{"gene":"PF3D7_0505800","run_date":"2026-09-07T10:55:52","timeline":{"discoveries":[{"year":2008,"finding":"P. falciparum SUMO (PF3D7_0505800) is expressed as a ~100-amino-acid protein that is conjugated to blood-stage parasite proteins; antibodies against PfSUMO detect conjugates ranging from ~40–250 kDa, localizing to the nucleus, parasite cytoplasm, and host-cell Maurer's clefts. FLAG-tagged PfSUMO confirmed these localizations by IFA and Western blot.","method":"Immunofluorescence microscopy, Western blot with anti-peptide antibodies, FLAG-tagged overexpression, LC-MS/MS after two-step immunoprecipitation (anti-FLAG / anti-PfSUMO)","journal":"Cellular microbiology","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — multiple orthogonal methods (IFA, WB, IP-MS) in a single lab; FLAG-tagging corroborates antibody results","pmids":["18547337"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2008,"finding":"PfSir2, a telomere-associated nuclear protein involved in var gene silencing, was identified as a sumoylation target of PF3D7_0505800 by LC-MS/MS analysis following two-step immunoprecipitation with anti-FLAG and anti-PfSUMO antibodies.","method":"Two-step immunoprecipitation (anti-FLAG / anti-PfSUMO) followed by LC-MS/MS","journal":"Cellular microbiology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — single-lab immunoprecipitation + MS identification, corroborated by two independent antibody reagents","pmids":["18547337"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2011,"finding":"SUMO/ubiquitin heterologous mixed conjugates (SUMO/UB) were detected on P. falciparum proteins, indicating that proteins sumoylated by PF3D7_0505800 can also be ubiquitylated, suggesting cross-regulation between the two pathways.","method":"MudPIT mass spectrometry combined with immunofluorescence and biochemical assays","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MS-based detection with complementary microscopy in a single lab; novel but not yet independently replicated","pmids":["21930698"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2011,"finding":"PfSENP1 (PFL1635w), a SUMO-specific protease, deconjugates substrates modified by PF3D7_0505800; its catalytic domain was recombinantly expressed and shown to have a unique cleavage-sequence preference relative to human SENPs using a positional scanning substrate library. Small-molecule inhibitors of PfSENP1 inhibited both recombinant protease activity and parasite replication.","method":"Recombinant protein expression, positional scanning substrate library profiling, small-molecule inhibitor assays, parasite growth inhibition assay","journal":"Chemistry & biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro enzymatic assay with substrate library and mutagenesis-level mechanistic characterization, complemented by cellular growth inhibition; single lab but multiple orthogonal biochemical methods","pmids":["21700207"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2013,"finding":"Sumoylation levels (conjugation by PF3D7_0505800) peak during mid-stages of the intra-erythrocyte developmental cycle, coinciding with hemoglobin consumption and elevated oxidative stress. In vitro reconstitution showed that P. falciparum E1 and E2 enzymes recognize and modify RanGAP1 (a mammalian SUMO substrate), but P. falciparum E1 and E2 fail to interact with cognate human E2 and E1 partners, respectively, due to divergent residues at the E1–E2 binding interface.","method":"Immunoblot analysis across intraerythrocytic stages, in vitro sumoylation assay (reconstitution with recombinant Pf E1/E2 and RanGAP1), structural analysis, binding studies, mutagenesis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution plus binding studies plus mutagenesis defining interface residues; single lab but multiple orthogonal methods","pmids":["23943616"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2015,"finding":"X-ray crystallography of the human SUMO E1 ubiquitin fold domain in complex with E2 (Ubc9) defined the E1–E2 interface; guided in silico modeling, mutagenesis, and in vitro biochemical studies of P. falciparum E1 and E2 enzymes identified surface residues explaining species-specific interactions with PF3D7_0505800-dependent sumoylation. SUMOylation was shown to play a protective role during oxidative stress at red blood cell stages.","method":"X-ray crystallography (human E1–E2 complex), in silico modeling, site-directed mutagenesis, in vitro biochemical assays, oxidative-stress treatment of parasites","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure plus mutagenesis plus in vitro functional assays; single lab with multiple rigorous