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

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

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

Previously published sex ratio data for <i>Hydrobates pelagicus</i>.

<p>Previously published sex ratio data for <i>Hydrobates pelagicus</i>.</p

Sex ratios of <i>Hydrobates pelagicus</i> adults and chicks in different locations and years.

<p>All samples were sexed using DNA extracted from feathers, except for the storm-killed birds in Portugal (sexed by dissection) and the chicks sampled in France (sexed using DNA extracted from faeces - see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046330#s2" target="_blank">Methods</a>).</p

Sex ratio of <i>Hydrobates pelagicus</i> controlled in different countries or re-trapped in Portugal.

<p>Sex ratio of <i>Hydrobates pelagicus</i> controlled in different countries or re-trapped in Portugal.</p