement. RESULTS: In vitro, crenezumab immunoprecipitated Aß oligomers from both synthetic Aß preparations and endogenous brain homogenates from PS2APP mice. In vivo studies in the PS2APP mouse showed that crenezumab localizes to regions surrounding the periphery of amyloid plaques in addition to the hippocampal mossy fibers. These regions around the plaques are reported to be enriched in oligomeric Aß, actively incorporate soluble Aß, and contribute to Aß-induced neurotoxicity and axonal dystrophy. In addition, crenezumab did not appear to bind to the dense core region of plaques or vascular amyloid. CONCLUSIONS: Crenezumab binds to multiple forms of amyloid ß (Aß), particularly oligomeric forms, and localizes to brain areas rich in Aß oligomers, including the halo around plaques and hippocampal mossy fibers, but not to vascular Aß. These insights highlight a unique mechanism of action for crenezumab of engaging Aß oligomers.


Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Anticorpos Monoclonais Humanizados/farmacologia , Encéfalo/efeitos dos fármacos , Animais , Encéfalo/metabolismo , Camundongos , Camundongos Transgênicos , Placa Amiloide/metabolismo , Ligação Proteica
10.
Sci Rep ; 9(1): 8833, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-31222070

RESUMO

In response to environmental and nutrient stress, adipose tissues must establish a new homeostatic state. Here we show that cold exposure of obese mice triggers an adaptive tissue remodeling in visceral adipose tissue (VAT) that involves extracellular matrix deposition, angiogenesis, sympathetic innervation, and adipose tissue browning. Obese VAT is predominated by pro-inflammatory M1 macrophages; cold exposure induces an M1-to-M2 shift in macrophage composition and dramatic changes in macrophage gene expression in both M1 and M2 macrophages. Antibody-mediated CSF1R blocking prevented the cold-induced recruitment of adipose tissue M2 macrophages, suggesting the role of CSF1R signaling in the process. These cold-induced effects in obese VAT are phenocopied by an administration of the FGF21-mimetic antibody, consistent with its action to stimulate sympathetic nerves. Collectively, these studies illuminate adaptive visceral adipose tissue plasticity in obese mice in response to cold stress and antibody-based metabolic therapy.


Assuntos
Adaptação Fisiológica , Anticorpos/farmacologia , Resposta ao Choque Frio , Gordura Intra-Abdominal/fisiologia , Animais , Movimento Celular , Fatores de Crescimento de Fibroblastos/imunologia , Macrófagos/citologia , Macrófagos/imunologia , Camundongos , Camundongos Obesos , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/imunologia , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Células Estromais/fisiologia
11.
Sci Rep ; 7(1): 12524, 2017 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-28970566

RESUMO

Fibroblast Activation Protein (FAP) is a membrane-bound serine protease whose expression is often elevated in activated fibroblasts associated with tissue remodeling in various common diseases such as cancer, arthritis and fibrosis. Like the closely related dipeptidyl peptidase DPPIV, the extracellular domain of FAP can be released into circulation as a functional enzyme, and limited studies suggest that the circulating level of FAP correlates with the degree of tissue fibrosis. Here we describe a novel homogeneous fluorescence intensity assay for circulating FAP activity based on a recently identified natural substrate, FGF21. This assay is unique in that it can effectively distinguish endopeptidase activity of FAP from that of other related enzymes such as prolyl endopeptidase (PREP) and was validated using Fap-deficient mice. Structural modeling was used to elucidate the mechanistic basis for the observed specificity in substrate recognition by FAP, but not by DPPIV or PREP. Finally, the assay was used to detect elevated FAP activity in human patients diagnosed with liver cirrhosis and to determine the effectiveness of a chemical inhibitor for FAP in mice. We propose that the assay presented here could thus be utilized for diagnosis of FAP-related pathologies and for the therapeutic development of FAP inhibitors.


Assuntos
Fatores de Crescimento de Fibroblastos/genética , Fibrose/genética , Gelatinases/genética , Cirrose Hepática/genética , Proteínas de Membrana/genética , Serina Endopeptidases/genética , Animais , Endopeptidases/genética , Fibroblastos/enzimologia , Fibroblastos/metabolismo , Fibrose/enzimologia , Fibrose/patologia , Regulação Enzimológica da Expressão Gênica , Humanos , Cirrose Hepática/enzimologia , Cirrose Hepática/patologia , Camundongos , Prolil Oligopeptidases , Especificidade por Substrato
12.
Structure ; 24(8): 1335-1345, 2016 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-27452404

RESUMO

The deubiquitinating enzyme USP7 has a pivotal role in regulating the stability of proteins involved in fundamental cellular processes of normal biology and disease. Despite the importance of USP7, the mechanisms underlying substrate recognition and catalytic activation are poorly understood. Here we present structural, biochemical, and biophysical analyses elucidating the molecular mechanism by which the C-terminal 19 amino acids of USP7 (residues 1084-1102) enhance the ubiquitin cleavage activity of the deubiquitinase (DUB) domain. Our data demonstrate that the C-terminal peptide binds the activation cleft in the catalytic domain and stabilizes the catalytically competent conformation of USP7. Additional structures of longer fragments of USP7, as well as solution studies, provide insight into full-length USP7, the role of the UBL domains, and demonstrate that both substrate recognition and deubiquitinase activity are highly regulated by the catalytic and noncatalytic domains of USP7, a feature that could be essential for the proper function of multi-domain DUBs.


