RESUMO
The contact system comprises a series of serine proteases that mediate procoagulant and proinflammatory activities via the intrinsic pathway of coagulation and the kallikrein-kinin system, respectively. Inhibition of Factor XIIa (FXIIa), an initiator of the contact system, has been demonstrated to lead to thrombo-protection and anti-inflammatory effects in animal models and serves as a potentially safer target for the development of antithrombotics. Herein, we describe the use of the Randomised Nonstandard Peptide Integrated Discovery (RaPID) mRNA display technology to identify a series of potent and selective cyclic peptide inhibitors of FXIIa. Cyclic peptides were evaluated in vitro, and three lead compounds exhibited significant prolongation of aPTT, a reduction in thrombin generation, and an inhibition of bradykinin formation. We also describe our efforts to identify the critical residues for binding FXIIa through alanine scanning, analogue generation, and via in silico methods to predict the binding mode of our lead cyclic peptide inhibitors.
Assuntos
Fator XIIa/antagonistas & inibidores , Peptídeos Cíclicos/química , RNA Mensageiro/metabolismo , Inibidores de Serina Proteinase/química , Sítios de Ligação , Fator XIIa/metabolismo , Biblioteca Gênica , Código Genético , Humanos , Concentração Inibidora 50 , Calicreínas/química , Calicreínas/metabolismo , Simulação de Dinâmica Molecular , Tempo de Tromboplastina Parcial , Peptídeos Cíclicos/metabolismo , Estabilidade Proteica , Tempo de Protrombina , Puromicina/química , RNA Mensageiro/química , Inibidores de Serina Proteinase/metabolismo , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Translation is an important point of regulation in protein synthesis. However, there is a limited number of methods available to measure global translation activity in yeast. Recently, O-propargyl-puromycin (OPP) labelling has been established for mammalian cells, but unmodified yeasts are unsusceptible to puromycin. RESULTS: We could increase susceptibility by using a Komagataella phaffii strain with an impaired ergosterol pathway (erg6Δ), but translation measurements are restricted to this strain background, which displayed growth deficits. Using surfactants, specifically Imipramine, instead, proved to be more advantageous and circumvents previous restrictions. Imipramine-supplemented OPP-labelling with subsequent flow cytometry analysis, enabled us to distinguish actively translating cells from negative controls, and to clearly quantify differences in translation activities in different strains and growth conditions. Specifically, we investigated K. phaffii at different growth rates, verified that methanol feeding alters translation activity, and analysed global translation in strains with genetically modified stress response pathways. CONCLUSIONS: We set up a simple protocol to measure global translation activity in yeast on a single cell basis. The use of surfactants poses a practical and non-invasive alternative to the commonly used ergosterol pathway impaired strains and thus impacts a wide range of applications where increased drug and dye uptake is needed.
Assuntos
Imipramina/farmacologia , Puromicina/análogos & derivados , Saccharomycetales/efeitos dos fármacos , Saccharomycetales/genética , Biossíntese de Proteínas , Puromicina/química , Puromicina/metabolismo , Saccharomycetales/metabolismo , Tensoativos/farmacologiaRESUMO
Puromycin and the Streptomyces alboniger-derived puromycin N-acetyltransferase (PAC) enzyme form a commonly used system for selecting stably transfected cultured cells. The crystal structure of PAC has been solved using X-ray crystallography, revealing it to be a member of the GCN5-related N-acetyltransferase (GNAT) family of acetyltransferases. Based on structures in complex with acetyl-CoA or the reaction products CoA and acetylated puromycin, four classes of mutations in and around the catalytic site were designed and tested for activity. Single-residue mutations were identified that displayed a range of enzymatic activities, from complete ablation to enhanced activity relative to wild-type (WT) PAC. Cell pools of stably transfected HEK293 cells derived using two PAC mutants with attenuated activity, Y30F and A142D, were found to secrete up to three-fold higher levels of a soluble, recombinant target protein than corresponding pools derived with the WT enzyme. A third mutant, Y171F, appeared to stabilise the intracellular turnover of PAC, resulting in an apparent loss of selection stringency. Our results indicate that the structure-guided manipulation of PAC function can be utilised to enhance selection stringency for the derivation of mammalian cell lines secreting elevated levels of recombinant proteins.
