Cloning and functional expression of Rpn1, a regulatory-particle non-ATPase subunit 1, of proteasome from Trypanosoma cruzi

Non-lysosomal protein degradation in eukaryotic cells involves a proteolytic complex referred to as 26S proteasome that consists of a 20S core particle and one or two 19S regulatory particles. We have cloned the gene RPN1 encoding Rpn1 (regulatory-particle non-ATPase subunit 1), one of the largest subunits of proteasome, from Trypanosoma cruzi. It contains 2712 bp and encodes 904 amino acid residues with a calculated molecular mass of 98.2 kDa and an isoelectric point of 5.2. The predicted amino acid sequence of the trypanosomatid Rpn1 shares 39.0 and 32.0% overall identities with human Rpn1 and Saccharomyces cerevisiae Nas1 (non-ATPase subunit 1), an Rpn1 homolog, respectively, while the sequence identities among T. cruzi, Plasmodium falciparum, and Entamoeba histolytica Rpn1 are approximately 30%. T. cruzi Rpn1 contains nine repeats of about 36 amino acid residues conserved in Rpn1s from various organisms. T. cruzi RPN1 is located on the 2300- and 1900-kb chromosomal DNA, displays a putative allelic variation as RPN1-1 and RPN1-2 with 98.8% identity between these two putative gene products, and is transcribed from both alleles at a comparable level throughout the three developmental stages of the parasite, epimastigotes, trypomastigotes, and amastigotes. The expression of the trypanosomatid Rpn1 in the temperature-sensitive nas1 yeast mutant rescued the growth defect at the restrictive temperature, indicating that Rpn1 functions as a Nas1 and probably assembles into the 19S regulatory particle of the yeast 26S proteasome.     

N,N′-Thiophene-substituted polyamine analogs inhibit mammalian host cell invasion and intracellular multiplication of Trypahosoma cruzi

We studied the effects of two, N,N′-thiophene-substituted polyamine analogs (MDL 28302 and MDL 29431) on the capacities of Trypanosoma cruzi, the etiologic agent of Chagas' disease, to invade and multiply within a mammalian host cell. Both compounds inhibited infectivity significantly in a time- and concentration-dependent manner. This inhibition resulted from a selective effect on the parasite, because pretreatment of T. cruzi but not host cell cultures with either MDL 28302 or MDL 29431 reduced infectivity. The parasite gradually recovered its infective capacity after removal of unincorporated polyamine analog, denoting the reversible nature of the inhibitory effect. Some biochemical modification of MDL 28302 and MDL 29431 appeared to be required for their inhibitory activities to be exerted, since the effects of these drugs on T. cruzi infectivity were abrogated by MDL 72527, a drug known to inhibit polyamine oxidase (PAO) activity specifically. Supporting the notion of that products of MDL 28302 and MDL 29431 oxidation by PAO were involved in the activity of these compounds was the finding that PAO competitive substrates (N¹Lacetylspermine and N¹-acetylspermidine) also abolished the inhibition of T. cruzi infectivity mediated by MDL 28302 or MDL 29431. However, we can not rule out that MDL 72527 and the PAO competitive substrates might have altered an alternative mechanism because no significant polyamine oxidase activity could be demonstrated in preparations of lysed or intact T. cruzi in assays monitoring conversion of [¹⁴C]spermine to [¹⁴C]spermidine. When either MDL 28302 or MDL 29431 was added to infected cell cultures, a marked reduction in the rate of intracellular parasite growth ensued. The significance of the finding that N,N′-thiophene-substituted polyamine analogs inhibit cell invasion and cytoplasmic replication by T. cruzi resides in the fact that this pathogenic parasite requires a cytoplasmic localization to replicate in mammalian hosts.     

Purification and characterization of a soluble nucleoside diphosphate kinase in Trypanosoma cruzi

