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ABSTRACT: In animals, inositol 1,4,5-trisphosphate receptors (IP(3) Rs) are ion channels that play a pivotal role in many biological processes by mediating Ca(2+) release from the endoplasmic reticulum. Here, we report the identification and characterization of a novel IP(3) R in the parasitic protist, Trypanosoma cruzi, the pathogen responsible for Chagas disease. DT40 cells lacking endogenous IP(3) R genes expressing T. cruzi IP(3) R (TcIP(3) R) exhibited IP(3) -mediated Ca(2+) release from the ER, and demonstrated receptor binding to IP(3) . TcIP(3) R was expressed throughout the parasite life cycle but the expression level was much lower in bloodstream trypomastigotes than in intracellular amastigotes or epimastigotes. Disruption of 2 of the 3 TcIP(3) R gene loci led to the death of the parasite, suggesting that IP(3) R is essential for T. cruzi. Parasites expressing reduced or increased levels of TcIP(3) R displayed defects in growth, transformation, and infectivity, indicating that TcIP(3) R is an important regulator of the parasite's life cycle. Furthermore, mice infected with T. cruzi expressing reduced levels of TcIP(3) R exhibited a reduction of disease symptoms, indicating that TcIP(3) R is an important virulence factor. Combined with the fact that the primary structure of TcIP(3) R has low similarity to that of mammalian IP(3) Rs, TcIP(3) R is a promising drug target for Chagas disease.
Molecular Microbiology 01/2013; · 5.01 Impact Factor
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Kunihiko Minakata,
Fumiyuki Takahashi,
Takeshi Nara, Muneaki Hashimoto,
Ken Tajima,
Akiko Murakami,
Fariz Nurwidya,
Suzu Yae,
Fumiaki Koizumi,
Hiroyuki Moriyama,
Kuniaki Seyama,
Kazuto Nishio,
Kazuhisa Takahashi
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ABSTRACT: Somatic mutations in the epidermal growth factor receptor (EGFR) gene, such as exon 19 deletion mutations, are important factors in determining therapeutic responses to gefitinib in non-small-cell lung cancer (NSCLC). However, some patients have activating mutations in EGFR and show poor responses to gefitinib. In this study, we examined three NSCLC cell lines, HCC827, PC9, and HCC2935, that expressed an EGFR exon 19 deletion mutation. All cells expressed mutant EGFR, but the PC9 and HCC2935 cells also expressed wild-type EGFR. The HCC827 cells were highly sensitive to gefitinib under both normoxia and hypoxia. However, the PC9 and HCC2935 cells were more resistant to gefitinib under hypoxic conditions compared to normoxia. Phosphorylation of EGFR and ERK was suppressed with gefitinib treatment to a lesser extent under hypoxia. The expression of transforming growth factor-α (TGFα) was dramatically upregulated under hypoxia, and the knockdown of TGFα or hypoxia-inducible factor-1α (HIF1α) reversed the resistance to gefitinib in hypoxic PC9 and HCC2935 cells. Finally, introduction of the wild-type EGFR gene into the HCC827 cells caused resistance to gefitinib under hypoxia. This phenomenon was also reversed by the knockdown of TGFα or HIF1α. Our results indicate that hypoxia causes gefitinib resistance in EGFR-mutant NSCLC through the activation of wild-type EGFR mediated by the upregulation of TGFα. The presence of wild-type and mutant EGFR along with tumor hypoxia are important factors that should be considered when treating NSCLC patients with gefitinib.
