RESUMO
Malaria during pregnancy is a major global health problem caused by infection with Plasmodium falciparum parasites. Severe effects arise from the accumulation of infected erythrocytes in the placenta. Here, erythrocytes infected by late blood-stage parasites adhere to placental chondroitin sulphate A (CS) via VAR2CSA-type P. falciparum erythrocyte membrane protein 1 (PfEMP1) adhesion proteins. Immunity to placental malaria is acquired through exposure and mediated through antibodies to VAR2CSA. Through evolution, the VAR2CSA proteins have diversified in sequence to escape immune recognition but retained their overall macromolecular structure to maintain CS binding affinity. This structural conservation may also have allowed development of broadly reactive antibodies to VAR2CSA in immune women. Here we show the negative stain and cryo-EM structure of the only known broadly reactive human monoclonal antibody, PAM1.4, in complex with VAR2CSA. The data shows how PAM1.4's broad VAR2CSA reactivity is achieved through interactions with multiple conserved residues of different sub-domains forming conformational epitope distant from the CS binding site on the VAR2CSA core structure. Thus, while PAM1.4 may represent a class of antibodies mediating placental malaria immunity by inducing phagocytosis or NK cell-mediated cytotoxicity, it is likely that broadly CS binding-inhibitory antibodies target other epitopes at the CS binding site. Insights on both types of broadly reactive monoclonal antibodies may aid the development of a vaccine against placental malaria.
Assuntos
Malária Falciparum , Malária , Humanos , Feminino , Gravidez , Antígenos de Protozoários , Malária Falciparum/parasitologia , Epitopos , Anticorpos Antiprotozoários , Anticorpos Monoclonais , Microscopia Crioeletrônica , Placenta/metabolismo , Plasmodium falciparum/metabolismo , Eritrócitos/parasitologia , Sulfatos de Condroitina/metabolismoRESUMO
Lineage-determining transcription factors (LD-TFs) drive the differentiation of progenitor cells into a specific lineage. In CD4+ T cells, T-bet dictates differentiation of the TH1 lineage, whereas GATA3 drives differentiation of the alternative TH2 lineage. However, LD-TFs, including T-bet and GATA3, are frequently co-expressed but how this affects LD-TF function is not known. By expressing T-bet and GATA3 separately or together in mouse T cells, we show that T-bet sequesters GATA3 at its target sites, thereby removing GATA3 from TH2 genes. This redistribution of GATA3 is independent of GATA3 DNA binding activity and is instead mediated by the T-bet DNA binding domain, which interacts with the GATA3 DNA binding domain and changes GATA3's sequence binding preference. This mechanism allows T-bet to drive the TH1 gene expression program in the presence of GATA3. We propose that redistribution of one LD-TF by another may be a common mechanism that could explain how specific cell fate choices can be made even in the presence of other transcription factors driving alternative differentiation pathways.
Assuntos
Fator de Transcrição GATA3 , Proteínas com Domínio T/metabolismo , Células Th2 , Animais , Linhagem da Célula , DNA/metabolismo , Fator de Transcrição GATA3/genética , Fator de Transcrição GATA3/metabolismo , Expressão Gênica , Camundongos , Proteínas com Domínio T/genética , Células Th2/citologia , Células Th2/metabolismoRESUMO
IGF2 mRNA-binding protein 1 (IMP1) is a key regulator of messenger RNA (mRNA) metabolism and transport in organismal development and, in cancer, its mis-regulation is an important component of tumour metastasis. IMP1 function relies on the recognition of a diverse set of mRNA targets that is mediated by the combinatorial action of multiple RNA-binding domains. Here, we dissect the structure and RNA-binding properties of two key RNA-binding domains of IMP1, KH1 and KH2, and we build a kinetic model for the recognition of RNA targets. Our data and model explain how the two domains are organized as an intermolecular pseudo-dimer and that the important role they play in mRNA target recognition is underpinned by the high RNA-binding affinity and fast kinetics of this KH1KH2-RNA recognition unit. Importantly, the high-affinity RNA-binding by KH1KH2 is achieved by an inter-domain coupling 50-fold stronger than that existing in a second pseudo-dimer in the protein, KH3KH4. The presence of this strong coupling supports a role of RNA re-modelling in IMP1 recognition of known cancer targets.
