RESUMO
Tumors contain various stromal cells, such as immune cells, endothelial cells, and fibroblasts, which contribute to the development of a tumor-specific microenvironment characterized by hypoxia and inflammation, and are associated with malignant progression. In this study, we investigated the activity of intratumoral hypoxia-inducible factor (HIF), which functions as a master regulator of the cellular response to hypoxia and inflammation. We constructed the HIF activity-monitoring reporter gene hypoxia-response element-Venus-Akaluc (HVA) that expresses the green fluorescent protein Venus and modified firefly luciferase Akaluc in a HIF activity-dependent manner, and created transgenic mice harboring HVA transgene (HVA-Tg). In HVA-Tg, HIF-active cells can be visualized using AkaBLI, an ultra-sensitive in vivo bioluminescence imaging technology that produces an intense near-infrared light upon reaction of Akaluc with the D-luciferin analog AkaLumine-HCl. By orthotopic transplantation of E0771, a mouse triple negative breast cancer cell line without a reporter gene, into HVA-Tg, we succeeded in noninvasively monitoring bioluminescence signals from HIF-active stromal cells as early as 8 days after transplantation. The HIF-active stromal cells initially clustered locally and then spread throughout the tumors with growth. Immunohistochemistry and flow cytometry analyses revealed that CD11b+ F4/80+ macrophages were the predominant HIF-active stromal cells in E0771 tumors. These results indicate that HVA-Tg is a useful tool for spatiotemporal analysis of HIF-active tumor stromal cells, facilitating investigation of the roles of HIF-active tumor stromal cells in tumor growth and malignant progression.
Assuntos
Células Endoteliais , Neoplasias , Camundongos , Animais , Células Estromais , Hipóxia , Hipóxia Celular , Inflamação , Imagem Óptica , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Linhagem Celular Tumoral , Microambiente TumoralRESUMO
Targeting less abundant amino acid residues on the protein surface may realize site-selective protein modification of natural proteins. The relative hydrophobicity of tyrosine combined with the π-π stacking tendency of the aromatic rings results in generally low accessibility. In this study, site-selective protein modification was achieved by targeting surface-exposed tyrosine residues without using a genetic encoding system. Tyrosine residues were modified with N-methylated luminol derivative under single-electron transfer (SET) reaction conditions. Horseradish peroxidase (HRP)-catalyzed SET and electrochemically activated SET modified surface-exposed tyrosine residues selectively. N-Methylated luminol derivative modified tyrosine residues more efficiently than 4-arylurazole under tyrosine click conditions using HRP and electrochemistry. Tyrosine residues that are evolutionarily exposed only in the complementarity-determining region (CDR) of an antibody were selectively modified by tyrosine click reactions. CDR-modified antibodies were applied to in vivo imaging and antibody-drug conjugated (ADC).
Assuntos
Proteínas/química , Tirosina/química , Animais , Sítios de Ligação , Biocatálise , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Química Click , Transporte de Elétrons , Peroxidase do Rábano Silvestre/metabolismo , Humanos , Luminol/química , Masculino , Metilação , Camundongos , Modelos Moleculares , Conformação ProteicaRESUMO
HIF-1 is regarded as a promising target for the drugs used in cancer chemotherapy, and creating readily accessible templates for the development of synthetic drug candidates that could inhibit HIF-1 transcriptional activity is an important pursuit. In this study, indeno[2,1-c]pyrazolones were designed as readily available synthetic inhibitors of HIF-1 transcriptional activity. Nine compounds were synthesized in 4-5 steps from commercially available starting materials. In evaluations of the ability to inhibit the hypoxia-induced transcriptional activity of HIF-1, compound 3c showed a higher level compared with that of known inhibitor, YC-1. The compound 3c suppressed HIF-1α protein accumulation without affecting the levels of HIF-1α mRNA.
