RESUMO
Recent advances in the field of proteomics have allowed extensive insights into the molecular regulations of the cell proteome. Specifically, this allows researchers to dissect a multitude of signaling arrays while targeting for the discovery of novel protein signatures. These approaches based on data mining are becoming increasingly powerful for identifying both potential disease mechanisms as well as indicators for disease progression and overall survival predictive and prognostic molecular markers for cancer. Furthermore, mass spectrometry (MS) integrations satisfy the ongoing demand for in-depth biomarker validation. For the purpose of this review, we will highlight the current developments based on MS sensitivity, to place quantitative proteomics into clinical settings and provide a perspective to integrate proteomics data for future applications in cancer precision medicine. We will also discuss malignancies associated with oncogenic viruses such as Acquire Immunodeficiency Syndrome (AIDS) and suggest novel mechanisms behind this phenomenon. Human Immunodeficiency Virus type-1 (HIV-1) proteins are known to be oncogenic per se, to induce oxidative and endoplasmic reticulum stresses, and to be released from the infected or expressing cells. HIV-1 proteins can act alone or in collaboration with other known oncoproteins, which cause the bulk of malignancies in people living with HIV-1 on ART.
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The bioactive peptide bradykinin obtained from cleavage of precursor kininogens activates the kinin-B2 receptor functioning in induction of inflammation and vasodilatation. In addition, bradykinin participates in kidney and cardiovascular development and neuronal and muscle differentiation. Here we show that kinin-B2 receptors are expressed throughout differentiation of murine C2C12 myoblasts into myotubes. An autocrine loop between receptor activation and bradykinin secretion is suggested, since bradykinin secretion is significantly reduced in the presence of the kinin-B2 receptor antagonist HOE-140 during differentiation. Expression of skeletal muscle markers and regenerative capacity were decreased after pharmacological inhibition or genetic ablation of the B2 receptor, while its antagonism increased the number of myoblasts in culture. In summary, the present work reveals to date no functions described for the B2 receptor in muscle regeneration due to the control of proliferation and differentiation of muscle precursor cells.
Assuntos
Diferenciação Celular , Músculo Esquelético/fisiologia , Mioblastos/citologia , Receptor B2 da Bradicinina/metabolismo , Regeneração , Animais , Biomarcadores/metabolismo , Bradicinina/metabolismo , Cardiotoxinas/administração & dosagem , Linhagem Celular , Proliferação de Células , Citoesqueleto/metabolismo , Deleção de Genes , Cininogênios/genética , Cininogênios/metabolismo , Camundongos Endogâmicos C57BL , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/metabolismo , Cadeias Pesadas de Miosina/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor B2 da Bradicinina/genéticaRESUMO
HIV-1 clades are known to be one of the key factors implicated in modulating HIV-associated neurocognitive disorders. HIV-1 B and C clades account for the majority of HIV-1 infections, clade B being the most neuropathogenic. The mechanisms behind HIV-mediated neuropathogenesis remain the subject of active research. We hypothesized that HIV-1 gp120 clade B and C proteins may exert differential proliferation, cell survival and NeuroAIDS effects in human astrocytoma cells via the Unfolded Protein Response, an endoplasmic reticulum- based cytoprotective mechanism. The differential effect of gp120 clade B and C was evaluated using for the first time a Tandem Mass Tag isobaric labeling quantitative proteomic approach. Flow cytometry analyses were performed for cell cycle and cell death identification. Among the proteins differentiated by HIV-1 gp120 proteins figure cytoskeleton, oxidative stress, UPR markers and numerous glycolytic metabolism enzymes. Our results demonstrate that HIV-1 gp120 B induced migration, proliferative and protective responses granted by the expression of GRP78, while HIV-1 gp120 C induced the expression of key inflammatory and pro-apoptotic markers. These novel findings put forward the first evidence that GRP78 is a key player in HIV-1 clade B and C neuropathogenic discrepancies and can be used as a novel target for immunotherapies.
