Inhibition of Plasmepsin V activity demonstrates its essential role in protein export, PfEMP1 display, and survival of malaria parasites

The malaria parasite Plasmodium falciparum exports several hundred proteins into the infected erythrocyte that are involved in cellular remodeling and severe virulence. The export mechanism involves the Plasmodium export element (PEXEL), which is a cleavage site for the parasite protease, Plasmepsin V (PMV). The PMV gene is refractory to deletion, suggesting it is essential, but definitive proof is lacking. Here, we generated a PEXEL-mimetic inhibitor that potently blocks the activity of PMV isolated from P. falciparum and Plasmodium vivax. Assessment of PMV activity in P. falciparum revealed PEXEL cleavage occurs cotranslationaly, similar to signal peptidase. Treatment of P. falciparum-infected erythrocytes with the inhibitor caused dose-dependent inhibition of PEXEL processing as well as protein export, including impaired display of the major virulence adhesin, PfEMP1, on the erythrocyte surface, and cytoadherence. The inhibitor killed parasites at the trophozoite stage and knockdown of PMV enhanced sensitivity to the inhibitor, while overexpression of PMV increased resistance. This provides the first direct evidence that PMV activity is essential for protein export in Plasmodium spp. and for parasite survival in human erythrocytes and validates PMV as an antimalarial drug target

Colorectal cancer cell line proteomes are representative of primary tumors and predict drug sensitivity

Proteomics holds promise for individualizing cancer treatment. We analyzed to what extent the proteomic landscape of human colorectal cancer (CRC) is maintained in established CRC cell lines and the utility of proteomics for predicting therapeutic responses. Proteomic and transcriptomic analyses were performed on 44 CRC cell lines, compared against primary CRCs (n=95) and normal tissues (n=60), and integrated with genomic and drug sensitivity data. Cell lines mirrored the proteomic aberrations of primary tumors, in particular for intrinsic programs. Tumor relationships of protein expression with DNA copy number aberrations and signatures of post-transcriptional regulation were recapitulated in cell lines. The 5 proteomic subtypes previously identified in tumors were represented among cell lines. Nonetheless, systematic differences between cell line and tumor proteomes were apparent, attributable to stroma, extrinsic signaling, and growth conditions. Contribution of tumor stroma obscured signatures of DNA mismatch repair identified in cell lines with a hypermutation phenotype. Global proteomic data showed improved utility for predicting both known drug-target relationships and overall drug sensitivity as compared with genomic or transcriptomic measurements. Inhibition of targetable proteins associated with drug responses further identified corresponding synergistic or antagonistic drug combinations. Our data provide evidence for CRC proteomic subtype-specific drug responses. Proteomes of established CRC cell line are representative of primary tumors. Proteomic data tend to exhibit improved prediction of drug sensitivity as compared with genomic and transcriptomic profiles. Our integrative proteogenomic analysis highlights the potential of proteome profiling to inform personalized cancer medicine

Oval Cell Numbers in Human Chronic Liver Diseases Are Directly Related to Disease Severity

The risk of developing hepatocellular carcinoma is significantly increased in patients with genetic hemochromatosis, alcoholic liver disease, or chronic hepatitis C infection. The precise mechanisms underlying the development of hepatocellular carcinoma in these conditions are not well understood. Stem cells within the liver, termed oval cells, are involved in the pathogenesis of hepatocellular carcinoma in animal models and may be important in the development of hepatocellular carcinoma in human chronic liver diseases. The aims of this study were to determine whether oval cells could be detected in the liver of patients with genetic hemochromatosis, alcoholic liver disease, or chronic hepatitis C, and whether there is a relationship between the severity of the liver disease and the number of oval cells. Oval cells were detected using histology and immunohistochemistry in liver biopsies from patients with genetic hemochromatosis, alcoholic liver disease, or chronic hepatitis C. Oval cells were not observed in normal liver controls. Oval cell numbers increased significantly with the progression of disease severity from mild to severe in each of the diseases studied. We conclude that oval cells are frequently found in subjects with genetic hemochromatosis, alcoholic liver disease, or chronic hepatitis C. There is an association between severity of liver disease and increase in the number of oval cells consistent with the hypothesis that oval cell proliferation is associated with increased risk for development of hepatocellular carcinoma in chronic liver disease

