| 2015 |
ASP5 is a Golgi-resident aspartyl protease that cleaves GRA16, GRA19, and GRA20 at their PEXEL-like (TEXEL) motifs; in the absence of ASP5, these GRAs fail to localize correctly to the parasitophorous vacuole membrane and GRA16/GRA24 are retained within the vacuolar space rather than reaching the host nucleus. |
Genetic deletion (Δasp5), epitope-tagging, immunofluorescence localization, and in vitro cleavage assays |
PLoS pathogens |
High |
26473595
|
| 2015 |
TgASP5 directly mediates proteolytic cleavage of GRA16 at its N-terminal PEXEL-like motif (RRLAE), and this cleavage is required for GRA16 export into the host cell; GRA24 export is also ASP5-dependent but its maturation/cleavage is ASP5-independent, indicating ASP5 has multiple export-related functions. |
N- and C-terminal epitope tagging to detect proteolysis, genetic deletion of TgASP5, western blot analysis of GRA16 processing |
Cellular microbiology |
High |
26270241
|
| 2015 |
ASP5 cleaves a highly constrained PEXEL-like motif (TEXEL, RRL↓ consensus) at both N- and C-terminal ends of substrate proteins; ASP5 is Golgi-resident and also controls trafficking of effectors that lack this motif. ASP5 is required for establishment of the intravacuolar nanotubular network and for recruitment of host mitochondria to the vacuole. |
Genetic deletion (Δasp5), mass spectrometry of cleavage products, immunofluorescence, electron microscopy, host transcriptome analysis |
eLife |
High |
26576949
|
| 2016 |
MYR1 requires TgASP5 for its proteolytic cleavage into two stable fragments, both of which localize to the parasitophorous vacuole and the PV membrane; MYR1 is essential for GRA16 and GRA24 translocation into the host cell. |
Whole-genome sequencing of mutants, genetic deletion, western blot for cleavage products, immunofluorescence localization |
mBio |
High |
26838724
|
| 2018 |
ASP5 substrates identified by TAILS N-terminomics are exclusively dense granule proteins; validated substrates reside within the PV or PVM (not exported to the host cytosol), including two kinases (WNG1, WNG2) and one phosphatase, and are not processed in Δasp5 parasites or when the RRL motif is mutated to ARL. |
Terminal Amine Isotopic Labeling of Substrates (TAILS) N-terminomics comparing WT and Δasp5, site-directed mutagenesis (RRL→ARL), immunofluorescence, mouse virulence model |
mBio |
High |
30377279
|
| 2018 |
RNA-Seq of host cells infected with Δasp5 versus Δmyr1 tachyzoites shows that MYR1 function does not require ASP5 cleavage, indicating that a subset of effectors require only MYR1 (not ASP5) for export; ASP5 and MYR1 thus define partially overlapping but distinct export requirements. |
RNA-Seq transcriptome comparison of RH-WT, RHΔmyr1, and RHΔasp5 infections of human foreskin fibroblasts |
mBio |
Medium |
29615509
|
| 2018 |
MYR1, which is cleaved by ASP5 into two disulfide-linked fragments, localizes to the PVM and is part of a novel protein complex with MYR2 and MYR3 required for GRA effector translocation; MYR3 but not MYR2 stably associates with MYR1, and all three are membrane-associated. |
Co-immunoprecipitation, immunofluorescence, western blot (disulfide linkage), genetic deletion, GRA16/GRA24 nuclear trafficking assays |
PLoS pathogens |
High |
29357375
|
| 2020 |
ASP5 is required for translocation of GRA16 into host cells and for upregulation of host c-Myc and cyclin E1; GRA44 and GRA45, two MYR1-interacting proteins that contain ASP5 processing motifs, are essential for effector translocation, although ASP5 processing at these sites appears nonessential for their translocation function. |
Affinity purification/Co-IP of MYR1, genetic deletion, GRA16 nuclear translocation assay, c-Myc and cyclin E1 upregulation assays |
mSphere |
Medium |
32075880
|
| 2022 |
In bradyzoites, ASP5 cleaves novel substrates identified by TAILS N-terminomics; Δasp5 bradyzoites are impaired in cyst wall formation in vitro and show significantly reduced cyst number and size in chronically infected mouse brains, establishing ASP5 as required for the establishment of the chronic infection niche. |
N-terminal amine isotopic labeling of substrates (TAILS) comparing WT and Δasp5 tachyzoites and bradyzoites, immunofluorescence, two-photon serial tomography of infected mouse brains |
Molecular microbiology |
High |
36210525
|
| 2024 |
Computational structural modeling of TgASP5 reveals a pepsin-like aspartyl protease active site; residues Ser505, Ala776, and Tyr689 in the S2 binding pocket confer specificity for Arg at the P2 position of the TEXEL substrate; the known inhibitor RRLStatine blocks the active site by forming a hydrogen bond with a catalytic aspartate. |
Molecular modeling, all-atom MD simulation, molecular docking, MM-PBSA binding energy calculations |
Journal of biomolecular structure & dynamics |
Low |
38424737
|
| 2025 |
GRA84 export to the host nucleus is dependent on TgASP5; GRA84 undergoes a separate N-terminal processing event (removing >90 amino acids) that is ASP5-independent, and mutagenesis shows this maturation is critical for transit across the vacuolar membrane via the MYR translocon. |
Genetic deletion of ASP5, site-directed mutagenesis of processing sites, epitope tagging, immunofluorescence localization |
Molecular biology of the cell |
Medium |
40464784
|
| 2018 |
Deletion of ASP5 or MYR1 does not alter the inhibition of nitric oxide production in activated J774-A1 or RAW 264.7 macrophages infected with T. gondii, indicating this immune evasion phenotype is ASP5/MYR1-independent. |
Genetic deletion of ASP5 and MYR1, measurement of NO production and iNOS expression in infected macrophage lines |
Frontiers in microbiology |
Medium |
30177926
|