{"gene":"PF3D7_0506900","run_date":"2026-09-07T10:55:52","timeline":{"discoveries":[{"year":2006,"finding":"PF3D7_0506900 (ROM4) was shown in a heterologous cleavage assay to be a functional rhomboid intramembrane protease capable of cleaving multiple P. falciparum adhesins (TRAP, CTRP, MTRAP, PFF0800c, EBA-175, BAEBL, JESEBL, MAEBL, Rh1, Rh2a, Rh2b, Rh4) through their transmembrane domains, and exhibited a novel substrate specificity distinct from canonical rhomboid proteases and from its paralog ROM1. Swapping the transmembrane domains between BAEBL and AMA1 switched relative ROM4/ROM1 preferences, demonstrating that specificity is encoded in the adhesin transmembrane domain.","method":"Heterologous rhomboid cleavage assay; transmembrane domain swap mutagenesis","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 1 / Strong — enzymatic activity demonstrated in vitro with substrate mutagenesis, multiple substrates tested, replicated across two independent 2006 studies","pmids":["17040128"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2006,"finding":"PF3D7_0506900 (ROM4) localizes to the merozoite plasma membrane and cleaves EBA-175 within its transmembrane domain during erythrocyte invasion; mutations in the EBA-175 TMD that abolish cleavage by ROM4 prevent parasite growth, establishing an essential role for ROM4-mediated intramembrane proteolysis in the parasite life cycle.","method":"Localization by immunofluorescence/fractionation; in vitro cleavage assay with TMD mutants; parasite growth assay","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct subcellular localization with functional consequence, enzymatic cleavage assay with mutagenesis, loss-of-function growth phenotype; replicated in companion 2006 paper","pmids":["17000879"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2011,"finding":"PfRh2a undergoes a cleavage event in its transmembrane region during merozoite invasion consistent with the activity of PF3D7_0506900 (ROM4), resulting in release of the ectodomain.","method":"Proteolytic cleavage analysis of PfRh2a in invasion assays; inference from TMD cleavage site consistent with ROM4 substrate specificity","journal":"PLoS pathogens","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — cleavage in transmembrane region reported with attribution to ROM4 but based on biochemical inference rather than direct enzymatic reconstitution or mutagenesis in this paper","pmids":["21698217"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2013,"finding":"PF3D7_0506900 (ROM4) is refractory to gene deletion in P. berghei, indicating an essential role during asexual blood-stage development.","method":"Systematic gene deletion screen (loss-of-function genetic analysis) in P. berghei","journal":"Molecular microbiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean genetic deletion attempt with clear phenotypic readout (refractory), single study but systematic across all 8 rhomboids providing strong internal controls","pmids":["23490234"],"is_preprint":false,"attribution":[{"species":"Plasmodium berghei","strain":""}]},{"year":2020,"finding":"PF3D7_0506900 (ROM4) exhibits atypical substrate specificity arising from 'steric exclusion': juxtamembrane residues prevent canonical rhomboid proteases from cleaving ROM4 substrates and vice versa. An engineered optimal substrate enhanced ROM4 proteolysis >10-fold; high-resolution structural data revealed that boronate warheads enhance inhibition >100-fold. A peptide boronate modeled on the 'super-substrate' carrying a steric-excluding residue selectively inhibited ROM4 but not human rhomboid proteolysis. A structurally distinct alpha-ketoamide also potently and selectively inhibited ROM4. Single-dose treatment with these inhibitors blocked blood-stage merozoite invasion and cleared parasitemia in culture, confirming that ROM4 activity is essential for host-cell invasion.","method":"Substrate engineering with in vitro proteolysis assays; high-resolution structural analysis; compound library screening; inhibitor treatment of P. falciparum blood-stage cultures with ultrastructural analysis","journal":"Cell chemical biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — enzymatic mechanism decoded with mutagenesis/substrate engineering, high-resolution structures obtained, multiple selective inhibitor classes validated, functional consequence (invasion block, parasite clearance) confirmed in culture","pmids":["32888502"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]}],"current_model":"PF3D7_0506900 (ROM4) is an intramembrane rhomboid serine protease that localizes to the P. falciparum merozoite plasma membrane and cleaves a broad set of invasion adhesins (including EBA-175, TRAP, CTRP, MTRAP, multiple EBAs, and Rh proteins) within their transmembrane domains through a 'steric exclusion' mechanism that confers parasite-selective substrate specificity; this proteolytic activity is essential for erythrocyte invasion and blood-stage parasite survival, and can be selectively blocked by engineered peptide boronates or alpha-ketoamide inhibitors that clear parasitemia in culture."