RESUMO
Glioma is a primary, malignant, and aggressive brain tumor in adults. To develop new therapeutic strategies for glioma, we must determine its underlying mechanisms. In the present study, we aimed to investigate the potential role of miR-1272-ADAM9-CDCP1 signaling in the progression of glioma. We found that ectopic expression of miR-1272 produced significant inhibitory effects on cell proliferation and migration and was associated with cell cycle G0/G1 arrest in A172 and SHG44 glioma cells. Using the luciferase reporter assay, we identified ADAM9 as a target of miR-1272. The expression of ADAM9 was markedly decreased or increased after overexpression or inhibition, respectively, of miR-1272 in glioma cells. Moreover, overexpression of ADAM9 reversed the inhibitory effects of miR-1272 on glioma cell progression. Furthermore, CDCP1 served as a potential downstream molecule of miR-1272/ADAM9 signaling in glioma and promoted the proliferation and migration of glioma. Results derived from clinical samples and online databases confirmed correlations between the expression of ADAM9 and CDCP1 and both the severity and prognosis of glioma. In conclusion, these results suggest that miR-1272 and CDCP1 may act as novel regulators in glioma. The miR-1272/ADAM9/CDCP1 pathway may serve as a potential candidate pathway for the prevention of glioma.
Assuntos
Proteínas ADAM/genética , Antígenos de Neoplasias/genética , Neoplasias Encefálicas/genética , Moléculas de Adesão Celular/genética , Glioma/genética , Proteínas de Membrana/genética , MicroRNAs/metabolismo , Proteínas ADAM/metabolismo , Antígenos de Neoplasias/metabolismo , Astrocitoma/genética , Astrocitoma/metabolismo , Astrocitoma/patologia , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Moléculas de Adesão Celular/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Progressão da Doença , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patologia , Glioma/metabolismo , Glioma/patologia , Humanos , Proteínas de Membrana/metabolismo , MicroRNAs/genética , Transdução de SinaisRESUMO
Accumulated evidence reveals that microRNAs play vital roles in various tumors, including gliomas. MiRNAs have been shown to participate in multiple cellular functions, including cell proliferation, migration and apoptosis. Here, we investigate the potential role of a novel miRNA, miR-6807-3p, in glioma. A quantitative reverse transcription-polymerase chain reaction (RT-qPCR) and western blot were applied to detect the expression of miR-6807-3p and its target molecule in glioma specimens and cultured cells. The direct targets of miR-6807-3p were predicted by bioinformatics software and were further verified by a luciferase reporter assay. The effects of miR-6807-3p on glioma cell proliferation, migration, cell apoptosis and the cell cycle of glioma cells were analyzed by the Cell-Counting Kit-8 (CCK-8) assay, a cell migration assay and flow cytometry assays. MiR-6807-3p was found to promote tumor growth and migration and inhibits apoptosis and cell cycle arrest in vitro, thus playing a tumor oncogenic role in the progression of glioma. Expression levels of miR-6807-3p were greatly upregulated in glioma specimens, and dachshund homolog 1 (DACH1) was ascertained as a direct target of miR-6807-3p, modulated by the expression of miR-6807-3p in glioma cells. Aberrant expression of DACH1 was associated with the clinical survival of glioma patients. Furthermore, overexpression of DACH1 rescued the promotive effects of miR-6807-3p in glioma. Based on these findings, a novel miR-6807-3p may act as a glioma enhancer by targeting DACH1.