Assuntos
Acetilcoenzima A/química , Acetiltransferases/ultraestrutura , Proteínas Recombinantes/ultraestrutura , Streptomyces/ultraestrutura , Acetilcoenzima A/genética , Acetilação , Acetiltransferases/química , Acetiltransferases/genética , Animais , Domínio Catalítico/genética , Linhagem Celular , Cristalografia por Raios X , Regulação da Expressão Gênica/genética , Células HEK293 , Humanos , Mutação/genética , Puromicina/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Streptomyces/enzimologiaRESUMO
We developed a cDNA TRAP display for the rapid selection of antibody-like proteins in various conditions. By modifying the original puromycin linker in the TRAP display, a monobody was covalently attached to the cDNA. As a proof-of-concept, we demonstrated a rapid model selection of an anti-EGFR1 monobody in a solution containing ribonuclease.
Assuntos
Anticorpos Monoclonais/química , DNA Complementar/química , Técnicas Biossensoriais , Humanos , Biblioteca de Peptídeos , Ligação Proteica , Puromicina/química , RNA Mensageiro/química , Ribonucleases/química , Sensibilidade e EspecificidadeRESUMO
Puromycin-hydrolizing peptidases have been described as members of the prolyl oligopeptidase peptidase family. These enzymes are present across all domains of life but still little is known of the homologs found in the pathogenic bacterium Mycobacterium tuberculosis. The crystal structure of a M. tuberculosis puromycin hydrolase peptidase has been determined at 3 Angstrom resolution, revealing a conserved prolyl oligopeptidase fold, defined by α/ß-hydrolase and ß-propeller domains with two distinctive loops that occlude access of large substrates to the active site. The enzyme displayed amino peptidase activity with a substrate specificity preference for hydrophobic residues in the decreasing order of phenylalanine, leucine, alanine and proline. The enzyme's active site is lined by residues Glu564 for the coordination of the substrates amino terminal moiety and His561, Val608, Tyr78, Trp306, Phe563 and Ty567 for the accommodation of hydrophobic substrates. The availability of a crystal structure for puromycin hydrolase of M. tuberculosis shall facilitate the development of inhibitors with therapeutic applications.
Assuntos
Aminopeptidases/química , Proteínas de Bactérias/química , Hidrolases/química , Mycobacterium tuberculosis/enzimologia , Prolil Oligopeptidases/química , Puromicina/química , Alanina/química , Alanina/metabolismo , Aminopeptidases/genética , Aminopeptidases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Hidrolases/genética , Hidrolases/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Cinética , Leucina/química , Leucina/metabolismo , Modelos Moleculares , Mycobacterium tuberculosis/química , Fenilalanina/química , Fenilalanina/metabolismo , Prolina/química , Prolina/metabolismo , Prolil Oligopeptidases/genética , Prolil Oligopeptidases/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Puromicina/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
Monitoring translational regulation in response to environmental signals is crucial for understanding cellular proteostasis. However, only limited approaches are currently available for quantifying acute changes in protein synthesis induced by stimuli. Recently, a clickable puromycin analogue, O-propargyl-puromycin (OPP), was developed and applied to label the C-termini of nascent polypeptide chains (NPCs). Following affinity purification via a click reaction, OPP allows for a proteomic analysis of NPCs. Despite its advantage, the affinity purification of NPCs using magnetic beads or resins inherently suffers from significant non-specific protein binding, which hinders accurate quantification of the nascent proteins. To address this issue, we employed dual-pulse labelling of NPCs with both OPP and stable isotope-labelled amino acids to distinguish bona fide NPCs from non-specific proteins, thereby enabling the accurate quantitative profiling of NPCs. We applied this method to dissecting translation responses upon transcriptional inhibition and quantified â¼3,000 nascent proteins. We found that the translation of a subset of ribosomal proteins (e.g. RPSA, RPLP0) as well as signalling proteins (e.g. BCAR3, EFNA1, DUSP1) was significantly repressed by transcription inhibition. Together, the present method provides an accurate and broadly applicable nascent proteome profiling for many biological applications at the level of translation.
Assuntos
Aminoácidos/metabolismo , Marcação por Isótopo/métodos , Espectrometria de Massas/métodos , Proteoma/metabolismo , Proteômica/métodos , Puromicina/análogos & derivados , Aminoácidos/química , Células HeLa , Humanos , Biossíntese de Proteínas , Proteoma/análise , Puromicina/químicaRESUMO
Here, we describe a protocol to detect and visualize protein synthesis by click-chemistry-based immunofluorescence in patient-derived organoids (PDOs) in vitro. The protocol uses O-propargyl puromycin (OPP), an analog of puromycin that enters the acceptor site of ribosomes and is incorporated into nascent polypeptides. OPP can be detected by a click chemistry reaction and can be combined with conventional antibody staining. We describe procedures for imaging intact organoids in 3D format or imaging sections of organoids from paraffin blocks. For complete details on the use and execution of this protocol, please refer to Morral, Stanisavljevic et al. (2020).
