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1.
Cell ; 182(2): 345-356.e16, 2020 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-32589945

RESUMO

Pathogenic clostridial species secrete potent toxins that induce severe host tissue damage. Paeniclostridium sordellii lethal toxin (TcsL) causes an almost invariably lethal toxic shock syndrome associated with gynecological infections. TcsL is 87% similar to C. difficile TcdB, which enters host cells via Frizzled receptors in colon epithelium. However, P. sordellii infections target vascular endothelium, suggesting that TcsL exploits another receptor. Here, using CRISPR/Cas9 screening, we establish semaphorins SEMA6A and SEMA6B as TcsL receptors. We demonstrate that recombinant SEMA6A can protect mice from TcsL-induced edema. A 3.3 Å cryo-EM structure shows that TcsL binds SEMA6A with the same region that in TcdB binds structurally unrelated Frizzled. Remarkably, 15 mutations in this evolutionarily divergent surface are sufficient to switch binding specificity of TcsL to that of TcdB. Our findings establish semaphorins as physiologically relevant receptors for TcsL and reveal the molecular basis for the difference in tissue targeting and disease pathogenesis between highly related toxins.


Assuntos
Toxinas Bacterianas/metabolismo , Clostridium sordellii/metabolismo , Semaforinas/metabolismo , Animais , Toxinas Bacterianas/química , Toxinas Bacterianas/toxicidade , Sítios de Ligação , Sistemas CRISPR-Cas/genética , Linhagem Celular , Microscopia Crioeletrônica , Edema/patologia , Edema/prevenção & controle , Feminino , Humanos , Pulmão/efeitos dos fármacos , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/uso terapêutico , Semaforinas/química , Semaforinas/genética
2.
Proc Natl Acad Sci U S A ; 116(14): 6812-6817, 2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30894493

RESUMO

Aberrant activation of Wnt/ß-catenin signaling occurs frequently in cancer. However, therapeutic targeting of this pathway is complicated by the role of Wnt in stem cell maintenance and tissue homeostasis. Here, we evaluated antibodies blocking 6 of the 10 human Wnt/Frizzled (FZD) receptors as potential therapeutics. Crystal structures revealed a common binding site for these monoclonal antibodies (mAbs) on FZD, blocking the interaction with the Wnt palmitoleic acid moiety. However, these mAbs displayed gastrointestinal toxicity or poor plasma exposure in vivo. Structure-guided engineering was used to refine the binding of each mAb for FZD receptors, resulting in antibody variants with improved in vivo tolerability and developability. Importantly, the lead variant mAb significantly inhibited tumor growth in the HPAF-II pancreatic tumor xenograft model. Taken together, our data demonstrate that anti-FZD cancer therapeutic antibodies with broad specificity can be fine-tuned to navigate in vivo exposure and tolerability while driving therapeutic efficacy.


Assuntos
Especificidade de Anticorpos , Antineoplásicos Imunológicos , Receptores Frizzled/antagonistas & inibidores , Neoplasias Pancreáticas , Engenharia de Proteínas , Animais , Especificidade de Anticorpos/genética , Especificidade de Anticorpos/imunologia , Antineoplásicos Imunológicos/imunologia , Antineoplásicos Imunológicos/farmacocinética , Antineoplásicos Imunológicos/farmacologia , Linhagem Celular Tumoral , Feminino , Receptores Frizzled/genética , Receptores Frizzled/imunologia , Células HEK293 , Humanos , Camundongos , Camundongos Nus , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
3.
J Biol Chem ; 292(42): 17290-17301, 2017 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-28842504

RESUMO

Clostridium difficile is a major nosocomial pathogen that produces two exotoxins, TcdA and TcdB, with TcdB thought to be the primary determinant in human disease. TcdA and TcdB are large, multidomain proteins, each harboring a cytotoxic glucosyltransferase domain that is delivered into the cytosol from endosomes via a translocation domain after receptor-mediated endocytosis of toxins from the cell surface. Although there are currently no known host cell receptors for TcdA, three cell-surface receptors for TcdB have been identified: CSPG4, NECTIN3, and FZD1/2/7. The sites on TcdB that mediate binding to each receptor are not defined. Furthermore, it is not known whether the combined repetitive oligopeptide (CROP) domain is involved in or required for receptor binding. Here, in a screen designed to identify sites in TcdB that are essential for target cell intoxication, we identified a region at the junction of the translocation and the CROP domains that is implicated in CSPG4 binding. Using a series of C-terminal truncations, we show that the CSPG4-binding site on TcdB extends into the CROP domain, requiring three short repeats for binding and for full toxicity on CSPG4-expressing cells. Consistent with the location of the CSPG4-binding site on TcdB, we show that the anti-TcdB antibody bezlotoxumab, which binds partially within the first three short repeats, prevents CSPG4 binding to TcdB. In addition to establishing the binding region for CSPG4, this work ascribes for the first time a role in TcdB CROPs in receptor binding and further clarifies the relative roles of host receptors in TcdB pathogenesis.