methods","pmids":["26697886"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2016,"finding":"NMR backbone and side-chain resonance assignments (1H, 13C, 15N) of recombinant P. falciparum SUMO (PF3D7_0505800) were determined by 2D and 3D heteronuclear NMR experiments, yielding predicted secondary structure information.","method":"2D and 3D heteronuclear NMR spectroscopy (HSQC, triple-resonance experiments)","journal":"Biomolecular NMR assignments","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — NMR structural characterization of purified recombinant protein; single lab, assignment only (no functional validation reported)","pmids":["27699617"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2020,"finding":"Mutations in SUMO-activating enzyme subunit 2 (part of the E1 enzyme that activates PF3D7_0505800) conferred high-level resistance to benzoxaborole AN13762, and introduction of this mutation by genome editing produced a similar resistance level, placing the PF3D7_0505800 sumoylation cascade in the mechanism of drug resistance.","method":"In vitro selection of resistant parasites, whole-genome sequencing, targeted introduction of resistance mutations by genetic engineering, IC50 determination","journal":"mBio","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis via mutation introduction with defined phenotype (resistance IC50 shift); single lab, single drug-target linkage","pmids":["31992618"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2022,"finding":"Biochemical and biophysical analyses demonstrated a unidirectional cross-species interaction: PF3D7_0505800 (Pf-SUMO) interacts with human E2 (Ubc9), whereas human SUMO1 fails to interact with Pf-E2. Surface-accessible aspartate residues of PF3D7_0505800 determine the efficacy and specificity of SUMO–Ubc9 interactions. Mutating critical N-terminal residues of Pf-Ubc9 to their human counterparts restored cross-species interactions by restoring electrostatic, π–π, and hydrophobic contacts.","method":"Biochemical binding assays, biophysical interaction measurements, site-directed mutagenesis of Pf-SUMO surface residues and Pf-Ubc9 N-terminus","journal":"Biophysical journal","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — mutagenesis combined with binding and biophysical assays identifying specific interface residues; single lab","pmids":["35331687"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]}],"current_model":"PF3D7_0505800 (PfSUMO) is a ~100-amino-acid small ubiquitin-related modifier in P. falciparum that is covalently conjugated to numerous nuclear and cytoplasmic proteins (including PfSir2) throughout the intraerythrocytic cycle, peaking at mid-stages under oxidative stress; its conjugation is executed by parasite-specific E1 and E2 enzymes whose interaction interface differs from human counterparts due to critical surface residues on both PfSUMO and Pf-E2, it can form heterologous SUMO/ubiquitin mixed conjugates, it is processed and deconjugated by the SUMO-specific protease PfSENP1 whose catalytic preference is distinct from human SENPs, and disruption of its activating enzyme confers resistance to the benzoxaborole antimalarial AN13762."},"narrative":{"mechanistic_narrative":"PF3D7_0505800 (PfSUMO) is a ~100-amino-acid small ubiquitin-related modifier in Plasmodium falciparum that is covalently conjugated to a broad set of blood-stage parasite proteins spanning the nucleus, parasite cytoplasm, and host-cell Maurer's clefts [PMID:18547337]. Among its identified substrates is PfSir2, a telomere-associated nuclear protein, linking PfSUMO conjugation to the parasite's chromatin regulatory machinery [PMID:18547337]. Sumoylation of parasite proteins is dynamic across the intraerythrocytic developmental cycle, peaking at mid-stages in concert with hemoglobin consumption and elevated oxidative stress, and PfSUMO conjugation is protective under oxidative challenge [PMID:23943616, PMID:26697886]. Conjugation is carried out by parasite-specific E1 and E2 (Pf-Ubc9) enzymes whose E1–E2 binding interface diverges from the human system; surface-accessible aspartate residues on PF3D7_0505800 and N-terminal residues of Pf-Ubc9 dictate the specificity and unidirectional cross-species interactions of the cascade [PMID:23943616, PMID:35331687]. PfSUMO-modified proteins can carry heterologous SUMO/ubiquitin mixed conjugates, indicating cross-regulation between the two modifier pathways [PMID:21930698], and conjugates are removed by the SUMO-specific protease PfSENP1, whose cleavage-sequence preference is distinct from human SENPs [PMID:21700207]. The pathway is pharmacologically relevant: mutation of the SUMO-activating E1 subunit confers resistance