peerj_json_files/PeerJ_Json_131.json

{
    "v1_Abstract": "Small Ubiquitin Like Modifier (SUMO) proteins are involved in many processes in eukaryotes. We here show that Trypanosoma brucei SUMO (Tb927.5.3210) modifies many proteins. The levels of SUMOylation were unaffected by temperature changes but were increased by severe oxidative stress. We obtained evidence that trypanosome homologues of the SUMO conjugating enzyme Ubc9 (Tb927.2.2460) and the SUMO-specific protease SENP (Tb927.9.2220) are involved in SUMOylation and SUMO removal, respectively. Introduction Small Ubiquitin Like Modifier (SUMO) proteins have been found in almost all eukaryotes. Conjugation of SUMO to target proteins alters their functions in multiple ways, and it is therefore central to a multitude of different cellular processes Like ubiquitin, SUMO is attached to its targets via 3 enzymatic steps [1, 2]. First, a SUMOspecific protease (SENP) removes 2-11 amino acids at the SUMO C-terminus, revealing a Cterminal di-glycine motif [3]. Next, SUMO is activated by the SUMO activation complex (E1 complex), which consists of two enzymes, Aos1/SAE1 (budding yeast/human) and Uba2/SAE2 [4, 5]. The C-terminal glycine of SUMO forms a thioester bond with a cysteine residue of Uba2. From there, it is transferred to a cysteine residue of the E2 SUMO conjugating enzyme (Ubc9) [6, 1",
    "v1_col_introduction": "introduction  : Small Ubiquitin Like Modifier (SUMO) proteins have been found in almost all eukaryotes. Conjugation of SUMO to target proteins alters their functions in multiple ways, and it is therefore central to a multitude of different cellular processes Like ubiquitin, SUMO is attached to its targets via 3 enzymatic steps [1, 2]. First, a SUMOspecific protease (SENP) removes 2-11 amino acids at the SUMO C-terminus, revealing a Cterminal di-glycine motif [3]. Next, SUMO is activated by the SUMO activation complex (E1 complex), which consists of two enzymes, Aos1/SAE1 (budding yeast/human) and Uba2/SAE2 [4, 5]. The C-terminal glycine of SUMO forms a thioester bond with a cysteine residue of Uba2. From there, it is transferred to a cysteine residue of the E2 SUMO conjugating enzyme (Ubc9) [6,\n11\n12\n13\n14\n15\n16\n17\n18\n19\n20\n21\n22\n23\n24\n25\n26\n27\nPeerJ reviewing PDF | (v2012:11:99:1:1:NEW 13 Sep 2013)\n7]. From the E2 conjugation enzyme, SUMO binds to a target lysine residue [1, 2]. This process is assisted by an E3 ligase. SUMO is usually attached as a monomer, although chain formation can occur [8]. SUMO is removed from its targets by a variety of peptidases called SENPs [9]. SENP regulation is critical for homeostasis [3, 10-12] and is also involved in responses to stresses such as heat shock and oxidation [10, 13]. SUMO is essential for growth in S. cerevisiae [4] but not in fission yeast [14] or Aspergillus [15]. Work on Chlamydomonas revealed that the abundance of SUMOylated proteins increases during heat shock and osmotic stress [16]. SUMO was also examined in Toxoplasma gondii [17] and Plasmodium falciparum [18]: in both cases many SUMOylated proteins were observed and identified by mass spectrometry, but details of the roles of SUMO in specific processes are not yet known. In the kinetoplastid Trypanosoma cruzi, the components of the SUMOylation machinery have been identified by BLAST search. Numerous SUMOylated bands were identified by Western blotting using both anti-SUMO antibody and detection of epitope-tagged SUMO. In addition, 236 potentially SUMOylated proteins were identified by tandem affinity purification and mass spectrometry[19], but unfortunately, a recent careful re-examination of the spectra could unambiguously identify only eight SUMOylated peptides on just seven proteins [20]. One of the targets identified by the tandem affinity purification, metacaspase 3, was confirmed by coimmunoprecipitation [19] although the SUMOylated peptide was not found [20]. The paraflagellar rod protein PFR1 (also called PAR3) was suggested as a SUMO target by Western blot analysis and in vitro SUMOylation [21] but again no SUMOylated peptide was found [20]. T. cruzi SUMO itself has a SUMOylation site and is able to polymerize [21]. Together these results suggest that many proteins are SUMOylated in T. cruzi, but purification is very difficult. Possibly, SUMO protease is very active and persists during purification procedures. The amino-acid sequence of Trypanosoma brucei SUMO (TbSUMO, Tb927.5.3210) is 37% identical with that of human SUMO-1 and the 3D structure (solved using NMR) is similar to those of yeast and mammalian SUMO [22]. It was shown by chemical shift analysis that TbSUMO interacts with human Ubc9. RNAi targeting SUMO in procyclic trypanosomes caused growth arrest and cell death, and HA-tagged SUMO was predominantly found in the T. brucei nucleus [23]. RNA intereference targeting SUMO in T. brucei caused growth arrest, followed by death, and antibodies to SUMO in bloodstream-form parasites recognised two prominent bands at around 55 and 60 kDa [24]. In this paper we describe preliminary functional characterisation of further components of the SUMOylation system in T. brucei and investigate the effects of various stresses on protein 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60\nPeerJ reviewing PDF | (v2012:11:99:1:1:NEW 13 Sep 2013)\nR ev ie w in g M an\nus cr ip t\nSUMOylation.",
    "v2_Abstract": "Small Ubiquitin Like Modifier (SUMO) proteins are involved in many processes in eukaryotes. We here show that Trypanosoma brucei SUMO (Tb927.5.3210) modifies many proteins and is required for bloodstream trypanosome growth. The levels of SUMOylation were unaffected by temperature changes but were increased by severe oxidative stress. We obtained evidence that trypanosome homologues of the SUMO conjugating enzyme Ubc9 (Tb927.2.2460) and the SUMO-specific protease SENP (Tb927.9.2220) are involved in SUMOylation and SUMO removal, respectively.",
    "v2_col_introduction": "introduction  : Small Ubiquitin Like Modifier (SUMO) proteins have been found in almost all eukaryotes. Conjugation of SUMO to target proteins alters their functions in multiple ways, and it is therefore central to a multitude of different cellular processes. Like ubiquitin, SUMO is attached to its targets via 3 enzymatic steps (Geiss-Friedlander and Melchior, 2007;Ulrich, 2009). First, a SUMO-specific protease (SENP) removes 2-11 amino acids at the SUMO C-terminus, revealing a C-terminal di-glycine motif (Kim and Baek, 2009). Next, SUMO is activated by the SUMO activating complex (E1 complex), which consists of two enzymes, Aos1/SAE1 (budding yeast/human) and Uba2/SAE2 (Desterro et al., 1999;Johnson et al., 1997). The C-terminal glycine of SUMO forms a thioester bond with a cysteine residue of Uba2. From there, it is transferred to a cysteine residue of the E2 SUMO conjugating enzyme (Ubc9) (Desterro et al., 1997;Johnson and Blobel, 1997). From the E2\nPre Prin ts Pre\nconjugating enzyme, SUMO is transferred to a target lysine residue (Geiss-Friedlander and Melchior, 2007;Ulrich, 2009). This process is assisted by an E3 ligase. SUMO is usually attached as a monomer, although chain formation can occur (Ulrich, 2008). SUMO is removed from its targets by a variety of peptidases called SENPs (Mukhopadhyay and Dasso, 2007). SENP regulation is critical for homeostasis (Au et al., 2009;Drag and Salvesen, 2008;Kim and Baek, 2009;Yeh, 2009) and is also involved in responses to stresses such as heat shock and oxidation (Au et al., 2009;Tempe et al., 2008). Most studies of SUMO so far have concentrated on multicellular eukaryotes and yeast. Outside these groups, work on Chlamydomonas revealed that the abundance of SUMOylated proteins increases during heat shock and osmotic stress (Wang et al., 2008). SUMO was also examined in Toxoplasma gondii (Braun et al., 2009) and Plasmodium falciparum (Issar et al., 2008): in both cases many SUMOylated proteins were observed and identified by mass spectrometry, but details of the roles of SUMO in specific processes are not yet known. The amino-acid sequence of Trypanosoma brucei SUMO (TbSUMO, Tb927.5.3210) is 37% identical with that of human SUMO-1 and the 3D structure (solved using NMR) is similar to those of yeast and mammalian SUMO. It was shown by chemical shift analysis that TbSUMO interacts with human Ubc9 (Shang et al., 2009). RNAi targeting SUMO in procyclic trypanosomes caused growth arrest and cell death, and HA-tagged SUMO was predominantly found in the T. brucei nucleus (Liao et al., 2010). Meanwhile, in the kinetoplastid Trypanosoma cruzi, possible SUMOylated proteins have been identified by tandem affinity purification and mass spectrometry (Bayona et al., 2011). SUMOylation of one of the identified targets, metacaspase 3, was confirmed by immunoprecipitation (Bayona et al., 2011). PFR1 (also called PAR3) was also identified as a SUMO target by Western blot analysis and in vitro SUMOylation (Annoura et al., 2012). T. cruzi SUMO itself has a SUMOylation site and is able to polymerize in vitro (Annoura et al., 2012). In this paper we describe preliminary functional characterisation of components of the SUMOylation system in T. brucei and investigate the effects of various stresses on protein SUMOylation.",