},"narrative":{"mechanistic_narrative":"PF3D7_0506900 (ROM4) is an intramembrane rhomboid serine protease that drives Plasmodium erythrocyte invasion by cleaving invasion adhesins within their transmembrane domains [PMID:17040128, PMID:17000879]. In a heterologous cleavage system it processes a broad set of P. falciparum adhesins—including TRAP, CTRP, MTRAP, EBA-175, multiple EBAs (BAEBL, JESEBL, MAEBL), and Rh proteins (Rh1, Rh2a, Rh2b, Rh4)—with a substrate specificity distinct from canonical rhomboids and from its paralog ROM1; transmembrane-domain swaps between BAEBL and AMA1 reassign ROM4/ROM1 preference, showing that specificity is encoded in the adhesin TMD [PMID:17040128]. ROM4 localizes to the merozoite plasma membrane, where it cleaves EBA-175 during invasion, and TMD mutations that abolish this cleavage block parasite growth, establishing its proteolytic activity as essential to the blood-stage life cycle [PMID:17000879]; consistent with this, the gene is refractory to deletion in P. berghei [PMID:23490234]. Its atypical specificity arises from a 'steric exclusion' mechanism in which juxtamembrane residues mutually exclude ROM4 and canonical rhomboids from each other's substrates; this feature enabled engineered peptide boronate and alpha-ketoamide inhibitors that selectively block ROM4 (but not human rhomboids), arrest merozoite invasion, and clear parasitemia in culture [PMID:32888502].","teleology":[{"year":2006,"claim":"It was unknown whether ROM4 was a functional protease and what it acted on; a heterologous assay established it as a rhomboid intramembrane protease with a distinct adhesin-directed substrate repertoire, defining its enzymatic identity.","evidence":"Heterologous rhomboid cleavage assay against multiple adhesins plus BAEBL/AMA1 transmembrane-domain swap mutagenesis","pmids":["17040128"],"confidence":"High","gaps":["Cleavage demonstrated in a heterologous cell system, not in the native parasite for all listed substrates","Which of the many candidate substrates are physiologically cleaved by ROM4 during invasion was not resolved"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2006,"claim":"The subcellular site and biological necessity of ROM4 activity were unknown; localization to the merozoite plasma membrane plus a growth-lethal EBA-175 TMD cleavage mutant established that ROM4-mediated intramembrane proteolysis is essential for the life cycle.","evidence":"Immunofluorescence/fractionation localization, in vitro cleavage with EBA-175 TMD mutants, and parasite growth assays","pmids":["17000879"],"confidence":"High","gaps":["Essentiality inferred from a substrate-side (EBA-175 TMD) mutation rather than direct ROM4 knockout","Timing of cleavage relative to invasion steps not precisely mapped"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2011,"claim":"Whether a ROM4-type transmembrane cleavage occurs on Rh-family adhesins during native invasion was open; PfRh2a was found to be cleaved in its transmembrane region with ectodomain release, consistent with ROM4 activity.","evidence":"Proteolytic cleavage analysis of PfRh2a in invasion assays, with attribution to ROM4 by TMD cleavage-site inference","pmids":["21698217"],"confidence":"Medium","gaps":["Attribution to ROM4 is biochemical inference, not direct enzymatic reconstitution or mutagenesis in this study","Functional consequence of Rh2a cleavage for invasion not established here"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2013,"claim":"Direct genetic evidence for ROM4 essentiality was lacking; a systematic rhomboid deletion screen in P. berghei found the gene refractory to deletion, confirming an essential role in asexual blood-stage development independent of substrate-side mutations.","evidence":"Systematic loss-of-function gene-deletion screen across all eight rhomboids