Mem Inst Oswaldo Cruz, Rio de Janeiro, 113(8) August 2018
Short communication

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

Cassiano Martin Batista1, Felipe Saad1, Stephane Pini Costa Ceccoti1, Iriane Eger2, Maurilio José Soares1,+

1Fundação Oswaldo Cruz-Fiocruz, Instituto Carlos Chagas, Laboratório de Biologia Celular, Curitiba, PR, Brasil
2Universidade Estadual de Ponta Grossa, Departamento de Biologia Geral, Ponta Grossa, PR, Brasil

DOI: 10.1590/0074-02760180086
93 views 78 downloads
ABSTRACT

Dynamic S-palmitoylation of proteins is the addition of palmitic acid by zDHHC palmitoyl transferases (PATs) and depalmitoylation by palmitoyl protein thioesterases (PPTs). A putative PAT (TcPAT1) has been previously identified in Trypanosoma cruzi, the etiological agent of Chagas disease. Here we analyse other 14 putative TcPATs and 2 PPTs in the parasite genome. T. cruzi cell lines expressing TcPATs and TcPPTs plus a FLAG tag at the C terminus were produced for most enzymes, with positive detection by indirect immunofluorescence. Overexpressed TcPATs were mostly found as single spots at the parasite anterior end, while the TcPPTs were dispersed throughout the parasite body.

Dynamic protein S-palmitoylation concerns the addition of palmitate to cysteines of the modified protein by zDHHC palmitoyl transferases (PATs) through thioester linkages and depalmitoylation by palmitoyl protein thioesterases (PPTs) (Conibear and Davis 2010 5 ). Protein S-palmitoylation cycles promote the insertion of target proteins into membranes, regulating their localisation and function (Linder and Deschenes 2003 15 ).

PATs are key transmembrane enzymes, with cysteine rich domains in the DHHC motif (CRD-DHHC domain), in addition to DPG and TTxE structural domains (Greaves and Chamberlain 2011 12 ). PATs are involved in diverse biological processes in several organisms, such as Homo sapiens cancer (Ducker et al. 2004 7 ) and neurological diseases (Young et al. 2012 22 , Cho and Park 2016 4 ), yeast endocytosis (Feng and Davis 2000 9 ), Cryptococcus neoformans virulence (Santiago-Tirado et al. 2015 21 ), Giardia lamblia encystation (Merino et al. 2014 18 ) and invasion in Apicomplexa (Frénal et al. 2013 10 ). TbPAT7 is responsible for flagellar localisation of calflagin in the trypanosomatid protozoan Trypanosoma brucei (Emmer et al. 2009 8 ).

PPTs belong to the serine hydrolases family, are less abundant in number than PATs and are characterised by the presence of a serine active site for hydrolysis of the substrate, being able to cleave amide, ester and thioester bounds (Long and Cravatt 2011 17 ).

Goldston et al. (2014) 11 identified, by in silico search, 15 PATs in the Trypanosoma cruzi genome, as opposed to 12 in T. brucei and 20 in Leishmania major. However, up to now only one PAT has been characterised in T. cruzi, the etiological agent of Chagas disease: TcHIP, or TcPAT1 (Batista et al. 2013 1 ). TcPAT1 is a 95.4 kDa Golgi protein expressed in different developmental stages of the parasite, with a modified DHYC motif (Batista et al. 2013 1 ). Such modified motif is functional in the homologue Akr1p enzyme of Saccharomyces cerevisae (Roth et al. 2002 20 ).

