{"gene":"PF3D7_0507500","run_date":"2026-09-07T10:55:52","timeline":{"discoveries":[{"year":1998,"finding":"SUB1 (PfSUB-1) is expressed in asexual blood stages of P. falciparum and the primary gene product undergoes post-translational processing during secretory transport; the putative mature protease (p47) accumulates in dense granules (secretory organelles) at the apical end of the merozoite, and is subsequently secreted in a truncated, soluble form following merozoite release and erythrocyte invasion.","method":"Western blot, immunofluorescence, subcellular fractionation, electron microscopy","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct localization by IFA and EM with biochemical fractionation, replicated in subsequent studies","pmids":["9722575"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2000,"finding":"SUB1 undergoes two autocatalytic processing steps: the primary translation product (82 kDa precursor) is first cleaved in the parasite endoplasmic reticulum to a 54 kDa form (p54); p54 then undergoes calcium-dependent intramolecular autocatalytic cleavage to produce the 47 kDa form (p47), with cleavage occurring between Asp251 and Ala252. Mutation of the predicted active-site serine abolished processing, confirming the enzyme is a serine protease. p54 is secreted in a non-covalent complex with its prodomain (p31).","method":"In vitro translation, baculovirus recombinant expression, site-directed mutagenesis, cross-linking, in vitro cleavage assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with active-site mutagenesis, replicated across multiple studies","pmids":["10617661"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2002,"finding":"Recombinant SUB1 (p54) requires high calcium for optimum activity, has an alkaline pH optimum, and displays a relaxed P1 substrate specificity (unable to efficiently cleave substrates with P1 leucine). Replacement of P4 valine with lysine abolished cleavage. Recombinant enzyme is indistinguishable from the parasite-derived enzyme. Homology modeling suggests at least three calcium ion-binding sites, three intramolecular disulfide bridges, and a single free cysteine in the S1 pocket.","method":"Recombinant protein expression and purification, enzyme kinetics with decapeptide and protein substrates, 3D homology modeling","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro biochemical characterization with multiple substrates and mutagenesis, replicated across labs","pmids":["12052828"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2002,"finding":"Autocatalytic activation of SUB1 involves cleavage of an Asp-Asn bond within the internal sequence motif (215)LVSADNIDIS(224). A synthetic decapeptide based on this cleavage site is efficiently cleaved by recombinant SUB1, enabling a fluorogenic rhodamine-based continuous assay of SUB1 proteolytic activity with ~30-fold fluorescence increase upon cleavage.","method":"Fluorogenic peptide substrate assay, NMR, time-resolved fluorescence measurements","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with defined substrate and biochemical characterization, single lab","pmids":["12356327"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2003,"finding":"The SUB1 propeptide (p31) possesses significant secondary structure and functions as a fast-binding, high-affinity inhibitor of the SUB1 catalytic domain. Inhibition of two bacterial subtilisins by p31 was 49–60-fold less effective, demonstrating specificity. Truncation of 11 residues from the p31 C-terminus substantially reduced but did not abolish inhibitory activity.","method":"Circular dichroism, enzyme kinetics (inhibition constants), truncation and point mutants of p31","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with mutagenesis and kinetic characterization, single lab","pmids":["12764150"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2005,"finding":"SUB1 contains six surface-associated insertion loops conserved in all Plasmodium SUB1 orthologues but absent from other subtilases. Individual deletion of loops I, II, IV, V, and VI impaired protein maturation and/or stability; loop III was dispensable. Specific substitutions within loop II inhibited both maturation and enzyme activity. Mutations in loops V and VI specifically blocked the second autocatalytic maturation step (p54→p47), establishing that the two processing steps have distinct structural requirements and that p47 conversion is not a prerequisite for trans-proteolytic activity.","method":"Deletion/substitution mutagenesis, insect cell expression system, Western blot analysis of processing","journal":"Molecular and biochemical parasitology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — systematic mutagenesis with functional readouts, single lab with multiple orthogonal deletion mutants","pmids":["16183148"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2007,"finding":"Just prior to egress, SUB1 is discharged from a previously unrecognized parasite organelle called the exoneme into the parasitophorous vacuole space. In the PV, SUB1 mediates proteolytic maturation of at least two members of the SERA family. Pharmacological blockade of SUB1 inhibits egress and abolishes the invasive capacity of released merozoites.","method":"Immunofluorescence, electron microscopy, pharmacological inhibition with protease inhibitors, Western blot for SERA processing","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct organelle localization by EM and IFA, substrate cleavage assay, functional pharmacological inhibition, replicated by subsequent studies","pmids":["18083098"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2008,"finding":"Using a chemical genetic screen with covalent serine/cysteine protease inhibitor library, SUB1 was identified as a primary regulator of erythrocyte rupture. Inhibition of SUB1 caused a block in proteolytic processing of SERA5 that correlated with the block in rupture. Additionally, DPAP3 inhibition reduced levels of mature SUB1, suggesting DPAP3 acts upstream of SUB1 maturation (though this role for DPAP3 was later refuted by genetic studies).","method":"Chemical genetic screen, protease inhibitor library, Western blot for SERA5 processing, parasite culture rupture assay","journal":"Nature chemical biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — chemical genetic screen identifying SUB1 as a rupture regulator, pharmacological evidence for SERA5 processing; DPAP3 upstream role was later disproven genetically","pmids":["18246061"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2009,"finding":"SUB1 has a further role in priming the merozoite for invasion by proteolytically maturing the MSP1/MSP6/MSP7 surface protein complex just before egress. Inhibition of SUB1 results in accumulation of unprocessed MSPs on the merozoite surface and significantly reduced erythrocyte invasion, demonstrating that SUB1 is a multifunctional processing protease essential for both egress and merozoite surface remodelling.","method":"Pharmacological SUB1 inhibition, Western blot for MSP1/MSP6/MSP7 processing, erythrocyte invasion assay","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct substrate identification with functional invasion assay, replicated across multiple studies","pmids":["19214190"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2010,"finding":"SUB1 mediates primary processing of MSP1 prior to egress; primary processing is ordered with the site closest to the C-terminal end of MSP1 cleaved last. Replacement of the secondary processing site (normally refractory to SUB1) with a SUB1-sensitive site is deleterious to parasite growth, demonstrating that correct spatiotemporal regulation of MSP1 maturation by SUB1 (and SUB2) is crucial for parasite viability.","method":"Conditional expression system, site-directed mutagenesis of MSP1 processing sites, parasite growth assays, Western blot","journal":"Molecular microbiology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — genetic manipulation of substrate cleavage site with functional growth readout, single lab","pmids":["20735778"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2011,"finding":"Proteomic and bioinformatic analysis of the late asexual blood stage proteome identified multiple previously unknown SUB1 substrates including RAP1 (rhoptry-associated protein 1) and MSRP2 (MSP7-related protein 2), establishing that SUB1 processes a broad range of merozoite, PV, and PVM proteins.","method":"Mass spectrometry-based proteomics, bioinformatic substrate motif analysis, Western blot validation","journal":"Infection and immunity","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MS-based substrate identification with validation, single lab","pmids":["21220481"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2012,"finding":"SUB1 directly cleaves SERA6 in the parasitophorous vacuole just prior to egress, converting it to an active cysteine protease. Mutations replacing the predicted catalytic Cys of SERA6, or blocking SERA6 processing by SUB1, could not be stably introduced into the parasite genomic locus, indicating SERA6 is essential and that processing by SUB1 is required for its function.","method":"Conditional mutagenesis, Western blot for SERA6 processing, in vitro enzyme activity assay, attempted genomic locus replacement","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic essentiality confirmed, direct SUB1 cleavage shown biochemically, activation of cysteine protease confirmed, replicated","pmids":["22984267"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2012,"finding":"Intracellular calcium elevation just before egress is required for SUB1 discharge from exonemes into the parasitophorous vacuole. Chelation of intracellular Ca2+ with BAPTA-AM, or inhibition of Ca2+ release from intracellular stores with a PLC inhibitor, blocks SUB1 discharge, inhibits SERA5 processing, and prevents PVM rupture and merozoite egress.","method":"Time-lapse video microscopy, Ca2+ chelators and PLC inhibitor pharmacology, Western blot for SUB1 discharge and SERA5 processing, immunofluorescence","journal":"Cellular microbiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pharmacological dissection with functional readouts, single lab","pmids":["23217145"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2013,"finding":"PfPKG (cGMP-dependent protein kinase) activity is required upstream of SUB1 discharge into the PV. Inhibition of PfPKG prevents SUB1 discharge, while stimulation of PfPKG by inhibiting parasite phosphodiesterase induces premature SUB1 discharge and egress of developmentally immature, non-invasive parasites.","method":"PfPKG inhibitor compound 2 (C2) treatment, phosphodiesterase inhibitor stimulation, Western blot for SUB1 discharge, live microscopy","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 2 / Strong — pharmacological epistasis with gain-of-function and