Assuntos
Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Clostridioides difficile/enzimologia , Glucosiltransferases/metabolismo , Proteínas de Membrana/metabolismo , Animais , Anticorpos Monoclonais/química , Anticorpos Neutralizantes/química , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Toxinas Bacterianas/antagonistas & inibidores , Toxinas Bacterianas/genética , Anticorpos Amplamente Neutralizantes , Células CHO , Células CACO-2 , Chlorocebus aethiops , Proteoglicanas de Sulfatos de Condroitina/genética , Clostridioides difficile/genética , Clostridioides difficile/patogenicidade , Cricetinae , Cricetulus , Glucosiltransferases/antagonistas & inibidores , Glucosiltransferases/genética , Células HEK293 , Humanos , Proteínas de Membrana/genética , Ligação Proteica , Domínios Proteicos
4.
Clin Cancer Res ; 29(4): 791-804, 2023 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-36441800

RESUMO

PURPOSE: Leukemia inhibitory factor (LIF) is a multifunctional cytokine with numerous reported roles in cancer and is thought to drive tumor development and progression. Characterization of LIF and clinical-stage LIF inhibitors would increase our understanding of LIF as a therapeutic target. EXPERIMENTAL DESIGN: We first tested the association of LIF expression with transcript signatures representing multiple processes regulating tumor development and progression. Next, we developed MSC-1, a high-affinity therapeutic antibody that potently inhibits LIF signaling and tested it in immune competent animal models of cancer. RESULTS: LIF was associated with signatures of tumor-associated macrophages (TAM) across 7,769 tumor samples spanning 22 solid tumor indications. In human tumors, LIF receptor was highly expressed within the macrophage compartment and LIF treatment drove macrophages to acquire immunosuppressive capacity. MSC-1 potently inhibited LIF signaling by binding an epitope that overlaps with the gp130 receptor binding site on LIF. MSC-1 showed monotherapy efficacy in vivo and drove TAMs to acquire antitumor and proinflammatory function in syngeneic colon cancer mouse models. Combining MSC-1 with anti-PD1 leads to strong antitumor response and a long-term tumor-free survival in a significant proportion of treated mice. CONCLUSIONS: Overall, our findings highlight LIF as a therapeutic target for cancer immunotherapy.


Assuntos
Neoplasias , Microambiente Tumoral , Animais , Humanos , Camundongos , Terapia de Imunossupressão , Fator Inibidor de Leucemia/genética , Fator Inibidor de Leucemia/metabolismo , Macrófagos/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Microambiente Tumoral/genética
5.
Cancer Cell ; 40(1): 53-69.e9, 2022 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-34971569

RESUMO

Pediatric cancers often mimic fetal tissues and express proteins normally silenced postnatally that could serve as immune targets. We developed T cells expressing chimeric antigen receptors (CARs) targeting glypican-2 (GPC2), a fetal antigen expressed on neuroblastoma (NB) and several other solid tumors. CARs engineered using standard designs control NBs with transgenic GPC2 overexpression, but not those expressing clinically relevant GPC2 site density (∼5,000 molecules/cell, range 1-6 × 103). Iterative engineering of transmembrane (TM) and co-stimulatory domains plus overexpression of c-Jun lowered the GPC2-CAR antigen density threshold, enabling potent and durable eradication of NBs expressing clinically relevant GPC2 antigen density, without toxicity. These studies highlight the critical interplay between CAR design and antigen density threshold, demonstrate potent efficacy and safety of a lead GPC2-CAR candidate suitable for clinical testing, and credential oncofetal antigens as a promising class of targets for CAR T cell therapy of solid tumors.


