RESUMO
Proteins of the sorting nexin (SNX) family present a modular structural architecture with a phox homology (PX) phosphoinositide (PI)-binding domain and additional PX structural domains, conferring to them a wide variety of vital eukaryotic cell's functions, from signal transduction to membrane deformation and cargo binding. Although SNXs are well studied in human and yeasts, they are poorly investigated in protists. Herein, is presented the characterization of the first SNX identified in Leishmania protozoan parasites encoded by the LdBPK_352470 gene. In silico secondary and tertiary structure prediction revealed a PX domain on the N-terminal half and a Bin/amphiphysin/Rvs (BAR) domain on the C-terminal half of this protein, with these features classifying it in the SNX-BAR subfamily of SNXs. We named the LdBPK_352470.1 gene product LdSNXi, as it is the first SNX identified in Leishmania (L.) donovani. Its expression was confirmed in L. donovani promastigotes under different cell cycle phases, and it was shown to be secreted in the extracellular medium. Using an in vitro lipid binding assay, it was demonstrated that recombinant (r) LdSNXi (rGST-LdSNXi) tagged with glutathione-S-transferase (GST) binds to the PtdIns3P and PtdIns4P PIs. Using a specific a-LdSNXi antibody and immunofluorescence confocal microscopy, the intracellular localization of endogenous LdSNXi was analyzed in L. donovani promastigotes and axenic amastigotes. Additionally, rLdSNXi tagged with enhanced green fluorescent protein (rLdSNXi-EGFP) was heterologously expressed in transfected HeLa cells and its localization was examined. All observed localizations suggest functions compatible with the postulated SNX identity of LdSNXi. Sequence, structure, and evolutionary analysis revealed high homology between LdSNXi and the human SNX2, while the investigation of protein-protein interactions based on STRING (v.11.5) predicted putative molecular partners of LdSNXi in Leishmania.
Assuntos
Leishmania , Humanos , Leishmania/genética , Células HeLa , Nexinas de Classificação/genética , Transdução de Sinais , Anticorpos , Glutationa TransferaseRESUMO
Kinetoplastea are free living and parasitic protists with unique features among Eukaryota. Pathogenic Kinetoplastea parasites (i.e., Trypanosoma and Leishmania spp.) undergo several developmental transitions essential for survival in their hosts. These transitions require membrane and cytoskeleton reorganizations that involve phosphoinositides (PIs). Phospholipids like PIs are key regulators of vital functions in all eukaryotes including signal transduction, protein transport and sorting, membrane trafficking, and cytoskeleton and membrane remodeling. A large repertoire of PI-metabolizing enzymes and PI-binding proteins/effectors carrying distinct PI-binding modules like the PX (phox homology) module could play significant roles in the life and virulence of pathogenic Kinetoplastea. The aim of this study was to retrieve the entire spectrum of Kinetoplastea protein sequences containing the PX module (PX-proteins), predict their structures, and identify in them evolutionary conserved and unique traits. Using a large array of bioinformatics tools, protein IDs from two searches (based on PFam's pHMM for PX domain (PF00787)) were combined, aligned, and utilized for the construction of a new Kinetoplastea_PX pHMM. This three-step search retrieved 170 PX-protein sequences. Structural domain configuration analysis identified PX, Pkinase, Lipocalin_5, and Vps5/BAR3-WASP domains and clustered them into five distinct subfamilies. Phylogenetic tree and domain architecture analysis showed that some domain architectures exist in proteomes of all Kinetoplastea spp., while others are genus-specific. Finally, amino acid conservation logos of the Kinetoplastea spp. and Homo sapiens PX domains revealed high evolutionary conservation in residues forming the critical structural motifs for PtdIns3P recognition. This study highlights the PX-Pkinase domain architecture as unique within Trypanosoma spp. and forms the basis for a targeted functional analysis of Kinetoplastea PX-proteins as putative targets for a rational design of anti-parasitic drugs.
Assuntos
Proteínas de Transporte , Fosfatidilinositóis , Humanos , Filogenia , Fosfatidilinositóis/metabolismo , Proteínas de Transporte/metabolismo , Eucariotos/metabolismo , Transdução de SinaisRESUMO
Olive oil phenols (OOPs) are associated with the prevention of many human cancers. Some of these have been shown to inhibit cell proliferation and induce apoptosis. However, no systematic comparative study exists for all the investigated compounds under the same conditions, due to difficulties in their isolation or synthesis. Herein are presented innovative methods for large-scale selective extraction of six major secoiridoids from olive oil or leaves enabling their detailed investigation. The cytotoxic/antiproliferative bioactivity of these six compounds was evaluated on sixteen human cancer cell lines originating from eight different tissues. Cell viability with half-maximal effective concentrations (EC50) was evaluated after 72 h treatments. Antiproliferative and pro-apoptotic effects were also assessed for the most bioactive compounds (EC50 ≤ 50 µM). Oleocanthal (1) showed the strongest antiproliferative/cytotoxic activity in most cancer cell lines (EC50: 9−20 µM). The relative effectiveness of the six OOPs was: oleocanthal (1) > oleuropein aglycone (3a,b) > ligstroside aglycone (4a,b) > oleacein (2) > oleomissional (6a,b,c) > oleocanthalic acid (7). This is the first detailed study comparing the bioactivity of six OOPs in such a wide array of cancer cell lines, providing a reference for their relative antiproliferative/cytotoxic effect in the investigated cancers.