to the benzoxaborole AN13762 [PMID:31992618].","teleology":[{"year":2008,"claim":"Whether P. falciparum possesses a functional SUMO system and where its conjugates act was unknown; demonstrating PfSUMO conjugation across parasite compartments established SUMO as an active post-translational modifier in blood-stage parasites and identified a chromatin-regulatory substrate.","evidence":"Immunofluorescence, Western blot with anti-peptide antibodies, FLAG-tagged overexpression, and two-step IP-LC-MS/MS in P. falciparum","pmids":["18547337"],"confidence":"Medium","gaps":["Full substrate repertoire beyond PfSir2 not defined","Functional consequence of PfSir2 sumoylation on var gene silencing not tested","Compartment-specific conjugation dynamics not resolved"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2011,"claim":"It was unclear how PfSUMO conjugates are removed and processed; recombinant characterization of PfSENP1 established a parasite SUMO-specific protease with a cleavage preference distinct from human SENPs, and its inhibitors block parasite replication, nominating deconjugation as a druggable node.","evidence":"Recombinant catalytic-domain expression, positional scanning substrate library profiling, small-molecule inhibitor and parasite growth assays","pmids":["21700207"],"confidence":"High","gaps":["In vivo substrate specificity of PfSENP1 not mapped","Whether inhibitor lethality is solely via SUMO deconjugation not established","Structure of PfSENP1 not determined"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2011,"claim":"Whether PfSUMO cross-talks with ubiquitin was unknown; detection of SUMO/ubiquitin mixed conjugates on parasite proteins established cross-regulation between the two modifier pathways.","evidence":"MudPIT mass spectrometry with complementary immunofluorescence and biochemical assays in P. falciparum","pmids":["21930698"],"confidence":"Medium","gaps":["Specific dual-modified substrates not identified","Functional outcome of mixed conjugates (degradation vs. stabilization) untested","Enzymes generating the mixed chains not defined"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2013,"claim":"The timing and enzymatic basis of PfSUMO conjugation were undefined; showing peak sumoylation at mid-cycle under oxidative stress and reconstituting Pf E1/E2 activity established stage-regulated conjugation and revealed a divergent E1–E2 interface that blocks cross-species enzyme pairing.","evidence":"Stage-resolved immunoblotting, in vitro sumoylation reconstitution with recombinant Pf E1/E2 and RanGAP1, structural analysis and mutagenesis","pmids":["23943616"],"confidence":"High","gaps":["Physiological Pf substrates of the reconstituted cascade not confirmed in vitro","Causal link between oxidative stress and conjugation peak not mechanistically resolved","In vivo requirement of the E1/E2 enzymes not tested by knockout"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2015,"claim":"The residue basis for species-specific SUMO enzyme interactions and the biological role of sumoylation were unclear; structure-guided modeling plus functional assays defined interface residues and demonstrated a protective role for sumoylation during oxidative stress at RBC stages.","evidence":"X-ray crystallography of human E1–E2, in silico modeling, site-directed mutagenesis, in vitro biochemistry, and oxidative-stress treatment of parasites","pmids":["26697886"],"confidence":"High","gaps":["Direct Pf E1–E2 structure not solved (human structure used as guide)","Downstream effectors mediating oxidative-stress protection not identified","Genetic loss-of-function confirmation of the protective role absent"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2020,"claim":"Whether the sumoylation cascade contributes to antimalarial drug action was unknown; identifying E1 (SUMO-activating enzyme subunit 2) mutations that confer AN13762 resistance placed the PF3D7_0505800 pathway in the mechanism of benzoxaborole resistance.","evidence":"In vitro resistance selection, whole-genome sequencing, genome-edited introduction of the mutation, and IC50 determination","pmids":["31992618"],"confidence":"Medium","gaps":["Whether PfSUMO conjugation itself (vs. E1 enzyme) is the drug target not resolved","Molecular mechanism of AN13762 action on the cascade not defined","Effect of resistance mutation on baseline sumoylation not reported"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2022,"claim":"The molecular determinants of PfSUMO's interaction with E2 were incompletely defined; biophysical dissection showed a unidirectional cross-species interaction driven by surface aspartates on PF3D7_0505800 and N-terminal residues of Pf-Ubc9.","evidence":"Biochemical binding and biophysical interaction assays with site-directed mutagenesis of Pf-SUMO surface residues and Pf-Ubc9 N-terminus","pmids":["35331687"],"confidence":"Medium","gaps":["Consequences of interface residues for in vivo conjugation efficiency untested","Co-structure of the Pf-SUMO–Pf-Ubc9 complex not determined","Whether these residues are exploitable for selective inhibitors unexplored"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":null,"claim":"The genome-wide PfSUMO substrate landscape and the phenotypic consequences of blocking PfSUMO conjugation in vivo remain undefined.