Cancer Science 08/2012; · 3.33 Impact Factor
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Takeshi Nara, Muneaki Hashimoto,
Hiroko Hirawake,
Chien-Wei Liao,
Yoshihisa Fukai,
Shigeo Suzuki,
Akiko Tsubouchi,
Jorge Morales,
Shinzaburo Takamiya,
Tsutomu Fujimura,
Hikari Taka,
Reiko Mineki,
Chia-Kwung Fan,
Daniel Ken Inaoka,
Masayuki Inoue,
Akiko Tanaka,
Shigeharu Harada,
Kiyoshi Kita,
Takashi Aoki
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ABSTRACT: The first 3 reaction steps of the de novo pyrimidine biosynthetic pathway are catalyzed by carbamoyl-phosphate synthetase II (CPSII), aspartate transcarbamoylase (ATC), and dihydroorotase (DHO), respectively. In eukaryotes, these enzymes are structurally classified into 2 types: (1) a CPSII-DHO-ATC fusion enzyme (CAD) found in animals, fungi, and amoebozoa, and (2) stand-alone enzymes found in plants and the protist groups. In the present study, we demonstrate direct intermolecular interactions between CPSII, ATC, and DHO of the parasitic protist Trypanosoma cruzi, which is the causative agent of Chagas disease. The 3 enzymes were expressed in a bacterial expression system and their interactions were examined. Immunoprecipitation using an antibody specific for each enzyme coupled with Western blotting-based detection using antibodies for the counterpart enzymes showed co-precipitation of all 3 enzymes. From an evolutionary viewpoint, the formation of a functional tri-enzyme complex may have preceded-and led to-gene fusion to produce the CAD protein. This is the first report to demonstrate the structural basis of these 3 enzymes as a model of CAD. Moreover, in conjunction with the essentiality of de novo pyrimidine biosynthesis in the parasite, our findings provide a rationale for new strategies for developing drugs for Chagas disease, which target the intermolecular interactions of these 3 enzymes.
Biochemical and Biophysical Research Communications 02/2012; 418(1):140-3. · 2.48 Impact Factor
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Muneaki Hashimoto,
Jorge Morales,
Yoshihisa Fukai,
Shigeo Suzuki,
Shinzaburo Takamiya,
Akiko Tsubouchi,
Syou Inoue,
Masayuki Inoue,
Kiyoshi Kita,
Shigeharu Harada,
Akiko Tanaka,
Takashi Aoki,
Takeshi Nara
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ABSTRACT: The intracellular parasitic protist Trypanosoma cruzi is the causative agent of Chagas disease in Latin America. In general, pyrimidine nucleotides are supplied by both de novo biosynthesis and salvage pathways. While epimastigotes-an insect form-possess both activities, amastigotes-an intracellular replicating form of T. cruzi-are unable to mediate the uptake of pyrimidine. However, the requirement of de novo pyrimidine biosynthesis for parasite growth and survival has not yet been elucidated. Carbamoyl-phosphate synthetase II (CPSII) is the first and rate-limiting enzyme of the de novo biosynthetic pathway, and increased CPSII activity is associated with the rapid proliferation of tumor cells. In the present study, we showed that disruption of the T. cruzi cpsII gene significantly reduced parasite growth. In particular, the growth of amastigotes lacking the cpsII gene was severely suppressed. Thus, the de novo pyrimidine pathway is important for proliferation of T. cruzi in the host cell cytoplasm and represents a promising target for chemotherapy against Chagas disease.
Biochemical and Biophysical Research Communications 12/2011; 417(3):1002-6. · 2.48 Impact Factor
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Ken Tajima,
Kayo Miura,
Toshiji Ishiwata,
Fumiyuki Takahashi,
Masataka Yoshioka,
Kunihiko Minakata,
Akiko Murakami,
Shinichi Sasaki,
Shinichiro Iwakami,
Takeshi Annoura, Muneaki Hashimoto,
Takeshi Nara,
Kazuhisa Takahashi
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ABSTRACT: The lung is one of the sites of granulomatous responses, which are characterized by the recruitment and organization of activated macrophages and lymphocytes. There have been several reports that have shown that some pulmonary granulomatous diseases, such as sarcoidosis and nontuberculous mycobacterial disease, are likely to be characterized by a preponderance in postmenopausal females. Although sex hormones have been shown to play an important role in the regulation of the immune system, the influence of sex hormones on pulmonary granuloma formation is still unclear. Objectives: The purpose of this study was to assess whether sex hormones are involved in granulomatous inflammation and to evaluate how sex hormones modulate this response in the lung.
Ovariectomized rats were used as an experimental postmenopausal model in which chronic pulmonary granulomatous inflammation was induced by intravenous injection of complete Freund's adjuvant.
Histological analysis of lung tissues demonstrated enhancement of granuloma formation in the ovariectomized group. Such enhanced granuloma formation was significantly associated with generalized Th1-biased cytokine production in the bronchoalveolar lavage fluid.
