RESUMO
UNLABELLED: Rotaviruses (RVs) enter cells through different endocytic pathways. Bovine rotavirus (BRV) UK uses clathrin-mediated endocytosis, while rhesus rotavirus (RRV) employs an endocytic process independent of clathrin and caveolin. Given the differences in the cell internalization pathway used by these viruses, we tested if the intracellular trafficking of BRV UK was the same as that of RRV, which is known to reach maturing endosomes (MEs) to infect the cell. We found that BRV UK also reaches MEs, since its infectivity depends on the function of Rab5, the endosomal sorting complex required for transport (ESCRT), and the formation of endosomal intraluminal vesicles (ILVs). However, unlike RRV, the infectivity of BRV UK was inhibited by knocking down the expression of Rab7, indicating that it has to traffic to late endosomes (LEs) to infect the cell. The requirement for Rab7 was also shared by other RV strains of human and porcine origin. Of interest, most RV strains that reach LEs were also found to depend on the activities of Rab9, the cation-dependent mannose-6-phosphate receptor (CD-M6PR), and cathepsins B, L, and S, suggesting that cellular factors from the trans-Golgi network (TGN) need to be transported by the CD-M6PR to LEs to facilitate RV cell infection. Furthermore, using a collection of UK × RRV reassortant viruses, we found that the dependence of BRV UK on Rab7, Rab9, and CD-M6PR is associated with the spike protein VP4. These findings illustrate the elaborate pathway of RV entry and reveal a new process (Rab9/CD-M6PR/cathepsins) that could be targeted for drug intervention. IMPORTANCE: Rotavirus is an important etiological agent of severe gastroenteritis in children. In most instances, viruses enter cells through an endocytic pathway that delivers the viral particle to vesicular organelles known as early endosomes (EEs). Some viruses reach the cytoplasm from EEs, where they start to replicate their genome. However, other viruses go deeper into the cell, trafficking from EEs to late endosomes (LEs) to disassemble and reach the cytoplasm. In this work, we show that most RV strains have to traffic to LEs, and the transport of endolysosomal proteases from the Golgi complex to LEs, mediated by the mannose-6-phosphate receptor, is necessary for the virus to exit the vesicular compartment and efficiently start viral replication. We also show that this deep journey into the cell is associated with the virus spike protein VP4. These findings illustrate the elaborate pathway of RV entry that could be used for drug intervention.
Assuntos
Catepsinas/metabolismo , Doenças dos Bovinos/enzimologia , Doenças dos Bovinos/virologia , Endossomos/virologia , Doenças dos Macacos/enzimologia , Receptor IGF Tipo 2/metabolismo , Infecções por Rotavirus/veterinária , Rotavirus/fisiologia , Animais , Catepsinas/genética , Bovinos , Doenças dos Bovinos/genética , Doenças dos Bovinos/metabolismo , Endossomos/enzimologia , Endossomos/metabolismo , Macaca mulatta , Camundongos , Doenças dos Macacos/genética , Doenças dos Macacos/metabolismo , Doenças dos Macacos/virologia , Receptor IGF Tipo 2/genética , Rotavirus/genética , Infecções por Rotavirus/enzimologia , Infecções por Rotavirus/metabolismo , Infecções por Rotavirus/virologia , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Internalização do VírusRESUMO
It is well known that the activation of mast cells due to the binding of mastoparan to the G(α) subunit of trimeric G proteins involves exocytosis regulation. However, experimental evidence in the literature indicates that mastoparan can also activate certain regulatory targets of exocytosis at the level of the mast cell endosomal membranes that have not yet been identified. Therefore, the aim of the present investigation was the proteomic identification of these targets. To achieve these objectives, mast cells were activated by the peptide Protopolybia MP-III, and the proteins of the endosomal membranes were converted to proteoliposomes using sonication. Proteins were separated from one another by affinity chromatography using proteoliposomes as analytes and Protopolybia MP III-immobilized Sepharose 4B resin as the ligand. This experimental approach, which used SDS-PAGE, in-gel trypsin digestion and proteomic analysis, permitted the identification of five endosomal proteins: Rho GTPase Cdc 42 and exocyst complex component 7 as components of the Ca(2+) -independent FcεRI-mediated exocytosis pathway, synaptosomal-associated protein 29, and GTP-binding protein Rab3D as components of the Ca(2+) -dependent FcεRI-mediated exocytosis pathway and Ras-related protein M-Ras, a protein that is related to the mediation of cell shaping and proliferation following exocytosis. The identification of the five proteins as targets of mastoparans may contribute in the near future to the use of this family of peptides as novel tools for dissecting the mechanism of exocytosis in mast cells.