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No conditional knockout/knockdown of PF3D7_0505800 reported","Comprehensive substrate proteome beyond PfSir2 not mapped","Direct 3D structure of PfSUMO (beyond NMR resonance assignments) not solved"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":[],"partners":["PFSIR2 (SUMOYLATION SUBSTRATE)","PF-E1 SUMO-ACTIVATING ENZYME","PF-UBC9 / PF-E2 SUMO-CONJUGATING ENZYME","PFSENP1 (SUMO-SPECIFIC PROTEASE)","SUMO-ACTIVATING ENZYME SUBUNIT 2 (E1)"],"other_free_text":["PfSUMO conjugation peaks at mid-intraerythrocytic stages coinciding with hemoglobin consumption and oxidative stress [#4].","PfSUMO forms heterologous SUMO/ubiquitin mixed conjugates, indicating cross-talk with the ubiquitin pathway [#2].","Pf E1/E2 enzymes fail to pair with human counterparts due to divergent interface residues; PfSUMO surface aspartates and Pf-Ubc9 N-terminal residues govern this specificity [#4, #8].","NMR backbone and side-chain resonance assignments of recombinant PfSUMO have been determined [#6].","Mutation of the E1 subunit (SUMO-activating enzyme subunit 2) confers benzoxaborole AN13762 resistance [#7]."]}},"prefetch_data":{},"corpus_meta":[{"pmid":"21930698","id":"PMC_21930698","title":"Unraveling the ubiquitome of the human malaria parasite.","date":"2011","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/21930698","citation_count":67,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["SUMO"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"18547337","id":"PMC_18547337","title":"Identification of a novel post-translational modification in Plasmodium falciparum: protein sumoylation in different cellular compartments.","date":"2008","source":"Cellular microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/18547337","citation_count":49,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["SUMO"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"21700207","id":"PMC_21700207","title":"Functional characterization of a SUMO deconjugating protease of Plasmodium falciparum using newly identified small molecule inhibitors.","date":"2011","source":"Chemistry & biology","url":"https://pubmed.ncbi.nlm.nih.gov/21700207","citation_count":42,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["SUMO","small ubiquitin-related modifier"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"31992618","id":"PMC_31992618","title":"Plasmodium falciparum Resistance to a Lead Benzoxaborole Due to Blocked Compound Activation and Altered Ubiquitination or Sumoylation.","date":"2020","source":"mBio","url":"https://pubmed.ncbi.nlm.nih.gov/31992618","citation_count":29,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["SUMO"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"23943616","id":"PMC_23943616","title":"Identification of biochemically distinct properties of the small ubiquitin-related modifier (SUMO) conjugation pathway in Plasmodium falciparum.","date":"2013","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/23943616","citation_count":23,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["SUMO","small ubiquitin-related modifier"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"26697886","id":"PMC_26697886","title":"Characterization and Structural Insights into Selective E1-E2 Interactions in the Human and Plasmodium falciparum SUMO Conjugation Systems.","date":"2015","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/26697886","citation_count":14,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["SUMO"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"34178724","id":"PMC_34178724","title":"Targeting SUMOylation in Plasmodium as a Potential Target for Malaria Therapy.","date":"2021","source":"Frontiers in cellular and infection microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/34178724","citation_count":10,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["SUMO"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"27631812","id":"PMC_27631812","title":"Detection of SUMOylation in Plasmodium