2022 04 07 487537v1 Full

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1 Trypanosoma cruzi PARP is enriched in the
2 nucleolus and is present in a thread connecting
3 nuclei during mitosis
5 María Laura Kevorkian1, Salomé C. Vilchez Larrea1,2*, Silvia H. Fernández Villamil1,3 *
7 1 Instituto de Investigaciones en Ingeniería Genética y Biología Molecular “Dr.
8 Héctor N. Torres”, Consejo Nacional de Investigaciones Científicas y Técnicas,
9 Ciudad Autónoma de Buenos Aires, Argentina.
10 2 Departamento de Fisiología, Biología Molecular y Celular, Facultad de
11 Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Autónoma
12 de Buenos Aires, Argentina.
13 3 Departamento de Química Biológica, Facultad de Farmacia y Bioquímica,
14 Universidad de Buenos Aires, Ciudad Autónoma de Buenos Aires, Argentina.
15
16 *Corresponding authors
17 Silvia H. Fernández Villamil (SFV)
18 E-mail address: silvia.villamil@gmail.com; s.villamil@ingebi.conicet.gov.ar
19 Salome Vilchez Larrea (SVL)
20 E-mail address: vilchez.ingebi@gmail.com

1
bioRxiv preprint doi: https://doi.org/10.1101/2022.04.07.487537; this version posted April 7, 2022. The copyright holder for this preprint
21 Abstract
22 Poly (ADP-ribose) polymerase (PARP) is responsible for the synthesis of ADP-
23 ribose polymers, which are involved in a wide range of cellular processes such as
24 preservation of genome integrity, DNA damage signaling and repair, molecular switch
25 between distinct cell death pathways and cell cycle progression. Previously, we
26 demonstrated that the only PARP present in T. cruzi migrates to the nucleus upon
27 genotoxic stimulus. In this work, we identify the N-terminal domain as being sufficient for
28 TcPARP nuclear localization and describe for the first time that TcPARP is enriched in the
29 parasite nucleolus. We also describe that TcPARP is present in a thread that connects two
30 dividing nuclei and co-localizes with nucleolar material and microtubules. Furthermore,
31 ADP-ribose polymers could also be detected in this wire during mitosis. These findings
32 represent a first approach to new potential TcPARP functions inside the nucleus and will
33 help understand its role well beyond the largely described DNA damage response protein
34 in trypanosomatids.
35
36 Keywords
37 Poly (ADP-ribose) polymerase; Trypanosoma cruzi; Nucleolus; ADP-ribose polymer;
38 mitotic spindle.
39
40 Competing interests
41 The authors declare no competing interests.
2
42 Introduction
43 Poly (ADP-ribose) polymerase (PARP) catalyzes the transfer of ADP-ribose units
44 from NAD+ to a target protein or an elongating polymer chain, a post-translational
45 modification known as PARylation (1). Enzymes able to carry out PARylation have been
46 described in all types of eukaryotic organisms, from mammals and plants to lower
47 eukaryotes, except for yeast. Orthologs have also been found in bacteria (2). In humans,
48 the PARP family consists of 18 members grouped into subfamilies. One of these
49 subfamilies, the “DNA-dependent” PARPs (PARP-1, PARP-2 and PARP-3), contain
50 domains capable of recognizing discontinued DNA, thus enabling the recruitment of
51 proteins involved in DNA repairing (3–5). The most extensively studied function of human
52 PARP-1 (hPARP-1) is its role in DNA damage signaling and repair. Nevertheless, over the
53 last years, the functions of poly (ADP-ribose) (PAR) have transcended from being solely
54 associated with genomic damage to being involved in diverse processes such as the
55 regulation of transcription factors, inflammation, cell death and survival, as well as different
56 pathologies (4,6–9).
57 Our research team has pioneered the study of PAR metabolism in trypanosomatids,
58 specially Trypanosoma cruzi, the agent responsible for Chagas´ disease (10–16). We
59 found a single 65 kDa PARP in T. cruzi, named TcPARP, which is expressed in the three
60 developmental stages of the parasite (11). TcPARP shares some structural features with
61 DNA-dependent PARPs, as three of their typical domains are present in the parasite’s
62 counterpart: WGR domain, central regulatory domain and catalytic domain. TcPARP lacks
63 the typical zinc-finger N-terminal PARP-1 domain but bears a shorter basic N-terminal
64 region, like hPARP-2 and hPARP-3 (11). PARP-1 has been identified as a nucleolar
65 protein in mammalian cells (17). TcPARP, however, is concentrated in the nucleus upon
3
66 stimuli such as DNA damage. As it occurs with PARP from other species, DNA breaks
67 induce TcPARP activation, leading to nuclear PAR production (13).
68 In early divergent unicellular eukaryotes, such as the members of the
69 Trypanosomatidae family, mechanisms involved in the nucleolar formation at the end of
70 closed mitosis are poorly understood. Nucleologenesis occurs as a continuous process
71 that does not require the concentration of nucleolar material within intermediate nuclear
72 bodies such as prenucleolar bodies, suggesting the direct transmission of preassembled
