RESUMO
Antibodies from hyperimmune monkey sera, selected by absorption to Plasmodium falciparum-infected erythrocytes, and elution at acidic pH, allowed us to characterize a novel parasite protein, Pfsbp1 (P. falciparum skeleton binding protein 1). Pfsbp1 is an integral membrane protein of parasite-induced membranous structures associated with the erythrocyte plasma membrane and referred to as Maurer's clefts. The carboxy-terminal domain of Pfsbp1, exposed within the cytoplasm of the host cell, interacts with a 35 kDa erythrocyte skeletal protein and might participate in the binding of the Maurer's clefts to the erythrocyte submembrane skeleton. Antibodies to the carboxy- and amino-terminal domains of Pfsbp1 labelled similar vesicular structures in the cytoplasm of Plasmodium chabaudi and Plasmodium berghei-infected murine erythrocytes, suggesting that the protein is conserved among malaria species, consistent with an important role of Maurer's cleft-like structures in the intraerythrocytic development of malaria parasites.
Assuntos
Proteínas de Transporte/metabolismo , Vesículas Citoplasmáticas/metabolismo , Membrana Eritrocítica/metabolismo , Eritrócitos/parasitologia , Proteínas de Membrana/metabolismo , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Animais , Proteínas Sanguíneas/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Transporte/isolamento & purificação , Cromatografia de Afinidade , Clonagem Molecular , Dosagem de Genes , Genes de Protozoários , Malária/parasitologia , Malária Falciparum/parasitologia , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/isolamento & purificação , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Plasmodium falciparum/genética , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , SaimiriRESUMO
The process of human erythrocyte invasion by Plasmodium falciparum parasites involves a calcium-dependent serine protease with properties consistent with a subtilisin-like activity. This enzyme achieves the last crucial maturation step of merozoite surface protein 1 (MSP1) necessary for parasite entry into the host erythrocyte. In eukaryotic cells, such processing steps are performed by subtilisin-like maturases, known as proprotein convertases. In an attempt to characterize the MSP1 maturase, we have identified a gene that encodes a P. falciparum subtilisin-like protease (PfSUB2) whose deduced active site sequence resembles more bacterial subtilisins. Therefore, we propose that PfSUB2 belongs to a subclass of eukaryotic subtilisins different from proprotein convertases. Pfsub2 is expressed during merozoite differentiation and encodes an integral membrane protein localized in the merozoite dense granules, a secretory organelle whose contents are believed to participate in a late step of the erythrocyte invasion. PfSUB2's subcellular localization, together with its predicted enzymatic properties, leads us to propose that PfSUB2 could be responsible for the late MSP1 maturation step and thus is an attractive target for the development of new antimalarial drugs.
Assuntos
Proteína 1 de Superfície de Merozoito/metabolismo , Plasmodium falciparum/enzimologia , Plasmodium falciparum/genética , Subtilisinas/genética , Subtilisinas/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Domínio Catalítico , Eritrócitos/parasitologia , Humanos , Dados de Sequência Molecular , Filogenia , Plasmodium falciparum/patogenicidade , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Subtilisinas/químicaRESUMO
Using reverse transcriptase-PCR and degenerate oligonucleotides derived from the active-site residues of subtilisin/kexin-like serine proteinases, we have identified a highly conserved and phylogenetically ancestral human, rat, and mouse type I membrane-bound proteinase called subtilisin/kexin-isozyme-1 (SKI-1). Computer databank searches reveal that human SKI-1 was cloned previously but with no identified function. In situ hybridization demonstrates that SKI-1 mRNA is present in most tissues and cells. Cleavage specificity studies show that SKI-1 generates a 28-kDa product from the 32-kDa brain-derived neurotrophic factor precursor, cleaving at an RGLT downward arrowSL bond. In the endoplasmic reticulum of either LoVo or HK293 cells, proSKI-1 is processed into two membrane-bound forms of SKI-1 (120 and 106 kDa) differing by the nature of their N-glycosylation. Late along the secretory pathway some of the membrane-bound enzyme is shed into the medium as a 98-kDa form. Immunocytochemical analysis of stably transfected HK293 cells shows that SKI-1 is present in the Golgi apparatus and within small punctate structures reminiscent of endosomes. In vitro studies suggest that SKI-1 is a Ca2+-dependent serine proteinase exhibiting a wide pH optimum for cleavage of pro-brain-derived neurotrophic factor.
