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1.
J Exp Clin Cancer Res ; 38(1): 353, 2019 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-31412953

RESUMO

BACKGROUND: Tubeimoside-I (TBM), a plant-derived bioactive compound, shows antitumor activity in different tumors and can enhance the efficacy of chemotherapeutic agents. However, the detail mechanism underlying remains to be elucidated. METHODS: The cytotoxic potential of TBM towards CRC cells was examined by CCK8 assay, colony formation, LDH release assay, flow cytometry method and Western blots. The ROS levels, autophagy, apoptosis, chemosensitivity to 5-FU or DOX, etc. were determined between control and TBM-treated CRC cells. RESULTS: In this study, we found that TBM could inhibit proliferation and induce apoptosis in colorectal cancer (CRC) cells. Intriguingly, TBM treatment could either promote autophagy initiation by ROS-induced AMPK activation, or block autophagy flux through inhibiting lysosomal hydrolytic enzymes, which leaded to massive impaired autophagylysosomes accumulation. Administration of autophagy initiation inhibitor (3-MA or selective ablation of autophagy related proteins) relieves TBM-induced CRC suppression, while combination use of autophagy flux inhibitor chloroquine (CQ) slightly augments TBM-induced cell death, suggesting that impaired autophagylysosomes accumulation contributes to TBM-induced growth inhibition in CRC cells. Notably, as an autophagy flux inhibitor, TBM works synergistically with 5-fluorouracil (5-FU) or doxorubicin (DOX) in CRC suppression. CONCLUSION: Together, our study provides new insights regarding the anti-tumor activity of TBM against CRC, and established potential applications of TBM for CRC combination therapies in clinic.


Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Medicamentos de Ervas Chinesas/farmacologia , Fagossomos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Saponinas/farmacologia , Triterpenos/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Fluoruracila/farmacologia , Humanos , Lisossomos/metabolismo , Proteólise/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
2.
Nat Chem Biol ; 15(9): 889-899, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31427817

RESUMO

Mycobacterium tuberculosis (Mtb) is the world's most deadly pathogen. Unlike less virulent mycobacteria, Mtb produces 1-tuberculosinyladenosine (1-TbAd), an unusual terpene nucleoside of unknown function. In the present study 1-TbAd has been shown to be a naturally evolved phagolysosome disruptor. 1-TbAd is highly prevalent among patient-derived Mtb strains, where it is among the most abundant lipids produced. Synthesis of TbAd analogs and their testing in cells demonstrate that their biological action is dependent on lipid linkage to the 1-position of adenosine, which creates a strong conjugate base. Furthermore, C20 lipid moieties confer passage through membranes. 1-TbAd selectively accumulates in acidic compartments, where it neutralizes the pH and swells lysosomes, obliterating their multilamellar structure. During macrophage infection, a 1-TbAd biosynthesis gene (Rv3378c) confers marked phagosomal swelling and intraphagosomal inclusions, demonstrating an essential role in regulating the Mtb cellular microenvironment. Although macrophages kill intracellular bacteria through phagosome acidification, Mtb coats itself abundantly with antacid.


Assuntos
Antiácidos/metabolismo , Lipídeos/biossíntese , Lipídeos/química , Mycobacterium tuberculosis/metabolismo , Fagossomos/metabolismo , Animais , Regulação Bacteriana da Expressão Gênica , Humanos , Concentração de Íons de Hidrogênio , Lisossomos , Macrófagos/metabolismo , Camundongos , Estrutura Molecular , Mycobacterium kansasii/genética , Prevalência
3.
Biochim Biophys Acta Mol Cell Res ; 1866(10): 1595-1607, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31301364