Assuntos
Antineoplásicos , Neoplasias , Olea , Humanos , Iridoides/farmacologia , Azeite de Oliva/farmacologia , Antineoplásicos/farmacologia , Neoplasias/tratamento farmacológico , Linhagem CelularRESUMO
Leishmaniases are cutaneous, mucocutaneous, and visceral diseases affecting humans and domesticated animals mostly in the tropical and subtropical areas of the planet. Host genetics have been widely investigated for their role in developing various infectious diseases. The SLC11A1 gene has been reported to play a role in neutrophil function and is associated with susceptibility to infectious and inflammatory diseases such as tuberculosis or rheumatoid arthritis. In the present meta-analysis, we investigate the genetic association of SLC11A1 polymorphisms with susceptibility to leishmaniasis. Genotypes and other risk-related data were collected from 13 case-control and family-based studies (after literature search). Conventional random-effects meta-analysis was performed using STATA 13. To pool case-control and family-based data, the weighted Stouffer's method was also applied. Eight polymorphisms were investigated: rs2276631, rs3731865, rs3731864, rs17221959, rs201565523, rs2279015, rs17235409, and rs17235416. We found that rs17235409 (D543N) and rs17235416 (1729 + 55del4) are significantly associated with a risk for cutaneous leishmaniasis (CL), whereas rs17221959, rs2279015, and rs17235409 are associated with visceral leishmaniasis (VL). Our results suggest that polymorphisms in SLC11A1 affect susceptibility to CL and VL. These findings open new pathways in understanding macrophage response to Leishmania infection and the genetic factors predisposing to symptomatic CL or VL that can lead to the usage of predictive biomarkers in populations at risk.
Assuntos
Proteínas de Transporte de Cátions/genética , Leishmaniose Cutânea/genética , Leishmaniose Visceral/genética , Polimorfismo de Nucleotídeo Único , Estudos de Casos e Controles , Proteínas de Transporte de Cátions/imunologia , Predisposição Genética para Doença , Genótipo , Humanos , Leishmania/fisiologia , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/parasitologia , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/parasitologia , Macrófagos/imunologia , Neutrófilos/imunologia , Fatores de Transcrição STAT/genética , Fatores de Transcrição STAT/imunologiaRESUMO
Sjögren's syndrome (SS) patients manifest high cell-free DNA (cf-DNA) levels in serum, associated with impaired DNaseI activity. Undegraded DNA may accumulate in tissues and act as an inflammasome-activating signal. Herein, we investigated the occurrence of aberrant DNA build-up in various biologic compartments of SS patients and its correlation with the activity of NLRP3 and AIM2 inflammasomes. For this purpose, we evaluated sera, PBMC, circulating monocytes and salivary glands (SG) from different SS patient subgroups and controls. We found that SS patients at high risk for lymphoma and those with established lymphoma display high serum cf-DNA levels, substantial extranuclear DNA accumulations in PBMC and SG tissues, a unique NLRP3 inflammasome gene signature in PBMC, and significantly increased serum IL-18 and ASC levels. In these patients, the circulating monocytes manifested NLRP3 inflammasome activation and increased response to NLRP3 stimuli, whereas SG-infiltrating macrophages exhibited signs of NLRP3 activation and pyroptosis. Cell-free nucleic acids isolated from patients' sera competently primed the activation of both NLRP3 and AIM2 inflammasomes in healthy monocytes. SS patients also manifested diminished DNaseI activity in serum and DNaseII expression in PBMC, which inversely correlated with indices of inflammasome activation. DNaseII gene-silencing in healthy monocytes led to cytoplasmic DNA deposition and activation of inflammasome-related genes and of caspase1. Our data reveal the occurrence of systemic NLRP3 inflammasome activation in severe SS, which is associated with widespread extranuclear accumulations of inflammagenic DNA and impaired DNA degradation. These findings can provide novel biomarkers and new therapeutic targets for the management of SS patients with adverse outcomes.