loss-of-function, replicated across studies","pmids":["23675297"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2013,"finding":"Using conditional mutagenesis in P. berghei, SUB1 was shown to be essential at the hepatic stage. SUB1-deficient parasites failed to rupture the parasitophorous vacuole membrane and egress from hepatocytes. Mechanically released SUB1-null parasites were not adequately primed and failed to establish blood stage infection in vivo.","method":"Conditional mutagenesis (stage-specific sub1 invalidation), intravital microscopy, in vivo mouse infection","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional genetic knockout with specific PVM rupture phenotype and in vivo validation","pmids":["24089525"],"is_preprint":false,"attribution":[{"species":"Plasmodium berghei","strain":""}]},{"year":2013,"finding":"Using P. berghei conditional mutagenesis and Flp recombinase deleters, SUB1 was shown to be essential for complete development of the liver stage schizont and production of hepatic merozoites, but not required for early intrahepatic growth.","method":"Conditional mutagenesis with Flp/FRT system, P. berghei rodent malaria model, liver stage phenotypic analysis","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional genetic knockout with stage-specific phenotypic analysis in rodent model","pmids":["24348254"],"is_preprint":false,"attribution":[{"species":"Plasmodium berghei","strain":""}]},{"year":2014,"finding":"Crystal structure of P. falciparum SUB1 at 2.25 Å in complex with its cognate prodomain revealed: (1) the structural basis for calcium dependence; (2) an unusual requirement to interact with substrate residues on both prime and non-prime sides of the scissile bond; (3) a solvent-exposed redox-sensitive disulfide bridge unique among subtilisins that likely acts as a regulator of protease activity.","method":"X-ray crystallography at 2.25 Å resolution, functional validation of calcium dependence and disulfide bridge","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — high-resolution crystal structure with functional validation, later replicated by independent structural studies","pmids":["24785947"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2014,"finding":"Crystal structure of full-length SUB1 from P. vivax revealed a bacterial-like catalytic domain in complex with a Plasmodium-specific prodomain that has a novel 'belt' architecture: an N-terminal insertion that embraces the catalytic domain to stabilize the pre-protease and undergoes calcium-dependent autoprocessing during activation. The 'belt' domain is dispensable for recombinant enzymatic activity but could not be deleted in P. berghei in vivo, indicating it is essential for parasite development.","method":"X-ray crystallography of PvSUB1, deletion mutagenesis in P. berghei, in vitro enzyme activity assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with in vivo genetic validation in rodent malaria model","pmids":["25204226"],"is_preprint":false,"attribution":[{"species":"Plasmodium vivax","strain":""},{"species":"Plasmodium berghei","strain":""}]},{"year":2015,"finding":"SUB1-mediated processing of MSP1 is important for parasite viability; processing modifies the secondary structure of MSP1 and activates its capacity to bind spectrin, the major component of the erythrocyte cytoskeleton. Parasites expressing an inefficiently processed MSP1 mutant show delayed egress, and merozoites lacking surface-bound MSP1 display a severe egress defect.","method":"Conditional mutagenesis, MSP1 mutant parasite lines, spectrin-binding assays, egress assays, electron microscopy","journal":"Cell host & microbe","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic manipulation of SUB1 cleavage site in MSP1 with multiple orthogonal functional readouts","pmids":["26468747"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2017,"finding":"Plasmepsin X (PMX) is essential for maturation of SUB1 in exoneme secretory vesicles; pharmacological inhibition of PMX prevents SUB1 maturation and consequently blocks both egress and invasion.","method":"Conditional knockdown of PMX, Western blot for SUB1 maturation, PMX inhibitor treatment, egress/invasion assays","journal":"Science (New York, N.Y.)","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional genetic knockdown with direct molecular readout of SUB1 processing, replicated by multiple subsequent studies","pmids":["29074774"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2018,"finding":"P. falciparum parasites lacking SUB1 undergo none of the morphological transformations that precede egress and fail to rupture the PVM. In contrast, SERA6-null parasites rupture the PVM normally but fail to rupture the RBC membrane. Complementation shows SERA6 is an enzyme requiring processing by SUB1 to function. RBCM rupture is associated with SERA6-dependent proteolytic cleavage of β-spectrin within its actin-binding domain. This establishes SUB1 and SERA6 as playing distinct, essential roles in a coordinated proteolytic cascade enabling sequential membrane rupture.","method":"Conditional knockout (SUB1-null and SERA6-null parasites), live microscopy, electron microscopy, complementation studies, Western blot for β-spectrin cleavage","journal":"Nature microbiology","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional genetic knockouts with precise phenotypic dissection, complementation, and molecular substrate identification","pmids":["29459732"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2018,"finding":"Live-cell microscopy with PV-targeted fluorescent proteins showed that abrupt rupture of the PV membrane and release of vacuolar contents are under control of PfPKG and SUB1, occurring ~1.5 minutes after the PV begins rounding (which is under control of parasite [Ca2+]i). This temporally positions SUB1 action downstream of PfPKG activation.","method":"Live-cell fluorescence microscopy with PV-targeted fluorescent proteins, specific egress inhibitors (PKG inhibitor, SUB1 inhibitor)","journal":"Cellular microbiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — live imaging with specific inhibitors, temporal resolution of pathway steps, single lab","pmids":["29900649"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2019,"finding":"In P. berghei, SUB1 is selectively expressed in mature male gametocytes, localizes to male osmiophilic bodies (secretory organelles involved in gamete egress), and is required for male gamete egress. The PbSUB1 substrate PbSERA3 is expressed in gametocytes and processed by PbSUB1 upon gametocyte activation.","method":"Immunofluorescence, electron microscopy, transgenic P. berghei lines deficient in PbSUB1 expression or enzyme activity, Western blot for PbSERA3 processing","journal":"Cellular microbiology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — genetic loss-of-function with specific phenotype, substrate processing confirmed, single lab","pmids":["30941868"],"is_preprint":false,"attribution":[{"species":"Plasmodium berghei","strain":""}]},{"year":2020,"finding":"The ER-resident CREC family protein PfERC is required for proteolytic maturation of SUB1. PfERC knockdown prevents proteolytic maturation of plasmepsin X, which is required for SUB1 cleavage. Knockdown of PfERC inhibits PVM rupture and downstream substrate processing, placing PfERC as an early regulator in the egress proteolytic cascade upstream of PMX and SUB1.","method":"Conditional knockdown of PfERC, double mutant parasites, Western blot for PMX and SUB1 maturation, egress assays","journal":"mBio","confidence":"High","confidence_rationale":"Tier 2 / Moderate — conditional knockdown with epistasis analysis using double mutants, multiple molecular readouts","pmids":["32098818"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2020,"finding":"SUB1 inhibitor profiling of P. falciparum parasites expressing conditionally reduced levels of PfSUB1 confirmed that inhibition of SUB1 activity is the mechanism by which peptidic boronic acid compounds (developed as SUB1 inhibitors) block parasite egress and replication.","method":"Conditionally regulated PfSUB1 parasite lines, peptidic boronic acid inhibitors, egress assays, parasite replication assays","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic validation using hypomorphic SUB1 lines with inhibitor, single lab","pmids":["33975947"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2021,"finding":"Activation of SERA6 involves a second autocatalytic step triggered by SUB1 cleavage, which requires interaction in multimolecular complexes with the PV-located protein cofactor MSA180—itself a SUB1 substrate. Genetic ablation of MSA180 mimics SERA6 disruption, producing a fatal block in β-spectrin cleavage and RBC rupture.","method":"Conditional genetic knockout of MSA180, Co-IP/pulldown for MSA180-SERA6 complex, Western blot for SERA6 maturation and β-spectrin cleavage, egress assays in P. falciparum and P. knowlesi","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis, complex identification, and functional assay across two Plasmodium species","pmids":["33932049"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""},{"species":"Plasmodium knowlesi","strain":""}]},{"year":2020,"finding":"A stand-alone propeptide-like protein, SUB1-ProM, is expressed in the parasitophorous vacuole of blood-stage P. falciparum where it acts as a fast-binding, potent inhibitor of SUB1 but not of SUB2 or other proteases examined, suggesting it may function as an endogenous regulator of SUB1 activity in the PV.","method":"Recombinant protein expression, enzyme inhibition kinetics, mass spectrometry, immunofluorescence localization","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — recombinant inhibition assays with localization, single lab; endogenous regulatory role inferred but not directly tested genetically","pmids":["31942933"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2023,"finding":"PMX activates SUB1 by directly cleaving both p31 (the inhibitory prodomain) and p54 (the intermediate catalytic domain). PMX cleavage sites on p31 were mapped by mass spectrometry. Conversion of p54 to p47 can be effected by cleavage at either SUB1 or PMX cleavage sites that are adjacent to one another. Once p31 is removed, p54 is fully functional inside parasites, establishing that conversion to p47 is dispensable for SUB1 activity. This reveals a novel mechanism for subtilisin activation in which relief of propiece inhibition is achieved via a heterologous protease.","method":"PMX knockdown in parasites, recombinant PMX and SUB1 in vitro cleavage assay, mass spectrometry mapping of cleavage sites, Western blot, parasite functional assays","journal":"mBio","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution of PMX-mediated SUB1 activation combined with parasite genetics; cleavage sites mapped; multiple orthogonal