Assuntos
Endossomos/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Mastócitos/metabolismo , Peptídeos/metabolismo , Proteômica , Venenos de Vespas/metabolismo , Sequência de Aminoácidos , Animais , Degranulação Celular , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Endossomos/enzimologia , Exocitose , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/isolamento & purificação , Peptídeos e Proteínas de Sinalização Intercelular , Masculino , Espectrometria de Massas , Mastócitos/citologia , Dados de Sequência Molecular , Peptídeos/síntese química , Peptídeos/química , Ratos , Venenos de Vespas/síntese química , Venenos de Vespas/química , Vespas/químicaRESUMO
BACKGROUND INFORMATION: Rab11 is a small GTPase that controls diverse intracellular trafficking pathways. However, the molecular machinery that regulates the participation of Rab11 in those different transport events is poorly understood. In resting cells, Rab11 localizes at the endocytic recycling compartment (ERC), whereas the different protein kinase C (PKC) isoforms display a cytosolic distribution. RESULTS: Sustained phorbol ester stimulation induces the translocation of the classical PKCα and PKCßII isoenzymes to the ERC enriched in Rab11, and results in transferrin recycling inhibition. In contrast, novel PKCε and atypical PKCζ isoenzymes neither redistribute to the perinucleus nor modify transferrin recycling transport after phorbol ester stimulation. Although several Rabs have been shown to be phosphorylated, there is to date no evidence indicating Rab11 as a kinase substrate. In this report, we show that Rab11 appears phosphorylated in vivo in phorbol ester-stimulated cells. A bioinformatic analysis of Rab11 allowed us to identify several high-probability Ser/Thr kinase phosphorylation sites. Our results demonstrate that classical PKC (PKCα and PKCßII but not PKCßI) directly phosphorylate Rab11 in vitro. In addition, novel PKCε and PKCη but not PKCδ isoenzymes also phosphorylate Rab11. Mass spectrometry analysis revealed that Ser 177 is the Rab11 residue to be phosphorylated in vitro by either PKCßII or PKCε. In agreement, the phosphomimetic mutant, Rab11 S177D, retains transferrin at the ERC in the absence of phorbol-12-myristate-13-acetate stimulus. CONCLUSIONS: This report shows for the first time that Rab11 is differentially phosphorylated by distinct PKC isoenzymes and that this post-translational modification might be a regulatory mechanism of intracellular trafficking.
