Evaluating the role of CRM1-mediated export for adenovirus gene expression

A complex of the Adenovirus (Ad) early region 1b 55-kDa (E1b-55kDa) and early region 4 ORF6 34-kDa (E4-34kDa) proteins promotes viral late gene expression. E1b-55kDa and E4-34kDa have leucine-rich nuclear export signals (NESs) similar to that of HIV Rev. It was proposed that E1b-55kDa and/or E4-34kDa might promote the export of Ad late mRNA via their Rev-like NESs, and the transport receptor CRM1. We treated infected cells with the cytotoxin leptomycin B to inhibit CRM1-mediated export; treatment initially delays the onset of late gene expression, but this activity completely recovers as the late phase progresses. We find that the E1b-55kDa NES is not required to promote late gene expression. Previous results showed that E4-34kDa-mediated late gene expression does not require an intact NES (J. Virol. 74 (2000), 6684-6688). Our results indicate that these Ad regulatory proteins promote late gene expression without intact NESs or active CRM1.     

Insights into IL-33 on inflammatory response during in vitro infection by Trypanosoma cruzi

Inflammatory and regulatory cytokines play an important role in the immunopathogenesis of Trypanosoma cruzi infection. Interleukin (IL)-33 is a member of the IL-1 superfamily of cytokines whose expression/production is upregulated following pro-inflammatory stimulation to alert the immune system in response to tissue stress or damage. The aim of this study was to evaluate the inflammatory profile induced in cultured J774 cells stimulated or not with IL-33 (10 ng/mL), with live parasites (1 × 10⁶ metacyclic trypomastigote forms) and/or total antigen, TcAg (100 µg/mL) and with both, IL-33 and TcAg/T. cruzi. The cultures were evaluated at 24 h and 48 h after addition of the stimuli. For this, the supernatants were collected for the measurement of TNF, IL-17, CCL2, and IL-10 by ELISA and of nitrite by the Griess method. TNF, IL-17, and CCL2 concentrations were elevated in the presence of TcAg or live T. cruzi parasites at 24 h, and the addition of IL-33 potentiated these effects at 48 h. In addition, the T. cruzi-amastigote forms reduced in those infected J774 cells stimulated with IL-33 at 48 h. In conclusion, the IL-33 elevated the production of the TNF, IL-17, and CCL2 in cultured J774 cells stimulated with T. cruzi and/or its antigen and reduced the intracellular parasites, providing impetus to new investigations on its potential actions on the parasite-induced inflammation.     

The BRO proteins of Bombyx mori nucleopolyhedrovirus are nucleocytoplasmic shuttling proteins that utilize the CRM1-mediated nuclear export pathway

The BRO proteins of Bombyx mori nucleopolyhedrovirus (BmNPV) display a biphasic pattern of intracellular localization during infection. At early times, they reside in the nucleus but then show both cytoplasmic and nuclear localization as the infection proceeds. Therefore, we examined the possibility of nuclear export. Using inhibitors, we reveal that BmNPV BRO proteins shuttle between the nucleus and cytoplasm. Mutations on the leucine-rich region of BRO proteins resulted in nuclear accumulation of transiently expressed proteins, suggesting that this region functions as a CRM1-dependent nuclear export signal (NES). On the contrary, mutant BRO-D with an altered NES did not show nuclear accumulation in infected cells, although protein production seemed to be reduced. RT-PCR analysis showed that the lower level of protein production was due to a reduction in RNA synthesis. Taken together, our results suggest that BRO proteins are nucleocytoplasmic shuttling proteins that utilize the CRM1-mediated nuclear export pathway.     