Assuntos
Pró-Proteína Convertases , Proteínas de Saccharomyces cerevisiae , Subtilisinas/genética , Subtilisinas/metabolismo , Sequência de Aminoácidos , Animais , Animais Recém-Nascidos , Linhagem Celular , Feminino , Humanos , Imuno-Histoquímica , Mamíferos , Camundongos , Dados de Sequência Molecular , Peso Molecular , Especificidade de Órgãos , Reação em Cadeia da Polimerase , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Subtilisinas/química , Transcrição Gênica , TransfecçãoRESUMO
mLIM3, a member of the LIM homeobox family, was recently demonstrated to be critical for proliferation and differentiation of the pituitary cell lineage. Using a pool of degenerate oligonucleotides we determined the DNA sequence ANNAGGAAA(T/C)GA(CIG)AA as the set preferentially recognized by mLIM3. A nearly identical sequence is found in the prolactin (PRL) promoter, within a 15-mer stretch from nucleotides (nts) -218 to -204 which is highly conserved between human, rat, and bovine. In order to test the hypothesis of a transcriptional effect of mLIM3 on the prolactin promoter, stable transfectants of mLIM3 cDNA in AtT20 tumor cells revealed that PRL mRNA expression was induced in 3 separate stable clones. Gel retardation experiments performed using nuclear extracts isolated from one of the AtT20/mLIM3 stable transfectants revealed affinity towards the 15-mer element of the PRL promoter. From these results, we propose that the PRL promoter element (nts -218 to -204) could be functional in vivo. Finally, we demonstrate that in AtT20 cells prolactin mRNA expression is not induced by the Pit-1/GHF-1 pathway and that growth hormone mRNA is not detected concomitantly with prolactin. We conclude that mLIM3 may play a key role in inducing PRL gene expression in lactotrophs by binding to a conserved motif close to a Pit-1/GHF-1 site within the proximal PRL promoter.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Hipófise/metabolismo , Prolactina/genética , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Bovinos , Diferenciação Celular , Linhagem Celular , Sequência Consenso , DNA/genética , DNA/metabolismo , Humanos , Proteínas com Homeodomínio LIM , Hipófise/citologia , Regiões Promotoras Genéticas , Ratos , Fator de Transcrição Pit-1RESUMO
During Plasmodium falciparum asexual intraerythrocytic development, the host's cell plasma membrane is modified by the insertion of parasite proteins. One or more of these modifications mediate the cytoadherence of infected erythrocytes to host vascular endothelium. However, these surface antigens can be the target of cytophilic antibodies which promote phagocytosis of the infected erythrocyte. It has been proposed that antibodies directed to epitopes rich in asparagine play an important role in this process, which has promoted efforts to isolate the corresponding gene(s). We describe here P. falciparum asparagine- and aspartate-rich protein 1 (PfAARP1), a new giant (circa 700-kDa) protein associated with the infected erythrocyte membrane which is rich in asparagine and aspartate residues due to the presence of nine blocks of repeats. Topology analysis predicts that PfAARP1 has multiple transmembrane domains and at least five external loops. Human antibodies immunopurified against a sequence composed exclusively of asparagine and aspartate amino acids derived from PfAARP1 label the surface of the infected erythrocyte, demonstrating that such motifs are exposed. Interestingly, external loop 4 of PfAARP1 contains repetitions of these residues, and their possible role as a target of cytophilic antibodies is discussed.
Assuntos
Membrana Eritrocítica/química , Proteínas de Membrana/análise , Plasmodium falciparum/química , Proteínas de Protozoários/análise , Sequência de Aminoácidos , Animais , Mapeamento Cromossômico , Membrana Eritrocítica/parasitologia , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Camundongos , Dados de Sequência Molecular , Peso Molecular , Fases de Leitura Aberta , Proteínas de Protozoários/química , Proteínas de Protozoários/genéticaRESUMO
We describe here a new Plasmodium falciparum antigen, asparagine and aspartate rich protein 2 (PfAARP2) of 150 kDa, which is encoded by a unique gene on chromosome 1. PfAARP2 is first expressed 12 h post-invasion and accumulates in trophozoites and schizonts. Immunofluorescence studies indicate that PfAARP2 is translocated into the red blood cell cytoplasm. The central region of Pfaarp2 contains blocks of repetitions encoding asparagine and aspartate residues, which define a new family of related genes dispersed on different chromosomes, and two members of this family have also been identified. Interestingly, the non-repeated N- and C-termini of PfAARP2 display significant similarity to two yeast and human predicted proteins, and its possible function is discussed.
