Activation of TGF-beta by Leishmania chagasi: importance for parasite survival in macrophages.
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TGF-beta is a potent regulatory cytokine that suppresses expression of inducible NO synthase and IFN-gamma, and suppresses Th1 and Th2 cell development. We examined whether functionally active TGF-beta is present in the local environment surrounding the invading protozoan Leishmania chagasi. Our prior data showed that TGF-beta levels are significantly increased in L. chagasi-infected mice. In the current study, we found TGF-beta was also abundant in bone marrows of humans with acute visceral leishmaniasis but not in those of uninfected controls. Furthermore, L. chagasi infection caused an increase in biologically active TGF-beta in human macrophage cultures without changing the total TGF-beta. Therefore, we investigated the means through which leishmania could augment activated but not total TGF-beta. Incubation of latent TGF-beta with Leishmania sp. promastigotes caused active TGF-beta to be released from the latent complex. In contrast, the nonpathogenic protozoan Crithidia fasciculata could not activate TGF-beta. TGF-beta activation by leishmania was prevented by inhibitors of cysteine proteases and by the specific cathepsin B inhibitor CA074. Physiologic concentrations of TGF-beta inhibited killing of intracellular L. chagasi in macrophages, although the phagocytosis-induced respiratory burst remained intact. In contrast, supraphysiologic concentrations of TGF-beta had no effect on parasite survival. We hypothesize that the combined effect of abundant TGF-beta stores at extracellular sites during infection, and the ability of the parasite to activate TGF-beta in its local environment, leads to high levels of active TGF-beta in the vicinity of the infected macrophage. Locally activated TGF-beta could, in turn, enhance parasite survival through its effects on innate and adaptive immune responses. (+info)
Tryparedoxins from Crithidia fasciculata and Trypanosoma brucei: photoreduction of the redox disulfide using synchrotron radiation and evidence for a conformational switch implicated in function.
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Tryparedoxin (TryX) is a member of the thioredoxin (TrX) fold family involved in the regulation of oxidative stress in parasitic trypanosomatids. Like TrX, TryX carries a characteristic Trp-Cys-Xaa-Xaa-Cys motif, which positions a redox-active disulfide underneath a tryptophan lid. We report the structure of a Crithidia fasciculata tryparedoxin isoform (CfTryX2) in two crystal forms and compare them with structures determined previously. Efforts to chemically generate crystals of reduced TryX1 were unsuccessful, and we carried out a novel experiment to break the redox-active disulfide, formed between Cys-40 and Cys-43, utilizing the intense x-radiation from a third generation synchrotron undulator beamline. A time course study of the S-S bond cleavage is reported with the structure of a TryX1 C43A mutant as the control. When freed from the constraints of a disulfide link to Cys-43, Cys-40 pivots to become slightly more solvent-accessible. In addition, we have determined the structure of Trypanosoma brucei TryX, which, influenced by the molecular packing in the crystal lattice, displays a significantly different orientation of the active site tryptophan lid. This structural change may be of functional significance when TryX interacts with tryparedoxin peroxidase, the final protein in the trypanothione-dependent peroxidase pathway. Comparisons with chloroplast TrX and its substrate fructose 1,6-bisphosphate phosphatase suggest that this movement may represent a general feature of redox regulation in the trypanothione and thioredoxin peroxidase pathways. (+info)
Presence of multiple mRNA cycling sequence element-binding proteins in Crithidia fasciculata.
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A consensus sequence present in the 5'- or 3'-untranslated regions of several Crithidia fasciculata messenger RNAs encoding proteins involved in DNA metabolism has been shown to be necessary for the periodic accumulation of these mRNAs during the cell cycle. A protein complex termed cycling sequence-binding protein (CSBP) has two subunits, CSBPA and CSBPB, and binds the consensus sequence with high specificity. The binding activity of CSBP was shown to vary during the cell cycle in parallel with the levels of putative target mRNAs. Although disruption of the CSBPA gene resulted in loss of both CSBPA and CSBPB, the putative target message levels still continued to vary during the cell cycle. The presence of an additional and distinct binding activity was revealed in these CSBPA null mutant cells. This activity, termed CSBP II, was also expressed in wild-type Crithidia cells. CSBP II has higher binding specificity for the cycling sequence element than the earlier described CSBP complex. Three polypeptides associated with purified CSBP II show specific binding to the cycling sequence. These proteins may represent a family of sequence-specific RNA-binding proteins involved in post-transcriptional regulation. (+info)
Sequence elements in both the intergenic space and the 3' untranslated region of the Crithidia fasciculata KAP3 gene are required for cell cycle regulation of KAP3 mRNA.
