The use of the green fluorescent protein to monitor and improve transfection in Trypanosoma cruzi

Ramirez, Marcel I.; Yamauchi, Lucy Megumi; Freitas, Lucio H.G de; Uemura, Hirotsugu; Schenkman, Sérgio

doi:10.1016/s0166-6851(00)00309-1

Cited by 33 publications

(38 citation statements)

References 17 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…As shown above, histone H1 in the late stages of cytokinesis and in G 1 cells appeared centrally located in the parasite nucleus, a region known to contain the nucleolus (19). To explore this issue, anti-histone H1 antibodies were used to label T. cruzi cells expressing only a short portion of the N terminus of histone H2B in fusion with GFP, previously described to be targeted to the nucleolus (18,38). As shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…T. cruzi epimastigote forms (strain Y) were cultivated in liver infusion tryptose medium supplemented with 10% fetal bovine serum at 28°C, as previously described (9). Parasites transfected with the plasmid p33H2b-GFP (38) were grown under the same conditions in the presence of 0.5 mg per ml of Geneticin (G418). Histones were extracted as described previously from precipitates of 5 ϫ 10 8 parasites kept at Ϫ70°C (13).…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Histone H1 of Trypanosoma cruzi Is Concentrated in the Nucleolus Region and Disperses upon Phosphorylation during Progression to Mitosis

2008

Self Cite

View full text Add to dashboard Cite

Phosphorylation of histone H1 is intimately related to the cell cycle progression in higher eukaryotes, reaching maximum levels during mitosis. We have previously shown that in the flagellated protozoan Trypanosoma cruzi, which does not condense chromatin during mitosis, histone H1 is phosphorylated at a single cyclin-dependent kinase site. By using an antibody that recognizes specifically the phosphorylated T. cruzi histone H1 site, we have now confirmed that T. cruzi histone H1 is also phosphorylated in a cell cycle-dependent manner. Differently from core histones, the bulk of nonphosphorylated histone H1 in G 1 and S phases of the cell cycle is concentrated in the central regions of the nucleus, which contains the nucleolus and less densely packed chromatin. When cells pass G 2 , histone H1 becomes phosphorylated and starts to diffuse. At the onset of mitosis, histone H1 phosphorylation is maximal and found in the entire nuclear space. As permeabilized parasites preferentially lose phosphorylated histone H1, we conclude that this modification promotes its release from less condensed and nucleolar chromatin after G 2 .The nucleosome, the basic chromatin unit, is assembled by wrapping DNA around an octamer formed by two copies of histone H2A, H2B, H3, and H4 proteins. A fifth histone, called histone H1, packs the chromatin by contacting internucleosomal DNA and the nucleosome particle (50). Histone H1 is formed by a globular domain flanked by a long unstructured C-terminal portion, comprising almost half of the protein, and by a short and also nonstructured N-terminal domain. The C-terminal domain is enriched in basic amino acids that interact with the negative phosphodiester charges of DNA through S/TPKK motifs (26). The globular portion contacts the core histones and the nucleosomal DNA (3), favoring chromatin compaction, which prevents the access of chromatin remodeling factors and therefore restricts transcription and replication (10). Fluorescence recovery after photobleaching experiments have shown that the chromatin residence time of histone H1 is much shorter than that of other histones, suggesting that histone H1 dynamically associates with the nucleosome particles, contributing to several nuclear processes involving chromatin condensation and decondensation (10, 31, 36). However, when histone H1 is absent, chromatin decondenses (20, 47).Histone H1 is phosphorylated at the N-and C-terminal domains in a cell cycle-dependent manner (22). The number of phosphorylated residues is small in G 1 phase and starts to increase when the cell progresses through S and G 2 , reaching a maximal level at mitosis (28). Histone H1 phosphorylation is required for DNA replication (24), and protein kinases that activate replication also promote histone H1 phosphorylation (1). Histone H1 phosphorylation affects the heterochromatin structure (23), DNA repair, chromatin remodeling, apoptosis, and cell aging (29,49). Histone H1 phosphorylation is also involved in chromosome condensation during mitosis. In the absence of histon...

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Histone H1 of Trypanosoma cruzi Is Concentrated in the Nucleolus Region and Disperses upon Phosphorylation during Progression to Mitosis

2008

Self Cite

View full text Add to dashboard Cite

show abstract

“…The fragments were cloned into pGEM-T Easy vector (Promega) and the expected constructs confirmed by restriction analysis and DNA sequencing. The fragments were removed by endonuclease reaction using XbaI and BamHI and inserted into p33 vector (30), which was previously digested with the same enzymes. To generate mutated TcSir2rp3 protein, we used two round of PCRs.…”

Section: Methodsmentioning

confidence: 99%

Characterization of Trypanosoma cruzi Sirtuins as Possible Drug Targets for Chagas Disease