Assuntos
Neoplasias Encefálicas/genética , Proteínas do Olho/genética , Glioma/genética , MicroRNAs/genética , Fatores de Transcrição/genética , Apoptose/fisiologia , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Carcinogênese/genética , Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Proteínas do Olho/biossíntese , Proteínas do Olho/metabolismo , Glioma/metabolismo , Glioma/patologia , Humanos , MicroRNAs/metabolismo , Fatores de Transcrição/biossíntese , Fatores de Transcrição/metabolismo , Regulação para CimaRESUMO
BACKGROUND AND OBJECTIVE: Glioma is one of the most aggressive and malignant cancers originating from the human brain. Increasing evidence suggests that aberrant expression of microRNAs (miRNAs) frequently occurs in glioma and miRNAs are critical regulators of glioma. miR-671 has recently been revealed to be a novel miRNA that plays a vital role in human glioblastoma multiforme. However, the functional role and underlying mechanisms of miR-671-3p require further analysis. MATERIALS AND METHODS: Western blot and fluorescence quantitative PCR were used to assess the expression of cytoskeleton-associated protein 4 (CKAP4) and miR-671-3p, respectively. A Cell Counting Kit-8 (CCK-8) assay and a Boyden chamber assay were used to detect the proliferative and migratory abilities of glioma cells. A luciferase assay was used to determine the target gene of miR-671-3p. Apoptosis was analyzed by flow cytometry. RESULTS: Our results revealed that overexpression of miR-671-3p promoted cell proliferation and migration in vitro. Meanwhile, forced expression of miR-671-3p reduced apoptosis. In contrast, inhibition of miR-671-3p had the opposite effects. We also identified CKAP4 to be a direct target of miR-671-3p. The expression levels of CKAP4 were decreased in clinical samples and inversely correlated with miR-671-3p expression levels. Ectopic expression of CKAP4 reversed the promotive activity of miR-671-3p in the proliferation and migration and enhanced apoptosis. CONCLUSION: Our study demonstrates that miR-671-3p is a predominant positive regulator of glioma progression, thus providing new insights into the molecular mechanisms of glioma development. The findings suggest that the miR-6713p/CKAP4 axis may serve as a potential therapeutic target or biomarker in glioma.
RESUMO
Secreted proteins encoded by mutated genes (mutant proteins) are a particularly rich source of biomarkers being not only components of the cancer secretome but also actually implicated in tumorigenesis. One of the challenges of proteomics-driven biomarker discovery research is that the bulk of secreted mutant proteins cannot be identified directly and quantified by mass spectrometry due to the lack of mutated peptide information in extant proteomics databases. Here we identify, using an integrated genomics and proteomics strategy (referred to iMASp - identification of Mutated And Secreted proteins), 112 putative mutated tryptic peptides (corresponding to 57 proteins) in the collective secretomes derived from a panel of 18 human colorectal cancer (CRC) cell lines. Central to this iMASp was the creation of Human Protein Mutant Database (HPMD), against which experimentally-derived secretome peptide spectra were searched. Eight of the identified mutated tryptic peptides were confirmed by RT-PCR and cDNA sequencing of RNA extracted from those CRC cells from which the mutation was identified by mass spectrometry. The iMASp technology promises to improve the link between proteomics and genomic mutation data thereby providing an effective tool for targeting tryptic peptides with mutated amino acids as potential cancer biomarker candidates. This article is part of a Special Issue entitled: Integrated omics.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias do Colo/metabolismo , Mutação , Proteínas de Neoplasias/metabolismo , Proteoma/metabolismo , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Bases de Dados de Proteínas , Humanos , Espectrometria de Massas , Proteínas de Neoplasias/genética , Proteoma/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Plasma membrane (PM) proteins are attractive therapeutic targets because of their accessibility to drugs. Although genes encoding PM proteins represent 20-30% of eukaryotic genomes, a detailed characterisation of their encoded proteins is underrepresented, due, to their low copy number and the inherent difficulties in their isolation and purification as a consequence of their high hydrophobicity. We describe here a strategy that combines two orthogonal methods to isolate and purify PM proteins from Madin Darby canine kidney (MDCK) cells. In this two-step method, we first used cationic colloidal silica (CCS) to isolate adherent (Ad) and non-adherent (nAd) PM fractions, and then subjected each fraction to Triton X-114 (TX-114) phase partitioning to further enrich for hydrophobic proteins. While CCS alone identified 255/757 (34%) membrane proteins, CCS/TX-114 in combination yielded 453/745 (61%). Strikingly, of those proteins unique to CCS/TX-114, 277/393 (70%) had membrane annotation. Further characterisation of the CCS/TX-114 data set using Uniprot and transmembrane hidden Markov model revealed that 306/745 (41%) contained one or more transmembrane domains (TMDs), including proteins with 25 and 17 TMDs. Of the remaining proteins in the data set, 69/439 (16%) are known to contain lipid modifications. Of all membrane proteins identified, 93 had PM origin, including proteins that mediate cell adhesion, modulate transmembrane ion transport, and cell-cell communication. These studies reveal that the application of CCS to first isolate Ad and nAd PM fractions, followed by their detergent-phase TX-114 partitioning, to be a powerful method to isolate low-abundance PM proteins, and a useful adjunct for in-depth cell surface proteome analyses.