\u3b2TrCP- and Rsk1/2-Mediated Degradation of BimEL Inhibits Apoptosis

The BimEL tumor suppressor is a potent proapop- totic BH3-only protein. We found that, in response to survival signals, BimEL was rapidly phosphory- lated on three serine residues in a conserved degron, facilitating binding and degradation via the F box protein bTrCP. Phosphorylation of the BimEL degron was executed by Rsk1/2 and promoted by the Erk1/2-mediated phosphorylation of BimEL on Ser69. Compared to wild-type BimEL, a BimEL phos- phorylation mutant unable to bind bTrCP was stabi- lized and consequently potent at inducing apoptosis by the intrinsic mitochondrial pathway. Moreover, although non-small cell lung cancer (NSCLC) cells often become resistant to gefitinib (a clinically rele- vant tyrosine kinase inhibitor that induces apoptosis through BimEL), silencing of either bTrCP or Rsk1/2 resulted in BimEL-mediated apoptosis of both gefiti- nib-sensitive and gefitinib-insensitive NSCLC cells. Our findings reveal that bTrCP promotes cell survival in cooperation with the ERK-RSK pathway by target- ing BimEL for degradation

Structure activity refinement of phenylsulfonyl piperazines as antimalarials that block erythrocytic invasion

The emerging resistance to combination therapies comprised of artemisinin derivatives has driven a need to identify new antimalarials with novel mechanisms of action. Central to the survival and proliferation of the malaria parasite is the invasion of red blood cells by Plasmodium merozoites, providing an attractive target for novel therapeutics. A screen of the Medicines for Malaria Venture Pathogen Box employing transgenic P. falciparum parasites expressing the nanoluciferase bioluminescent reporter identified the phenylsulfonyl piperazine class as a specific inhibitor of erythrocyte invasion. Here, we describe the optimization and further characterization of the phenylsulfonyl piperazine class. During the optimization process we defined the functionality required for P. falciparum asexual stage activity and determined the alpha-carbonyl S-methyl isomer was important for antimalarial potency. The optimized compounds also possessed comparable activity against multidrug resistant strains of P. falciparum and displayed weak activity against sexual stage gametocytes. We determined that the optimized compounds blocked erythrocyte invasion consistent with the asexual activity observed and therefore the phenylsulfonyl piperazine analogues described could serve as useful tools for studying Plasmodium erythrocyte invasion.William Nguyena, Madeline G. Dansc, Anna Ngoa, Maria R. Ganchevaf, Ornella Romeof, Sandra Duffy ... et al

WEHI-916 is lethal to <i>P. falciparum</i> 3D7.

<p>(A) Dose-response curves of <i>P. falciparum</i> 3D7 in the presence of 916, 024, or 025. EC₅₀ values are shown. (B) Parasitemia measured at 72 h (<i>y</i>-axis) following drug treatment at rings (30 min postinvasion) and replacement of the medium with inhibitor-free medium (wash-out) at the time intervals shown (<i>x</i>-axis). (C) Parasitemia at 72 h (<i>y</i>-axis) after replacement of inhibitor-free medium with media containing compounds at the intervals shown (<i>x</i>-axis). Parasitemia was determined by FACS in (A–C) and is relative to DMSO treatment in (B) and (C). Concentrations are as follows: 916, 024, 025 (15 µM); CQ, chloroquine (150 ng/ml); ART, artemisinin (100 ng/ml). Error bars in (A–C) are mean ±SEM from duplicate experiments. (D) Light micrographs of Giemsa-stained parasites 16 and 32 h after drug treatment at early rings (15 µM). 916-treated parasites failed to develop into trophozoites and did not recover. Ring parasites treated with E-64 (10 µM) <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001897#pbio.1001897-terKuile1" target="_blank">[22]</a> contained swollen food vacuoles (arrow) due to inhibition of proteases involved in hemoglobin degradation; however, treatment with DMSO, 916, 024, or 025 did not cause swelling. Swelling was quantified using 500 infected cells per condition in duplicate. Scale bar is 6 µm.</p

PMV conservation and expression.

