RESUMO
BACKGROUND AND AIMS: Pancreatic cancer is one of the most lethal malignancies, partly due to resistance to conventional chemotherapy. The chemoresistance of malignant tumors is associated with epithelial-mesenchymal transition (EMT) and the stemness of cancer cells. The aim of this study is to investigate the availability and functional mechanisms of trefoil factor family 1 (TFF1), a tumor-suppressive protein in pancreatic carcinogenesis, to treat pancreatic cancer. METHODS: To investigate the role of endogenous TFF1 in human and mice, specimens of human pancreatic cancer and genetically engineered mouse model of pancreatic cancer (KPC/TFF1KO; Pdx1-Cre/LSL-KRASG12D/LSL-p53R172H/TFF1-/-) were analyzed by immunohistochemistry (IHC). To explore the efficacy of extracellular administration of TFF1, recombinant and chemically synthesized TFF1 were administered to pancreatic cancer cell lines, a xenograft mouse model and a transgenic mouse model. RESULTS: The deficiency of TFF1 was associated with increased EMT of cancer cells in mouse models of pancreatic cancer, KPC. The expression of TFF1 in cancer cells was associated with better survival rate of the patients who underwent chemotherapy, and loss of TFF1 deteriorated the benefit of gemcitabine in KPC mice. Extracellular administration of TFF1 inhibited gemcitabine-induced EMT, Wnt pathway activation and cancer stemness, eventually increased apoptosis of pancreatic cancer cells in vitro. In vivo, combined treatment of gemcitabine and subcutaneous administration of TFF1 arrested tumor growth in xenograft mouse model and resulted in the better survival of KPC mice by inhibiting EMT and cancer stemness. CONCLUSION: These results indicate that TFF1 can contribute to establishing a novel strategy to treat pancreatic cancer patients by enhancing chemosensitivity.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Transição Epitelial-Mesenquimal , Células-Tronco Neoplásicas , Neoplasias Pancreáticas , Fator Trefoil-1 , Animais , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/genética , Fator Trefoil-1/metabolismo , Fator Trefoil-1/genética , Humanos , Camundongos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Linhagem Celular Tumoral , Proteínas Supressoras de Tumor/metabolismo , Proteínas Supressoras de Tumor/genética , Ensaios Antitumorais Modelo de Xenoenxerto , Gencitabina , Camundongos Transgênicos , Feminino , Masculino , Proliferação de Células/efeitos dos fármacos , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Desoxicitidina/uso terapêutico , Apoptose/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacosRESUMO
Chemical protein synthesis can provide well-defined modified proteins. Herein, we report the chemical synthesis of plant-derived cysteine-rich secretory proteins and late-stage derivatization of the synthetic proteins. The syntheses were achieved with distinct chemoselective amide bond forming reactions - EPF2 by native chemical ligation (NCL), epidermal patterning factor (EPF) 1 by the α-ketoacid-hydroxylamine (KAHA) ligation, and fluorescent functionalization of their folded variants by potassium acyltrifluoroborate (KAT) ligation. The chemically synthesized EPFs exhibit bioactivity on stomatal development in Arabidopsis thaliana. Comprehensive synthesis of EPF derivatives allowed us to identify suitable fluorescent variants for bioimaging of the subcellar localization of EPFs.
RESUMO
The synthesis of secreted cysteine-rich proteins (CRPs) is a long-standing challenge due to protein aggregation and premature formation of inter- and intramolecular disulfide bonds. Chemical synthesis provides reduced CRPs with a higher purity, which is advantageous for folding and isolation. Herein, we report the chemical synthesis of pollen tube attractant CRPs Torenia fournieri LURE (TfLURE) and Torenia concolor LURE (TcLURE) and their chimeric analogues via α-ketoacid-hydroxylamine (KAHA) ligation. The bioactivity of chemically synthesized TfLURE protein was shown to be comparable to E. coli expressed recombinant protein through in vitro assay. The convergent protein synthesis approach is beneficial for preparing these small protein variants efficiently.
