Affinage · Toxoplasma gondii

SUB1

subtilisin SUB1 · UniProt S8F5S3

Annotated
2026-09-07
8 papers in source corpus 6 papers cited in narrative 6 extracted findings

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

TgSUB1 is a GPI-anchored subtilisin-type serine protease of Toxoplasma gondii that drives host-cell attachment, gliding motility, and invasion by proteolytically trimming secreted microneme adhesins on the parasite surface (PMID:11564738, PMID:20678172). It is trafficked to the micronemes rather than the surface via its prodomain, which is sufficient to redirect a reporter to this compartment only when correctly folded in the full-length protein (PMID:18532988). TgSUB1 matures through sequential cleavages—an initial event in the ER followed by secretion-triggered secondary processing—and this activation depends on the upstream endosome-like-compartment cathepsin C-like protease TgCPC1, whose deletion abolishes TgSUB1 maturation and globally impairs surface trimming of invasion and egress effectors (PMID:11564738, PMID:37326431). Upon secretion, TgSUB1 cleaves the microneme proteins MIC2, MIC4, and M2AP, with its GPI anchor contributing to full activity; Δsub1 parasites show reduced attachment, gliding, invasion, and virulence in mice (PMID:20678172). Protease activity is held in check by TgMIC5, an endogenous inhibitor that mimics a subtilisin prodomain and is itself retained on the plasma membrane through physical interaction with membrane-anchored TgSUB1 (PMID:22896704). TgSUB1 expression is positioned downstream of TgTKL1 kinase activity, which is required for normal TgSUB1 transcription and hence microneme protein processing and invasion (PMID:39475314).

Mechanistic history

Synthesis pass · year-by-year structured walk · 6 steps
  1. 2001 High

    Established that TgSUB1 is a micronemal subtilisin protease that matures by staged proteolysis, defining it as a secreted processing enzyme rather than a surface receptor.

    Evidence Cloning, subcellular fractionation, secretion-stimulation assays, and inhibitor studies (brefeldin A, serine protease inhibitors) in T. gondii tachyzoites

    PMID:11564738

    Open questions at the time
    • Substrates of TgSUB1 not yet identified
    • The protease responsible for maturation cleavages not defined
  2. 2008 High

    Resolved how a GPI-anchored protein is routed to micronemes rather than the surface, showing the prodomain encodes the micronemal trafficking signal.

    Evidence Deletion constructs and chimeric reporter proteins with subcellular localization readouts in T. gondii

    PMID:18532988

    Open questions at the time
    • The trafficking receptor/machinery recognizing the folded prodomain is unknown
    • Does not address enzymatic substrates
  3. 2010 High

    Defined the physiological function of TgSUB1 by showing it trims specific microneme adhesins and is required for motility, invasion, and virulence.

    Evidence Δsub1 knockout with full-length and ΔGPI complementation, surface processing, gliding, invasion assays, and mouse virulence model

    PMID:20678172

    Open questions at the time
    • Exact cleavage sites and consensus recognition motif not mapped
    • Whether adhesin trimming is directly catalyzed by TgSUB1 versus a downstream protease not fully separated
  4. 2012 High

    Identified an endogenous regulatory mechanism, showing TgMIC5 inhibits TgSUB1 by prodomain mimicry and tethers to the membrane via the protease.

    Evidence NMR structure of TgMIC5, in vitro protease inhibition assays, and co-localization/interaction studies in T. gondii

    PMID:22896704

    Open questions at the time
    • Co-crystal/complex structure of TgMIC5 bound in the TgSUB1 active site not determined
    • Physiological consequence of losing TgMIC5 inhibition in vivo not tested
  5. 2023 High

    Placed TgSUB1 activation downstream of the cathepsin C-like protease TgCPC1, establishing the upstream maturation step in the secretory pathway.

    Evidence Genetic deletion of TgCPC1 with western-blot analysis of TgSUB1 maturation and microneme processing, invasion and egress phenotyping in T. gondii

    PMID:37326431

    Open questions at the time
    • Whether TgCPC1 directly cleaves TgSUB1 or acts via an intermediate not resolved
    • Cleavage sites during maturation not mapped
  6. 2024 Medium

    Connected upstream kinase signaling to TgSUB1 by showing TgTKL1 activity is required for normal TgSUB1 transcription and hence microneme processing.