    "v1_text": "results and discussion : Many proteins are SUMOylated in T. brucei To detect SUMOylated proteins, an antibody was raised to His-tagged TbSUMO produced in E. coli. (For details of all plasmid constructs see Table 1.) The anti-T.brucei SUMO antibody was unfortunately insufficiently specific. Although it recognised purified recombinant SUMO, it detected several bands, but not monomeric SUMO, in bloodstream- and procyclic-form cell extracts and the banding pattern was not affected by SUMO RNAi. Next, in bloodstream-form trypanosomes, we integrated a sequence encoding a tandem affinity purification tag (TAP-tag) N-terminally in frame with one allele of SUMO. The TAP-SUMO was seen as a ~40kDa band (Figure 1A); the expected size was 33.5 kDa, comprising 12.5 kDa SUMO + 21 kDa tag. Many SUMOylated proteins were present, with a prominent band at 100kDa, which, without the tag, would correpond to an 80kDa SUMOylated conjugate. In T. cruzi also, using either HA-tagged or untagged SUMO, many SUMOylated bands were seen in addition to monomeric SUMO [31]. The pattern that we observed, however, bears no resemblance to the two prominent bands at 55 and 60 kDa that were previously described for bloodstream-form T. brucei using an anti-SUMO antibody [24]. There are several possible explanations for the discrepancy. Our TAP-SUMO may have impaired function due to the tag, or the 100kDa band could actually be equivalent to the 55KDa band (but with very aberrant migration). Alternatively the previously-published pattern could have been affected by antibody cross-reactions with abundant proteins. Since, in the published results, the antibody staining was abolished after 72h RNAi, the last possibility seems unlikely. It cannot, however, be ruled out since no evidence was presented that the antibody recognised native monomeric SUMO on the Western blots. Also the RNAi had depleted the SUMO RNA within 24h but the signal on the blots was unchanged after 48h RNAi despite extensive morphological degeneration [24]. Our pattern of TAP-SUMO-conjugated proteins was unaffected by heat shock (Figure 1A) or by treatment for 12h or 24h with a sub-lethal level (100 \u00b5M) of phloretin [32] to partially inhibit glucose import (not shown). The bloodstream forms used for these experiments are not able to differentiate into growing procyclic forms, but can undergo some early steps of differentiation after addition of cis-aconitate and transfer to 27\u00b0C. When we did this in the TAP-SUMOexpressing line, the banding pattern remained largely unchanged, but one band migrating at 90 kDa reproducibly disappeared (Figure 1B, marked with a star). In accordance with this result, a changing SUMOylation pattern during differentiation was found in T. cruzi [21]. Our 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 PeerJ reviewing PDF | (v2012:11:99:1:1:NEW 13 Sep 2013) R ev ie w in g M an us cr ip t experiments only detected the most abundant SUMOylated proteins and it is quite possible that less abundant proteins show regulated SUMOylation. In procyclic forms (Figure 1C), we integrated a sequence encoding a V5 epitope tag upstream of the open reading frame [26]. We expected monomeric V5-SUMO to migrate at 13 kDa. This was not reproducibly seen, but we did sometimes see a band or bands running at 20 kDa (Figure 1C). In contrast, slower mobility bands were always present, in particular a prominent double band just below 100 kDa. Comparison of the patterns from bloodstream and procyclic forms (by manipulating the photographs to allow for the sizes of the tags, not shown) suggested that the patterns of abundant SUMOylated proteins were similar in both forms. The SUMOylation pattern in procyclics was unaffected by temperature changes (1h incubations, Figure 1C lanes 7-10). SUMOylation increases after oxidative stress In mammalian cells, peroxide concentrations of 1mM and lower inhibit SUMOylation [33] through formation of a disulfide bond between the catalytic domains of the E2 enzyme Ubc9 and the E1 complex subunit Uba2. In contrast, in trypanosomes, oxidative stress increased the abundance of SUMOylated protein, even at relatively low peroxide levels (32 \u00b5M, 1h incubation; Figure 1C). We do not know the reason for this discrepancy: the trypanosome E1 and E2 enzymes may differ such that the dimerization cannot occur, or the dimerization in mammalian cells may be caused by a specific regulatory process that is absent in trypanosomes. Peroxide concentrations above 1 mM in Saccharomyces cerevisiae [34], or 10mM in mammalian cells [33, 35], increase SUMOylation, probably by inhibiting the SENP proteases [33, 36]. Trypanosomes probably react at lower peroxide concentrations because they are much more susceptible to oxidative stress than mammalian cells and yeast: the EC50 of hydrogen peroxide for bloodstream T. brucei is 223\u00b5M [37], and we found that procyclic trypanosomes were killed by concentrations above 250\u00b5M. Failure to purify SUMOylated proteins from T. brucei extracts We made multiple attempts to purify the SUMOylated proteins from trypanosome extracts, using TAP-, His- and V5 tags and a variety of protocols. As previously reported for T. cruzi, all of these attempts failed [21]. First, we attempted tandem affinity purification. SUMOylation was stable for 2h at 4\u00b0C in the lysis buffer, in which standard protease inhibitor mix and N-ethyl-maleimide (20 mM, NEM) were included in order to inhibit SUMO proteases. Although NEM was removed before the TEV 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 PeerJ reviewing PDF | (v2012:11:99:1:1:NEW 13 Sep 2013) R ev ie w in g M an us cr ip t protease cleavage step, we were unable to elute the tagged preteins from the column. A one-step immunoprecipitation, using V5-tagged SUMO, also yielded no specific protein pattern because only 5% of the V5-tagged SUMO bound to the beads. As SUMO binds covalently to its targets, it might be better to use His-tagged SUMO so that SUMOylated proteins can be purified under denaturating conditions. Role of SUMOylation in T. brucei Reciprocal BLASTp searches using yeast and human sequences, and comparison with T.cruzi [19] revealed several putative homologues of Uba2 and Aos1, the enzymes forming the E1 complex, and also of the E2 enzyme Ubc9 (Table 2). Since SUMO E1 and E2 enzymes resemble those for ubiquitination, the specificities of these proteins is unclear. There were four possible E3 ligases, consistent with the need to regulate SUMOylation of different targets separately. However, only one SENP was found. This is surprising, given that the function of SENPs include both the processing and the removal of SUMO, but the same was reported for T.cruzi [19]. As previously reported [24], RNAi targeting SUMO in bloodstream trypanosomes halted growth 2 days after RNAi induction (Figure 2A). We too observed numerous defects in cell division, which is normal in growth-arrested trypanosomes and does not by itself constitute evidence of a role of SUMO in regulating the cell cycle. RNAi in procyclic forms expressing V5-SUMO gave only a transient decrease in V5-SUMO (on day 2 after induction) although the RNA was clearly decreased; in two independent clones, the doubling time increased from 12.6 h to 14.5 h and 15.1 h (not shown). Liao et al [23] observed stronger growth inhibtion. We first targeted the putative SUMO protease, SENP (Tb09.160.0970/ Tb927.9.2220). RNAi had hardly any effect on cell growth (doubling time increase of only 0.3 h, not shown), but there was a strong increase in the abundance of SUMO modification (Figure 2B, lanes 1-4), confirming that the Tb927.9.2220 protein is important for SUMO removal in trypanosomes. Given this increase in SUMOylation, we speculate that a different enzyme might be involved in the activation of SUMO prior to transfer to the E1 conjugating enzyme. Alternatively, much lower levels of SENP activity may be needed for initial SUMO processing than for SUMO removal. Next, we targeted the possible E2 conjugating enzyme UBC9 (Tb927.2.2460) in procyclic trypanosomes expressing V5-SUMO. A UBC9 RNAi line grew slower than the parent line, even in the absence of tetracycline, and there was only marginal slowing after tetracycline addition (not shown). We did not check the mRNA levels: the mRNA is present at less than one copy per cell so it would be difficult to detect even before RNAi [38]. However, there was a reproducible 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 PeerJ reviewing PDF | (v2012:11:99:1:1:NEW 13 Sep 2013) R ev ie w in g M an us cr ip t decrease in SUMOylation (Figure 2B, lanes 5-9) after RNAi. figure legends : Figure 1 Protein modification by tagged SUMO A. Effect of temperature on the pattern of modification with TAP-SUMO in bloodstream trypanosomes containing the plasmid pHD2020. Lane 1: cells without TAP-SUMO. Lanes 2,3,4: The cells were incubated for 1 h at the indicated temperatures. The antibody used for detection was PAP: peroxidase anti-peroxidase antibody (binds to the IgG-binding domain of the TAP tag). B. Effect of differentiation conditions on the pattern of TAP-SUMOylated proteins. Bloodstream trypanosomes were isolated at 6 x 105 cells/ml (L, lower density, lane 1) or 2.5 x 106 cells/ml (H, 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 PeerJ reviewing PDF | (v2012:11:99:1:1:NEW 13 Sep 2013) R ev ie w in g M an us cr ip t higher density, lane 2). Cis-aconitate was added to the higher-density cells and the culture incubated at 37\u00b0C for 17h (lanes 3-6) [39]. Then, the culture was centrifuged and resuspended in procyclic-form medium at 27\u00b0C (lanes 7 & 8). C. Effect of oxidative stress and temperature stress on the pattern of V5-SUMO modification in procyclic trypanosomes. Parasites were transfected with pHD2021 to V5-in situ tag SUMO at the N-terminus [26]. Lanes 1 and 7 are controls. Lanes 2-6: Cells with V5-in situ-tagged SUMO were incubated for 1 h with 15.6, 31.2, 62.5, 125 or 250 \u00b5M hydrogen peroxide. Lanes 8-10: incubation for 1 h at the indicated temperatures. Proteins were detected with anti-V5; as a control, a monoclonal antibody to tubulin (TUB) (from K. Gull) was used. Figure 2 A. Effect of RNAi targeting SUMO on growth of bloodstream-form trypanosomes. RNAi was induced by addition of tetracycline and growth followed daily, with dilution as required to keep the cell density below 1 x 106/ml. B. The effect of RNAi targeting TbUBC9 and TbSENP on SUMOylation in procyclic trypanosomes. Trypanosomes expressing T7 polymerase and the tet repressor [27] were transfected with pHD 2021 and pHD2038 or pHD2037. RNA interference was induced with tetracycline (100 ng/ml in the absence of other selective drugs) for the times shown and the patterns of SUMOylation assayed by Western blotting. 