Assuntos
Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/isolamento & purificação , Membrana Celular/genética , Proteínas de Membrana/genética , Proteínas de Membrana/isolamento & purificação , Proteômica/métodos , Animais , Western Blotting , Cátions/metabolismo , Moléculas de Adesão Celular/metabolismo , Fracionamento Celular , Membrana Celular/química , Membrana Celular/metabolismo , Células Cultivadas , Coloides/metabolismo , Bases de Dados de Proteínas , Cães , Feminino , Interações Hidrofóbicas e Hidrofílicas , Transporte de Íons/genética , Rim/citologia , Espectrometria de Massas , Proteínas de Membrana/metabolismo , Octoxinol , Polietilenoglicóis/metabolismo , Análise Serial de Proteínas/métodos , Estrutura Terciária de Proteína , Proteoma/química , Proteoma/genética , Proteoma/metabolismo , Dióxido de Silício/metabolismoRESUMO
Integral membrane proteins (IMPs) mediate several cellular functions including cell adhesion, ion and nutrient transport, and cell signalling. IMPs are typically hard to isolate and purify due to their hydrophobic nature and low cellular abundance, however, microsomes are small lipid vesicles rich in IMPs, which form spontaneously when cells are mechanically disrupted. In this study, we have employed mouse liver microsomes as a model for optimising a method for IMP isolation and characterisation. Microsomes were collected by differential centrifugation, purified with sodium carbonate, and subjected to GeLC-MS/MS analysis. A total of 1124 proteins were identified in the microsome fraction, with 47% (524/1124) predicted by TMHMM to contain at least one transmembrane domain (TMD). The ability of phase partitioning using the detergent Triton X-114 (TX-114) to further enrich for membrane proteins was evaluated. Microsomes were subjected to successive rounds of solubility-based phase separation, with proteins partitioning into the aqueous phase, detergent phase, or TX-114-insoluble pellet fraction. GeLC-MS/MS analysis of the three TX-114 fractions identified 1212 proteins, of which 146 were not detected in the un-fractionated microsome sample. Conspicuously, IMPs partitioned to the detergent phase, with 56% (435/770) of proteins identified in that fraction containing at least one TMD. GO Slim characterisation of the microsome proteome revealed enrichment of proteins from the endoplasmic reticulum, mitochondria, Golgi apparatus, endosome, and cytoplasm. Further, enzymes including monooxygenases were well represented with 35 cytochrome P450 identifications (CYPs 1A2, 2A5, 2A12, 2B10, 2C29, 2C37, 2C39, 2C44, 2C50, 2C54. 2C67, 2C68, 2C70, 2D10, 2D11, 2D22, 2D26, 2D9, 2E1, 2F2, 2J5, 2U1, 3A11, 3A13, 3A25, 4A10, 4A12A, 4A12B, 4F13, 4F14, 4F15, 4V3, 51,7B1, and 8B1). Evaluation of biological processes showed enrichment of proteins involved in fatty acid biosynthesis and elongation, as well as steroid synthesis. In addition, transport proteins including 24 members of the Rab family of GTPases were identified. Comparison of this dataset with the current mouse liver microsome proteome contributes an additional 648 protein identifications, of which 50% (326/648) contain at least one TMD.
Assuntos
Fracionamento Químico/métodos , Proteínas de Membrana/isolamento & purificação , Microssomos Hepáticos/metabolismo , Polietilenoglicóis/química , Animais , Camundongos , Octoxinol , ProteomaRESUMO
Epithelial-mesenchymal transition (EMT) describes a process whereby polarized epithelial cells with restricted migration transform into elongated spindle-shaped mesenchymal cells with enhanced motility and invasiveness. Although there are some molecular markers for this process, including the down-regulation of E-cadherin, our understanding of plasma membrane (PM) and associated proteins involved in EMT is limited. To specifically explore molecular alterations occurring at the PM, we used the cationic colloidal silica isolation technique to purify PM fractions from epithelial Madin-Darby canine kidney cells during Ras/TGF-ß-mediated EMT. Proteins in the isolated membrane fractions were separated by one-dimensional SDS-PAGE and subjected to nano-LC-MS/MS-based protein identification. In this study, the first membrane protein analysis of an EMT model, we identified 805 proteins and determined their differential expression using label-free spectral counting. These data reveal that Madin-Darby canine kidney cells switch from cadherin-mediated to integrin-mediated adhesion following Ras/TGF-ß-mediated EMT. Thus, during the EMT process, E-cadherin, claudin 4, desmoplakin, desmoglein-2, and junctional adhesion molecule A were down-regulated, whereas integrins α6ß1, α3ß1, α2ß1, α5ß1, αVß1, and αVß3 along with their extracellular ligands collagens I and V and fibronectin had increased expression levels. Conspicuously, Wnt-5a expression was elevated in cells undergoing EMT, and transient Wnt-5a siRNA silencing attenuated both cell migration and invasion in these cells. Furthermore, Wnt-5a expression suppressed canonical Wnt signaling induced by Wnt-3a. Wnt-5a may act through the planar cell polarity pathway of the non-canonical Wnt signaling pathway as several of the components and modulators (Wnt-5a, -5b, frizzled 6, collagen triple helix repeat-containing protein 1, tyrosine-protein kinase 7, RhoA, Rac, and JNK) were found to be up-regulated during Ras/TGF-ß-mediated EMT.