73 nucleolar complexes to daughter nuclei at the end of mitosis. Interestingly, in T. cruzi the
74 presence of a thread connecting the two dividing nuclei has been reported (18).
75 In this work, we identify the N-terminal domain as being sufficient for TcPARP
76 nuclear localization and describe for the first time that TcPARP is enriched in the parasite
77 nucleolus. We also report that both TcPARP and PAR are present in a thread that
78 connects two dividing nuclei and co-localize with nucleolar proteins. These results could
79 extend PAR role well beyond the largely described DNA damage response protein in
80 trypanosomatids.
81
82 Materials and methods
83 Parasite cultures
84 Trypanosoma cruzi epimastigotes (CL Brener strain) were cultured at 28 °C in liver
85 infusion tryptose (LIT) medium (5 g.L-1 liver infusion, 5 g.L-1 bacto-tryptose, 68 mM NaCl,
86 5.3 mM KCl, 22 mM Na2HPO4, 0.2% (w/v) glucose and 0.002% (w/v) hemin)
87 supplemented with 10% (v/v) fetal bovine serum, 100 U.mL-1 penicillin and 100 mg.L-1
88 streptomycin.
89
4
90 Plasmid constructions
91 Several sets of primers were used for the amplification of different TcPARP protein domain
92 combinations by PCR using a plasmid bearing a copy of T. cruzi PARP gene (GenBank:
93 DQ061295) as a template (11). PCR products were cloned into a pGEM-T Easy vector
94 and sub-cloned into a pRIBOTEX vector to overexpress the different TcPARP constructs
95 tagged with HA. TcPARP full-length was cloned into pTEX-GFP vector. T. cruzi
96 epimastigotes of the CL Brener strain were transfected with the above-mentioned
97 construct as previously described (13). Stable cell lines were achieved after 60 days of
98 treatment with 500 μg mL-1 of G418. Full-length TcPARP encompasses from amino acid 1
99 to 592; ΔN from 127 to 592; ΔNW from 212 to 592; ΔRC from 1 to 211; ΔWRC from 1 to
100 126 and ΔNRC from 127 to 211. The recombinant proteins were overexpressed in T. cruzi
101 epimastigotes and the presence of chimeric proteins was confirmed by Western Blot (S1A
102 Fig). Growth curves of transgenic parasites showed no significant differences compared to
103 wild type epimastigotes (S1B and S1C Figs).
104
105 SDS–PAGE and Western blot analysis
106 Whole cell lysate of transgenic epimastigotes was quantified using the Bradford method.
107 Proteins (40 µg) were run on 10% SDS–PAGE gel and transferred to an Amersham
108 Hybond-ECL nitrocellulose membrane (GE healthcare), according to the manufacturer’s
109 instructions. Immunodetection of TcPARP constructs was carried out using a 1:1000
110 dilution of rat polyclonal antibody directed against HA, influenza virus hemagglutinin
111 (Roche), followed by 1:4000 anti-rat horseradish peroxidase (HRP) conjugated antibody.
112 For full-length TcPARP-GFP, anti GFP (B-2): sc-9996 - Santa Cruz Biotechnology
113 antibody was used. The signal was detected with the Western Lightning Plus-ECL kit
114 (PerkinElmer).
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115 Immunofluorescence
116 For immunolocalization of TcPARP, transgenic T. cruzi epimastigotes (107 parasites ml-1)
117 were treated with 300 µM H2O2 for 10 min, fixed for 20 min with 4% (w/v) formaldehyde in
118 PBS, permeabilized with fresh PBS – 0.1% Triton X-100 and blocked for 1 h at room
119 temperature with 3% (w/v) BSA in PBS. For full-length TcPARP, an anti-GFP antibody
120 1:500 was used and TcPARP constructs were detected with 1:500 rat polyclonal antibody
121 directed against HA, followed by 1:500 Alexa Fluor 488 goat anti-rat IgG antibody (Sigma-
122 Aldrich) conjugated antibody. The nucleolus was identified using 1:100 L1C6 monoclonal
123 antibody and mouse monoclonal anti α-Tubulin (Sigma-Aldrich) for α-Tubulin. Alexa Fluor
124 594 goat anti-mouse IgG (Sigma-Aldrich) conjugated antibody was used as a secondary
125 antibody. PAR binding reagent was rabbit polyclonal anti PAR (BD PharmingenTM). Nuclei
126 were stained with 2 µg ml-1 DAPI (Sigma) in PBS. Coverslips were mounted with
127 VectaShield® and then visualized using an Olympus BX41/FV300 or Leica SPE confocal
128 microscope. Control images, without primary antibodies, were taken in an analogous
129 condition, during the same microscopy session.
130
131 Results
132 TcPARP is enriched in the nucleolus of epimastigotes
133 As already known from previous research, subcellular localization of TcPARP is altered
134 in response to genotoxic stress, which stimulates TcPARP to re-localize and accumulate in
135 the nucleus in the epimastigote form of the parasite (13). Constructs with different
136 arrangements of TcPARP domains were designed and the recombinant proteins were
137 overexpressed in T. cruzi epimastigotes. Actively growing cells were examined for the
138 distribution of the recombinant proteins after treatment with 300 µM H2O2 for 10 minutes,
6
139 which has been shown to induce nuclear translocation of endogenous TcPARP (11).
140 Nuclear localization was observed for full length TcPARP and the constructs bearing the N-
141 terminal domain, under control conditions or after H2O2 treatment. However, TcPARP