Assuntos
Biomarcadores/sangue , Ácidos Nucleicos Livres/sangue , Inflamassomos/metabolismo , Leucócitos Mononucleares/imunologia , Linfoma/imunologia , Glândulas Salivares/imunologia , Síndrome de Sjogren/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Morte Celular , Ácidos Nucleicos Livres/imunologia , Células Cultivadas , Degradação Necrótica do DNA , Fragmentação do DNA , Progressão da Doença , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Feminino , Humanos , Interleucina-18/metabolismo , Linfoma/diagnóstico , Masculino , Pessoa de Meia-Idade , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Risco , Síndrome de Sjogren/diagnóstico , Adulto JovemRESUMO
Acid ecto-phosphatase activity has been implicated in Leishmania donovani promastigote virulence. In the present study, we report data contributing to the molecular/structural and functional characterization of the L. donovani LdMAcP (L. donovani membrane acid phosphatase), member of the histidine acid phosphatase (HAcP) family. LdMAcP is membrane-anchored and shares high sequence identity with the major secreted L. donovani acid phosphatases (LdSAcPs). Sequence comparison of the LdMAcP orthologues in Leishmania sp. revealed strain polymorphism and species specificity for the L. donovani complex, responsible for visceral leishmaniasis (Khala azar), proposing thus a potential value of LdMAcP as an epidemiological or diagnostic tool. The extracellular orientation of the LdMAcP catalytic domain was confirmed in L. donovani promastigotes, wild-type (wt) and transgenic overexpressing a recombinant LdMAcP-mRFP1 (monomeric RFP1) chimera, as well as in transiently transfected mammalian cells expressing rLdMAcP-His. For the first time it is demonstrated in the present study that LdMAcP confers tartrate resistant acid ecto-phosphatase activity in live L. donovani promastigotes. The latter confirmed the long sought molecular identity of at least one enzyme contributing to this activity. Interestingly, the L. donovani rLdMAcP-mRFP1 promastigotes generated in this study, showed significantly higher infectivity and virulence indexes than control parasites in the infection of J774 mouse macrophages highlighting thereby a role for LdMAcP in the parasite's virulence.
Assuntos
Fosfatase Ácida/química , Fosfatase Ácida/metabolismo , Leishmania donovani/enzimologia , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Fosfatase Ácida/genética , Animais , Linhagem Celular , Sequência Conservada , Genes de Protozoários , Células HeLa , Humanos , Leishmania/enzimologia , Leishmania/genética , Leishmania/patogenicidade , Leishmania donovani/genética , Leishmania donovani/patogenicidade , Macrófagos/parasitologia , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Filogenia , Proteínas de Protozoários/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Especificidade da Espécie , VirulênciaRESUMO
Colivelin (CL), first reported in 2005, is the most potent member of the humanin family of neuroprotective peptides with in vitro and in vivo rescuing action against insults associated with Alzheimer's disease (AD). The objective of the present work is the design, synthesis and characterization of specific CL derivatives that can be used as molecular probes in the investigation of the unknown mechanism of CL action. Within this framework, three CL derivatives bearing suitable tags, i.e., the fluorescent moiety FITC, the streptavidin-counterpart biotinyl-group, and the (99m)Tc-radiometal chelating unit dimethylGly-Ser-Cys, were developed and subsequently applied in biological evaluation experiments. Specifically, the FITC-labeled derivative of CL was used in confocal microscopy, where specific binding at the periphery of F11 cells was observed; the biotin-labeled derivative of CL was used in an in-house developed ELISA-type assay, where specific and concentration-dependent binding with the ß-amyloid peptide of AD was shown; finally, the (99m)Tc-radiolabeled derivative of CL was used in in vivo biodistribution studies in healthy Swiss Albino mice, where 0.58% of the radioactivity administered was measured in the mouse brain 2min after injection. The above first successful applications of the CL probes demonstrate their potential to contribute in the field of neuroprotective peptides.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/síntese química , Peptídeos e Proteínas de Sinalização Intracelular/farmacologia , Sondas Moleculares/química , Fármacos Neuroprotetores/síntese química , Fármacos Neuroprotetores/farmacologia , Tecnécio , Sequência de Aminoácidos , Animais , Técnicas de Química Sintética , Desenho de Fármacos , Gânglios Espinais/citologia , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/farmacocinética , Masculino , Camundongos , Microscopia de Fluorescência , Dados de Sequência Molecular , Neurônios/citologia , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/farmacocinética , Ratos , Tecnécio/químicaRESUMO
