RESUMO
Helminths cause a number of medical and agricultural problems and are a major cause of parasitic infections in humans, animals and plants. Comparative analysis of helminth genes and genomes are important to understand the genomic biodiversity and evolution of parasites and their hosts in terms of different selective pressures in their habitats. The interactions between the infective organisms and their hosts are mediated in large part by secreted proteins, known collectively as the "secretome". Proteins secreted by parasites are able to modify a host's environment and modulate their immune system. The composition and function of this set of proteins varies depending on the ecology, lifestyle and environment of an organism. The present study aimed to predict, in silico, the secretome in 44 helminth species including Nematoda (31 species) and Platyhelminthes (13 species) and, understand the diversity and evolution of secretomes. Secretomes from plant helminths range from 7.6% (943 proteins) to 13.9% (2,077 proteins) of the filtered proteome with an average of 10.2% (1,412 proteins) and from free-living helminths range from 4.4% (870 proteins) to 13% (3,121 proteins) with an average of 9.8% (2,126 proteins), respectively, and thus are considerably larger secretomes in relation to animal helminth secretomes which range from 4.2% (431 proteins) to 11.8% (2,419 proteins) of the proteomes, with an average of 7.1% (804 proteins). Across 44 secretomes in different helminth species, we found five conserved domains: (i) PF00014 (Kunitz/Bovine pancreatic trypsin inhibitor domain), (ii) PF00046 (Homeobox domain), (iii) PF00188 (cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 proteins), (iv) PF00085 (Thioredoxin) and (v) PF07679 (Immunoglobulin I-set domain). Our results detected secreted proteins associated with invasion, infection, adhesion and immunoregulation processes as protease inhibitors and cytokines, among other functions. In summary, this study will contribute towards the understanding of host-parasite interactions and possibly identify new molecular targets for the treatment or diagnosis of helminthiases.
Assuntos
Proteínas de Helminto/metabolismo , Helmintos/metabolismo , Animais , Biodiversidade , Sequência Conservada , Genoma , Proteínas de Helminto/química , Proteínas de Helminto/fisiologia , Helmintos/classificação , Helmintos/genética , Helmintos/fisiologia , Interações Hospedeiro-Parasita , Estilo de Vida , Filogenia , Plantas/parasitologia , Domínios Proteicos , Sinais Direcionadores de Proteínas/fisiologia , Especificidade da EspécieRESUMO
This study tested the hypothesis that the IFN-γ R1 287-YVSLI-91 intracellular motif regulates its endocytosis. IFN-γ exerts its biological activities by interacting with a specific cell-surface RC composed of two IFN-γ R1 and two IFN-γ R2 chains. Following IFN-γ binding and along with the initiation of signal transduction, the ligand and IFN-γ R1 are internalized. Two major types of consensus-sorting signals are described in receptors, which are rapidly internalized from the plasma membrane to intracellular compartments: tyrosine-based and dileucine-based internalization motifs. Transfection of HEK 293 cells and IFN-γ R1-deficient fibroblasts with WT and site-directed, mutagenesis-generated mutant IFN-γ R1 expression vectors helped us to identify region IFN-γ R1 287-YVSLI-291 as the critical domain required for IFN-γ-induced IFN-γ R1 internalization and Y287 and LI290-291 as part of a common structure essential for receptor endocytosis and function. This new endocytosis motif, YxxLI, shares characteristics of tyrosine-based and dileucine-based internalization motifs and is highly conserved in IFN-γ Rs across species. The IFN-γ R1 270-LI-271 dileucine motif, previously thought to be involved in this receptor endocytosis, showed to be unnecessary for receptor endocytosis.
Assuntos
Endocitose/imunologia , Leucina/química , Leucina/metabolismo , Receptores de Interferon/química , Receptores de Interferon/metabolismo , Motivos de Aminoácidos/imunologia , Sequência de Aminoácidos , Sequência Conservada/imunologia , Células HEK293 , Humanos , Dados de Sequência Molecular , Sinais Direcionadores de Proteínas/fisiologia , Receptores de Interferon/genética , Tirosina/química , Tirosina/metabolismo , Receptor de Interferon gamaRESUMO
The presence of apoplastic proteins without predicted signal peptide in the gene sequence suggests the existence of protein secretion independent of the ER/Golgi classical route. In animals, one of the pathways proposed for alternative protein secretion involves the release of exosomes to the extracellular space. Although this pathway has not been dissected in plants some indirect evidence is emerging. We have reported that apoplastic fractions of sunflower seeds contain exosome-like vesicles. Besides, these vesicles are enriched in the lectin Helja, which is immunolocalized in the extracellular space even if it the protein has no predicted signal peptide. Here we show that Helja is not glycosylated and its secretion is insensitive to brefeldin A, two of the major characteristics to discard ER/Golgi-mediated protein transport. Moreover, the levels of Helja in sunflower extracellular vesicles are not affected by brefeldin A treatment. Our results suggest that Helja could be exported through an exosome-mediated pathway and point out that this mechanism may be responsible for the secretion of at least part of the leaderless proteins detected in the extracellular compartment of plants.