Assuntos
Antígenos de Protozoários/genética , Plasmodium falciparum/genética , Plasmodium falciparum/imunologia , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Sequência Conservada , Primers do DNA/genética , DNA Complementar/genética , DNA de Protozoário/genética , Evolução Molecular , Expressão Gênica , Genes de Protozoários , Humanos , Dados de Sequência Molecular , Família Multigênica , Plasmodium falciparum/metabolismo , Reação em Cadeia da Polimerase , Proteínas de Protozoários/metabolismo , Sequências Repetitivas de Ácido Nucleico , Homologia de Sequência de AminoácidosRESUMO
LIM-homeodomain proteins are important in cell lineage specification and possibly mediate transcriptional processes in eukaryotes. During the screening of a mouse pituitary cDNA library, we isolated a partial cDNA coding for a novel gene product that exhibited a predicted amino-terminal sequence similar to the homeobox of LIM-homeodomain-containing proteins. Reverse transcriptase-polymerase chain reactions (RT-PCR) performed on mouse pituitary mRNA using degenerate oligonucleotides based on the conserved LIM-domain sequences, allowed the extension of the 5' end of the sequence. The composite 2.2-kb cDNA structure predicts a 400-amino-acid-long novel mouse (m) protein, called mLIM-3. This name was chosen since within the 59-amino-acid homeodomain, it exhibits 97% sequence identity to a recently reported Xenopus homologue xLIM-3. The gene coding for mLIM-3 maps to the murine chromosome 2, most probably within the 2B band. Based on sequence characteristics, we suggest that LIM-3 belongs to a distinct subfamily of LIM-containing homeoproteins. Ontogeny studies using in situ hybridization demonstrated that mLIM-3 transcripts can be detected on embryonic day 11 (e11) in the primordium of the hypophysis. Following a maximum between e12 and e14, lower levels persisted into adulthood, where mLIM-3 was expressed primarily in the anterior and intermediate lobes of the pituitary. These results were confirmed by Northern blot analysis in adult mice which revealed a 2.4-kb pituitary mRNA transcript. mLIM-3 transcripts were also detected in pituitary cell lines such as the somatotrophs GH3 and GH4C1, the gonadotroph alpha T3-1, and the corticotroph AtT-20 cells, but not in 20 other cell lines derived from peripheral, endocrine, and neural tissues. Starting from e11, we also observed a transient expression of mLIM-3 in the ventral part of the spinal cord, pons, and medulla oblongata, reaching a maximum at e13 and from p7 onward, the expression of this transcript is no longer detectable. mLIM-3 is also expressed in the pineal gland with high levels observed at e20. These data suggest a potential role for mLIM-3 in the transcriptional regulation of certain genes during morphogenesis and/or maintenance of the differentiated state of the pituitary, motor neurons, and pineal gland.
Assuntos
Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Hipófise/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Sistema Nervoso Central/embriologia , Sistema Nervoso Central/metabolismo , Mapeamento Cromossômico , Clonagem Molecular , DNA Complementar/genética , Desenvolvimento Embrionário e Fetal , Epitélio/metabolismo , Feminino , Proteínas com Homeodomínio LIM , Masculino , Camundongos , Dados de Sequência Molecular , Hipófise/embriologia , RNA Mensageiro/análise , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Fatores de TranscriçãoRESUMO
Three enzymes have been described in malaria merozoites: a serine-protease and two phospholipases. The parasite serine-protease is necessary for parasite entry into the red blood cell. This enzyme is synthesized by intraerythrocytic schizonts as a glycolipid-anchored membrane precursor, harbouring a preformed serine-protease active site but no detectable proteolytic activity. Detection of the enzymatic activity correlates with the solubilisation of the enzyme by a parasite glycolipid-specific phospholipase C in merozoites. A third enzyme has been detected with glycolipid-degrading activity, presumably a lipase A. These activities participate in a biochemical cascade originating with the attachment of the merozoite to the red blood cell, including the translocation of the phospholipase C to the membrane-bound protease, the solubilisation/activation of the protease and its secretion at the erythrocyte/parasite junction and ending with the entry of the parasite into the host cell. Both the phospholipase C and the lipase A might generate secondary messages in the merozoite. Our current knowledge concerning these enzymes is presented.