Endogenous presentation of a nuclear antigen on MHC class II by autophagy in the absence of CRM1-mediated nuclear export

Accumulating evidence suggests that intracellular antigens are endogenously presented on MHC class II, but it is still unknown whether antigens within different subcellular compartments are presented with similar efficiency, and via the same or different pathways. We have previously shown that endogenous MHC class II presentation of the cytosolic bacterial antigen neomycin phosphotransferase II (NeoR) is mediated by autophagy. Here, we addressed whether secluding NeoR from this cytoplasmic pathway by directing the protein into the cell nucleus (NucNeoR) would affect antigen presentation. Unexpectedly, NucNeoR was presented at least as efficiently as the cytosolic version of the antigen. Furthermore, presentation of NucNeoR was also dependent on autophagocytosis and lysosomal processing, indicating that both antigens were presented via the same pathway. Inhibition of CRM1-mediated nuclear export did not impede antigen presentation, indicating that NucNeoR gained access to this autophagy-dependent MHC class II presentation pathway by a CRM1-independent route. Thus, this endogenous presentation pathway broadens the spectrum of intracellular antigens surveyed by CD4(+) T cells by efficiently sampling cytoplasmic as well as nuclear antigens.     

Involvement of CRM1, a nuclear export receptor, in mRNA export in mammalian cells and fission yeast

BACKGROUND: CRM1, an evolutionarily conserved protein, was shown to be a receptor for leucine-rich nuclear export signal (NES)-dependent protein transport. In lower eukaryotes CRM1 is reported to be required for the export of mRNA, however, involvement of the NES-dependent transport pathway in mRNA export in higher eukaryotes has not been established. RESULTS: We have found that treatment of mammalian cells with leptomycin B (LMB), a specific inhibitor of CRM1, induces the nuclear accumulation of endogenous mRNA, probably due to the inhibition of its export. In fission yeasts, the nuclear accumulation of mRNA also occurred in cells treated with LMB or in a temperature-sensitive crm1 mutant at a restrictive temperature. A synthetic mRNA that was injected into the nucleus of mammalian cultured cells was exported from the nucleus within 5 h. This export was inhibited by both wheat germ agglutinin and a temperature of 4 degrees C. Importantly, this mRNA export was inhibited by LMB or by an excess amount of the NES peptide-conjugates. LMB treatment, on the other hand, rapidly induced the nuclear entry of RanBP1, a factor involved in the active nucleocytoplasmic transport, although the treatment did not interfere with a nuclear localization signal-dependent transport system within 7 h. CONCLUSION: These results suggest that CRM1 is involved in mRNA export in eukaryotic cells.     

Alternative splicing of LYT1 transcripts in Trypanosoma cruzi

As a result of alternative trans splicing, three distinct LYT1 mRNAs are produced in Trypanosoma cruzi, two encoding the full-length LYT1 protein and the third encoding a truncated LYT1 protein lacking a possible signal sequence. Analysis of the three mRNAs in different developmental forms of the parasite revealed that the alternative processing events were regulated differently during the parasite life cycle.     

出核因子CRM1及p27在胶质瘤中的表达

目的 探讨出核因子CRM1、p27 10位丝氨酸(Ser10)磷酸化及p27蛋白在胶质瘤中的表达、相互关系及意义.方法 免疫组织化学SP法检测70例胶质瘤和10例非肿瘤对照脑组织标本中CRM1、p27Ser10磷酸化形式及p27蛋白的表达,Western blot检测6例新鲜胶质瘤标本中相应蛋白的表达.结果 CRM1及p27Ser10磷酸化形式在对照脑组织中表达不明显,在低级别胶质瘤中表达较少,在高级别胶质瘤中表达较多,两两比较差异均有统计学意义(P<0.01).p27在对照脑组织中表达明显,其表达水平随肿瘤级别增高而降低,差异有统计学意义(P<0.01).Western blot结果显示CRM1、p27Ser10磷酸化形式在胶质瘤中的表达水平与肿瘤细胞的恶性程度相关.相关分析显示:胶质瘤中CRM1蛋白表达与p27蛋白表达呈负相关(r=0.727,P<0.01),与p27Ser10磷酸化形式呈正相关(rs=0.954,P<0.01),与增殖指标Ki-67表达呈正相关(rs=0.799,P<0.01);p27Ser10磷酸化形式与p27蛋白表达呈负相关(rs=-0.744,P<0.01),与Ki-67表达呈正相关(rs=0.785,P<0.01).结论 在胶质瘤中高表达的CRM1可能通过识别并结合高表达的p27Ser10磷酸化形式,促进p27的出核降解,使p27表达降低,失去对细胞周期的调控,从而促进胶质瘤的恶性进展和增殖.     