Assuntos
Química Click/métodos , Imunofluorescência/métodos , Proteínas/análise , Puromicina/análogos & derivados , Técnicas de Cultura de Células/métodos , Neoplasias Colorretais/metabolismo , Humanos , Organoides/metabolismo , Biossíntese de Proteínas/fisiologia , Puromicina/química , Ribossomos/metabolismo , Coloração e Rotulagem/métodosRESUMO
Puromycin sensitive aminopeptidase (PSA or NPEPPS) is a M1 class aminopeptidase is selectively inhibited by the natural product puromycin, an aminonucleoside antibiotic produced by the bacterium Streptomyces alboniger. The molecular basis for this selective inhibition has not been understood well. Here, we report the basis for selectivity of puromycin using biochemical, structural and molecular modeling tools on four different M1 family enzymes including human PSA. Except for PSA, the other three enzymes were not inhibited. Instead, the peptide bond in the puromycin is hydrolyzed to O-methyl-L-tyrosine (OMT) and puromycin aminonucleoside (PAN). Neither of the hydrolyzed products, individually or together inhibit any of the four enzymes. Crystal structure of ePepN using crystals that are incubated with puromycin contained the hydrolyzed products instead of intact puromycin. On the other hand, intact puromycin molecule was observed in the crystal structure of the inactive mutant ePepN (E298A)-puromycin complex. Surprisingly, puromycin does not enter the active site of the mutant enzyme but binds near the entrance. Comparison of puromycin binding region in ePepN mutant enzyme and molecular modeling studies suggest that PSA might be inhibited by similar mode of binding there by blocking the entrance of the active site.
Assuntos
Modelos Moleculares , Antígeno Prostático Específico/antagonistas & inibidores , Conformação Proteica , Puromicina/química , Sequência de Aminoácidos/genética , Escherichia coli/genética , Humanos , Cinética , Masculino , Antígeno Prostático Específico/química , Antígeno Prostático Específico/genética , Antígeno Prostático Específico/ultraestrutura , Puromicina/farmacologia , Especificidade por Substrato/genéticaRESUMO
Protein synthesis is quickly and tightly regulated in cells to adapt to the ever-changing extracellular and intracellular environment. Accurate quantitation of rapid protein synthesis changes can provide insights into protein functions and cellular activities, but it is very challenging to achieve because of the lack of effective analysis methods. Here, we developed an effective mass spectrometry-based method named quantitative O-propargyl-puromycin tagging (QOT) by integrating O-propargyl-puromycin (OPP) labeling, bioorthogonal chemistry, and multiplexed proteomics for global and quantitative analysis of rapid protein synthesis. The current method enables us to accurately quantitate rapid changes of newly synthesized proteins because, unlike amino acids and their analogs, OPP can be utilized by the ribosome immediately without being activated and conjugated to tRNA, and thus cell starvation or pretreatment is not required. This method was applied to quantitate rapid changes of protein synthesis in THP-1 macrophages treated with lipopolysaccharide (LPS). For 15-min labeling, >3000 proteins were quantitated, and the synthesis of 238 proteins was significantly altered, including transcription factors and cytokines. The results demonstrated that protein synthesis was modulated to facilitate protein secretion in macrophages in response to LPS. Considering the importance of protein synthesis, this method can be extensively applied to investigate rapid changes of protein synthesis in the biological and biomedical research fields.
Assuntos
Proteínas/análise , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Humanos , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Proteínas/síntese química , Proteômica , Puromicina/análogos & derivados , Puromicina/química , Células THP-1RESUMO
Nucleic Acid Aptamers (NAAs) are a class of synthetic DNA or RNA molecules that bind specifically to their target. We recently introduced an aptamer termed R1.2 against membrane Immunoglobulin M (mIgM) expressing B-cell neoplasms using Ligand Guided Selection (LIGS). While LIGS-generated aptamers are highly specific, their lower affinity prevents aptamers from being used for translational applications. Highly specific aptamers with higher affinity can increase targetability, boosting the application of aptamers as diagnostic and therapeutic molecules. Herein, we report that dimerization of R1.2, an aptamer generated from LIGS, leads to high affinity variants without compromising the specificity. Three dimeric aptamer analogues with variable linker lengths were designed to evaluate the effect of linker length in affinity. The optimized dimeric R1.2 against cultured B-cell neoplasms, four donor B-cell samples and mIgM-positive Waldenström's Macroglobulinemia (WM) showed specificity. Furthermore, confocal imaging of dimeric aptamer and anti-IgM antibody in purified B-cells suggests co-localization. Binding assays against IgM knockout Burkitt's Lymphoma cells utilizing CRISPR/Cas9 further validated specificity of dimeric R1.2. Collectively, our findings show that LIGS-generated aptamers can be re-engineered into dimeric aptamers with high specificity and affinity, demonstrating wide-range of applicability of LIGS in developing clinically practical diagnostic and therapeutic aptamers.
