SUMOylation in Trypanosoma brucei

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 (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′ 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 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, Vadivelu & Field, 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 5 × 10⁴ cells/ml, with a maximum density of 1.5–2 × 10⁶ cells/ml. Procyclics were diluted to 5 × 10⁵, with a maximum density of 5 × 10⁶. Tetracycline was added to a final concentration of 0.25 µg/ml to induce expression from tetracycline-regulated promoters.

For differentiation, bloodstream-form cells were grown to 1.5–2 × 10⁶ cells/ml, then cis-aconitate was added to a concentration of 6 mM. The cells were grown for 16 h at 37°C then transferred to 27°C. Inhibition of glucose transport was achieved by adding phloretin to a concentration of 100 µM. For oxidative stress, H₂O₂ was added to procyclic trypanosomes to final concentrations of 250 µM, 125 µM, 62.5 µM, 31.25 µM or 15.6 µM; the cells were harvested after an incubation time of 1 h.

Tandem affinity purification

For each tandem affinity purification 4–5 × 10⁹ cells were harvested at 4°C and washed twice with ice-cold PBS containing 20 mM N-ethyl maleimide (NEM). Bloodstream cells were harvested at a density of 2 × 10⁶ cells/ml, procyclic cells at a density of 5 × 10⁶ cells/ml. The cell pellets were snap-frozen in liquid nitrogen and stored at −80°C. Cell breakage was performed in 6 ml breakage buffer (10 mM Tris-Cl, 10 mM NaCl, 0.1% NP40, 20 mM 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 13,000 g for 30 min at 4°C to remove the cell debris. Then NaCl was added to a final concentration of 150 mM. The purification was done according to Puig et al. (2001). 20 mM 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

2.5–5 × 10⁹ procyclic cells were harvested at a density of approximately 5 × 10⁶ cells/ml, washed twice with ice-cold PBS containing 10 mM NEM and 10 mM iodoacetamide (IAA) and snap-frozen in liquid nitrogen and stored at −80°C. For use, the cell pellet was resuspended in 1 ml lysis buffer (10 mM Tris-C, 10 mM NaCl, 0.1% NP40, 1% SDS, complete protease inhibitor (Roche), 10 mM NEM, 10 mM IAA, pH = 7.5). Cells were passed the cells 20–25 times through a 21 gauge needle. The lysate was spun at 13,000 g for 30 min at 4°C to remove the cell debris and was diluted 1:10 in IP100 (10 mM Tris-Cl, 100 mM NaCl, 0.1% NP40, complete protease inhibitor (Roche), 10 mM NEM, 10 mM IAA).

Before immunoprecipitation, the lysate was incubated with protein A sepharose for 1 h on a rotary shaker at 4°C to absorb non-specifically binding proteins. The supernatant was then added to 50 µl α-V5 beads (self-made or from Sigma). The lysate was incubated for 3 h on a rotary shaker at 4°C. The beads were washed seven times with IP 100. Elution was done by incubating the beads twice with 125 µl IP buffer mixed with 25 µl V5 peptide for 30 min, then boiling in 4 × Laemmli buffer.

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 ∼40 kDa band (Fig. 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 100 kDa, which, without the tag, would correspond to an 80 kDa SUMOylated conjugate. In T. cruzi also, using either HA-tagged or untagged SUMO, many SUMOylated bands were seen in addition to monomeric SUMO (Bayona et al., 2011). 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 (Obado et al., 2011). There are several possible explanations for the discrepancy. Our TAP-SUMO may have impaired function due to the tag, or the 100 kDa 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 72 h 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 24 h but the signal on the blots was unchanged after 48 h RNAi despite extensive morphological degeneration (Obado et al., 2011).

Our pattern of TAP-SUMO-conjugated proteins was unaffected by heat shock (Fig. 1A) or by treatment for 12 h or 24 h with a sub-lethal level (100 µM) of phloretin (Haanstra et al., 2011) 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°C. 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 (Fig. 1B, marked with a star). 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 less abundant proteins show regulated SUMOylation.

In procyclic forms (Fig. 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 (Fig. 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 (1 h incubations, Fig. 1C lanes 7–10).

SUMOylation increases after oxidative stress

In mammalian cells, peroxide concentrations of 1 mM and lower inhibit SUMOylation (Bossis & 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 (32 µM, 1 h incubation; Fig. 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 (Zhou, Ryan & Zhou, 2004), or 10 mM in mammalian cells (Bossis & Melchior, 2006; Saitoh & Hinchey, 2000), increase SUMOylation, probably by inhibiting the SENP proteases (Bossis & 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 µM (Krieger et al., 2000), and we found that procyclic trypanosomes were killed by concentrations above 250 µM.

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 (Annoura et al., 2012).

First, we attempted tandem affinity purification. SUMOylation was stable for 2 h at 4°C 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 protease cleavage step, we were unable to elute the tagged proteins 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 (Bayona et al., 2011) 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 (Bayona et al., 2011).

As previously reported (Obado et al., 2011), RNAi targeting SUMO in bloodstream trypanosomes halted growth 2 days after RNAi induction (Fig. 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. (2010) observed stronger growth inhibition.

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 (Fig. 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 (Manful, Fadda & Clayton, 2011). However, there was a reproducible decrease in SUMOylation (Fig. 2B, lanes 5–9) after RNAi.