methods","pmids":["37036362"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2024,"finding":"Crystal structure of full-length P. falciparum SUB1 demonstrated that the atypical Plasmodium-specific SUB1 prodomain (PD) directly promotes assembly of inactive enzyme homodimers at acidic pH, whereas SUB1 is primarily monomeric at neutral pH. This establishes a pH-dependent autoinhibition mechanism: the PD-driven dimeric form keeps SUB1 inactive in acidic exonemes, while PMX processing and subsequent exposure to neutral pH in the PV allows monomeric, active SUB1.","method":"X-ray crystallography of full-length PfSUB1, analytical ultracentrifugation, biophysical measurements of pH-dependent dimerization, biochemical activity assays","journal":"mBio","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure combined with biophysical and biochemical validation of pH-dependent dimerization mechanism","pmids":["38386597"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2024,"finding":"Biochemical and enzymatic analysis confirmed that both p31 and p31-p54 are largely dimeric under acidic conditions (as they would be exposed to PMX), and that sequential PMX cleavage of p31 results in rapid loss of the capacity of p31 to inhibit SUB1 catalytic activity. Direct exposure of recombinant p31-p54 complex to PMX activates SUB1 activity, confirming PMX-mediated prodomain cleavage as the activation mechanism.","method":"Analytical ultracentrifugation, enzyme kinetics, mass spectrometry mapping of PMX cleavage sites, in vitro SUB1 activity assay after PMX treatment","journal":"Biochimica et biophysica acta. General subjects","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with defined cleavage-site mapping and direct activation demonstrated, single lab","pmids":["38969256"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2023,"finding":"Crystal structures of P. vivax SUB1 catalytic domain (PvS1Cat), alone and in complex with the α-ketoamide inhibitor MAM-117, showed that the catalytic serine of SUB1 forms a covalent bond with the α-keto group of the inhibitor. The catalytic groove of SUB1 underwent significant structural changes when bound to the peptidomimetic inhibitor, particularly in the S4 pocket, and P1'/P2' residues of the inhibitor contribute important stabilizing interactions.","method":"X-ray crystallography of PvSUB1 catalytic domain in apo and inhibitor-bound states","journal":"Acta crystallographica. Section D, Structural biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — high-resolution crystal structures of covalent enzyme-inhibitor complex revealing structural basis of inhibition, single lab","pmids":["37428845"],"is_preprint":false,"attribution":[{"species":"Plasmodium vivax","strain":""}]},{"year":2024,"finding":"Crystal structures of PvSUB1 in complex with α-ketoamide inhibitor compound 8 revealed a covalent bond at the catalytic serine and extended interactions to P5, with two additional hydrogen bonds compared to the reference compound. Structure-activity relationships established that hydrophobicity improvements at multiple positions enhanced PfSUB1 inhibition (IC50 values down to 10–12 nM for PvSUB1 and PfSUB1).","method":"X-ray crystallography, enzyme inhibition assays (IC50), parasite egress assays","journal":"European journal of medicinal chemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — crystal structure with inhibitor and functional enzyme assays, single lab","pmids":["38503166"],"is_preprint":false,"attribution":[{"species":"Plasmodium vivax","strain":""},{"species":"Plasmodium falciparum","strain":""}]},{"year":2018,"finding":"Plasmepsin X inhibitors (peptidomimetic compounds) inhibit maturation of SUB1 and also inhibit parasite egress from erythrocytes, further supporting that PMX-mediated maturation of SUB1 is the mechanism of antimalarial action of PMX inhibitors.","method":"Plasmepsin inhibitor treatment of P. falciparum cultures, Western blot for SUB1 maturation, egress assay","journal":"European journal of medicinal chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pharmacological evidence linking PMX inhibition to SUB1 maturation block, consistent with genetic studies","pmids":["30529637"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]}],"current_model":"SUB1 (PF3D7_0507500) is an essential subtilisin-like serine protease that is synthesized as an 82 kDa precursor, undergoes autocatalytic cleavage in the ER to a p54 form non-covalently associated with its inhibitory prodomain (p31), and is then stored in specialized secretory organelles called exonemes in an inactive, pH-dependent homodimeric state; upon activation—triggered by PfPKG/Ca2+ signaling and mediated by the aspartic protease plasmepsin X (PMX), which cleaves and destroys the inhibitory p31 prodomain—monomeric active SUB1 (p47) is discharged into the parasitophorous vacuole where it acts as a master processing protease that cleaves multiple substrates including SERA5, SERA6 (activating it as a cysteine protease to drive RBC membrane rupture via β-spectrin cleavage), MSP1/MSP6/MSP7 (remodelling the merozoite surface for invasion), RAP1, MSRP2, and MSA180, thereby orchestrating sequential rupture of the PVM and RBC membrane for merozoite egress; SUB1 also plays essential roles in hepatic stage egress and male gamete egress in Plasmodium berghei."},"narrative":{"mechanistic_narrative":"SUB1 (PF3D7_0507500) is an essential Plasmodium subtilisin-like serine protease that acts as the master trigger of a proteolytic cascade governing merozoite egress and invasion in asexual blood stages [PMID:18083098, PMID:19214190]. The enzyme is synthesized as an 82 kDa precursor that undergoes autocatalytic maturation during secretory transport—first cleaved in the ER to a p54 form and then, via calcium-dependent intramolecular autocatalysis between Asp251 and Ala252, to p47—with mutation of the catalytic serine abolishing all processing, confirming its serine-protease mechanism [PMID:10617661, PMID:12052828]. Mature enzyme is stored in specialized apical secretory organelles termed exonemes and is discharged into the parasitophorous vacuole (PV) just before egress [PMID:9722575, PMID:18083098]. Discharge is controlled by an upstream signaling axis in which PfPKG activity and a Ca2+/PLC-dependent step are both required to release SUB1 into the PV [PMID:23217145, PMID:23675297, PMID:29900649]. Within exonemes SUB1 is held inactive: its Plasmodium-specific prodomain (p31) is a fast-binding, high-affinity inhibitor that drives assembly of inactive homodimers at acidic pH, and activation is achieved not by autoprocessing but by the aspartic protease plasmepsin X (PMX), which directly cleaves and inactivates the inhibitory prodomain, liberating active enzyme upon exposure to neutral PV pH [PMID:12764150, PMID:37036362, PMID:38386597]. Once active, SUB1 processes a broad substrate repertoire: it matures SERA5 and activates SERA6 as a cysteine protease that cleaves β-spectrin to rupture the RBC membrane, and it remodels the merozoite surface by cleaving the MSP1/MSP6/MSP7 complex (enabling MSP1 spectrin binding) as well as RAP1, MSRP2 and MSA180 [PMID:18083098, PMID:19214190, PMID:21220481, PMID:22984267, PMID:26468747, PMID:29459732, PMID:33932049]. SUB1-null parasites fail the morphological changes preceding egress and cannot rupture the PVM, establishing SUB1 and its downstream substrate SERA6 as sequential, non-redundant effectors of PVM then RBC-membrane rupture [PMID:29459732]. Beyond the blood stage, SUB1 is essential in Plasmodium berghei for hepatic-stage schizont maturation and PVM rupture and for male gamete egress from osmiophilic bodies [PMID:24089525, PMID:24348254, PMID:30941868]. Crystal structures of P. falciparum and P. vivax SUB1 define its calcium-dependent bacterial-like catalytic domain, its unusual prodomain 'belt' architecture, a redox-sensitive surface disulfide, and the covalent binding mode of α-ketoamide inhibitors, making SUB1 a validated antimalarial drug target [PMID:24785947, PMID:25204226, PMID:38386597, PMID:37428845].","teleology":[{"year":1998,"claim":"Established that SUB1 is an asexual blood-stage protease that is post-translationally processed during secretory transport and stored apically, framing it as a regulated secretory enzyme rather than a constitutive one.","evidence":"Western blot, immunofluorescence, subcellular fractionation and EM in P. falciparum","pmids":["9722575"],"confidence":"High","gaps":["Function of the secreted enzyme not yet defined","Substrates unknown","Trigger for secretion not identified"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2000,"claim":"Defined the two-step autocatalytic maturation pathway and proved SUB1 is a serine protease, explaining how the precursor becomes an active enzyme.","evidence":"In vitro translation, recombinant expression, active-site mutagenesis and cleavage mapping (Asp251/Ala252) in P. falciparum","pmids":["10617661"],"confidence":"High","gaps":["In vivo trigger for calcium-dependent second cleavage not established","Physiological substrates still unknown"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2002,"claim":"Characterized SUB1 enzymology—calcium dependence, alkaline pH optimum, and P4/P1 substrate preferences—providing the specificity rules needed to predict natural substrates and design inhibitors.","evidence":"Recombinant enzyme kinetics with peptide and protein substrates, fluorogenic assay development, and homology modeling in P. falciparum","pmids":["12052828","12356327"],"confidence":"High","gaps":["Natural substrates in the parasite not yet identified","Structural basis of specificity inferred from modeling only"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2003,"claim":"Showed the prodomain (p31) is a specific high-affinity fast-binding inhibitor, revealing how SUB1 is kept latent before deployment.","evidence":"Circular dichroism, inhibition kinetics, and truncation/point mutants of p31 in P. falciparum","pmids":["12764150"],"confidence":"High","gaps":["How prodomain inhibition is relieved in vivo not known","Structural basis of prodomain binding not resolved"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2005,"claim":"Identified Plasmodium-specific surface loops required for maturation and activity and showed the two processing steps have distinct structural requirements, indicating p47 conversion is not required for trans-proteolytic activity.","evidence":"Systematic deletion/substitution mutagenesis with insect-cell expression and processing readouts in P. falciparum","pmids":["16183148"],"confidence":"High","gaps":["Single expression system","In vivo relevance of loop functions not tested genetically"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2007,"claim":"Discovered exonemes and placed SUB1 at the top of an egress pathway—discharged into the PV where it matures SERA-family proteins and enables merozoite egress and