falciparum.","date":"2016","source":"Methods in molecular biology (Clifton, N.J.)","url":"https://pubmed.ncbi.nlm.nih.gov/27631812","citation_count":3,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["SUMO"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"27699617","id":"PMC_27699617","title":"Backbone and side-chain resonance assignments of Plasmodium falciparum SUMO.","date":"2016","source":"Biomolecular NMR assignments","url":"https://pubmed.ncbi.nlm.nih.gov/27699617","citation_count":1,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["SUMO"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"35331687","id":"PMC_35331687","title":"Implications of critical node-dependent unidirectional cross-talk of Plasmodium SUMO pathway proteins.","date":"2022","source":"Biophysical journal","url":"https://pubmed.ncbi.nlm.nih.gov/35331687","citation_count":1,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["SUMO"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":7746,"output_tokens":2780,"usd":0.032469,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":8339,"output_tokens":3904,"usd":0.069648,"stage2_stop_reason":"end_turn"},"total_usd":0.102117,"stage1_batch_id":"msgbatch_01DbiymDmubkPpYvdbAu5a7V","stage2_batch_id":"msgbatch_012TbrWvMRH129T7PQ4SmvrJ","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2008,\n      \"finding\": \"P. falciparum SUMO (PF3D7_0505800) is expressed as a ~100-amino-acid protein that is conjugated to blood-stage parasite proteins; antibodies against PfSUMO detect conjugates ranging from ~40–250 kDa, localizing to the nucleus, parasite cytoplasm, and host-cell Maurer's clefts. FLAG-tagged PfSUMO confirmed these localizations by IFA and Western blot.\",\n      \"method\": \"Immunofluorescence microscopy, Western blot with anti-peptide antibodies, FLAG-tagged overexpression, LC-MS/MS after two-step immunoprecipitation (anti-FLAG / anti-PfSUMO)\",\n      \"journal\": \"Cellular microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — multiple orthogonal methods (IFA, WB, IP-MS) in a single lab; FLAG-tagging corroborates antibody results\",\n      \"pmids\": [\"18547337\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"PfSir2, a telomere-associated nuclear protein involved in var gene silencing, was identified as a sumoylation target of PF3D7_0505800 by LC-MS/MS analysis following two-step immunoprecipitation with anti-FLAG and anti-PfSUMO antibodies.\",\n      \"method\": \"Two-step immunoprecipitation (anti-FLAG / anti-PfSUMO) followed by LC-MS/MS\",\n      \"journal\": \"Cellular microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — single-lab immunoprecipitation + MS identification, corroborated by two independent antibody reagents\",\n      \"pmids\": [\"18547337\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"SUMO/ubiquitin heterologous mixed conjugates (SUMO/UB) were detected on P. falciparum proteins, indicating that proteins sumoylated by PF3D7_0505800 can also be ubiquitylated, suggesting cross-regulation between the two pathways.\",\n      \"method\": \"MudPIT mass spectrometry combined with immunofluorescence and biochemical assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MS-based detection with complementary microscopy in a single lab; novel but not yet independently replicated\",\n      \"pmids\": [\"21930698\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"PfSENP1 (PFL1635w), a SUMO-specific protease, deconjugates substrates modified by PF3D7_0505800; its catalytic domain was recombinantly expressed and shown to have a unique cleavage-sequence preference relative to human SENPs using a positional scanning substrate library. Small-molecule inhibitors of PfSENP1 inhibited both recombinant protease activity and parasite replication.\",\n      \"method\": \"Recombinant protein expression, positional scanning substrate library profiling, small-molecule inhibitor assays, parasite growth inhibition assay\",\n      \"journal\": \"Chemistry & biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro enzymatic assay with substrate library and mutagenesis-level mechanistic characterization, complemented by cellular growth inhibition; single lab but multiple orthogonal biochemical methods\",\n      \"pmids\": [\"21700207\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Sumoylation levels (conjugation by PF3D7_0505800) peak during mid-stages of the intra-erythrocyte developmental cycle, coinciding with hemoglobin consumption and elevated oxidative stress. In vitro reconstitution showed that P. falciparum E1 and E2 enzymes recognize and modify RanGAP1 (a mammalian SUMO substrate), but P. falciparum E1 and E2 fail to interact with cognate human E2 and E1 partners, respectively, due to divergent residues at the E1–E2 binding interface.