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 PeerJ reviewing PDF | (v2012:11:99:1:1:NEW 13 Sep 2013) R ev ie w in g M an us cr ip t Figure 1 Figure 1 Protein modification by tagged SUMO A. Effect of temperature on the pattern of modification with TAP-SUMO in bloodstream trypanosomes containing the plasmid pHD2020. Lane 1: cells without TAP-SUMO. Lanes 2,3,4: The cells were incubated for 1 h at the indicated temperatures. The antibody used for detection was PAP: peroxidase anti-peroxidase antibody (binds to the IgG-binding domain of the TAP tag). B. Effect of differentiation conditions on the pattern of TAP-SUMOylated proteins. Bloodstream trypanosomes were isolated at 6 x 105 cells/ml (L, lower density, lane 1) or 2.5 x 106 cells/ml (H, higher density, lane 2). Cis-aconitate was added to the higher-density cells and the culture incubated at 37\u00b0C for 17h (lanes 3-6) [ 39 ] . Then, the culture was centrifuged and resuspended in procyclic- form medium at 27\u00b0C (lanes 7 & 8). C. Effect of oxidative stress and temperature stress on the pattern of V5-SUMO modification in procyclic trypanosomes. Parasites were transfected with pHD2021 to V5-in situ tag SUMO at the N-terminus [ 26 ] . Lanes 1 and 7 are controls. Lanes 2-6: Cells with V5-in situ-tagged SUMO were incubated for 1 h with 15.6, 31.2, 62.5, acknowledgements : We thank Keith Gull (University of Oxford) for the anti-tubulin antibody, J. Haanstra and B. Bakker (Utrecht) for communicating phloretin results and Frauke Melchior (ZMBH) for advice. DD was supported by Sonderforschungsbereich 544 of the Deutsche Forschungsgemeinschaft. methods : conclusions : We confirmed the functions of the trypanosome SENP and UBC9 genes, and could show that SUMO modifies many trypanosome proteins. The pattern of SUMOylation was surprisingly unresponsive to stress and also appeared not to be strongly developmentally regulated. table legends : Table 1 Plasmids used in this work (not all results described in text) Table 2 Trypanosome genes potentially involved in SUMOylation. Genes were identified by reciprocal BLASTp. Only genes giving a yeast SUMO pathway enzyme as the best match are included. The putative PIAS homologues each have the expected RING domain and the single SENP has a cysteine protease domain. plasmids : For the N-terminal in situ TAP tag, a part of the TbSUMO open reading frame (ORF) was amplified using the following primers fw: 5'-gac aag ctt ccg cca ccg acg aac cca ctc ata ac-3' rv: 5'-gtc gat atc tca tgt ctg ctc cac cat cgc-3' and cloned into the p2676 vector [25] using Hind III and EcoR V. For the N-terminal V5 in situ tag, a part of the TbSUMO ORF was amplified (fw: 5' - gac ctc gag gac gaa ccc act cat-3', rv; 5' - gac ggg ccc tca cgc cat gca cca-3'), as well as a part of the 5\u2019 untranslated region (UTR) (fw: 5'- gac ccg cgg tgt cct tgt ggt tac gt-3' rv: 5'-gac tct aga aag agg aag tcg ggg ag-3'). The ORF and UTR fragments were cloned into a vector containing the V5-tag and the Blasticidin resistance as described in [26] using Apa I and Xho I for the ORF and Sac II and Xba I for the UTR fragment. For the RNAi constructs, portions of the open reading frames of the targeted genes were amplified and cloned into p2T7TA blue[27]. The following primers were used: for TbSUMO fw: 5'-ggg ggt acc gac gaa ccc act cat aac-3' rv: 5'-ccc aag ctt tca cgc cat gca cca aag 3'; for Tb09.160.0970 (TbSENP) fw: 5'-cag acg act cac tat cgc ca-3', rv:5'-tgc gct caa atg ttg ttc tc-3' and for Tb927.2.2460 (TbUBC9) fw: 5'-tag ctc agt cac gcc tac ga-3'rv:5'-aca cac gaa atg gct ctt cc-3'. The primers were designed using RNAit [28]. trypanosome culture : Trypanosoma brucei strain Lister 427 expressing the tet repressor, with or without T7 polymerase, were used throughout [27], with culturing and transfecting of trypanosomes as previously described [29]. For growth studies, bloodstream-form cells were diluted to a starting concentration of 5x104 cells/ml, with a maximum density of 1.5-2x106 cells/ml. Procyclics were diluted to 5x105, with a maximum density of 5x106. Tetracycline was added to a final concentration of 0.25\u00b5g/ml to induce expression from tetracycline-regulated promoters. For differentiation, bloodstream-form cells were grown to 1.5-2x106 cells/ml, then cis-aconitate was added to a concentration of 6mM. The cells were grown for 16h at 37\u00b0C then transferred to 27\u00b0C. Inhibition of glucose transport was achieved by adding phloretin to a concentration of 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 PeerJ reviewing PDF | (v2012:11:99:1:1:NEW 13 Sep 2013) R ev ie w in g M an us cr ip t 100\u00b5M. For oxidative stress, H2O2 was added to procyclic trypanosomes to final concentrations of 250\u00b5M, 125\u00b5M, 62,5\u00b5M, 31,25\u00b5M or 15,6\u00b5M; the cells were harvested after an incubation time of 1h. tandem affinity purification : For each tandem affinity purification 4-5x109 cells were harvested at 4\u00b0C and washed twice with ice-cold PBS containing 20mM N-ethyl maleimide (NEM). Bloodstream cells were harvested at a density of 2x106 cells/ml, procyclic cells at a density of 5 x106 cells/ml. The cell pellets were snap-frozen in liquid nitrogen and stored at -80\u00b0C. Cell breakage was performed in 6ml breakage buffer (10mM Tris-Cl, 10mM NaCl, 0.1% NP40, 20mM NEM, one tablet of complete inhibitor (without EDTA, Roche) pH=7.8) by passing the cells 20-25 times through a 21 gauge needle. The lysate was spun at 13,000g for 30 min at 4\u00b0C to remove the cell debris. Then NaCl was added to a final concentration of 150mM. The purification was done according to [30]. 20mM NEM was added to all the buffers, except during the wash and elution step of the IgG beads and during the TEV cleavage, as NEM inhibits TEV protease. v5 immunoprecipitation : 2.5-5x109 procyclic cells were harvested at a density of approximately 5 x106 cells/ml, washed twice with ice-cold PBS containing 10mM NEM and 10mM iodoacetamide (IAA) and snapfrozen in liquid nitrogen and stored at -80\u00b0C. For use, the cell pellet was resuspended in 1ml lysis buffer (10mM Tris-C, 10mM NaCl, 0.1% NP40, 1% SDS, complete protease inhibitor (Roche), 10mM NEM, 10mM IAA, pH=7.5). Cells were passed the cells 20-25 times through a 21 gauge needle. The lysate was spun at 13,000g for 30 min at 4\u00b0C to remove the cell debris and was diluted 1:10 in IP100 (10mM Tris-Cl, 100mM NaCl, 0.1% NP40, complete protease inhibitor (Roche), 10mM NEM, 10mM IAA). Before immunoprecipitation, the lysate was incubated with protein A sepharose for 1 hour on a rotary shaker at 4\u00b0C to absorb non-specifically binding proteins. The supernatant was than added to 50\u00b5l \u03b1-V5 beads (self-made or from Sigma). The lysate was incubated for 3h on a rotary shaker at 4\u00b0C. The beads were washed seven times with IP 100. Elution was done by incubating the beads twice with 125\u00b5l IP buffer mixed with 25\u00b5l V5 peptide for 30min, then boiling in 4x Laemmli buffer. 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 PeerJ reviewing PDF | (v2012:11:99:1:1:NEW 13 Sep 2013) R ev ie w in g M an us cr ip t 125 or 250 \u00b5m hydrogen peroxide. lanes 8-10: incubation for 1 h at the indicated : temperatures. Proteins were detected with anti-V5; as a control, a monoclonal antibody to tubulin (TUB) (from K. Gull) was used. PeerJ reviewing PDF | (v2012:11:99:1:1:NEW 13 Sep 2013) R ev ie w in g M an us cr ip t Figure 2 Figure 2 A. Effect of RNAi targeting SUMO on growth of bloodstream-form trypanosomes. RNAi was induced by addition of tetracycline and growth followed daily, with dilution as required to keep the cell density below 1 x 106/ml. B. The effect of RNAi targeting TbUBC9 and TbSENP on SUMOylation in procyclic trypanosomes. Trypanosomes expressing T7 polymerase and the tet repressor [ 27 ] were transfected with pHD 2021 and pHD2038 or pHD2037. RNA interference was induced with tetracycline (100 ng/ml in the absence of other selective drugs) for the times shown and the patterns of SUMOylation assayed by Western blotting. PeerJ reviewing PDF | (v2012:11:99:1:1:NEW 13 Sep 2013) R ev ie w in g M an us cr ip t Table 1(on next page) Table 1 Plasmids used in this work (not all results described in text). PeerJ reviewing PDF | (v2012:11:99:1:1:NEW 13 Sep 2013) R ev ie w in g M an us cr ip t Table 1 pl as mi d Descri ption cloning strategy pH D2 02 0 SUMO /TAP in-situ tag (Ntermina l) A part of the SUMO ORF was amplified using the following primers fw: 5'-gac aag ctt ccg cca ccg acg aac cca ctc ata ac-3' rv: 5'-gtc gat atc tca tgt ctg ctc cac cat cgc-3' and cloned into the p2676 [25] using Hind III and Eco RV pH D2 02 1 SUMO /V5 insitu tag (N- termina l) The SUMO ORF was amplified using the following primers: fw: 5' - gac ctc gag gac gaa ccc act cat-3' rv; 5' - gac ggg ccc tca cgc cat gca cca-3'. A fragment of the UTR was amplified using the following primers: fw: 5'- gac ccg cgg tgt cct tgt ggt tac gt-3' rv: 5'-gac tct aga aag agg aag tcg ggg ag-3'. The ORF was cloned into the Bla V5 vector [26] using Apa I and Xho I, the 5\u2019 UTR using Sac II and Xba I. pH D2 02 2 SUMO knockout A fragment of the SUMO 3' UTR was amplified using the following primers: fw: 5'- gac tct aga cat aag tgc gcg tag tgg-3' rv: 5'- gtc ccg cgg gca aac gac cgc aga agt-3'. A 5'-UTR fragment was amplified using the following primers: fw: 5'-cac tcg agc cct cat atc cac atc ctc a-3' rv: 5'- gtc aag ctt cgt ggg ctc aga aat gaa-3'. 