Localization and Developmental Regulation of a Dispersed Gene Family 1 Protein in Trypanosoma cruzi

The dispersed gene family 1 (DGF-1) is the fifth largest gene family in the Trypanosoma cruzi genome, with over 500 members (11). Many of the predicted DGF-1 protein products have several transmembrane domains and N-glycosylation and phosphorylation sites and were thought to localize in the plasma membrane. Here, we report that affinity-purified antibodies against a region of one of these proteins (DGF-1.2) localized it intracellularly in different stages of the parasite. DGF-1.2 is more abundant in the amastigote stage than in trypomastigotes and epimastigotes, as detected by immunofluorescence and Western blot analyses. The protein changed localization during intracellular or extracellular differentiation from the trypomastigote to the amastigote stage, where it finally localized to small bodies in close contact with the inner side of the amastigote plasma membrane. DGF-1.2 did not colocalize with markers of other subcellular organelles, such as acidocalcisomes, glycosomes, reservosomes, lipid droplets, or endocytic vesicles. During extracellular differentiation, the protein was detected in the culture medium from 0 to 22 h, peaking at 14 h. The presence of DGF-1.2 in the differentiation culture medium was confirmed by mass spectrometry analysis. Finally, when epimastigotes were subjected to starvation, there was a decrease in the labeling of the cells and, in Western blots, the appearance of bands of lower molecular mass, suggesting its cleavage. These results represent the first report of direct immunodetection and developmental expression and secretion of a DGF-1 protein.Trypanosoma cruzi is the causative agent of Chagas disease, an endemic illness affecting between 16 and 18 million people in North, Central, and South America for which no vaccine or satisfactory treatment is available (22). During its life cycle, the parasite goes through different stages in the vector (epimastigotes and metacyclic trypomastigotes) and in the mammalian host (amastigotes and bloodstream trypomastigotes). As part of its survival strategy in these varying environments, the parasite has developed a large repertoire of multigene families (9, 11, 12, 16). Among these families, the dispersed gene family 1 (DGF-1) has approximately 565 copies, ranging from 6 to 10 kbp, dispersed throughout the parasite genome (11). The members of the DGF-1 family encode proteins that share 85 to 95% sequence identity (11). Wincker and colleagues first identified clones bearing a common repeated sequence from a T. cruzi genome library (24) and later described the nucleotide sequence of a representative gene (DGF-1.1) (23). They concluded, from in silico studies, that DGF-1 genes encoded putative cell surface proteins (23). In 2005, Kim and colleagues (16) described a new member of this family (DGF-1.2) located in the subtelomeric region of a T. cruzi chromosome surrounded by mainly two kinds of sequences: genes encoding the trans-sialidase (TS) and retrotransposon hot spot (RHS) protein families. The sizes of the open reading frames (ORFs) of DGF-1 genes and their abundance in the T. cruzi genome suggested that they are essential sequences for parasite survival. Furthermore, the existence of some telomeric DGF-1 copies that were always flanked by pseudogenes suggested that these genes have been subjected to strong selective pressure and, as a consequence, that they should be expressed at some life cycle stage of the parasite (16).A glycopeptide shared by several members of the DGF-1 family was recently detected in a glycoproteomic study of T. cruzi trypomastigotes, demonstrating that at least a DGF-1 family member protein is actually expressed and N-glycosylated (3). We also detected a number of peptides corresponding to several DGF-1 family member proteins in a proteomic study of acidocalcisome fractions of epimastigotes of T. cruzi (R. Docampo, J. A. Atwood, R. Tarleton, and R. Orlando, unpublished data). However, this family of proteins has no known orthologs in other species, even in trypanosomatids, and little is known about their localization, expression patterns, and functions in T. cruzi.In the present work, we prepared affinity-purified antibodies against a peptide of the DGF-1.2 protein and investigated its subcellular localization by immunofluorescence microscopy. We also used mass spectrometry (MS) to identify specific peptides recognized by anti-DGF-1.2 antibodies by using fingerprinting analysis.We found that the antibodies preferentially labeled amastigotes. The localization of the protein was in intracellular bodies and not on the cell surface and changed during amastigote development. During the in vitro trypomastigote-to-amastigote transition, we detected continuous secretion of DGF-1.2 into the medium, peaking at 14 h. Anti DGF-1 antibodies that recognized the intracellular protein in both differentiation forms also recognized the secreted protein from trypomastigotes and amastigotes. Finally, when epimastigotes were subjected to starvation, there was a decrease in labeling of the cells and the appearance of defined bands of smaller molecular mass in Western blots, suggesting its cleavage.     