\",\n      \"method\": \"Immunoblot analysis across intraerythrocytic stages, in vitro sumoylation assay (reconstitution with recombinant Pf E1/E2 and RanGAP1), structural analysis, binding studies, mutagenesis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution plus binding studies plus mutagenesis defining interface residues; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"23943616\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"X-ray crystallography of the human SUMO E1 ubiquitin fold domain in complex with E2 (Ubc9) defined the E1–E2 interface; guided in silico modeling, mutagenesis, and in vitro biochemical studies of P. falciparum E1 and E2 enzymes identified surface residues explaining species-specific interactions with PF3D7_0505800-dependent sumoylation. SUMOylation was shown to play a protective role during oxidative stress at red blood cell stages.\",\n      \"method\": \"X-ray crystallography (human E1–E2 complex), in silico modeling, site-directed mutagenesis, in vitro biochemical assays, oxidative-stress treatment of parasites\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure plus mutagenesis plus in vitro functional assays; single lab with multiple rigorous methods\",\n      \"pmids\": [\"26697886\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"NMR backbone and side-chain resonance assignments (1H, 13C, 15N) of recombinant P. falciparum SUMO (PF3D7_0505800) were determined by 2D and 3D heteronuclear NMR experiments, yielding predicted secondary structure information.\",\n      \"method\": \"2D and 3D heteronuclear NMR spectroscopy (HSQC, triple-resonance experiments)\",\n      \"journal\": \"Biomolecular NMR assignments\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — NMR structural characterization of purified recombinant protein; single lab, assignment only (no functional validation reported)\",\n      \"pmids\": [\"27699617\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Mutations in SUMO-activating enzyme subunit 2 (part of the E1 enzyme that activates PF3D7_0505800) conferred high-level resistance to benzoxaborole AN13762, and introduction of this mutation by genome editing produced a similar resistance level, placing the PF3D7_0505800 sumoylation cascade in the mechanism of drug resistance.\",\n      \"method\": \"In vitro selection of resistant parasites, whole-genome sequencing, targeted introduction of resistance mutations by genetic engineering, IC50 determination\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis via mutation introduction with defined phenotype (resistance IC50 shift); single lab, single drug-target linkage\",\n      \"pmids\": [\"31992618\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Biochemical and biophysical analyses demonstrated a unidirectional cross-species interaction: PF3D7_0505800 (Pf-SUMO) interacts with human E2 (Ubc9), whereas human SUMO1 fails to interact with Pf-E2. Surface-accessible aspartate residues of PF3D7_0505800 determine the efficacy and specificity of SUMO–Ubc9 interactions. Mutating critical N-terminal residues of Pf-Ubc9 to their human counterparts restored cross-species interactions by restoring electrostatic, π–π, and hydrophobic contacts.\",\n      \"method\": \"Biochemical binding assays, biophysical interaction measurements, site-directed mutagenesis of Pf-SUMO surface residues and Pf-Ubc9 N-terminus\",\n      \"journal\": \"Biophysical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — mutagenesis combined with binding and biophysical assays identifying specific interface residues; single lab\",\n      \"pmids\": [\"35331687\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    }\n  ],\n  \"current_model\": \"PF3D7_0505800 (PfSUMO) is a ~100-amino-acid small ubiquitin-related modifier in P. falciparum that is covalently conjugated to numerous nuclear and cytoplasmic proteins (including PfSir2) throughout the intraerythrocytic cycle, peaking at mid-stages under oxidative stress; its conjugation is executed by parasite-specific E1 and E2 enzymes whose interaction interface differs from human counterparts due to critical surface residues on both PfSUMO and Pf-E2, it can form heterologous SUMO/ubiquitin mixed conjugates, it is processed and deconjugated by the SUMO-specific protease PfSENP1 whose catalytic preference is distinct from human SENPs, and disruption of its activating enzyme confers resistance to the benzoxaborole antimalarial AN13762.