Assuntos
Desenho de Fármacos , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Pirazolonas/farmacologia , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células HeLa , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Estrutura Molecular , Pirazolonas/síntese química , Pirazolonas/química , Relação Estrutura-Atividade , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/genéticaRESUMO
Lung metastasis is a major cause of mortality in patients with osteosarcoma (OS). A better understanding of the molecular mechanism of OS lung metastasis may facilitate development of new therapeutic strategies to prevent the metastasis. We have established high- and low-metastatic sublines (LM8-H and LM8-L, respectively) from Dunn OS cell line LM8 by using in vivo image-guided screening. Among the genes whose expression was significantly increased in LM8-H compared to LM8-L, the transcription factor lymphoid enhancer-binding factor 1 (LEF1) was identified as a factor that promotes LM8-H cell extravasation into the lungs. To identify downstream effectors of LEF1 that are involved in OS lung metastasis, 13 genes were selected based on LM8 microarray data and genomewide meta-analysis of a public database for OS patients. Among them, the cytoglobin (Cygb) gene was identified as a key effector in promoting OS extravasation into the lungs. CYGB overexpression increased the extravasation ability of LM8-L cells, whereas knocking out the Cygb gene in LM8-H cells reduced this ability. Our results showed a novel LEF1-CYGB axis in OS lung metastasis and may provide a new way of developing therapeutic strategies to prevent OS lung metastasis.
Assuntos
Neoplasias Ósseas/patologia , Globinas/fisiologia , Neoplasias Pulmonares/secundário , Fator 1 de Ligação ao Facilitador Linfoide/fisiologia , Osteossarcoma/patologia , Animais , Linhagem Celular Tumoral , Citoglobina , Globinas/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3HRESUMO
We report here a new class of collagen-binding peptides, cyclic collagen-mimetic peptides (cCMPs), that efficiently hybridize with the triple-helix-forming portions of collagen. cCMPs are composed of two parallel collagen-like (Xaa-Yaa-Gly)n strands with both termini tethered by covalent linkages. Enzyme-linked immunosorbent assays and western blotting analysis showed that cCMPs exhibit more potent affinity toward collagen than reported collagen-binding peptides and can specifically detect different collagen polypeptides in a mixture of proteins. Collagen secreted from cultured cells was detected by confocal microscopy with fluorescein-labeled cCMP. The cCMP is also shown to detect sensitively folding intermediates in the endoplasmic reticulum, something that was difficult to visualize with conventional collagen detectors. Molecular-dynamics simulations suggested that a cCMP forms a more stably hybridized product than its single-chain counterpart; this could explain why cCMP has higher affinity toward denatured collagen. These results indicate the usefulness of cCMPs as tools for detecting denatured collagen.
Assuntos
Colágeno/análise , Fluoresceína/química , Corantes Fluorescentes/química , Peptídeos Cíclicos/química , Sequência de Aminoácidos , Animais , Western Blotting , Linhagem Celular , Ensaio de Imunoadsorção Enzimática , Camundongos , Microscopia ConfocalRESUMO
Pancreatic cancer is one of the most lethal digestive system cancers with a 5-year survival rate of 4-7%. Despite extensive efforts, recent chemotherapeutic regimens have provided only limited benefits to pancreatic cancer patients. Gemcitabine and TS-1, the current standard-of-care chemotherapeutic drugs for treatment of this severe cancer, have a low response rate. Hypoxia is one of the factors contributing to treatment resistance. Specifically, overexpression of hypoxia-inducible factor, a master transcriptional regulator of cell adaption to hypoxia, is strongly correlated with poor prognosis in many human cancers. TAT-ODD-procaspase-3 (TOP3) is a protein prodrug that is specifically processed and activated in hypoxia-inducible factor-active cells in cancers, leading to cell death. Here, we report combination therapies in which TOP3 was combined with gemcitabine or TS-1. As monotherapy, gemcitabine and TS-1 showed a limited effect on hypoxic and starved pancreatic cancer cells, whereas co-treatment with TOP3 successfully overcame this limitation in vitro. Furthermore, combination therapies of TOP3 with these drugs resulted in a significant improvement in survival of orthotopic pancreatic cancer models involving the human pancreatic cancer cell line SUIT-2. Overall, our study indicates that the combination of TOP3 with current chemotherapeutic drugs can significantly improve treatment outcome, offering a promising new therapeutic option for patients with pancreatic cancer.