In the model filamentous fungus Aspergillus nidulans, PilA and PilB, two homologues of the Saccharomyces cerevisiae eisosome proteins Pil1/Lsp1, and SurG, a strict orthologue of Sur7, are assembled and form tightly packed structures in conidiospores. As A. nidulans differs in its reproduction pattern from the Saccharomycotina in that it has the ability to reproduce through two different types of spores, conidiospores and ascospores, the products of the asexual and the sexual cycle respectively, we investigated the eisosome distribution and localization during the sexual cycle. Our results show that core eisosome proteins PilA, PilB and SurG are not expressed in hülle cells or early ascospores, but are expressed in mature ascospores. All eisosomal proteins form punctate structures at the membrane of late ascospores. In mature but quiescent ascospores, PilA forms static punctate structures at the plasma membrane. PilB also was observed at the ascospore membrane as well, with higher concentration at the areas where the two halves of ascospores are joined together. Finally, SurG was localized both at the membrane of ascospores and perinuclearly. In germlings originating from ascospores the punctate structures were shown to be composed only of PilA. PilB is diffused in the cytoplasm and SurG was located in vacuoles and endosomes. This altered localization is identical to that found in germlings originated from conidiospores. In germinated ascospores PilA foci did not colocalise with the highly mobile and transient peripheral punctate structures of AbpA, a marker for sites of clathrin-mediated endocytosis. Deletions of each one or all the three core eisosomal genes do not affect viability or germination of ascospores. In the presence of myriocin - a specific inhibitor of sphingolipid biosynthesis - PilA-GFP foci of ascospore germlings were less numerous and their distribution was significantly altered, suggesting a correlation between PilA foci and sphingolipid biosynthesis.
Assuntos
Aspergillus nidulans/metabolismo , Membrana Celular/metabolismo , Proteínas Fúngicas/metabolismo , Aspergillus nidulans/genética , Proteínas Fúngicas/genética , Deleção de Genes , Expressão Gênica , Regulação Fúngica da Expressão Gênica , Espaço Intracelular/metabolismo , Fenótipo , Ligação Proteica , Transporte Proteico , Esfingolipídeos/metabolismo , Esporos Fúngicos/genética , Esporos Fúngicos/metabolismoRESUMO
Several individual olive oil phenols (OOPs) and their secoiridoid derivatives have been shown to exert anti-proliferative and pro-apoptotic activity in treatments of human cancer cell lines originating from several tissues. This study evaluated the synergistic anti-proliferative/cytotoxic effects of five olive secoiridoid derivatives (oleocanthal, oleacein, oleuropein aglycone, ligstroside aglycone and oleomissional) in all possible double combinations and of total phenolic extracts (TPEs) on eleven human cancer cell lines representing eight cell-culture-based cancer models. Individual OOPs were used to treat cells for 72 h in half of their EC50 values for each cell line and their synergistic, additive or antagonistic interactions were evaluated by calculating the coefficient for drug interactions (CDI) for each double combination of OOPs. Olive oil TPEs of determined OOPs' content, originating from three different harvests of autochthonous olive cultivars in Greece, were evaluated as an attempt to investigate the efficacy of OOPs to reduce cancer cell numbers as part of olive oil consumption. Most combinations of OOPs showed strong synergistic effect (CDIs < 0.9) in their efficacy, whereas TPEs strongly impaired cancer cell viability, better than most individual OOPs tested herein, including the most resistant cancer cell lines evaluated.
Assuntos
Antineoplásicos , Neoplasias , Olea , Humanos , Antineoplásicos/uso terapêutico , Iridoides/farmacologia , Neoplasias/tratamento farmacológico , Azeite de Oliva/uso terapêutico , Fenóis/análise , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Linhagem Celular TumoralRESUMO
Polyols are compounds that play various physiological roles in plants. Here we present the identification of four cDNA clones of the model legume Lotus japonicus, encoding proteins of the monosaccharide transporter-like (MST) superfamily that share significant homology with previously characterized polyol transporters (PLTs). One of the transporters, named LjPLT4, was characterized functionally after expression in yeast. Transport assays revealed that LjPLT4 is a xylitol-specific H(+)-symporter (K (m), 0.34 mM). In contrast to the previously characterized homologues, LjPLT4 was unable to transport other polyols, including mannitol, sorbitol, myo-inositol and galactitol, or any of the monosaccharides tested. Interestingly, some monosaccharides, including fructose and xylose, inhibited xylitol uptake, although no significant uptake of these compounds was detected in the LjPLT4 transformed yeast cells, suggesting interactions with the xylitol binding site. Subcellular localization of LjPLT4-eYFP fusions expressed in Arabidopsis leaf epidermal cells indicated that LjPLT4 is localized in the plasma membrane. Real-time RT-PCR revealed that LjPLT4 is expressed in all major plant organs, with maximum transcript accumulation in leaves correlating with maximum xylitol levels there, as determined by GC-MS. Thus, LjPLT4 is the first plasma membrane xylitol-specific H(+)-symporter to be characterized in plants.