Assuntos
Membrana Celular/metabolismo , Epitélio/metabolismo , Rim/metabolismo , Mesoderma/metabolismo , Proteômica/métodos , Fator de Crescimento Transformador beta/metabolismo , Proteínas Wnt/metabolismo , Proteínas ras/metabolismo , Animais , Movimento Celular , Cromatografia Líquida/métodos , Coloides/química , Cães , Espectrometria de Massas/métodos , Microscopia de Fluorescência/métodos , Proteoma , Transdução de SinaisRESUMO
Epithelial-mesenchymal transition (EMT) describes a process whereby immotile epithelial cells escape structural constraints imposed by cellular architecture and acquire a phenotype characteristic of migratory mesenchymal cells. Implicated in carcinoma progression and metastasis, EMT has been the focus of several recent proteomics-based studies aimed at identifying new molecular players. To gain insights into extracellular mediators associated with EMT, we conducted an extensive proteomic analysis of the secretome from MDCK cells following oncogenic Ras-induced EMT (21D1 cells). Using Orbitrap technology and a label-free quantitative approach, differential expression of several secreted modulators were revealed. Proteomic findings were further substantiated by mRNA transcript expression analysis with 71% concordance. MDCK cells undergoing Ras-induced EMT remodel the extracellular matrix (ECM) via diminished expression of basement membrane constituents (collagen type IV and laminin 5), up-regulation of extracellular proteases (MMP-1, kallikreins -6 and -7), and increased production and secretion of ECM constituents (SPARC, collagen type I, fibulins -1 and -3, biglycan, and decorin). Collectively, these findings suggest that hierarchical regulation of a subset of extracellular effectors may coordinate a biological response during EMT that enhances cell motility. Transient silencing of MMP-1 in 21D1 cells via siRNA-mediated knockdown attenuated cell migration. Many of the secretome proteins identified broaden our understanding of the EMT process.
Assuntos
Movimento Celular , Células Epiteliais/citologia , Matriz Extracelular/metabolismo , Mesoderma/citologia , Proteína Oncogênica p21(ras)/fisiologia , Animais , Transformação Celular Neoplásica , Células Cultivadas , Cães , Perfilação da Expressão Gênica , Microscopia Confocal , Microscopia de Fluorescência , Proteína Oncogênica p21(ras)/genética , RNA Mensageiro/genéticaRESUMO
OBJECTIVE: To examine the effects of Han's acupoint and nerve stimulator (HANS) electroacupuncture on the expression of NPY in periaqueductal grey (PAG) of heroin addicted rats. METHODS: Heroin was injected subcutaneously according to the principle of daily increasing dose in rats of experimented group. The ability of special learning and memory were tested by Morris water maze; The expression of NPY in PAG of rat were detected by immunohistochemistry. RESULTS: (1) Escape latency and searching distance in heroin-addiction group were significantly increased compared with those of normal group during the place navigation test (P < 0.05). However, in acupuncture group, escape latency and searching distance was obviously shortened compared with those of heroin-addiction group (P < 0.05). The exploring time and distance of original platform area in proportion to the total distance in heroin-addiction group significantly decreased compared with those of normal group during spatial probe test (P < 0.05). The exploring time and distance of original platform area in proportion to the total distance in acupuncture group was increased compared with those in heroin-addiction group (P < 0.01). (2) The expression of NPY of heroin-addiction group was lower than that in normal group in PAG, while those of acupuncture group was higher than that in the heroin-addiction group (P < 0.05). CONCLUSION: The learning and memory induced by heroin-addiction could be reversed and the expression of NPY in PAG was increased by HANS in rats.