A soluble nucleoside diphosphate kinase (NDP kinase) was purified and characterized in epimastigote forms of Trypanosoma cruzi. The enzyme was purified by affinity chromatography on Blue-agarose and Q-Sepharose columns and by FPLC on a Superose 12 column. A membrane-associated NDP kinase was identified which accounts for 30% of total enzymatic activity. Western blot analysis of the soluble NDP kinase revealed a 16.5-kDa monomer recognized by polyclonal antibodies to NDP kinase from Dictyostelium discoideum, Candida albicans or human. Most of the T. cruzi NDP kinase is found in the cell as a hexamer composed of 16.5-kDa monomers. The K_m values of the enzyme for ATP, GDP and dTDP were 0.2 ± 0.008 mM, 0.125 ± 0.012 mM and 0.4 ± 0.009 mM, respectively. The parasite enzyme was stable, remained active at 65°C and was found to tolerate up to 2.5 M urea. The 16.5-kDa subunit was phosphorylated with [γ-³²P]ATP or thiophosphorylated with [³⁵S]GTPγS. The incubation of the ³²P-labelled phosphoenzyme with unlabelled nucleoside 5′-diphosphates resulted in the formation of ³²P-labelled nucleoside 5′-triphosphates without strict base specificity, indicating that the reaction mechanism of the T. cruzi enzyme is the same as reported for other NDP kinases. When the phosphoenzyme was incubated with a mixture of nucleoside 5′-diphosphates, GTP was preferentially formed.     

Comparative analysis of genomic sequences suggests that Trypanosoma cruzi CL Brener contains two sets of non-intercalated repeats of satellite DNA that correspond to T. cruzi I and T. cruzi II types

Approximately 10% of the Trypanosoma cruzi genome is formed by a satellite DNA, composed by 195-bp repeats organized in 30 ± 10 kb clusters in some, but not all chromosomes. Here, the satellite DNA of six representative T. cruzi strains was sequenced and used for phylogenetic inference. The results show that CL Brener contains satellite repeats from T. cruzi I and T. cruzi II strains, although type II sequences are more abundant. The presence of types I and II sequences extends previous propositions that genetic exchange between the two major T. cruzi lineages have occurred in CL Brener, although our data accommodate alternative scenarios of hybridization within T. cruzi II, as proposed by others. Altogether, present data suggest a complex origin for CL Brener. Sequence analysis of satellites isolated from chromosomal bands indicates that satellite DNA sequences are not chromosome specific. Neighbor analysis of in tandem satellite DNAs containing up to five repeats shows that each cluster contains only one type of sequence. Consequently, clusters with intercalated types I and II repeats were not found. We propose that the CL Brener genome contains large pieces of satellite DNA originated mainly from chromosomes of T. cruzi II with introgression of T. cruzi I lineage.     

Differential tissue distribution of diverse clones of Trypanosoma cruzi in infected mice.

Chagas disease, caused by the protozoan Trypanosoma cruzi, presents variable clinical course but the phenomena underlying this variability remain largely unknown. T. cruzi has a clonal population structure and infecting strains are often multiclonal. T. cruzi genetic variability could be a determinant of differential tissue tropism or distribution and consequently of the clinical forms of the disease. We tested this hypothesis by using low-stringency single specific primer polymerase chain reaction (LSSP-PCR) to type genetically the parasites in tissues of experimental infected mice. BALB/c mice were simultaneously inoculated with two different T. cruzi populations (JG strain and Col1.7G2 clone). Doubly infected animals showed clear differential tissue distribution for the two populations (chronic phase). Our results indicate a significant influence of the genetic polymorphism of infecting T. cruzi populations in the pathogenesis of chronic Chagas disease.     

Inhibitory action of the antitumor agent lonidamine on mitochondrial respiration of Trypanosoma cruzi and T. brucei

The antitumor and antispermatogenic agent lonidamine inhibits Trypanosoma cruzi epimastigotes growth in culture with an ID₅₀ around 80 μM. The main site of action appears to be the mitochondria, where the rate of uncoupled respiration was inhibited in 50% at a similar lonidamine concentration (50 μM). Hexokinase (the other point where lonidamine inhibits tumor energy metabolism) was not sensitive to this drug. Lonidamine also inhibited uncoupled respiration in T. brucei procyclic trypomastigotes, suggesting a common mechanism of action with T. cruzi. When lonidamine was added to T. brucei trypomastigotes, there was little effect on the CN-insensitive respiration, demonstrating that at least in T. brucei glycolysis is not affected by the drug.     

