MOESM2 of Murine Gbp1 and Gbp2 are ubiquitinated independent of Toxoplasma gondii infection

Additional file 2: Table S1. List of all ubiquitinated protein sites identified in Toxoplasma-infected MEFs. Maxquant processed data taken from the modifiedpeptides.txt output table. Log2 normalised ratios for the indicated peptide sequences are displayed

MOESM1 of Murine Gbp1 and Gbp2 are ubiquitinated independent of Toxoplasma gondii infection

Additional file 1. Additional Methods

p97/VCP targets Toxoplasma gondii vacuoles for parasite restriction in interferon-stimulated human cells

Infection with the parasite Toxoplasma gondii leads to production of interferon gamma (IFNγ) that stimulates cells to upregulate defense proteins targeting the parasite for cell intrinsic elimination or growth restriction. Various host defense mechanisms operate at the parasitophorous vacuole (PV) in different human cell types leading to PV disruption, acidification, or membrane envelopment. Ubiquitin and p62 are players in all human host control mechanisms of Toxoplasma, but other unifying proteins have not been identified. Here, we show that p97/valosin-containing protein (VCP), as well as its associated proteins ANKRD13A and UBXD1 control Toxoplasma infection while recruited to the PV in IFNγ-stimulated endothelial cells. Convergent deposition of ANKRD13A, p97/VCP, and UBXD1 onto the same vacuole is dependent on vacuolar ubiquitination and observed within 2 h post-infection. ANKRD13A, p97/VCP, and UBXD1 all drive the acidification mechanism of the vacuole, which is the IFNγ-dependent control pathway of Toxoplasma in endothelial cells. We assessed p97/VCP in Toxoplasma control in various human cells and demonstrate that p97/VCP is a universal IFNγ-dependent host restriction factor targeting the Toxoplasma PV in epithelial (HeLa) and endothelial cells (human umbilical vein endothelial cells), fibroblasts (human foreskin fibroblast), and macrophages (THP1)

Labelling of cysteine cathepsins in mammalian cells.

(A) Lysates from RAW macrophages were treated with 1 μM of the indicated ABPs for 30 min. (B) Live RAW cells were treated with 1 μM of the indicated ABPs for 3 h. Samples were run on a SDS-PAGE gel, and in-gel fluorescence measured using a fluorescence scanner. The identity of the different cysteine cathepsins are indicated with different coloured arrowheads.</p

Novel broad-spectrum activity-based probes to profile malarial cysteine proteases.

Clan CA cysteine proteases, also known as papain-like proteases, play important roles throughout the malaria parasite life cycle and are therefore potential drug targets to treat this disease and prevent its transmission. In order to study the biological function of these proteases and to chemically validate some of them as viable drug targets, highly specific inhibitors need to be developed. This is especially challenging given the large number of clan CA proteases present in Plasmodium species (ten in Plasmodium falciparum), and the difficulty of designing selective inhibitors that do not cross-react with other members of the same family. Additionally, any efforts to develop antimalarial drugs targeting these proteases will also have to take into account potential off-target effects against the 11 human cysteine cathepsins. Activity-based protein profiling has been a very useful tool to determine the specificity of inhibitors against all members of an enzyme family. However, current clan CA proteases broad-spectrum activity-based probes either target endopeptidases or dipeptidyl aminopeptidases, but not both subfamilies efficiently. In this study, we present a new series of dipeptydic vinyl sulfone probes containing a free N-terminal tryptophan and a fluorophore at the P1 position that are able to label both subfamilies efficiently, both in Plasmodium falciparum and in mammalian cells, thus making them better broad-spectrum activity-based probes. We also show that some of these probes are cell permeable and can therefore be used to determine the specificity of inhibitors in living cells. Interestingly, we show that the choice of fluorophore greatly influences the specificity of the probes as well as their cell permeability

Labelling of cysteine proteases in parasite lysates.

(A) Merozoite lysates diluted 1:10 in acetate buffer were treated for 1 h with 1–1000 nM of the indicated ABPs. For the highest ABP concentration, samples were also pre-treated for 30 min with 1 μM of the DPAP3 inhibitor SAK1, which results in the loss of labelling of the three isoforms of DPAP3 running at 120, 95, and 42 kDa. (B-D) Lysates collected at merozoite (B), trophozoite (C), or schizont (D) stages were diluted in acetate buffer (pH 5.5), pre-treated for 30 min with DMSO or 10 μM of different known covalent inhibitors of DPAP1 (SAK2), DPAP3 (SAK1 or W-hPG-VS), the FPs (E64), or the negative control compound D-W-hPG-VS. This was followed by 1 h labelling with the different ABPs at 0.1 μM except for DCG04 that was used at 1 μM concentration. (A-D) The fluorescent bands corresponding to DPAP1, DPAP3, FP1, and FP2/3 are indicated by blue, red, light green, and dark green arrowheads, respectively. Two additional biological replicates of these experiments are shown in S3 Fig.</p

Structures of inhibitors and ABPs.

(A) Structures of inhibitors used in this study. All the new ABPs described in this paper were synthesized by conjugating different azido-tags (B, fluorophore or D, a biotin/TAMRA bifunctional tag) to the alkyne group of W-hPG-VS. (C) Structures of previously published ABPs used in this study for comparison purposes.</p

Comparison of labelling profiles of W-sCy5-VS and W-BF-VS.

(A) Schizont lysates were treated for 1 h either with 0.5 μM of W-sCy5-VS, W-BF-VS, or a mixture of both probes, each at 0.5 μM (Mix). After running the samples in a SDS-PAGE gel, the gel was scanned either in the Cy5 and Cy3 channels. The composite image shows very similar labelling profiles for both probes and a clear co-migration of the labelled bands in the Mix sample. (B) Coomassie staining of the gel shown in A showing equal protein loading. (C) Quantification of the labelling profiles for each probe by densitometry. Fluorescent intensity vs. migration distance (Rf) is shown. The position of FP2/3 and DPAP1 are indicated in A and C. Two additional biological replicates of this experiment are shown in S5 Fig.</p

Top ten enriched proteins using the W-BF-VS probe.

Top ten enriched proteins using the W-BF-VS probe.</p

Labelling of cysteine protease in live parasites.

Very mature schizonts were diluted ten-fold in RPMI and treated with different concentrations of probes for 1 h. Samples were run on a SDS-PAGE gel, and the labelled proteins detected using at fluorescence scanner. Bands corresponding to DPAP1, DPAP3, FP1, and FP2/3 are indicated by blue, red, light green, and dark green arrowheads, respectively. Two additional biological replicates of this experiment are shown in S4 Fig.</p