Assuntos
Eletroacupuntura , Dependência de Heroína/metabolismo , Neuropeptídeo Y/metabolismo , Substância Cinzenta Periaquedutal/metabolismo , Animais , Masculino , Aprendizagem em Labirinto , Memória , Ratos , Ratos WistarRESUMO
Dipeptide-derived compounds, bearing various P2 residues and a diaryl pyrrolidin-2-yl phosphonate at the P1 position, were evaluated as dipeptidyl peptidase 8 (DPP8) inhibitors. With these products, irreversible inhibition of DPP8 was observed. To obtain inhibitors with an improved activity and selectivity profile, a set of selected analogues containing a diaryl isoindolin-1-ylphosphonate at P1 was synthesized and evaluated. Within this latter series, compound 2e was shown to be a potent, irreversible inhibitor of DPP8, demonstrating very low affinity for DPP IV and DPP II.
Assuntos
Dipeptidases/antagonistas & inibidores , Dipeptídeos/síntese química , Isoindóis/síntese química , Organofosfonatos/síntese química , Pirrolidinas/síntese química , Dipeptidases/química , Dipeptídeos/química , Isoindóis/química , Cinética , Organofosfonatos/química , Pirrolidinas/química , Relação Estrutura-AtividadeRESUMO
DPP8 belongs to the family of prolyl dipeptidases, which are capable of cleaving the peptide bond after a penultimate proline residue. Unlike DPP-IV, a drug target for type II diabetes, no information is available on the crystal structure of DPP8, the regulation of its enzymatic activity, or its substrate specificity. In this study, using analytical ultracentrifugation and native gel electrophoresis, we show that the DPP8 protein is predominantly dimeric when purified or in the cell extracts. Four conserved residues in the C-terminal loop of DPP8 (Phe(822), Val(833), Tyr(844), and His(859)), corresponding to those located at the dimer interface of DPP-IV, were individually mutated to Ala. Surprisingly, unlike DPP-IV, these single-site mutations abolished the enzymatic activity of DPP8 without disrupting its quaternary structure, indicating that dimerization itself is not sufficient for the optimal enzymatic activity of DPP8. Moreover, these mutations not only decreased k(cat), as did the corresponding DPP-IV mutations, but also dramatically increased K(m). We further show that the K(m) effect is independent of the substrate assayed. Finally, we identified the distinctive and strict substrate selectivity of DPP8 for hydrophobic or basic residues at the P2 site, which is in sharp contrast to the much less discriminative substrate specificity of DPP-IV. Our study has identified the residues absolutely required for the optimal activity of DPP8 and its unique substrate specificity. This study extends the functional importance of the C-terminal loop to the whole family of prolyl dipeptidases.
Assuntos
Dipeptidases/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Primers do DNA , Dimerização , Dipeptidases/química , Dipeptidases/genética , Dipeptidases/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
Dipeptidyl peptidase IV (DPP-IV) inhibitors are expected to become a new type of antidiabetic drugs. Most known DPP-IV inhibitors often resemble the dipeptide cleavage products, with a proline mimic at the P1 site. As off-target inhibitions of DPP8 and/or DPP9 have shown profound toxicities in the in vivo studies, it is important to develop selective DPP-IV inhibitors for clinical usage. To achieve this, a new class of 2-[3-[[2-[(2S)-2-cyano-1-pyrrolidinyl]-2-oxoethyl]amino]-1-oxopropyl]-based DPP-IV inhibitors was synthesized. SAR studies resulted in a number of DPP-IV inhibitors, having IC(50) values of <50 nM with excellent selectivity over both DPP8 (IC(50) > 100 microM) and DPP-II (IC(50) > 30 microM). Compound 21a suppressed the blood glucose elevation after an oral glucose challenge in Wistar rats and also inhibited plasma DPP-IV activity for up to 4 h in BALB/c mice. The results show that compound 21a possesses in vitro and in vivo activities comparable to those of NVP-LAF237 (4), which is in clinical development.