Assuntos
Ubiquitina Tiolesterase/química , Ubiquitina/química , Sequência de Aminoácidos , Domínio Catalítico , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Humanos , Cinética , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Termodinâmica , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo , Peptidase 7 Específica de Ubiquitina
13.
Sci Rep ; 6: 39374, 2016 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-27996029

RESUMO

Accumulation of amyloid-ß (Aß) peptides and amyloid plaque deposition in brain is postulated as a cause of Alzheimer's disease (AD). The precise pathological species of Aß remains elusive although evidence suggests soluble oligomers may be primarily responsible for neurotoxicity. Crenezumab is a humanized anti-Aß monoclonal IgG4 that binds multiple forms of Aß, with higher affinity for aggregated forms, and that blocks Aß aggregation, and promotes disaggregation. To understand the structural basis for this binding profile and activity, we determined the crystal structure of crenezumab in complex with Aß. The structure reveals a sequential epitope and conformational requirements for epitope recognition, which include a subtle but critical element that is likely the basis for crenezumab's versatile binding profile. We find interactions consistent with high affinity for multiple forms of Aß, particularly oligomers. Of note, crenezumab also sequesters the hydrophobic core of Aß and breaks an essential salt-bridge characteristic of the ß-hairpin conformation, eliminating features characteristic of the basic organization in Aß oligomers and fibrils, and explains crenezumab's inhibition of aggregation and promotion of disaggregation. These insights highlight crenezumab's unique mechanism of action, particularly regarding Aß oligomers, and provide a strong rationale for the evaluation of crenezumab as a potential AD therapy.

14.
EBioMedicine ; 2(7): 730-43, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-26288846

RESUMO

Dissipating excess calories as heat through therapeutic stimulation of brown adipose tissues (BAT) has been proposed as a potential treatment for obesity-linked disorders. Here, we describe the generation of a humanized effector-less bispecific antibody that activates fibroblast growth factor receptor (FGFR) 1/ßKlotho complex, a common receptor for FGF21 and FGF19. Using this molecule, we show that antibody-mediated activation of FGFR1/ßKlotho complex in mice induces sustained energy expenditure in BAT, browning of white adipose tissue, weight loss, and improvements in obesity-associated metabolic derangements including insulin resistance, hyperglycemia, dyslipidemia and hepatosteatosis. In mice and cynomolgus monkeys, FGFR1/ßKlotho activation increased serum high-molecular-weight adiponectin, which appears to contribute over time by enhancing the amplitude of the metabolic benefits. At the same time, insulin sensitization by FGFR1/ßKlotho activation occurs even before the onset of weight loss in a manner that is independent of adiponectin. Together, selective activation of FGFR1/ßKlotho complex with a long acting therapeutic antibody represents an attractive approach for the treatment of type 2 diabetes and other obesity-linked disorders through enhanced energy expenditure, insulin sensitization and induction of high-molecular-weight adiponectin.


Assuntos
Tecido Adiposo Marrom/metabolismo , Anticorpos Biespecíficos/farmacologia , Insulina/farmacologia , Proteínas de Membrana/agonistas , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/agonistas , Adiponectina/metabolismo , Tecido Adiposo Marrom/efeitos dos fármacos , Animais , Linhagem Celular , Metabolismo Energético/efeitos dos fármacos , Fatores de Crescimento de Fibroblastos/farmacologia , Células HEK293 , Humanos , Proteínas Klotho , Macaca fascicularis , Masculino , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Obesos , Ligação Proteica/efeitos dos fármacos , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Termogênese/efeitos dos fármacos , Redução de Peso/efeitos dos fármacos
15.
PLoS One ; 9(7): e102695, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25029443

RESUMO

Receptor tyrosine kinase-like orphan receptors (ROR) 1 and 2 are atypical members of the receptor tyrosine kinase (RTK) family and have been associated with several human diseases. The vertebrate RORs contain an ATP binding domain that deviates from the consensus amino acid sequence, although the impact of this deviation on catalytic activity is not known and the kinase function of these receptors remains controversial. Recently, ROR2 was shown to signal through a Wnt responsive, ß-catenin independent pathway and suppress a canonical Wnt/ß-catenin signal. In this work we demonstrate that both ROR1 and ROR2 kinase domains are catalytically deficient while CAM-1, the C. elegans homolog of ROR, has an active tyrosine kinase domain, suggesting a divergence in the signaling processes of the ROR family during evolution. In addition, we show that substitution of the non-consensus residues from ROR1 or ROR2 into CAM-1 and MuSK markedly reduce kinase activity, while restoration of the consensus residues in ROR does not restore robust kinase function. We further demonstrate that the membrane-bound extracellular domain alone of either ROR1 or ROR2 is sufficient for suppression of canonical Wnt3a signaling, and that this domain can also enhance Wnt5a suppression of Wnt3a signaling. Based on these data, we conclude that human ROR1 and ROR2 are RTK-like pseudokinases.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Evolução Molecular , Modelos Moleculares , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/genética , Transdução de Sinais/genética , Sequência de Aminoácidos , Animais , Caenorhabditis elegans , Catálise , Células HEK293 , Humanos , Immunoblotting , Luciferases , Dados de Sequência Molecular , Fosforilação , Estrutura Terciária de Proteína , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/química , Alinhamento de Sequência , Especificidade da Espécie
16.
Virology ; 401(1): 49-60, 2010 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-20206959