Assuntos
Exossomos/metabolismo , Espaço Extracelular/metabolismo , Helianthus/metabolismo , Lectinas de Plantas/metabolismo , Proteínas de Plantas/metabolismo , Brefeldina A/farmacologia , Retículo Endoplasmático/fisiologia , Glicosilação , Complexo de Golgi/fisiologia , Helianthus/efeitos dos fármacos , Sinais Direcionadores de Proteínas/fisiologia , Inibidores da Síntese de Proteínas/farmacologia , Transporte Proteico , Sementes/efeitos dos fármacos , Sementes/metabolismoRESUMO
Transketolase has been characterized in Leishmania mexicana. A gene encoding this enzyme was identified and cloned. The gene was expressed in Escherichia coli and the protein was purified and characterized. An apparent K(m) of 2.75 mM for ribose 5-phosphate was determined. X-ray crystallography was used to determine the three-dimensional structure of the enzyme to a resolution of 2.2 A (1 A identical with 0.1 nm). The C-terminus of the protein contains a type-1 peroxisome-targeting signal, suggestive of a possible glycosomal subcellular localization. Subcellular localization experiments performed with promastigote forms of the parasite revealed that the protein was predominantly cytosolic, although a significant component of the total activity was associated with the glycosomes. Transketolase is thus the first enzyme of the nonoxidative branch of the pentose phosphate pathway whose presence has been demonstrated in a peroxisome-like organelle.
Assuntos
Leishmania mexicana/química , Leishmania mexicana/enzimologia , Transcetolase/metabolismo , Sequência de Aminoácidos/genética , Animais , Clonagem Molecular , Cristalografia por Raios X/métodos , DNA de Protozoário/genética , Leishmania mexicana/crescimento & desenvolvimento , Microcorpos/química , Microcorpos/enzimologia , Dados de Sequência Molecular , Peroxissomos/química , Peroxissomos/enzimologia , Sinais Direcionadores de Proteínas/fisiologia , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Transcetolase/biossíntese , Transcetolase/química , Transcetolase/genéticaRESUMO
The biogenesis of organelles and the maintenance of cell functions in multi-compartmentalized plant cells require a specific protein delivery mechanism to ensure efficient and effective translocation of proteins to their respective destinations. Increasing numbers of studies demonstrate that some proteins are targeted simultaneously to more than one compartment by a range of mechanisms, involving composite targeting sequences and/or transcriptional and translational controls. Recent data indicate that the final destination of a protein might respond to changes in the environment; this underlines the complexity of cell engineering that is required to localize a protein.
Assuntos
Cloroplastos/metabolismo , Mitocôndrias/metabolismo , Proteínas de Plantas/metabolismo , Compartimento Celular , Proteínas de Plantas/genética , Sinais Direcionadores de Proteínas/genética , Sinais Direcionadores de Proteínas/fisiologia , Transporte ProteicoRESUMO
Membrane proteins exit the endoplasmic reticulum (ER) in COPII-transport vesicles. ER export is a selective process in which transport signals present in the cytoplasmic tail (CT) of cargo membrane proteins must be recognized by coatomer proteins for incorporation in COPII vesicles. Two classes of ER export signals have been described for type I membrane proteins, the diacidic and the dihydrophobic motifs. Both motifs participate in the Sar1-dependent binding of Sec23p-Sec24p complex to the CTs during early steps of cargo selection. However, information concerning the amino acids in the CTs that interact with Sar1 is lacking. Herein, we describe a third class of ER export motif, [RK](X)[RK], at the CT of Golgi resident glycosyltransferases that is required for these type II membrane proteins to exit the ER. The dibasic motif is located proximal to the transmembrane border, and experiments of cross-linking in microsomal membranes and of binding to immobilized peptides showed that it directly interacts with the COPII component Sar1. Sar1GTP-bound to immobilized peptides binds Sec23p. Collectively, the present data suggest that interaction of the dibasic motif with Sar1 participates in early steps of selection of Golgi resident glycosyltransferases for transport in COPII vesicles.
Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Retículo Endoplasmático/metabolismo , Glicosiltransferases/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Sinais Direcionadores de Proteínas/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Animais , Células Cultivadas , Clonagem Molecular , Cricetinae , Cricetulus , Dados de Sequência Molecular , Mutação , Estrutura Terciária de Proteína/fisiologia , Proteínas/metabolismo , Análise de Sequência de Proteína , Proteínas de Transporte VesicularRESUMO
The targeting of proteins to cell organelles and membranes, or of proteins destined to secretion, is coordinated by signal sequences located at the 5'-end of their respective genes. A signal sequence trap system was envisaged in which a truncated version of the yeast acid phosphatase pho5 gene lacking the start codon and signal sequence could serve as a reporter gene. A fraction enriched in 5'-end fragments obtained by PCR from a potato guard-cell cDNA library was cloned in frame to the acid phosphatase gene and the acid phosphatase activity was assayed directly in yeast colonies grown on selective medium. Putative signal sequences targeting the acid phosphatase to the membrane or to the outside of the cell were used to screen the cDNA bank in order to recover the original full-size sequence which gave rise to the signal sequence. Two unknown sequences displaying marked tissue-specific expression were retrieved, one of them (YE139) with a higher expression level in green buds and stem cells, and the other one (YE290) with a higher expression level in androceum, gyneceum, and roots. The limitations of the system are further analyzed using other sequences as control.