Assuntos
Proteínas Proto-Oncogênicas c-myc/genética , RNA Mensageiro/química , Proteínas de Ligação a RNA/química , Sequência de Aminoácidos , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Cinética , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Gentamicin is an aminoglycoside widely used in treatments of, in particular, enterococcal, mycobacterial, and severe Gram-negative bacterial infections. Large doses of gentamicin cause nephrotoxicity and ototoxicity, entering the cell via the receptor megalin. Until now, no structural information has been available to describe the interaction with gentamicin in atomic detail, and neither have any three-dimensional structures of domains from the human megalin receptor been solved. To address this gap in our knowledge, we have solved the NMR structure of the 10th complement type repeat of human megalin and investigated its interaction with gentamicin. Using NMR titration data in HADDOCK, we have generated a three-dimensional model describing the complex between megalin and gentamicin. Gentamicin binds to megalin with low affinity and exploits the common ligand binding motif previously described (Jensen, G. A., Andersen, O. M., Bonvin, A. M., Bjerrum-Bohr, I., Etzerodt, M., Thogersen, H. C., O'Shea, C., Poulsen, F. M., and Kragelund, B. B. (2006) J. Mol. Biol. 362, 700-716) utilizing the indole side chain of Trp-1126 and the negatively charged residues Asp-1129, Asp-1131, and Asp-1133. Binding to megalin is highly similar to gentamicin binding to calreticulin. We discuss the impact of this novel insight for the future structure-based design of gentamicin antagonists.
Assuntos
Gentamicinas/química , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/química , Simulação de Acoplamento Molecular , Motivos de Aminoácidos , Gentamicinas/metabolismo , Humanos , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Estrutura Terciária de Proteína , Sequências Repetitivas de Aminoácidos , Relação Estrutura-AtividadeRESUMO
The lipoprotein receptor (LR) family constitutes a large group of structurally closely related receptors with broad ligand-binding specificity. Traditionally, ligand binding to LRs has been anticipated to involve merely the complement type repeat (CR)-domains omnipresent in the family. Recently, this dogma has transformed with the observation that ß-propellers of some LRs actively engage in complex formation too. Based on an in-depth decomposition of current structures and sequences, we suggest that exploitation of the ß-propellers as binding targets depends on receptor subgroups. In particular, we highlight the shutter mechanism of ß-propellers as a general recognition motif for NxI-containing ligands, and we present indications that the generalized ß-propeller-induced ligand release mechanism is not applicable for the larger LRs. For the giant LR members, we present evidence that their ß-propellers may also actively engage in ligand binding. We therefore advocate for an increased focus on solving the structure-function relationship of this group of important biological receptors.
Assuntos
Receptores de Lipoproteínas/química , Sequência de Aminoácidos , Humanos , Concentração de Íons de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Homologia Estrutural de ProteínaRESUMO
Malaria infected erythrocytes utilize the parasite protein VAR2CSA to bind to a unique presentation of chondroitin sulfate (CS) for their placenta specific tropism. Interestingly, many cancers express a similar form of CS, thereby termed oncofetal CS (ofCS). The distinctive tropism of malaria infected erythrocytes and the identification of oncofetal CS, therefore, represent potentially potent tools for cancer targeting. Here we describe an intriguing drug delivery platform that effectively mimics infected erythrocytes and their specificity for ofCS. We used a lipid catcher-tag conjugation system for the functionalization of erythrocyte membrane-coated drug carriers with recombinant VAR2CSA (rVAR2). We show that these malaria mimicking erythrocyte nanoparticles (MMENPs) loaded with docetaxel (DTX) specifically target and kill melanoma cells in vitro. We further demonstrate effective targeting and therapeutic efficacy in a xenografted melanoma model. These data thus provide a proof of concept for the use of a malaria biomimetic for tumor targeted drug delivery. Given the broad presentation of ofCS found across various types of malignancies, this biomimetic may therefore show potential as a broadly targeted cancer therapy against multiple tumor indications.