142 constructs which lacked the N-terminal domain did not localize to this organelle even after
143 genotoxic stress, showing a scattered pattern throughout the cytoplasm (S2). This result
144 indicates that the N-terminal domain is necessary for the nuclear localization of the protein.
145 Notoriously, nucleolar enrichment of the recombinant proteins that bear the N-
146 terminal domain (TcPARPΔWRC, TcPARPΔRC and TcPARP-FL) in overexpressing
147 epimastigotes could be observed (Fig 1). A higher intranuclear fluorescence density of the
148 anti-tag signal was detected within the area with reduced DAPI staining. This region
149 corresponds to the nucleolus, which has low DNA content. In order to confirm this
150 localization in transgenic epimastigotes, a co-localization assay was carried out using the
151 nucleolus marker L1C6, which recognizes a nucleolar antigen both in T. brucei and T. cruzi
152 (19). Figure 1A shows the location of L1C6 in the nucleolar structure of CL Brener wild type
153 epimastigotes. The signal corresponding to TcPARP-FL overlaps with the labeling of L1C6
154 inside the nucleus of the parasites, which asserts the sub-nuclear localization of this
155 protein. The same phenomenon could be observed for truncated TcPARP constructs,
156 including the N-terminal domain, where the fluorescent signal co-localizes with nucleolus
157 marker L1C6 (Fig 1B). We noticed that in some parasites, both TcPARP (full length or
158 truncated versions) and L1C6 nucleolar marker were also found in the nucleoplasm. In
159 Drosophila it has been reported that PARP-1 depletion resulted in a mis-localization of
160 nucleolar specific proteins and loss of nucleolar structure (17). To determine whether
161 TcPARP enzymatic activity and the presence of PAR are essentials for maintaining
162 nucleolar integrity in T. cruzi, we inhibited TcPARP activity by adding the NAD+ analogue, 3
163 aminobenzamide (3AB) or the more selective TcPARP inhibitor, Olaparib (20). We did not
164 detect delocalization of the nucleolar marker in the presence PARP inhibitors, ruling out this
7
165 hypothesis (S3 Fig). Other possibility would be that the high transcriptional activity
166 generates the delocalization of nucleolar markers. To test this hypothesis, we
167 overexpressed a non-nuclear protein such as PI3K TcVps34 in epimastigotes, both in
168 absence or in the presence of cycloheximide. We did not observe differences in the
169 overexpressing parasites in comparison to the control, except for a slight decrease in
170 nucleolar marker in cycloheximide treated parasites. The dispersion of L1C6 could
171 therefore be attributed to epimastigotes in stationary phase as described by Gluenz et al.
172 (21).
173 Fig 1. Co-localization of TcPARP constructs with nucleolus marker L1C6. A) Indirect
174 immunofluorescence (IF) of Wild Type or TcPARP-FL overexpressing epimastigotes. B) IF
175 of epimastigotes overexpressing the empty vector pRIBOTEX, TcPARP ΔWRC or TcPARP
176 ΔRC. In both cases, detection of nucleolar region was performed using an antibody
177 directed against L1C6 and the TcPARP construct with the corresponding anti-tag antibody.
178 Bar: 5 μm.
179
180 TcPARP is present in a connecting wire between nuclei of
181 dividing epimastigotes
182 T. cruzi epimastigotes undergoing the cellular division process show the presence
183 of a thread connecting the two dividing nuclei (18), which is associated to nucleolar
184 proteins. We confirmed co-localization of TcPARP with nucleolar proteins in this narrow
185 link between both nuclei by indirect immunofluorescence. The presence of L1C6 could be
186 identified in the entire structure of the wire between cores, co-localizing with the truncated
187 TcPARPΔWRC version of TcPARP or the complete protein (Fig 2). Co-localization of
188 TcPARP with tubulin in the connecting wire is shown in Fig 3, which implies the presence
8
189 of TcPARP in the mitotic spindle during nuclear segregation and brings forward possible
190 new functions for TcPARP that need further study.
191 Fig 2. Co-localization of TcPARP with nucleolar material in the connecting wire.
192 Indirect immunofluorescence of epimastigotes overexpressing TcPARP-FL or the N-
193 terminal domain of TcPARP (TcPARP ΔWRC). Nucleolar proteins were evidenced with
194 antibody directed against L1C6. Recombinant TcPARP-FL and chimeric peptides were
195 detected with the correspondent anti-tag antibody. Bar: 5 μm.
196 Fig 3. Co-localization of TcPARP in connecting wire with tubulin thread. Indirect
197 immunofluorescence of epimastigotes overexpressing TcPARP-FL or the N-terminal
198 domain of TcPARP (TcPARP ΔWRC and TcPARP ΔRC). Tubulin was detected by an
199 antibody directed against α-Tubulin and recombinant variants of TcPARP with the
200 correspondent anti-tag antibody. Bar: 5 μm.
201
202 PARylated proteins are present in the connecting thread
203 We investigated if TcPARP was catalytically active in the thread. The presence of