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"PF3D7_0505800 (PfSUMO) is a ~100-amino-acid small ubiquitin-related modifier in Plasmodium falciparum that is covalently conjugated to a broad set of blood-stage parasite proteins spanning the nucleus, parasite cytoplasm, and host-cell Maurer's clefts [#0]. Among its identified substrates is PfSir2, a telomere-associated nuclear protein, linking PfSUMO conjugation to the parasite's chromatin regulatory machinery [#1]. Sumoylation of parasite proteins is dynamic across the intraerythrocytic developmental cycle, peaking at mid-stages in concert with hemoglobin consumption and elevated oxidative stress, and PfSUMO conjugation is protective under oxidative challenge [#4, #5]. Conjugation is carried out by parasite-specific E1 and E2 (Pf-Ubc9) enzymes whose E1–E2 binding interface diverges from the human system; surface-accessible aspartate residues on PF3D7_0505800 and N-terminal residues of Pf-Ubc9 dictate the specificity and unidirectional cross-species interactions of the cascade [#4, #8]. PfSUMO-modified proteins can carry heterologous SUMO/ubiquitin mixed conjugates, indicating cross-regulation between the two modifier pathways [#2], and conjugates are removed by the SUMO-specific protease PfSENP1, whose cleavage-sequence preference is distinct from human SENPs [#3]. The pathway is pharmacologically relevant: mutation of the SUMO-activating E1 subunit confers resistance to the benzoxaborole AN13762 [#7].\",\n  \"teleology\": [\n    {\n      \"year\": 2008,\n      \"claim\": \"Whether P. falciparum possesses a functional SUMO system and where its conjugates act was unknown; demonstrating PfSUMO conjugation across parasite compartments established SUMO as an active post-translational modifier in blood-stage parasites and identified a chromatin-regulatory substrate.\",\n      \"evidence\": \"Immunofluorescence, Western blot with anti-peptide antibodies, FLAG-tagged overexpression, and two-step IP-LC-MS/MS in P. falciparum\",\n      \"pmids\": [\"18547337\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Full substrate repertoire beyond PfSir2 not defined\", \"Functional consequence of PfSir2 sumoylation on var gene silencing not tested\", \"Compartment-specific conjugation dynamics not resolved\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"It was unclear how PfSUMO conjugates are removed and processed; recombinant characterization of PfSENP1 established a parasite SUMO-specific protease with a cleavage preference distinct from human SENPs, and its inhibitors block parasite replication, nominating deconjugation as a druggable node.\",\n      \"evidence\": \"Recombinant catalytic-domain expression, positional scanning substrate library profiling, small-molecule inhibitor and parasite growth assays\",\n      \"pmids\": [\"21700207\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo substrate specificity of PfSENP1 not mapped\", \"Whether inhibitor lethality is solely via SUMO deconjugation not established\", \"Structure of PfSENP1 not determined\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Whether PfSUMO cross-talks with ubiquitin was unknown; detection of SUMO/ubiquitin mixed conjugates on parasite proteins established cross-regulation between the two modifier pathways.\",\n      \"evidence\": \"MudPIT mass spectrometry with complementary immunofluorescence and biochemical assays in P. falciparum\",\n      \"pmids\": [\"21930698\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Specific dual-modified substrates not identified\", \"Functional outcome of mixed conjugates (degradation vs. stabilization) untested\", \"Enzymes generating the mixed chains not defined\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"The timing and enzymatic basis of PfSUMO conjugation were undefined; showing peak sumoylation at mid-cycle under oxidative stress and reconstituting Pf E1/E2 activity established stage-regulated conjugation and revealed a divergent E1–E2 interface that blocks cross-species enzyme pairing.\",\n      \"evidence\": \"Stage-resolved immunoblotting, in vitro sumoylation reconstitution with recombinant Pf E1/E2 and RanGAP1, structural analysis and mutagenesis\",\n      \"pmids\": [\"23943616\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological Pf substrates of the reconstituted cascade not confirmed in vitro\", \"Causal link between oxidative stress and conjugation peak not mechanistically resolved\", \"In vivo requirement of the E1/E2 enzymes not tested by knockout\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"The residue basis for species-specific SUMO enzyme interactions and the biological role of sumoylation were unclear; structure-guided modeling plus functional assays defined interface residues and demonstrated a protective role for sumoylation during oxidative stress at RBC stages.