Assuntos
Lotus/genética , Simportadores/genética , Xilitol/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Membrana Celular/metabolismo , Clonagem Molecular , DNA Complementar/genética , Lotus/metabolismo , Folhas de Planta/genética , Folhas de Planta/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Simportadores/metabolismoRESUMO
The Hepatitis C virus (HCV) NS4B protein, a multispanning endoplasmic reticulum (ER) membrane protein, generates intracellular rearrangements of ER-derived membranes, essential for HCV replication. In this study, we characterized NS4B elements involved in the process of targeting, association and retention in the ER membrane. We investigated the localization and membrane association of a number of C- or N-terminal NS4B deletions expressed as GFP chimeras by biochemical and fluorescence microscopy techniques. A second set of GFP-NS4B chimeras containing the plasma membrane ecto-ATPase CD39 at the C-terminus of each NS4B deletion mutant was used to further examine the role of N-terminal NS4B sequences in ER retention. Several structural elements, besides the first two transmembrane domains (TMs), within the NS4B N-terminal half (residues 1-130) were found to mediate association of the NS4B-GFP chimeras with ER membranes. Both TM1 and TM2 are required for ER anchoring and retention but are not sufficient for ER retention. Sequences upstream of TM1 are also required. These include two putative amphipathic alpha-helices and a Leucine Rich Repeat-like motif, a sequence highly conserved in all HCV genotypes. The N-terminal 55peptidic sequence, containing the 1st amphipathic helix, mediates association of the 55N-GFP chimera with cellular membranes including the ER, but is dispensable for ER targeting of the entire NS4B molecule. Importantly, the C-terminal 70peptidic sequence can associate with membranes positive for ER markers in the absence of any predicted TMs. In conclusion, HCV NS4B targeting and retention in the ER results from the concerted action of several NS4B structural elements.
Assuntos
Retículo Endoplasmático/metabolismo , Hepacivirus/fisiologia , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/fisiologia , Motivos de Aminoácidos/fisiologia , Antígenos CD/genética , Antígenos CD/metabolismo , Apirase/genética , Apirase/metabolismo , Linhagem Celular Tumoral , Retículo Endoplasmático/genética , Retículo Endoplasmático/virologia , Humanos , Mapeamento de Peptídeos/métodos , Estrutura Terciária de Proteína/fisiologia , Proteínas não Estruturais Virais/genéticaRESUMO
The intracellular protozoan parasites of the Leishmania genus are responsible for Leishmaniases, vector borne diseases with a wide range of clinical manifestations. Leishmania (L.) donovani causes visceral leishmaniasis (kala azar), the most severe of these diseases. Along their biological cycle, Leishmania parasites undergo distinct developmental transitions including metacyclogenesis and differentiation of metacyclic promastigotes (MPs) to amastigotes. Metacyclogenesis inside the phlebotomine sandfly host's midgut converts the procyclic dividing promastigotes to non-dividing infective MPs eventually injected into the skin of mammalian hosts and phagocytosed by macrophages where the MPs are converted inside modified phagolysosomes to the intracellular amastigotes. These developmental transitions involve dramatic changes in cell size and shape and reformatting of the flagellum requiring thus membrane and cytoskeleton remodeling in which phosphoinositide (PI) signaling and metabolism must play central roles. This study reports on the LDBPK_220120.1 gene, the L. donovani ortholog of LmjF.22.0250 from L. major that encodes a phosphatase from the "Atypical Lipid Phosphatases" (ALPs) enzyme family. We confirmed the expression of the LDBPK_220120.1 gene product in both L. donovani promastigotes and axenic amastigotes and showed that it behaves in vitro as a Dual Specificity P-Tyr and monophosphorylated [PI(3)P and PI(4)P] PI phosphatase and therefore named it LdTyrPIP_22 (Leishmaniad onovani Tyrosine PI Phosphatase, gene locus at chromosome 22). By immunofluorescence confocal microscopy we localized the LdTyrPIP_22 in several intracellular sites in the cell body of L. donovani promastigotes and amastigotes and in the flagellum. A temperature and pH shift from 25°C to 37°C and from pH 7 to 5.5, induced a pronounced recruitment of LdTyrPIP_22 epitopes to the flagellar pocket and a redistribution around the nucleus. These results suggest possible role(s) for this P-Tyr/PI phosphatase in the regulation of processes initiated or upregulated by this temperature/pH shift that contribute to the developmental transition from MPs to amastigotes inside the mammalian host macrophages.