Assuntos
Desoxicitidina/análogos & derivados , Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Pró-Fármacos/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Silicatos/farmacologia , Titânio/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Linhagem Celular Tumoral , Desoxicitidina/administração & dosagem , Desoxicitidina/farmacologia , Fluoruracila/farmacologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pró-Fármacos/farmacologia , Proteínas Recombinantes de Fusão/farmacologia , Silicatos/administração & dosagem , Análise de Sobrevida , Titânio/administração & dosagem , Hipóxia Tumoral , GencitabinaRESUMO
Bone metastasis is a multistep process that includes cancer cell dissemination, colonization, and metastatic growth. Furthermore, this process involves complex, reciprocal interactions between cancer cells and the bone microenvironment. Bone resorption is known to be involved in both osteolytic and osteoblastic bone metastasis. However, the precise roles of the bone resorption in the multistep process of osteoblastic bone metastasis remain unidentified. In this study, we show that bone resorption plays important roles in cancer cell colonization during the initial stage of osteoblastic bone metastasis. We applied bioluminescence/X-ray computed tomography multimodal imaging that allows us to spatiotemporally analyze metastasized cancer cells and bone status in osteoblastic bone metastasis models. We found that treatment with receptor activator of factor-κB ligand (RANKL) increased osteoblastic bone metastasis when given at the same time as intracardiac injection of cancer cells, but failed to increase metastasis when given 4 days after cancer cell injection, suggesting that RANKL-induced bone resorption facilitates growth of cancer cells colonized in the bone. We show that insulin-like growth factor-1 released from the bone during bone resorption and hypoxia-inducible factor activity in cancer cells cooperatively promoted survival and proliferation of cancer cells in bone marrow. These results suggest a mechanism that bone resorption and hypoxic stress in the bone microenvironment cooperatively play an important role in establishing osteoblastic metastasis.
Assuntos
Neoplasias Ósseas/secundário , Reabsorção Óssea/metabolismo , Reabsorção Óssea/patologia , Fator 1 Induzível por Hipóxia/metabolismo , Osteossarcoma/secundário , Ligante RANK/farmacologia , Somatomedinas/metabolismo , Animais , Células da Medula Óssea/patologia , Hipóxia Celular , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Ativação Enzimática , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transplante de Neoplasias , Fator de Crescimento Transformador beta/metabolismo , Transplante Heterólogo , Microambiente TumoralRESUMO
Migrasomes are extracellular vesicles that form on the retraction fibers of migrating cells. In this study, we report the formation of migrasome-like vesicles enriched in tetraspanin 4 and containing cytoplasmic components in response to hypoosmotic stress. When migrating cells were subjected to hypoosmotic stress, vesicles with a size distribution of 0.5 to 2 µm formed on the retraction fibers, and vanished in a few minutes. The vesicles are rich in cholesterol, and their number was reduced when cells were pretreated with lipoprotein-deficient serum. The formation of migrasome-like vesicles upon hypoosmotic stress may provide biophysical cues regarding the cellular response to this external stimulus in cells and tissues.
Assuntos
Organelas , Pressão Osmótica , Citoplasma , CitosolRESUMO
Epitope binning, an approach for grouping antibodies based on epitope similarities, is a critical step in antibody drug discovery. However, conventional methods are complex, involving individual antibody production. Here, we established Epitope Binning-seq, an epitope binning platform for simultaneously analyzing multiple antibodies. In this system, epitope similarity between the query antibodies (qAbs) displayed on antigen-expressing cells and a fluorescently labeled reference antibody (rAb) targeting a desired epitope is analyzed by flow cytometry. The qAbs with epitope similar to the rAb can be identified by next-generation sequencing analysis of fluorescence-negative cells. Sensitivity and reliability of this system are confirmed using rAbs, pertuzumab and trastuzumab, which target human epidermal growth factor receptor 2. Epitope Binning-seq enables simultaneous epitope evaluation of 14 qAbs at various abundances in libraries, grouping them into respective epitope bins. This versatile platform is applicable to diverse antibodies and antigens, potentially expediting the identification of clinically useful antibodies.
