RESUMEN
Background: In pregnancy, Plasmodium falciparum parasites express the surface antigen VAR2CSA, which mediates adherence of red blood cells to chondroitin sulfate A (CSA) in the placenta. VAR2CSA antibodies are generally acquired during infection in pregnancy and are associated with protection from placental malaria. We observed previously that men and children in Colombia also had antibodies to VAR2CSA, but the origin of these antibodies was unknown. Here, we tested whether infection with Plasmodium vivax is an alternative mechanism of acquisition of VAR2CSA antibodies. Methods: We analyzed sera from nonpregnant Colombians and Brazilians exposed to P. vivax and monoclonal antibodies raised against P. vivax Duffy binding protein (PvDBP). Cross-reactivity to VAR2CSA was characterized by enzyme-linked immunosorbent assay, immunofluorescence assay, and flow cytometry, and antibodies were tested for inhibition of parasite binding to CSA. Results: Over 50% of individuals had antibodies that recognized VAR2CSA. Affinity-purified PvDBP human antibodies and a PvDBP monoclonal antibody recognized VAR2CSA, showing that PvDBP can give rise to cross-reactive antibodies. Importantly, the monoclonal antibody inhibited parasite binding to CSA, which is the primary in vitro correlate of protection from placental malaria. Conclusions: These data suggest that PvDBP induces antibodies that functionally recognize VAR2CSA, revealing a novel mechanism of cross-species immune recognition to falciparum malaria.
Asunto(s)
Antígenos de Protozoos/inmunología , Antígenos de Superficie/inmunología , Reacciones Cruzadas/inmunología , Malaria Falciparum/inmunología , Malaria Vivax/inmunología , Plasmodium falciparum/inmunología , Plasmodium vivax/inmunología , Proteínas Protozoarias/inmunología , Receptores de Superficie Celular/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Antiprotozoarios/sangre , Niño , Sulfatos de Condroitina , Colombia , Eritrocitos/parasitología , Euterios/inmunología , Femenino , Humanos , Inmunidad , EmbarazoRESUMEN
Malaria in pregnancy can cause serious adverse outcomes for the mother and the fetus. However, little is known about the effects of submicroscopic infections (SMIs) in pregnancy, particularly in areas where Plasmodium falciparum and Plasmodium vivax cocirculate. A cohort of 187 pregnant women living in Puerto Libertador in northwest Colombia was followed longitudinally from recruitment to delivery. Malaria was diagnosed by microscopy, reverse transcription-quantitative PCR (RT-qPCR), and placental histopathology. Gestational age, hemoglobin concentration, VAR2CSA-specific IgG levels, and adhesion-blocking antibodies were measured during pregnancy. Statistical analyses were performed to evaluate the impact of SMIs on birth weight and other delivery outcomes. Twenty-five percent of women (45/180) were positive for SMIs during pregnancy. Forty-seven percent of infections (21/45) were caused by P. falciparum, 33% were caused by P. vivax, and 20% were caused by mixed Plasmodium spp. Mixed infections of P. falciparum and P. vivax were associated with lower gestational age at delivery (P = 0.0033), while other outcomes were normal. Over 60% of women had antibodies to VAR2CSA, and there was no difference in antibody levels between those with and without SMIs. The anti-adhesion function of these antibodies was associated with protection from SMI-related anemia at delivery (P = 0.0086). SMIs occur frequently during pregnancy, and while mixed infections of both P. falciparum and P. vivax were not associated with a decrease in birth weight, they were associated with significant risk of preterm birth. We propose that the lack of adverse delivery outcomes is due to functional VAR2CSA antibodies that can protect pregnant women from SMI-related anemia.