Assuntos
Leishmania donovani , Animais , Leishmania donovani/genética , Lipídeos , Fosfatos de Fosfatidilinositol , Monoéster Fosfórico Hidrolases/genética , Especificidade por SubstratoRESUMO
Ran-GTPase regulates multiple cellular processes such as nucleocytoplasmic transport, mitotic spindle assembly, nuclear envelope assembly, cell-cycle progression and the mitotic checkpoint. The leishmanial Ran protein, in contrast with its mammalian counterpart which is predominately nucleoplasmic, is localized at the nuclear rim. The aim of the present study was to characterize the LdRan (Leishmania donovani Ran) orthologue with an emphasis on the Ran-histone association. LdRan was found to be developmentally regulated, expressed 3-fold less in the amastigote stage. LdRan overexpression caused a growth defect linked to a delayed S-phase progression in promastigotes as for its mammalian counterpart. We report for the first time that Ran interacts with a linker histone, histone H1, in vitro and that the two proteins co-localize at the parasite nuclear rim. Interaction of Ran with core histones H3 and H4, creating in metazoans a chromosomal Ran-GTP gradient important for mitotic spindle assembly, is speculative in Leishmania spp., not only because this parasite undergoes a closed mitosis, but also because the main localization of LdRan is different from that of core histone H3. Interaction of Ran with the leishmanial linker histone H1 (LeishH1) suggests that this association maybe involved in modulation of pathways other than those documented for the metazoan Ran-core histone association.
Assuntos
Núcleo Celular/metabolismo , Histonas/metabolismo , Leishmania donovani/metabolismo , Proteínas de Protozoários/metabolismo , Proteína ran de Ligação ao GTP/metabolismo , Ciclo Celular , Citometria de Fluxo , Immunoblotting , Leishmania donovani/genética , Microscopia Confocal , Ligação Proteica , Proteínas de Protozoários/genética , Transfecção , Proteína ran de Ligação ao GTP/genéticaRESUMO
Acid ecto-phosphatases are enzymes that hydrolyze phosphomonoesters in the acidic pH range with their active sites facing the extacellular medium. Their activities can be measured in living cells. In bacteria and protozoan pathogens, acid ecto-phosphatases have been associated with the survival of intracellular pathogens within phagocytes through inhibition of the respiratory burst, suggesting that they act as virulence factors. Extracellular acid phosphatase activity in Leishmania (L.) donovani has been associated with the degree of promastigote virulence/infectivity. The levels of acid ecto-phosphatase activity in different Leishmania sp or even strains of the same species vary and this has been linked to their virulence. It may also be related to their ability to survive and multiply in the insect host. Acid phosphatase enzymatic activity can be measured in crude membrane fractions and in membrane fractions enriched in plasma membrane, however, in these cases, the intracellular acid phosphatases, mainly localized in lysosomes, contribute to the final result. Therefore, measuring phosphatase activity at the surface of live cells in acidic pH range is the only accurate way to measure acid ecto-phosphatase activity. This assay is performed at 25 °C or 37 °C for 30 min using as substrate the generic phosphatase substrate p-nitrophenyl phosphate (pNPP), in a citrate buffer, with or without sodium tartrate (L(+)-tartaric acid), as histidine acid phosphatases are classified according to their sensitivity to tartate inhibition. The steps of the protocol consist of pelleting cells in suspension, in this case Leishmania promastigotes, washing twice with HEPES buffer, resuspending the cells in the substrate reaction mixture and terminating the reaction by the addition of 0.5 N NaOH. The cells are removed by centrifugation and the absorbance of the reaction product (p-nitrophenolate=pNP) in the supernatant is measured at 405 nm. The enzymatic activity (A405 values) is normalized for the mean number of cells/ml used for each independent experiment.
RESUMO
Recombinant expression systems for mammalian membrane transport proteins are often limited by insufficient yields to support structural studies, inadequate post-translational processing and problems related with improper membrane targeting or cytotoxicity. Use of alternative expression systems and optimization of expression/purification protocols are constantly needed. In this work, we explore the applicability of the laboratory strain LEXSY of the ancient eukaryotic microorganism Leishmania tarentolae as a new expression system for mammalian nucleobase permeases of the NAT/NCS2 (Nucleobase-Ascorbate Transporter/Nucleobase-Cation Symporter-2) family. We achieved the heterologous expression of the purine-pyrimidine permease rSNBT1 from Rattus norvegicus (tagged at C-terminus with a red fluorescent protein), as confirmed by confocal microscopy and biochemical analysis of the subcellular fractions enriched in membrane proteins. The cDNA of rSNBT1 has been subcloned in a pLEXSY-sat-mrfp1vector and used to generate transgenic L. tarentolae-rsnbt1-mrfp1 strains carrying the pLEXSY-sat-rsnbt1-mrfp1 plasmid either episomally or integrated in the chromosomal DNA. The chimeric transporter rSNBT1-mRFP1 is targeted to the ER and the plasma membrane of the L. tarentolae promastigotes. The transgenic strains are capable of transporting nucleobases that are substrates of rSNBT1 but also of the endogenous L. tarentolae nucleoside/nucleobase transporters. A dipyridamole-resistant Na+-dependent fraction of uptake is attributed to the exogenously expressed rSNBT1.