Assuntos
Malária Falciparum , Malária , Melanoma , Humanos , Antígenos de Protozoários/metabolismo , Biomimética , Sulfatos de Condroitina/metabolismo , Eritrócitos/metabolismo , Malária Falciparum/metabolismo , Plasmodium falciparumRESUMO
BACKGROUND: The malaria protein VAR2CSA binds oncofetal chondroitin sulfate (ofCS), a unique chondroitin sulfate, expressed on almost all mammalian cancer cells. Previously, we produced a bispecific construct targeting ofCS and human T cells based on VAR2CSA and anti-CD3 (V-aCD3Hu). V-aCD3Hu showed efficacy against xenografted tumors in immunocompromised mice injected with human immune cells at the tumor site. However, the complex effects potentially exerted by the immune system as a result of the treatment cannot occur in mice without an immune system. Here we investigate the efficacy of V-aCD3Mu as a monotherapy and combined with immune checkpoint inhibitors in mice with a fully functional immune system. METHODS: We produced a bispecific construct consisting of a recombinant version of VAR2CSA coupled to an anti-murine CD3 single-chain variable fragment. Flow cytometry and ELISA were used to check cell binding capabilities and the therapeutic effect was evaluated in vitro in a killing assay. The in vivo efficacy of V-aCD3Mu was then investigated in mice with a functional immune system and established or primary syngeneic tumors in the immunologically "cold" 4T1 mammary carcinoma, B16-F10 malignant melanoma, the pancreatic KPC mouse model, and in the immunologically "hot" CT26 colon carcinoma model. RESULTS: V-aCD3Mu had efficacy as a monotherapy, and the combined treatment of V-aCD3Mu and an immune checkpoint inhibitor showed enhanced effects resulting in the complete elimination of solid tumors in the 4T1, B16-F10, and CT26 models. This anti-tumor effect was abscopal and accompanied by a systemic increase in memory and activated cytotoxic and helper T cells. The combined treatment also led to a higher percentage of memory T cells in the tumor without an increase in regulatory T cells. In addition, we observed partial protection against re-challenge in a melanoma model and full protection in a breast cancer model. CONCLUSIONS: Our findings suggest that V-aCD3Mu combined with an immune checkpoint inhibitor renders immunologically "cold" tumors "hot" and results in tumor elimination. Taken together, these data provide proof of concept for the further clinical development of V-aCD3 as a broad cancer therapy in combination with an immune checkpoint inhibitor.
Assuntos
Anticorpos Biespecíficos , Carcinoma , Melanoma Experimental , Humanos , Camundongos , Animais , Sulfatos de Condroitina/farmacologia , Sulfatos de Condroitina/metabolismo , Memória Imunológica , Inibidores de Checkpoint Imunológico , Melanoma Experimental/tratamento farmacológico , Carcinoma/tratamento farmacológico , Anticorpos Biespecíficos/farmacologia , Anticorpos Biespecíficos/uso terapêutico , Linhagem Celular Tumoral , Mamíferos/metabolismoRESUMO
The Streptococcus pyogenes cysteine protease SpeB (streptococcal pyrogenic exotoxin B) is important for the invasive potential of the bacteria, but its production is down-regulated following systemic infection. This prompted us to investigate if SpeB potentiated the host immune response after systemic spreading. Addition of SpeB to human plasma increased plasma-mediated bacterial killing and prolonged coagulation time through the intrinsic pathway of coagulation. This effect was independent of the enzymatic activity of SpeB and was mediated by a non-covalent medium-affinity binding and modification of the serpin A1AT (α-1 antitrypsin). Consequently, addition of A1AT to plasma increased bacterial survival. Sequestration of A1AT by SpeB led to enhanced contact system activation, supported by increased bacterial growth in prekallikrein deficient plasma. In a mouse model of systemic infection, administration of SpeB reduced significantly bacterial dissemination. The findings reveal an additional layer of complexity to host-microbe interactions that may be of benefit in the treatment of severe bacterial infections.