Assuntos
Endossomos/enzimologia , Proteína Quinase C/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Biologia Computacional , Citosol/efeitos dos fármacos , Citosol/metabolismo , Endossomos/efeitos dos fármacos , Células HeLa , Humanos , Isoenzimas/metabolismo , Espectrometria de Massas , Fosforilação , Plasmídeos , Processamento de Proteína Pós-Traducional , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Serina/metabolismo , Especificidade por Substrato , Acetato de Tetradecanoilforbol/farmacologia , Transfecção , Transferrina/antagonistas & inibidores , Transferrina/metabolismo , Proteínas rab de Ligação ao GTP/genéticaRESUMO
Translational control mediated by phosphorylation of the alpha subunit of the eukaryotic initiation factor 2 (eIF2alpha) is central to stress-induced programs of gene expression. Trypanosomatids, important human pathogens, display differentiation processes elicited by contact with the distinct physiological milieu found in their insect vectors and mammalian hosts, likely representing stress situations. Trypanosoma brucei, the agent of African trypanosomiasis, encodes three potential eIF2alpha kinases (TbeIF2K1 to -K3). We show here that TbeIF2K2 is a transmembrane glycoprotein expressed both in procyclic and in bloodstream forms. The catalytic domain of TbeIF2K2 phosphorylates yeast and mammalian eIF2alpha at Ser51. It also phosphorylates the highly unusual form of eIF2alpha found in trypanosomatids specifically at residue Thr169 that corresponds to Ser51 in other eukaryotes. T. brucei eIF2alpha, however, is not a substrate for GCN2 or PKR in vitro. The putative regulatory domain of TbeIF2K2 does not share any sequence similarity with known eIF2alpha kinases. In both procyclic and bloodstream forms TbeIF2K2 is mainly localized in the membrane of the flagellar pocket, an organelle that is the exclusive site of exo- and endocytosis in these parasites. It can also be detected in endocytic compartments but not in lysosomes, suggesting that it is recycled between endosomes and the flagellar pocket. TbeIF2K2 location suggests a relevance in sensing protein or nutrient transport in T. brucei, an organism that relies heavily on posttranscriptional regulatory mechanisms to control gene expression in different environmental conditions. This is the first membrane-associated eIF2alpha kinase described in unicellular eukaryotes.
Assuntos
Membrana Celular/enzimologia , Flagelos/enzimologia , Trypanosoma brucei brucei/citologia , Trypanosoma brucei brucei/enzimologia , eIF-2 Quinase/metabolismo , Sequência de Aminoácidos , Animais , Endossomos/enzimologia , Fator de Iniciação 2 em Eucariotos/metabolismo , Glicosilação , Humanos , Membranas Intracelulares/enzimologia , Estágios do Ciclo de Vida , Mamíferos , Dados de Sequência Molecular , Fosforilação , Fosfotreonina/metabolismo , Estrutura Terciária de Proteína , Transporte Proteico , Proteínas de Protozoários/metabolismo , Saccharomyces cerevisiae/metabolismo , Trypanosoma brucei brucei/crescimento & desenvolvimento , eIF-2 Quinase/químicaRESUMO
During invasion of nonphagocytic cells by Trypanosoma cruzi (T. cruzi), host cell lysosomes are recruited to the plasma membrane attachment site followed by lysosomal enzyme secretion. The membrane trafficking events involved in invasion have not been delineated. We demonstrate here that T. cruzi invasion of nonphagocytic cells was completely abolished by overexpression of a dominant negative mutant of dynamin. Likewise, overexpression of a dominant negative mutant of Rab5, the rate-limiting GTPase for endocytosis, resulted in reduced infection rates compared with cells expressing Rab5 wild-type. Moreover, cells expressing the activated mutant of Rab5 experienced higher infection rates. A similar pattern was also observed when Rab7-transfected cells were examined. Confocal microscopy experiments showed that parasites colocalized with green fluorescent protein-Rab5-positive early endosomes after 5 min of invasion. These data clearly indicate that newly forming T. cruzi phagosomes first interact with an early endosomal compartment and subsequently with other late component markers before lysosomal interaction occurs.
Assuntos
Endossomos/enzimologia , GTP Fosfo-Hidrolases/fisiologia , Trypanosoma cruzi/patogenicidade , Proteínas rab de Ligação ao GTP/fisiologia , Proteínas rab5 de Ligação ao GTP/fisiologia , Animais , Células CHO , Cricetinae , Dinaminas , Endocitose , Células HeLa , Interações Hospedeiro-Parasita , Humanos , Lisossomos/química , Microscopia Confocal , Modelos Biológicos , Fagócitos/fisiologia , Vacúolos/enzimologia , Vacúolos/parasitologia , proteínas de unión al GTP Rab7RESUMO
Primary cultures of heart muscle cells provide powerful tools for cardiac cell biological research that permits both physiological and biochemical approaches. In the present study we analyzed the endocytosis of cardiac cells and presented morphological characterization of the endocytic machinery using markers, which enabled us to follow the fluid-phase, receptor-mediated endocytosis and the internalization of large particles. Our results demonstrated the route of the internalized cargo to early endosomes followed or not by its discharge in the late compartments. We also confirmed the ability of cardiac muscle cells to ingest large particles such as the mannosylated ligand zymosan A, and even internalize whole eukaryotic cells such as the protozoan parasite Trypanosoma cruzi. Since endocytosis is involved in many important cellular functions, the present work contributes to the knowledge of possible additional roles played by cardiac muscle cells besides their well known ability to act as physically energetic cells in the body, constantly contracting without tiring.