Assuntos
Leishmania/genética , Proteínas de Transporte de Nucleobases/genética , Engenharia de Proteínas/métodos , Animais , Animais Geneticamente Modificados , Transporte Biológico/genética , Transporte de Íons , Leishmania/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Transporte de Nucleobases/metabolismo , Purinas , Pirimidinas , Ratos , Sódio/metabolismo , Simportadores/metabolismoRESUMO
l-dopa decarboxylase (DDC) that catalyzes the biosynthesis of bioactive amines, such as dopamine and serotonin, is expressed in the nervous system and peripheral tissues, including the liver, where its physiological role remains unknown. Recently, we reported a physical and functional interaction of DDC with the major signaling regulator phosphoinosite-3-kinase (PI3K). Here, we provide compelling evidence for the involvement of DDC in viral infections. Studying dengue (DENV) and hepatitis C (HCV) virus infection in hepatocytes and HCV replication in liver samples of infected patients, we observed a negative association between DDC and viral replication. Specifically, replication of both viruses reduced the levels of DDC mRNA and the ~120 kDa SDS-resistant DDC immunoreactive functional complex, concomitant with a PI3K-dependent accumulation of the ~50 kDa DDC monomer. Moreover, viral infection inhibited PI3K-DDC association, while DDC did not colocalize with viral replication sites. DDC overexpression suppressed DENV and HCV RNA replication, while DDC enzymatic inhibition enhanced viral replication and infectivity and affected DENV-induced cell death. Consistently, we observed an inverse correlation between DDC mRNA and HCV RNA levels in liver biopsies from chronically infected patients. These data reveal a novel relationship between DDC and Flaviviridae replication cycle and the role of PI3K in this process.
Assuntos
Dengue/metabolismo , Dopa Descarboxilase/metabolismo , Hepatite C/metabolismo , Fígado/enzimologia , Replicação Viral , Animais , Linhagem Celular Tumoral , Chlorocebus aethiops , Dengue/virologia , Vírus da Dengue/patogenicidade , Vírus da Dengue/fisiologia , Dopa Descarboxilase/genética , Hepacivirus/patogenicidade , Hepacivirus/fisiologia , Hepatite C/virologia , Humanos , Fígado/virologia , Fosfatidilinositol 3-Quinases/metabolismo , Células VeroRESUMO
Through the progressive accumulation of genetic and epigenetic alterations in cellular physiology, non-small-cell lung cancer (NSCLC) evolves in distinct steps involving mutually exclusive oncogenic mutations in K-Ras or EGFR along with inactivating mutations in the p53 tumor suppressor. Herein, we show two independent in vivo lung cancer models in which CHUK/IKK-α acts as a major NSCLC tumor suppressor. In a novel transgenic mouse strain, wherein IKKα ablation is induced by tamoxifen (Tmx) solely in alveolar type II (AT-II) lung epithelial cells, IKKα loss increases the number and size of lung adenomas in response to the chemical carcinogen urethane, whereas IKK-ß instead acts as a tumor promoter in this same context. IKKα knockdown in three independent human NSCLC lines (independent of K-Ras or p53 status) enhances their growth as tumor xenografts in immune-compromised mice. Bioinformatics analysis of whole transcriptome profiling followed by quantitative protein and targeted gene expression validation experiments reveals that IKKα loss can result in the up-regulation of activated HIF-1-α protein to enhance NSCLC tumor growth under hypoxic conditions in vivo.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Quinase I-kappa B/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/patologia , Animais , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Receptores ErbB/genética , Feminino , Perfilação da Expressão Gênica , Xenoenxertos , Humanos , Quinase I-kappa B/deficiência , Neoplasias Pulmonares/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Regulação para Cima , Proteínas ras/genéticaRESUMO
The hepatitis C virus (HCV) genome possesses an open reading frame overlapping the core gene in the +1 frame (core+1 ORF). Initial studies, mainly in rabbit reticulocyte lysates, indicated that the HCV-1 core+1 ORF is expressed by a -2/+1 frameshift at codons 8-11 during translation elongation of the viral polyprotein, resulting in a protein known as alternative reading frame protein (ARFP), frameshift (F), or core+1. However, subsequent investigation, based on reporter constructs carrying portions of the core+1 ORF, suggested the function of alternative mechanisms for core+1 expression in mammalian cells, including translation initiation from internal codons 85/87 or 26. Because results from these studies have been variable, we sought to re-evaluate expression of the core+1 ORF using constructs carrying the complete core+1 coding sequence fused to GFP or LUC. We showed here that codons 85/87 serve as the predominant initiation sites for internal translation initiation of core+1 ORF in Huh-7 and Huh-7/T7 mammalian cells, which support nuclear or cytoplasmic transcription, respectively. We also showed that internal translation initiation can occur concomitantly with the expression of the core+1/F protein that is produced artificially in Huh-7 or naturally in Huh-7/T7 cells. Furthermore, translation of core+1 ORF is not significantly affected by the presence of the HCV IRES element. The core+1/S-GFP protein is cytoplasmic and exhibits an ER distribution similar to that of the core+1/F-GFP protein.