204 PAR was evaluated in epimastigotes that overexpressed the complete TcPARP or the
205 domains responsible for its nuclear localization. Basal levels of PAR are low and therefore
206 hardly detectable by the methods commonly employed (11), therefore, we subjected the
207 parasites to a genotoxic stimulus in order to enhance PAR production. Figure 4 shows the
208 presence of PAR in the nucleus of H2O2-treated parasites; PAR production co-localizing
209 with the TcPARP constructs is highlighted in the thread that connects the dividing
210 epimastigotes. PAR localization showed the same pattern observed in epimastigotes
211 expressing the empty vector pRIBOTEX, or in wild type parasites (Fig 4). This
9
212 demonstrates that PARylated proteins are present in this structure as a consequence of
213 endogenous TcPARP activity.
214 Fig 4. Presence of PAR in the connecting wire between nuclei of dividing
215 epimastigotes. Upper panel: Indirect immunofluorescence (IF) of epimastigotes bearing
216 the empty vector pRIBOTEX or overexpressing the TcPARPΔWRC or TcPARPΔRC
217 constructs, after 300 μM H2O2 treatment for 10 minutes. Lower panel: IF of wild type
218 epimastigotes or TcPARP-FL overexpressing parasites after 300 μM H2O2 treatment for 10
219 minutes. In both cases, PAR was detected using with a polyclonal antibody directed against
220 PAR chains and recombinant variants of TcPARP with the correspondent anti-tag antibody.
221 Bar: 5 μm.
222 Discussion
223 In Trypanosoma cruzi, TcPARP translocates to the nucleus under genotoxic stress
224 and in this work we demonstrate that the N-terminal domain is sufficient for this
225 translocation. Despite that no canonical NLS has been found, roughly 30% of N-terminal
226 amino acids are basic (13), which indicates the possible presence of a nuclear localization
227 signal within this region. Interestingly, the TcPARP N-terminal contains six repeats of the
228 amino acid sequence AAKKA, which is present in T. cruzi histone H2A and in all H1, which
229 could mediate the DNA binding necessary for TcPARP functions. A T. cruzi importin α with
230 the classical structure of ARM repeats, was recently reported (22). This protein was
231 functional as a nuclear transport factor in these parasites, supporting the idea that this
232 import machinery was an early acquisition of the eukaryotic cell. Numerous authors have
233 described the sub-nuclear localization of PARP-1 in Drosophila and in mammalian cells
234 (17,23–27), where up to 40% of PARP-1 is enriched in the nucleolus. Our finding that
235 TcPARP localizes in the nucleolus of T. cruzi epimastigotes is consistent with that data.
10
236 Altogether, our results demonstrate that the N-terminal domain of TcPARP is involved in
237 nuclear and nucleolar targeting.
238 In Trypanosoma cruzi closed mitosis occurs, in which the chromatin does not
239 condense and the nucleolus is not clearly identified. The nucleolar structure persists
240 throughout closed mitosis, where the nucleolus is elongated and, finally, divided into two
241 structures (28). Observation of trypanosomes expressing various fusion proteins allowed
242 visualization of mitotic cells connected by a thin thread (29). In dividing cells, we found that
243 TcPARP was located in the connecting wire that separates the daughter nuclei, co-
244 localizing with tubulin and the nucleolar marker. As determined by DAPI staining, nuclear
245 DNA seems to be excluded from the thread indicating a late stage in mitosis. This
246 observation implicates that TcPARP, like other nucleolar components, forms part of the
247 connecting thread after chromosomal segregation. Among the PARP functions in
248 mammalian nucleolus, Raemaekers (2003) identified the modification of NuSAP1 (nucleolar
249 protein associated with spindle 1), a protein involved in the mitotic spindle formation, which
250 concentrates in the nucleolus. In metaphase and early anaphase, NuSAP is redistributed,
251 co-localizing with and stabilizing the spindle microtubules (30). Both NuSAP and tubulin are
252 targets of PARylation (31). Similarly, NUMA1 (nuclear mitotic apparatus protein 1), which
253 participates in the clustering of microtubules into poles as a prerequisite for bipolar spindle
254 organization, is associated with PAR. Its PARylation and PAR-binding property may
255 promote correct assembly of bipolar spindles by crosslinking NUMA1 molecules between
256 spindle poles (32). Thus, the PARylation of spindle proteins associated with microtubules
257 plays an important role in the assembly and function of the mitotic spindle, where PARP
258 could work as a Microtubules Associated Protein. In addition to this function, PAR has been
259 postulated as a structural component of the spindle that contributes to creating the force
260 exerted by microtubules to elongate the spindle and separate chromosomes (33).
261 Furthermore, ECT2 (epithelial cell trans-forming sequence 2 oncogene), necessary for the
11
262 control of cytokinesis, is recruited by PARylated α-tubulin to the spindle during metaphase