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Curcumin, an extract from the turmeric rhizome (Curcuma longa), is known to exhibit anti-inflammatory, antioxidant, chemopreventive and antitumoral activities against aggressive and recurrent cancers. Accumulative data indicate that curcumin may induce cancer cell death. However, the detailed mechanism underlying its pro-apoptotic and anti-cancer effects remains to be elucidated. In the present study, we examined the signaling pathways triggered by curcumin, specifically, the exact molecular mechanisms of curcumin-induced apoptosis in highly metastatic human prostate cancer cells. The effect of curcumin was evaluated using for the first time in prostate cancer, a gel-free shotgun quantitative proteomic analysis coupled with Tandem Mass Tag isobaric labeling-based-signaling networks. Results were confirmed at the gene expression level by qRT-PCR and at the protein expression level by western blot and flow cytometry. Our findings revealed that curcumin induced an Endoplasmic Reticulum stress-mediated apoptosis in PC3. The mechanisms by which curcumin promoted cell death in these cells were associated with cell cycle arrest, increased reactive oxygen species, autophagy and the Unfolded Protein Response. Furthermore, the upregulation of ER stress was measured using key indicators of ER stress: Glucose-Regulated Protein 78, Inositol-Requiring Enzyme 1 alpha, Protein Disulfide isomerase and Calreticulin. Chronic ER stress induction was concomitant with the upregulation of pro-apoptotic markers (caspases 3,9,12) and Poly (ADP-ribose) polymerase. The downregulated proteins include anti-apoptotic and anti-tumor markers, supporting their curcumin-induced pro-apoptotic role in prostate cancer cells. Taken together, these data suggest that curcumin may serve as a promising anticancer agent by inducing a chronic ER stress mediated cell death and activation of cell cycle arrest, UPR, autophagy and oxidative stress responses.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Curcumina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Calreticulina/genética , Calreticulina/metabolismo , Caspase 3/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Regulação para Baixo/efeitos dos fármacos , Chaperona BiP do Retículo Endoplasmático , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Masculino , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/metabolismo , Proteômica , Regulação para Cima/efeitos dos fármacosRESUMO
A plethora of prior studies has linked HIV-1-infected and immune activated brain mononuclear phagocytes (MP; blood borne macrophages and microglia) to neuronal dysfunction. These are modulated by N-methyl-D-aspartate receptor (NMDAR) antagonists and supporting their relevance for HIV-1-associated nervous system disease. The role of NMDAR subsets in HIV-1-induced neuronal injury, nonetheless, is poorly understood. To this end, we investigated conditioned media from HIV-1gp120-treated human monocyte-derived-macrophages (MDM) for its abilities to affect NMDAR-mediated excitatory postsynaptic currents (EPSC(NMDAR)) in rat hippocampal slices. Bath application of gp120-treated MDM-conditioned media (MCM) produced an increase of EPSC(NMDAR). In contrast, control (untreated) MCM had limited effects on EPSC(NMDAR). Testing NR2A NMDAR (NR2AR)-mediated EPSC (EPSC(NR2AR)) and NR2B NMDAR (NR2BR)-mediated EPSC (EPSC(NR2BR)) for MCM showed significant increased EPSC(NR2BR) when compared to EPSC(NR2AR) enhancement. When synaptic NR2AR-mediated EPSC was blocked by bath application of MK801 combined with low frequency stimulations, MCM retained its ability to enhance EPSC(NMDAR) evoked by stronger stimulations. This suggested that increase in EPSC(NMDAR) was mediated, in part, through extra-synaptic NR2BR. Further analyses revealed that the soluble factors with low (<3 kD) to medium (3-10 kD) molecular weight mediated the observed increases in EPSC(NMDAR). The link between activation of NR2BRs and HIV-1gp120 MCM for neuronal injury was demonstrated by NR2BR but not NR2AR blockers. Taken together, these results indicate that macrophage secretory products induce neuronal injury through extra-synaptic NR2BRs.
Assuntos
Antagonistas de Aminoácidos Excitatórios/farmacologia , Potenciais Pós-Sinápticos Excitadores/fisiologia , Proteína gp120 do Envelope de HIV/toxicidade , HIV-1/fisiologia , Macrófagos/fisiologia , Receptores de N-Metil-D-Aspartato/fisiologia , Animais , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Feminino , HIV-1/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Hipocampo/fisiologia , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Masculino , Técnicas de Cultura de Órgãos , Gravidez , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/antagonistas & inibidoresRESUMO
HCV-infected "speedball" users (n = 30) were selected from an original cohort of 400 intravenous drug users for cytokine analysis. Cytokine concentrations (TNF-alpha, IL-1beta, IL-6, IFN-gamma, IL-2, IL-4, IL-10 and IL-12) were determined in plasma and peripheral blood mononuclear cells (PBMC) cultures derived ex vivo from these patients. In addition, lymphocyte proliferation was measured in 49 HCV-positive "speedball" users. TNF-alpha, IL-6, IFN-gamma, IL-2, IL-4, IL-10, IL-12 cytokines and not IL-1beta were significantly increased in plasma from HCV-positive "speedball" users compared with healthy controls. Except for IL-10, all other cytokines measured were augmented in phytohemagglutinin-stimulated PBMC cultures from HCV-positive "speedball" users. Likewise, overproduction of cytokines TNF-alpha, IL-1beta, IL-6 and IFN-gamma, was consistently detected when PBMC cultures from HCV-positive "speedball" users were stimulated with a biological response modifier. However, HCV-infected "speedball" users showed significant reduction in lymphoproliferative activity. Compared with healthy subjects, there was a consistent overproduction of both TH1 and TH2 type cytokines in the plasma and PBMC's of HCV-infected "speedball" users. Furthermore, there was a persistent reduction of lymphoproliferative activity in this group. These immunologic abnormalities, coupled with the range of response between the two TH-types in HCV-infected "speedball" users, suggest impairment in the regulatory mechanism of the TH1-TH2 system.