Assuntos
Dipeptidil Peptidase 4/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Isoquinolinas/farmacologia , Pirrolidinonas/farmacologia , Administração Oral , Animais , Glicemia/efeitos dos fármacos , Dipeptidases/antagonistas & inibidores , Dipeptidil Peptidase 4/sangue , Dipeptidil Peptidases e Tripeptidil Peptidases/antagonistas & inibidores , Avaliação Pré-Clínica de Medicamentos , Tolerância a Medicamentos , Inibidores Enzimáticos/síntese química , Glucose/administração & dosagem , Glucose/antagonistas & inibidores , Humanos , Técnicas In Vitro , Isoquinolinas/síntese química , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Conformação Molecular , Pirrolidinonas/síntese química , Ratos , Ratos Wistar , Relação Estrutura-Atividade , Fatores de TempoRESUMO
The synthesis and structure-activity relationship studies of novel indole derivatives as peroxisome proliferator-activated receptor (PPAR) agonists are reported. Indole, a drug-like scaffold, was studied as a core skeleton for the acidic head part of PPAR agonists. The structural features (acidic head, substitution on indole, and linker) were optimized first, by keeping benzisoxazole as the tail part, based on binding and functional activity at PPARgamma protein. The variations in the tail part, by introducing various heteroaromatic ring systems, were then studied. In vitro evaluation led to identification of a novel series of indole compounds with a benzisoxazole tail as potent PPAR agonists with the lead compound 14 (BPR1H036) displaying an excellent pharmacokinetic profile in BALB/c mice and an efficacious glucose lowering activity in KKA(y) mice. Structural biology studies of 14 showed that the indole ring contributes strong hydrophobic interactions with PPARgamma and could be an important moiety for the binding to the protein.
Assuntos
Indóis/síntese química , Receptores Ativados por Proliferador de Peroxissomo/agonistas , 1-Metil-3-Isobutilxantina/farmacologia , Células 3T3-L1 , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Cristalização , Cristalografia , Desoxiglucose/metabolismo , Dexametasona/farmacologia , Desenho de Fármacos , Humanos , Indóis/farmacocinética , Indóis/farmacologia , Insulina/farmacologia , Isoxazóis/síntese química , Isoxazóis/farmacocinética , Isoxazóis/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Receptores Ativados por Proliferador de Peroxissomo/farmacologia , Relação Estrutura-AtividadeRESUMO
To find potent and selective inhibitors of dipeptidyl peptidase IV (DPP-IV), we synthesized a series of 2-cyanopyrrolidine with P2-site 4-substituted glutamic acid derivatives and tested their activities against DPP-IV, DPP8, and DPP-II. Analogues that incorporated a bulky substituent at the first carbon position of benzylamine or isoquinoline showed over 30-fold selectivity for DPP-IV over both DPP8 and DPP-II. From structure-activity relationship studies, we speculate that the S2 site of DPP8 might be similar to that of DPP-IV, while DPP-IV inhibitor with N-substituted glycine in the P2 site and/or with a moiety involving in hydrophobic interaction with the side chain of Phe357 might provide a better selectivity for DPP-IV over DPP8.
Assuntos
Dipeptidil Peptidases e Tripeptidil Peptidases/antagonistas & inibidores , Inibidores Enzimáticos/síntese química , Ácido Glutâmico/análogos & derivados , Inibidores de Adenosina Desaminase , Sítios de Ligação , Dipeptidases/antagonistas & inibidores , Dipeptidil Peptidase 4 , Inibidores Enzimáticos/farmacologia , Ácido Glutâmico/síntese química , Ácido Glutâmico/farmacologia , Glicoproteínas/antagonistas & inibidores , Humanos , Interações Hidrofóbicas e Hidrofílicas , Concentração Inibidora 50 , Modelos Moleculares , Ligação Proteica , Pirrolidinas/síntese química , Pirrolidinas/farmacologia , Relação Estrutura-AtividadeRESUMO
The Bacillus megaterium mbgA gene encodes a lactose-hydrolyzing beta-galactosidase. An AraC/XylS-type activator BgaR can activate mbgA transcription in response to lactose. In this report, we show by various deletion analyses and point mutagenesis analyses that an inverted repeat centered at position -60.5 relative to the mbgA transcriptional initiation site is the cis-acting element responsible for lactose induction of mbgA expression.