invasion.","evidence":"IFA/EM organelle localization, SERA processing Western blots, and pharmacological inhibition of egress in P. falciparum","pmids":["18083098"],"confidence":"High","gaps":["Signal driving exoneme discharge not defined","Full substrate repertoire unknown"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2008,"claim":"Chemical genetics confirmed SUB1 as a primary regulator of erythrocyte rupture via SERA5 processing; a proposed upstream role for DPAP3 in SUB1 maturation was later refuted genetically.","evidence":"Covalent protease inhibitor library screen with rupture and SERA5 processing readouts in P. falciparum","pmids":["18246061"],"confidence":"Medium","gaps":["Pharmacological, not genetic, evidence","DPAP3 upstream claim disproven by later work"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2009,"claim":"Extended SUB1's role from egress to invasion by showing it matures the MSP1/MSP6/MSP7 surface complex, establishing SUB1 as a multifunctional processing protease.","evidence":"Pharmacological inhibition with MSP processing and invasion assays in P. falciparum","pmids":["19214190"],"confidence":"High","gaps":["Functional consequence of MSP maturation not yet mechanistically defined","Reliance on inhibitors"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2010,"claim":"Demonstrated that ordered, spatiotemporally correct MSP1 processing by SUB1 is required for viability, showing that miscued cleavage is deleterious.","evidence":"Conditional expression and MSP1 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falciparum","pmids":["22984267"],"confidence":"High","gaps":["SERA6 downstream target not yet identified at this stage"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2012,"claim":"Placed intracellular Ca2+/PLC signaling immediately upstream of SUB1 discharge, connecting a second messenger to the proteolytic egress program.","evidence":"Time-lapse microscopy with BAPTA-AM and PLC inhibitor and discharge/processing readouts in P. falciparum","pmids":["23217145"],"confidence":"Medium","gaps":["Molecular Ca2+ effector for discharge not identified","Pharmacological evidence only"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2013,"claim":"Established PfPKG as an upstream master switch whose activity is necessary and, when forced, sufficient to drive premature SUB1 discharge and egress.","evidence":"PfPKG inhibitor (C2) and phosphodiesterase-inhibitor gain-of-function with discharge readouts and live microscopy in P. falciparum","pmids":["23675297"],"confidence":"High","gaps":["Direct PfPKG substrates linking to discharge not defined"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2013,"claim":"Genetic knockout in P. berghei proved SUB1 is essential across the life cycle—required for hepatic-stage PVM rupture, schizont maturation and merozoite priming, not merely a blood-stage enzyme.","evidence":"Conditional/Flp-FRT mutagenesis with intravital microscopy and in vivo infection in P. berghei","pmids":["24089525","24348254"],"confidence":"High","gaps":["Hepatic-stage substrates not defined","Cross-species substrate conservation not fully mapped"],"attribution":[{"species":"Plasmodium berghei","strain":""}]},{"year":2014,"claim":"Crystal structures of P. falciparum and P. vivax SUB1 provided the structural basis for calcium dependence, the bacterial-like catalytic fold, a redox-sensitive disulfide, and the essential Plasmodium-specific prodomain 'belt' architecture.","evidence":"X-ray crystallography with functional validation, plus belt-deletion mutagenesis in P. berghei","pmids":["24785947","25204226"],"confidence":"High","gaps":["Structure did not resolve the in vivo activation trigger","Oligomeric state not addressed"],"attribution":[{"species":"Plasmodium falciparum","strain":""},{"species":"Plasmodium vivax","strain":""},{"species":"Plasmodium berghei","strain":""}]},{"year":2015,"claim":"Linked SUB1-mediated MSP1 maturation to a concrete molecular function—activating MSP1 spectrin binding—and to egress kinetics, explaining why correct MSP1 processing matters.","evidence":"MSP1 processing-site mutant lines, spectrin-binding, egress assays and EM in P. falciparum","pmids":["26468747"],"confidence":"High","gaps":["Precise contribution of MSP1-spectrin binding to membrane destabilization not fully resolved"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2017,"claim":"Identified plasmepsin X as essential for SUB1 maturation, revealing a heterologous protease acting upstream of SUB1 in exonemes.","evidence":"PMX conditional knockdown and inhibitor treatment with SUB1 maturation Westerns and egress/invasion assays in P. falciparum","pmids":["29074774"],"confidence":"High","gaps":["Molecular mechanism of PMX action on SUB1 not yet defined at this stage"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2018,"claim":"Conditional knockouts dissected the cascade into sequential membrane-rupture events: SUB1-null parasites fail PVM rupture, while SERA6 (activated by SUB1) cleaves β-spectrin to rupture the RBC membrane.","evidence":"SUB1-null and SERA6-null lines with live/EM imaging, complementation and β-spectrin cleavage Westerns in P. falciparum; live PV-marker imaging positioning SUB1 downstream of PfPKG","pmids":["29459732","29900649"],"confidence":"High","gaps":["How SUB1 itself contributes to PVM rupture mechanistically not fully 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falciparum","strain":""}]},{"year":2020,"claim":"Identified endogenous SUB1 inhibitors—SUB1-ProM in the PV—suggesting native regulation of active SUB1 beyond prodomain removal.","evidence":"Recombinant inhibition kinetics, mass spectrometry and localization in P. falciparum; also boronic-acid inhibitor validation using conditional SUB1 lines","pmids":["31942933","33975947"],"confidence":"Medium","gaps":["Endogenous regulatory role of SUB1-ProM inferred, not tested genetically","Boronic acid on-target validation single lab"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2021,"claim":"Revealed that SERA6 activation requires assembly with the SUB1 substrate MSA180 into multimolecular complexes, adding a cofactor layer to the terminal RBC-rupture step.","evidence":"MSA180 conditional knockout, Co-IP, SERA6/β-spectrin cleavage and egress assays in P. falciparum and P. knowlesi","pmids":["33932049"],"confidence":"High","gaps":["Structural basis of the SERA6-MSA180 complex not resolved"],"attribution":[{"species":"Plasmodium falciparum","strain":""},{"species":"Plasmodium knowlesi","strain":""}]},{"year":2023,"claim":"Defined the molecular activation mechanism: PMX directly cleaves the inhibitory p31 prodomain (and p54), relieving propeptide inhibition, and showed p54→p47 conversion is dispensable for activity—a novel subtilisin activation route via a heterologous protease.","evidence":"PMX knockdown, recombinant PMX/SUB1 cleavage assays, MS site mapping and parasite functional assays in P. falciparum; also PvSUB1 covalent inhibitor structures","pmids":["37036362","37428845"],"confidence":"High","gaps":["How neutral-pH transition and PMX cleavage are temporally coupled in vivo not fully resolved"],"attribution":[{"species":"Plasmodium falciparum","strain":""},{"species":"Plasmodium vivax","strain":""}]},{"year":2024,"claim":"Completed the autoinhibition model: the full-length structure and biophysics show the prodomain drives inactive homodimers 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exoneme discharge unresolved","Functional roles of many identified SUB1 substrates untested","Endogenous SUB1-ProM regulatory role not tested genetically"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":["SUB1 p54–prodomain (p31) non-covalent inhibitory complex","pH-dependent inactive SUB1 homodimer"],"partners":["PLASMEPSIN X (PMX) — ACTIVATING PROTEASE THAT CLEAVES THE SUB1 PRODOMAIN","PFPKG (CGMP-DEPENDENT PROTEIN KINASE) — UPSTREAM SIGNALING REGULATOR OF SUB1 DISCHARGE","PFERC — ER CREC-FAMILY PROTEIN REQUIRED UPSTREAM OF PMX/SUB1 MATURATION","SUB1-PROM — ENDOGENOUS PV PRODOMAIN-LIKE INHIBITOR OF SUB1","SERA5, SERA6 (AND PBSERA3) — SUB1 SUBSTRATES","MSP1/MSP6/MSP7 COMPLEX — SUB1 SUBSTRATES","RAP1, MSRP2, MSA180 — SUB1 SUBSTRATES"],"other_free_text":["Synthesized as an 82 kDa precursor, autocatalytically processed to p54 (ER) then to p47 (calcium-dependent, Asp251/Ala252); 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Mutation of the predicted active-site serine abolished processing, confirming the enzyme is a serine protease. p54 is secreted in a non-covalent complex with its prodomain (p31).\",\n      \"method\": \"In vitro translation, baculovirus recombinant expression, site-directed mutagenesis, cross-linking, in vitro cleavage assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with active-site mutagenesis, replicated across multiple studies\",\n      \"pmids\": [\"10617661\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Recombinant SUB1 (p54) requires high calcium for optimum activity, has an alkaline pH optimum, and displays a relaxed P1 substrate specificity (unable to efficiently cleave substrates with P1 leucine). Replacement of P4 valine with lysine abolished cleavage. Recombinant enzyme is indistinguishable from the parasite-derived enzyme. Homology modeling suggests at least three calcium ion-binding sites, three intramolecular disulfide bridges, and a single free cysteine in the S1 pocket.\",\n      \"method\": \"Recombinant protein expression and purification, enzyme kinetics with decapeptide and protein substrates, 3D homology modeling\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro biochemical characterization with multiple substrates and mutagenesis, replicated across labs\",\n      \"pmids\": [\"12052828\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Autocatalytic activation of SUB1 involves cleavage of an Asp-Asn bond within the internal sequence motif (215)LVSADNIDIS(224). A synthetic decapeptide based on this cleavage site is efficiently cleaved by recombinant SUB1, enabling a fluorogenic rhodamine-based continuous assay of SUB1 proteolytic activity with ~30-fold fluorescence increase upon cleavage.\",\n      \"method\": \"Fluorogenic peptide substrate assay, NMR, time-resolved fluorescence measurements\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with defined substrate and biochemical characterization, single lab\",\n      \"pmids\": [\"12356327\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"The SUB1 propeptide (p31) possesses significant secondary structure and functions as a fast-binding, high-affinity inhibitor of the SUB1 catalytic domain. Inhibition of two bacterial subtilisins by p31 was 49–60-fold less effective, demonstrating specificity. Truncation of 11 residues from the p31 C-terminus substantially reduced but did not abolish inhibitory activity.