Assuntos
Epitopos , Humanos , Epitopos/imunologia , Análise de Sequência de DNA/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Animais , Receptor ErbB-2/imunologia , Receptor ErbB-2/genética , Citometria de Fluxo/métodos , Trastuzumab/imunologia , Mapeamento de Epitopos/métodos , Anticorpos/imunologia , Anticorpos/genética , Anticorpos Monoclonais Humanizados/imunologiaRESUMO
Myeloid-derived suppressor cell (MDSC)-like adherent cells (MLACs) are a recently identified CD11b+ F4/80- myeloid cell subset that can infiltrate tumors early in development and promote their growth. Because of these functions, MLACs play an important role in establishing an immunosuppressive tumor microenvironment (TME). However, the lack of MLAC-specific markers has hampered further characterization of this cell type. This study identifies the gene signature of MLACs by analyzing RNA-sequencing (RNA-seq) and public single-cell RNA-seq data, revealing that MLACs are an independent cell population that are distinct from other intratumoral myeloid cells. After combining proteome analysis of membrane proteins with RNA-seq data, H2-Ab1 and CD11c are indicated as marker proteins that can support the isolation of MLAC subsets from CD11b+ F4/80- myeloid cells by fluorescence-activated cell sorting. The CD11b+ F4/80- H2-Ab1+ and CD11b+ F4/80- CD11c+ MLAC subsets represent approximately half of the MLAC population that is isolated based on their adhesion properties and possess gene signatures and functional properties similar to those of the MLAC population. Additionally, membrane proteome analysis suggests that MLACs express highly heterogeneous surface proteins. This study facilitates an integrated understanding of heterogeneous intratumoral myeloid cells, as well as the molecular and cellular details of the development of an immunosuppressive TME.
Assuntos
Células Supressoras Mieloides , Células Supressoras Mieloides/metabolismo , Proteoma/metabolismo , Células Mieloides , Citometria de Fluxo , Linhagem Celular TumoralRESUMO
Triple-negative breast cancer (TNBC) is an aggressive and highly heterogenous disease with no well-defined therapeutic targets. Treatment options are thus limited and mortality is significantly higher compared with other breast cancer subtypes. Mammary gland tissue-resident macrophages (MGTRMs) are found to be the most abundant stromal cells in early TNBC before angiogenesis. We therefore aimed to explore novel therapeutic approaches for TNBC by focusing on MGTRMs. Local depletion of MGTRMs in mammary gland fat pads the day before TNBC cell transplantation significantly reduced tumor growth and tumor-associated macrophage (TAM) infiltration in mice. Furthermore, local depletion of MGTRMs at the site of TNBC resection markedly reduced recurrence and distant metastases, and improved chemotherapy outcomes. This study demonstrates that MGTRMs are a major TAM resource and play pivotal roles in the growth and malignant progression of TNBC. The results highlight a possible novel anti-cancer approach targeting tissue-resident macrophages.
Assuntos
Neoplasias de Mama Triplo Negativas , Humanos , Animais , Camundongos , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Macrófagos Associados a Tumor , Linhagem Celular TumoralRESUMO
Cancer cells inevitably interact with neighboring host tissue-resident cells during the process of metastatic colonization, establishing a metastatic niche to fuel their survival, growth, and invasion. However, the underlying mechanisms in the metastatic niche are yet to be fully elucidated owing to the lack of methodologies for comprehensively studying the mechanisms of cell-cell interactions in the niche. Here, we improve a split green fluorescent protein (GFP)-based genetically encoded system to develop secretory glycosylphosphatidylinositol-anchored reconstitution-activated proteins to highlight intercellular connections (sGRAPHIC) for efficient fluorescent labeling of tissue-resident cells that neighbor on and putatively interact with cancer cells in deep tissues. The sGRAPHIC system enables the isolation of metastatic niche-associated tissue-resident cells for their characterization using a single-cell RNA sequencing platform. We use this sGRAPHIC-leveraged transcriptomic platform to uncover gene expression patterns in metastatic niche-associated hepatocytes in a murine model of liver metastasis. Among the marker genes of metastatic niche-associated hepatocytes, we identify Lgals3, encoding galectin-3, as a potential pro-metastatic factor that accelerates metastatic growth and invasion.
