Identification of biochemically distinct properties of the small ubiquitin-related modifier (SUMO) conjugation pathway in Plasmodium falciparum

Abstract

Small ubiquitin-related modifiers (SUMOs) are post-translationally conjugated to other proteins and are thereby essential regulators of a wide range of cellular processes. Sumoylation, and enzymes of the sumoylation pathway, are conserved in the malaria causing parasite, Plasmodium falciparum. However, the specific functions of sumoylation in P. falciparum, and the degree of functional conservation between enzymes of the human and P. falciparum sumoylation pathways, have not been characterized. Here, we demonstrate that sumoylation levels peak during midstages of the intra-erythrocyte developmental cycle, concomitant with hemoglobin consumption and elevated oxidative stress. In vitro studies revealed that P. falciparum E1- and E2-conjugating enzymes interact effectively to recognize and modify RanGAP1, a model mammalian SUMO substrate. However, in heterologous reactions, P. falciparum E1 and E2 enzymes failed to interact with cognate human E2 and E1 partners, respectively, to modify RanGAP1. Structural analysis, binding studies, and functional assays revealed divergent amino acid residues within the E1-E2 binding interface that define organism-specific enzyme interactions. Our studies identify sumoylation as a potentially important regulator of oxidative stress response during the P. falciparum intra-erythrocyte developmental cycle, and define E1 and E2 interactions as a promising target for development of parasite-specific inhibitors of sumoylation and parasite replication.

Keywords: Crystal Structure; Malaria; Plasmodium; Sumo; Sumoylation; Ubiquitin-conjugating Enzyme (Ubc).

FIGURE 1.

The sumoylation pathway is conserved in P. falciparum. a, SUMO is conjugated to proteins through an enzymatic cascade involving E1-activating and E2-conjugating enzymes and E3 ligases. Deconjugation is catalyzed by isopeptidases. b, enzymes of the P. falciparum sumoylation pathway identified by sequence similarity searches (% identity to human proteins is indicated). Gene ID and predicted molecular weights (MW) were obtained from PlasmoDB. c, amino acid sequence alignment of SP-RING domains from human (Hs), S. cerevisiae (Sc), Schizosaccharomyces pombe (Sp), and Plasmodium falciparum (Pf) SUMO E3 ligases, as well as RING domains from c-Cbl and BRCA1. Sequence conservation is highlighted in gray and black. The SP-RING domain is indicated by the top bar and Zn²⁺ coordinating cysteine and histidine residues are indicated by black dots. Cysteine and histidine residues unique to SP-RING domains are indicated with asterisks.

FIGURE 2.

Sumoylation is differentially regulated during P. falciparum red blood cell stages. a, uninfected red blood cell lysate (RBC), 3D7 parasite lysate, and recombinant PfSUMO, HsSUMO-1, and HsSUMO-2, were analyzed using mAb 7E11 (α-PfSUMO). To demonstrate antibody specificity, lower portions of the blot were also probed with mAbs 21C7 (α-HsSUMO-1) and 8A2 (α-HsSUMO-2). b, immunoblot analysis of stage-specific P. falciparum parasite lysates using mAb 7E11 (α-PfSUMO). Detection of GFP-BCCP and Coomassie Blue staining of the immunoblot membrane are included as loading controls. c, indirect immunofluorescence microscopy of synchronized 3D7 parasites probed with anti-PfSUMO mAb 7E11 and anti-acyl carrier protein (ACP) antibodies. DNA was detected using DAPI. Bar indicates 2 μm.

FIGURE 3.