RESUMO

The rapid and precise clearance of apoptotic cells (efferocytosis) involves a series of phagocytic processes through which apoptotic cells are recognized, engulfed, and degraded within phagocytes. The Rho-family GTPases critically rearrange the cytoskeleton for these phagocytic processes, but we know little about the mechanisms by which regulatory proteins control the spatiotemporal activities of the Rho-family GTPases. Here, we identify ArhGAP12 as a functional GTPase-activating protein (GAP) of Rac1 during Stabilin-2 mediated efferocytosis. ArhGAP12 constitutively forms a complex with the phosphatidylserine receptor, Stabilin-2, via direct interaction with the downstream protein, GULP, but is released from the complex when Stabilin-2 interacts with apoptotic cells. When the phagocytic cup is closed and the apoptotic cell is surrounded by the phagosomal membrane, ArhGAP12 localizes to the phagocytic cup via a specific interaction with phosphatidylinositol-4,5-bisphosphate, which is transiently biosynthesized in the phagocytic cup. Down-regulation of ArhGAP12 results in sustained Rac1 activity, arrangement of F-actin, and delayed phagosome-lysosome fusion. Our results collectively suggest that ArhGAP12 carries dual roles in Stabilin-2 mediated efferocytosis: it binds to GULP/Stabilin-2 and switches off Rac1 basal activity and switches on the Rac1 by releasing itself from the complex. In addition, the spatiotemporal membrane targeting of ArhGAP12 inactivates Rac1 in a time-specific and spatially coordinated manner to orchestrate phagosome maturation. This may shed light on how other RhoGAPs spatiotemporally inactivate Rac or Cdc42 during phagocytosis by various cells, in different circumstances.


Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Fagocitose/fisiologia , Fagossomos/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Moléculas de Adesão Celular Neuronais/farmacologia , Linhagem Celular , Regulação para Baixo , Proteínas Ativadoras de GTPase/genética , Técnicas de Silenciamento de Genes , Humanos , Lisossomos/metabolismo , Camundongos , Fagócitos , Fagocitose/efeitos dos fármacos , Fosfatidilinositol 4,5-Difosfato/metabolismo
4.
Methods Mol Biol ; 1982: 301-312, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31172480

RESUMO

Phagosomal ROS generation is critical for our immune defense against microbial infections. Quantitative assessment of phagosomal ROS production is required to understand the complex relationship between the phagocyte and the microbe, in particular for pathogens that resist phagosomal destruction. ROS detection is difficult due to the transient nature of the reactive species and their multiple interactions with the environment. Direct labeling of phagocytic prey with a ROS-sensitive dye allows to target the dye into the phagosome and to follow the kinetics of phagosomal ROS production on a single phagosome base. Here we describe the basic labeling procedure, the quality assessment, and the imaging technique to achieve this kinetic analysis.


Assuntos
Fagossomos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Citometria de Fluxo , Fluoresceínas/química , Cinética , Imagem Molecular/métodos , NADPH Oxidases/metabolismo , Fagocitose , Coloração e Rotulagem , Imagem com Lapso de Tempo , Leveduras/metabolismo
5.
Cells ; 8(6)2019 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-31242668

RESUMO

Mitochondria-associated ER membranes (MAMs) are crucial for lipid transport and synthesis, calcium exchange, and mitochondrial functions, and they also act as signaling platforms. These contact sites also play a critical role in the decision between autophagy and apoptosis with far reaching implications for cell fate. Vascular smooth muscle cell (VSMC) apoptosis accelerates atherogenesis and the progression of advanced lesions, leading to atherosclerotic plaque vulnerability and medial degeneration. Though the successful autophagy of damaged mitochondria promotes VSMC survival against pro-apoptotic atherogenic stressors, it is unknown whether MAMs are involved in VSMC mitophagy processes. Here, we investigated the role of the multifunctional MAM protein phosphofurin acidic cluster sorting protein 2 (PACS-2) in regulating VSMC survival following a challenge by atherogenic lipids. Using high-resolution confocal microscopy and proximity ligation assays, we found an increase in MAM contacts as in PACS-2-associated MAMs upon stimulation with atherogenic lipids. Correspondingly, the disruption of MAM contacts by PACS-2 knockdown impaired mitophagosome formation and mitophagy, thus potentiating VSMC apoptosis. In conclusion, our data shed new light on the significance of the MAM modulatory protein PACS-2 in vascular cell physiopathology and suggest MAMs may be a new target to modulate VSMC fate and favor atherosclerotic plaque stability.