RESUMEN
Diacylglycerol acyltranferase-2 (DGAT2) is a resident protein of the endoplasmic reticulum that catalyzes the synthesis of triacylglycerol. When lipid droplet formation is stimulated by incubating cells with fatty acids, DGAT2 becomes concentrated around the surface of cytosolic lipid droplets. Using confocal microscopy and directed mutagenesis, we have identified a 17-amino acid sequence in the C-terminal region of DGAT2 that is necessary and sufficient for targeting DGAT2 to lipid droplets. When this region was deleted, DGAT2 remained in the ER and did not target to lipid droplets. Fusing this sequence to mCherry directed the fluorescent reporter to lipid droplets. Similarly, when the corresponding region of monoacylglycerol acyltransferase-2 (MGAT2) was replaced with this sequence, MGAT2 was also targeted to lipid droplets. Lastly, we demonstrated that DGAT2 in ER membranes is continuous with lipid droplets. We propose a new model whereby DGAT2 remains in the ER during lipid droplet formation via it's transmembrane domains and interacts with nascent lipid droplets via its C-terminal lipid droplet interacting domain as they expand.
Asunto(s)
Diacilglicerol O-Acetiltransferasa/metabolismo , Retículo Endoplásmico/metabolismo , Gotas Lipídicas/metabolismo , N-Acetilglucosaminiltransferasas/metabolismo , Secuencia de Aminoácidos , Animales , Transporte Biológico , Células COS , Chlorocebus aethiops , Diacilglicerol O-Acetiltransferasa/química , Diacilglicerol O-Acetiltransferasa/genética , Diglicéridos/metabolismo , Retículo Endoplásmico/química , Expresión Génica , Genes Reporteros , Células HEK293 , Humanos , Gotas Lipídicas/química , Metabolismo de los Lípidos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , N-Acetilglucosaminiltransferasas/química , N-Acetilglucosaminiltransferasas/genética , Señales de Clasificación de Proteína , Transporte de Proteínas , Triglicéridos/metabolismo , Proteína Fluorescente RojaRESUMEN
Acyl CoA:1,2-diacylglycerol acyltransferase (DGAT)-2 is an integral membrane protein that catalyzes triacylglycerol (TG) synthesis using diacylglycerol and fatty acyl CoA as substrates. DGAT2 resides in the endoplasmic reticulum (ER), but when cells are incubated with fatty acids, DGAT2 interacts with lipid droplets presumably to catalyze localized TG synthesis for lipid droplet expansion. Previous studies have shown that DGAT2 interacts with proteins that synthesize its fatty acyl CoA substrates. In this study, we provide additional evidence that DGAT2 is present in a protein complex. Using a chemical cross-linker, disuccinimidyl suberate (DSS), we demonstrated that DGAT2 formed a dimer and was also part of a protein complex of â¼ 650 kDa, both in membranes and on lipid droplets. Using co-immunoprecipitation experiments and an in situ proximity ligation assay, we found that DGAT2 interacted with monoacylglycerol acyltransferase (MGAT)-2, an enzyme that catalyzes the synthesis of diacylglycerol. Deletion mutagenesis showed that the interaction with MGAT2 was dependent on the two transmembrane domains of DGAT2. No significant interaction of DGAT2 with lipin1, another enzyme that synthesizes diacylglycerol, could be detected. When co-expressed in cells, DGAT2 and MGAT2 co-localized in the ER and on lipid droplets. Co-expression also resulted in increased TG storage compared with expression of DGAT2 or MGAT2 alone. Incubating McArdle rat hepatoma RH7777 cells with 2-monoacylglycerol caused DGAT2 to translocate to lipid droplets. This also led to the formation of large cytosolic lipid droplets, characteristic of DGAT2, but not DGAT1, and indicated that DGAT2 can utilize monoacylglycerol-derived diacylglycerol. These findings suggest that the interaction of DGAT2 and MGAT2 serves to channel lipid substrates efficiently for TG biosynthesis.