Assuntos
Eritrócitos/parasitologia , Lipase/sangue , Malária/sangue , Plasmodium/fisiologia , Serina Endopeptidases/sangue , Fosfolipases Tipo C/sangue , Animais , Malária/enzimologia , Plasmodium/enzimologiaRESUMO
Three enzymes have been described in malaria merozoites: a serine-protease and two phospholipases. The parasite serine-protease is necessary for parasite entry into the red blood cell. This enzyme is synthesized by intraerythrocytic schizonts as a glycolipid-anchored membrane precursor, harbouring a performed serine-protease active site but not detectable proteolytic activity. Detection of the enzymatic activity correlates with the solubilisation of the enzyme by a parasite glycolipid-specific phospholipase C in merozoites. A third enzyme has been detected with glycolipid-degrading activity, presumably a lipase A. These activities participate in a biochemical cascade originating with the attachment of the merozoite to the red blood cell, including the translocation of the phospholipase C to the membrane-bound protease, the solubilisation/activation of the protease and its secretion at the erytrocyte/parasite junction and ending with the entry of the parasite into the host cell. Both the phospholipase C and the lipase A might generate secondary messages in the merozoite. Our current knowledge concerning these enzymes is presented
Assuntos
Eritrócitos , Lipase/metabolismo , Malária/enzimologia , Serina Proteases/metabolismo , Fosfolipases Tipo C/metabolismo , DNA , Ácidos Graxos , Fosfatidilinositóis/metabolismo , Glicolipídeos/metabolismo , Microscopia Eletrônica , Plasmodium falciparumAssuntos
Mapeamento Cromossômico , Proteínas de Choque Térmico/genética , Plasmodium chabaudi/genética , Proteínas de Protozoários/genética , Sequências Repetitivas de Ácido Nucleico , Sequência de Aminoácidos , Animais , Sequência de Bases , Códon/genética , Dados de Sequência Molecular , Fases de Leitura Aberta , Plasmodium berghei/genética , Reação em Cadeia da Polimerase , Homologia de Sequência de AminoácidosAssuntos
Antígenos de Protozoários/genética , DNA de Protozoário/química , Genes de Protozoários , Plasmodium falciparum/genética , Sequência de Aminoácidos , Animais , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/imunologia , Sequência de Bases , Western Blotting , Éxons , Imunofluorescência , Regulação da Expressão Gênica , Biblioteca Gênica , Íntrons , Dados de Sequência Molecular , Fases de Leitura Aberta , Plasmodium falciparum/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologiaRESUMO
To define the role of malaria parasite enzymes during the process of erythrocyte invasion, we have developed an in vitro serum-free invasion assay of mouse erythrocytes by purified Plasmodium chabaudi merozoites. The sensitivity of a merozoite-specific serine protease (p68) to various inhibitors and the effect of these inhibitors on invasion indicate a crucial role for p68. The substrate specificity of the purified enzyme has been partially defined using fluorogenic peptides. Consistent with this, in vitro incubation of mouse erythrocytes with the merozoite enzyme led to the cleavage of band 3 protein. The possible implication of erythrocyte band 3 truncation for the successful entry of the merozoite into the erythrocyte is discussed.
Assuntos
Eritrócitos/parasitologia , Plasmodium chabaudi/enzimologia , Serina Endopeptidases/metabolismo , Animais , Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Membrana Eritrocítica/metabolismo , Camundongos , Peso Molecular , Inibidores de Proteases/farmacologia , Serina Endopeptidases/química , Especificidade por SubstratoRESUMO
The C-terminal domain (CTD) of RNA polymerase II (RNAP) has an essential function in the regulation of transcription. The CTD of the human malaria parasite, Plasmodium falciparum, differs dramatically from that of higher eukaryotes. To determine whether this is a general feature of malarial parasites, we have analysed the CTD of the distantly related rodent malaria parasite P.berghei. The CTDs of the two parasites enzymes are very similar in amino acid composition and contain the basic structure of most eukaryotic CTDs, which is a tandem repeat of a heptapeptide (SPTSPSY). The CTD of P.berghei differs, however, in three aspects from the CTD of P.falciparum and other eukaryotes. First, both domains show a divergence from the consensus sequence at position 6 of the heptapeptide repeat. The Ser6 is always substituted, with a bias for lysine. The latter substitution might increase the binding efficiency to the DNA template. Second, the rodent and human malarial CTDs contain a 3' extension of, respectively, 66 or 67 amino acid residues. This tail-piece is unique among eukaryotes. Third, the enlargement of the CTD of the human parasite by six heptapeptide repeats is most likely generated by a recent amplification of a specific repeat unit.