These results indicate that sex hormones play an important role in pulmonary granuloma formation by altering the Th1 responses.
Respiration 04/2011; 81(6):491-8. · 2.26 Impact Factor
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ABSTRACT: Glycosomes are peroxisome-related organelles containing glycolytic enzymes that have been found only in kinetoplastids. We show here that a glycolytic enzyme is compartmentalized in diplonemids, the sister group of kinetoplastids. We found that, similar to kinetoplastid aldolases, the fructose 1,6-bisphosphate aldolase of Diplonema papillatum possesses a type 2-peroxisomal targeting signal. Western blotting showed that this aldolase was present predominantly in the membrane/organellar fraction. Immunofluorescence analysis showed that this aldolase had a scattered distribution in the cytosol, suggesting its compartmentalization. In contrast, orotidine-5'-monophosphate decarboxylase, a non-glycolytic glycosomal enzyme in kinetoplastids, was shown to be a cytosolic enzyme in D. papillatum. Since euglenoids, the earliest diverging branch of Euglenozoa, do not possess glycolytic compartments, these findings suggest that the routing of glycolytic enzymes into peroxisomes may have occurred in a common ancestor of diplonemids and kinetoplastids, followed by diversification of these newly established organelles in each of these euglenozoan lineages.
Protist 03/2011; 162(3):482-9. · 3.14 Impact Factor
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ABSTRACT: While some intracellular bacterial and viral proteins secreted into host cell possess ubiquitin ligase (E3) activity for their profit, it has not been reported whether intracellular parasites secrete such molecules. We identified a gene that encodes a protein containing a secretory signal peptide and a RING finger domain in the intracellular protozoan parasite, Trypanosoma cruzi. This gene was specific to T. cruzi and was designated spring (secretory protein with RING finger domain). An in vitro ubiquitination assay showed that SPRING possessed E3 activity in a RING finger domain-dependent manner. SPRING could utilize human ubiquitin-activating enzymes (E2), UbcH5 and UbcH13. Although SPRING was found to be a secretory protein, the signal peptide-cleaved mature form of SPRING was localized in the nucleus of host cells, indicating that SPRING may function in the host cell nuclei. Yeast two-hybrid screening identified 52 putative SPRING interactors in HeLa cells, suggesting that SPRING affects the stability or function of a number of host proteins. Furthermore, a co-immunoprecipitation assay showed that breast cancer-associated protein 3 interacted with SPRING, as well as being ubiquitinated by SPRING in vitro. These findings are the first to show that this protozoan parasite secretes an ubiquitin ligase-related protein into host cells.
Cellular Microbiology 09/2009; 12(1):19-30. · 5.46 Impact Factor
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ABSTRACT: Cyclic AMP-protein kinase A (PKA) signaling is important for the growth and differentiation of Trypanosoma cruzi. Immunofluorescence suggests that PKA can associate with the plasma membrane of trypomastigotes. We found that the PKA regulatory subunit interacts with several P-type ATPases. These P-type ATPases may play a role in anchoring PKA to the plasma membrane in T. cruzi.
The American journal of tropical medicine and hygiene 07/2009; 80(6):941-3. · 2.59 Impact Factor
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ABSTRACT: We reported previously that Ascaris suum cytochrome b5, specifically expressed in this nematode at the adult stage and dually localized in extracellular perienteric fluid and hypodermis, is involved in both perienteric NADH-methemoglobin and cytosolic NADH-metmyoglobin reduction, where cytochrome b5 functions as an electron carrier between NADH-mediated cytochrome b5 reductase and substrates, methemo(myo)globins to reduce the nonfunctional globins back to functional ferrous hemo(myo)globins. To further characterize NADH-methemo(myo)globin reductase systems, the midpoint potentials of A. suum perienteric hemoglobin and body wall myoglobin, as well as the affinities of Ascaris methemoglobin and metmyoglobin toward cytochrome b5, were evaluated using potentiometric titration and surface plasmon resonance techniques, respectively. Midpoint potentials of +7.2 mV and +19.5 mV were obtained for Ascaris perienteric hemoglobin and body wall myoglobin, respectively. The affinities of Ascaris perienteric methemoglobin and body wall metmyoglobin toward the nematode cytochrome b5 were comparable to that for mammalian hemoglobin and cytochrome b5; association constants were 0.585 x 10(3) M(-1) and 2.32 x 10(3) M(-1), respectively, with rapid equilibration kinetics. These observations highlight the physiological importance of A. suum perienteric NADH-methemoglobin and cytosolic metmyoglobin reductase systems. Differential roles of A. suum perienteric hemoglobin and body wall myoglobin are also discussed from the viewpoint of oxygen homeostasis under hypoxic conditions.