\",\n      \"method\": \"Circular dichroism, enzyme kinetics (inhibition constants), truncation and point mutants of p31\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with mutagenesis and kinetic characterization, single lab\",\n      \"pmids\": [\"12764150\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"SUB1 contains six surface-associated insertion loops conserved in all Plasmodium SUB1 orthologues but absent from other subtilases. Individual deletion of loops I, II, IV, V, and VI impaired protein maturation and/or stability; loop III was dispensable. Specific substitutions within loop II inhibited both maturation and enzyme activity. Mutations in loops V and VI specifically blocked the second autocatalytic maturation step (p54→p47), establishing that the two processing steps have distinct structural requirements and that p47 conversion is not a prerequisite for trans-proteolytic activity.\",\n      \"method\": \"Deletion/substitution mutagenesis, insect cell expression system, Western blot analysis of processing\",\n      \"journal\": \"Molecular and biochemical parasitology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — systematic mutagenesis with functional readouts, single lab with multiple orthogonal deletion mutants\",\n      \"pmids\": [\"16183148\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Just prior to egress, SUB1 is discharged from a previously unrecognized parasite organelle called the exoneme into the parasitophorous vacuole space. In the PV, SUB1 mediates proteolytic maturation of at least two members of the SERA family. Pharmacological blockade of SUB1 inhibits egress and abolishes the invasive capacity of released merozoites.\",\n      \"method\": \"Immunofluorescence, electron microscopy, pharmacological inhibition with protease inhibitors, Western blot for SERA processing\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct organelle localization by EM and IFA, substrate cleavage assay, functional pharmacological inhibition, replicated by subsequent studies\",\n      \"pmids\": [\"18083098\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Using a chemical genetic screen with covalent serine/cysteine protease inhibitor library, SUB1 was identified as a primary regulator of erythrocyte rupture. Inhibition of SUB1 caused a block in proteolytic processing of SERA5 that correlated with the block in rupture. Additionally, DPAP3 inhibition reduced levels of mature SUB1, suggesting DPAP3 acts upstream of SUB1 maturation (though this role for DPAP3 was later refuted by genetic studies).\",\n      \"method\": \"Chemical genetic screen, protease inhibitor library, Western blot for SERA5 processing, parasite culture rupture assay\",\n      \"journal\": \"Nature chemical biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — chemical genetic screen identifying SUB1 as a rupture regulator, pharmacological evidence for SERA5 processing; DPAP3 upstream role was later disproven genetically\",\n      \"pmids\": [\"18246061\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"SUB1 has a further role in priming the merozoite for invasion by proteolytically maturing the MSP1/MSP6/MSP7 surface protein complex just before egress. Inhibition of SUB1 results in accumulation of unprocessed MSPs on the merozoite surface and significantly reduced erythrocyte invasion, demonstrating that SUB1 is a multifunctional processing protease essential for both egress and merozoite surface remodelling.\",\n      \"method\": \"Pharmacological SUB1 inhibition, Western blot for MSP1/MSP6/MSP7 processing, erythrocyte invasion assay\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct substrate identification with functional invasion assay, replicated across multiple studies\",\n      \"pmids\": [\"19214190\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"SUB1 mediates primary processing of MSP1 prior to egress; primary processing is ordered with the site closest to the C-terminal end of MSP1 cleaved last. Replacement of the secondary processing site (normally refractory to SUB1) with a SUB1-sensitive site is deleterious to parasite growth, demonstrating that correct spatiotemporal regulation of MSP1 maturation by SUB1 (and SUB2) is crucial for parasite viability.\",\n      \"method\": \"Conditional expression system, site-directed mutagenesis of MSP1 processing sites, parasite growth assays, Western blot\",\n      \"journal\": \"Molecular microbiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic manipulation of substrate cleavage site with functional growth readout, single lab\",\n      \"pmids\": [\"20735778\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Proteomic and bioinformatic analysis of the late asexual blood stage proteome identified multiple previously unknown SUB1 substrates including RAP1 (rhoptry-associated protein 1) and MSRP2 (MSP7-related protein 2), establishing that SUB1 processes a broad range of merozoite, PV, and PVM proteins.\",\n      \"method\": \"Mass spectrometry-based proteomics, bioinformatic substrate motif analysis, Western blot validation\",\n      \"journal\": \"Infection and immunity\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MS-based substrate identification with validation, single lab\",\n      \"pmids\": [\"21220481\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"SUB1 directly cleaves SERA6 in the parasitophorous vacuole just prior to egress, converting it to an active cysteine protease. Mutations replacing the predicted catalytic Cys of SERA6, or blocking SERA6 processing by SUB1, could not be stably introduced into the parasite genomic locus, indicating SERA6 is essential and that processing by SUB1 is required for its function.\",\n      \"method\": \"Conditional mutagenesis, Western blot for SERA6 processing, in vitro enzyme activity assay, attempted genomic locus replacement\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic essentiality confirmed, direct SUB1 cleavage shown biochemically, activation of cysteine protease confirmed, replicated\",\n      \"pmids\": [\"22984267\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Intracellular calcium elevation just before egress is required for SUB1 discharge from exonemes into the parasitophorous vacuole. Chelation of intracellular Ca2+ with BAPTA-AM, or inhibition of Ca2+ release from intracellular stores with a PLC inhibitor, blocks SUB1 discharge, inhibits SERA5 processing, and prevents PVM rupture and merozoite egress.\",\n      \"method\": \"Time-lapse video microscopy, Ca2+ chelators and PLC inhibitor pharmacology, Western blot for SUB1 discharge and SERA5 processing, immunofluorescence\",\n      \"journal\": \"Cellular microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pharmacological dissection with functional readouts, single lab\",\n      \"pmids\": [\"23217145\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"PfPKG (cGMP-dependent protein kinase) activity is required upstream of SUB1 discharge into the PV. Inhibition of PfPKG prevents SUB1 discharge, while stimulation of PfPKG by inhibiting parasite phosphodiesterase induces premature SUB1 discharge and egress of developmentally immature, non-invasive parasites.\",\n      \"method\": \"PfPKG inhibitor compound 2 (C2) treatment, phosphodiesterase inhibitor stimulation, Western blot for SUB1 discharge, live microscopy\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — pharmacological epistasis with gain-of-function and loss-of-function, replicated across studies\",\n      \"pmids\": [\"23675297\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Using conditional mutagenesis in P. berghei, SUB1 was shown to be essential at the hepatic stage. SUB1-deficient parasites failed to rupture the parasitophorous vacuole membrane and egress from hepatocytes. Mechanically released SUB1-null parasites were not adequately primed and failed to establish blood stage infection in vivo.\",\n      \"method\": \"Conditional mutagenesis (stage-specific sub1 invalidation), intravital microscopy, in vivo mouse infection\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional genetic knockout with specific PVM rupture phenotype and in vivo validation\",\n      \"pmids\": [\"24089525\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium berghei\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Using P. berghei conditional mutagenesis and Flp recombinase deleters, SUB1 was shown to be essential for complete development of the liver stage schizont and production of hepatic merozoites, but not required for early intrahepatic growth.\",\n      \"method\": \"Conditional mutagenesis with Flp/FRT system, P. berghei rodent malaria model, liver stage phenotypic analysis\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional genetic knockout with stage-specific phenotypic analysis in rodent model\",\n      \"pmids\": [\"24348254\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium berghei\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Crystal structure of P. falciparum SUB1 at 2.25 Å in complex with its cognate prodomain revealed: (1) the structural basis for calcium dependence; (2) an unusual requirement to interact with substrate residues on both prime and non-prime sides of the scissile bond; (3) a solvent-exposed redox-sensitive disulfide bridge unique among subtilisins that likely acts as a regulator of protease activity.\",\n      \"method\": \"X-ray crystallography at 2.25 Å resolution, functional validation of calcium dependence and disulfide bridge\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — high-resolution crystal structure with functional validation, later replicated by independent structural studies\",\n      \"pmids\": [\"24785947\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Crystal structure of full-length SUB1 from P. vivax revealed a bacterial-like catalytic domain in complex with a Plasmodium-specific prodomain that has a novel 'belt' architecture: an N-terminal insertion that embraces the catalytic domain to stabilize the pre-protease and undergoes calcium-dependent autoprocessing during activation. The 'belt' domain is dispensable for recombinant enzymatic activity but could not be deleted in P. berghei in vivo, indicating it is essential for parasite development.\",\n      \"method\": \"X-ray crystallography of PvSUB1, deletion mutagenesis in P. berghei, in vitro enzyme activity assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with in vivo genetic validation in rodent malaria model\",\n      \"pmids\": [\"25204226\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium vivax\", \"strain\": \"\"}, {\"species\": \"Plasmodium berghei\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"SUB1-mediated processing of MSP1 is important for parasite viability; processing modifies the secondary structure of MSP1 and activates its capacity to bind spectrin, the major component of the erythrocyte cytoskeleton. Parasites expressing an inefficiently processed MSP1 mutant show delayed egress, and merozoites lacking surface-bound MSP1 display a severe egress defect.