1 PeerJ reviewing PDF | (v2012:11:99:1:1:NEW 13 Sep 2013) R ev ie w in g M an us cr ip t The 3'-UTR fragment was cloned into pHD1748 (Blasticidin resistance cassette in polylinker) using Xba I and Sac II, the 5' UTR fragment using Xho I and Hind III. pH D2 02 3 SUMO knockout The Blasticidin resistence was removed from pHD2022 using Hind III and Eco RI and replaced by a Puromycin resistence which was acquired by digesting pHD1747 with the same enzymes. pH D2 02 4 SUMO RNAi The SUMO ORF was amplified using the following primers: fw: 5'-ggg ggt acc gac gaa ccc act cat aac-3' rv: 5'-ccc aag ctt tca cgc cat gca cca aag -3' and cloned into p2T7TAblue [27] pH D2 02 5 HisSUMO The SUMO ORF was amplified using the following primers: fw: 5'- gag ggt acc gac gaa ccc act cat aac-3' rv: 5'-ccc aag ctt tca cgc cat gca cca aag-3' and cloned into pQEA38 using Kpn I and Hind III. pQEA38 is an expression vector with ten His tags and a TEV cleavage site, modified from pQTEV (AY243506), from the lab of D. G\u00f6rlich (then at ZMBH). pH D2 02 6 SUMO /His insitu tag (N- termina l) A 5' UTR fragment was amplified using the following primers: fw: 5'- gac ccg cgg tgt cct tgt ggt tac gt-3' rv: 5'- gac tct aga aag agg aag tcg ggg ag-3' and cloned into the Bla V5 vector using Sac II and Xba I. Then the V5 tag was removed using Eco NI and Xho I. The SUMO ORF and the His-tag sequence were cut out of pHD 2025 using Eco RI and Hind III. The vector and the tagged ORF were blunted using the Klenow fragment and ligated. pH D2 03 7 TbSEN P RNAi A fragment of the SENP ORF was amplified using the following primers: fw: 5'-cag acg act cac tat cgc ca-3' rv:5'-tgc gct caa atg ttg ttc tc-3' and cloned into the p2T7TAblue vector pH TbUB A fragment of the UBC9 ORF was amplified using the following PeerJ reviewing PDF | (v2012:11:99:1:1:NEW 13 Sep 2013) R ev ie w in g M an us cr ip t D2 03 8 C9 RNAi primers fw: 5'-tag ctc agt cac gcc tac ga-3' rv:5'-aca cac gaa atg gct ctt cc-3' and cloned into the p2T7TAblue vector PeerJ reviewing PDF | (v2012:11:99:1:1:NEW 13 Sep 2013) R ev ie w in g M an us cr ip t Table 2(on next page) Table 2 Trypanosome genes potentially involved in SUMOylation. Genes were identified by reciprocal BLASTp. Only genes giving a yeast SUMO pathway enzyme as the best match are included. The putative PIAS homologues each have the expected RING domain and the single SENP has a cysteine protease domain. PeerJ reviewing PDF | (v2012:11:99:1:1:NEW 13 Sep 2013) R ev ie w in g M an us cr ip t Table 2 Enzyme Function Tb homologue Name Aos1 E1 complex Tb11.02.5410 AOS1 Uba2 E1 complex Tb927.5.3430 UBA2 Ubc9 E2 complex Tb927.2.2460 UBC9 PIAS4/Siz1 E3 ligase Tb09.211.2400 PIAS4/Siz1 E3 ligase Tb927.2.4420 PIAS4/Nfi1 E3 ligase Tb11.01.8710 PIAS1/Siz1 E3 ligase Tb927.6.4830 SUMO1/Ulp2 SENP Tb09.160.0970 SENP 1 PeerJ reviewing PDF | (v2012:11:99:1:1:NEW 13 Sep 2013) R ev ie w in g M an us cr ip t",
    "v2_text": "results and discussion : Many proteins are SUMOylated in T. brucei To detect SUMOylated proteins, an antibody was raised to His-tagged TbSUMO produced in E. coli. The anti-T.brucei SUMO antibody was unfortunately insufficiently specific. Although it recognised purified recombinant SUMO, it detected several bands, but not monomeric SUMO, in bloodstream- and procyclic-form cell extracts and the banding pattern was not affected by SUMO RNAi (not shown). Next, in bloodstream-form trypanosomes, we integrated a sequence encoding a tandem affinity purification tag (TAP-tag) N-terminally in frame with one allele of SUMO. The TAP-SUMO was seen as a ~40kDa band (Figure 1A); the expected size was 33.5 kDa, comprising 12.5 kDa SUMO + 21 kDa tag. In addition, many SUMOylated proteins were present, with a prominent band above 100kDa. The pattern was unaffected by heat shock (Figure 1A) or by treatment for 12h or 24h with a sub-lethal level (100 \u00b5M) of phloretin (Haanstra et al., 2011) to partially inhibit Pre Prin ts Pre Prin ts glucose import (not shown). The bloodstream forms used for these experiments are not able to differentiate into growing procyclic forms, but can undergo some early steps of differentiation after addition of cis-aconitate and transfer to 27\u00b0C. When we did this in the TAP-SUMO-expressing line, the banding pattern remained largely unchanged, but one band migrating at 90 kDa reproducibly disappeared (Figure 1B). In accordance with this result, a changing SUMOylation pattern during differentiation was found in T. cruzi (Annoura et al., 2012). Our experiments only detected the most abundant SUMOylated proteins and it is quite possible that many more, less abundant, proteins show regulated SUMOylation. In procyclic forms (Figure 1C), we integrated a sequence encoding a V5 epitope tag upstream of the open reading frame (Shen et al., 2001). We expected monomeric V5-SUMO to migrate at 13 kDa. This was not reproducibly seen, but we did sometimes see a band or bands running at 20 kDa (Figure 1C). In contrast, slower mobility bands were always present, in particular a prominent double band just below 100 kDa. Comparison of the patterns from bloodstream and procyclic forms (by manipulating the photographs to allow for the sizes of the tags, not shown) suggested that the patterns of abundant SUMOylated proteins were similar in both forms. The SUMOylation pattern in procyclics was unaffected by temperature changes (1h incubations, Figure 1C lanes 7-10). SUMOylation increases after oxidative stress In mammalian cells, peroxide concentrations of 1mM and lower inhibit SUMOylation (Bossis and Melchior, 2006) through formation of a disulfide bond between the catalytic domains of the E2 enzyme Ubc9 and the E1 complex subunit Uba2. In contrast, in trypanosomes, oxidative stress increased the abundance of SUMOylated protein, even at relatively low peroxide levels (15,6\u00b5M or 31,25\u00b5M, 1h incubation; Figure 1C, lane 2 and 3). We do not know the reason for this discrepancy: the trypanosome E1 and E2 enzymes may differ such that the dimerization cannot occur, or the dimerization in mammalian cells may be caused by a specific regulatory process that is absent in trypanosomes. Peroxide concentrations above 1 mM in Saccharomyces cerevisiae (Zhou et al., 2004), or 10mM in mammalian cells (Bossis and Melchior, 2006;Saitoh and Hinchey, 2000), increase SUMOylation, probably by inhibiting the SENP proteases (Bossis and Melchior, 2006;Xu et al., 2008). Trypanosomes probably react at lower peroxide concentrations because they are much more susceptible to oxidative stress than mammalian cells and yeast: the EC50 of hydrogen peroxide for bloodstream T. brucei is 223\u00b5M (Krieger et al., 2000), and Pre Prin ts Pre Prin ts we found that procyclic trypanosomes were killed by concentrations above 250\u00b5M. Failure to purify SUMOylated proteins from T. brucei extracts We made multiple attempts to purify the SUMOylated proteins from trypanosome extracts, using TAP- and V5-tags and a variety of protocols. As previously reported for T. cruzi, all of these attempts failed (Annoura et al., 2012). First, we attempted tandem affinity purification, as previously successfully applied to the exosome (Estevez et al., 2001). SUMOylation was stable for 2h at 4\u00b0C in TAP lysis buffer, as observed by Western blot (not shown). Both protease inhibitor and N-ethyl-maleimide (NEM, 20 mM) were included in the lysis buffer in order to inhibit SUMO proteases. However, NEM was removed by washing before the TEV cleavage step, as it inhibits the TEV protease. Eluates from cells expressing TAP-tagged SUMO gave exactly the same pattern on SDS-PAGE as eluates from cells expressing TAP alone and SUMO was not visible. Further analysis indicated that the majority of TAP-tagged SUMO had bound to the IgG beads, but it was never recovered. Additional bands were however not seen after analysis of the boiled beads. A one-step immunoprecipitation, using V5 tagged SUMO, also yielded no specific protein pattern. Western blot analysis, however, showed that only about 5% of the V5-tagged SUMO bound to the beads, suggesting that the problem lay with poor binding of the tagged protein to the beads. One reason for the failure of the purification of SUMO might be that we failed to inhibit SUMO proteases. As SUMO binds covalently to its targets, it should be possible to purify SUMOylated proteins under denaturating conditions, with all proteases completely inactive during the purification.Therefore, expression of His-tagged SUMO in trypanosomes, would allow purification on nickel agarose beads, in the presence of urea. Bayona et al. (Bayona et al., 2011) have, in contrast to our experience and that of Annoura et al., reported purification of SUMOylated T. cruzi proteins. They started with ten times as many parasites, included TLCK, iodoacetamide, and PMSF as protease inhibitors, and used a tandem affinity approach with His and HA tags, without TEV protease cleavage and with a control extract lacking tagged protein. The list of putatively SUMOylated proteins included most highly abundant proteins, so - although the authors attempted quantitation by comparing spectral counts, and did the experiments in triplicate - it is unclear how many of the identified proteins really are SUMOylated. Nevertheless, they could confirm that a very small proportion of metacaspase 3 is indeed SUMOylated. Pre Prin ts Pre Prin ts Role of SUMOylation in T. brucei To identify enzymes involved in SUMOylation, we performed reciprocal BLASTp searches using yeast and human sequences, as well as comparing the results to the homologues found in T.cruzi (Bayona et al., 2011). We found several putative homologues of Uba2 and Aos1, the enzymes forming the E1 complex, and also for the E2 enzyme Ubc9 (Table 2). Since SUMO E1 and E2 enzymes resemble those for ubiquitination, the specificities of these proteins is unclear. There were four possible E3 ligases, consistent with the need to regulate SUMOylation of different targets separately. However, only one SENP was found. This is surprising, given that the function of SENPs include both the processing and the removal of SUMO. The same was reported for T.cruzi (Bayona et al., 2011). RNAi targeting SUMO in bloodstream trypanosomes halted growth 2 days after RNAi induction (Figure 2A). The RNAi was confirmed by Northern blot (not shown). We observed numerous defects in cell division, which is normal in growth-arrested trypanosomes and does not constitute evidence of a role of SUMO in regulating the cell cycle. RNAi in procyclic forms expressing V5-SUMO gave only a transient decrease in V5-SUMO (on day 2 after induction); in two independent clones, the doubling time increased from 12.6 h to 14.5 h and 15.1 h (not shown). Liao et al (Liao et al., 2010), in contrast, observed stronger growth inhibition. Thus in trypanosomes, as in S. cerevisiae (Johnson et al., 1997), SUMO is essential for growth. In contrast, SUMO is not essential in fission yeast (Tanaka et al., 1999) or