Assuntos
Compartimento Celular/fisiologia , Endocitose/fisiologia , Endossomos/enzimologia , Miócitos Cardíacos/enzimologia , Transporte Proteico/fisiologia , Animais , Biomarcadores , Adesão Celular/fisiologia , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Células Cultivadas , Feto , Interações Hospedeiro-Parasita/fisiologia , Membranas Intracelulares/metabolismo , Membranas Intracelulares/ultraestrutura , Camundongos , Microscopia Eletrônica , Miócitos Cardíacos/ultraestrutura , Trypanosoma cruzi/metabolismo , Trypanosoma cruzi/patogenicidade , Trypanosoma cruzi/ultraestrutura , Zimosan/metabolismoRESUMO
Giardia lamblia, a primitive eukaryotic cell, lacks organelles such as mitochondria, peroxisomes, and a typical Golgi complex and presents a system of vesicles located below the plasma membrane. We used fluorescence and electron microscopy to better characterize the peripheral vesicles. Incubation of living cells with acridine orange showed that the peripheral vesicles correspond to an acidic compartment. Incubation with lucifer yellow, and with horseradish peroxidase, showed labeling of the peripheral vesicles even after several hours. Acid phosphatase was localized in the endoplasmic reticulum and in most of the peripheral vesicles. On the other hand, glucose 6-phosphatase, an endoplasmic reticulum marker, was observed in the endoplasmic reticulum cisternae and in some peripheral vesicles. A similar labeling pattern was observed using the zinc iodide technique, which reveals SH-containing proteins. Three-dimensional reconstruction and electron microscopy tomography of cells stained for acid phosphatase and glucose-6-phosphatase revealed the connection between some vesicles and profiles of the endoplasmic reticulum. Taken together, our observations suggest that trophozoites of G. lamblia present an endosomal-lysosomal system concentrated in a single system, the peripheral vesicles, which may represent an ancient organellar system that later on subdivided into compartments such as early and late endosomes and lysosomes.
Assuntos
Endossomos/ultraestrutura , Giardia lamblia/ultraestrutura , Lisossomos/ultraestrutura , Fosfatase Ácida/metabolismo , Laranja de Acridina/metabolismo , Animais , Biomarcadores/análise , Retículo Endoplasmático/ultraestrutura , Endossomos/enzimologia , Histocitoquímica , Peroxidase do Rábano Silvestre/metabolismo , Isoquinolinas/metabolismo , Lisossomos/enzimologia , Microscopia Eletrônica , Microscopia de Fluorescência , TomografiaRESUMO
In a previous publication, we described the purification of a membrane-bound acid phosphatase of Leishmania mexicana as a heterogeneously N-glycosylated protein of an apparent molecular mass of 70000-72000 expressed in both the promastigote and the amastigote stage of the parasite [19]. Screening of a genomic DNA library of L. mexicana with degenerate oligonucleotides designed according to the NH2-terminus of the protein led to the cloning of the lmmbap gene, which is present in one copy per haploid genome. The open reading frame predicts a protein of 516 amino acids composed of a signal sequence, a large hydrophilic region, a trans-membrane alpha-helix and a short cytoplasmic tail. The sequence of the hydrophilic region is homologous to acid phosphatases from other organisms. While in wild-type promastigotes, the acid phosphatase is located in the endosomal/lysosomal compartment between the flagellar pocket and the nucleus, overexpression leads to its abundant exposure on the cell surface. In cells transfected with a construct lacking the region corresponding to the trans-membrane and the cytoplasmic parts, the resulting altered acid phosphatase is efficiently secreted into the culture medium. The potential of this system for studies on membrane trafficking in kinetoplastid organisms is discussed.