It has been recently shown that dynamic protein S-palmitoylation is involved in life cycle progression and virulence in some pathogenic protozoa (Brown et al. 2017 3 ). However, no evidence of global PATs or PPTs expression has been yet reported in T. cruzi. Thus, aim of this work was to verify the expression of dynamic protein S- palmitoylation enzymes in T. cruzi Dm28c (Contreras et al. 1988 6 ) epimastigote forms. An in silico search for PATs was made in the T. cruzi genomic data base (TritrypDB), in parallel with nucleotide BLAST alignment (nBlast-NCBI, Bethesda, MD, USA) of T. cruzi genes with the well characterised S. cerevisiae PAT genes that encode for Erf2 (with DHHC-CRD motif) (Lobo et al. 2002 16 ) and Akr1p (with DHYC-CRD motif) (Roth et al. 2002 20 ). As a result, 15 PATs genes were found, identical to that formerly identified by Goldston et al. (2014) 11 . Size of these genes varied from 768 (TcPAT7) to 2610 (TcPAT1) base pairs and the resulting protein products were between 30 and 95.4 kDa. Sequence identity between the TcPATs was very low, between 14.2% and 26.83%, as assessed using multiple alignment with Clustal Omega (EMBL-EBI, Cambridgeshire, UK). The softwares TMHMM Server v. 2.0 (Center for Biological Sequence Analysis, CBS, Lyngby, Denmark) and Phyre2 (Kelley et al. 2015 13 ) were used to predict transmembrane regions and calculate 3D protein models, respectively. It could be determined that these proteins had three (TcPATs 2 and 6) to seven (TcPAT5) transmembrane domains. By using pFAM software (Sanger Institute, Cambridge, UK) to predict protein domains, it was found that only TcPAT1 had the DHYC motif, while the number of cysteines close to the DHHC/DHYC motif varied from 5 to 9. Only TcPAT4, TcPAT10 and TcPAT14 had both DPG and TTxE structural motifs. On the other hand, TcPATs 5 and 9 had only the DPG motif, while TcPATs 1, 7 and 8 had only the TTxE motif (Fig. 1).

All TcPATs showed similar predicted 3D models, except for TcPAT1 (larger and with ankyrin repeats). TcPPTs 1 and 2 were very different from each other. All 3D models had 100% confidence (Fig. 2).

Aiming to produce transfectant cell lines of T. cruzi epimastigotes expressing TcPATs plus a FLAG tag at the C terminus (FLAGC tagged TcPAT), the genes were amplified using specific primers (Table I) with recombination sites for the Gateway cloning platform (Thermo Fischer Scientific, Waltham, MA, USA) by using the entry plasmid vector pDONR 221 and the destination T. cruzi vector pTcGWFLAGC (Batista et al. 2010 2 , Kugeratski et al. 2015 14 ). All genes were cloned, except TcPAT6 and TcPAT1 (already characterised). Three-day-old T. cruzi epimastigotes were transfected with a Gene Pulser XCell BIORAD electroporator (BIORAD Inc., Hercules, CA, USA), selected with 500 µg.mL-1 G418 and maintained with 250 µg.mL-1 of the same antibiotic, as previously described (Batista et al. 2010 2 ). Twelve resistant cell lines could be selected, with the exception of TcPAT4.

For subcellular localisation by indirect immunofluorescence assays (IFA), T. cruzi transfectants werewashed twice in PBS, fixed for 10 min with 4% paraformaldehyde, adhered to 0.1% poly-L-lysine coated coverslips, permeabilised with 0.5% Triton/PBS, and incubated for one hour at 37ºC using a mouse anti-flag antibody (Sigma-Aldrich St. Louis, MO, USA) diluted 1:4000 in incubation buffer (PBS pH 7.4 containing 1.5% bovine serum albumin). After three washes in PBS, the samples were incubated in the same conditions with a secondary goat anti-mouse antibody coupled to AlexaFluor 594 (Thermo Fischer Scientific, Waltham, MA, USA) diluted 1:600 in incubation buffer. The samples were washed three times with PBS, incubated for 5 min with 1.3 nM Hoechst 33342 (Sigma-Aldrich St. Louis, MO, USA) and the coverslips were mounted with Prolong Gold antifading agent (Thermo Fischer Scientific, Waltham, MA, USA). The slides were observed in a Nikon Eclipse E600 epifluorescence microscope.

As a result, TcPATs 3, 5, 8, 11, 12, 14 and 15 were located as single dots at the anterior region of the parasite, close to the kinetoplast and the flagellar pocket (Fig. 3). Interestingly, most PATs with four transmembrane domains (five out of seven) showed this pattern. The positive reaction was frequently found lateral to the kinetoplast, which suggests Golgi, flagellar pocket or contractile vacuole localisation. TcPAT2 labeling appeared as strong dots distributed throughout the cell body, suggestive of localisation in some cytoplasmic organelle (Fig. 3). TcPAT13 presented a stronger labeling at the perinuclear region (Fig. 3). These patterns were expected, since PATs are usually found at the endoplasmic reticulum, Golgi and plasma membranes (Ohno et al. 2006 19 ). No positive reaction was detected for TcPATs 7, 9, 10 (Fig. 3). Transcriptomic data from TritrypDB indicate that TcPATs 7 and 9 are expressed in metacyclic trypomastigotes, but not in epimastigotes. Therefore, gene expression of these two enzymes (and possibly also TcPAT10) can be down-regulated in epimastigotes. In summary, these results indicated that at least nine TcPATs could be overexpressed in T. cruzi epimastigotes.