Post-translational modifications of Trypanosoma cruzi histone H4

Histone tails provide sites for a variety of post-translational modifications implicated in the control of gene expression and chromatin assembly. As both histones and control of gene expression in trypanosomes are highly divergent compared to most eukaryotes, post-translational modifications of Trypanosoma cruzi histones were investigated. After in vivo incubation of live parasites with radiolabeled precursors, histone H4 mainly incorporates [³H]-acetyl, and to a lesser extent [³H]-methyl residues. In contrast, histone H3 preferentially incorporates [³H]-methyl residues. The modifications of histone H4 were further characterized by mass spectrometry. MALDI-TOF–TOF-MS analysis revealed that peptides from histone H4 amino-terminus, obtained by either endoproteinase Glu-C or endoproteinase Arg-C digestion, contain isoforms with 14 and 42 Da additions, suggesting the presence of simultaneous acetylations and/or methylations. Tandem mass spectrometry analysis demonstrated that the N-terminal alanine is methylated, and lysine residues at positions 4, 10, 14 and 57 are acetylated; lysine at position 18 is mono-methylated, while arginine at position 53 is dimethylated. Immunoblotting analyses using specific antibodies raised against synthetic and acetylated peptides of T. cruzi histone H4 indicate that lysine 4 is acetylated in the majority of histone H4, while other acetylations at the N-terminus portion of histone H4 are less abundant.     

A proton pumping pyrophosphatase in the Golgi apparatus and plasma membrane vesicles of Trypanosoma cruzi

The proton pumping pyrophosphatase (H⁺-PPase) is an enzyme that has been identified in membranes of plant vacuoles, in the Golgi complex of plants and Chlamydomonas reinhardtii, and more recently in acidocalcisomes of different trypanosomatids and apicomplexan parasites. Immunofluorescence and immunoelectron microscopy studies using antibodies against the plant enzyme also suggested a plasma membrane localization in different stages of Trypanosoma cruzi. In this report we provide immunogold electron microscopy evidence of the presence of the H⁺-PPase in the Golgi complex and plasma membrane of epimastigotes of T. cruzi. Pyrophosphate promoted acidification of plasma membrane vesicles as determined using acridine orange. This activity was stimulated by K⁺ ions, inhibited by the pyrophosphate analogs imidodiphosphate (IDP) and aminomethylenediphosphonate (AMDP) by KF, NaF and DCCD, and it had different responses to ions and inhibitors as compared with the activity present in acidocalcisomes. Surface localization of the H⁺-PPase was confirmed by experiments using biotinylation of cell surface proteins and immunoprecipitation with antibodies against H⁺-PPase. Taken together, these results are consistent with the presence of a functional H⁺-PPase in the plasma membrane of these parasites.     

Chemiluminescence enhancement by trypanocidal drugs and by inhibitors of antioxidant enzymes in Trypanosoma cruzi

The spontaneous emission of chemiluminescence by Trypanosoma cruzi epimastigotes was 133 ± 5 counts s⁻¹ (mg protein)⁻¹. The measured intracellular steady state concentration of hydrogen peroxide in the same cells was 1.5 ± 0.5 μM. These two values are about 12- and 15-times higher than the corresponding ones for isolated rat hepatocytes. The intracellular steady state concentrations of superoxide radical and hydrogen peroxide were apparently increased by inhibiting superoxide dismutase (with diethyldithiocarbamate or KCN addition) and by the addition of two different trypanocidal agents (β-lapachone and nifurtimox) capable of intracellular redox cycling and in each case an increased chemiluminescence was observed. Depletion of intracellular reduced non-protein SH groups by 80% increased 3-fold the chemiluminescence of T. cruzi cells. It is apparent that both an increase in the intracellular steady state concentration of superoxide anion or hydrogen peroxide and a decrease in the level of reduced SH groups lead to an increase in the level of peroxy radicals which are the precursor species for light emission.     

The humoral immune response to the kinetoplastid membrane protein-11 in patients with American Leishmaniasis and Chagas disease: prevalence of IgG subclasses and mapping of epitopes

The kinetoplastid membrane protein-11 (KMP-11) is a major target of the humoral immune response during Leishmania-infections. The majority of sera from visceral leishmaniasis, mucocutaneous leishmaniasis and even some cutaneous leishmaniasis patients contain detectable IgG antibodies against KMP-11. We also provide evidence that this protein may act as a potent antigen in T. cruzi infections, since most Chagas sera show immunological cross-reactivity. Therefore, KMP-11 cannot be used as a specific diagnostical tool for the serodiagnosis of leishmaniasis in those regions where both, Leishmania and T. cruzi infections overlap geographically. When analyzing the subclass specificity of the antibody response to KMP-11 we observed the following order of reactivity: IgG1 >>IgG3 >IgG2 >IgG4, which is similiar to that seen in crude parasite extract. The mapping of antigenic determinants by using synthetic 20-mer peptides revealed the existence of predominantly conformational epitopes in leishmaniasis, while 50% of sera from Chagas patients reacted with a particular KMP-11 peptide. These results therefore suggest the presence of disease-specific B-cell epitopes.     

