Subcellular localisation of FLAG tagged enzymes of the dynamic protein S-palmitoylation cycle of Trypanosoma cruzi epimastigotes

Batista, Cassiano Martin; Saad, Felipe; Ceccoti, Stephane Pini Costa; Eger, Iriane; Soares, Maurílio J.

doi:10.1590/0074-02760180086

Cited by 4 publications

(6 citation statements)

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“…The repertoire of T. cruzi palmitoyl transferases (PATs) was first checked and 15 putative proteins were found [ 27 ], which agrees with Goldston and coworkers [ 28 ]. All respective genes, except for TcPAT6, were successfully isolated by PCR, thus indicating that T. cruzi has the palmitoylation machinery in its genome.…”

Section: Discussionsupporting

confidence: 62%

“…All respective genes, except for TcPAT6, were successfully isolated by PCR, thus indicating that T. cruzi has the palmitoylation machinery in its genome. Indeed, dynamic S-palmitoylation machinery could be expressed in T. cruzi epimastigotes [ 27 ]. Therefore, 2-BP could be exploited to gain some information on this lipid modification in this parasite, since it has been already demonstrated that 2-BP inhibits some PATs in T. brucei [ 19 ].…”

Section: Discussionmentioning

confidence: 99%

“…The large vesicles close to the Golgi complex that were observed by transmission electron microscopy could be a result of inhibition of palmitoylation, leading to accumulation of depalmitoylated proteins in vesicles at the trans-Golgi network, since it is known that the Golgi compartment acts as a hub for palmitoylation [ 51 ]. Moreover, mostly overexpressed FLAG-tagged TcPATs were found as single spots at the parasite anterior end, which could be the Golgi complex [ 27 ]. It is possible that the vesicles observed close to the Golgi could leave the flagellar pocket by exocytosis, thus forming the extracellular material observed by scanning electron microscopy.…”

Section: Discussionmentioning

confidence: 99%

“…DNA sequences of fourteen TcPATs (PATs 2–15) were identified and used for primer design [ 27 ] with subsequent amplification by PCR, using genomic DNA (gDNA) of T. cruzi (Dm28c) epimastigotes. gDNA was extracted from three-day-old culture epimastigotes by a phenol-chloroform method [ 33 ].…”

Section: Methodsmentioning

confidence: 99%

“…Two T. cruzi proteins are known to be palmitoylated: TcFCaBP [ 24 ], which is involved in parasite motility, and TcPI-PLC [ 25 ], which is involved in evading the host immune system. A putative PAT has been identified in this protozoan (TcHIP/TcPAT1), localized in the Golgi complex of different life stages [ 26 ] and other nine could be overexpressed in epimastigotes, being mostly located at the anterior end of the parasites [ 27 ]. However, other still unidentified proteins should be also palmitoylated in T. cruzi , and palmitoylation is probably involved in diverse biological functions.…”

Section: Introductionmentioning

confidence: 99%

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Treatment of Trypanosoma cruzi with 2-bromopalmitate alters morphology, endocytosis, differentiation and infectivity