Assuntos
Aptâmeros de Nucleotídeos/química , Linfócitos B/metabolismo , Epitopos/química , Linfoma de Burkitt/metabolismo , Sistemas CRISPR-Cas , Células Cultivadas , Dimerização , Células HEK293 , Humanos , Imunoglobulina M/química , Lentivirus/genética , Leucócitos Mononucleares/citologia , Ligantes , Linfoma de Células B/metabolismo , Plasmídeos/metabolismo , Ligação Proteica , Engenharia de Proteínas , Puromicina/química , Técnica de Seleção de Aptâmeros , Temperatura , Macroglobulinemia de Waldenstrom/metabolismoRESUMO
The isolation and structure elucidation of four new naturally occurring amino-nucleoside [puromycins B-E (1-4)] metabolites from a Himalayan isolate ( Streptomyces sp. PU-14-G, isolated from the Bara Gali region of northern Pakistan) is reported. Consistent with prior reports, comparative antimicrobial assays revealed the need for the free 2â³-amine for anti-Gram-positive bacteria and antimycobacterial activity. Similarly, comparative cancer cell line cytotoxicity assays highlighted the importance of the puromycin-free 2â³-amine and the impact of 3'-nucleoside substitution. These studies extend the repertoire of known naturally occurring puromycins and their corresponding SAR. Notably, 1 represents the first reported naturally occurring bacterial puromycin-related metabolite with a 3'- N-amino acid substitution that differs from the 3'- N-tyrosinyl of classical puromycin-type natural products. This discovery suggests the biosynthesis of 1 in Streptomyces sp. PU-14G may invoke a uniquely permissive amino-nucleoside synthetase and/or multiple synthetases and sets the stage for further studies to elucidate, and potentially exploit, new biocatalysts for puromycin chemoenzymatic diversification.
Assuntos
Nucleosídeos/metabolismo , Puromicina/química , Puromicina/isolamento & purificação , Streptomyces/metabolismo , Bactérias Gram-Positivas/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Mycobacterium/efeitos dos fármacos , Paquistão , Puromicina/biossíntese , Puromicina/farmacologiaRESUMO
α-Ketoglutarate (αKG) is a key node in many important metabolic pathways. The αKG analog N-oxalylglycine (NOG) and its cell-permeable prodrug dimethyloxalylglycine (DMOG) are extensively used to inhibit αKG-dependent dioxygenases. However, whether NOG interference with other αKG-dependent processes contributes to its mode of action remains poorly understood. Here we show that, in aqueous solutions, DMOG is rapidly hydrolyzed, yielding methyloxalylglycine (MOG). MOG elicits cytotoxicity in a manner that depends on its transport by monocarboxylate transporter 2 (MCT2) and is associated with decreased glutamine-derived tricarboxylic acid-cycle flux, suppressed mitochondrial respiration and decreased ATP production. MCT2-facilitated entry of MOG into cells leads to sufficiently high concentrations of NOG to inhibit multiple enzymes in glutamine metabolism, including glutamate dehydrogenase. These findings reveal that MCT2 dictates the mode of action of NOG by determining its intracellular concentration and have important implications for the use of (D)MOG in studying αKG-dependent signaling and metabolism.