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Transtornos Relacionados ao Uso de Cocaína/epidemiologia , Transtornos Relacionados ao Uso de Cocaína/imunologia , Citocinas/imunologia , Hepatite C , Dependência de Heroína/epidemiologia , Dependência de Heroína/imunologia , Leucócitos Mononucleares/imunologia , Adolescente , Adulto , Proliferação de Células , Feminino , Infecções por HIV/epidemiologia , Infecções por HIV/imunologia , Infecções por HIV/virologia , Hepatite B/epidemiologia , Hepatite B/imunologia , Hepatite B/virologia , Hepatite C/epidemiologia , Hepatite C/imunologia , Hepatite C/virologia , Humanos , Masculino , Células Th1/imunologia , Células Th2/imunologiaRESUMO
Bacterial effector proteins delivered into eukaryotic cells via bacterial type III secretion systems are important virulence factors in plant-pathogen interactions. Type III secretion systems have been found in Rhizobium species that form symbiotic, nitrogen-fixing associations with legumes. One such bacterium, Rhizobium sp. NGR234, secretes a number of type III effectors, including nodulation outer protein L (NopL, formerly y4xL). Here, we show that expression of nopL in tobacco (Nicotiana tabacum) prevents full induction of pathogenesis-related (PR) defense proteins. Transgenic tobacco plants that express nopL and were infected with potato virus Y (necrotic strain 605) exhibited only very low levels of chitinase (class I) and beta-1,3-glucanase (classes I and III) proteins. Northern-blot analysis indicated that expression of nopL in plant cells suppresses transcription of PR genes. Treatment with ethylene counteracted the effect of NopL on chitinase (class I). Transgenic Lotus japonicus plants that expressed nopL exhibited delayed development and low chitinase levels. In vitro experiments showed that NopL is a substrate for plant protein kinases. Together, these data suggest that NopL, when delivered into the plant cell, modulates the activity of signal transduction pathways that culminate in activation of PR proteins.
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Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Rhizobium/metabolismo , Transdução de Sinais/fisiologia , Quitinases/metabolismo , Etilenos/farmacologia , Regulação Bacteriana da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Teste de Complementação Genética , Glucana 1,3-beta-Glucosidase/metabolismo , Imunidade Inata/genética , Lotus/enzimologia , Lotus/genética , Lotus/microbiologia , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Doenças das Plantas/virologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Vírus de Plantas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas , Proteínas Serina-Treonina Quinases/metabolismo , Rhizobium/genética , Transdução de Sinais/genética , Especificidade por Substrato , Nicotiana/enzimologia , Nicotiana/genética , Nicotiana/virologia , Transcrição GênicaRESUMO
Bacterial pathogens use type III secretion systems (TTSSs) to deliver virulence factors into eukaryotic cells. These effectors perturb host-defence responses, especially signal transduction pathways. A functional TTSS was identified in the symbiotic, nitrogen-fixing bacterium Rhizobium sp. NGR234. NopL (formerly y4xL) of NGR234 is a putative symbiotic effector that modulates nodulation in legumes. To test whether NopL could interact with plant proteins, in vitro phosphorylation experiments were performed using recombinant nopL protein purified from Escherichia coli as well as protein extracts from Lotus japonicus and tobacco plants. NopL serves as a substrate for plant protein kinases as well as purified protein kinase A. Phosphorylation of NopL was inhibited by the Ser/Thr kinase inhibitor K252a as well as by PD98059, a mitogen-activated protein (MAP) kinase kinase inhibitor. It thus seems likely that, after delivery into the plant cell, NopL modulates MAP kinase pathways.