Assuntos
Retículo Endoplasmático/metabolismo , Mitocôndrias/metabolismo , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/metabolismo , Fagossomos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Morte Celular , Humanos , Lipoproteínas LDL , Camundongos , Modelos Biológicos
6.
Artigo em Inglês | MEDLINE | ID: mdl-31245297

RESUMO

Phosphatidylinositol (PtdIns) metabolism is indispensable in eukaryotes. Phosphoinositides (PIs) are phosphorylated derivatives of PtdIns and consist of seven species generated by reversible phosphorylation of the inositol moieties at the positions 3, 4, and 5. Each of the seven PIs has a unique subcellular and membrane domain distribution. In the enteric protozoan parasite Entamoeba histolytica, it has been previously shown that the PIs phosphatidylinositol 3-phosphate (PtdIns3P), PtdIns(4,5)P2, and PtdIns(3,4,5)P3 are localized to phagosomes/phagocytic cups, plasma membrane, and phagocytic cups, respectively. The localization of these PIs in E. histolytica is similar to that in mammalian cells, suggesting that PIs have orthologous functions in E. histolytica. In contrast, the conservation of the enzymes that metabolize PIs in this organism has not been well-documented. In this review, we summarized the full repertoire of the PI kinases and PI phosphatases found in E. histolytica via a genome-wide survey of the current genomic information. E. histolytica appears to have 10 PI kinases and 23 PI phosphatases. It has a panel of evolutionarily conserved enzymes that generate all the seven PI species. However, class II PI 3-kinases, type II PI 4-kinases, type III PI 5-phosphatases, and PI 4P-specific phosphatases are not present. Additionally, regulatory subunits of class I PI 3-kinases and type III PI 4-kinases have not been identified. Instead, homologs of class I PI 3-kinases and PTEN, a PI 3-phosphatase, exist as multiple isoforms, which likely reflects that elaborate signaling cascades mediated by PtdIns(3,4,5)P3 are present in this organism. There are several enzymes that have the nuclear localization signal: one phosphatidylinositol phosphate (PIP) kinase, two PI 3-phosphatases, and one PI 5-phosphatase; this suggests that PI metabolism also has conserved roles related to nuclear functions in E. histolytica, as it does in model organisms.


Assuntos
Entamoeba histolytica/enzimologia , Entamoeba histolytica/metabolismo , Fosfatidilinositóis/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , 1-Fosfatidilinositol 4-Quinase/metabolismo , Animais , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Fagossomos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Monoéster Fosfórico Hidrolases/classificação , Fosfotransferases (Aceptor do Grupo Álcool)/classificação , Isoformas de Proteínas , Transdução de Sinais
7.
Tuberculosis (Edinb) ; 116S: S19-S27, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31078419

RESUMO

We have recently reported that in vitro and intracellular organic peroxide stress oxidizes OhrR of Mycobacterium smegmatis and that the oxidized OhrR consequently derepresses the expression of Ohr. Here we demonstrate that the OhrR-Ohr system is highly useful for the expression of recombinant mycobacterial proteins and also for the delivery of Mycobacterium tuberculosis (Mtb) antigens to the phagosomal compartments. Recombinant M. smegmatis strains, which bear plasmid constructs to express Ohr2-T85BCFP and Ohr2-MtrA, showed expression of fusion proteins upon induction with t-butyl hydroperoxide (t-BHP) in a dose dependent manner. The M. smegmatis expressed Ohr2-T85BCFP fusion could be affinity purified by adding a 9x histidine tag to the C-terminal end of the fusion protein. Further, mouse bone marrow derived macrophages (BMDMs) infected with either recombinant M. smegmatis or BCG strains with ohr2-T85BCFP construct showed expression of T85BCFP protein without any exogenously added inducer. In addition, BMDMs infected with either recombinant BCG or Mtb with ohr2-T85BCFP construct could effectively deliver the antigens to T-cells at higher levels than strains bearing the control plasmid alone. Altogether, these results suggest that the OhrR-Ohr system is a novel inducible system to study the biology and pathogenesis of mycobacteria.


Assuntos
Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Mycobacterium smegmatis/metabolismo , Mycobacterium tuberculosis/metabolismo , Fagossomos/metabolismo , Proteínas Repressoras/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Células Cultivadas , Regulação Bacteriana da Expressão Gênica , Histidina/metabolismo , Macrófagos/metabolismo , Macrófagos/microbiologia , Camundongos Endogâmicos C57BL , Mycobacterium bovis/genética , Mycobacterium bovis/metabolismo , Mycobacterium smegmatis/genética , Mycobacterium tuberculosis/genética , Oligopeptídeos/metabolismo , Fagossomos/genética , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/genética
8.
Int Rev Immunol ; 38(2): 57-69, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31117900