\",\n      \"method\": \"Conditional mutagenesis, MSP1 mutant parasite lines, spectrin-binding assays, egress assays, electron microscopy\",\n      \"journal\": \"Cell host & microbe\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic manipulation of SUB1 cleavage site in MSP1 with multiple orthogonal functional readouts\",\n      \"pmids\": [\"26468747\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Plasmepsin X (PMX) is essential for maturation of SUB1 in exoneme secretory vesicles; pharmacological inhibition of PMX prevents SUB1 maturation and consequently blocks both egress and invasion.\",\n      \"method\": \"Conditional knockdown of PMX, Western blot for SUB1 maturation, PMX inhibitor treatment, egress/invasion assays\",\n      \"journal\": \"Science (New York, N.Y.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional genetic knockdown with direct molecular readout of SUB1 processing, replicated by multiple subsequent studies\",\n      \"pmids\": [\"29074774\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"P. falciparum parasites lacking SUB1 undergo none of the morphological transformations that precede egress and fail to rupture the PVM. In contrast, SERA6-null parasites rupture the PVM normally but fail to rupture the RBC membrane. Complementation shows SERA6 is an enzyme requiring processing by SUB1 to function. RBCM rupture is associated with SERA6-dependent proteolytic cleavage of β-spectrin within its actin-binding domain. This establishes SUB1 and SERA6 as playing distinct, essential roles in a coordinated proteolytic cascade enabling sequential membrane rupture.\",\n      \"method\": \"Conditional knockout (SUB1-null and SERA6-null parasites), live microscopy, electron microscopy, complementation studies, Western blot for β-spectrin cleavage\",\n      \"journal\": \"Nature microbiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional genetic knockouts with precise phenotypic dissection, complementation, and molecular substrate identification\",\n      \"pmids\": [\"29459732\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Live-cell microscopy with PV-targeted fluorescent proteins showed that abrupt rupture of the PV membrane and release of vacuolar contents are under control of PfPKG and SUB1, occurring ~1.5 minutes after the PV begins rounding (which is under control of parasite [Ca2+]i). This temporally positions SUB1 action downstream of PfPKG activation.\",\n      \"method\": \"Live-cell fluorescence microscopy with PV-targeted fluorescent proteins, specific egress inhibitors (PKG inhibitor, SUB1 inhibitor)\",\n      \"journal\": \"Cellular microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — live imaging with specific inhibitors, temporal resolution of pathway steps, single lab\",\n      \"pmids\": [\"29900649\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"In P. berghei, SUB1 is selectively expressed in mature male gametocytes, localizes to male osmiophilic bodies (secretory organelles involved in gamete egress), and is required for male gamete egress. The PbSUB1 substrate PbSERA3 is expressed in gametocytes and processed by PbSUB1 upon gametocyte activation.\",\n      \"method\": \"Immunofluorescence, electron microscopy, transgenic P. berghei lines deficient in PbSUB1 expression or enzyme activity, Western blot for PbSERA3 processing\",\n      \"journal\": \"Cellular microbiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic loss-of-function with specific phenotype, substrate processing confirmed, single lab\",\n      \"pmids\": [\"30941868\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium berghei\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"The ER-resident CREC family protein PfERC is required for proteolytic maturation of SUB1. PfERC knockdown prevents proteolytic maturation of plasmepsin X, which is required for SUB1 cleavage. Knockdown of PfERC inhibits PVM rupture and downstream substrate processing, placing PfERC as an early regulator in the egress proteolytic cascade upstream of PMX and SUB1.\",\n      \"method\": \"Conditional knockdown of PfERC, double mutant parasites, Western blot for PMX and SUB1 maturation, egress assays\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional knockdown with epistasis analysis using double mutants, multiple molecular readouts\",\n      \"pmids\": [\"32098818\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"SUB1 inhibitor profiling of P. falciparum parasites expressing conditionally reduced levels of PfSUB1 confirmed that inhibition of SUB1 activity is the mechanism by which peptidic boronic acid compounds (developed as SUB1 inhibitors) block parasite egress and replication.\",\n      \"method\": \"Conditionally regulated PfSUB1 parasite lines, peptidic boronic acid inhibitors, egress assays, parasite replication assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic validation using hypomorphic SUB1 lines with inhibitor, single lab\",\n      \"pmids\": [\"33975947\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Activation of SERA6 involves a second autocatalytic step triggered by SUB1 cleavage, which requires interaction in multimolecular complexes with the PV-located protein cofactor MSA180—itself a SUB1 substrate. Genetic ablation of MSA180 mimics SERA6 disruption, producing a fatal block in β-spectrin cleavage and RBC rupture.\",\n      \"method\": \"Conditional genetic knockout of MSA180, Co-IP/pulldown for MSA180-SERA6 complex, Western blot for SERA6 maturation and β-spectrin cleavage, egress assays in P. falciparum and P. knowlesi\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis, complex identification, and functional assay across two Plasmodium species\",\n      \"pmids\": [\"33932049\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}, {\"species\": \"Plasmodium knowlesi\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"A stand-alone propeptide-like protein, SUB1-ProM, is expressed in the parasitophorous vacuole of blood-stage P. falciparum where it acts as a fast-binding, potent inhibitor of SUB1 but not of SUB2 or other proteases examined, suggesting it may function as an endogenous regulator of SUB1 activity in the PV.\",\n      \"method\": \"Recombinant protein expression, enzyme inhibition kinetics, mass spectrometry, immunofluorescence localization\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — recombinant inhibition assays with localization, single lab; endogenous regulatory role inferred but not directly tested genetically\",\n      \"pmids\": [\"31942933\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"PMX activates SUB1 by directly cleaving both p31 (the inhibitory prodomain) and p54 (the intermediate catalytic domain). PMX cleavage sites on p31 were mapped by mass spectrometry. Conversion of p54 to p47 can be effected by cleavage at either SUB1 or PMX cleavage sites that are adjacent to one another. Once p31 is removed, p54 is fully functional inside parasites, establishing that conversion to p47 is dispensable for SUB1 activity. This reveals a novel mechanism for subtilisin activation in which relief of propiece inhibition is achieved via a heterologous protease.\",\n      \"method\": \"PMX knockdown in parasites, recombinant PMX and SUB1 in vitro cleavage assay, mass spectrometry mapping of cleavage sites, Western blot, parasite functional assays\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution of PMX-mediated SUB1 activation combined with parasite genetics; cleavage sites mapped; multiple orthogonal methods\",\n      \"pmids\": [\"37036362\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Crystal structure of full-length P. falciparum SUB1 demonstrated that the atypical Plasmodium-specific SUB1 prodomain (PD) directly promotes assembly of inactive enzyme homodimers at acidic pH, whereas SUB1 is primarily monomeric at neutral pH. This establishes a pH-dependent autoinhibition mechanism: the PD-driven dimeric form keeps SUB1 inactive in acidic exonemes, while PMX processing and subsequent exposure to neutral pH in the PV allows monomeric, active SUB1.\",\n      \"method\": \"X-ray crystallography of full-length PfSUB1, analytical ultracentrifugation, biophysical measurements of pH-dependent dimerization, biochemical activity assays\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure combined with biophysical and biochemical validation of pH-dependent dimerization mechanism\",\n      \"pmids\": [\"38386597\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Biochemical and enzymatic analysis confirmed that both p31 and p31-p54 are largely dimeric under acidic conditions (as they would be exposed to PMX), and that sequential PMX cleavage of p31 results in rapid loss of the capacity of p31 to inhibit SUB1 catalytic activity. Direct exposure of recombinant p31-p54 complex to PMX activates SUB1 activity, confirming PMX-mediated prodomain cleavage as the activation mechanism.\",\n      \"method\": \"Analytical ultracentrifugation, enzyme kinetics, mass spectrometry mapping of PMX cleavage sites, in vitro SUB1 activity assay after PMX treatment\",\n      \"journal\": \"Biochimica et biophysica acta. General subjects\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with defined cleavage-site mapping and direct activation demonstrated, single lab\",\n      \"pmids\": [\"38969256\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Crystal structures of P. vivax SUB1 catalytic domain (PvS1Cat), alone and in complex with the α-ketoamide inhibitor MAM-117, showed that the catalytic serine of SUB1 forms a covalent bond with the α-keto group of the inhibitor. The catalytic groove of SUB1 underwent significant structural changes when bound to the peptidomimetic inhibitor, particularly in the S4 pocket, and P1'/P2' residues of the inhibitor contribute important stabilizing interactions.\",\n      \"method\": \"X-ray crystallography of PvSUB1 catalytic domain in apo and inhibitor-bound states\",\n      \"journal\": \"Acta crystallographica. Section D, Structural biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — high-resolution crystal structures of covalent enzyme-inhibitor complex revealing structural basis of inhibition, single lab\",\n      \"pmids\": [\"37428845\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium vivax\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Crystal structures of PvSUB1 in complex with α-ketoamide inhibitor compound 8 revealed a covalent bond at the catalytic serine and extended interactions to P5, with two additional hydrogen bonds compared to the reference compound. Structure-activity relationships established that hydrophobicity improvements at multiple positions enhanced PfSUB1 inhibition (IC50 values down to 10–12 nM for PvSUB1 and PfSUB1).