TcrPDEA1, a cAMP-specific phosphodiesterase with atypical pharmacological properties from Trypanosoma cruzi

Cyclic nucleotide phosphodiesterases (PDEs) catalyze the degradation of cAMP and cGMP, and regulate a variety of cellular processes by controlling the levels of these second messengers. We have previously described the presence of both a calcium-stimulated adenylyl cyclase and two membrane-bound cAMP-specific PDEs (one of them strongly associated to the flagellum and the other one with a possible vesicular localization) in Trypanosoma cruzi. Here we report the identification and characterization of TcrPDEA1, a singular phosphodiesterase of T. cruzi which is resistant to the typical phosphodiesterase inhibitors, such as IBMX, papaverine and theofylline. TcrPDEA1 is a single copy gene that encodes a 620-amino acid protein, which is grouped with PDE1 family members, mainly with its kinetoplastid orthologs. TcrPDEA1 was able to complement a mutant yeast strain deficient in PDE genes, demonstrating that this enzyme is a functional phosphodiesterase. TcrPDEA1 is specific for cAMP with a high K(m) value (191.1+/-6.5 microM). Cyclic GMP neither activates the enzyme nor competes as a substrate. In addition, calcium-calmodulin did not affect the kinetic parameters and, as its counterpart in T. brucei, magnesium showed to be crucial for its activity and stability. Although TcrPDEA1 function remains unclear, its presence points out the high complexity of the cAMP signaling in trypanosomatids and the possible compartmentalization of the enzymes involved in the cAMP pathway.     

Two casein kinase 1 isoforms are differentially expressed in Trypanosoma cruzi

The cDNAs for two casein kinase 1 (CK1) homologues, TcCK1.1 and TcCK1.2, have been isolated from Trypanosoma cruzi. Both isoforms showed strong identity with other known CK1s. Their corresponding genes encode proteins of 312- and 330-amino acid residues with apparent molecular weights of 16 and 37 kDa, respectively. TcCK1.1 is a two-copy gene while TcCK1.2 is tandemly repeated, an arrangement not yet found in any other CK1. TcCK1.1 has been overexpressed in Escherichia coli and the recombinant protein exhibited properties characteristic of the CK1 family. Northern blot indicated that both TcCK1s are expressed differentially during the life stages of the parasite: the isoform TcCK1.1 shows low levels of mRNA expression in epimastigotes and increased expression in trypomastigotes while TcCK1.2 presents an augmented expression in amastigotes as compared with the other two life stages of the parasite. The CK1-like activity of amastigotes and trypomastigotes is significantly higher than that of epimastigotes and, independent of the life stage of the parasite, a constitutive activity is observed which, in the epimastigote forms, is found predominantly in the microsomal fraction. Also in the epimastigote forms, the CK1-like activity increases in the log phase of growth of the parasites, and, through synchronization studies, this activity has been most conspicuously circumscribed to the S and M phases of the cell cycle.     

Myocardial expression of endothelin-1 in murine Trypanosoma cruzi infection.