Parasitology International 05/2009; 58(3):278-84. · 2.13 Impact Factor
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ABSTRACT: Death receptor-mediated host cell apoptosis, a defense strategy for elimination by the immune system of parasite-infected cells, is inhibited by Trypanosoma cruzi, the causative agent of Chagas' disease. It has previously been reported by us that, in infected cells, T. cruzi upregulates and exploits cFLIP(L), a mammalian inhibitor of death receptor signaling. Here it is shown that ubiquitination of cFLIP(L,) leading to proteasomal degradation, is inhibited in parasite-infected cells. The extent of expression of Itch, a protein thought to be an ubiquitin ligase for cFLIP(L), was found to be equivalent in T. cruzi-infected and in uninfected cells. However, co-immunoprecipitation analysis showed that the interaction between cFLIP(L) and Itch is strongly inhibited in T. cruzi-infected cells. This unique parasite strategy, which has not been reported in any other pathogen-infected cells, may allow the host cell to accumulate cFLIP(L), eventually resulting in the inhibition of apoptosis of T. cruzi-infected cells.
Microbiology and Immunology 12/2008; 52(11):539-43. · 1.30 Impact Factor
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ABSTRACT: The anaerobic parasitic nematode Ascaris suum has an oxygen-avid hemoglobin in the perienteric fluid, the biological function of which remains elusive. Here, we report that Ascaris cytochrome b5 is expressed specifically in the intestinal parasitic stage and is secreted into the perienteric fluid, thus co-localizing with Ascaris hemoglobin. We also found that cytochrome b5 reduces Ascaris non-functioning ferric methemoglobin more efficiently than mammalian methemoglobin. Furthermore, a computer graphics model of the electron transfer complex between Ascaris cytochrome b5 and Ascaris hemoglobin strongly suggested that these two proteins are physiological redox partners. Nitric oxide has been reported to react easily with oxygen captured in hemoglobin to form nitrate, but not toxic free radicals, which may result in production of methemoglobin for the cytochrome b5 to regenerate functional ferrous hemoglobin. Therefore, our findings suggest that Ascaris cytochrome b5 is a key redox partner of Ascaris hemoglobin, which acts as an antioxidant.
Archives of Biochemistry and Biophysics 04/2008; 471(1):42-9. · 2.93 Impact Factor
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ABSTRACT: As Trypanosoma cruzi, the etiological agent of Chagas disease, multiplies in the cytoplasm of nucleated host cells, infection with this parasite is highly likely to affect host cells. We performed an exhaustive transcriptome analysis of T. cruzi-infected HeLa cells using an oligonucleotide microarray containing probes for greater than 47,000 human gene transcripts. In comparison with uninfected cells, those infected with T. cruzi showed greater than threefold up-regulation of 41 genes and greater than threefold down-regulation of 23 genes. Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) of selected, differentially expressed genes confirmed the microarray data. Many of these up- and down-regulated genes were related to cellular proliferation, including seven up-regulated genes encoding proliferation inhibitors and three down-regulated genes encoding proliferation promoters, strongly suggesting that T. cruzi infection inhibits host cell proliferation, which may allow more time for T. cruzi to replicate and produce its intracellular nests. These findings provide new insight into the molecular mechanisms by which intracellular T. cruzi infection influences the host cell, leading to pathogenicity.