RESUMO

Phagosome-lysosome (P-L) fusion is one of the central immune-effector responses of host. It is known that phagosome maturation process is associated with numerous signaling cascades and among these, important role of calcium (Ca2+) signaling has been realized recently. Ca2+ plays key roles in actin rearrangement, activation of NADPH oxidase and protein kinase C (PKC). Involvement of Ca2+ in these cellular processes directs phagosomal maturation process. Some of the intracellular pathogens have acquired the strategies to modulate Ca2+ associated pathways to block P-L fusion process. In this review we have described the mechanism of Ca2+ signals that influence P-L fusion by controlling ROS, actin and PKC signaling cascades. We have also discussed the strategies implemented by the intracellular pathogens to manipulate Ca2+ signaling to consequently subvert P-L fusion. A detail study of factors associated in manipulating Ca2+ signaling may provide new insights for the development of therapeutic tools for more effective treatment options against infectious diseases.


Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Fagocitose , Fagossomos/metabolismo , Actinas/metabolismo , Animais , Citotoxicidade Imunológica , Humanos , Espaço Intracelular , Lisossomos/metabolismo , Macrófagos/metabolismo , Fagocitose/imunologia , Fagossomos/imunologia , Proteína Quinase C/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais
9.
PLoS One ; 14(3): e0213150, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30830942

RESUMO

In nature, many plants or their extracted compounds have been found to possess anti-inflammatory features and therapeutic properties against infectious as well as non-infectious diseases, including cancer. In this study, we analysed the immunomodulatory effects on innate immune cells of hydroalcoholic extract from Origanum vulgare L. ssp. hirtum (HyE-Ov), a plant traditionally known for its anti-oxidative properties. The effects of HyE-Ov were tested on human monocyte derived dendritic cells (DC), type-1 (M1) and type-2 macrophages (M2) infected with M. bovis Bacille Calmette-Guérin (BCG), used as a model of persistent intracellular bacterium. DC, M1 and M2 treated with HyE-Ov significantly enhanced their mycobactericidal activity, which was associated with phagosomal acidification in M1 and M2 and increase of phagosomal, but not mitochondrial ROS production in M1, M2, and DC. Treatment of BCG-infected DC with HyE-Ov significantly reduced TNF-α and IL-12 production and increased TGF-ß synthesis. Finally, experiments were repeated using eight different HPLC fractions of HyE-Ov. Results showed that the capability to activate anti-microbial and anti-inflammatory response is shared by different fractions, suggesting that diverse bioactive molecules are present within the hydroalcoholic extract. Altogether, these results show that HyE-Ov promotes anti-mycobacterial innate immunity and limits inflammatory response in vitro and suggest that this plant extract may be exploitable as phytocomplex or nutraceutical for novel host-directed therapeutic approaches.


Assuntos
Álcoois/farmacologia , Anti-Infecciosos/farmacologia , Anti-Inflamatórios/farmacologia , Células Dendríticas/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Mycobacterium bovis/efeitos dos fármacos , Origanum/química , Álcoois/química , Anti-Infecciosos/química , Anti-Inflamatórios/química , Células Cultivadas , Células Dendríticas/citologia , Células Dendríticas/imunologia , Células Dendríticas/microbiologia , Voluntários Saudáveis , Humanos , Imunidade Inata/efeitos dos fármacos , Interleucina-2/metabolismo , Macrófagos/citologia , Macrófagos/imunologia , Macrófagos/microbiologia , Mycobacterium bovis/patogenicidade , Fagossomos/metabolismo , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Fator de Crescimento Transformador beta/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
10.
Dev Cell ; 49(2): 251-266.e8, 2019 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-30880001

RESUMO

In neurons, defects in autophagosome clearance have been associated with neurodegenerative disease. Yet, the mechanisms that coordinate trafficking and clearance of synaptic autophagosomes are poorly understood. Here, we use genetic screens and in vivo imaging in single neurons of C. elegans to identify mechanisms necessary for clearance of synaptic autophagosomes. We observed that autophagy at the synapse can be modulated in vivo by the state of neuronal activity, that autophagosomes undergo UNC-16/JIP3-mediated retrograde transport, and that autophagosomes containing synaptic material mature in the cell body. Through forward genetic screens, we then determined that autophagosome maturation in the cell body depends on the protease ATG-4.2, but not the related ATG-4.1, and that ATG-4.2 can cleave LGG-1/Atg8/GABARAP from membranes. Our studies revealed that ATG-4.2 is specifically necessary for the maturation and clearance of autophagosomes and that defects in transport and ATG-4.2-mediated maturation genetically interact to enhance abnormal accumulation of autophagosomes in neurons.