in P. berghei","pmids":["23490234"],"confidence":"Medium","gaps":["Refractoriness to deletion does not itself define the essential substrate or step","Shown in P. berghei; direct knockout essentiality in P. falciparum not established here"],"attribution":[{"species":"Plasmodium berghei","strain":""}]},{"year":2020,"claim":"The structural basis of ROM4's parasite-selective specificity and its druggability were unknown; a 'steric exclusion' mechanism was decoded and exploited to build selective inhibitors that block invasion, validating ROM4 as an antimalarial target.","evidence":"Substrate engineering with in vitro proteolysis, high-resolution structural analysis, compound screening, and inhibitor treatment of blood-stage cultures with ultrastructural readout","pmids":["32888502"],"confidence":"High","gaps":["In vivo efficacy of the inhibitors beyond culture not addressed","Resistance liability and off-target profile across the parasite proteome not defined"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":null,"claim":"The precise set of native, physiologically-cleaved ROM4 substrates during invasion and the consequences of a direct P. falciparum ROM4 knockout remain to be defined.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No direct conditional ROM4 knockout in P. falciparum reported in the corpus","The in vivo hierarchy among the many candidate adhesin substrates is unresolved"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":[],"partners":["EBA-175 (SUBSTRATE)","TRAP (SUBSTRATE)","CTRP (SUBSTRATE)","MTRAP (SUBSTRATE)","PFF0800C (SUBSTRATE)","BAEBL (SUBSTRATE)","JESEBL (SUBSTRATE)","MAEBL (SUBSTRATE)","RH1 (SUBSTRATE)","RH2A (SUBSTRATE)","RH2B (SUBSTRATE)","RH4 (SUBSTRATE)"],"other_free_text":["Rhomboid intramembrane serine protease cleaving adhesins within their transmembrane domains [#0, #1].","Substrate specificity is encoded in the adhesin transmembrane domain, demonstrated by BAEBL/AMA1 TMD swaps that reassign ROM4 vs ROM1 preference [#0].","'Steric exclusion' mechanism: juxtamembrane residues mutually exclude ROM4 and canonical rhomboids from each other's substrates, conferring parasite-selective specificity [#4].","Essential for asexual blood-stage development and erythrocyte invasion; refractory to deletion in P. berghei [#3] and growth-lethal when EBA-175 TMD cleavage is abolished in P. falciparum [#1].","Selectively inhibited by engineered peptide boronates and an alpha-ketoamide that block merozoite invasion and clear parasitemia in culture [#4]."]}},"prefetch_data":{},"corpus_meta":[{"pmid":"17040128","id":"PMC_17040128","title":"Two Plasmodium rhomboid proteases preferentially cleave different adhesins implicated in all invasive stages of malaria.","date":"2006","source":"PLoS pathogens","url":"https://pubmed.ncbi.nlm.nih.gov/17040128","citation_count":180,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["PFE0340c","PfROM4"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"17000879","id":"PMC_17000879","title":"Intramembrane proteolysis mediates shedding of a key adhesin during erythrocyte invasion by the malaria parasite.","date":"2006","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/17000879","citation_count":150,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["PfROM4"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"23490234","id":"PMC_23490234","title":"Loss-of-function analyses defines vital and redundant functions of the Plasmodium rhomboid protease family.","date":"2013","source":"Molecular microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/23490234","citation_count":41,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["ROM4"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"21698217","id":"PMC_21698217","title":"Plasmodium falciparum merozoite invasion is inhibited by antibodies that target the PfRh2a and b binding domains.","date":"2011","source":"PLoS pathogens","url":"https://pubmed.ncbi.nlm.nih.gov/21698217","citation_count":40,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["PfROM4"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"32888502","id":"PMC_32888502","title":"Designed Parasite-Selective Rhomboid Inhibitors Block Invasion and Clear Blood-Stage Malaria.","date":"2020","source":"Cell chemical biology","url":"https://pubmed.ncbi.nlm.nih.gov/32888502","citation_count":21,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["PfROM4"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"39448193","id":"PMC_39448193","title":"Plasmodium