RESUMO
Oryza longistaminata, a wild rice, vegetatively reproduces and forms a networked clonal colony consisting of ramets connected by rhizomes. Although water, nutrients, and other molecules can be transferred between ramets via the rhizomes, inter-ramet communication in response to spatially heterogeneous nitrogen availability is not well understood. We studied the response of ramet pairs to heterogeneous nitrogen availability using a split hydroponic system that allowed each ramet root to be exposed to different conditions. Ammonium uptake was compensatively enhanced in the sufficient-side root when roots of the ramet pairs were exposed to ammonium-sufficient and ammonium-deficient conditions. Comparative transcriptome analysis revealed that a gene regulatory network for effective ammonium assimilation and amino acid biosynthesis was activated in the sufficient-side roots. Allocation of absorbed nitrogen from the nitrogen-sufficient to the nitrogen-deficient ramets was rather limited. Nitrogen was preferentially used for newly growing axillary buds on the sufficient-side ramets. Biosynthesis of trans-zeatin (tZ), a cytokinin, was upregulated in response to the nitrogen supply, but tZ appeared not to target the compensatory regulation. Our results also implied that the O. longistaminata putative ortholog of rice (Oryza sativa) C-terminally encoded peptide1 plays a role as a nitrogen-deficient signal in inter-ramet communication, providing compensatory upregulation of nitrogen assimilatory genes. These results provide insights into the molecular basis for efficient growth strategies of asexually proliferating plants growing in areas where the distribution of ammonium ions is spatially heterogeneous.
Assuntos
Compostos de Amônio , Oryza , Compostos de Amônio/metabolismo , Citocininas/metabolismo , Perfilação da Expressão Gênica , Nitrogênio/metabolismo , Oryza/genética , Oryza/metabolismo , Raízes de Plantas/metabolismoRESUMO
E2 conjugating enzymes are the key catalytic actors in the transfer of ubiquitin, SUMO, and other ubiquitin-like modifiers to their substrate proteins. Their high rates of transfer and promiscuous binding complicate studies of their interactions and binding partners. To access specific, covalently linked conjugates of the SUMO E2 conjugating enzyme Ubc9, we prepared synthetic variants bearing site-specific non-native modifications including the following: (1) replacement of Cys93 to 2,3-diaminopropionic acid to form the amide-linked stable E2-SUMO conjugate, which is known to have high affinity for E3 ligases; (2) a photoreactive group (diazirine) to trap E3 ligases upon UV irradiation; and (3) an N-terminal biotin for purification and detection. To construct these Ubc9 variants in a flexible, convergent manner, we combined the three leading methods: native chemical ligation (NCL), α-ketoacid-hydroxylamine (KAHA) ligation, and serine/threonine ligation (STL). Using the synthetic proteins, we demonstrated the selective formation of Ubc9-SUMO conjugates and the trapping of an E3 ligase (RanBP2) to form the stable, covalently linked SUMO1-Ubc9-RanBP2 ternary complex. The powerful combination of ligation methods-which minimizes challenges of functional group manipulations-will enable chemical probes based on E2 conjugating enzymes to trap E3 ligases and facilitate the synthesis of other protein classes.
Assuntos
Sumoilação , Enzimas de Conjugação de Ubiquitina/síntese química , Células HEK293 , Humanos , Chaperonas Moleculares/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismoRESUMO
Palladium-catalyzed dehydration of primary amides to nitriles efficiently proceeds under mild, aqueous conditions via the use of dichloroacetonitrile as a water acceptor. A key to the design of this transfer dehydration catalysis is the identification of an efficient water acceptor, dichloroacetonitrile, that preferentially reacts with amides over other polar functional groups with the aid of the Pd catalyst and makes the desired scheme exergonic, thereby driving the dehydration.