et al. 2018

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BackgroundThe palmitate analogue 2-bromopalmitate (2-BP) is a non-selective membrane tethered cysteine alkylator of many membrane-associated enzymes that in the last years emerged as a general inhibitor of protein S-palmitoylation. Palmitoylation is a post-translational protein modification that adds palmitic acid to a cysteine residue through a thioester linkage, promoting membrane localization, protein stability, regulation of enzymatic activity, and the epigenetic regulation of gene expression. Little is known on such important process in the pathogenic protozoan Trypanosoma cruzi, the etiological agent of Chagas disease.ResultsThe effect of 2-BP was analyzed on different developmental forms of Trypanosoma cruzi. The IC50/48 h value for culture epimastigotes was estimated as 130 μM. The IC50/24 h value for metacyclic trypomastigotes was 216 nM, while for intracellular amastigotes it was 242 μM and for cell derived trypomasigotes was 262 μM (IC50/24 h). Our data showed that 2-BP altered T. cruzi: 1) morphology, as assessed by bright field, scanning and transmission electron microscopy; 2) mitochondrial membrane potential, as shown by flow cytometry after incubation with rhodamine-123; 3) endocytosis, as seen after incubation with transferrin or albumin and analysis by flow cytometry/fluorescence microscopy; 4) in vitro metacyclogenesis; and 5) infectivity, as shown by host cell infection assays. On the other hand, lipid stress by incubation with palmitate did not alter epimastigote growth, metacyclic trypomastigotes viability or trypomastigote infectivity.ConclusionOur results indicate that 2-BP inhibits key cellular processes of T. cruzi that may be regulated by palmitoylation of vital proteins and suggest a metacyclic trypomastigote unique target dependency during the parasite development.Electronic supplementary materialThe online version of this article (10.1186/s12860-018-0170-3) contains supplementary material, which is available to authorized users.

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Section: Discussionsupporting

confidence: 62%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Treatment of Trypanosoma cruzi with 2-bromopalmitate alters morphology, endocytosis, differentiation and infectivity

et al. 2018

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Advances in the cellular biology, biochemistry, and molecular biology of acidocalcisomes

Docampo

2024

Microbiol Mol Biol Rev

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SUMMARY Acidocalcisomes are organelles conserved during evolution and closely related to the so-called volutin granules of bacteria and archaea, to the acidocalcisome-like vacuoles of yeasts, and to the lysosome-related organelles of animal species. All these organelles have in common their acidity and high content of polyphosphate and calcium. They are characterized by a variety of functions from storage of phosphorus and calcium to roles in Ca 2+ signaling, osmoregulation, blood coagulation, and inflammation. They interact with other organelles through membrane contact sites or by fusion, and have several enzymes, pumps, transporters, and channels.

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Acidocalcisome localization of membrane transporters and enzymes in Trypanosoma brucei

Huang,

Docampo

2024

Microbiol Spectr

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Acidocalcisomes of Trypanosoma brucei are membrane-bounded organelles characterized by their acidity and high content of polyphosphate and cations, like calcium and magnesium. They have important roles in cation and phosphorus storage, osmoregulation, autophagy initiation, calcium signaling, and virulence. Acidocalcisomes of T. brucei possess several membrane transporters, pumps, and channels, some of which were identified by proteomic and immunofluorescence analyses and validated as acidocalcisome proteins by their colocalization with the acidocalcisome marker vacuolar proton pyrophosphatase (VP1). Here, we report that a set of membrane transporters and enzymes, which were proposed to be present in acidocalcisomes by the morphological appearance of tagged proteins, colocalize with VP1, validating their character as acidocalcisome proteins. IMPORTANCE Acidocalcisomes are acidic organelles rich in polyphosphate and calcium present in a variety of eukaryotes and important for osmoregulation and calcium signaling. Several proteins were postulated to localize to acidocalcisomes based on their morphological characteristics. We provide validation of the localization of ten10 acidocalcisome proteins by their co-localization with enzymatic markers. These findings reveal the roles of acidocalcisomes in the storage of toxic metals, and the presence of enzymes involved in palmitoylation and polyphosphate metabolism.

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Subcellular localisation of FLAG tagged enzymes of the dynamic protein S-palmitoylation cycle of Trypanosoma cruzi epimastigotes

Cited by 4 publications

References 22 publications

Treatment of Trypanosoma cruzi with 2-bromopalmitate alters morphology, endocytosis, differentiation and infectivity

Treatment of Trypanosoma cruzi with 2-bromopalmitate alters morphology, endocytosis, differentiation and infectivity

Advances in the cellular biology, biochemistry, and molecular biology of acidocalcisomes

Acidocalcisome localization of membrane transporters and enzymes in Trypanosoma brucei

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