In order to characterise the TcPPTs, a genomic data search was performed as described above, and two genes were identified (Table II). TcPPT1 is an 843 base pairs gene and the product (30.2 kDa) is homologue to H. sapiens acyl-protein thioesterase-1 (APT1) and lysophospholipase genes, which are involved in cytosolic and lysosomal protein depalmitoylation (Long and Cravatt 2011 17 ). TcPPT2 is a 951 base pairs gene and the product (35.5 kDa) is homologue to H. sapiens acyl-protein thioesterase-2 (APT2), involved in cytosolic depalmitoylation (Long and Cravatt 2011 17 ). Primers were then designed for isolation and amplification of these genes (Table II).

The same steps described above for TcPATs were used to produce T. cruzi cell lines expressing TcPPTs plus a FLAG tag at the C terminus (FLAGC tagged TcPPTs). Resistant cell lines expressing TcPPT1 and TcPPT2 were selected with 500 µg.mL-1 G418. After IFA in the same conditions as described above, both TcPPTs showed strong labeling dispersed through the cell body, suggesting a cytoplasmic localisation (Fig. 3), indicating that T. cruzi epimastigotes overexpressed both TcPPTs, in the expected cytoplasmic localisation.

In conclusion, our data indicate that a dynamic protein S-palmitoylation machinery (nine PATS and two PPTs) could be overexpressed in T. cruzi. Future studies will be crucial to determine the importance of this machinery for the parasite survival. Palmitoylation and depalmitoylation of proteins can play an important role in this parasite, in events as diverse as nutrition, protein traffic, differentiation, host-cell interaction and infection establishment.

 

ACKNOWLEDGEMENTS

 

To the Program for Technological Development in Tools for Health-PDTIS-FIOCRUZ for use of its facility (Confocal and Electronic Microscopy Platform RPT07C) at the Instituto Carlos Chagas/Fiocruz-PR, Brazil.

 

AUTHORS' CONTRIBUTION

 

CMB planned the experiments, designed the PATs primers, performed part of the cloning experiment, selected Trypanosoma cruzi cell lines and wrote the first manuscript draft; FS performed cloning and IFAs; SC made PPTs primer design and cloning; IE helped to plan the experiments and revised the manuscript; MJS conceived the study and edited the final form of the manuscript. All authors read and approved the final manuscript.