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mRNA levels of several Crithidia fasciculata genes involved in DNA metabolism have previously been found to cycle as cells progress through the cell cycle. Octamer consensus sequences in the 5' untranslated regions (5' UTRs) of these transcripts were shown to be required for cycling of these mRNAs. The KAP3 gene encodes a kinetoplast histone H1-like DNA binding protein, and its mRNA levels cycle in parallel with those of the kinetoplast DNA topoisomerase (TOP2), dihydrofolate reductase-thymidylate synthase (DHFR-TS), and the large subunit of the nuclear single-stranded DNA binding protein (RPA1). KAP3 mRNA contains two octamer consensus sequences in its 3' UTR but none in its 5' UTR. Mutation of these octamer sequences was not sufficient to prevent cycling of a sequence-tagged KAP3 mRNA expressed from a plasmid. Mutation of an octamer sequence contained on the precursor transcript but not on the mRNA, in addition to mutation of the two octamer sequences in the 3' UTR, was necessary to abolish cycling of the mRNA. The requirement for a sequence not present on the mature mRNA indicates that regulation of the mRNA levels by the octamer sequences occurs at or prior to splicing of the transcript. Incompletely processed RNAs containing octamer sequences were also found to accumulate during the cell cycle when the mRNA levels were lowest. These RNA species hybridize to both the KAP3 coding sequence and that of the downstream drug resistance gene, indicating a lack of processing within the intergenic region separating these genes. We propose a cell cycle-dependent interference in transcript processing mediated by octamer consensus sequences as a mechanism contributing to the cycling of such transcripts. (+info)
Purification of glutathionylspermidine and trypanothione synthetases from Crithidia fasciculata.
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Two enzymes involved in the biosynthesis of the trypanosomatid-specific dithiol trypanothione-glutathionylspermidine (Gsp) synthetase and trypanothione (TSH) synthetase--have been identified and purified individually from Crithidia fasciculata. The Gsp synthetase has been purified 93-fold and the TSH synthetase 52-fold to apparent homogeneity from a single DEAE fraction that contained both activities. This constitutes the first indication that the enzymatic conversion of two glutathione molecules and one spermidine to the N1,N8-bis(glutathionyl)spermidine (TSH) occurs in two discrete enzymatic steps. Gsp synthetase, which has a kcat of 600/min, shows no detectable TSH synthetase activity, whereas TSH synthetase does not make any detectable Gsp and has a kcat of 75/min. The 90-kDa Gsp synthetase and 82-kDa TSH synthetase are separable on phenyl Superose and remain separated on gel filtration columns in high salt (0.8 M NaCl). Active complexes can be formed under low to moderate salt conditions (0.0-0.15 M NaCl), consistent with a functional complex in vivo. (+info)
83-kilodalton heat shock proteins of trypanosomes are potent peptide-stimulated ATPases.
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A Crithidia fasciculata 83-kDa protein purified during a separate study of C. fasciculata trypanothione synthetase was shown to have ATPase activity and to belong to the hsp90 family of stress proteins. Because no ATPase activity has previously been reported for the hsp90 class, ATP utilization by C. fasciculata hsp83 was characterized: this hsp83 has an ATPase kcat of 150 min-1 and a Km of 60 microM, whereas the homologous mammalian hsp90 binds ATP but has no ATPase activity. Crithidia fasciculata hsp83 undergoes autophosphorylation on serine and threonine at a rate constant of 3.3 x 10(-3) min-1. Similar analysis was performed on recombinant Trypanosoma cruzi hsp83, and comparable ATPase parameters were obtained (kcat = 100 min-1, Km = 80 microM, kautophosphorylation = 6.3 x 10(-3) min-1). The phosphoenzyme is neither on the ATPase hydrolytic pathway nor does it affect ATPase catalytic efficiency. Both C. fasciculata and T. cruzi hsp83 show up to fivefold stimulation of ATPase activity by peptides of 6-24 amino acids. (+info)
Kinetoplast-associated DNA topoisomerase in Crithidia fasciculata: crosslinking of mitochondrial topoisomerase II to both minicircles and maxicircles in cells treated with the topoisomerase inhibitor VP16.
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The mitochondrial DNA of the trypanosomatid Crithidia fasciculata consists of thousands of copies of a 2.5 kb minicircle and a small number of 37kb maxicircles catenated into a single enormous network. Treatment of C. fasciculata with the type II DNA topoisomerase inhibitor VP16 produces cleavable complexes of a type II DNA topiosomerase with both minicircles and maxicircles. A combined Southern and Western blot analysis of the cleaved DNA species released from the network by SDS treatment has identified topollmt, the kinetoplast-associated topisomerase, in covalent complexes with linear forms of minicircle and maxicircle DNAs. These results directly implicate topollmt in the topological reactions required for the duplication of the kinetoplast network. (+info)
A single-stranded DNA-binding protein from Crithidia fasciculata recognizes the nucleotide sequence at the origin of replication of kinetoplast DNA minicircles.
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A sequence-specific single-stranded DNA-binding protein from the trypanosomatid protozoan Crithidia fasciculata binds to a sequence of 12 nucleotides located at the origin of replication of kinetoplast DNA minicircles. This sequence, termed the universal minicircle sequence (UMS), is conserved in the kinetoplast DNA minicircles among species of the family Trypanosomatidae. The purified protein binds specifically to the heavy strand of the DNA at this site, which consists of the sequence 5'-GGGGTTGGTGTA-3'. Binding analyses using mutated UMS dodecamers have revealed the significant contribution of each of the individual residues at the binding site, with the exception of the 3'-terminal adenine residue, to the generation of specific protein-DNA complexes. The possible role of this sequence-specific single-stranded DNA-binding protein in replication of kinetoplast DNA minicircles and the relation of the UMS to chromosomal telomeric sequences are discussed. (+info)