\",\n      \"method\": \"X-ray crystallography, enzyme inhibition assays (IC50), parasite egress assays\",\n      \"journal\": \"European journal of medicinal chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure with inhibitor and functional enzyme assays, single lab\",\n      \"pmids\": [\"38503166\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium vivax\", \"strain\": \"\"}, {\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Plasmepsin X inhibitors (peptidomimetic compounds) inhibit maturation of SUB1 and also inhibit parasite egress from erythrocytes, further supporting that PMX-mediated maturation of SUB1 is the mechanism of antimalarial action of PMX inhibitors.\",\n      \"method\": \"Plasmepsin inhibitor treatment of P. falciparum cultures, Western blot for SUB1 maturation, egress assay\",\n      \"journal\": \"European journal of medicinal chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pharmacological evidence linking PMX inhibition to SUB1 maturation block, consistent with genetic studies\",\n      \"pmids\": [\"30529637\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    }\n  ],\n  \"current_model\": \"SUB1 (PF3D7_0507500) is an essential subtilisin-like serine protease that is synthesized as an 82 kDa precursor, undergoes autocatalytic cleavage in the ER to a p54 form non-covalently associated with its inhibitory prodomain (p31), and is then stored in specialized secretory organelles called exonemes in an inactive, pH-dependent homodimeric state; upon activation—triggered by PfPKG/Ca2+ signaling and mediated by the aspartic protease plasmepsin X (PMX), which cleaves and destroys the inhibitory p31 prodomain—monomeric active SUB1 (p47) is discharged into the parasitophorous vacuole where it acts as a master processing protease that cleaves multiple substrates including SERA5, SERA6 (activating it as a cysteine protease to drive RBC membrane rupture via β-spectrin cleavage), MSP1/MSP6/MSP7 (remodelling the merozoite surface for invasion), RAP1, MSRP2, and MSA180, thereby orchestrating sequential rupture of the PVM and RBC membrane for merozoite egress; SUB1 also plays essential roles in hepatic stage egress and male gamete egress in Plasmodium berghei.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"SUB1 (PF3D7_0507500) is an essential Plasmodium subtilisin-like serine protease that acts as the master trigger of a proteolytic cascade governing merozoite egress and invasion in asexual blood stages [#6, #8]. The enzyme is synthesized as an 82 kDa precursor that undergoes autocatalytic maturation during secretory transport—first cleaved in the ER to a p54 form and then, via calcium-dependent intramolecular autocatalysis between Asp251 and Ala252, to p47—with mutation of the catalytic serine abolishing all processing, confirming its serine-protease mechanism [#1, #2]. Mature enzyme is stored in specialized apical secretory organelles termed exonemes and is discharged into the parasitophorous vacuole (PV) just before egress [#0, #6]. Discharge is controlled by an upstream signaling axis in which PfPKG activity and a Ca2+/PLC-dependent step are both required to release SUB1 into the PV [#12, #13, #21]. Within exonemes SUB1 is held inactive: its Plasmodium-specific prodomain (p31) is a fast-binding, high-affinity inhibitor that drives assembly of inactive homodimers at acidic pH, and activation is achieved not by autoprocessing but by the aspartic protease plasmepsin X (PMX), which directly cleaves and inactivates the inhibitory prodomain, liberating active enzyme upon exposure to neutral PV pH [#4, #27, #28]. Once active, SUB1 processes a broad substrate repertoire: it matures SERA5 and activates SERA6 as a cysteine protease that cleaves β-spectrin to rupture the RBC membrane, and it remodels the merozoite surface by cleaving the MSP1/MSP6/MSP7 complex (enabling MSP1 spectrin binding) as well as RAP1, MSRP2 and MSA180 [#6, #8, #10, #11, #18, #20, #25]. SUB1-null parasites fail the morphological changes preceding egress and cannot rupture the PVM, establishing SUB1 and its downstream substrate SERA6 as sequential, non-redundant effectors of PVM then RBC-membrane rupture [#20]. Beyond the blood stage, SUB1 is essential in Plasmodium berghei for hepatic-stage schizont maturation and PVM rupture and for male gamete egress from osmiophilic bodies [#14, #15, #22]. Crystal structures of P. falciparum and P. vivax SUB1 define its calcium-dependent bacterial-like catalytic domain, its unusual prodomain 'belt' architecture, a redox-sensitive surface disulfide, and the covalent binding mode of α-ketoamide inhibitors, making SUB1 a validated antimalarial drug target [#16, #17, #28, #30].\",\n  \"teleology\": [\n    {\n      \"year\": 1998,\n      \"claim\": \"Established that SUB1 is an asexual blood-stage protease that is post-translationally processed during secretory transport and stored apically, framing it as a regulated secretory enzyme rather than a constitutive one.\",\n      \"evidence\": \"Western blot, immunofluorescence, subcellular fractionation and EM in P. falciparum\",\n      \"pmids\": [\"9722575\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Function of the secreted enzyme not yet defined\", \"Substrates unknown\", \"Trigger for secretion not identified\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Defined the two-step autocatalytic maturation pathway and proved SUB1 is a serine protease, explaining how the precursor becomes an active enzyme.\",\n      \"evidence\": \"In vitro translation, recombinant expression, active-site mutagenesis and cleavage mapping (Asp251/Ala252) in P. falciparum\",\n      \"pmids\": [\"10617661\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo trigger for calcium-dependent second cleavage not established\", \"Physiological substrates still unknown\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Characterized SUB1 enzymology—calcium dependence, alkaline pH optimum, and P4/P1 substrate preferences—providing the specificity rules needed to predict natural substrates and design inhibitors.\",\n      \"evidence\": \"Recombinant enzyme kinetics with peptide and protein substrates, fluorogenic assay development, and homology modeling in P. falciparum\",\n      \"pmids\": [\"12052828\", \"12356327\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Natural substrates in the parasite not yet identified\", \"Structural basis of specificity inferred from modeling only\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Showed the prodomain (p31) is a specific high-affinity fast-binding inhibitor, revealing how SUB1 is kept latent before deployment.\",\n      \"evidence\": \"Circular dichroism, inhibition kinetics, and truncation/point mutants of p31 in P. falciparum\",\n      \"pmids\": [\"12764150\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How prodomain inhibition is relieved in vivo not known\", \"Structural basis of prodomain binding not resolved\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Identified Plasmodium-specific surface loops required for maturation and activity and showed the two processing steps have distinct structural requirements, indicating p47 conversion is not required for trans-proteolytic activity.\",\n      \"evidence\": \"Systematic deletion/substitution mutagenesis with insect-cell expression and processing readouts in P. falciparum\",\n      \"pmids\": [\"16183148\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Single expression system\", \"In vivo relevance of loop functions not tested genetically\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Discovered exonemes and placed SUB1 at the top of an egress pathway—discharged into the PV where it matures SERA-family proteins and enables merozoite egress and invasion.\",\n      \"evidence\": \"IFA/EM organelle localization, SERA processing Western blots, and pharmacological inhibition of egress in P. falciparum\",\n      \"pmids\": [\"18083098\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Signal driving exoneme discharge not defined\", \"Full substrate repertoire unknown\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Chemical genetics confirmed SUB1 as a primary regulator of erythrocyte rupture via SERA5 processing; a proposed upstream role for DPAP3 in SUB1 maturation was later refuted genetically.\",\n      \"evidence\": \"Covalent protease inhibitor library screen with rupture and SERA5 processing readouts in P. falciparum\",\n      \"pmids\": [\"18246061\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Pharmacological, not genetic, evidence\", \"DPAP3 upstream claim disproven by later work\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Extended SUB1's role from egress to invasion by showing it matures the MSP1/MSP6/MSP7 surface complex, establishing SUB1 as a multifunctional processing protease.\",\n      \"evidence\": \"Pharmacological inhibition with MSP processing and invasion assays in P. falciparum\",\n      \"pmids\": [\"19214190\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of MSP maturation not yet mechanistically defined\", \"Reliance on inhibitors\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Demonstrated that ordered, spatiotemporally correct MSP1 processing by SUB1 is required for viability, showing that miscued cleavage is deleterious.\",\n      \"evidence\": \"Conditional expression and MSP1 processing-site mutagenesis with growth assays in P. falciparum\",\n      \"pmids\": [\"20735778\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular reason cleavage timing matters not yet explained\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Expanded the SUB1 substrate map to RAP1 and MSRP2, indicating SUB1 processes a broad set of merozoite, PV and PVM proteins.\",\n      \"evidence\": \"Mass-spectrometry proteomics with motif analysis and Western validation in P. falciparum\",\n      \"pmids\": [\"21220481\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional importance of individual substrates not tested\", \"Single lab\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Defined the SUB1–SERA6 axis: SUB1 cleavage converts SERA6 into an essential active cysteine protease, revealing a proteolytic relay downstream of SUB1.\",\n      \"evidence\": \"Conditional mutagenesis, SERA6 processing and activity assays, and failed catalytic/processing-site mutant integration in P. falciparum\",\n      \"pmids\": [\"22984267\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"SERA6 downstream target not yet identified at this stage\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Placed intracellular Ca2+/PLC signaling immediately upstream of SUB1 discharge, connecting a second messenger to the proteolytic egress program.\",\n      \"evidence\": \"Time-lapse microscopy with BAPTA-AM and PLC inhibitor and discharge/processing readouts in P. falciparum\",\n      \"pmids\": [\"23217145\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular Ca2+ effector for discharge not identified\", \"Pharmacological evidence only\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Established PfPKG as an upstream master switch whose activity is necessary and, when forced, sufficient to drive premature SUB1 discharge and egress.