\",\n      \"evidence\": \"X-ray crystallography of human E1–E2, in silico modeling, site-directed mutagenesis, in vitro biochemistry, and oxidative-stress treatment of parasites\",\n      \"pmids\": [\"26697886\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct Pf E1–E2 structure not solved (human structure used as guide)\", \"Downstream effectors mediating oxidative-stress protection not identified\", \"Genetic loss-of-function confirmation of the protective role absent\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Whether the sumoylation cascade contributes to antimalarial drug action was unknown; identifying E1 (SUMO-activating enzyme subunit 2) mutations that confer AN13762 resistance placed the PF3D7_0505800 pathway in the mechanism of benzoxaborole resistance.\",\n      \"evidence\": \"In vitro resistance selection, whole-genome sequencing, genome-edited introduction of the mutation, and IC50 determination\",\n      \"pmids\": [\"31992618\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether PfSUMO conjugation itself (vs. E1 enzyme) is the drug target not resolved\", \"Molecular mechanism of AN13762 action on the cascade not defined\", \"Effect of resistance mutation on baseline sumoylation not reported\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"The molecular determinants of PfSUMO's interaction with E2 were incompletely defined; biophysical dissection showed a unidirectional cross-species interaction driven by surface aspartates on PF3D7_0505800 and N-terminal residues of Pf-Ubc9.\",\n      \"evidence\": \"Biochemical binding and biophysical interaction assays with site-directed mutagenesis of Pf-SUMO surface residues and Pf-Ubc9 N-terminus\",\n      \"pmids\": [\"35331687\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Consequences of interface residues for in vivo conjugation efficiency untested\", \"Co-structure of the Pf-SUMO–Pf-Ubc9 complex not determined\", \"Whether these residues are exploitable for selective inhibitors unexplored\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The genome-wide PfSUMO substrate landscape and the phenotypic consequences of blocking PfSUMO conjugation in vivo remain undefined.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No conditional knockout/knockdown of PF3D7_0505800 reported\", \"Comprehensive substrate proteome beyond PfSir2 not mapped\", \"Direct 3D structure of PfSUMO (beyond NMR resonance assignments) not solved\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0031386\", \"supporting_discovery_ids\": [0, 4, 5]},\n      {\"term_id\": \"GO:0019789\", \"supporting_discovery_ids\": [1, 4]},\n      {\"term_id\": \"GO:0044388\", \"supporting_discovery_ids\": [4, 8]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"GO:0005737\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0020030\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0016925\", \"supporting_discovery_ids\": [0, 4, 5]},\n      {\"term_id\": \"GO:0016926\", \"supporting_discovery_ids\": [3]},\n      {\"term_id\": \"GO:0006979\", \"supporting_discovery_ids\": [4, 5]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"PfSir2 (sumoylation substrate)\",\n      \"Pf-E1 SUMO-activating enzyme\",\n      \"Pf-Ubc9 / Pf-E2 SUMO-conjugating enzyme\",\n      \"PfSENP1 (SUMO-specific protease)\",\n      \"SUMO-activating enzyme subunit 2 (E1)\"\n    ],\n    \"other_free_text\": [\n      \"PfSUMO conjugation peaks at mid-intraerythrocytic stages coinciding with hemoglobin consumption and oxidative stress [#4].\",\n      \"PfSUMO forms heterologous SUMO/ubiquitin mixed conjugates, indicating cross-talk with the ubiquitin pathway [#2].\",\n      \"Pf E1/E2 enzymes fail to pair with human counterparts due to divergent interface residues; PfSUMO surface aspartates and Pf-Ubc9 N-terminal residues govern this specificity [#4, #8].\",\n      \"NMR backbone and side-chain resonance assignments of recombinant PfSUMO have been determined [#6].\",\n      \"Mutation of the E1 subunit (SUMO-activating enzyme subunit 2) confers benzoxaborole AN13762 resistance [#7].\"\n    ]\n  }\n}\n```","audit_flag":null,"strains":[{"strain":"Pf3D7","gene_id":"PF3D7_0505800","name":null,"product":"small ubiquitin-related modifier","uniprot_acc":null,"chromosome":null,"start_pos":null},{"strain":"PbANKA","gene_id":"PBANKA_1105400","name":null,"product":"small ubiquitin-related modifier, putative","uniprot_acc":null,"chromosome":null,"start_pos":null},{"strain":"PkH","gene_id":"PKNH_1027800","name":null,"product":"small ubiquitin-related modifier, putative","uniprot_acc":null,"chromosome":null,"start_pos":null}],"gene_symbol":"SUMO","evaluation":null}