Assuntos
Proteínas de Bactérias/metabolismo , Exotoxinas/metabolismo , alfa 1-Antitripsina/metabolismo , Animais , Proteínas de Bactérias/genética , Quimiotaxia , Exotoxinas/genética , Humanos , Leucócitos Mononucleares , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Ligação Proteica , Infecções Estreptocócicas/metabolismo , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenesRESUMO
Proteoglycans are proteins that are modified with glycosaminoglycan chains. Chondroitin sulfate proteoglycans (CSPGs) are currently being exploited as targets for drug-delivery in various cancer indications, however basic knowledge on how CSPGs are internalized in tumor cells is lacking. In this study we took advantage of a recombinant CSPG-binding lectin VAR2CSA (rVAR2) to track internalization and cell fate of CSPGs in tumor cells. We found that rVAR2 is internalized into cancer cells via multiple internalization mechanisms after initial docking on cell surface CSPGs. Regardless of the internalization pathway used, CSPG-bound rVAR2 was trafficked to the early endosomes in an energy-dependent manner but not further transported to the lysosomal compartment. Instead, internalized CSPG-bound rVAR2 proteins were secreted with exosomes to the extracellular environment in a strictly chondroitin sulfate-dependent manner. In summary, our work describes the cell fate of rVAR2 proteins in tumor cells after initial binding to CSPGs, which can be further used to inform development of rVAR2-drug conjugates and other therapeutics targeting CSPGs.
Assuntos
Proteoglicanas de Sulfatos de Condroitina/metabolismo , Lectinas/metabolismo , Neoplasias/metabolismo , Transporte Proteico , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Endossomos/metabolismo , Exossomos/metabolismo , Humanos , Ligação Proteica , Proteínas Recombinantes/metabolismoRESUMO
Lineage plasticity of prostate cancer is associated with resistance to androgen receptor (AR) pathway inhibition (ARPI) and supported by a reactive tumor microenvironment. Here we show that changes in chondroitin sulfate (CS), a major glycosaminoglycan component of the tumor cell glycocalyx and extracellular matrix, is AR-regulated and promotes the adaptive progression of castration-resistant prostate cancer (CRPC) after ARPI. AR directly represses transcription of the 4-O-sulfotransferase gene CHST11 under basal androgen conditions, maintaining steady-state CS in prostate adenocarcinomas. When AR signaling is inhibited by ARPI or lost during progression to non-AR-driven CRPC as a consequence of lineage plasticity, CHST11 expression is unleashed, leading to elevated 4-O-sulfated chondroitin levels. Inhibition of the tumor cell CS glycocalyx delays CRPC progression, and impairs growth and motility of prostate cancer after ARPI. Thus, a reactive CS glycocalyx supports adaptive survival and treatment resistance after ARPI, representing a therapeutic opportunity in patients with advanced prostate cancer.
Assuntos
Neoplasias de Próstata Resistentes à Castração , Androgênios , Sulfatos de Condroitina , Glicocálix/metabolismo , Humanos , Masculino , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Transdução de Sinais , Microambiente TumoralRESUMO
Placental malaria can have severe consequences for both mother and child and effective vaccines are lacking. Parasite-infected red blood cells sequester in the placenta through interaction between parasite-expressed protein VAR2CSA and the glycosaminoglycan chondroitin sulfate A (CS) abundantly present in the intervillous space. Here, we report cryo-EM structures of the VAR2CSA ectodomain at up to 3.1 Å resolution revealing an overall V-shaped architecture and a complex domain organization. Notably, the surface displays a single significantly electropositive patch, compatible with binding of negatively charged CS. Using molecular docking and molecular dynamics simulations as well as comparative hydroxyl radical protein foot-printing of VAR2CSA in complex with placental CS, we identify the CS-binding groove, intersecting with the positively charged patch of the central VAR2CSA structure. We identify distinctive conserved structural features upholding the macro-molecular domain complex and CS binding capacity of VAR2CSA as well as divergent elements possibly allowing immune escape at or near the CS binding site. These observations will support rational design of second-generation placental malaria vaccines.
Assuntos
Antígenos de Protozoários/química , Antígenos de Protozoários/metabolismo , Sulfatos de Condroitina/metabolismo , Malária Falciparum/complicações , Placenta/parasitologia , Complicações Parasitárias na Gravidez/metabolismo , Complicações Parasitárias na Gravidez/parasitologia , Sequência de Aminoácidos , Antígenos de Protozoários/genética , Microscopia Crioeletrônica , Feminino , Humanos , Evasão da Resposta Imune , Malária Falciparum/metabolismo , Malária Falciparum/parasitologia , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Mutagênese , Placenta/imunologia , Placenta/metabolismo , Plasmodium falciparum/genética , Plasmodium falciparum/imunologia , Plasmodium falciparum/patogenicidade , Gravidez , Ligação Proteica , Domínios ProteicosRESUMO
As an immune evasion and survival strategy, the Plasmodium falciparum malaria parasite has evolved a protein named VAR2CSA. This protein mediates sequestration of infected red blood cells in the placenta through the interaction with a unique carbohydrate abundantly and exclusively present in the placenta. Cancer cells were found to share the same expression of this distinct carbohydrate, termed oncofetal chondroitin sulfate on their surface. In this study we have used a protein conjugation system to produce a bispecific immune engager, V-aCD3, based on recombinant VAR2CSA as the cancer targeting moiety and an anti-CD3 single-chain variable fragment linked to a single-chain Fc as the immune engager. Conjugation of these two proteins resulted in a single functional moiety that induced immune mediated killing of a broad range of cancer cells in vitro and facilitated tumor arrest in an orthotopic bladder cancer xenograft model.