263 as a prerequisite for functional cytokinesis and completion of mitosis (34). Then, the
264 nucleolar location of TcPARP and associated PARylated proteins during mitosis could be
265 involved in spindle formation, not only through the PARylation of nucleolar proteins but also
266 as a result of their structural functions. The possible participation of TcPARP in the
267 nucleolus as an enzyme related to the formation of the mitotic spindle requires deeper
268 investigation. Thus, TcPARP could be performing a wide array of new roles, leaving aside
269 the notion that this enzyme is functional only in the nucleus.
270
271 Acknowledgments
272 The authors are grateful to Dr. Daniel Sanchez, Instituto de Investigaciones
273 Biotecnológicas (IIB), Universidad Nacional de San Martín – CONICET, for providing L1C6
274 antibody. We are also grateful to Dr. Alejandra Schoijet, Instituto de Investigaciones en
275 Ingeniería Genética y Biología Molecular “Dr. Héctor N. Torres”, for the PI3K TcVps34
276 epimastigotes.
277
278 References
279 1. Kamaletdinova T, Fanaei-Kahrani Z, Wang ZQ. The Enigmatic Function of PARP1:
280 From PARylation Activity to PAR Readers. Cells. 2019;8(12):1–20.
281 2. Perina, D., Mikoč, A., Ahel, J., Ćetković, H., Žaja, R., Ahel, I., 2014. Distribution of
282 protein poly(ADP-ribosyl)ation systems across all domains of life. DNA Repair
283 (Amst). https://doi.org/10.1016/j.dnarep.2014.05.003
12
284 3. Hottiger, M.O., Hassa, P.O., Lu, B., Schu, H., 2010. Toward a unified nomenclature
285 for mammalian ADP-ribosyltransferases. Trends Biochem. Sci.
286 https://doi.org/10.1016/j.tibs.2009.12.003
287 4. Vyas S, Chesarone-Cataldo M, Todorova T, Huang Y-HH, Chang P. A systematic
288 analysis of the PARP protein family identifies new functions critical for cell
289 physiology. Nat Commun [Internet]. 2013;4:2240.
290 5. Gupte R, Liu Z, Kraus WL. Parps and adp-ribosylation: Recent advances linking
291 molecular functions to biological outcomes. Genes Dev. 2017;31(2):101–26.
292 6. Bartolomei G, Leutert M, Manzo M, Baubec T, Hottiger MO. Analysis of Chromatin
293 ADP-Ribosylation at the Genome-wide Level and at Specific Loci by ADPr-ChAP.
294 Mol Cell [Internet]. 2016;61(3):474–85. Available from:
295 http://dx.doi.org/10.1016/j.molcel.2015.12.025
296 7. Burkle A, Virag L. Poly(ADP-ribose): PARadigms and PARadoxes. Mol Asp Med
297 [Internet]. 2013;34(6):1046–65.
298 8. De Vos M, Schreiber V, Dantzer F. The diverse roles and clinical relevance of
299 PARPs in DNA damage repair: Current state of the art. Biochem Pharmacol
300 [Internet]. 2012 Jul [cited 2017 Jul 29];84(2):137–46.
301 9. Curtin NJ, Szabo C. Poly(ADP-ribose) polymerase inhibition: past, present and
302 future. Nat Rev Drug Discov [Internet]. 2020;19(10):711–36.
303 http://dx.doi.org/10.1038/s41573-020-0076-6
304 10. Podestá D, Garcı́a-Herreros MI, Cannata JJB, Stoppani AOM, Fernández Villamil
305 SH, Purification and properties of poly(ADP-ribose)polymerase from Crithidia
306 fasciculata. Automodification and poly(ADP-ribosyl)ation of DNA topoisomerase I.
307 Mol Biochem Parasitol [Internet]. 2004 Jun [cited 2011 May 23];135(2):211–9.
13
308 11. Fernandez Villamil SH, Baltanas R, Alonso GD, Vilchez Larrea SC, Torres HN,
309 Flawia MM. TcPARP: A DNA damage-dependent poly(ADP-ribose) polymerase from
310 Trypanosoma cruzi. Int J Parasitol [Internet]. 2008 Mar;38(3–4):277–87.
311 12. Villamil SF, Podesta D, Molina Portela MDP, Stoppani A, Characterization of
312 poly(ADP-ribose)polymerase from Crithidia fasciculata: enzyme inhibition by beta-
313 lapachone. Mol Biochem Parasitol [Internet]. 2001 Jul;115(2):249–56.
314 13. Vilchez Larrea SC, Alonso GD, Schlesinger M, Torres HN, Flawia MM, Fernandez
315 Villamil SH, et al. Poly(ADP-ribose) polymerase plays a differential role in DNA
316 damage-response and cell death pathways in Trypanosoma cruzi. Int J Parasitol
317 [Internet]. 2011 Dec 22 [cited 2010 Dec 31];41(3):405–16.
318 14. Schlesinger M, Vilchez Larrea SC, Haikarainen T, Narwal M, Venkannagari H,
319 Flawiá MM, et al. Disrupted ADP-ribose metabolism with nuclear Poly (ADP-ribose)
320 accumulation leads to different cell death pathways in presence of hydrogen
321 peroxide in procyclic Trypanosoma brucei. Parasit Vectors [Internet]. 2016;9(1):173.
322 15. Haikarainen T, Schlesinger M, Obaji E, Fernández Villamil SH, Lehtiö L. Structural
323 and Biochemical Characterization of Poly-ADP-ribose Polymerase from
324 Trypanosoma brucei. Sci Rep [Internet]. 2017;7(1):3642.
325 16. Fernández Villamil SH, Vilchez Larrea SC. Poly(ADP-ribose) metabolism in human
326 parasitic protozoa. Acta Trop [Internet]. 2020;208(April):105499.
327 17. Boamah EK, Kotova E, Garabedian M, Jarnik M, Tulin A V. Poly(ADP-Ribose)
328 Polymerase 1 (PARP-1) Regulates Ribosomal Biogenesis in Drosophila Nucleoli.
329 PLoS Genet. 2012;8(1):1002442.
330 18. Nepomuceno-Mejía T, Lara-Martínez R, Hernández R, Segura-Valdez MDL,
331 Jiménez-García LF. Nucleologenesis in Trypanosoma cruzi. Microsc Microanal.