Assuntos
Glipicanas/imunologia , Imunoterapia Adotiva , Neuroblastoma/tratamento farmacológico , Receptores de Antígenos de Linfócitos T/metabolismo , Animais , Linhagem Celular Tumoral , Glipicanas/metabolismo , Humanos , Imunoterapia/métodos , Neuroblastoma/patologia , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
6.
ACS Omega ; 6(38): 24553-24561, 2021 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-34604637

RESUMO

Cleistanthus collinus leaf extracts are consumed for suicidal purposes in southern India. The boiled decoction is known to be more toxic than the fresh leaf juice. Although several compounds have been isolated and their toxicity tested, controversy remains as to which compounds are responsible for the high level of toxicity of C. collinus. We report herein that cleistanthoside A is the major toxin in the boiled aqueous extract of fresh leaves and causes death in rats in small doses. The toxicity of the boiled extract prepared in the manner described can be attributed entirely to cleistanthoside A. Cleistanthin A could also be isolated from the boiled extract, albeit in trace amounts. As hypotension not responding to vasoconstrictors is the cause of death in patients who have consumed the boiled extract, effects of cleistanthoside A on the determinants of blood pressure, namely, force of cardiac contraction and vascular resistance, were tested in isolated organ experiments. Cleistanthoside A has a direct vasoconstrictor effect; however, it inhibits ventricular contractility. Therefore, the notion that the shock in C. collinus poisoning is of vascular origin must be considered carefully, and the possibility of cardiogenic shock must be studied. We present the crystal structure of cleistanthin A and show the potency of fast NMR methods (NOAH4-BSCN-NUS) in the full spectral assignment of cleistanthoside A as a real-world sample of a natural product. We also compare the results of the NOAH4-BSCN-NUS NMR experiments with conventional NMR methods.

7.
Cell Rep Med ; 2(7): 100344, 2021 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-34337560

RESUMO

Glypican 2 (GPC2) is a MYCN-regulated, differentially expressed cell-surface oncoprotein and target for immune-based therapies in neuroblastoma. Here, we build on GPC2's immunotherapeutic attributes by finding that it is also a highly expressed, MYCN-driven oncoprotein on small-cell lung cancers (SCLCs), with significantly enriched expression in both the SCLC and neuroblastoma stem cell compartment.By solving the crystal structure of the D3-GPC2-Fab/GPC2 complex at 3.3 Å resolution, we further illustrate that the GPC2-directed antibody-drug conjugate (ADC; D3-GPC2-PBD), that links a human GPC2 antibody (D3) to DNA-damaging pyrrolobenzodiazepine (PBD) dimers, binds a tumor-specific, conformation-dependent epitope of the core GPC2 extracellular domain. We then show that this ADC induces durable neuroblastoma and SCLC tumor regression via induction of DNA damage, apoptosis, and bystander cell killing, notably with no signs of ADC-induced in vivo toxicity. These studies provide preclinical data to support the clinical translation of ADCs targeting GPC2.


Assuntos
Epitopos/química , Epitopos/metabolismo , Glipicanas/imunologia , Imunoconjugados/farmacologia , Neoplasias Pulmonares/patologia , Neuroblastoma/patologia , Carcinoma de Pequenas Células do Pulmão/patologia , Animais , Efeito Espectador/efeitos dos fármacos , Compartimento Celular , Morte Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Dano ao DNA , Feminino , Humanos , Camundongos Endogâmicos C57BL , Camundongos SCID , Proteína Proto-Oncogênica N-Myc/metabolismo , Proteínas Oncogênicas/metabolismo , Conformação Proteica
8.
Acta Crystallogr F Struct Biol Commun ; 71(Pt 6): 688-96, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26057797