Assuntos
Eritrócitos/metabolismo , Malária Falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Sulfatos de Condroitina/imunologia , Sulfatos de Condroitina/metabolismo , Feminino , Humanos , Malária/imunologia , Malária/metabolismo , Malária Falciparum/imunologia , Placenta/metabolismo , Plasmodium falciparum/metabolismo , Gravidez , Proteínas de Protozoários/imunologia , Proteínas Recombinantes/metabolismoRESUMO
Broad-spectrum therapeutics in non-small cell lung cancer (NSCLC) are in demand. Most human solid tumors express proteoglycans modified with distinct oncofetal chondroitin sulfate (CS) chains that can be detected and targeted with recombinant VAR2CSA (rVAR2) proteins and rVAR2-derived therapeutics. Here, we investigated expression and targetability of oncofetal CS expression in human NSCLC. High oncofetal CS expression is associated with shorter disease-free survival and poor overall survival of clinically annotated stage I and II NSCLC patients (n = 493). Oncofetal CS qualifies as an independent prognosticator of NSCLC in males and smokers, and high oncofetal CS levels are more prevalent in EGFR/KRAS wild-type cases, as compared to mutation cases. NSCLC cell lines express oncofetal CS-modified proteoglycans that can be specifically detected and targeted by rVAR2 proteins in a CSA-dependent manner. Importantly, a novel VAR2-drug conjugate (VDC-MMAE) efficiently eliminates NSCLC cells in vitro and in vivo. In summary, oncofetal CS is a prognostic biomarker and an actionable glycosaminoglycan target in NSCLC.
RESUMO
Vaccines pave the way out of the SARS-CoV-2 pandemic. Besides mRNA and adenoviral vector vaccines, effective protein-based vaccines are needed for immunization against current and emerging variants. We have developed a virus-like particle (VLP)-based vaccine using the baculovirus-insect cell expression system, a robust production platform known for its scalability, low cost, and safety. Baculoviruses were constructed encoding SARS-CoV-2 spike proteins: full-length S, stabilized secreted S, or the S1 domain. Since subunit S only partially protected mice from SARS-CoV-2 challenge, we produced S1 for conjugation to bacteriophage AP205 VLP nanoparticles using tag/catcher technology. The S1 yield in an insect-cell bioreactor was â¼11 mg/liter, and authentic protein folding, efficient glycosylation, partial trimerization, and ACE2 receptor binding was confirmed. Prime-boost immunization of mice with 0.5 µg S1-VLPs showed potent neutralizing antibody responses against Wuhan and UK/B.1.1.7 SARS-CoV-2 variants. This two-component nanoparticle vaccine can now be further developed to help alleviate the burden of COVID-19. IMPORTANCE Vaccination is essential to reduce disease severity and limit the transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Protein-based vaccines are useful to vaccinate the world population and to boost immunity against emerging variants. Their safety profiles, production costs, and vaccine storage temperatures are advantageous compared to mRNA and adenovirus vector vaccines. Here, we use the versatile and scalable baculovirus expression vector system to generate a two-component nanoparticle vaccine to induce potent neutralizing antibody responses against SARS-CoV-2 variants. These nanoparticle vaccines can be quickly adapted as boosters by simply updating the antigen component.