14
332 2016;22(3):621–9.
333 19. Názer E, Sánchez DO. Nucleolar Accumulation of RNA Binding Proteins Induced by
334 ActinomycinD Is Functional in Trypanosoma cruzi and Leishmania mexicana but Not
335 in T . brucei. PLoS One. 2011;6(8):6–10.
336 20. Vilchez Larrea SC, Haikarainen T, Narwal M, Schlesinger M, Venkannagari H,
337 Flawia MM, et al. Inhibition of poly(ADP-ribose) polymerase interferes with
338 Trypanosoma cruzi infection and proliferation of the parasite. PLoS One.
339 2012;7(9):e46063.
340 21. Gluenz E, Taylor MC, Kelly JM. The Trypanosoma cruzi metacyclic-specific protein
341 Met-III associates with the nucleolus and contains independent amino and carboxyl
342 terminal targeting elements. Vol. 37, International Journal for Parasitology. 2007. p.
343 617–25.
344 22. Canela-Pérez I, López-Villaseñor I, Cevallos AM, Hernández R. Trypanosoma cruzi
345 Importin α: ability to bind to a functional classical nuclear localization signal of the
346 bipartite type. Vol. 119, Parasitology Research. 2020. p. 3899–907.
347 23. Guetg C, Santoro R. Formation of nuclear heterochromatin: the nucleolar point of
348 view. Epigenetics. 2012 Aug;7(8):811–4.
349 24. Meder VS, Boeglin M, de Murcia G, Schreiber V. PARP-1 and PARP-2 interact with
350 nucleophosmin/B23 and accumulate in transcriptionally active nucleoli. J Cell Sci.
351 2005;118(Pt 1):211–22.
352 25. Ryu KW, Kim DS, Kraus WL. New facets in the regulation of gene expression by
353 ADP-ribosylation and poly(ADP-ribose) polymerases. Chem Rev.
354 2015;115(6):2453–81.
15
355 26. Schreiber V, Dantzer F, Ame J-CC, de Murcia G. Poly(ADP-ribose): novel functions
356 for an old molecule. Nat Rev Mol Cell Biol [Internet]. 2006 Jul;7(7):517–28.
357 27. Boamah EK, Kotova E, Garabedian M, Jarnik M, Tulin A V. Ribosomal Biogenesis in
358 Drosophila Nucleoli. PLoS Genet. 2012;8(1):1–14.
359 28. Martínez-Calvillo S, Florencio-Martínez LE, Nepomuceno-Mejía T. Nucleolar
360 Structure and Function in Trypanosomatid Protozoa. Cells. 2019;8(5):421.
361 29. Marchetti MA, Tschudi C, Kwon H, Wolin SL, Ullu E. Import of proteins into the
362 trypanosome nucleus and their distribution at karyokinesis. J Cell Sci.
363 2000;906:899–906.
364 30. Raemaekers T, Ribbeck K, Beaudouin J, Annaert W, Van Camp M, Stockmans I, et
365 al. NuSAP, a novel microtubule-associated protein involved in mitotic spindle
366 organization. J Cell Biol. 2003;162(6):1017–29.
367 31. Gagné J-P, Isabelle M, Lo KS, Bourassa S, Hendzel MJ, Dawson VL, et al.
368 Proteome-wide identification of poly(ADP-ribose) binding proteins and poly(ADP-
369 ribose)-associated protein complexes. Nucleic Acids Res [Internet]. 2008
370 Dec;36(22):6959–76.
371 32. Slade D. Mitotic functions of poly(ADP-ribose) polymerases. Biochem Pharmacol
372 [Internet]. 2019;167(March):33–43.
373 33. Kim MY, Zhang T, Kraus WL. Poly(ADP-ribosyl)ation by PARP-1: “PAR-laying”
374 NAD+ into a nuclear signal. Genes Dev [Internet]. 2005;19(17):1951–67.
375 34. Li M, Bian C, Yu X. Poly(ADP-ribosyl)ation is recognized by ECT2 during mitosis.
376 Cell Cycle. 2014;13(18):2944–51.
377 Supporting information