Aspergillus (Szewczyk et al., 2008). Next, we targeted the possible E2 conjugating enzyme UBC9 (Tb927.2.2460) in procyclic trypanosomes expressing V5-SUMO A UBC9 RNAi line grew slower than the parent line, even in the absence of tetracycline (not shown). There was only marginal slowing after tetracycline addition and the decrease in SUMOylation was reproducible but moderate (Figure 2B, lanes 5-9), suggesting that substantial levels of UBC9 activity remained. Finally, we targeted the putative SUMO protease, SENP (Tb09.160.0970/ Tb927.9.2220). RNAi had hardly any effect on cell growth (doubling time increase of only 0.3 h, not shown), but there was reproducibly a clear increase in the abundance of SUMO modification (Figure 2B, lanes 1-4), confirming that the identified gene indeed is important for SUMO removal in trypanosomes. It is paradoxical that only one SUMO protease was identified, since the activity is needed for both activation and removal of SUMO. Since RNAi targeting SENP resulted in a general increase in SUMOylation, we speculate that a different enzyme might be involved in the activation of SUMO prior to transfer to the E1 conjugating enzyme. Alternatively, Pre Prin ts Pre Prin ts much lower levels of SENP activity may be needed for the initial SUMO processing than for SUMO removal. figure legends : Figure 1 Protein modification by tagged SUMO A. Effect of temperature on the pattern of modification with TAP-SUMO in bloodstream trypanosomes containing the plasmid pHD2020. Lane 1: cells without TAP-SUMO. Lanes 2,3,4: The cells were incubated for 1 h at the indicated temperatures. The antibody used for detection was PAP: peroxidase anti-peroxidase antibody (binds to the IgG-binding domain of the TAP tag). B. Effect of differentiation conditions on the pattern of TAP-SUMOylated proteins. Bloodstream trypanosomes were isolated at 6 x 105 cells/ml (L, lower density, lane 1) or 2.5 x 106 cells/ml (H, higher density, lane 2). Cis-aconitate was added to the higher-density cells and the culture incubated at 37\u00b0C for 17h (lanes 3-6) (Queiroz et al., 2009). Then, the culture was centrifuged and resuspended in procyclic-form Pre Prin ts Pre Prin ts medium at 27\u00b0C (lanes 7 & 8). The asterisk marks the lane disappearing during the treatment. The arrow marks the lane corresponding to the unblound SUMO protein. C. Effect of oxidative stress and temperature stress on the pattern of V5-SUMO modification in procyclic trypanosomes. Parasites were transfected with pHD2021 to V5-in situ tag SUMO at the N-terminus (Shen et al., 2001). Lanes 1 and 7 are controls. Lanes 2-6: Cells with V5-in situ-tagged SUMO were incubated for 1 h with 15.6, 31.2, 62.5, 125 or 250 \u00b5M hydrogen peroxide. Lanes 8-10: incubation for 1 h at the indicated temperatures. Proteins were detected with anti-V5 (AbD Serotech); as a control, a monoclonal antibody to tubulin (KMX1) (from K. Gull) was used. Figure 2 Roles of SUMO, UBC9 and SENP A. Effect of RNAi targeting SUMO on growth of bloodstream-form trypanosomes. RNAi was induced by addition of tetracycline and growth followed daily, with dilution as required to keep the cell density below 1 x 106/ml. B. The effect of RNAi targeting TbUBC9 and TbSENP on SUMOylation in procyclic trypanosomes. Trypanosomes expressing T7 polymerase and the tet repressor (Alibu et al., 2004) were transfected with pHD 2021 and pHD2038 or pHD2037. RNA interference was induced with tetracycline (100 ng/ml in the absence of other selective drugs) for the times shown and the patterns of SUMOylation assayed by Western blotting. Pre Prin ts Pre Prin ts Figure 1 Protein modification by tagged SUMO Pre Prin ts Pre Prin ts Figure 2 Roles of SUMO, UBC9 and SENP Pre Prin ts Pre Prin ts acknowledgements : We thank Keith Gull (University of Oxford) for the anti-tubulin antibody J. Haanstra and B. Bakker (Utrecht) for communicating phloretin results and Frauke Melchior (ZMBH) for advice. DD was supported by Sonderforschungsbereich 544 of the Deutsche Forschungsgemeinschaft. methods : conclusions : We confirmed the functions of the trypanosome SENP and UBC9 genes, and could show that SUMO modifies many trypanosome proteins. The pattern of SUMOylation was surprisingly unresponsive to stress and also appeared not to be strongly developmentally regulated. tables : Table 1 Trypanosome genes potentially involved in SUMOylation. Genes were identified by reciprocal BLASTp. Only genes giving a yeast SUMO pathway enzyme as the best match are included. The putative PIAS homologues each have the expected RING domain and the single SENP has a cysteine protease domain. Enzyme Function Tb homologue Name Aos1 E1 complex Tb11.02.5410 AOS1 Uba2 E1 complex Tb927.5.3430 UBA2 Ubc9 E2 complex Tb927.2.2460 UBC9 PIAS4/Siz1 E3 ligase Tb09.211.2400 PIAS4/Siz1 E3 ligase Tb927.2.4420 PIAS4/Nfi1 E3 ligase Tb11.01.8710 PIAS1/Siz1 E3 ligase Tb927.6.4830 SUMO1/Ulp2 SENP Tb09.160.0970 SENP plasmids : For the N-terminal in situ TAP tag, a part of the TbSUMO open reading frame (ORF) was amplified using the following primers fw: 5'-gac aag ctt ccg cca ccg acg aac cca Pre Prin ts Pre Prin ts ctc ata ac-3' rv: 5'-gtc gat atc tca tgt ctg ctc cac cat cgc-3' and cloned into the p2676 vector (Kelly et al., 2007) using Hind III and EcoR V. For the N-terminal V5 in situ tag, a part of the TbSUMO ORF was amplified (fw: 5' - gac ctc gag gac gaa ccc act cat-3', rv: 5' - gac ggg ccc tca cgc cat gca cca-3'), as well as a part of the 5\u2019 untranslated region (UTR) (fw: 5'- gac ccg cgg tgt cct tgt ggt tac gt-3' rv: 5'-gac tct aga aag agg aag tcg ggg ag-3'). The ORF and UTR fragments were cloned into a vector containing the V5-tag and the Blasticidin resistance as described in (Shen et al., 2001) using Apa I and Xho I for the ORF and Sac II and Xba I for the UTR fragment. For the expression of recombinant TbSUMO in E.coli, the ORF was amplified with the following primers: fw: 5'- gag ggt acc gac gaa ccc act cat aac-3' rv: 5'-ccc aag ctt tca cgc cat gca cca aag-3' and cloned into pQEA38 using Kpn I and Hind III. pQEA38 is an expression vector with ten His tags and a TEV cleavage site, modified from pQTEV (AY243506), from the lab of D. G\u00f6rlich (then at ZMBH). Protein expression was done using BL21 cells. Protein purification was done under native conditions, using Ni-NTA agarose beads (Qiagen) according to the Qiagen manual. For the RNAi constructs, portions of the open reading frames of the targeted genes were amplified and cloned into p2T7TA blue(Alibu et al., 2004). The following primers were used: for TbSUMO fw: 5'-ggg ggt acc gac gaa ccc act cat aac-3' rv: 5'-ccc aag ctt tca cgc cat gca cca aag 3'; for Tb09.160.0970 (TbSENP) fw: 5'-cag acg act cac tat cgc ca-3', rv:5'-tgc gct caa atg ttg ttc tc-3' and for Tb927.2.2460 (TbUBC9) fw: 5'-tag ctc agt cac gcc tac ga-3'rv:5'-aca cac gaa atg gct ctt cc-3'. The primers were designed using RNAit (Redmond et al., 2003). trypanosome culture : Trypanosoma brucei strain Lister 427 expressing the tet repressor, with or without T7 polymerase, were used throughout (Alibu et al., 2004), with culturing and transfecting of trypanosomes as previously described (van Deursen et al., 2001). For growth studies, bloodstream-form cells were diluted to a starting concentration of 5x104 cells/ml, with a maximum density of 1.5-2x106 cells/ml. Procyclics were diluted to 5x105, with a maximum density of 5x106. Tetracycline was added to a final concentration of 0.25\u00b5g/ml to induce expression from tetracycline-regulated promoters. For differentiation, bloodstream-form cells were grown to 1.5-2x106 cells/ml, then cis-aconitate was added to a concentration of 6mM. The cells were grown for 16h at 37\u00b0C then transferred to 27\u00b0C. Inhibition of glucose transport was achieved by adding phloretin to a concentration of 100\u00b5M. For oxidative stress, H2O2 was added Pre Prin ts Pre Prin ts to procyclic trypanosomes to final concentrations of 250\u00b5M, 125\u00b5M, 62,5\u00b5M, 31,25\u00b5M or 15,6\u00b5M; the cells were harvested after an incubation time of 1h. tandem affinity purification : For each tandem affinity purification approximately 5x109 cells were harvested at 4\u00b0C and washed twice with ice-cold PBS containing 20mM N-ethyl maleimide (NEM). Bloodstream cells were harvested at a density of 2x106 cells/ml, procyclic cells at a density of 5 x106 cells/ml. The cell pellets were snap-frozen in liquid nitrogen and stored at -80\u00b0C. Cell breakage was performed in 6ml breakage buffer (10mM Tris-Cl, 10mM NaCl, 0,1% IGEPAL, 20mM NEM, one tablet of complete protease inhibitor (without EDTA, Roche) pH=7.8) by passing the cells 20-25 times through a 21 gauge needle. The lysate was spun at 16,200g for 30 min at 4\u00b0C to remove the cell debris. Then NaCl was added to a final concentration of 150mM. The purification was done according to (Puig et al., 2001). 20mM NEM was added to all the buffers, except during the wash and elution step of the IgG beads and during the TEV cleavage, as NEM inhibits TEV protease. v5 immunoprecipitation : Anti-V5 antibodies (AbD serotech) were diluted in 1ml 1xPBS and incubated with protein A sepharose (1mg antibody per mg beads) for 1h at room temperature. Then the bound antibody was coupled to the column according to (Harlow and Lane, 1999). The beads were washed five times with PBS before use. 2.5-5x109 procyclic cells a density of approximately 5 x106cells/ml were harvested, washed twice with ice-cold PBS containing 10mM NEM and 10mM iodoacetamide (IAA) and snap-frozen in liquid nitrogen and stored at -80\u00b0C. For use, the cell pellet was resuspended in 1ml lysis buffer (10mM Tris-C, 10mM NaCl, 0,1% IGEPAL, 1% SDS, complete protease inhibitor (Roche), 10mM NEM, 10mM IAA, pH=7.5). Cells were passed the cells 20-25 times through a 21 gauge needle. The lysate was spun at 16,200g for 30 min at 4\u00b0C to remove the cell debris and was diluted 1:10 in IP100 (10mM Tris-Cl, 100mM NaCl, 0,1% IGEPAL, complete protease inhibitor (Roche), 10mM NEM, 10mM IAA). Before immunoprecipitation, the lysate was incubated with protein A sepharose for 1 hour on a rotary shaker at 4\u00b0C to absorb non-specifically binding proteins. The supernatant was than added to 50\u00b5l \u03b1-V5 beads (self-made or from Sigma). The lysate was incubated for 3h on a rotary shaker at 4\u00b0C. The beads were washed seven times with IP100. Elution was done by incubating the beads twice with 125\u00b5l Pre Prin ts Pre Prin ts IP buffer mixed with 25\u00b5l V5 peptide (2mg/ml) for 30min, then were boiled in 4x Laemmli buffer. Eluates were TCA-precipitated and loaded on a 15% SDS gel, which was stained with silver stain. Purification of metabolically labelled proteins A small scale purification of metabolic labelled proteins was done to test the efficiency of the V5 pull-down. About 6x107 procyclic cells were spun down at 600g, transferred to a 1.5ml Eppendorf tube and washed once with PBS. The cells were resuspended in 400ml labelling medium, as described in (Clayton, 1987). Then 12\u00b5l 35S methionine solution (120\u00b5Ci) was added. The cells were incubated for 1h at 30\u00b0C. Then the cells were pelleted and washed twice with PBS containing 10mM NEM and 10mM IAA. The purification was done according to the already described protocols, with slight modifications. 