Assuntos
Códon de Iniciação , Hepacivirus/metabolismo , Fases de Leitura Aberta/fisiologia , Biossíntese de Proteínas , Proteínas do Core Viral/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Linhagem Celular Tumoral , Códon , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Luciferases/genética , Luciferases/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fases de Leitura Aberta/genética , Plasmídeos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Proteínas do Core Viral/química , Proteínas do Core Viral/genéticaRESUMO
Recent studies have suggested the existence of a novel protein of hepatitis C virus (HCV) encoded by an ORF overlapping the core gene in the +1 frame (core+1 ORF). Two alternative translation mechanisms have been proposed for expression of the core+1 ORF of HCV-1a in cultured cells; a frameshift mechanism within codons 8-11, yielding a protein known as core+1/F, and/or translation initiation from internal codons in the core+1 ORF, yielding a shorter protein known as core+1/S. To date, the main evidence for the expression of this protein in vivo has been the specific humoral and cellular immune responses against the protein in HCV-infected patients, inasmuch as its detection in biopsies or the HCV infectious system remains elusive. In this study, we characterized the expression properties of the HCV-1a core+1 protein in mammalian cells in order to identify conditions that facilitate its detection. We showed that core+1/S is a very unstable protein, and that expression of the core protein in addition to proteosome activity can downregulate its intracellular levels. Also, we showed that in the Huh-7/T7 cytoplasmic expression system the core+1 ORF from the HCV-1 isolate supports the synthesis of both the core+1/S and core+1/F proteins. Finally, immunofluorescence and subcellular fractionation analyses indicated that core+1/S and core+1/F are cytoplasmic proteins with partial endoplasmic reticulum distribution in interphase cells, whereas in dividing cells they also localize to the microtubules of the mitotic spindle.
Assuntos
Líquido Intracelular/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas do Core Viral/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Immunoblotting , Microscopia de Fluorescência , Complexo de Endopeptidases do Proteassoma/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Proteínas do Core Viral/genéticaRESUMO
The catalytic core of methionyl-tRNA synthetase (MetRS) is conserved among all life kingdoms but, depending on species origin, is often linked to non-catalytic domains appended to its N- or C-terminus. These domains usually contribute to protein-protein or protein-tRNA interactions but their exact biological role and evolutionary purpose is poorly understood. Yeast MetRS contains an N-terminal appendix that mediates its interaction with the N-terminal part of Arc1p. Association with Arc1p controls the subcellular distribution of MetRS. Furthermore, the C-terminal part of Arc1p harbors a conserved tRNA-binding domain (TRBD) required for the Arc1p-dependent stimulation of the catalytic activity of MetRS. The same TRBD is found directly fused to catalytic domains of plant and nematode MetRS as well as human tyrosyl-tRNA synthetase. To investigate the purpose of Arc1p-MetRS complex formation in yeast, we tested the ability of TRBD to assist the function of MetRS independently of Arc1p. We attached the TRBD directly to the C-terminus of the MetRS catalytic core (MC) by constructing the chimeric protein MC-TRBD. The effect of MC-TRBD expression on yeast cell growth as well as its localization and in vitro aminoacylation activity were analyzed and compared to that of MC alone or wild-type MetRS, both in the absence or presence of Arc1p. We show that MC-TRBD exhibits improved enzymatic activity and can effectively substitute the MetRS-Arc1p binary complex in vivo. Moreover, MC-TRBD, being exclusively cytoplasmic, also mimics the MetRS-Arc1p complex in terms of subcellular localization. Our results suggest that the sole role of the N-terminal appended domain of yeast MetRS is to mediate the indirect association with the TRBD, which, nevertheless, can also function effectively in vivo when directly fused to the catalytic MetRS core.