Asunto(s)
Aciltransferasas/genética , Diacilglicerol O-Acetiltransferasa/genética , Hepatocitos/enzimología , Triglicéridos/biosíntesis , Aciltransferasas/metabolismo , Animales , Células COS , Línea Celular Tumoral , Chlorocebus aethiops , Reactivos de Enlaces Cruzados/química , Diacilglicerol O-Acetiltransferasa/metabolismo , Retículo Endoplásmico/química , Retículo Endoplásmico/metabolismo , Regulación de la Expresión Génica , Células HEK293 , Hepatocitos/citología , Humanos , Gotas Lipídicas/química , Gotas Lipídicas/metabolismo , Monoglicéridos/metabolismo , Unión Proteica , Multimerización de Proteína , Ratas , Transducción de Señal , Succinimidas/químicaRESUMEN
Acyl CoA:diacylglycerol acyltransferase-2 (DGAT2) is an integral membrane protein that catalyzes the synthesis of triacylglycerol (TG). DGAT2 is present in the endoplasmic reticulum (ER) and also localizes to lipid droplets when cells are stimulated with oleate. Previous studies have shown that DGAT2 can interact with membranes and lipid droplets independently of its two transmembrane domains, suggesting the presence of an additional membrane binding domain. In order to identify additional membrane binding regions, we confirmed that DGAT2 has only two transmembrane domains and demonstrated that the loop connecting them is present in the ER lumen. Increasing the length of this short loop from 5 to 27 amino acids impaired the ability of DGAT2 to localize to lipid droplets. Using a mutagenesis approach, we were able to identify a stretch of amino acids that appears to have a role in binding DGAT2 to the ER membrane. Our results confirm that murine DGAT2 has only two transmembrane domains but also can interact with membranes via a previously unidentified helical domain containing its active site.
Asunto(s)
Diacilglicerol O-Acetiltransferasa/metabolismo , Retículo Endoplásmico/metabolismo , Triglicéridos/química , Animales , Células COS , Fraccionamiento Celular , Membrana Celular/química , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Chlorocebus aethiops , Diacilglicerol O-Acetiltransferasa/química , Diacilglicerol O-Acetiltransferasa/genética , Retículo Endoplásmico/química , Retículo Endoplásmico/efectos de los fármacos , Expresión Génica , Células HEK293 , Humanos , Ratones , Mutagénesis Sitio-Dirigida , Ácido Oléico/farmacología , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Triglicéridos/biosíntesisRESUMEN
Triacylglycerol (TG) is the major form of stored energy in eukaryotic organisms and is synthesized by two distinct acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes, DGAT1 and DGAT2. Both DGAT enzymes reside in the endoplasmic reticulum (ER), but DGAT2 also co-localizes with mitochondria and lipid droplets. In this report, we demonstrate that murine DGAT2 is part of a multimeric complex consisting of several DGAT2 subunits. We also identified the region of DGAT2 responsible for its localization to the ER. A DGAT2 mutant lacking both its transmembrane domains, although still associated with membranes, was absent from the ER and instead localized to mitochondria. Unexpectedly, this mutant was still active and capable of interacting with lipid droplets to promote TG storage. Additional experiments indicated that the ER targeting signal was present in the first transmembrane domain (TMD1) of DGAT2. When fused to a fluorescent reporter, TMD1, but not TMD2, was sufficient to target mCherry to the ER. Finally, the interaction of DGAT2 with lipid droplets was dependent on the C terminus of DGAT2. DGAT2 mutants, in which regions of the C terminus were either truncated or specific regions were deleted, failed to co-localize with lipid droplets when cells were oleate loaded to stimulate TG synthesis. Our findings demonstrate that DGAT2 is capable of catalyzing TG synthesis and promote its storage in cytosolic lipid droplets independent of its localization in the ER.