    Evidence CRISPR-Cas9 kinase-dead TgTKL1 mutant, transcriptomic profiling, microneme processing and invasion assays in T. gondii

    PMID:39475314

    Open questions at the time
    • TgSUB1 regulation is an indirect finding in a TgTKL1-focused study
    • Whether the effect on TgSUB1 is direct transcriptional control or indirect not established

Open questions

Synthesis pass · forward-looking unresolved questions
  • The precise substrate cleavage-site specificity of TgSUB1 and the structural basis of TgMIC5 inhibition in the active site remain to be defined.
  • No mapped cleavage consensus for MIC2/MIC4/M2AP
  • No co-structure of TgSUB1 with substrate or inhibitor
  • Direct versus indirect nature of TgCPC1-mediated maturation unresolved

Mechanism profile

Synthesis pass · controlled-vocabulary classification
Partners
M2AP (SUBSTRATE)MIC2 (SUBSTRATE)MIC4 (SUBSTRATE)TGCPC1 (UPSTREAM CATHEPSIN C-LIKE ACTIVATING PROTEASE)TGMIC5 (ENDOGENOUS PRODOMAIN-MIMIC INHIBITOR)TGTKL1 (UPSTREAM KINASE REGULATING TGSUB1 TRANSCRIPTION)
Complex memberships
TgSUB1–TgMIC5 inhibitory complex on the parasite plasma membrane

Evidence

Reading pass · 6 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2001 TgSUB1 is a subtilisin-type serine protease localized to micronemes of T. gondii tachyzoites. It undergoes initial cleavage in the endoplasmic reticulum and is then transported to micronemes. Upon stimulation of microneme secretion, TgSUB1 is further cleaved into smaller products that are secreted; this secondary processing is inhibited by brefeldin A and serine protease inhibitors. Cloning, subcellular fractionation, secretion stimulation assays, inhibitor studies (brefeldin A, serine protease inhibitors) The Journal of biological chemistry High 11564738
2008 TgSUB1 is a GPI-anchored protein that is nonetheless targeted to micronemes rather than the parasite surface. The prodomain of TgSUB1 is responsible for trafficking to micronemes and is sufficient to target a reporter protein to micronemes, but only when correctly folded in the context of the full-length protein. Deletion constructs, chimeric reporter proteins, subcellular localization by imaging/fractionation Traffic (Copenhagen, Denmark) High 18532988
2010 TgSUB1 is required for proteolytic trimming of secreted microneme proteins MIC2, MIC4, and M2AP on the parasite surface. Loss of TgSUB1 (Δsub1) causes defects in cell attachment, gliding motility, and invasion rates. The GPI anchor of TgSUB1 contributes to full activity, as a GPI-anchor-deleted form only partially restores processing. Δsub1 parasites are less virulent in mice. Genetic knockout (Δsub1), complementation with full-length and ΔGPI constructs, surface processing assays, gliding and invasion assays, mouse virulence model Cellular microbiology High 20678172
2012 TgMIC5 inhibits TgSUB1 protease activity by mimicking a subtilisin prodomain, with a flexible C-terminal peptide proposed to insert into the active site. TgMIC5 is approximately 50-fold more potent an inhibitor of TgSUB1 than the small molecule ALLN. TgMIC5 is retained on the parasite plasma membrane through physical interaction with the membrane-anchored TgSUB1. NMR structure determination of TgMIC5, in vitro protease activity assays comparing inhibitor potencies, co-localization and physical interaction studies The Journal of biological chemistry High 22896704
2023 Deletion of the endosome-like compartment (ELC)-residing cathepsin C-like protease TgCPC1 completely blocks the activation (maturation) of TgSUB1, demonstrating that TgCPC1 acts upstream of TgSUB1 in the secretory pathway. Loss of TgSUB1 activation globally impairs surface trimming of key micronemal invasion and egress effectors. Genetic deletion of TgCPC1, western blot analysis of TgSUB1 maturation, microneme protein processing assays, invasion and egress phenotypic analysis mBio High 37326431
2024 Kinase-inactive mutation of TgTKL1 leads to downregulation of TgSUB1 transcription, which in turn causes defects in post-exocytosis processing of micronemal proteins and host-cell invasion, placing TgTKL1 kinase activity upstream of TgSUB1 expression in the invasion regulatory pathway. CRISPR-Cas9 kinase-dead mutant generation, transcriptomic profiling, microneme protein processing assays, invasion assays mSphere Medium 39475314

Source papers

Stage 0 corpus · 8 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2001 A conserved subtilisin-like protein TgSUB1 in microneme organelles of Toxoplasma gondii. The Journal of biological chemistry 75 11564738
2010 Toxoplasma gondii protease TgSUB1 is required for cell surface processing of micronemal adhesive complexes and efficient adhesion of tachyzoites. Cellular microbiology 58 20678172
2008 The prodomain of Toxoplasma gondii GPI-anchored subtilase TgSUB1 mediates its targeting to micronemes. Traffic (Copenhagen, Denmark) 23 18532988
2012 Microneme protein 5 regulates the activity of Toxoplasma subtilisin 1 by mimicking a subtilisin prodomain. The Journal of biological chemistry 18 22896704
2011 Identification of Toxoplasma gondii SUB1 antigen as a marker for acute infection by use of an innovative evaluation method. Journal of clinical microbiology 9 21543561
2023 A cathepsin C-like protease mediates the post-translation modification of Toxoplasma gondii secretory proteins for optimal invasion and egress. mBio 4 37326431
2024 Kinase function of TgTKL1 is essential for its role in Toxoplasma propagation and pathogenesis. mSphere 2 39475314
2023 A cathepsin C-like protease post-translationally modifies Toxoplasma gondii secretory proteins for optimal invasion and egress. bioRxiv : the preprint server for biology 0 36712013