MIC5

TgMIC5 is a parvulin-family peptidyl-prolyl isomerase-like protein of Toxoplasma gondii micronemes that regulates the surface proteolysis of micronemal invasion proteins during host-cell entry (PMID:11087916, PMID:16980407). It is synthesized as a preproprotein cleaved by signal peptidase and then matured post-Golgi to a ~22 kDa form that is stored in the micronemes and secreted (PMID:11087916). The propeptide functions as a forward-targeting element required for exit from the ER and correct micronemal delivery, acting through an aliphatic residue (Leu at position +1) and functionally interchangeable with propeptides of other MICs and of Eimeria tenella MIC5, indicating a conserved aliphatic-residue-dependent targeting mechanism (PMID:17995454, PMID:21438967). Once secreted onto the parasite surface, MIC5 restrains proteolytic trimming of micronemal proteins (and GRA1): its genetic ablation enhances processing, and complementation or the inhibitor ALLN reverses this phenotype (PMID:16980407). The structural and mechanistic basis is that TgMIC5 mimics a subtilisin prodomain — its NMR structure includes a flexible C-terminal peptide poised to insert into the active site of the subtilisin protease TgSUB1 — acting as a potent inhibitor (~50-fold over ALLN) and being retained at the plasma membrane through direct physical interaction with membrane-anchored TgSUB1 (PMID:22896704).

1. 2000 High

   Establishing that MIC5 is a parvulin-family PPIase-like protein processed from a preproprotein and stored in micronemes defined it as a secreted micronemal protein of the invasion machinery.

   Evidence Molecular cloning, cell fractionation, secretion assays, immunofluorescence and immunoelectron microscopy in T. gondii tachyzoites

   PMID:11087916

   Open questions at the time

   • Molecular function of the protein on the surface not yet defined
   • Substrate or binding partner unknown at this stage
2. 2006 High

   Genetic ablation showed MIC5 suppresses surface proteolytic trimming of micronemal proteins, revealing a regulatory role over an invasion-associated protease rather than an adhesive one.

   Evidence Targeted gene knockout, complementation, ALLN inhibitor treatment, and surface biotinylation/western analysis of processing in T. gondii

   PMID:16980407

   Open questions at the time

   • Could not distinguish direct protease inhibition from substrate occlusion
   • Identity of the responsible protease (then termed MPP2) unresolved
   • Mechanism of surface retention despite lacking membrane signals unexplained
3. 2008 High

   Deletion and swap experiments demonstrated the propeptide is a forward-targeting element required for ER exit and micronemal delivery, explaining how proMIC5 is correctly routed.

   Evidence Deletion and site-directed mutagenesis, propeptide-swap with M2AP, pulse-chase/secretion assays and fractionation in T. gondii

   PMID:17995454

   Open questions at the time

   • Precise trafficking receptor recognizing the propeptide not identified
   • Post-Golgi maturation protease not defined
4. 2011 High

   Point mutagenesis pinpointed a Leu at position +1 as the key determinant of microneme targeting and showed the mechanism is conserved across MICs and across species, refining the targeting model.

   Evidence N-terminal deletion and point mutagenesis, cross-species propeptide swaps with EtMIC5, immunofluorescence trafficking assays in T. gondii

   PMID:21438967

   Open questions at the time

   • How the aliphatic residue is read out mechanistically remains unknown
   • The sorting machinery that recognizes it is uncharacterized
5. 2012 High

   The NMR structure and enzymatic assays resolved the mechanism: MIC5 mimics a subtilisin prodomain to directly and potently inhibit TgSUB1 and is retained on the surface via binding to it, unifying the earlier trimming and surface-retention observations.

   Evidence NMR structure determination, in vitro TgSUB1 protease activity assay with IC50 quantification, and co-immunoprecipitation in T. gondii

   PMID:22896704

   Open questions at the time

   • Whether the C-terminal peptide inserts into the TgSUB1 active site not directly visualized
   • In vivo consequences of the MIC5–TgSUB1 interaction for invasion efficiency not quantified
   • Relationship of PPIase-like fold to any isomerase activity unresolved

• Whether TgMIC5 possesses genuine peptidyl-prolyl isomerase enzymatic activity, and how its protease-inhibitory role integrates quantitatively into host-cell invasion, remain open.
• No demonstration of PPIase catalytic activity in the timeline
• Contribution to invasion efficiency in vivo not measured