Assuntos
Aminoácidos Dicarboxílicos/química , Ácidos Cetoglutáricos/química , Transportadores de Ácidos Monocarboxílicos/metabolismo , Trifosfato de Adenosina/química , Animais , Fenômenos Bioquímicos , Bovinos , Linhagem Celular Tumoral , Ciclo do Ácido Cítrico , Perfilação da Expressão Gênica , Glutamina/metabolismo , Humanos , Hidrólise , Concentração Inibidora 50 , Células MCF-7 , Metabolômica , Camundongos , Mitocôndrias/metabolismo , Oxigênio/química , Puromicina/química , Transdução de Sinais , Ácidos Tricarboxílicos/químicaRESUMO
Ribosome profiling, or Ribo-seq, is based on large-scale sequencing of RNA fragments protected from nuclease digestion by ribosomes. Thanks to its unique ability to provide positional information about ribosomes flowing along transcripts, this method can be used to shed light on mechanistic aspects of translation. However, current Ribo-seq approaches lack the ability to distinguish between fragments protected by either ribosomes in active translation or inactive ribosomes. To overcome this possible limitation, we developed RiboLace, a method based on an original puromycin-containing molecule capable of isolating active ribosomes by means of an antibody-free and tag-free pull-down approach. RiboLace is fast, works reliably with low amounts of input material, and can be easily and rapidly applied both in vitro and in vivo, thereby generating a global snapshot of active ribosome footprints at single nucleotide resolution.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Ribossomos/metabolismo , Animais , Linhagem Celular , Humanos , Camundongos , Microesferas , Puromicina/análogos & derivados , Puromicina/síntese química , Puromicina/química , RNA Mensageiro/metabolismoRESUMO
Single-domain antibody (e.g., Nanobody, VHH antibody) is a promising scaffold for therapeutic and diagnostic reagents. To expand the range of target molecules, in vitro selection using cell-free display technologies such as cDNA display is useful and powerful because of their huge libraries and robust stability. We provide technical details for in vitro selection of single-domain antibodies using cDNA display.
Assuntos
Técnicas de Visualização da Superfície Celular/métodos , DNA Complementar/metabolismo , Anticorpos de Domínio Único/metabolismo , Animais , Reagentes de Ligações Cruzadas/química , DNA Complementar/genética , Luz , Puromicina/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Coelhos , Transcrição GênicaRESUMO
Probing biomolecular motion beyond a single nucleotide is technically challenging but fundamentally significant. We have developed super-resolution force spectroscopy (SURFS) with 0.5 pN force resolution and revealed that the ribosome moves by half a nucleotide upon the formation of the pre-translocation complex, which is beyond the resolution of other techniques.
Assuntos
Movimento (Física) , RNA Mensageiro/química , Ribossomos/química , Sondas de DNA/química , Nucleotídeos/genética , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/genética , Conformação Proteica , Puromicina/química , RNA Mensageiro/genética , Ribossomos/genética , Espectinomicina/química , Análise Espectral/instrumentação , Análise Espectral/métodos , Ondas UltrassônicasRESUMO
Hydrogen peroxide (H2O2) is a central reactive oxygen species (ROS) that contributes to diseases from obesity to cancer to neurodegeneration but is also emerging as an important signaling molecule. We now report a versatile histochemical approach for detection of H2O2 that can be employed across a broad range of cell and tissue specimens in both healthy and disease states. We have developed a first-generation H2O2-responsive analogue named Peroxymycin-1, which is based on the classic cell-staining molecule puromycin and enables covalent staining of biological samples and retains its signal after fixation. H2O2-mediated boronate cleavage uncages the puromycin aminonucleoside, which leaves a permanent and dose-dependent mark on treated biological specimens that can be detected with high sensitivity and precision through a standard immunofluorescence assay. Peroxymycin-1 is selective and sensitive enough to image both exogenous and endogenous changes in cellular H2O2 levels and can be exploited to profile resting H2O2 levels across a panel of cell lines to distinguish metastatic, invasive cancer cells from less invasive cancer and nontumorigenic counterparts, based on correlations with ROS status. Moreover, we establish that Peroxymycin-1 is an effective histochemical probe for in vivo H2O2 analysis, as shown through identification of aberrant elevations in H2O2 levels in liver tissues in a murine model of nonalcoholic fatty liver disease, thus demonstrating the potential of this approach for studying disease states and progression associated with H2O2. This work provides design principles that should enable development of a broader range of histochemical probes for biological use that operate via activity-based sensing.