16
378 S1A Fig. Constructs bearing different combinations of TcPARP domains expressed
379 in transgenic epimastigotes. Left Panel: Schematic Diagram of truncated TcPARP
380 recombinant proteins made through the combination of different domains and fused to HA
381 tag. TcPARP full length was tagged with GFP. Numbers under each diagram indicate
382 amino acid positions. Theoretical molecular weight was calculated considering the
383 molecular weight of HA (1 kDa) or GFP (27 kDa). Right panel: Western Blot on total
384 extract of T. cruzi epimastigotes transfected with plasmids bearing the indicated
385 constructs, using the appropriate antibody against the tag. The arrows indicate the
386 expected molecular weigth corresponding to the expression of the different constructs.
387 S1B Fig. Parasite growth curves. Cultures of CL Brener strain-epimastigotes in the
388 exponential growth phase were collected by centrifugation at 3000g/5min at room
389 temperature, washed with PBS and suspended in LIT (6.106 parasites ml-1). Parasite
390 growth was measured daily by 600 nm absorbance readings in 96-wellmicroplates.
391 S1B Fig. Relative growth. Abs600nm at day 4 was normalized to the initial value (Abs t0).
392
393 S2 Fig. Subcellular location of TcPARP constructs. Indirect immunofluorescence of T.
394 cruzi epimastigotes (CL Brener strain) that overexpress TcPARP-FL or different
395 combinations of protein domains, under basal conditions (Control) or treated with 300 μM
396 hydrogen peroxide (H2O2) for 10 minutes. (A) TcPARP-FL. (B) TcPARPΔN. (C)
397 TcPARPΔNW. (D) TcPARPΔRC. (E) TcPARPΔWRC. (F) TcPARPΔNRC. Bar: 10 μm
398
399 S3 Fig. Dispersion of the nucleolus. Actively growing wild type epimastigotes
400 preincubated for 1 h in the presence of the NAD+ analogue, 3 aminobenzamide (3AB), or
401 the TcPARP inhibitor, Olaparib; and PI3K TcVps34 overexpressing parasites
17
402 (Overexpressants) in the presence or absence of cycloheximide, were fixed and labeled
403 with L1C6 antibody. Bar: 5 μm
18