5\u00b5l \u03b1-V5-tagged beads were used. Cell lysis was done by keeping the cells on ice for 45min, with occasional vortexing. The samples were incubated on a rotary shaker rotated at 4\u00b0C for 3,5h. The beads were spun down and washed five times with 1ml IP100. The supernatant of the V5-tagged cells was given on 5\u00b5l new \u03b1-V5-tagged beads and rotated at 4\u00b0C for additional 1.5h. The proteins were eluted with 5\u00b5l V5 peptide (2mg/ml). To yield additional proteins, the beads were boiled in 2x Laemmli buffer.",
    "url": "https://peerj.com/articles/181/reviews/",
    "review_1": "Alla Kostyukova \u00b7 Sep 26, 2013 \u00b7 Academic Editor\nACCEPT\nFont size in some parts of the manuscript is smaller than it should be. If it is still a result of transferring the doc file to the pdf file, please, inform the production staff about this problem.",
    "review_2": "Alla Kostyukova \u00b7 Sep 9, 2013 \u00b7 Academic Editor\nMINOR REVISIONS\nYou don't need to do any additional experiments but you should revise your discussion and address other reviewer's comments.",
    "review_3": "Reviewer 1 \u00b7 Sep 8, 2013\nBasic reporting\nThe article provides sufficient introduction and background to support the experimental design of the study. Formatting of Figures appears appropriate and the article seems to conform with template information.\n\nMinor comments:\nIn the abstract, it should be clarified that the donor (AEDANS) is bound to the tropomyosin isoforms.\nIn the first paragraph of the introduction (line 18), S1 should be introduced as being the S1 subfragment of myosin which may not be clear to an audience not familiar with the field of research.\nTypo in line 75: Change \u2018Than cDNA from the first\u2026, to \u2018Then, cDNA from the first\u2026\u2019\nTypo in line 186: Change \u2018the donor-acceptor distance the absence\u2019 to \u2018the donor-acceptor distance in the absence\u2019\nReference \u2018Barua et al., 2013 (lines 281-283) needs volume and page information\nExperimental design\nThe research question is clearly defined and directly links this study with the findings of the authors from a previous study. The approach and experimental design has been validated by the previous study. The research question is a logical consequence from their previous findings. The method are described with sufficient detail.\nValidity of the findings\nIn the present study, Slivinska and Moraczewska determined isoform-specific localisation of tropomyosin C-termini along actin filaments and muscle myosin S1 subfragment-dependent changes in the localisation of the C-termini. This work is a direct follow-up study where the authors have tested the properties of the N-termini of the same tropomyosin isoforms. The current study reveals interesting information on the displacement of the C-termini of low- and high molecular weight tropomyosins in the presence of myosin S1 binding. These findings provide significant experimental evidence for an isoform specific mechanism by which different tropomyosins regulate the access of actin-associated proteins to the filaments.\nThe recognition of tropomyosins as \u2018gate keepers\u2019 of actin filaments is an emerging concept in the field of studying the regulation of cellular architecture. A large diversity in the regulation of actin-associated proteins by tropomyosins has particularly been explored previously for non-muscle tropomyosins. In this context it would be interesting to test differences in the S1 subfragment-dependent C-terminus displacement of tropomyosins for which isoform-specific regulation of the interaction between actin-associated proteins and the filament has been shown in different cell types. This includes for example the different properties of the TPM1 gene product TmBr3 (containing exons 1b and 9c) and the TPM3 gene product Tm5NM1 (containing exon 1b and 9d) (Bryce et al., 2003). Similarly, it would be interesting to test the effect of non-muscle myosin S1 (e.g. non-muscle myosin IIb S1) on displacement of the tropomyosin C-Termini due to the different binding properties of the muscle and non-muscle S1.\nAdditional comments\nno comments\nCite this review as\nAnonymous Reviewer (2013) Peer Review #1 of \"Structural differences between C-terminal regions of tropomyosin isoforms (v0.1)\". PeerJ https://doi.org/10.7287/peerj.181v0.1/reviews/1",
    "review_4": "Dmitrii Levitsky \u00b7 Sep 5, 2013\nBasic reporting\nThe manuscript 647 by \u015aliwi\u0144ska and Moraczewska describes the influence of the C-terminal sequence of tropomyosin (C-terminal exons 9a or 9d) on the distances between this part of Tm bound to F-actin and Cys374 of actin. For this purpose, four Tm isoforms with different C-terminal exons, 9a or 9d, were used, and the distances between fluorescence donor attached to Cys residue introduced into this Tm exon (Cys 269 or Cys 232) and acceptor attached to Cys374 of actin were measured by FRET in two different states of Tm on the surface of actin filament (closed and open).\n\nThis paper continues the previous paper of the authors (Sliwinska et al. (2011) Cytoskeleton 68, 300-312 ). Both papers are very similar (and sometimes even identical) in respect to Tm isoforms and the methods used, with the only difference that in the previous paper the fluorescence donor was attached to Cys residue in the N-terminal part of Tm, whereas in the present work it was introduced into the Tm C-terminal part. Taken together, the results of both studies show that the distances between Tm end-to-end overlap region and actin are different for Tm isoforms, and their changes in response to myosin S1 binding are individual for each isoform. Generally, I agree with authors\u2019 conclusion that the Tm isoforms are differently oriented on actin filament and the degree of Tm shift in response to the filament activation by myosin S1 binding (i.e. transition from closed to open state) is individual for each type of the studied isoform.\nExperimental design\nThe FRET experiments are carefully performed and clearly described, and the results are well explained.\nValidity of the findings\nThe work is mostly novel, although the main aspects of the research have been previously published by the authors (see above).\nAdditional comments\nn my opinion, the paper could be quite suitable for publication in PeerJ after some corrections according to the following comments.\n\n1). First line in the abstract: \u201cTropomyosins are actin regulatory proteins...\u201d\nIt seems to me, \u201cTropomyosins are actin-binding regulatory proteins..\u201d is more correct.\n\n2). Results, line 102: \u201c...illustration of the overlap regions are shown in Fig. 1.\u201d\nIt should be either \u201d...illustration of the overlap regions is shown in Fig. 1\u201d or \u201c...illustrations of the overlap regions are shown in Fig. 1.\u201d\n\n3). Table 1, upper line (exon 9a, red): it should be A/C, but not A/.\n\n4). The English needs to be edited carefully, and the formatting of the text should be improved (e.g., on lines 87-89, 185, 193-197, and 258-263 the size of the letters is too small).\nCite this review as\nLevitsky D (2013) Peer Review #2 of \"Structural differences between C-terminal regions of tropomyosin isoforms (v0.1)\". PeerJ https://doi.org/10.7287/peerj.181v0.1/reviews/2",
    "pdf_1": "https://peerj.com/articles/181v0.2/submission",
    "pdf_2": "https://peerj.com/articles/181v0.1/submission",