Assuntos
Autofagossomos/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Autofagia/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Cisteína Proteases/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Doenças Neurodegenerativas/metabolismo , Neurônios/metabolismo , Fagossomos/metabolismo , Isoformas de Proteínas , Transdução de Sinais/fisiologia , Sinapses/metabolismo
11.
Front Immunol ; 10: 188, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30881356

RESUMO

The phagosome microenvironment maintains enzyme activity and function. Here we compared the phagosomal pH of human neutrophils, monocytes, dendritic cells (DC), and monocyte-derived cells. An unexpected observation was the striking difference in phagosomal environment between the three monocytes subsets. Classical monocytes and neutrophils exhibited alkaline phagosomes, yet non-classical monocytes had more acidic phagosomes, while intermediate monocytes had a phenotype in-between. We next investigated the differences between primary naïve DC vs. in vitro monocyte-derived DC (MoDC) and established that both these cells had acidic phagosomal environments. Across all phagocytes, alkalinization was dependent upon the activity of the NADPH oxidase activity, demonstrated by the absence of NADPH oxidase from a patient with chronic granulomatous disease (CGD) or the use of a pharmacological inhibitor, diphenylene iodonium (DPI). Interestingly, MoDC stimulated with bacterial lipopolysaccharide had increased phagosomal pH. Overall, the increase in alkalinity within the phagosome was associated with increased oxidase activity. These data highlight the heterogeneous nature and potential function of phagocytic vacuoles within the family of mononuclear phagocytes.


Assuntos
Microambiente Celular , Neutrófilos/metabolismo , Fagócitos/metabolismo , Fagossomos/metabolismo , Biomarcadores , Microambiente Celular/imunologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Imunofenotipagem , Lisossomos/metabolismo , Monócitos/imunologia , Monócitos/metabolismo , NADPH Oxidases/metabolismo , Neutrófilos/imunologia , Oxirredução , Fagócitos/imunologia , Fagocitose
12.
J Biosci ; 44(1)2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30837375

RESUMO

In today's era tuberculosis is a major threat to human population. The lethality of this disease is caused by very efficiently thrived bacteria Mycobacterium tuberculosis (M. tuberculosis). Ca2+ plays crucial role in maintenance of cellular homeostasis. Bacilli survival in human alveolar macrophages majorly depends on disruption in Ca2+ signaling. Bacilli sustainability in phagosome lies in the interruption of phagolysosomal fusion, which is possible because of low intracellular Ca2+ concentration. Bacilli contain various Ca2+ binding proteins which help in regulation of Ca2+ signaling for its own benefit. For the survival of pathogen, it requires alteration in normal Ca2+ concentration in healthy cell. In this review we aim to find the various Ca2+ binding domains which are present in several Ca2+ binding proteins of M. tuberculosis and variety of roles played by Ca2+ to survive bacilli within host cell. This manuscript emphasizes the Ca2+ binding domains present in PE_PGRS group of gene family and their functionality in M. tuberculosis survival and pathogenesis.


Assuntos
Cálcio/metabolismo , Interações Hospedeiro-Patógeno , Mycobacterium tuberculosis/metabolismo , Tuberculose/microbiologia , Sinalização do Cálcio/genética , Humanos , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/microbiologia , Mycobacterium tuberculosis/patogenicidade , Fagossomos/metabolismo , Fagossomos/microbiologia , Transdução de Sinais/genética , Tuberculose/metabolismo , Tuberculose/patologia
13.
Cells ; 8(3)2019 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-30857122

RESUMO

The small GTPase, Rab7a, and the regulators of its GDP/GTP-binding status were shown to have roles in both endocytic membrane traffic and autophagy. Classically known to regulate endosomal retrograde transport and late endosome-lysosome fusion, earlier work has indicated a role for Rab7a in autophagosome-lysosome fusion as well as autolysosome maturation. However, as suggested by recent findings on PTEN-induced kinase 1 (PINK1)-Parkin-mediated mitophagy, Rab7a and its regulators are critical for the correct targeting of Atg9a-bearing vesicles to effect autophagosome formation around damaged mitochondria. This mitophagosome formation role for Rab7a is dependent on an intact Rab cycling process mediated by the Rab7a-specific guanine nucleotide exchange factor (GEF) and GTPase activating proteins (GAPs). Rab7a activity in this regard is also dependent on the retromer complex, as well as phosphorylation by the TRAF family-associated NF-κB activator binding kinase 1 (TBK1). Here, we discuss these recent findings and broadened perspectives on the role of the Rab7a network in PINK1-Parkin mediated mitophagy.