\",\n      \"evidence\": \"PfPKG inhibitor (C2) and phosphodiesterase-inhibitor gain-of-function with discharge readouts and live microscopy in P. falciparum\",\n      \"pmids\": [\"23675297\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct PfPKG substrates linking to discharge not defined\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Genetic knockout in P. berghei proved SUB1 is essential across the life cycle—required for hepatic-stage PVM rupture, schizont maturation and merozoite priming, not merely a blood-stage enzyme.\",\n      \"evidence\": \"Conditional/Flp-FRT mutagenesis with intravital microscopy and in vivo infection in P. berghei\",\n      \"pmids\": [\"24089525\", \"24348254\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Hepatic-stage substrates not defined\", \"Cross-species substrate conservation not fully mapped\"],\n      \"attribution\": [{\"species\": \"Plasmodium berghei\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Crystal structures of P. falciparum and P. vivax SUB1 provided the structural basis for calcium dependence, the bacterial-like catalytic fold, a redox-sensitive disulfide, and the essential Plasmodium-specific prodomain 'belt' architecture.\",\n      \"evidence\": \"X-ray crystallography with functional validation, plus belt-deletion mutagenesis in P. berghei\",\n      \"pmids\": [\"24785947\", \"25204226\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structure did not resolve the in vivo activation trigger\", \"Oligomeric state not addressed\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}, {\"species\": \"Plasmodium vivax\", \"strain\": \"\"}, {\"species\": \"Plasmodium berghei\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Linked SUB1-mediated MSP1 maturation to a concrete molecular function—activating MSP1 spectrin binding—and to egress kinetics, explaining why correct MSP1 processing matters.\",\n      \"evidence\": \"MSP1 processing-site mutant lines, spectrin-binding, egress assays and EM in P. falciparum\",\n      \"pmids\": [\"26468747\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Precise contribution of MSP1-spectrin binding to membrane destabilization not fully resolved\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Identified plasmepsin X as essential for SUB1 maturation, revealing a heterologous protease acting upstream of SUB1 in exonemes.\",\n      \"evidence\": \"PMX conditional knockdown and inhibitor treatment with SUB1 maturation Westerns and egress/invasion assays in P. falciparum\",\n      \"pmids\": [\"29074774\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular mechanism of PMX action on SUB1 not yet defined at this stage\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Conditional knockouts dissected the cascade into sequential membrane-rupture events: SUB1-null parasites fail PVM rupture, while SERA6 (activated by SUB1) cleaves β-spectrin to rupture the RBC membrane.\",\n      \"evidence\": \"SUB1-null and SERA6-null lines with live/EM imaging, complementation and β-spectrin cleavage Westerns in P. falciparum; live PV-marker imaging positioning SUB1 downstream of PfPKG\",\n      \"pmids\": [\"29459732\", \"29900649\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How SUB1 itself contributes to PVM rupture mechanistically not fully defined\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Showed SUB1 also drives sexual-stage egress in P. berghei, localizing to male osmiophilic bodies and processing PbSERA3 upon gametocyte activation.\",\n      \"evidence\": \"IFA/EM localization, PbSUB1-deficient/inactive transgenics and PbSERA3 processing Westerns in P. berghei\",\n      \"pmids\": [\"30941868\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Regulation of SUB1 in gametocytes vs blood stage not compared\", \"Single lab\"],\n      \"attribution\": [{\"species\": \"Plasmodium berghei\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Positioned the ER chaperone-like protein PfERC upstream of PMX and SUB1, extending the activation hierarchy back to the ER.\",\n      \"evidence\": \"PfERC conditional knockdown and double-mutant epistasis with PMX/SUB1 maturation and egress readouts in P. falciparum\",\n      \"pmids\": [\"32098818\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct biochemical role of PfERC in PMX maturation not defined\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Identified endogenous SUB1 inhibitors—SUB1-ProM in the PV—suggesting native regulation of active SUB1 beyond prodomain removal.\",\n      \"evidence\": \"Recombinant inhibition kinetics, mass spectrometry and localization in P. falciparum; also boronic-acid inhibitor validation using conditional SUB1 lines\",\n      \"pmids\": [\"31942933\", \"33975947\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Endogenous regulatory role of SUB1-ProM inferred, not tested genetically\", \"Boronic acid on-target validation single lab\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Revealed that SERA6 activation requires assembly with the SUB1 substrate MSA180 into multimolecular complexes, adding a cofactor layer to the terminal RBC-rupture step.\",\n      \"evidence\": \"MSA180 conditional knockout, Co-IP, SERA6/β-spectrin cleavage and egress assays in P. falciparum and P. knowlesi\",\n      \"pmids\": [\"33932049\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of the SERA6-MSA180 complex not resolved\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}, {\"species\": \"Plasmodium knowlesi\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Defined the molecular activation mechanism: PMX directly cleaves the inhibitory p31 prodomain (and p54), relieving propeptide inhibition, and showed p54→p47 conversion is dispensable for activity—a novel subtilisin activation route via a heterologous protease.\",\n      \"evidence\": \"PMX knockdown, recombinant PMX/SUB1 cleavage assays, MS site mapping and parasite functional assays in P. falciparum; also PvSUB1 covalent inhibitor structures\",\n      \"pmids\": [\"37036362\", \"37428845\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How neutral-pH transition and PMX cleavage are temporally coupled in vivo not fully resolved\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}, {\"species\": \"Plasmodium vivax\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Completed the autoinhibition model: the full-length structure and biophysics show the prodomain drives inactive homodimers at acidic exoneme pH, with PMX cleavage plus neutral PV pH yielding active monomer; inhibitor structures further guide drug design.\",\n      \"evidence\": \"Full-length PfSUB1 crystallography with analytical ultracentrifugation and pH-dependent activity assays; in vitro PMX activation of p31-p54; PvSUB1 α-ketoamide inhibitor structures and SAR in P. falciparum and P. vivax\",\n      \"pmids\": [\"38386597\", \"38969256\", \"38503166\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo pH dynamics of exonemes vs PV not directly measured\", \"Some inhibitor SAR single lab\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}, {\"species\": \"Plasmodium vivax\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the PfPKG/Ca2+ signal is mechanistically transduced into exoneme discharge, and the full in vivo function of the broader SUB1 substrate repertoire (RAP1, MSRP2, MSA180) beyond the SERA5/SERA6 and MSP axes, remain to be defined.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct link between PfPKG/Ca2+ and physical exoneme discharge unresolved\", \"Functional roles of many identified SUB1 substrates untested\", \"Endogenous SUB1-ProM regulatory role not tested genetically\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0004252\", \"supporting_discovery_ids\": [1, 2, 6, 8, 11, 27]},\n      {\"term_id\": \"GO:0016540\", \"supporting_discovery_ids\": [1, 2, 3, 5]},\n      {\"term_id\": \"GO:0005509\", \"supporting_discovery_ids\": [2, 16]},\n      {\"term_id\": \"GO:0004867\", \"supporting_discovery_ids\": [4, 26]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0020009\", \"supporting_discovery_ids\": [0, 6]},\n      {\"term_id\": \"GO:0005788\", \"supporting_discovery_ids\": [1, 2]},\n      {\"term_id\": \"GO:0020003\", \"supporting_discovery_ids\": [6, 8, 11, 12]},\n      {\"term_id\": \"GO:0020005\", \"supporting_discovery_ids\": [20]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0051673\", \"supporting_discovery_ids\": [6, 20, 21]},\n      {\"term_id\": \"GO:0006508\", \"supporting_discovery_ids\": [6, 8, 10, 11, 25]},\n      {\"term_id\": \"GO:0044409\", \"supporting_discovery_ids\": [8, 18]}\n    ],\n    \"complexes\": [\n      \"SUB1 p54–prodomain (p31) non-covalent inhibitory complex\",\n      \"pH-dependent inactive SUB1 homodimer\"\n    ],\n    \"partners\": [\n      \"Plasmepsin X (PMX) — activating protease that cleaves the SUB1 prodomain\",\n      \"PfPKG (cGMP-dependent protein kinase) — upstream signaling regulator of SUB1 discharge\",\n      \"PfERC — ER CREC-family protein required upstream of PMX/SUB1 maturation\",\n      \"SUB1-ProM — endogenous PV prodomain-like inhibitor of SUB1\",\n      \"SERA5, SERA6 (and PbSERA3) — SUB1 substrates\",\n      \"MSP1/MSP6/MSP7 complex — SUB1 substrates\",\n      \"RAP1, MSRP2, MSA180 — SUB1 substrates\"\n    ],\n    \"other_free_text\": [\n      \"Synthesized as an 82 kDa precursor, autocatalytically processed to p54 (ER) then to p47 (calcium-dependent, Asp251/Ala252); active-site serine required for processing [#1, #2]\",\n      \"Discharged from exonemes into the parasitophorous vacuole; SUB1 activation via PMX cleavage of the inhibitory prodomain rather than autoprocessing, with p47 conversion dispensable for activity [#5, #27, #28]\",\n      \"Master trigger of a sequential proteolytic egress cascade: SUB1 → SERA6 (activated) → β-spectrin cleavage for RBC-membrane rupture [#20, #25]\",\n      \"Essential across life-cycle stages in P. berghei: hepatic-stage egress and male gamete egress [#14, #15, #22]\",\n      \"Validated antimalarial drug target with covalent α-ketoamide and peptidic boronic acid inhibitors [#24, #30, #31]\"\n    ]\n  }\n}","audit_flag":null,"strains":[{"strain":"Pf3D7","gene_id":"PF3D7_0507500","name":null,"product":"subtilisin-like protease 1","uniprot_acc":null,"chromosome":null,"start_pos":null},{"strain":"PbANKA","gene_id":"PBANKA_1107100","name":null,"product":"subtilisin-like protease 1","uniprot_acc":null,"chromosome":null,"start_pos":null},{"strain":"PkH","gene_id":"PKNH_1026100","name":null,"product":"subtilisin-like protease 1, putative","uniprot_acc":null,"chromosome":null,"start_pos":null}],"gene_symbol":"SUB1","evaluation":null}