    "review_5": "Reviewer 3 \u00b7 Sep 1, 2013\nBasic reporting\nIn this paper the authors have used FRET to study the relationship of the C terminus of tropomyosin to Cys 374 on actin in Tm isoforms that would have different overlap complexes, and the cooperativity of myosin binding. The rationale is sound: there are structures of the overlap regions of tropomyosins, and models for actin-Tm based on Tm models and structures that do not contain the overlap region. Since the ends are known to be so important for Tm function and a source of isoform diversity (work from Dr. Morazcewsaka\u2019s lab and others), this is a reasonable approach and significant goal. Cysteine mutations were introduced just outside of the overlap region in four different Tms and shown to have little effect on the function of the Tm. The relative affinities are consistent with her previous work (Moraczewska et al., 1999) but not cited, and the fluorescence quenching is consistent with the affinities. The appropriate controls were carried out.\nThe FRET distances were determined for four Tms with sequences encoded by four different combinations of N-terminal and C-terminal sequences encoded by alternatively-expressed exons. Three occur naturally, the fourth is a synthetic construct (1b9a). While Tm2, Tm5a and 1b9a are all alpha Tm (TPM1) sequences, that used for the skTm is not clear: unacetylated alpha TM binds poorly; the citation is in a paper that studied gamma skTm (TPM3) that is 285 residues long due to an extra Met at the N terminus. This needs to be clarified, and also any differences from the common exons in the alpha-Tm encoded forms. The calculated FRET distances for the long forms from the donor on Cys374 on actin were smaller than for the short Tm isoforms. The cooperativity of the response from S1 binding was cooperative in all forms, but the probe in long forms moved further from the donor, while that on the short forms moved closer.\nExperimental design\nsee above\nValidity of the findings\nWith structures and models now available, a value of FRET studies is the opportunity to interpret the observed FRET distances in terms of these models. This is where the manuscripts falls short, and it is quite confusing at best. The data used to make actin-Tm based on 3D reconstructions do not contain information about the axial position of Tm on actin. Previously published work from the Lehrer and Miki laboratories include computational studies to try to use the FRET data in positioning Tm on actin, considering the Tm probe could have FRET with the donor on more than one actin in the filament. The models in the present paper integrate the present FRET results into a model, rather than using the data as a way to test the model. Undoubtedly the FRET data are consistent with more than one model, given the size of the FRET distances.\nCartoons in Figure 4, that derive from published models (but it is not clear which one or how), are especially confusing, it that the authors have inferred that the complex between the N and C termini of 1b9d dissociates on actin, leading to different positions of the ends on actin (i.e. no continuous cable along the actin filament), and failure to interact with the expected sites on actin, D25, K326 and K238. This is especially surprising given the published results that the 1b9a ends form a tighter complex than 1b9d (based on model peptides). The interpretation of the ends coming apart is on the basis of trying to explain the long FRET value for the C terminus, compared to the others, and the shorter value of the N-terminal receptors (previously-published work). Is there another possible interpretation? Some of the reported difficulties working with 1b9a raise concern. Also, why is there no cartoon of stTm for comparison?\nThe observation that the N terminus determines the direction of movement in relation to the actin donor when myosin binds is particularly interesting. With the available of a molecular model of Tm on actin in the presence of myosin (Behrmann et al., 2012), some further discussion of this result would be worthwhile.\nAdditional comments\nSome other comments. The discussions of the structures in the PDB is a bit misleading. The Greenfield and Frye structures are very similar, despite being different genes and different methods (NMR and X-ray). The dynamics showing that the overlap complex is flexible come from the Greenfield structure and were confirmed by Frye in comparing their two structures. Also, it is the Greenfield work that showed the C-terminus opens up when forming a complex with the N terminus. Frye et al. did not determine the structure of the ends alone, and the other published structures of the C terminus from the Cohen and Maeda labs are of complexes of the C terminus with itself, in an antiparallel manner. The Murekami structure is completely unrelated to and inconsistent with those determined by Greenfield and Frye. In the overlap complexes, the C-terminal helices are not parallel; they are in the solution structure of the C terminus alone, but the chains splay apart when they form a complex with the N terminus in all the structures.\nThere is discussion about the myosin cooperativity, but not that myosin increases the affinity of Tm for actin (and visa versa). In 162 ff, this should be mentioned.\nThe paper needs to be reviewed carefully for English usage, particularly the use of articles. Also, the use of the term \u201corientation\u201d is misleading. In a polar structure such as the actin filament, the first reaction is that the orientation of the ends relative to each other is variable; better to use \u201cposition\u201d, I think that is what is suggested.\nIn summary, the data seem valid, but the interpretation is incomplete. Without incorporating the data into existing models in a relevant way, the value of the data significantly diminishes.\nCite this review as\nAnonymous Reviewer (2013) Peer Review #3 of \"Structural differences between C-terminal regions of tropomyosin isoforms (v0.1)\". PeerJ https://doi.org/10.7287/peerj.181v0.1/reviews/3",
    "all_reviews": "Review 1: Alla Kostyukova \u00b7 Sep 26, 2013 \u00b7 Academic Editor\nACCEPT\nFont size in some parts of the manuscript is smaller than it should be. If it is still a result of transferring the doc file to the pdf file, please, inform the production staff about this problem.\nReview 2: Alla Kostyukova \u00b7 Sep 9, 2013 \u00b7 Academic Editor\nMINOR REVISIONS\nYou don't need to do any additional experiments but you should revise your discussion and address other reviewer's comments.\nReview 3: Reviewer 1 \u00b7 Sep 8, 2013\nBasic reporting\nThe article provides sufficient introduction and background to support the experimental design of the study. Formatting of Figures appears appropriate and the article seems to conform with template information.\n\nMinor comments:\nIn the abstract, it should be clarified that the donor (AEDANS) is bound to the tropomyosin isoforms.\nIn the first paragraph of the introduction (line 18), S1 should be introduced as being the S1 subfragment of myosin which may not be clear to an audience not familiar with the field of research.\nTypo in line 75: Change \u2018Than cDNA from the first\u2026, to \u2018Then, cDNA from the first\u2026\u2019\nTypo in line 186: Change \u2018the donor-acceptor distance the absence\u2019 to \u2018the donor-acceptor distance in the absence\u2019\nReference \u2018Barua et al., 2013 (lines 281-283) needs volume and page information\nExperimental design\nThe research question is clearly defined and directly links this study with the findings of the authors from a previous study. The approach and experimental design has been validated by the previous study. The research question is a logical consequence from their previous findings. The method are described with sufficient detail.\nValidity of the findings\nIn the present study, Slivinska and Moraczewska determined isoform-specific localisation of tropomyosin C-termini along actin filaments and muscle myosin S1 subfragment-dependent changes in the localisation of the C-termini. This work is a direct follow-up study where the authors have tested the properties of the N-termini of the same tropomyosin isoforms. The current study reveals interesting information on the displacement of the C-termini of low- and high molecular weight tropomyosins in the presence of myosin S1 binding. These findings provide significant experimental evidence for an isoform specific mechanism by which different tropomyosins regulate the access of actin-associated proteins to the filaments.\nThe recognition of tropomyosins as \u2018gate keepers\u2019 of actin filaments is an emerging concept in the field of studying the regulation of cellular architecture. A large diversity in the regulation of actin-associated proteins by tropomyosins has particularly been explored previously for non-muscle tropomyosins. In this context it would be interesting to test differences in the S1 subfragment-dependent C-terminus displacement of tropomyosins for which isoform-specific regulation of the interaction between actin-associated proteins and the filament has been shown in different cell types. This includes for example the different properties of the TPM1 gene product TmBr3 (containing exons 1b and 9c) and the TPM3 gene product Tm5NM1 (containing exon 1b and 9d) (Bryce et al., 2003). Similarly, it would be interesting to test the effect of non-muscle myosin S1 (e.g. non-muscle myosin IIb S1) on displacement of the tropomyosin C-Termini due to the different binding properties of the muscle and non-muscle S1.\nAdditional comments\nno comments\nCite this review as\nAnonymous Reviewer (2013) Peer Review #1 of \"Structural differences between C-terminal regions of tropomyosin isoforms (v0.1)\". PeerJ https://doi.org/10.7287/peerj.181v0.1/reviews/1\nReview 4: Dmitrii Levitsky \u00b7 Sep 5, 2013\nBasic reporting\nThe manuscript 647 by \u015aliwi\u0144ska and Moraczewska describes the influence of the C-terminal sequence of tropomyosin (C-terminal exons 9a or 9d) on the distances between this part of Tm bound to F-actin and Cys374 of actin. For this purpose, four Tm isoforms with different C-terminal exons, 9a or 9d, were used, and the distances between fluorescence donor attached to Cys residue introduced into this Tm exon (Cys 269 or Cys 232) and acceptor attached to Cys374 of actin were measured by FRET in two different states of Tm on the surface of actin filament (closed and open).\n\nThis paper continues the previous paper of the authors (Sliwinska et al. (2011) Cytoskeleton 68, 300-312 ). Both papers are very similar (and sometimes even identical) in respect to Tm isoforms and the methods used, with the only difference that in the previous paper the fluorescence donor was attached to Cys residue in the N-terminal part of Tm, whereas in the present work it was introduced into the Tm C-terminal part. Taken together, the results of both studies show that the distances between Tm end-to-end overlap region and actin are different for Tm isoforms, and their changes in response to myosin S1 binding are individual for each isoform. Generally, I agree with authors\u2019 conclusion that the Tm isoforms are differently oriented on actin filament and the degree of Tm shift in response to the filament activation by myosin S1 binding (i.e. transition from closed to open state) is individual for each type of the studied isoform.\nExperimental design\nThe FRET experiments are carefully performed and clearly described, and the results are well explained.