Chagas' disease, caused by Trypanosoma cruzi, is an important cause of myocarditis and chronic cardiomyopathy and is accompanied by microvascular spasm and myocardial ischemia. We reported previously that infection of cultured endothelial cells with T. cruzi increased the synthesis of biologically active endothlein-1 (ET-1). In the present study, we examined the role of ET-1 in the cardiovascular system of CD1 mice infected with the Brazil strain of T. cruzi and C57BL/6 mice infected with the Tulahuen strain during acute infection. In the myocardium of infected mice myonecrosis and multiple pseudocysts were observed. There was also an intense vasculitis of the aorta, coronary artery, smaller myocardial vessels and the endocardial endothelium. Immunohistochemistry studies employing anti-ET-1 antibody revealed increased expression of ET-1 that was most intense in the endocardial and vascular endothelium. Elevated levels of mRNA for preproET-1, endothelin converting enzyme and ET-1 were observed in the same myocardial samples. Plasma ET-1 levels were significantly elevated in infected CD1 mice 10-15 days post infection. These observations suggest that increased levels of ET-1 are a consequence of the initial invasion of the cardiovascular system and provide a mechanism for infection-associated myocardial dysfunction.     

Morbillivirus nucleoprotein possesses a novel nuclear localization signal and a CRM1-independent nuclear export signal

Morbilliviruses, which belong to the Mononegavirales, replicate its RNA genome in the cytoplasm of the host cell. However, they also form characteristic intranuclear inclusion bodies, consisting of nucleoprotein (N), in infected cells. To analyze the mechanisms of nucleocytoplasmic transport of N protein, we characterized the nuclear localization (NLS) and nuclear export (NES) signals of canine distemper virus (CDV) N protein by deletion mutation and alanine substitution of the protein. The NLS has a novel leucine/isoleucine-rich motif (TGILISIL) at positions 70-77, whereas the NES is composed of a leucine-rich motif (LLRSLTLF) at positions 4-11. The NLS and NES of the N proteins of other morbilliviruses, that is, measles virus (MV) and rinderpest virus (RPV), were also analyzed. The NLS of CDV-N protein is conserved at the same position in MV-N protein, whereas the NES of MV-N protein is located in the C-terminal region. The NES of RPV-N protein is also located at the same position as CDV-N protein, whereas the NLS motif is present not only at the same locus as CDV-N protein but also at other sites. Interestingly, the nuclear export of all these N proteins appears to proceed via a CRM1-independent pathway.     

A new cruzipain-mediated pathway of human cell invasion by Trypanosoma cruzi requires trypomastigote membranes

The intracellular protozoan Trypanosoma cruzi causes Chagas' disease, a chronic illness associated with cardiomyopathy and digestive disorders. This pathogen invades mammalian cells by signaling them through multiple transduction pathways. We previously showed that cruzipain, the main cysteine protease of T. cruzi, promotes host cell invasion by activating kinin receptors. Here, we report a cruzipain-mediated invasion route that is not blocked by kinin receptor antagonists. By testing different strains of T. cruzi, we observed a correlation between the level of cruzipain secreted by trypomastigotes and the capacity of the pathogen to invade host cells. Consistent with a role for cruzipain, the cysteine protease inhibitor N-methylpiperazine-urea-Phe-homophenylalanine-vinylsulfone-benzene impaired the invasion of human smooth muscle cells by strains Dm28c and X10/6 but not by the G isolate. Cruzipain-rich supernatants of Dm28c trypomastigotes enhanced the infectivity of isolate G parasites twofold, an effect which was abolished by the cysteine protease inhibitor l-trans-epoxysuccinyl-leucylamido-(4-guanidino)butane and by thapsigargin, a drug that induces depletion of the intracellular Ca(2+) stores. The enhancement due to Dm28 supernatants was abolished upon cruzipain immunodepletion, and the activity was restored by purified cruzipain. In contrast, supernatants from isolate G trypomastigotes (with low levels of cruzipain) or supernatants from Dm28c epimastigotes or purified cruzipain alone did not enhance parasite invasion, indicating that the protease is required but not sufficient to engage this invasion pathway. We provide evidence that activation of this pathway requires cruzipain-mediated processing of a trypomastigote molecule associated with parasite-shed membranes. Our results couple cruzipain to host cell invasion through a kinin-independent route and further suggest that high-level cruzipain expression may contribute to parasite infectivity.     