Moretti

Augusto

Clemente

et al. 2015

Antimicrob Agents Chemother

Self Cite

View full text Add to dashboard Cite

c Acetylation of lysine is a major posttranslational modification of proteins and is catalyzed by lysine acetyltransferases, while lysine deacetylases remove acetyl groups. Among the deacetylases, the sirtuins are NAD ؉ -dependent enzymes, which modulate gene silencing, DNA damage repair, and several metabolic processes. As sirtuin-specific inhibitors have been proposed as drugs for inhibiting the proliferation of tumor cells, in this study, we investigated the role of these inhibitors in the growth and differentiation of Trypanosoma cruzi, the agent of Chagas disease. We found that the use of salermide during parasite infection prevented growth and initial multiplication after mammalian cell invasion by T. cruzi at concentrations that did not affect host cell viability. In addition, in vivo infection was partially controlled upon administration of salermide. There are two sirtuins in T. cruzi, TcSir2rp1 and TcSir2rp3. By using specific antibodies and cell lines overexpressing the tagged versions of these enzymes, we found that TcSir2rp1 is localized in the cytosol and TcSir2rp3 in the mitochondrion. TcSir2rp1 overexpression acts to impair parasite growth and differentiation, whereas the wild-type version of TcSir2rp3 and not an enzyme mutated in the active site improves both. The effects observed with TcSir2rp3 were fully reverted by adding salermide, which inhibited TcSir2rp3 expressed in Escherichia coli with a 50% inhibitory concentration (IC 50 ) ؎ standard error of 1 ؎ 0.5 M. We concluded that sirtuin inhibitors targeting TcSir2rp3 could be used in Chagas disease chemotherapy.

show abstract

“…Wild-type and mutant forms of TcPRL-1 open reading frames (ORFs) were digested and subcloned into the T. cruzi expression vector pRIBOTEX (19) fused to the C terminus of the green fluorescent protein (GFP). A total of 50 g of QUIAGEN purified DNA was used to transfect T. cruzi CL Brener as previously described (27). Stable transfectants were selected and expanded in 500 g of G418/ml and screened by observation under a fluorescence microscope.…”

Section: Methodsmentioning

confidence: 99%

Characterization of Farnesylated Protein Tyrosine Phosphatase TcPRL-1 from Trypanosoma cruzi

et al. 2005

View full text Add to dashboard Cite

Protein tyrosine kinases and phosphatases play important roles in the regulation of cell growth, development, and differentiation. We report here the identification in Trypanosoma cruzi of a gene (TcPRL-1) encoding a protein tyrosine phosphatase. The predicted protein (TcPRL-1) shares ca. 35% identity with the mammalian protein tyrosine phosphatase known as phosphatase of regenerating liver 1 (PRL-1). Four copies of this protein tyrosine phosphatase are present in the T. cruzi genome, and Northern blot assays showed a transcript of ϳ750 bases. TcPRL-1 was detected by Western blot analysis only in amastigote extracts as a 21-kDa protein.TcPRL-1 was expressed in Escherichia coli, and its phosphatase activity was determined by using p-nitrophenylphosphate and a phosphorylated protein as substrates. In contrast to other PRLs, TcPRL-1 activity was not affected by pentamidine, and it was inhibited by very low concentrations of o-vanadate. TcPRL-1 has a C-terminal CAAX motif (CAVM) and is farnesylated in vitro by T. cruzi epimastigote extracts and in vivo according to the transfection results. After transfection of T. cruzi with a vector that expresses TcPRL-1 as a C-terminal fusion to green fluorescent protein, GFP-TcPRL-1 was detected in the endocytic pathway of epimastigotes, amastigotes, and trypomastigotes by colocalization with cruzipain and concanavalin A. Interestingly, a mutant form without the CAAX motif localized to the cytoplasm, in contrast to its mammalian counterparts that localize to the nucleus. The results of these studies on TcPRL-1 reveal that, even though the animal and parasite PRLs share similar kinetic properties, their susceptibilities to inhibitors, as well as their localization, are distinct, implying that they may be involved in different cellular processes.Phosphorylation of proteins in specific tyrosyl residues is a major control mechanism for several processes such as normal cell growth, differentiation, metabolism, cell cycle, cell migration, and gene expression, among others (16). The levels of cellular protein phosphorylation in tyrosine residues are controlled by the activities of both protein tyrosine kinases and phosphatases. Although protein tyrosine phosphatases (PTPs) were initially believed to have housekeeping roles, it is now obvious that they are highly regulated and specific enzymes (33).PTPs form a large superfamily of enzymes with more than 100 members. The hallmark that defines the PTP superfamily is the presence of the signature motif (or active site), HCX 2 GX 2 R in the catalytic domain. PTPs can be divided according to their sequence homology and substrate specificity in tyrosine-specific phosphatases and dual-specific phosphatases. The latter class of phosphatases are the only that cleave phosphoester bonds in proteins that contain phosphotyrosine (pTyr), as well as phosphoserine and phosphothreonine.The phosphatase of regenerating liver (PRL) phosphatases represents a new class of PTP. These phosphatases, in mammals, belong to a family composed by at least three mem...

show abstract

The use of the green fluorescent protein to monitor and improve transfection in Trypanosoma cruzi

Cited by 33 publications

References 17 publications

Histone H1 of Trypanosoma cruzi Is Concentrated in the Nucleolus Region and Disperses upon Phosphorylation during Progression to Mitosis

Histone H1 of Trypanosoma cruzi Is Concentrated in the Nucleolus Region and Disperses upon Phosphorylation during Progression to Mitosis

Characterization of Trypanosoma cruzi Sirtuins as Possible Drug Targets for Chagas Disease

Characterization of Farnesylated Protein Tyrosine Phosphatase TcPRL-1 from Trypanosoma cruzi

Contact Info

Product

Resources

About