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bioRxiv preprint doi: https://doi.org/10.1101/2022.04.07.487537; this version posted April 7, 2022.

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1 Trypanosoma cruzi PARP is enriched in the

2 nucleolus and is present in a thread connecting

3 nuclei during mitosis

5 María Laura Kevorkian1, Salomé C. Vilchez Larrea1,2*, Silvia H. Fernández Villamil1,3 *

7 1 Instituto de Investigaciones en Ingeniería Genética y Biología Molecular “Dr.

8 Héctor N. Torres”, Consejo Nacional de Investigaciones Científicas y Técnicas,

9 Ciudad Autónoma de Buenos Aires, Argentina.

10 2 Departamento de Fisiología, Biología Molecular y Celular, Facultad de

11 Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Autónoma

12 de Buenos Aires, Argentina.

13 3 Departamento de Química Biológica, Facultad de Farmacia y Bioquímica,

14 Universidad de Buenos Aires, Ciudad Autónoma de Buenos Aires, Argentina.

17 Silvia H. Fernández Villamil (SFV)

18 E-mail address: silvia.villamil@gmail.com; s.villamil@ingebi.conicet.gov.ar

19 Salome Vilchez Larrea (SVL)

20 E-mail address: vilchez.ingebi@gmail.com

22 Poly (ADP-ribose) polymerase (PARP) is responsible for the synthesis of ADP-

37 Poly (ADP-ribose) polymerase; Trypanosoma cruzi; Nucleolus; ADP-ribose polymer;

41 The authors declare no competing interests.

43 Poly (ADP-ribose) polymerase (PARP) catalyzes the transfer of ADP-ribose units

44 from NAD+ to a target protein or an elongating polymer chain, a post-translational

49 subfamilies, the “DNA-dependent” PARPs (PARP-1, PARP-2 and PARP-3), contain

50 domains capable of recognizing discontinued DNA, thus enabling the recruitment of

67 induce TcPARP activation, leading to nuclear PAR production (13).

68 In early divergent unicellular eukaryotes, such as the members of the

69 Trypanosomatidae family, mechanisms involved in the nucleolar formation at the end of

70 closed mitosis are poorly understood. Nucleologenesis occurs as a continuous process

72 bodies such as prenucleolar bodies, suggesting the direct transmission of preassembled

82 Materials and methods

84 Trypanosoma cruzi epimastigotes (CL Brener strain) were cultured at 28 °C in liver

96 epimastigotes of the CL Brener strain were transfected with the above-mentioned

103 wild type epimastigotes (S1B and S1C Figs).

105 SDS–PAGE and Western blot analysis

108 Hybond-ECL nitrocellulose membrane (GE healthcare), according to the manufacturer’s

129 condition, during the same microscopy session.

132 TcPARP is enriched in the nucleolus of epimastigotes

146 terminal domain (TcPARPΔWRC, TcPARPΔRC and TcPARP-FL) in overexpressing

166 generates the delocalization of nucleolar markers. To test this hypothesis, we

167 overexpressed a non-nuclear protein such as PI3K TcVps34 in epimastigotes, both in

171 therefore be attributed to epimastigotes in stationary phase as described by Gluenz et al.

174 immunofluorescence (IF) of Wild Type or TcPARP-FL overexpressing epimastigotes. B) IF

178 Bar: 5 μm.

180 TcPARP is present in a connecting wire between nuclei of

181 dividing epimastigotes

190 new functions for TcPARP that need further study.

192 Indirect immunofluorescence of epimastigotes overexpressing TcPARP-FL or the N-

195 detected with the correspondent anti-tag antibody. Bar: 5 μm.

197 immunofluorescence of epimastigotes overexpressing TcPARP-FL or the N-terminal

200 correspondent anti-tag antibody. Bar: 5 μm.

202 PARylated proteins are present in the connecting thread

213 endogenous TcPARP activity.

215 epimastigotes. Upper panel: Indirect immunofluorescence (IF) of epimastigotes bearing

216 the empty vector pRIBOTEX or overexpressing the TcPARPΔWRC or TcPARPΔRC

221 Bar: 5 μm.

237 nuclear and nucleolar targeting.

279 1. Kamaletdinova T, Fanaei-Kahrani Z, Wang ZQ. The Enigmatic Function of PARP1:

280 From PARylation Activity to PAR Readers. Cells. 2019;8(12):1–20.

283 (Amst). https://doi.org/10.1016/j.dnarep.2014.05.003

285 for mammalian ADP-ribosyltransferases. Trends Biochem. Sci.

287 4. Vyas S, Chesarone-Cataldo M, Todorova T, Huang Y-HH, Chang P. A systematic

289 physiology. Nat Commun [Internet]. 2013;4:2240.

5 María Laura Kevorkian1, Salomé C. Vilchez Larrea1,2, Silvia H. Fernández Villamil1,3