Assuntos
Neoplasias Hepáticas , Humanos , Camundongos , Animais , Neoplasias Hepáticas/metabolismo , Hepatócitos/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Matriz Extracelular/metabolismo , Comunicação CelularRESUMO
We developed a novel near-infrared (NIR) fluorescent probe, GPU-167, for in vivo imaging of tumor hypoxia. GPU-167 comprises a tricarbocyanine dye as an NIR fluorophore and two 2-nitroimidazole moieties as exogenous hypoxia markers that undergo bioreductive activation and then selective entrapment in hypoxic cells. After treatment with GPU-167, tumor cells contained significantly higher levels of fluorescence in hypoxia than in normoxia. In vivo fluorescence imaging specifically detected GPU-167 in tumors 24 h after administration. Ex vivo analysis revealed that fluorescence showed a strong correlation with hypoxia inducible factor (HIF)-1 active hypoxic regions. These data suggest that GPU-167 is a promising in vivo optical imaging probe for tumor hypoxia.
Assuntos
Carbocianinas/química , Hipóxia Celular , Corantes Fluorescentes/química , Neoplasias/patologia , Nitroimidazóis/química , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Animais , Camundongos , Camundongos NusRESUMO
A near-infrared fluorochrome, GPU-311, was designed, synthesized and evaluated for its application in non-invasive imaging of tumor hypoxia. Efficient synthesis was achieved by nucleophilic substitution and click chemistry ring using the bifunctional tetraethylene glycol linker 2 containing thiol and azide groups for the conjugation of the propargylated nitroimidazole 1 and the heptamethine cyanine dye 3 bearing a 2-chloro-1-cyclohexenyl ring. GPU-311 exhibited long excitation and emission wavelength (Ex/Em=785/802 nm) and a decent quantum yield (0.05). The water solubility and hydrophilicity of GPU-311 increased. After in vitro treatment of SUIT-2/HRE-Luc pancreatic cancer cells with GPU-311, a higher level of fluorescence was observed selectively in hypoxia than in normoxia. However, in vivo fluorescence imaging of a mouse xenograft model after GPU-311 administration revealed inadequate accumulation of GPU-311 in tumors due to its rapid elimination through the liver.
Assuntos
Corantes Fluorescentes/síntese química , Neoplasias/diagnóstico , Animais , Carbocianinas/química , Hipóxia Celular , Linhagem Celular Tumoral , Fluorescência , Corantes Fluorescentes/química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Camundongos , Neoplasias/metabolismo , Neoplasias/patologia , Nitroimidazóis/química , Solubilidade , Espectroscopia de Luz Próxima ao Infravermelho/métodosRESUMO
In this study, we proposed a droplet-based valveless microfluidic system that has the necessary functions to perform the binding, washing, eluting, and collecting processes of phage-display screening against spheroids, which can be expected to present a similar repertoire and number of membrane proteins as in vivo. Although spheroids have much larger sizes than single cells, spheroids are difficult to manipulate through manual operation. The proposed microfluidic system actively controls the position and velocity of droplets using a camera, three air pumps, and three liquid pumps to perform the processes for phage-display screening. The cross section of the microchannel is large in width and height for the passage of spheroids. Valves that can close such a large cross-sectional microchannel are not readily available. Thus, we proposed valveless flow control using liquid pumps. In addition, the proposed microfluidic system involves complex flow channels with airflow subchannels to perform phage-display screening. For washing, nonspecific-binding phages remaining in the flow channels must be minimized. The proposed microfluidic system can perform selective blocking and flush washing. Selective blocking can prevent the airflow channels from becoming hydrophilic with blocking liquid, and flush washing can flush phages remaining in the flow channel. We experimentally verified the functions of the developed microfluidic device based on the proposed system.
