| 1998 |
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. |
Western blot, immunofluorescence, subcellular fractionation, electron microscopy |
The Journal of biological chemistry |
High |
9722575
|
| 2000 |
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). |
In vitro translation, baculovirus recombinant expression, site-directed mutagenesis, cross-linking, in vitro cleavage assay |
The Journal of biological chemistry |
High |
10617661
|
| 2002 |
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. |
Recombinant protein expression and purification, enzyme kinetics with decapeptide and protein substrates, 3D homology modeling |
The Journal of biological chemistry |
High |
12052828
|
| 2002 |
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. |
Fluorogenic peptide substrate assay, NMR, time-resolved fluorescence measurements |
Biochemistry |
High |
12356327
|
| 2003 |
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. |
Circular dichroism, enzyme kinetics (inhibition constants), truncation and point mutants of p31 |
The Journal of biological chemistry |
High |
12764150
|
| 2005 |
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. |
Deletion/substitution mutagenesis, insect cell expression system, Western blot analysis of processing |
Molecular and biochemical parasitology |
High |
16183148
|
| 2007 |
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. |
Immunofluorescence, electron microscopy, pharmacological inhibition with protease inhibitors, Western blot for SERA processing |
Cell |
High |
18083098
|
| 2008 |
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). |
Chemical genetic screen, protease inhibitor library, Western blot for SERA5 processing, parasite culture rupture assay |
Nature chemical biology |
Medium |
18246061
|
| 2009 |
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. |
Pharmacological SUB1 inhibition, Western blot for MSP1/MSP6/MSP7 processing, erythrocyte invasion assay |
The EMBO journal |
High |
19214190
|
| 2010 |
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. |
Conditional expression system, site-directed mutagenesis of MSP1 processing sites, parasite growth assays, Western blot |
Molecular microbiology |
High |
20735778
|
| 2011 |
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. |
Mass spectrometry-based proteomics, bioinformatic substrate motif analysis, Western blot validation |
Infection and immunity |
Medium |
21220481
|
| 2012 |
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. |
Conditional mutagenesis, Western blot for SERA6 processing, in vitro enzyme activity assay, attempted genomic locus replacement |
The Journal of biological chemistry |
High |
22984267
|
| 2012 |
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. |
Time-lapse video microscopy, Ca2+ chelators and PLC inhibitor pharmacology, Western blot for SUB1 discharge and SERA5 processing, immunofluorescence |
Cellular microbiology |
Medium |
23217145
|
| 2013 |
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. |
PfPKG inhibitor compound 2 (C2) treatment, phosphodiesterase inhibitor stimulation, Western blot for SUB1 discharge, live microscopy |
PLoS pathogens |
High |
23675297
|
| 2013 |
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. |
Conditional mutagenesis (stage-specific sub1 invalidation), intravital microscopy, in vivo mouse infection |
The Journal of biological chemistry |
High |
24089525
|
| 2013 |
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. |
Conditional mutagenesis with Flp/FRT system, P. berghei rodent malaria model, liver stage phenotypic analysis |
PLoS pathogens |
High |
24348254
|
| 2014 |
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. |
X-ray crystallography at 2.25 Å resolution, functional validation of calcium dependence and disulfide bridge |
Nature communications |
High |
24785947
|
| 2014 |
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. |
X-ray crystallography of PvSUB1, deletion mutagenesis in P. berghei, in vitro enzyme activity assays |
Nature communications |
High |
25204226
|
| 2015 |
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. |
Conditional mutagenesis, MSP1 mutant parasite lines, spectrin-binding assays, egress assays, electron microscopy |
Cell host & microbe |
High |
26468747
|
| 2017 |
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. |
Conditional knockdown of PMX, Western blot for SUB1 maturation, PMX inhibitor treatment, egress/invasion assays |
Science (New York, N.Y.) |
High |
29074774
|
| 2018 |
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. |
Conditional knockout (SUB1-null and SERA6-null parasites), live microscopy, electron microscopy, complementation studies, Western blot for β-spectrin cleavage |
Nature microbiology |
High |
29459732
|
| 2018 |
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. |
Live-cell fluorescence microscopy with PV-targeted fluorescent proteins, specific egress inhibitors (PKG inhibitor, SUB1 inhibitor) |
Cellular microbiology |
Medium |
29900649
|
| 2019 |
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. |
Immunofluorescence, electron microscopy, transgenic P. berghei lines deficient in PbSUB1 expression or enzyme activity, Western blot for PbSERA3 processing |
Cellular microbiology |
High |
30941868
|
| 2020 |
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. |
Conditional knockdown of PfERC, double mutant parasites, Western blot for PMX and SUB1 maturation, egress assays |
mBio |
High |
32098818
|
| 2020 |
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. |
Conditionally regulated PfSUB1 parasite lines, peptidic boronic acid inhibitors, egress assays, parasite replication assays |
Proceedings of the National Academy of Sciences of the United States of America |
Medium |
33975947
|
| 2021 |
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. |
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 |
The EMBO journal |
High |
33932049
|
| 2020 |
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. |
Recombinant protein expression, enzyme inhibition kinetics, mass spectrometry, immunofluorescence localization |
The Biochemical journal |
Medium |
31942933
|
| 2023 |
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. |
PMX knockdown in parasites, recombinant PMX and SUB1 in vitro cleavage assay, mass spectrometry mapping of cleavage sites, Western blot, parasite functional assays |
mBio |
High |
37036362
|
| 2024 |
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. |
X-ray crystallography of full-length PfSUB1, analytical ultracentrifugation, biophysical measurements of pH-dependent dimerization, biochemical activity assays |
mBio |
High |
38386597
|
| 2024 |
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. |
Analytical ultracentrifugation, enzyme kinetics, mass spectrometry mapping of PMX cleavage sites, in vitro SUB1 activity assay after PMX treatment |
Biochimica et biophysica acta. General subjects |
High |
38969256
|
| 2023 |
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. |
X-ray crystallography of PvSUB1 catalytic domain in apo and inhibitor-bound states |
Acta crystallographica. Section D, Structural biology |
High |
37428845
|
| 2024 |
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). |
X-ray crystallography, enzyme inhibition assays (IC50), parasite egress assays |
European journal of medicinal chemistry |
Medium |
38503166
|
| 2018 |
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. |
Plasmepsin inhibitor treatment of P. falciparum cultures, Western blot for SUB1 maturation, egress assay |
European journal of medicinal chemistry |
Medium |
30529637
|