Synthetic lethality of rpn11-1 rpn10Δ is linked to altered proteasome assembly and activity

An rpn11-1 temperature-sensitive mutant shows defect in proteolysis, mitochondrial function and proteasome assembly. The Rpn11 protein is a proteasome subunit that deubiquitinates proteolytic substrates. Multiubiquitinated proteins interact with proteasome receptors, such as Rpn10, which intriguingly is also required for promoting proteasome stability. We report here that Rpn10 binds Rpn11, and genetic studies revealed synthetic lethality of an rpn11-1 rpn10Δ double mutant. The carboxy-terminus of Rpn11 is critical for function, as deletion of 7 C-terminal residues prevented suppression of rpn11-1 rpn10Δ. Native gel electrophoresis showed increased levels of the proteasome 20S catalytic particle in rpn11-1 rpn10Δ, and altered assembly. The inviability of rpn11-1 rpn10Δ was suppressed by rpn10^uim, a mutant that can bind the proteasome, but not multiubiquitin chains. rpn10^uim reduced the levels of free 20S, and increased formation of intact proteasomes. In contrast, rpn10^vwa, which binds multiubiquitin chains but not the proteasome, failed to suppress rpn11-1 rpn10Δ. Moreover, high levels of multiubiquitinated proteins were bound to rpn10^vwa, but were not delivered to the proteasome. Based on these findings, we propose that the lethality of rpn11-1 rpn10Δ results primarily from altered proteasome integrity. It is conceivable that Rpn10/Rpn11 interaction couples proteasome assembly to substrate binding.     

The 19S complex of the proteasome regulates nucleotide excision repair in yeast

Previous studies suggest that the amino-terminal ubiquitin-like (ubl) domain of Rad23 protein can recruit the proteasome for a stimulatory role during nucleotide excision repair in the yeast Saccharomyces cerevisiae. In this report, we show that the 19S regulatory complex of the yeast proteasome can affect nucleotide excision repair independently of Rad23 protein. Strains with mutations in 19S regulatory subunits (but not 20S subunits) of the proteasome promote partial recovery of nucleotide excision repair in vivo in rad23 deletion mutants, but not in other nucleotide excision repair-defective strains tested. In addition, a strain that expresses a temperature-degradable ATPase subunit of the 19S regulatory complex manifests a dramatically increased rate of nucleotide excision repair in vivo. These data indicate that the 19S regulatory complex of the 26S proteasome can negatively regulate the rate of nucleotide excision repair in yeast and suggest that Rad23 protein not only recruits the 19S regulatory complex, but also can mediate functional interactions between the 19S regulatory complex and the nucleotide excision repair machinery. The 19S regulatory complex of the yeast proteasome functions in nucleotide excision repair independent of proteolysis.     

Galactofuranose-containing glycoconjugates of epimastigote and trypomastigote forms of Trypanosoma cruzi

Antiserum to LPPG, a lipopeptidophosphoglycan originally described on the surface of Trypanosoma cruzi epimastigotes of the Y strain, and antibodies to furanoic galactose (gal_f) were obtained in rabbits. A micromethod for the extraction and purification of LPPG from a limited amount of parasites is described. Analysis by Western blots of the purified glycoconjugate probed with both antisera confirmed the presence of gal_f-containing LPPG-like molecules in 10 different strains and clones of T. cruzi. An analogous approach indicated that trypomastigotes also contain LPPG-like components. Quantitation experiments allowed to calculate an average value of 1.0 × 10⁷ LPPG molecules per epimastigote cell and 0.16 × 10⁷ LPPG-like molecules per trypomastigote cell.

Immunoelectron microscopy has shown a homogeneous distribution of LPPG on the surface of epimastigotes. The trypomastigote population, however, is highly heterogeneous with no more than 15% of the parasites being labeled by the anti-LPPG serum. Intense labeling has also been found in vesicles inside the epimastigote and trypomastigote forms. The distribution of gal_f epitopes among glycoconjugates of epimastigotes and trypomastigotes was further investigated. It was shown that gal_f units in epimastigotes are bound to low molecular mass compounds which co-migrate with LPPG whereas in trypomastigotes they have been found in both low molecular mass LPPG-like molecules and glycoproteins of 80–90 kDa. Direct chemical evidence for the presence of gal_f residues in the N-linked oligosaccharide chains of these surface glycoproteins has been obtained. Finally, the natural antigenicity of LPPG and gal_f in chronic Chagas' disease was investigated. It was found that all chronic chagasic sera investigated recognize this glycoconjugate and that an important part of such recognition can be attributed to gal_f residues. Furthermore, no correlation among reactivity to LPPG, strain zymodeme and clinical forms of the disease was found.     