RESUMO
Cancer recurrence due to tumor cell quiescence after therapy and long-term remission is associated with cancer-related death. Previous studies have used cell models that are unable to return to a proliferative state; thus, the transition between quiescent and proliferative states is not well understood. Here, we report monolayer cancer cell models wherein the human non-small cell lung carcinoma cell line H2228 and pancreatic cancer cell line AsPC-1 can be reversibly induced to a quiescent state under hypoxic and serum-starved (HSS) conditions. Transcriptome and metabolome dual-omics profiles of these cells were compared with those of the human lung adenocarcinoma cell line A549, which was unable to enter a quiescent state under HSS conditions. The quiescence-inducible cells had substantially lower intracellular pyruvate and ATP levels in the quiescent state than in the proliferative state, and their response to sudden demand for energy was dramatically reduced. Furthermore, in quiescence-inducible cells, the transition between quiescent and proliferative states of these cells was regulated by the balance between the proliferation-promoting Ras and Rap1 signaling and the suppressive AGE/RAGE signaling. These cell models elucidate the transition between quiescent and proliferative states, allowing the development of drug-screening systems for quiescent tumor cells.
Assuntos
Quinase do Linfoma Anaplásico , Antígenos de Neoplasias , Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Proteínas Quinases Ativadas por Mitógeno , Neoplasias Pancreáticas , Receptor para Produtos Finais de Glicação Avançada , Células A549 , Quinase do Linfoma Anaplásico/genética , Quinase do Linfoma Anaplásico/metabolismo , Antígenos de Neoplasias/metabolismo , Hipóxia Celular , Proliferação de Células/genética , Proliferação de Células/fisiologia , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/metabolismo , Recidiva Local de Neoplasia/patologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Receptores Proteína Tirosina Quinases/metabolismo , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Transdução de Sinais , Neoplasias PancreáticasRESUMO
The microenvironment of solid tumors is characterized by low pO(2) that is well below physiological levels. Intratumoral hypoxia is a major factor contributing to cancer progression and is exacerbated as a result of oxygen consumption by rapidly proliferating tumor cells near blood vessels, poor lymphatic drainage resulting in high interstitial pressure, and irregular blood supply through immature tumor vasculature. Hypoxia-inducible factor-1 (HIF-1) is the main transcription factor that regulates cellular responses to hypoxia. Cellular changes induced by HIF-1 are extremely important targets for cancer therapy. Therefore, targeting strategies to counteract HIF-1-active cells are essential for cancer therapy. In this study, we introduce a novel strategy for targeting HIF-1-active cells.
Assuntos
Descoberta de Drogas/métodos , Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Hipóxia/fisiopatologia , Terapia de Alvo Molecular/métodos , Neoplasias/tratamento farmacológico , Microambiente Tumoral/efeitos dos fármacos , Animais , Citostáticos/farmacologia , Citostáticos/uso terapêutico , Humanos , Modelos Biológicos , Imagem Molecular/métodos , Oxirredução , Pró-Fármacos/farmacologia , Transplante HeterólogoRESUMO
Optimisation of protein binders relies on laborious screening processes. Investigation of sequence-function relationships of protein binders is particularly slow, since mutants are purified and evaluated individually. Here we developed peptide barcoding, a high-throughput approach for accurate investigation of sequence-function relationships of hundreds of protein binders at once. Our approach is based on combining the generation of a mutagenised nanobody library fused with unique peptide barcodes, the formation of nanobody-antigen complexes at different ratios, their fine fractionation by size-exclusion chromatography and quantification of peptide barcodes by targeted proteomics. Applying peptide barcoding to an anti-GFP nanobody as a model, we successfully identified residues important for the binding affinity of anti-GFP nanobody at once. Peptide barcoding discriminated subtle changes in KD at the order of nM to sub-nM. Therefore, peptide barcoding is a powerful tool for engineering protein binders, enabling reliable one-pot evaluation of sequence-function relationships.