Assuntos
Peróxido de Hidrogênio/análise , Hepatopatia Gordurosa não Alcoólica/diagnóstico por imagem , Puromicina/análogos & derivados , Puromicina/análise , Puromicina/química , Animais , Dieta Hiperlipídica/efeitos adversos , Corantes Fluorescentes/química , Células HeLa , Histocitoquímica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Estrutura Molecular , Hepatopatia Gordurosa não Alcoólica/induzido quimicamente , Coloração e Rotulagem , Células Tumorais CultivadasRESUMO
Accurate measurement of global and specific protein synthesis rates is becoming increasingly important, especially in the context of biotechnological applications such as process modeling or selection of production cell clones. While quantification of total protein translation across whole cell populations is easily achieved, methods that are capable of tracking population dynamics at the single-cell level are still lacking. To address this need, we apply O-propargyl-puromycin (OPP) labeling to assess total protein synthesis in single recombinant Chinese hamster ovary (CHO) cells by flow cytometry. Thereby we demonstrate that global protein translation rates slightly increase with progression through the cell cycle during exponential growth. Stable CHO cell lines producing recombinant protein display similar levels of total protein synthesis as their parental CHO host cell line. Global protein translation does not correlate with intracellular product content of three model proteins, but the host cell line with high transient productivity has a higher OPP signal. This indicates that production cell lines with increased overall protein synthesis capacity can be identified by our method at the single-cell level. In conclusion, OPP-labeling allows rapid and reproducible assessment of global protein synthesis in single CHO cells, and can be multiplexed with DNA staining or any type of immunolabeling of specific proteins or markers for organelles.
Assuntos
Células Clonais/citologia , Puromicina/análogos & derivados , Proteínas Recombinantes/análise , Análise de Célula Única/métodos , Animais , Células CHO , Células Clonais/metabolismo , Cricetulus , Citometria de Fluxo , Biossíntese de Proteínas , Puromicina/química , Proteínas Recombinantes/químicaRESUMO
Substantial progress has been described in the study of puromycin and its analogs for antibiotic properties. However, the peptidase inhibitory activity of related analogs has not been explored as extensively. Specifically, inhibiting aminopeptidases for achieving antitumor effect has been sparsely investigated. Herein, we address this challenge by reporting the synthesis of a series of analogs based on the structural template of puromycin. We also present exhaustive biochemical and in vitro analyses in support of our thesis. Analyzing the structure-activity relationship revealed a steric requirement for maximum potency. Effective inhibitors of Puromycin-Sensitive Aminopeptidase (PSA) are disclosed here. These potential therapeutic agents display superior in vitro antitumor potency against two leukemic cell lines, as compared to known inhibitors of aminopeptidases.
Assuntos
Aminopeptidases/antagonistas & inibidores , Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Neoplasias Hematológicas/tratamento farmacológico , Puromicina/farmacologia , Aminopeptidases/metabolismo , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Células HL-60 , Neoplasias Hematológicas/metabolismo , Neoplasias Hematológicas/patologia , Humanos , Estrutura Molecular , Puromicina/síntese química , Puromicina/química , Relação Estrutura-Atividade , Células VeroRESUMO
We have developed a versatile antibody-assisted strategy for the imaging and profiling of newly synthesized proteomes in a cell-specific manner. This strategy remained highly selective even in heterogeneous co-cultured cells, thus enabling labeling and enrichment of nascent proteomes from targeted cells without the need for physical separation.
Assuntos
Imagem Molecular , Proteoma/análise , Proteoma/biossíntese , Puromicina/análogos & derivados , Puromicina/análise , Animais , Anticorpos/imunologia , Células CHO , Linhagem Celular Tumoral , Técnicas de Cocultura , Cricetulus , Humanos , Camundongos , Células NIH 3T3 , Proteoma/química , Puromicina/síntese química , Puromicina/químicaRESUMO
BACKGROUND/AIM: Organ transplant patients treated with the immunosuppressive drug cyclosporine A often present malignant kidney tumors. Cyclosporine A can promote oncogenesis in a cell-intrinsic manner by increasing the production of vascular endothelial growth factor (VEGF). MATERIALS AND METHODS: We explored the impact of cyclosporine A and the role of the unfolded protein response (UPR) on three human renal cell carcinoma (RCC) cell lines under normoxic and hypoxic (1% O2) conditions. RESULTS: Cyclosporine A regulated the expression of VEGF at the post-transcriptional level. Cyclosporine A induced the inositol requiring enzyme-1α (IRE1α) arm of the UPR and stabilized neosynthesized proteins in RCC cells. Toyocamycin, an inhibitor of IRE1α, abolished the clonogenic growth of RCC cells and reduced induction of VEGF by cyclosporine A under hypoxia. CONCLUSION: Our findings highlight the impact of cyclosporine A on the proteostasis of RCC cells, and suggest the potential therapeutic interest of targeting the UPR against tumors arising in the context of organ transplantation.