The major cysteine proteinase (cruzipain) from Trypanosoma cruzi is encoded by multiple polymorphic tandemly organized genes located on different chromosomes.

We demonstrate that cruzipain, the major cysteine proteinase of Trypanosoma cruzi epimastigotes, is encoded by a large number of tandemly arranged genes. Restriction enzyme analysis of 20 clones containing complete repeat units of the gene, as well as sequencing of 2 of these clones, and comparison with previously published partial sequences, indicated that the sequence is conserved among the repeat units, although polymorphisms clearly exist. The repeat units contain an intergenic region of 528 bp and coding regions for pre- and pro-enzyme, a central domain and a C-terminal extension. The predicted amino acid sequences of these regions indicated a sequence identity of 30, 60, 70 and 36%, respectively, when the T. cruzi sequence was compared with the sequence of a similar cysteine proteinase from Trypanosoma brucei [14]. Studies by pulsed field gel electrophoresis, complemented with restriction analysis, indicated that the clusters are located on 2 4 different chromosomes in several parasite isolates.     

Absence of Trypanosoma cruzi myocardial infection reactivation in Chagas'' heart transplant

The risk of Trypanosoma cruzi myocardial infection reactivation after immunosuppressive therapy has led to precluding heart transplantation as a therapeutic procedure for patients with end-stage Chagas' heart disease. We report a case of an orthotopic heart transplantation in a 43-year-old critically ill chagasic patient with an uneventful postoperative period. He was treated with azathioprine and cyclosporine to control graft rejection and showed no reactivation of the chagasic infection. One year following surgery, the patient is doing well. Nonsteroidal therapy appears not to reactivate T. cruzi infection in transplant chagasic patients.     

Primary sequence of a putative non-ATPase subunit of the 26S proteasome from Entamoeba histolytica is similar to the human and yeast S2 subunit

The cDNA coding for a non-ATPase S2 subunit of the 26S proteasome from Entamoeba histolytica was cloned from a cDNA library (EhS2). The open reading frame has 2529 bp and the deduced amino acid sequence encodes a protein with a calculated molecular mass of 92,000 Da. EhS2 has 29–35% identity with the three other known S2 subunit sequences of yeasts and humans. Received: 23 August 1998 / Accepted: 2 November 1998     

Repression of tumor necrosis factor-β expression by the human immunodeficiency virus type-1 tat protein in central nervous system-derived glial cells

HIV-1 Tat is a potent transactivator that stimulates expression from the HIV-1 LTR, from certain cellular gene promoters and from several heterologous viral promoters. Previous reports show that HIV-1 Tat transactivates tumor necrosis factor-β (TNF-β) promoter-directed gene expression in lymphocytic and monocytic cell lines and further demonstrate that a ‘TAR-like structure' downstream of the TNF-β promoter is essential for Tat activity. The ability of Tat to activate TNF-β may have profound effects as TNF has been shown to be a potent activator of HIV-1 gene expression and an important immunomodulatory and growth regulatory factor. The studies presented herein demonstrate a novel finding where HIV-1 Tat specifically represses (>10-fold) TNF-β promoter-directed gene expression in central nervous system-derived glial cells. Amino acid residues 2 to 36 of HIV-1 Tat are required for TNF-β repression. Tat repression of TNF-β, a factor which upregulates HIV-1 gene expression, suggests a novel mechanism whereby HIV-1 is able to establish latent infection of glial cells that present no detectable virions and/or viral antigens.     

Protective immunity induced by a Trypanosoma cruzi soluble extract antigen in experimental Chagas' disease. Role of interferon gamma

CBA/J mice can be protected against lethal infection with Trypanosoma cruzi by treatment using T. cruzi soluble extract antigen (TCSE). In vivo administration of TCSE (400 μg/mouse) into naive mice increased the cellular proliferative response to Con A and elevated the levels of IFN-γ. The production of IFN-γ was extremely important in controlling the replication of the parasite since the protective activity of TCSE was completely abrogated by in vivo treatment with an anti IFN-γ neutralizing antibody. These results suggest that depending on the level, cytokine production results in the control of replication of the parasite in experimental Chagas'disease.