REFERENCES
01. Batista CM, Kalb LC, Moreira CM, Batista GT, Eger I, Soares MJ. Identification and subcellular localization of TcHIP, a putative Golgi zDHHC palmitoyl transferase of Trypanosoma cruzi. Exp Parasitol. 2013(1); 134: 52-60.
02. Batista M, Marchini FK, Celedon PA, Fragoso SP, Probst CM, Preti H, et al. A high-throughput cloning system for reverse genetics in Trypanosoma cruzi. BMC Microbiol. 2010; 10: 259.
03. Brown RW, Sharma AI, Engman DM. Dynamic protein S-palmitoylation mediates parasites life cycle progression and diverse mechanisms of virulence. Crit Rev Biochem Mol Biol. 2017; 52(2): 145-62.
04. Cho E, Park M. Palmitoylation in Alzheimer’s disease and other neurodegenerative diseases. Pharmacol Res. 2016; 111: 133-51.
05. Conibear E, Davis NG. Palmitoylation and depalmitoylation dynamics at a glance. J Cell Sci. 2010; 123(23); 4007-10.
06. Contreras VT, Araujo-Jorge TC, Bonaldo MC, Thomaz N, Barbosa HS, Meirelles MNSL, et al. Biological aspects of the DM28c clone of Trypanosoma cruzi after metaciclogenesis in chemically defined media. Mem Inst Oswaldo Cruz. 1988; 83(1): 123-33.
07. Ducker CE, Stettler EM, French KJ, Upson JJ, Smith CD. Huntingtin interacting protein 14 is an oncogenic human protein: palmitoyl acyltransferase. Oncogene. 2004; 23(57): 9230-7.
08. Emmer BT, Souther C, Toriello KM, Olson CL, Epting CL, Engman DM. Identification of a palmiytoyl acyltransferase required for protein sorting to the flagellar membrane. J Cell Sci. 2009; 122(6): 867-74.
09. Feng Y, Davis NG. Akr1p and the type I casein kinases act prior to the ubiquitination step of yeast endocytosis: Akr1p is required for kinase localization to the plasma membrane. Mol Cel Biol. 2000; 20(14): 5350-9.
10. Frénal K, Tay CL, Mueller C, Bushell ES, Jia Y, Graindorge A, et al. Global analysis of apliconplexan protein S-acyl transferases reveals an enzyme essential for evasion. Traffic. 2013; 14(8): 895-911.
11. Goldston AM, Sharma AI, Paul KS, Engman DM. Acylation in trypanosomatids: an essential process and potential drug target. Trends Parasitol. 2014; 30(7): 350-60.
12. Greaves J, Chamberlain LH. DHHC palmitoyl transferases: substrates interactions and (patho)physiology. Trends Biochem Sci. 2011; 36(5): 245-53.
13. Kelley LA, Mazulin S, Yates CM, Was MN, Sternberg MJE. The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc. 2015; 10(6): 845-58.
14. Kugeratski FG, Batista M, Inoue AH, Ramos BD, Krieger MA, Marchini FK. pTcGW plasmid vectors 1.1 version: a versatile tool for Trypanosoma cruzi gene characterisation. Mem Inst Oswaldo Cruz. 2015; 110(5): 687-90.
15. Linder ME, Deschenes RJ. New insights into the mechanisms of protein palmitoylation. Biochemistry. 2003; 42(15): 4311-7.
16. Lobo S, Greentree WK, Linder ME, Deschenes RJ. Identification of a Ras palmitoyltransferase in Saccharomyces cerevisiae. J Biol Chem. 2002; 277(43): 41268-73.
17. Long JZ, Cravatt BF. The metabolic serine hydrolases and their functions in mammalian physiology and disease. Chem Rev. 2011; 111(10): 6022-63.
18. Merino MC, Zamponi N, Vranych CV, Touz MC, Rópolo AS. Identification of Giardia lamblia DHHC proteins and the role of protein S-palmitoylation in the encystation process. PloS Negl Trop Dis. 2014; 8(7): e2997.
19. Ohno Y, Kihara A, Sano T, Igarashi Y. Intracellular localization and tissue-specific distribution of human and yeast DHHC cysteine-rich domain-containing proteins. Biochim Biophys Acta. 2006; 1761(4): 474-83.
20. Roth AF, Feng Y, Chen L, Davis NG. The yeast DHHC cysteine-rich domain protein Akr1p is a palmitoyl transferase. J Cell Biol. 2002; 159(1): 23-8.
21. Santiago-Tirado FH, Peng T, Yang M, Hang HC, Doering TL. A single protein S-acyl transferase acts through diverse substrates to determine Cryptococcal morphology, stress tolerance, and pathogenic outcome. PLoS Pathog. 2015; 11(5): e1004908.
22. Young FB, Butland SL, Sanders SS, Sutton LM, Hayden MR. Putting proteins in their place: palmitoylation in Huntington disease and other neuropsychiatric diseases. Prog Neurobiol. 2012; 97(2): 220-38.

Financial support: CNPq, CAPES and Fiocruz.
+ Corresponding author: maurilio.soares@fiocruz.br
Received 16 February 2018
Accepted 2 May 2018

Our Location

Memórias do Instituto Oswaldo Cruz

Av. Brasil 4365, Castelo Mourisco 
sala 201, Manguinhos, 21040-900 
Rio de Janeiro, RJ, Brazil

Tel.: +55-21-2562-1222

This email address is being protected from spambots. You need JavaScript enabled to view it.

Support Program

logo fiocruz logo governo
logo faperj logo cnpq marca capes