Asunto(s)
Diacilglicerol O-Acetiltransferasa/metabolismo , Retículo Endoplásmico/enzimología , Metabolismo de los Lípidos/fisiología , Triglicéridos/biosíntesis , Animales , Células COS , Chlorocebus aethiops , Citosol/metabolismo , Diacilglicerol O-Acetiltransferasa/genética , Retículo Endoplásmico/genética , Células HEK293 , Humanos , Ratones , Mutación , Estructura Terciaria de Proteína , Triglicéridos/genéticaRESUMEN
Acyl CoA:diacylglycerol acyltransferase (DGAT) is an integral membrane protein of the endoplasmic reticulum that catalyzes the synthesis of triacylglycerols. Two DGAT enzymes have been identified (DGAT1 and DGAT2) with unique roles in lipid metabolism. DGAT1 is a multifunctional acyltransferase capable of synthesizing diacylglycerol, retinyl, and wax esters in addition to triacylglycerol. Here, we report the membrane topology for murine DGAT1 using protease protections assays and indirect immunofluorescence in conjunction with selective permeabilization of cellular membranes. Topology models based on prediction algorithms suggested that DGAT1 had eight transmembrane domains. In contrast, our data indicate that DGAT1 has three transmembrane domains with the N terminus oriented toward the cytosol. The C-terminal region of DGAT1, which accounts for â¼50% of the protein, is present in the endoplasmic reticulum lumen and contains a highly conserved histidine residue (His-426) that may be part of the active site. Mutagenesis of His-426 to alanine impaired the ability of DGAT1 to synthesize triacylglycerols as well as retinyl and wax esters in an in vitro acyltransferase assay. Finally, we show that the N-terminal domain of DGAT1 is not required for the catalytic activity of DGAT1 but, instead, may be involved in regulating enzyme activity and dimer/tetramer formation.
Asunto(s)
Diacilglicerol O-Acetiltransferasa/química , Diacilglicerol O-Acetiltransferasa/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Dominio Catalítico , Línea Celular , Diacilglicerol O-Acetiltransferasa/genética , Histidina , Ratones , Datos de Secuencia Molecular , Multimerización de Proteína , Estructura Terciaria de Proteína , Alineación de Secuencia , Triglicéridos/metabolismoRESUMEN
Transcriptional elongation regulator 1 (TCERG1) is a nuclear protein that participates in multiple events that include regulating the elongation of RNA polymerase II and coordinating transcription and pre-mRNA processing. More recently, we showed that TCERG1 is also a specific inhibitor of the transcription factor CCAAT enhancer binding protein α (C/EBPα). Interestingly, the inhibition of C/EBPα by TCERG1 is associated with the relocalization of TCERG1 from the nuclear speckle compartment to the pericentromeric regions where C/EBPα resides. In the present study, we examined additional aspects of C/EBPα-induced redistribution of TCERG1. Using several mutants of C/EBPα, we showed that C/EBPα does not need to be transcriptionally competent or have anti-proliferative activity to induce TCERG1 relocalization. Moreover, our results show that C/EBPα does not need to be localized to the pericentromeric region in order to relocalize TCERG1. This conclusion was illustrated through the use of a V296A mutant of C/EBPα, which is incapable of binding to the pericentromeric regions of heterochromatin and thus takes on a dispersed appearance in the nucleus. This mutant retained the ability to redistribute TCERG1, however in this case the redistribution was from the nuclear speckle pattern to the dispersed phenotype of C/EBPα V296A. Moreover, we showed that TCERG1 was still able to inhibit the activity of the V296A mutant. While we previously hypothesized that TCERG1 might inhibit C/EBPα by keeping it sequestered at the pericentromeric regions, our new findings indicate that TCERG1 can inhibit C/EBPα activity regardless of the latter's location in the nucleus.