proteases and their role in development of Malaria vaccines.","date":"2024","source":"Advances in parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/39448193","citation_count":0,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["ROM4"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":5608,"output_tokens":1652,"usd":0.020802,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":6774,"output_tokens":3031,"usd":0.054823,"stage2_stop_reason":"end_turn"},"total_usd":0.075625,"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\": 2006,\n      \"finding\": \"PF3D7_0506900 (ROM4) was shown in a heterologous cleavage assay to be a functional rhomboid intramembrane protease capable of cleaving multiple P. falciparum adhesins (TRAP, CTRP, MTRAP, PFF0800c, EBA-175, BAEBL, JESEBL, MAEBL, Rh1, Rh2a, Rh2b, Rh4) through their transmembrane domains, and exhibited a novel substrate specificity distinct from canonical rhomboid proteases and from its paralog ROM1. Swapping the transmembrane domains between BAEBL and AMA1 switched relative ROM4/ROM1 preferences, demonstrating that specificity is encoded in the adhesin transmembrane domain.\",\n      \"method\": \"Heterologous rhomboid cleavage assay; transmembrane domain swap mutagenesis\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — enzymatic activity demonstrated in vitro with substrate mutagenesis, multiple substrates tested, replicated across two independent 2006 studies\",\n      \"pmids\": [\"17040128\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"PF3D7_0506900 (ROM4) localizes to the merozoite plasma membrane and cleaves EBA-175 within its transmembrane domain during erythrocyte invasion; mutations in the EBA-175 TMD that abolish cleavage by ROM4 prevent parasite growth, establishing an essential role for ROM4-mediated intramembrane proteolysis in the parasite life cycle.\",\n      \"method\": \"Localization by immunofluorescence/fractionation; in vitro cleavage assay with TMD mutants; parasite growth assay\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct subcellular localization with functional consequence, enzymatic cleavage assay with mutagenesis, loss-of-function growth phenotype; replicated in companion 2006 paper\",\n      \"pmids\": [\"17000879\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"PfRh2a undergoes a cleavage event in its transmembrane region during merozoite invasion consistent with the activity of PF3D7_0506900 (ROM4), resulting in release of the ectodomain.\",\n      \"method\": \"Proteolytic cleavage analysis of PfRh2a in invasion assays; inference from TMD cleavage site consistent with ROM4 substrate specificity\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — cleavage in transmembrane region reported with attribution to ROM4 but based on biochemical inference rather than direct enzymatic reconstitution or mutagenesis in this paper\",\n      \"pmids\": [\"21698217\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"PF3D7_0506900 (ROM4) is refractory to gene deletion in P. berghei, indicating an essential role during asexual blood-stage development.\",\n      \"method\": \"Systematic gene deletion screen (loss-of-function genetic analysis) in P. berghei\",\n      \"journal\": \"Molecular microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean genetic deletion attempt with clear phenotypic readout (refractory), single study but systematic across all 8 rhomboids providing strong internal controls\",\n      \"pmids\": [\"23490234\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium berghei\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"PF3D7_0506900 (ROM4) exhibits atypical substrate specificity arising from 'steric exclusion': juxtamembrane residues prevent canonical rhomboid proteases from cleaving ROM4 substrates and vice versa. An engineered optimal substrate enhanced ROM4 proteolysis >10-fold; high-resolution structural data revealed that boronate warheads enhance inhibition >100-fold. A peptide boronate modeled on the 'super-substrate' carrying a steric-excluding residue selectively inhibited ROM4 but not human rhomboid proteolysis. A structurally distinct alpha-ketoamide also potently and selectively inhibited ROM4. Single-dose treatment with these inhibitors blocked blood-stage merozoite invasion and cleared parasitemia in culture, confirming that ROM4 activity is essential for host-cell invasion.