Human and P. falciparum SUMO E1 and E2 enzyme interactions are distinct. a, P. falciparum enzymes modify RanGAP1 at the consensus site lysine. In vitro transcribed and translated wild type RanGAP1 (WT) or consensus sumoylation site mutants (E528A and K526A), were incubated with recombinant human (Hs) or P. falciparum (Pf)-conjugating enzymes. Conjugation was detected by autoradiography. b, human and P. falciparum E1-conjugating enzymes activate both human and P. falciparum SUMOs. Increasing concentrations of E1-activating enzymes were incubated with human or P. falciparum GFP-SUMO in the presence of ATP and reaction products were separated by non-reducing SDS-PAGE and analyzed by immunoblot analysis. The asterisk indicates a nonspecific GFP-SUMO-2 band. c, P. falciparum and human SUMOs are interchangeable. SUMO conjugation assays were performed in the presence of the indicated recombinant human or P. falciparum proteins. Reaction products were analyzed by immunoblot analysis. d, human and P. falciparum SUMO E2 enzymes are not interchangeable. SUMO conjugation assays were performed in the presence of the indicated recombinant human or P. falciparum proteins. Reaction products were analyzed by immunoblot analysis. e, E2∼SUMO thioesters are not formed in reactions containing heterologous E1 enzymes. The indicated recombinant proteins were incubated in the presence of ATP and reaction products were separated by non-reducing SDS-PAGE and analyzed by immunoblot analysis.

FIGURE 4.

HsUbc9 and PfUbc9 interact specifically with human and P. falciparum E1-activating enzymes, respectively. Representative isotherms of ITC binding data for (a) HsUba2^Ufd and HsUbc9, (b) PfUba2^Ufd and PfUbc9, (c) HsUba2^Ufd and PfUbc9, and (d) PfUba2^Ufd and HsUbc9.

FIGURE 5.

Crystallographic structure of PfUbc9. a, ribbon diagram of the determined structure of PfUbc9 with secondary structures comprising the predicted E1 interface numbered. The active site cysteine is highlighted in yellow. b and c, electrostatic surface representations of the α1 helix and β1-β2 loop regions of PfUbc9 and HsUbc9. Electrostatic potentials were calculated using APBS (52) and colored from positive (+5kT/e; blue) to negative (−5kT/e; red). d, specific divergent residues in the α1 helix and β1-β2 loop region of HsUbc9 and PfUbc9 are indicated.

FIGURE 6.

Human and P. falciparum Uba2^Ufd and Ubc9 interfaces are divergent. a, sequence alignment of S. cerevisiae (Sc) Uba2^Ufd sequence, with corresponding regions from human (Hs) and P. falciparum (Pf) Uba2. Secondary structure is indicated with arrows for β sheets, and dashed closed lines for α-helices. Residues shown to be important for interactions between S. cerevisiae Uba2^Ufd and Ubc9 are indicated in blue (19). Conserved residues are highlighted in green, and similarity is highlighted in yellow, with shading corresponding to degree of shared identity between the sequences. b, sequence alignment of S. cerevisiae, human and P. falciparum Ubc9. Residues involved in the S. cerevisiae Uba2^Ufd and Ubc9 interaction, but which have diverged between human and P. falciparum Ubc9, are highlighted as black asterisks. Conservation is indicated as above. c–e, comparison of determined interactions between S. cerevisiae Uba2^Ufd and Ubc9, and predicted interactions between the human and P. falciparum E1 and E2 enzyme interfaces.

FIGURE 7.

Ubc9 chimeras restore E1 and E2 interaction. a, sequence of N-terminal residues of PfUbc9, HsUbc9, and humanized PfUbc9 chimeras. HsUbc9-specific residues are highlighted in dark red. Residues predicted to be critical for E1-E2 interactions are indicated in blue. Secondary structure is illustrated, as described above. b, GST-RanGAP1 conjugation assays comparing low and high PfUbc9 chimera concentrations. Reactions were performed in the presence of human E1-activating enzyme and human SUMO-1. Products were analyzed by immunoblot analysis with anti-GST antibodies.

FIGURE 8.

Ubc9 chimeras affect human and P. falciparum E1 enzyme interactions. a, schematic of wild type and chimeric Ubc9 proteins. b, assays were performed in the presence of human GFP-tagged SUMO-1, human E1-activating enzyme, and the indicted Ubc9 protein. c, assays were performed in the presence of human GFP-tagged SUMO-1, P. falciparum E1-activating enzyme, and the indicated Ubc9 protein. Reaction products were detected by immunoblot analysis with anti-GFP antibodies.