Asunto(s)
Proteína alfa Potenciadora de Unión a CCAAT/metabolismo , Centrómero/metabolismo , Heterocromatina/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Factores de Elongación Transcripcional/metabolismo , Animales , Proteína alfa Potenciadora de Unión a CCAAT/genética , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Células COS , Puntos de Control del Ciclo Celular , Núcleo Celular/metabolismo , Chlorocebus aethiops , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Microscopía Fluorescente , Mutación Missense , Transporte de ProteínasRESUMEN
Transcription elongation regulator 1 (TCERG1) is an inhibitor of transcriptional elongation, and interacts with transcription and splicing factors, suggesting that it assists in coupling and coordinating these two processes. Recently we showed that TCERG1 possesses an additional activity, that being to repress the transactivation and anti-proliferative activities of the transcription factor CCAAT/Enhancer Binding Protein alpha (C/EBPalpha). In the present study, we provide evidence that TCERG1 functions as an inhibitor of C/EBPalpha rather than a transcriptional co-repressor. This conclusion was based on reporter gene experiments demonstrating that TCERG1 was able to reverse not only C/EBPalpha-mediated transactivation of promoter activity, but also C/EBPalpha-mediated transrepression of a promoter which is inhibited by C/EBPalpha. These observations, along with our previous findings that TCERG1 inhibits cellular proliferation conferred by C/EBPalpha, support the relabeling of TCERG1 as an inhibitor C/EBPalpha. Using mutants of TCERG1, we showed that the inhibitory activity lies in the amino terminal region. Because C/EBPalpha and TCERG1 have been shown to occupy different subnuclear compartments, we examined whether nuclear relocalization of either protein was involved in the inhibition of C/EBPalpha by TCERG1. Using confocal microscopy, we showed that TCERG1 localizes to nuclear speckles in the absence of C/EBPalpha. However, when co-expressed with C/EBPalpha, TCERG1 localizes to pericentromeric sites where C/EBPalpha resides. Nuclear redistribution of TCERG1 is required for its inhibitory activity, since mutants that did not display nuclear relocalization also lacked C/EBPalpha-inhibitory activity. We propose that TCERG1 inhibits C/EBPalpha activity by keeping it retained in inactive, pericentromeric heterochromatin.
Asunto(s)
Proteína alfa Potenciadora de Unión a CCAAT/metabolismo , Núcleo Celular/metabolismo , Proliferación Celular , Expresión Génica/fisiología , Transactivadores/metabolismo , Animales , Células COS , Línea Celular , Chlorocebus aethiops , Humanos , Microscopía Confocal , Transporte de Proteínas/fisiología , Transcripción Genética , Factores de Elongación TranscripcionalRESUMEN
Many pathogens evolve extensive genetic variation in virulence proteins as a strategy to evade host immunity. This poses a significant challenge for the host to develop broadly neutralizing antibodies. In Plasmodium falciparum, we show that a mechanism to circumvent this challenge is to elicit antibodies to cryptic epitopes that are not under immune pressure. We previously discovered that antibodies to the Plasmodium vivax invasion protein, PvDBP, cross-react with P. falciparum VAR2CSA, a distantly related virulence factor that mediates placental malaria. Here, we describe the molecular mechanism underlying this cross-species immunity. We identified an epitope in subdomain 1 (SD1) within the Duffy binding-like (DBL) domain of PvDBP that gives rise to cross-reactive antibodies to VAR2CSA and show that human antibodies affinity purified against a synthetic SD1 peptide block parasite adhesion to chondroitin sulfate A (CSA) in vitro The epitope in SD1 is subdominant and highly conserved in PvDBP, and in turn, SD1 antibodies target cryptic epitopes in P. falciparum VAR2CSA. The epitopes in VAR2CSA recognized by vivax-derived SD1 antibodies (of human and mouse origin) are distinct from those recognized by VAR2CSA immune serum. We mapped two peptides in the DBL5ε domain of VAR2CSA that are recognized by SD1 antibodies. Both peptides map to regions outside the immunodominant sites, and antibodies to these peptides are not elicited following immunization with VAR2CSA or natural infection with P. falciparum in pregnancy, consistent with the cryptic nature of these target epitopes.IMPORTANCE In this work, we describe a molecular mechanism of heterologous immunity between two distant species of Plasmodium Our results suggest a mechanism that subverts the classic parasite strategy of presenting highly polymorphic epitopes in surface antigens to evade immunity to that parasite. This alternative immune pathway can be exploited to protect pregnant women from falciparum placental malaria by designing vaccines to cryptic epitopes that elicit broadly inhibitory antibodies against variant parasite strains.