\",\n      \"method\": \"Substrate engineering with in vitro proteolysis assays; high-resolution structural analysis; compound library screening; inhibitor treatment of P. falciparum blood-stage cultures with ultrastructural analysis\",\n      \"journal\": \"Cell chemical biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — enzymatic mechanism decoded with mutagenesis/substrate engineering, high-resolution structures obtained, multiple selective inhibitor classes validated, functional consequence (invasion block, parasite clearance) confirmed in culture\",\n      \"pmids\": [\"32888502\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    }\n  ],\n  \"current_model\": \"PF3D7_0506900 (ROM4) is an intramembrane rhomboid serine protease that localizes to the P. falciparum merozoite plasma membrane and cleaves a broad set of invasion adhesins (including EBA-175, TRAP, CTRP, MTRAP, multiple EBAs, and Rh proteins) within their transmembrane domains through a 'steric exclusion' mechanism that confers parasite-selective substrate specificity; this proteolytic activity is essential for erythrocyte invasion and blood-stage parasite survival, and can be selectively blocked by engineered peptide boronates or alpha-ketoamide inhibitors that clear parasitemia in culture.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"PF3D7_0506900 (ROM4) is an intramembrane rhomboid serine protease that drives Plasmodium erythrocyte invasion by cleaving invasion adhesins within their transmembrane domains [#0, #1]. In a heterologous cleavage system it processes a broad set of P. falciparum adhesins—including TRAP, CTRP, MTRAP, EBA-175, multiple EBAs (BAEBL, JESEBL, MAEBL), and Rh proteins (Rh1, Rh2a, Rh2b, Rh4)—with a substrate specificity distinct from canonical rhomboids and from its paralog ROM1; transmembrane-domain swaps between BAEBL and AMA1 reassign ROM4/ROM1 preference, showing that specificity is encoded in the adhesin TMD [#0]. ROM4 localizes to the merozoite plasma membrane, where it cleaves EBA-175 during invasion, and TMD mutations that abolish this cleavage block parasite growth, establishing its proteolytic activity as essential to the blood-stage life cycle [#1]; consistent with this, the gene is refractory to deletion in P. berghei [#3]. Its atypical specificity arises from a 'steric exclusion' mechanism in which juxtamembrane residues mutually exclude ROM4 and canonical rhomboids from each other's substrates; this feature enabled engineered peptide boronate and alpha-ketoamide inhibitors that selectively block ROM4 (but not human rhomboids), arrest merozoite invasion, and clear parasitemia in culture [#4].\",\n  \"teleology\": [\n    {\n      \"year\": 2006,\n      \"claim\": \"It was unknown whether ROM4 was a functional protease and what it acted on; a heterologous assay established it as a rhomboid intramembrane protease with a distinct adhesin-directed substrate repertoire, defining its enzymatic identity.\",\n      \"evidence\": \"Heterologous rhomboid cleavage assay against multiple adhesins plus BAEBL/AMA1 transmembrane-domain swap mutagenesis\",\n      \"pmids\": [\"17040128\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Cleavage demonstrated in a heterologous cell system, not in the native parasite for all listed substrates\",\n        \"Which of the many candidate substrates are physiologically cleaved by ROM4 during invasion was not resolved\"\n      ],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"The subcellular site and biological necessity of ROM4 activity were unknown; localization to the merozoite plasma membrane plus a growth-lethal EBA-175 TMD cleavage mutant established that ROM4-mediated intramembrane proteolysis is essential for the life cycle.\",\n      \"evidence\": \"Immunofluorescence/fractionation localization, in vitro cleavage with EBA-175 TMD mutants, and parasite growth assays\",\n      \"pmids\": [\"17000879\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Essentiality inferred from a substrate-side (EBA-175 TMD) mutation rather than direct ROM4 knockout\",\n        \"Timing of cleavage relative to invasion steps not precisely mapped\"\n      ],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Whether a ROM4-type transmembrane cleavage occurs on Rh-family adhesins during native invasion was open; PfRh2a was found to be cleaved in its transmembrane region with ectodomain release, consistent with ROM4 activity.