Assuntos
Anticorpos Neutralizantes/metabolismo , Nanopartículas/metabolismo , SARS-CoV-2/metabolismo , Animais , COVID-19/imunologia , Feminino , Glicosilação , Camundongos , Camundongos Endogâmicos BALB C , SARS-CoV-2/imunologia , Células Sf9 , Vacinas Virais/imunologiaRESUMO
The rapid development of a SARS-CoV-2 vaccine is a global priority. Here, we develop two capsid-like particle (CLP)-based vaccines displaying the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. RBD antigens are displayed on AP205 CLPs through a split-protein Tag/Catcher, ensuring unidirectional and high-density display of RBD. Both soluble recombinant RBD and RBD displayed on CLPs bind the ACE2 receptor with nanomolar affinity. Mice are vaccinated with soluble RBD or CLP-displayed RBD, formulated in Squalene-Water-Emulsion. The RBD-CLP vaccines induce higher levels of serum anti-spike antibodies than the soluble RBD vaccines. Remarkably, one injection with our lead RBD-CLP vaccine in mice elicits virus neutralization antibody titers comparable to those found in patients that had recovered from COVID-19. Following booster vaccinations, the virus neutralization titers exceed those measured after natural infection, at serum dilutions above 1:10,000. Thus, the RBD-CLP vaccine is a highly promising candidate for preventing COVID-19.
Assuntos
Anticorpos Neutralizantes/imunologia , Vacinas contra COVID-19/imunologia , Capsídeo/imunologia , Ligação Proteica/imunologia , SARS-CoV-2/imunologia , Enzima de Conversão de Angiotensina 2 , Animais , Anticorpos Antivirais/imunologia , COVID-19/prevenção & controle , Feminino , Humanos , Imunogenicidade da Vacina , Cinética , Camundongos , Camundongos Endogâmicos BALB C , Ligação Proteica/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Testes Sorológicos , Glicoproteína da Espícula de Coronavírus/imunologiaRESUMO
Diffuse gliomas are the most common primary malignant brain tumor. Although extracranial metastases are rarely observed, recent studies have shown the presence of circulating tumor cells (CTCs) in the blood of glioma patients, confirming that a subset of tumor cells are capable of entering the circulation. The isolation and characterization of CTCs could provide a non-invasive method for repeated analysis of the mutational and phenotypic state of the tumor during the course of disease. However, the efficient detection of glioma CTCs has proven to be challenging due to the lack of consistently expressed tumor markers and high inter- and intra-tumor heterogeneity. Thus, for this field to progress, an omnipresent but specific marker of glioma CTCs is required. In this article, we demonstrate how the recombinant malaria VAR2CSA protein (rVAR2) can be used for the capture and detection of glioma cell lines that are spiked into blood through binding to a cancer-specific oncofetal chondroitin sulfate (ofCS). When using rVAR2 pull-down from glioma cells, we identified a panel of proteoglycans, known to be essential for glioma progression. Finally, the clinical feasibility of this work is supported by the rVAR2-based isolation and detection of CTCs from glioma patient blood samples, which highlights ofCS as a potential clinical target for CTC isolation.
Assuntos
Antígenos de Protozoários/farmacologia , Biomarcadores Tumorais/sangue , Neoplasias Encefálicas/diagnóstico , Separação Celular/métodos , Glioma/diagnóstico , Células Neoplásicas Circulantes/metabolismo , Neoplasias Encefálicas/metabolismo , Contagem de Células/métodos , Linhagem Celular Tumoral , Proteoglicanas de Sulfatos de Condroitina/sangue , Glioma/metabolismo , Humanos , Estudo de Prova de Conceito , Proteínas Recombinantes/farmacologiaRESUMO
Plasmodium falciparum engineer infected erythrocytes to present the malarial protein, VAR2CSA, which binds a distinct type chondroitin sulfate (CS) exclusively expressed in the placenta. Here, we show that the same CS modification is present on a high proportion of malignant cells and that it can be specifically targeted by recombinant VAR2CSA (rVAR2). In tumors, placental-like CS chains are linked to a limited repertoire of cancer-associated proteoglycans including CD44 and CSPG4. The rVAR2 protein localizes to tumors in vivo and rVAR2 fused to diphtheria toxin or conjugated to hemiasterlin compounds strongly inhibits in vivo tumor cell growth and metastasis. Our data demonstrate how an evolutionarily refined parasite-derived protein can be exploited to target a common, but complex, malignancy-associated glycosaminoglycan modification.