RESUMO
Compounds targeting the circadian clock have been identified as potential treatments for clock-related diseases, including cancer. Our cell-based phenotypic screen revealed uncharacterized clock-modulating compounds. Through affinity-based target deconvolution, we identified GO289, which strongly lengthened circadian period, as a potent and selective inhibitor of CK2. Phosphoproteomics identified multiple phosphorylation sites inhibited by GO289 on clock proteins, including PER2 S693. Furthermore, GO289 exhibited cell type-dependent inhibition of cancer cell growth that correlated with cellular clock function. The x-ray crystal structure of the CK2α-GO289 complex revealed critical interactions between GO289 and CK2-specific residues and no direct interaction of GO289 with the hinge region that is highly conserved among kinases. The discovery of GO289 provides a direct link between the circadian clock and cancer regulation and reveals unique design principles underlying kinase selectivity.
Assuntos
Carcinoma de Células Renais/metabolismo , Proliferação de Células/efeitos dos fármacos , Relógios Circadianos/efeitos dos fármacos , Ritmo Circadiano/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Neoplasias Renais/metabolismo , Animais , Proteínas CLOCK/metabolismo , Carcinoma de Células Renais/patologia , Caseína Quinase II/antagonistas & inibidores , Linhagem Celular Tumoral , Cristalografia por Raios X , Células HEK293 , Humanos , Neoplasias Renais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fosforilação/efeitos dos fármacosRESUMO
Lipopolysaccharide (LPS) is a potent virulence factor of pathogenic Gram-negative bacteria. To better understand the role of LPS in host-pathogen interactions and to elucidate the antigenic and immunogenic properties of LPS inner core region, a collection of well-defined L-glycero-D-manno-heptose (Hep) and 3-deoxy-α-D-manno-oct-2-ulosonic acid (Kdo)-containing inner core oligosaccharides is required. To address this need, we developed a diversity-oriented approach based on a common orthogonal protected disaccharide Hep-Kdo. Utilizing this new approach, we synthesized a range of LPS inner core oligosaccharides from a variety of pathogenic bacteria including Y. pestis, H. influenzae, and Proteus that cause plague, meningitis, and severe wound infections, respectively. Rapid access to these highly branched core oligosaccharides relied on elaboration of the disaccharide Hep-Kdo core as basis for the elongation with various flexible modules including unique Hep and 4-amino-4-deoxy-ß-L-arabinose (Ara4N) monosaccharides and branched Hep-Hep disaccharides. A regio- and stereoselective glycosylation of Kdo 7,8-diol was key to selective installation of the Ara4N moiety at the 8-hydroxyl group of Kdo moiety of the Hep-Kdo disaccharide. The structure of the LPS inner core oligosaccharides was confirmed by comparison of (1)H NMR spectra of synthetic antigens and isolated fragments. These synthetic LPS core oligosaccharides can be covalently bound to carrier proteins via the reducing end pentyl amine linker, to explore their antigenic and immunogenic properties as well as potential applications such as diagnostic tools and vaccines.
Assuntos
Bactérias Gram-Negativas/química , Lipopolissacarídeos/síntese química , Oligossacarídeos/síntese química , Fatores de Virulência/química , Antígenos de Bactérias/química , Proteínas de Transporte/química , Cromatografia em Camada Fina , Mapeamento de Epitopos , Haemophilus influenzae/química , Espectroscopia de Ressonância Magnética , Proteus/química , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrofotometria Ultravioleta , Yersinia pestis/químicaRESUMO
D-(+)-Camphor forms the enamine 2 with piperidine. Compound 2 adds HB(C(6)F(5))(2) at the enamine carbon atom C3 to form a Lewis acid/Lewis base adduct (exo-/endo-isomers of 3). Exposure of 3 to dihydrogen (2.5 bar, room temperature) leads to heterolytic splitting of H(2) to form the H(+)/H(-) addition products (4, two diastereoisomers) of the "invisible" frustrated Lewis pairs (5, two diastereoisomers) that were apparently generated in situ by enamine hydroboration under equilibrium conditions.
RESUMO
We disclosed structural dynamics hidden behind a series of aminoorganoboron (AOB) compounds that involved a recombinant of covalent, dative, and hydrogen bonds. A combination process occurred via reorganizing elements and bonds between the two major structures (open and closed), which were chemically switchable through precise adjustment of either acidic or basic conditions. The structural dynamics favoring an open structure seem to be more important for catalysis, as represented by methanolysis of activated amides.