Genomic variation in Trypanosoma cruzi clonal cultures

 Spontaneous changes in restriction DNA profiles and pulsed-field gel electrophoresis (PFGE) patterns, along with a concomitant loss of infectivity, were observed in infective clones of Trypanosoma cruzi strain Y either following a number of passages during the exponential growth phase or after subcloning in liver infusion tryptone (LIT) medium using as the probe a genomic fragment of the parasite (pMYP16), indicating naturally occurring rearrangements of DNA sequences. No variation could be detected when the genomic DNA was probed with conserved T. cruzi tubulin and actin genes. There was no correlation between such rearrangements and the life-cycle forms of the parasites, since trypomastigote forms showed the same karyotype and hybridization patterns as did epimastigote forms. The variations observed could be reverted and infectivity, recovered after inoculation of the parasites in newborn mice. Received: 10 March 1995 / Accepted: 1 August 1995     

Incorporation of sialic acid into Trypanosoma cruzi macromolecules. A proposal for a new metabolic route

Sialo- and asialoglycoconjugates were isolated from Trypanosoma cruzi epimastigotes and their composition determined. Sialoglycoconjugates bound to wheat germ agglutinin (WGA)-Sepharose and were precipitated by concanavalin A, Wistaria floribunda hemagglutinin and WGA. Asialoglycoconjugate bound to concanavalin A-Sepharose and precipitated with concanavalin-A and W. floribunda hemagglutinin but not with WGA. Cells grown in the presence of fetal calf serum were agglutinated by WGA but not by peanut agglutinin. The reverse was true for cells grown without fetal calf serum. Neuraminidase-treated cells incorporated sialic acid or its 7-carbon analog, 5-acetamido-3,5-dideoxy-L-arabino-2-heptulosonic acid (AcNeu7) from sialylated compounds such as fetuin or sialyl-lactose but did not incorporate free sialic acid. Restoration of the WGA sialylreceptors in neuraminidase-treated cells, as determined by cell agglutination with WGA, was also obtained by incubation with fetuin or sialyl-lactose but not with free sialic acid. Moreover, restoration of agglutinability by WGA in neuraminidase-treated cells or cells grown in medium without fetal calf serum occurred equally well in energy-rich or energy-depleted cells. A transglycosilase reaction for sialic acid incorporation in T. cruzi epimastigotes is suggested.     

Trypanosoma cruzi infection in B-cell-deficient rats.

The effect of neonatally initiated injections of anti-mu rabbit antiserum on immunity of rats against Trypanosoma cruzi infection was investigated in vivo. Anti-mu treatment resulted in a loss of immunoglobulin M (IgM) and IgG2a synthesis and, subsequently, of antibody production. These rats so treated were shown to be significantly more susceptible to the acute phase of the infection than the control rats treated with normal rabbit serum, as measured by increased parasitemia and mortality. These results indicate the essential role of antibodies, probably in association with complement or effector cells or both, in immunity to acute Chagas' disease.     

Multiple DNA-dependent RNA polymerases in Trypanosoma cruzi

DNA-dependant RNA polymerase solubilized from cultured Trypanosoma cruzi epimastigotes was chromatographed on A-25 Sephadex and gave a single peak of activity. Subsequent phosphocellulose chromatography revealed two peaks of RNA polymerase activity. These peaks have different sensitivities to the toxin alpha-amanitin. The first peak is 50% inhibited by 17.8 micrograms ml-1 amanitin while the second peak is 50% inhibited by an amanitin concentration of 44.6 micrograms ml-1. The activity of both peaks is blocked by actinomycin D, but is unaffected by rifampicin. Each peak is stimulated by Mn2+, and is optimal with single stranded DNA as a template.