\",\n      \"evidence\": \"Proteolytic cleavage analysis of PfRh2a in invasion assays, with attribution to ROM4 by TMD cleavage-site inference\",\n      \"pmids\": [\"21698217\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Attribution to ROM4 is biochemical inference, not direct enzymatic reconstitution or mutagenesis in this study\",\n        \"Functional consequence of Rh2a cleavage for invasion not established here\"\n      ],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Direct genetic evidence for ROM4 essentiality was lacking; a systematic rhomboid deletion screen in P. berghei found the gene refractory to deletion, confirming an essential role in asexual blood-stage development independent of substrate-side mutations.\",\n      \"evidence\": \"Systematic loss-of-function gene-deletion screen across all eight rhomboids in P. berghei\",\n      \"pmids\": [\"23490234\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Refractoriness to deletion does not itself define the essential substrate or step\",\n        \"Shown in P. berghei; direct knockout essentiality in P. falciparum not established here\"\n      ],\n      \"attribution\": [{\"species\": \"Plasmodium berghei\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"The structural basis of ROM4's parasite-selective specificity and its druggability were unknown; a 'steric exclusion' mechanism was decoded and exploited to build selective inhibitors that block invasion, validating ROM4 as an antimalarial target.\",\n      \"evidence\": \"Substrate engineering with in vitro proteolysis, high-resolution structural analysis, compound screening, and inhibitor treatment of blood-stage cultures with ultrastructural readout\",\n      \"pmids\": [\"32888502\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"In vivo efficacy of the inhibitors beyond culture not addressed\",\n        \"Resistance liability and off-target profile across the parasite proteome not defined\"\n      ],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The precise set of native, physiologically-cleaved ROM4 substrates during invasion and the consequences of a direct P. falciparum ROM4 knockout remain to be defined.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"No direct conditional ROM4 knockout in P. falciparum reported in the corpus\",\n        \"The in vivo hierarchy among the many candidate adhesin substrates is unresolved\"\n      ],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0004252\", \"supporting_discovery_ids\": [0, 1, 4]},\n      {\"term_id\": \"GO:0004175\", \"supporting_discovery_ids\": [0, 1, 2, 4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [1]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0044409\", \"supporting_discovery_ids\": [0, 1, 4]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"EBA-175 (substrate)\",\n      \"TRAP (substrate)\",\n      \"CTRP (substrate)\",\n      \"MTRAP (substrate)\",\n      \"PFF0800c (substrate)\",\n      \"BAEBL (substrate)\",\n      \"JESEBL (substrate)\",\n      \"MAEBL (substrate)\",\n      \"Rh1 (substrate)\",\n      \"Rh2a (substrate)\",\n      \"Rh2b (substrate)\",\n      \"Rh4 (substrate)\"\n    ],\n    \"other_free_text\": [\n      \"Rhomboid intramembrane serine protease cleaving adhesins within their transmembrane domains [#0, #1].\",\n      \"Substrate specificity is encoded in the adhesin transmembrane domain, demonstrated by BAEBL/AMA1 TMD swaps that reassign ROM4 vs ROM1 preference [#0].\",\n      \"'Steric exclusion' mechanism: juxtamembrane residues mutually exclude ROM4 and canonical rhomboids from each other's substrates, conferring parasite-selective specificity [#4].\",\n      \"Essential for asexual blood-stage development and erythrocyte invasion; refractory to deletion in P. berghei [#3] and growth-lethal when EBA-175 TMD cleavage is abolished in P. falciparum [#1].\",\n      \"Selectively inhibited by engineered peptide boronates and an alpha-ketoamide that block merozoite invasion and clear parasitemia in culture [#4].\"\n    ]\n  }\n}\n```","audit_flag":null,"strains":[{"strain":"Pf3D7","gene_id":"PF3D7_0506900","name":null,"product":"rhomboid protease ROM4","uniprot_acc":null,"chromosome":null,"start_pos":null},{"strain":"PbANKA","gene_id":"PBANKA_1106500","name":null,"product":"rhomboid protease ROM4","uniprot_acc":null,"chromosome":null,"start_pos":null},{"strain":"PkH","gene_id":"PKNH_1026700","name":null,"product":"rhomboid protease ROM4, putative","uniprot_acc":null,"chromosome":null,"start_pos":null}],"gene_symbol":"ROM4","evaluation":null}