Assuntos
Fagossomos/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Humanos , Lisossomos/metabolismo , Modelos Biológicos , Fosforilação
14.
Future Microbiol ; 14: 293-313, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30757918

RESUMO

AIM: To investigate the formation of Mycobacterium avium membrane vesicles (MVs) within macrophage phagosomes. MATERIALS & METHODS: A phagosome model was utilized to characterize proteomics and lipidomics of MVs. A click chemistry-based enrichment assay was employed to examine the presence of MV proteins in the cytosol of host cells. RESULTS: Exposure to metals at concentrations present in phagosomes triggers formation of bacterial MVs. Proteomics identified several virulence factors, including enzymes involved in the cell wall synthesis, lipid and fatty acid metabolism. Some of MV proteins were also identified in the cytosol of infected macrophages. MVs harbor dsDNA. CONCLUSION: M. avium produces MVs within phagosomes. MVs carry products with potential roles in modulation of host immune defenses and intracellular survival.


Assuntos
Macrófagos/metabolismo , Infecções por Mycobacterium/microbiologia , Mycobacterium avium/metabolismo , Fagossomos/metabolismo , Vesículas Transportadoras/metabolismo , Proteínas de Bactérias/análise , Proteínas de Bactérias/metabolismo , Ácidos Graxos/análise , Ácidos Graxos/metabolismo , Humanos , Macrófagos/química , Mycobacterium avium/química , Fagossomos/química , Proteômica , Vesículas Transportadoras/química
15.
PLoS One ; 14(2): e0212202, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30763357

RESUMO

Cyclic Adenosine 3',5'-monophosphate (cAMP) is a key second messenger known to directly regulate not only the protein kinase A (PKA) activity but also other important molecules such as the exchange protein activated by cAMP (EPAC), which is as a guanine nucleotide exchange factor (GEF) of the low molecular weight GTPase, Rap2. Coxiella burnetii is a Gram negative bacterium that survives and grows in a large Coxiella replicative vacuole (CRV), which displays lysosomal and autophagic features. In this report, we present evidence that both, EPAC and its downstream effector Rap2b, were recruited to the CRV. The transient over-expression of the Rap2b wt protein, but not its inactive mutant Rap2b ΔAAX, markedly inhibited the development of the large CRV. Additionally, Rap2b wtinhibited the fusion of early Coxiella phagosomes with the fully developed CRV, indicating that homotypic fusion events are altered in the presence of high levels of Rap2b wt. Likewise, the fusion of endosome/lysosomal compartments (heterotypic fusions) with the large CRV was also affected by the over-expression of this GTPase. Interestingly, cell overexpression of Rap2b wt markedly decreased the levels of the v-SNARE, Vamp7, suggesting that this down-regulation impairs the homotypic and heterotypic fusions events of the Coxiella vacuole.


Assuntos
Coxiella burnetii/fisiologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Febre Q/metabolismo , Vacúolos/microbiologia , Proteínas rap de Ligação ao GTP/metabolismo , Animais , Células CHO , Cricetulus , AMP Cíclico/metabolismo , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Fusão de Membrana , Fagossomos/metabolismo , Fagossomos/microbiologia , Febre Q/microbiologia , Vacúolos/metabolismo , Células Vero
16.
Mol Cell Proteomics ; 18(5): 909-922, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30808727