Asunto(s)
Anticuerpos Antiprotozoarios/inmunología , Antígenos de Protozoos/inmunología , Epítopos/inmunología , Inmunidad Heteróloga , Plasmodium falciparum/inmunología , Plasmodium vivax/inmunología , Proteínas Protozoarias/inmunología , Receptores de Superficie Celular/inmunología , Animales , Brasil , Adhesión Celular , Sulfatos de Condroitina/metabolismo , Colombia , Reacciones Cruzadas , Mapeo Epitopo , Humanos , Malaria Falciparum/inmunología , Malaria Vivax/inmunología , Ratones , Uganda , Factores de Virulencia/inmunologíaRESUMEN
We describe a novel one-step reverse transcriptase real-time PCR (direct RT-PCR) for Plasmodium falciparum malaria parasites that amplifies RNA targets directly from blood. We developed the assay to identify gametocyte-specific transcripts in parasites from patient blood samples, as a means of monitoring malaria parasite transmission in field settings. To perform the test, blood is added directly to a master mix in PCR tubes and analyzed by real-time PCR. The limit of detection of the assay on both conventional and portable real-time PCR instruments was 100 parasites/mL for 18S rRNA, and 1,000 parasites/mL for asexual (PFE0065W) and gametocyte (PF14_0367, PFGEXP5) mRNA targets. The usefulness of this assay in field studies was explored in samples from individuals living in a high-transmission region in Cameroon. The sensitivity and specificity of the assay compared with a standard two-step RT-PCR was 100% for 18S rRNA on both conventional and portable instruments. For PF14_0367, the sensitivity and specificity were 85.7% and 70.0%, respectively, on the conventional instrument and 78.6% and 90%, respectively, on the portable instrument. The concordance for assays run on the two instruments was 100% for 18S rRNA, and 79.2% for PF14_0367, with most discrepancies resulting from samples with low transcript levels. The results show asexual and sexual stage RNA targets can be detected directly from blood samples in a simple one-step test on a field-friendly instrument. This assay may be useful for monitoring malaria parasite transmission potential in elimination settings, where sensitive diagnostics are needed to evaluate the progress of malaria eradication initiatives.
Asunto(s)
ADN Protozoario/genética , Malaria Falciparum/sangre , Malaria Falciparum/transmisión , Técnicas de Diagnóstico Molecular/métodos , Plasmodium falciparum/genética , Plasmodium falciparum/aislamiento & purificación , ARN Ribosómico 18S/genética , Camerún , Humanos , Malaria Falciparum/parasitología , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y EspecificidadRESUMEN
The inhibition or dysregulation of apoptosis plays an intimate role in the initiation and progression of cancer by confounding normal tissue homeostasis. We currently do not have a clinical method to assess apoptosis induced by cancer therapies. Phosphatidylserine (PS) is an attractive target for imaging apoptosis because it is on the exterior of the apoptotic cells and PS externalization is an early marker of apoptosis. PS-binding peptides are an attractive option for developing an imaging probe to detect apoptosis using positron emission tomography. In this study, four peptides were evaluated for PS-binding characteristics using a plate-based assay system, a liposome mimic of cell membrane PS presentation, and a cell assay of apoptosis. This work also describes two screening techniques to enable researchers to identify and optimize compounds that bind to PS. The results of our study indicate that all four peptides bind to PS and are specific to apoptotic cells. Two of the peptides in particular that have an additional cysteine residue are good potential candidates for development into imaging probes because they bind to PS with high affinity and specificity and they can be easily radiolabelled with (18)F.