Assuntos
Bacillus megaterium/genética , Regulação Bacteriana da Expressão Gênica , Lactose/fisiologia , Regiões Promotoras Genéticas , beta-Galactosidase/genética , Bacillus megaterium/enzimologia , Bacillus megaterium/crescimento & desenvolvimento , Sequência de Bases , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Genoma Bacteriano , Óperon Lac , Lactose/metabolismo , Dados de Sequência Molecular , beta-Galactosidase/metabolismoRESUMO
DPP8 is a prolyl dipeptidase homologous to DPP-IV, which is a drug target for Type II diabetes. The biological function of DPP8 is not known. To identify potent and selective chemical compounds against DPP8, we have synthesized a series of isoquinoline and isoindoline derivatives and have tested their inhibitory activity against DPP8, DPP-IV and DPP-II. Isoindoline derivatives were found to be more potent DPP8 inhibitors than isoquinoline derivatives. Isoindoline with a 1-(4,4'-difluor-benzhydryl)-piperazine group at the P2 site was observed to be a very potent DPP8 inhibitor, having an IC(50) value of 14nM with at least a 2500-fold selectivity over either DPP-IV or DPP-II. From SAR results, we speculate that the S1 site of DPP8 may be larger than that of DPP-IV, which would allow the accommodation of larger C-terminal residues, such as isoquinoline or isoindoline.
Assuntos
Dipeptidases/antagonistas & inibidores , Indóis/síntese química , Indóis/farmacologia , Sítios de Ligação , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Humanos , Concentração Inibidora 50 , Isoquinolinas/síntese química , Isoquinolinas/farmacologia , Relação Estrutura-AtividadeRESUMO
DPP-IV is a prolyl dipeptidase, cleaving the peptide bond after the penultimate proline residue. It is an important drug target for the treatment of type II diabetes. DPP-IV is active as a dimer, and monomeric DPP-IV has been speculated to be inactive. In this study, we have identified the C-terminal loop of DPP-IV, highly conserved among prolyl dipeptidases, as essential for dimer formation and optimal catalysis. The conserved residue His750 on the loop contributes significantly for dimer stability. We have determined the quaternary structures of the wild type, H750A, and H750E mutant enzymes by several independent methods including chemical cross-linking, gel electrophoresis, size exclusion chromatography, and analytical ultracentrifugation. Wild-type DPP-IV exists as dimers both in the intact cell and in vitro after purification from human semen or insect cells. The H750A mutation results in a mixture of DPP-IV dimer and monomer. H750A dimer has the same kinetic constants as those of the wild type, whereas the H750A monomer has a 60-fold decrease in kcat. Replacement of His750 with a negatively charged Glu (H750E) results in nearly exclusive monomers with a 300-fold decrease in catalytic activity. Interestingly, there is no dynamic equilibrium between the dimer and the monomer for all forms of DPP-IVs studied here. This is the first study of the function of the C-terminal loop as well as monomeric mutant DPP-IVs with respect to their enzymatic activities. The study has important implications for the discovery of drugs targeted to the dimer interface.
Assuntos
Adenosina Desaminase/química , Adenosina Desaminase/genética , Dipeptidil Peptidase 4/química , Dipeptidil Peptidase 4/genética , Glicoproteínas/química , Glicoproteínas/genética , Adenosina Desaminase/metabolismo , Inibidores de Adenosina Desaminase , Sequência de Aminoácidos , Animais , Baculoviridae/genética , Sequência de Bases , Linhagem Celular , Sequência Conservada , DNA Complementar/genética , Dimerização , Dipeptidil Peptidase 4/metabolismo , Glicoproteínas/antagonistas & inibidores , Glicoproteínas/metabolismo , Humanos , Técnicas In Vitro , Cinética , Masculino , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Estrutura Quaternária de Proteína , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sais , Homologia de Sequência de AminoácidosRESUMO
DPP8 is a new member of the prolyl dipeptidases, many of which have important biological functions in vivo. DPP8 catalyzes the cleavage at the carboxyl side of the proline residue at the penultimate position. To study its structure and biochemical properties, we have overexpressed the human DPP8 protein in baculovirus infected Sf9 cells. The protein is soluble and can be purified to homogeneity. Using the chromogenic H-Gly-Pro-pNA as the substrate, a kinetic study shows that purified DPP8 is active and has a similar kcat value as that of DPP-IV, a prolyl dipeptidase that is a drug target for type II diabetes. The kinetic constants of DPP8 are also determined for other chromogenic substrates, and the results indicate that DPP8 has substrate preference at both the P1 and P2 sites. The expression system provides means of better understanding the structure, catalytic mechanism, and biological function of DPP8 protein.