Parasitology Research 04/2008; 102(4):715-22. · 2.15 Impact Factor
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ABSTRACT: Maintaining low intracellular sodium concentrations is vital for almost all organisms. Na(+) efflux is generally governed by P-type ATPases, Na(+)/K(+)-ATPase in animals and Na(+)-ATPase, called ENA, in fungi and plants. Trypanosoma cruzi, which parasitizes mammalian cells, must undergo drastic adaptations to high Na(+) concentrations outside and low Na(+) concentrations inside host cells. However, T. cruzi Na(+) efflux pumps have not been identified. We report here the cloning and characterization of the gene encoding Na(+)-ATPase in T cruzi, which resembled fungal and plant ENAs, termed TcENA. TcENA was a plasma membrane protein expressed throughout the parasite life cycle. The transcription level of TcENA was higher in insect stage epimastigotes and blood stream trypomastigotes than in intracellular amastigotes, probably reflecting the high Na(+) concentration outside the host cells. Biochemical analysis of TcENA expressed heterologously in mammalian cells demonstrated, for the fist time, that the ATPase activity of TcENA is stimulated by both Na(+) and K(+) and is insensitive to ouabain, a specific inhibitor of Na(+)/K(+)-ATPases. Furthermore, epimastigotes overproducing TcENA showed increased tolerance to high Na(+) stress. Our findings suggest that TcENA acts as a sodium pump and provide insights into the regulation of ion homeostasis in the parasitic protist.
Biochimica et Biophysica Acta 07/2006; 1758(6):738-46. · 4.66 Impact Factor
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ABSTRACT: Cytochrome b5 of the body wall of adult Ascaris suum, a porcine parasitic nematode, is a soluble protein that lacks a C-terminal membrane-anchoring domain, but possesses an N-terminal pre-sequence of 30 amino acids. During the maturation of cytochrome b5, the N-terminal pre-sequence is proteolytically cleaved to form the mature protein of 82 amino acid residues. A. suum cytochrome b5 is a basic protein containing more lysine residues and exhibiting a higher midpoint redox potential than its mammalian counterparts. We developed an expression system for the production of the recombinant nematode cytochrome b5, which is chemically and functionally identical with the native protein. Using this recombinant protein, we have determined the X-ray crystal structure of A. suum cytochrome b5 at 1.8 A (1 A=0.1 nm) resolution, and we have shown that this protein is involved in the reduction of nematode body-wall metmyoglobin. The crystal structure of A. suum cytochrome b5 consists of six alpha-helices and five beta-strands. It differs from its mammalian counterparts by having a head-to-tail disulphide bridge, as well as a four-residue insertion in the vicinity of the sixth ligating histidine, which forms an additional alpha-helix, alpha4A, between helices alpha4 and alpha5. A. suum cytochrome b5 exists predominantly as a haem-orientation B isomer. Furthermore, the haem plane is rotated approx. 80 degrees relative to the axis formed by haem-Fe and N atoms of the two histidine residues that are ligated to haem-Fe. The charge distribution around the haem crevice of A. suum cytochrome b5 is remarkably different from that of mammalian cytochrome b5 in that the nematode protein bears positively charged lysine residues surrounding the haem crevice. Using immunohistochemistry, we found that A. suum cytochrome b5 is present in the nematode hypodermis. Based on this histochemical and structural information, the physiological function of A. suum cytochrome b5 and its interaction with nematode metmyoglobin can be hypothesized.
Biochemical Journal 04/2006; 394(Pt 2):437-47. · 4.90 Impact Factor
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ABSTRACT: Dihydroorotate dehydrogenase (DHOD) is the fourth enzyme in the de novo pyrimidine biosynthetic pathway and is essential in Trypanosoma cruzi, the parasitic protist causing Chagas' disease. T. cruzi and human DHOD have different biochemical properties, including the electron acceptor capacities and cellular localization, suggesting that T. cruzi DHOD may be a potential chemotherapeutic target against Chagas' disease. Here, we report nucleotide sequence polymorphisms of T. cruzi DHOD genes and the kinetic properties of the recombinant enzymes. T. cruzi Tulahuen strain possesses three DHODgenes: DHOD1 and DHOD2, involved in the pyrimidine biosynthetic (pyr) gene cluster on an 800 and a 1000 kb chromosomal DNA, respectively, and DHOD3, located on an 800 kb DNA. The open reading frames of all three DHOD genes are comprised of 942 bp, and encode proteins of 314 amino acids. The three DHOD genes differ by 26 nucleotides, resulting in replacement of 8 amino acid residues. In contrast, all residues critical for constituting the active site are conserved among the three proteins. Recombinant T. cruzi DHOD1 and DHOD2 expressed in E. coli possess similar enzymatic properties, including optimal pH, optimal temperature, Vmax, and Km for dihydroorotate and fumarate. In contrast, DHOD3 had a higher Vmax and Km for both substrates. Orotate competitively inhibited all three DHOD enzymes to a comparable level. These results suggest that, despite their genetic variations, kinetic properties of the three T. cruziDHODs are conserved. Our findings facilitate further exploitation of T. cruzi DHOD inhibitors, as chemotherapeutic agents against Chagas' disease.