RESUMO

Heat-shock protein 90 (Hsp90) is an ATP-dependent molecular chaperone that is essential for the normal functioning of eukaryotic cells. It plays crucial roles in cell signalling, cell-cycle control and in maintaining proteome integrity and protein homeostasis. In plants, Hsp90s are required for normal plant growth and development. Hsp90s are observed to be upregulated in response to various abiotic and biotic stresses and are also involved in immune responses in plants. Although there are several studies elucidating the physiological role of Hsp90s in plants, their molecular mechanism of action is still unclear. In this study, biochemical characterization of an Hsp90 protein from rice (Oryza sativa; OsHsp90) has been performed and the crystal structure of its N-terminal domain (OsHsp90-NTD) was determined. The binding of OsHsp90 to its substrate ATP and the inhibitor 17-AAG was studied by fluorescence spectroscopy. The protein also exhibited a weak ATPase activity. The crystal structure of OsHsp90-NTD was solved in complex with the nonhydrolyzable ATP analogue AMPPCP at 3.1 Å resolution. The domain was crystallized by cross-seeding with crystals of the N-terminal domain of Hsp90 from Dictyostelium discoideum, which shares 70% sequence identity with OsHsp90-NTD. This is the second reported structure of a domain of Hsp90 from a plant source.


Assuntos
Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/química , Benzoquinonas/química , Proteínas de Choque Térmico HSP90/química , Lactamas Macrocíclicas/química , Oryza/química , Proteínas de Plantas/química , Sequência de Aminoácidos , Cristalografia por Raios X , Dictyostelium/química , Dictyostelium/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Oryza/metabolismo , Proteínas de Plantas/antagonistas & inibidores , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Espectrometria de Fluorescência , Eletricidade Estática
9.
Sci Rep ; 5: 17015, 2015 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-26599366

RESUMO

The involvement of Hsp90 in progression of diseases like cancer, neurological disorders and several pathogen related conditions is well established. Hsp90, therefore, has emerged as an attractive drug target for many of these diseases. Several small molecule inhibitors of Hsp90, such as geldanamycin derivatives, that display antitumor activity, have been developed and are under clinical trials. However, none of these tested inhibitors or drugs are peptide-based compounds. Here we report the first crystal structure of a peptide bound at the ATP binding site of the N-terminal domain of Hsp90. The peptide makes several specific interactions with the binding site residues, which are comparable to those made by the nucleotide and geldanamycin. A modified peptide was designed based on these interactions. Inhibition of ATPase activity of Hsp90 was observed in the presence of the modified peptide. This study provides an alternative approach and a lead peptide molecule for the rational design of effective inhibitors of Hsp90 function.


Assuntos
Proteínas de Choque Térmico HSP90/química , Peptídeos/química , Sequência de Aminoácidos , Benzoquinonas/química , Benzoquinonas/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Lactamas Macrocíclicas/química , Lactamas Macrocíclicas/metabolismo , Simulação de Dinâmica Molecular , Nucleotídeos/química , Nucleotídeos/metabolismo , Peptídeos/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação
10.
J Mol Biol ; 383(1): 24-35, 2008 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-18718841

RESUMO

Cytosolic heat shock protein 90 (Hsp90) has been implicated in diverse biological processes such as protein folding, cell cycle control, signal transduction, development, and morphological evolution. Model systems available for studying Hsp90 function either allow ease of manipulation for biochemical studies or facilitate a phenomenological study of its role in influencing phenotype. In this work, we have explored the use of the cellular slime mold Dictyostelium discoideum to examine cellular functions of Hsp90 in relation to its multicellular development. In addition to cloning, purification, biochemical characterization, and examination of its crystal structure, our studies, using a pharmacological inhibitor of Hsp90, demonstrate a role for the cytoplasmic isoform (HspD) in D. discoideum development. Inhibition of HspD function using geldanamycin (GA) resulted in delayed aggregation and arrest of D. discoideum development at the 'mound' stage. Crystal structure of the amino-terminal domain of HspD showed a binding pocket similar to that described for yeast Hsp90. Fluorescence spectroscopy, as well as GA-coupled beads affinity pulldown, confirmed a specific interaction between HspD and GA. The results presented here provide an important insight into the function of HspD in D. discoideum development and emphasize the potential of the cellular slime mold to serve as an effective model for studying the many roles of Hsp90 at cellular and organismal levels.


Assuntos
Dictyostelium/crescimento & desenvolvimento , Dictyostelium/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Proteínas de Protozoários/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Benzoquinonas/farmacologia , Sítios de Ligação , Clonagem Molecular , Cristalização , DNA de Protozoário/genética , Dictyostelium/efeitos dos fármacos , Dictyostelium/genética , Genes de Protozoários , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/química , Proteínas de Choque Térmico HSP90/genética , Cinética , Lactamas Macrocíclicas/farmacologia , Modelos Moleculares , Fenótipo , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade da Espécie
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