\nValidity of the findings\nThe work is mostly novel, although the main aspects of the research have been previously published by the authors (see above).\nAdditional comments\nn my opinion, the paper could be quite suitable for publication in PeerJ after some corrections according to the following comments.\n\n1). First line in the abstract: \u201cTropomyosins are actin regulatory proteins...\u201d\nIt seems to me, \u201cTropomyosins are actin-binding regulatory proteins..\u201d is more correct.\n\n2). Results, line 102: \u201c...illustration of the overlap regions are shown in Fig. 1.\u201d\nIt should be either \u201d...illustration of the overlap regions is shown in Fig. 1\u201d or \u201c...illustrations of the overlap regions are shown in Fig. 1.\u201d\n\n3). Table 1, upper line (exon 9a, red): it should be A/C, but not A/.\n\n4). The English needs to be edited carefully, and the formatting of the text should be improved (e.g., on lines 87-89, 185, 193-197, and 258-263 the size of the letters is too small).\nCite this review as\nLevitsky D (2013) Peer Review #2 of \"Structural differences between C-terminal regions of tropomyosin isoforms (v0.1)\". PeerJ https://doi.org/10.7287/peerj.181v0.1/reviews/2\nReview 5: Reviewer 3 \u00b7 Sep 1, 2013\nBasic reporting\nIn this paper the authors have used FRET to study the relationship of the C terminus of tropomyosin to Cys 374 on actin in Tm isoforms that would have different overlap complexes, and the cooperativity of myosin binding. The rationale is sound: there are structures of the overlap regions of tropomyosins, and models for actin-Tm based on Tm models and structures that do not contain the overlap region. Since the ends are known to be so important for Tm function and a source of isoform diversity (work from Dr. Morazcewsaka\u2019s lab and others), this is a reasonable approach and significant goal. Cysteine mutations were introduced just outside of the overlap region in four different Tms and shown to have little effect on the function of the Tm. The relative affinities are consistent with her previous work (Moraczewska et al., 1999) but not cited, and the fluorescence quenching is consistent with the affinities. The appropriate controls were carried out.\nThe FRET distances were determined for four Tms with sequences encoded by four different combinations of N-terminal and C-terminal sequences encoded by alternatively-expressed exons. Three occur naturally, the fourth is a synthetic construct (1b9a). While Tm2, Tm5a and 1b9a are all alpha Tm (TPM1) sequences, that used for the skTm is not clear: unacetylated alpha TM binds poorly; the citation is in a paper that studied gamma skTm (TPM3) that is 285 residues long due to an extra Met at the N terminus. This needs to be clarified, and also any differences from the common exons in the alpha-Tm encoded forms. The calculated FRET distances for the long forms from the donor on Cys374 on actin were smaller than for the short Tm isoforms. The cooperativity of the response from S1 binding was cooperative in all forms, but the probe in long forms moved further from the donor, while that on the short forms moved closer.\nExperimental design\nsee above\nValidity of the findings\nWith structures and models now available, a value of FRET studies is the opportunity to interpret the observed FRET distances in terms of these models. This is where the manuscripts falls short, and it is quite confusing at best. The data used to make actin-Tm based on 3D reconstructions do not contain information about the axial position of Tm on actin. Previously published work from the Lehrer and Miki laboratories include computational studies to try to use the FRET data in positioning Tm on actin, considering the Tm probe could have FRET with the donor on more than one actin in the filament. The models in the present paper integrate the present FRET results into a model, rather than using the data as a way to test the model. Undoubtedly the FRET data are consistent with more than one model, given the size of the FRET distances.\nCartoons in Figure 4, that derive from published models (but it is not clear which one or how), are especially confusing, it that the authors have inferred that the complex between the N and C termini of 1b9d dissociates on actin, leading to different positions of the ends on actin (i.e. no continuous cable along the actin filament), and failure to interact with the expected sites on actin, D25, K326 and K238. This is especially surprising given the published results that the 1b9a ends form a tighter complex than 1b9d (based on model peptides). The interpretation of the ends coming apart is on the basis of trying to explain the long FRET value for the C terminus, compared to the others, and the shorter value of the N-terminal receptors (previously-published work). Is there another possible interpretation? Some of the reported difficulties working with 1b9a raise concern. Also, why is there no cartoon of stTm for comparison?\nThe observation that the N terminus determines the direction of movement in relation to the actin donor when myosin binds is particularly interesting. With the available of a molecular model of Tm on actin in the presence of myosin (Behrmann et al., 2012), some further discussion of this result would be worthwhile.\nAdditional comments\nSome other comments. The discussions of the structures in the PDB is a bit misleading. The Greenfield and Frye structures are very similar, despite being different genes and different methods (NMR and X-ray). The dynamics showing that the overlap complex is flexible come from the Greenfield structure and were confirmed by Frye in comparing their two structures. Also, it is the Greenfield work that showed the C-terminus opens up when forming a complex with the N terminus. Frye et al. did not determine the structure of the ends alone, and the other published structures of the C terminus from the Cohen and Maeda labs are of complexes of the C terminus with itself, in an antiparallel manner. The Murekami structure is completely unrelated to and inconsistent with those determined by Greenfield and Frye. In the overlap complexes, the C-terminal helices are not parallel; they are in the solution structure of the C terminus alone, but the chains splay apart when they form a complex with the N terminus in all the structures.\nThere is discussion about the myosin cooperativity, but not that myosin increases the affinity of Tm for actin (and visa versa). In 162 ff, this should be mentioned.\nThe paper needs to be reviewed carefully for English usage, particularly the use of articles. Also, the use of the term \u201corientation\u201d is misleading. In a polar structure such as the actin filament, the first reaction is that the orientation of the ends relative to each other is variable; better to use \u201cposition\u201d, I think that is what is suggested.\nIn summary, the data seem valid, but the interpretation is incomplete. Without incorporating the data into existing models in a relevant way, the value of the data significantly diminishes.\nCite this review as\nAnonymous Reviewer (2013) Peer Review #3 of \"Structural differences between C-terminal regions of tropomyosin isoforms (v0.1)\". PeerJ https://doi.org/10.7287/peerj.181v0.1/reviews/3\nReview 6: \n",
    "LLM_extracted_review": "[\"Review 1: Font size in some parts of the manuscript is smaller than it should be. If it is still a result of transferring the doc file to the pdf file, please, inform the production staff about this problem.\\n\\nReview 2: You don't need to do any additional experiments but you should revise your discussion and address other reviewer's comments.\\n\\nReview 3: In the abstract, it should be clarified that the donor (AEDANS) is bound to the tropomyosin isoforms. In the first paragraph of the introduction (line 18), S1 should be introduced as being the S1 subfragment of myosin which may not be clear to an audience not familiar with the field of research. Typo in line 75: Change \u2018Than cDNA from the first\u2026, to \u2018Then, cDNA from the first\u2026\u2019 Typo in line 186: Change \u2018the donor-acceptor distance the absence\u2019 to \u2018the donor-acceptor distance in the absence\u2019. Reference \u2018Barua et al., 2013 (lines 281-283) needs volume and page information.\\n\\nReview 4: The English needs to be edited carefully, and the formatting of the text should be improved (e.g., on lines 87-89, 185, 193-197, and 258-263 the size of the letters is too small). First line in the abstract: \u201cTropomyosins are actin regulatory proteins...\u201d It seems to me, \u201cTropomyosins are actin-binding regulatory proteins..\u201d is more correct. Results, line 102: \u201c...illustration of the overlap regions are shown in Fig. 1.\u201d It should be either \u201d...illustration of the overlap regions is shown in Fig. 1\u201d or \u201c...illustrations of the overlap regions are shown in Fig. 1.\u201d Table 1, upper line (exon 9a, red): it should be A/C, but not A/.\\n\\nReview 5: The calculated FRET distances for the long forms from the donor on Cys374 on actin were smaller than for the short Tm isoforms. The cooperativity of the response from S1 binding was cooperative in all forms, but the probe in long forms moved further from the donor, while that on the short forms moved closer. The data used to make actin-Tm based on 3D reconstructions do not contain information about the axial position of Tm on actin. The interpretation of the ends coming apart is on the basis of trying to explain the long FRET value for the C terminus, compared to the others, and the shorter value of the N-terminal receptors (previously-published work). The discussions of the structures in the PDB is a bit misleading. The paper needs to be reviewed carefully for English usage, particularly the use of articles. Also, the use of the term \u201corientation\u201d is misleading. In a polar structure such as the actin filament, the first reaction is that the orientation of the ends relative to each other is variable; better to use \u201cposition\u201d, I think that is what is suggested.\"]"
}