Parasitology International 04/2006; 55(1):11-6. · 2.13 Impact Factor
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ABSTRACT: To determine the molecular mechanism by which apoptosis is inhibited in Trypanosoma cruzi-infected host cells, we used human cDNA apoptosis chips to compare the gene expression profiles in response with 'death ligands target' (Fas) stimulation in infected and uninfected cells. Of the 164 apoptosis-related genes examined, 20, including those encoding both pro- and anti-apoptotic proteins, were highly up-regulated in the infected group. Genes encoding caspases and apoptosis inhibitors were optimally expressed 10-30 min after induction of apoptosis, whereas genes involved in transcriptional regulation and cell proliferation were up-regulated after 2-24 h. These results suggest that host anti-apoptotic gene(s) may play a crucial role in the inhibition of Fas-mediated apoptosis in T. cruzi-infected cells.
International Journal for Parasitology 01/2006; 35(14):1587-94. · 3.39 Impact Factor
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ABSTRACT: Intracellular persistence of the protozoan parasite, Trypanosoma cruzi, is an aggravating cause of Chagas' disease, involving that the protozoan infection specifically inhibits death receptor-mediated apoptosis of host cells. Here we demonstrate that the parasite dramatically up-regulates cellular FLICE inhibitory protein (c-FLIP), the only known mammalian inhibitor specific for death receptor signaling, in infected cells by an unusual, posttranscriptional stabilization of the short-lived protein. We also show that c-FLIP is accumulated in T. cruzi-infected mouse heart muscle cells in vivo. Stimulation of death receptor Fas in infected cells induces recruitment of c-FLIP to block the procaspase-8 activation at the most upstream caspase cascade. c-FLIP knock-down with a small interfering RNA significantly restores Fas-mediated apoptosis in infected cells. Taken together, our findings indicate that T. cruzi posttranscriptionally up-regulates and exploits host c-FLIP for the inhibition of death-inducing signal, a mechanism that may allow parasites to persist in host cells.
Molecular Biology of the Cell 09/2005; 16(8):3521-8. · 4.94 Impact Factor
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ABSTRACT: The cytochrome b(5) of the body wall of adult Ascaris suum, a porcine parasitic nematode, is a novel type of cytochrome b(5). It is a soluble protein that lacks the COOH-terminal membrane-anchoring domain found in erythrocyte cytochrome b(5), but possesses an NH(2)-terminal extension (presequence) of 30 amino acids that are missing from the 82-residue protein purified from the nematode tissues [Yu, Y., Yamasaki, H., Kita, K., and Takamiya, S., 1996, Arch. Biochem. Biophys. 328, 165-172]. The nematode cytochrome b(5) is, therefore, probably synthesized as a precursor protein whose presequence is cleaved to form a mature protein, but the localization of the mature protein is still unknown. To investigate the processing of the putative precursor protein, the wild-type precursor of nematode cytochrome b(5) with a complete presequence (b5wt) and its NH(2) terminus-truncated derivatives, b5Delta18 and b5Delta28, with 18 and 28 residues deleted, respectively, were expressed using pET-28a(+) vector in Escherichia coli. As expected, all transformants, tb5wt, tb5Delta18, and tb5Delta28, produced recombinant proteins with a histidine-tagged NH(2)-terminal extension. However, only the recombinant protein with the full-length presequence, produced in tb5wt, was correctly processed and transported to the periplasm, from which the majority of the induced product was purified as a mature protein chemically and functionally identical to the native protein purified from the nematode body wall. These results clearly show that the nematode histidine-tagged presequence functions as a signal peptide in E. coli.
Archives of Biochemistry and Biophysics 06/2003; 413(2):253-61. · 2.93 Impact Factor