RESUMO

Dendritic cells (DCs) are professional phagocytes that use innate sensing and phagocytosis to internalize and degrade self as well as foreign material, such as pathogenic bacteria, within phagosomes. These intracellular compartments are equipped to generate antigenic peptides that serve as source for antigen presentation to T cells initiating adaptive immune responses. The phagosomal proteome of DCs is only partially studied and is highly dynamic as it changes during phagosome maturation, when phagosomes sequentially interact with endosomes and lysosomes. In addition, the activation status of the phagocyte can modulate the phagosomal composition and is able to shape phagosomal functions.In this study, we determined spatiotemporal changes of the proteome of DC phagosomes during their maturation and compared resting and lipopolysaccharide (LPS)-stimulated bone marrow-derived DCs by label-free, quantitative mass spectrometry. Ovalbumin-coupled latex beads were used as phagocytosis model system and revealed that LPS-treated DCs show decreased recruitment of proteins involved in phagosome maturation, such as subunits of the vacuolar proton ATPase, cathepsin B, D, S, and RAB7. In contrast, those phagosomes were characterized by an increased recruitment of proteins involved in antigen cross-presentation, e.g. different subunits of MHC I molecules, the proteasome and tapasin, confirming the observed increase in cross-presentation efficacy in those cells. Further, several proteins were identified that were not previously associated with phagosomal functions. Hierarchical clustering of phagosomal proteins demonstrated that their acquisition to DC phagosomes is not only dependent on the duration of phagosome maturation but also on the activation state of DCs. Thus, our study provides a comprehensive overview of how DCs alter their phagosome composition in response to LPS, which has profound impact on the initiation of efficient immune responses.


Assuntos
Células Dendríticas/metabolismo , Lipopolissacarídeos/farmacologia , Fagossomos/metabolismo , Proteoma/metabolismo , Animais , Células Dendríticas/efeitos dos fármacos , Cinética , Camundongos Endogâmicos C57BL , Fagossomos/efeitos dos fármacos , Proteômica , Fatores de Tempo
17.
Int J Mol Sci ; 20(2)2019 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-30650615

RESUMO

Epidemiological data from the Center of Disease Control (CDC) and the World Health Organization (WHO) statistics in 2017 show that 10.0 million people around the world became sick with tuberculosis. Mycobacterium tuberculosis (MTB) is an intracellular parasite that mainly attacks macrophages and inhibits their apoptosis. It can become a long-term infection in humans, causing a series of pathological changes and clinical manifestations. In this review, we summarize innate immunity including the inhibition of antioxidants, the maturation and acidification of phagolysosomes and especially the apoptosis and autophagy of macrophages. Besides, we also elaborate on the adaptive immune response and the formation of granulomas. A thorough understanding of these escape mechanisms is of major importance for the prevention, diagnosis and treatment of tuberculosis.


Assuntos
Evasão da Resposta Imune , Mycobacterium tuberculosis/imunologia , Animais , Apoptose , Autofagia , Humanos , Estresse Oxidativo , Fagossomos/metabolismo
18.
Endocrinology ; 160(3): 484-503, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30649248

RESUMO

The biology of transport of spermatids and spermatid adhesion across the seminiferous epithelium during the epithelial cycle remains largely unexplored. Nonetheless, studies have implicated the role of motor proteins in these cellular events. In this article, we report findings to unravel the role of myosin VIIa, an F-actin-based barbed (+)-end-directed motor protein, to support cellular transport and adhesion in the testis. Using RNA interference to knock down myosin VIIa in Sertoli cells cultured in vitro as a study model was shown to perturb the Sertoli cell tight junction permeability barrier, mediated through disorganization of actin- or microtubule (MT)-based cytoskeletons owing to disruptive changes on the spatiotemporal expression of F-actin or MT-regulatory proteins. Consistent with these in vitro findings, knockdown of myosin VIIa in the testis in vivo also induced disorganization of the actin- and MT-based cytoskeletons across the seminiferous epithelium, mediated by disruptive changes in the spatiotemporal expression of actin- and MT-based regulatory proteins. More important, the transport of spermatids and organelles across the epithelium, as well as cell adhesion, was grossly disrupted. For instance, step 19 spermatids failed to be transported to the adluminal compartment near the tubule lumen to undergo spermiation; in this manner, step 19 spermatids were persistently detected in stage IX and XII tubules, intermingling with step 9 and 12 spermatids, respectively. Also, phagosomes were detected near the tubule lumen in stage I to III tubules when they should have been degraded near the base of the seminiferous epithelium via the lysosomal pathway. In summary, myosin VIIa motor protein was crucial to support cellular transport and adhesion during spermatogenesis.