RESUMO

The vaccinia H5 protein has been implicated in several steps of virus replication including DNA synthesis, postreplicative gene transcription, and virion morphogenesis. Our recent mapping of mutants in the consolidated Condit-Dales collection identified a temperature-sensitive vaccinia mutant in the H5R gene (Dts57). We demonstrate here that Dts57 has a DNA negative phenotype, strongly suggesting a direct role for H5 in DNA replication. We used a temperature shift protocol to determine the impact of H5 temperature sensitivity on postreplicative gene expression and observed changes in the pattern of postreplicative viral mRNA metabolism consistent with a role of H5 in postreplicative transcription. Finally, using a rifampicin release temperature shift protocol, we show that H5 is involved in multiple steps of virion morphogenesis. These data demonstrate directly that H5 plays roles in DNA replication, transcription and morphogenesis in vivo.


Assuntos
Proteínas de Ligação a DNA/fisiologia , Vaccinia virus/fisiologia , Proteínas Virais/fisiologia , Replicação do DNA , DNA Viral/biossíntese , Humanos , Transcrição Gênica , Vírion/fisiologia , Replicação Viral
17.
J Biol Chem ; 283(9): 5267-75, 2008 Feb 29.
Artigo em Inglês | MEDLINE | ID: mdl-18089571

RESUMO

The mRNAs encoding the vaccinia virus F17 protein and the cowpox A-type inclusion protein are known to possess sequence-homogeneous 3' ends, generated by a post-transcriptional cleavage event. By using partially purified extracts, we have previously shown that the same factor probably cleaves both the F17 and A-type inclusion protein transcripts and that the cleavage factor is either virus-coded or virus-induced during the post-replicative phase of virus replication. In this study, we have purified the cleavage factor from vaccinia-infected HeLa cells using column chromatography and gel filtration. The factor eluted from the gel filtration column with an apparent molecular mass of approximately 440 kDa. Mass spectrometric analyses of the proteins present in the peak active fractions revealed the presence of at least one vaccinia protein with a high degree of certainty, the H5R gene product. To extend this finding, extracts were prepared from HeLa cells infected with vaccinia virus overexpressing His-tagged H5, chromatographed on a nickel affinity column, and eluted using an imidazole gradient. Cleavage activity eluted with the peak of His-tagged H5. Gel filtration of the affinity-purified material further demonstrated that cleavage activity and His-tagged H5 co-chromatographed with an apparent molecular mass of 463 kDa. We therefore conclude that H5 is specifically associated with post-transcriptional cleavage of F17R transcripts. In addition, we show that dephosphorylation of a cleavage competent extract with a nonspecific phosphatase abolishes cleavage activity implying a role for phosphorylation in cleavage activity.


Assuntos
Processamento Pós-Transcricional do RNA/fisiologia , Proteínas de Ligação a RNA/isolamento & purificação , Proteínas de Ligação a RNA/metabolismo , Vaccinia virus/metabolismo , Proteínas Virais/isolamento & purificação , Proteínas Virais/metabolismo , Células HeLa , Humanos , Corpos de Inclusão Viral/genética , Corpos de Inclusão Viral/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Proteínas de Ligação a RNA/genética , Transcrição Gênica/fisiologia , Vaccinia virus/genética , Proteínas Virais/genética , Replicação Viral/fisiologia
18.
Virology ; 375(1): 213-22, 2008 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-18314155

RESUMO

Complementation analysis of the combined Condit/Dales collection of vaccinia virus temperature-sensitive mutants has been reported (Lackner, C.A., D'Costa, S.M., Buck, C., Condit, R.C., 2003. Complementation analysis of the Dales collection of vaccinia virus temperature-sensitive mutants. Virology 305, 240-259), however not all complementation groups have previously been assigned to single genes on the viral genome. We have used marker rescue to map at least one representative of each complementation group to a unique viral gene. The final combined collection contains 124 temperature-sensitive mutants affecting 38 viral genes, plus five double mutants.


Assuntos
Mapeamento Cromossômico , Mutação , Vaccinia virus/genética , Animais , Linhagem Celular , Chlorocebus aethiops , Genes Virais , Teste de Complementação Genética , Temperatura Alta , Análise de Sequência de DNA , Ensaio de Placa Viral
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