Assuntos
Antígenos de Protozoários/genética , Sulfatos de Condroitina/metabolismo , Melanoma Experimental/terapia , Placenta/metabolismo , Proteínas Recombinantes/administração & dosagem , Neoplasias Cutâneas/terapia , Animais , Antígenos de Protozoários/metabolismo , Linhagem Celular Tumoral , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Feminino , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Receptores de Hialuronatos/metabolismo , Melanoma Experimental/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Terapia de Alvo Molecular , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Especificidade de Órgãos , Gravidez , Proteínas Recombinantes/farmacologia , Neoplasias Cutâneas/metabolismoRESUMO
The prolactin receptor (PRLR) is activated by binding of prolactin in a 2:1 complex, but the activation mechanism is poorly understood. PRLR has a conserved WSXWS motif generic to cytokine class I receptors. We have determined the nuclear magnetic resonance solution structure of the membrane proximal domain of the human PRLR and find that the tryptophans of the motif adopt a T-stack conformation in the unbound state. By contrast, in the hormone bound state, a Trp/Arg-ladder is formed. The conformational change is hormone-dependent and influences the receptor-receptor dimerization site 3. In the constitutively active, breast cancer-related receptor mutant PRLR(I146L), we observed a stabilization of the dimeric state and a change in the dynamics of the motif. Here we demonstrate a structural link between the WSXWS motif, hormone binding, and receptor dimerization and propose it as a general mechanism for class 1 receptor activation.
Assuntos
Receptores de Citocinas/química , Receptores da Prolactina/química , Motivos de Aminoácidos , Sítios de Ligação , Dicroísmo Circular , Humanos , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Tamanho da Partícula , Prolactina/química , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , TitulometriaRESUMO
Invasive infections of Streptococcus pyogenes are dependent on the cysteine protease streptococcal pyrogenic exotoxin B. Previous structures of the enzyme have not disclosed the proper active-site configuration. Here, the crystal structure of the mature enzyme is presented to 1.55 A, disclosing a homodimer. A serine from one subunit inserts into the active site of the other to donate to the oxyanion hole and coordinates the ligand proximal to the active-site cysteine. Dimerization is unique to the mature form and is clearly a prerequisite for catalysis. The present structure supports a tripartite switch system that is triggered upon dimerization and substrate binding: (1) liberation of the active-site histidine from an inactive configuration, (2) relocation of residues blocking the substrate binding pockets and (3) repositioning of two active-site tryptophans to settle in the active configuration. Based on the present structure, the active site of clan CA cysteine proteases is expanded and a detailed mechanism of the deacylation mechanism is proposed. The results may have applications for the development of protease inhibitors specific to bacterial cysteine proteases.
Assuntos
Proteínas de Bactérias/química , Cisteína Endopeptidases/química , Exotoxinas/química , Multimerização Proteica , Streptococcus pyogenes/enzimologia , Acilação , Domínio Catalítico , Cristalografia por Raios X , Modelos Moleculares , Estrutura Secundária de ProteínaRESUMO
The utility of pseudocontact shifts in the structure refinement of metalloproteins has been evaluated using a native, paramagnetic Cu(2+) metalloprotein, plastocyanin from Anabaena variabilis (A.v.), as a model protein. First, the possibility of detecting signals of nuclei spatially close to the paramagnetic metal ion is investigated using the WEFT pulse sequence in combination with the conventional TOCSY and (1)H-(15)N HSQC sequences. Second, the importance of the electrical charge of the metal ion for the determination of correct pseudocontact shifts from the obtained chemical shifts is evaluated. Thus, using both the Cu(+) plastocyanin and Cd(2+)-substituted plastocyanin as the diamagnetic references, it is found that the Cd(2+)-substituted protein with the same electrical charge of the metal ion as the paramagnetic Cu(2+) plastocyanin provides the most appropriate diamagnetic reference signals. Third, it is found that reliable pseudocontact shifts cannot be obtained from the chemical shifts of the (15)N nuclei in plastocyanin, most likely because these shifts are highly dependent on even minor differences in the structure of the paramagnetic and diamagnetic proteins. Finally, the quality of the obtained (1)H pseudocontact shifts, as well as the possibility of improving the accuracy of the obtained structure, is demonstrated by incorporating the shifts as restraints in a refinement of the solution structure of A.v. plastocyanin. It is found that incorporation of the pseudocontact shifts enhances the precision of the structure in regions with only few NOE restraints and improves the accuracy of the overall structure.