Assuntos
Junções Aderentes/metabolismo , Epitélio Seminífero/fisiologia , Células de Sertoli/fisiologia , Espermatogênese , Actinas/metabolismo , Animais , Adesão Celular , Citoesqueleto/metabolismo , Masculino , Fagossomos/metabolismo , Interferência de RNA , Ratos Sprague-Dawley , Espermátides , Proteínas de Junções Íntimas/metabolismo
19.
Proc Natl Acad Sci U S A ; 116(4): 1289-1298, 2019 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-30622175

RESUMO

Macropinocytosis and phagocytosis are evolutionarily conserved forms of bulk endocytosis used by cells to ingest large volumes of fluid and solid particles, respectively. Both processes are regulated by Ras signaling, which is precisely controlled by mechanisms involving Ras GTPase activating proteins (RasGAPs) responsible for terminating Ras activity on early endosomes. While regulation of Ras signaling during large-scale endocytosis in WT Dictyostelium has been, for the most part, attributed to the Dictyostelium ortholog of human RasGAP NF1, in commonly used axenic laboratory strains, this gene is mutated and inactive. Moreover, none of the RasGAPs characterized so far have been implicated in the regulation of Ras signaling in large-scale endocytosis in axenic strains. In this study, we establish, using biochemical approaches and complementation assays in live cells, that Dictyostelium IQGAP-related protein IqgC interacts with active RasG and exhibits RasGAP activity toward this GTPase. Analyses of iqgC - and IqgC-overexpressing cells further revealed participation of this GAP in the regulation of both types of large-scale endocytosis and in cytokinesis. Moreover, given the localization of IqgC to phagosomes and, most prominently, to macropinosomes, we propose IqgC acting as a RasG-specific GAP in large-scale endocytosis. The data presented here functionally distinguish IqgC from other members of the Dictyostelium IQGAP family and call for repositioning of this genuine RasGAP outside of the IQGAP group.


Assuntos
Dictyostelium/metabolismo , Endocitose/fisiologia , Proteínas de Protozoários/metabolismo , Proteínas Ativadoras de ras GTPase/metabolismo , Sequência de Aminoácidos , Citocinese/fisiologia , Humanos , Fagocitose/fisiologia , Fagossomos/metabolismo , Pinocitose/fisiologia , Alinhamento de Sequência , Transdução de Sinais/fisiologia , Proteínas ras/metabolismo
20.
Mol Cell Biol ; 39(6)2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30602494

RESUMO

Autophagy is a process of lysosomal self-degradation of cellular components by forming autophagosomes. Autophagosome formation is an essential process in autophagy and is fine-tuned by various autophagy-related gene (ATG) products, including ATG5, ATG12, and ATG16. Although several reports have shown that numerous factors affect multiple levels of gene regulation to orchestrate cellular autophagy, the detailed mechanism of autophagosome formation still needs further investigation. In this study, we demonstrate that the RNA binding protein HuR (human antigen R) performs an essential function in autophagosome formation. We observe that HuR silencing leads to inhibition of autophagosome formation and autophagic flux in liver cells. Ribonucleoprotein immunoprecipitation (RIP) assay allows the identification of ATG5, ATG12, and ATG16 mRNAs as the direct targets of HuR. We further show that HuR mediates the translation of ATG5, ATG12, and ATG16 mRNAs by binding to their 3' untranslated regions (UTRs). In addition, we show that HuR expression positively correlates with the levels of ATG5 and ATG12 in hepatocellular carcinoma (HCC) cells. Collectively, our results suggest that HuR functions as a pivotal regulator of autophagosome formation by enhancing the translation of ATG5, ATG12, and ATG16 mRNAs and that augmented expression of HuR and ATGs may participate in the malfunction of autophagy in HCC cells.


Assuntos
Autofagossomos/metabolismo , Proteínas Relacionadas à Autofagia/biossíntese , Carcinoma Hepatocelular/metabolismo , Proteína Semelhante a ELAV 1/metabolismo , Neoplasias Hepáticas/metabolismo , Autofagia/genética , Autofagia/fisiologia , Proteína 12 Relacionada à Autofagia/genética , Proteína 12 Relacionada à Autofagia/metabolismo , Proteína 5 Relacionada à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Proteína Semelhante a ELAV 1/genética , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Proteínas Associadas aos Microtúbulos/metabolismo , Fagossomos/metabolismo , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
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