Assuntos
Proteínas de Fluorescência Verde/metabolismo , Fragmentos de Peptídeos/metabolismo , Engenharia de Proteínas/métodos , Anticorpos de Domínio Único/metabolismo , Proteínas de Fluorescência Verde/genética , Humanos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Biblioteca de Peptídeos , Ligação Proteica , Proteômica , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/genética , Anticorpos de Domínio Único/imunologiaRESUMO
Triangular Au nanoplates (TrAuNPls) possessing strong plasmonic properties can be used as photothermal agents in cancer therapy. However, the controlled preparation of such morphologies typically requires harsh synthetic conditions. Biomolecules offer an alternative route to developing biocompatible synthetic protocols. In particular, peptides offer a novel route for inorganic synthesis under ambient conditions. Herein, using the previously isolated peptide, ASHQWAWKWE, for Au nanoparticle (AuNP) synthesis, the conditions for preparing TrAuNPls via a one-pot synthetic process of mixing HAuCl4 and peptides at room temperature were investigated to effectively obtain particles possessing near-infrared absorbance for non-invasive optical diagnosis and phototherapy. By adjusting the peptide concentration, the size and property of TrAuNPls were controlled under neutral pH conditions. The synthesised particles showed potential as photothermal therapeutic agents in vitro. In addition, peptide characterisation using B3 derivatives revealed the importance of the third amino acid histidine in morphological regulation and potential circular Au nanoplates (AuNPl) synthesis with ASEQWAWKWE and ASAQWAWKWE peptides. These findings provide not only an easy and green synthetic method for TrAuNPls and circular AuNPls, but also some insight to help elucidate the regulation of peptide-based nanoparticle synthesis for use in cancer therapy. STATEMENT OF SIGNIFICANCE: Biological molecules have received increasing attention as a vehicle to synthesise inorganic materials with specific properties under ambient conditions; particularly, short peptides have the potential to control the synthesis of nanoscale materials with tailored functions. Here, the application of a previously isolated peptide was assessed in synthesising Au nanoparticles containing decahedral and triangular nanoplates with near-infrared absorbance. The size and absorbance peaks of the triangular nanoplates observed were peptide concentration-dependent. In addition, these fine-tuned triangular nanoplates exhibited potential as a phototherapeutic agent. Moreover, the peptide derivatives indicated the possibility of synthesising circular nanoplates. These findings may offer insight into development of new techniques for synthesising functional nanoparticles having biological applications using non-toxic molecules under mild conditions stituted in the original B3 peptide is underlined.
Assuntos
Ouro , Nanopartículas Metálicas , Peptídeos , FototerapiaRESUMO
Small antibody mimetics that contain high-affinity target-binding peptides can be lower cost alternatives to monoclonal antibodies (mAbs). We have recently developed a method to create small antibody mimetics called FLuctuation-regulated Affinity Proteins (FLAPs), which consist of a small protein scaffold with a structurally immobilized target-binding peptide. In this study, to further develop this method, we established a novel screening system for FLAPs called monoclonal antibody-guided peptide identification and engineering (MAGPIE), in which a mAb guides selection in two manners. First, antibody-guided design allows construction of a peptide library that is relatively small in size, but sufficient to identify high-affinity binders in a single selection round. Second, in antibody-guided screening, the fluorescently labeled mAb is used to select mammalian cells that display FLAP candidates with high affinity for the target using fluorescence-activated cell sorting. We demonstrate the reliability and efficacy of MAGPIE using daclizumab, a mAb against human interleukin-2 receptor alpha chain (CD25). Three FLAPs identified by MAGPIE bound CD25 with dissociation constants of approximately 30 nM as measured by biolayer interferometry without undergoing affinity maturation. MAGPIE can be broadly adapted to any mAb to develop small antibody mimetics.