<p>(A) Structure and size of PMVHA proteins used in this study. Catalytic dyad residues DTG/DSG are shown including Asp to Ala mutations* in red. TM, transmembrane domain. (B) Immunoblot of infected erythrocytes with α-HA antibodies shows expression of PMVHA proteins in <i>P. falciparum</i>. Sizes indicate that the signal peptides were removed (PfPMVHA, 69.1 kDa; PvPMVHA, 60.9 kDa). (C) Immunoblotting of infected erythrocytes with rabbit α-PfPMV antibodies (Rα-PfPMV) validates they are specific for PfPMV. Endogenous PfPMV is the lower band (lanes 1, 3, 4, 5), and the larger band corresponds to 3× HA-tagged PfPMV (lanes 2, 4). Aldolase is a loading control in (B) and (C) and shows slight overloading of some lanes compared to others. (D, Top) Immunofluorescence micrographs show rabbit α-PfPMV antibodies (Rα-PfPMV, green) label PfPMV in the ER. Colocalizations were performed with mouse α-PfPMV antibodies (Mα-PfPMV, red), shown previously to label PMV in the ER <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001897#pbio.1001897-Klemba1" target="_blank">[16]</a>. (Middle) α-HA antibodies (red) label PfPMVHA (Top) and PvPMVHA (Bottom) in the parasite ER. (Bottom) α-HA antibodies (red) label PvPMVHA in the ER, as shown by clocalization with ERC (green). (E) Immunopurified PfPMVHA and PvPMVHA cleave KAHRP peptides containing the PEXEL sequence RTLAQ but not peptides containing point mutations R>K, L>I, or RL>A. Pf and Pv PMVmutHA proteins with catalytic D>A mutations did not cleave the KAHRP RTLAQ peptide. (F) Overexpression of PfPMVmutHA from episomes in <i>P. falciparum</i> 3D7 impairs growth relative to expression of a similar episomal construct encoding a mini PfEMP1HA reporter (miniVarHA). Parasites expressing episomes were selected on 5 nM WR99210 (WR). Two starting inocula were used in triplicate wells, and parasitaemia was determined at 72 h. *<i>p</i><.0001 (<i>t</i> test). Data are mean ± SEM from duplicate experiments.</p

WEHI-916 blocks PEXEL cleavage in <i>P. falciparum</i>.

<p>(A) Immunoblotting with α-GFP antibodies shows dose-dependent inhibition of PEXEL cleavage of PfEMP3-GFP in parasites after 5 h 916 treatment at the indicated concentrations. Uncleaved protein (black arrow), PEXEL-cleaved protein (blue arrow), and degraded chimera in food vacuole (GFP only) are labeled. PfEMP3-GFP R>A PEXEL mutant is shown as a size control. (B) Immunoblot shows time-dependent inhibition of PEXEL cleavage in parasites. HSP70 is a loading control in (A) and (B) and densitometry of the uncleaved band in each lane is shown below the blots in (A) and (B). (C) Immunoblot shows 916 treatment (20 and 50 µM for 5 h) causes accumulation of uncleaved PfEMP3-GFP and KAHRP-GFP (black arrow), but 024 and 025 have no effect. R>A PEXEL mutant size controls are shown. Signal peptide-cleaved species of KAHRP-GFP, but not PfEMP3-GFP, can be seen (red arrow). (D) Immunoblotting shows no defect (black arrow indicates the predicted size of uncleaved protein; ∼30 kDa) in signal peptide cleavage (red arrow; 27 kDa) of SERA5s-GFP, which lacks a PEXEL, following treatment with 916, 024, or 025 (20 µM for 5 h). Aldolase was a loading control in (C) and (D). (E) ³⁵S-Methionine/Cysteine labeling of PfEMP3-GFP in parasites reveals the rapid rate of translation, ER import, and N-terminal processing in <i>P. falciparum</i>. Uncleaved (black arrow), signal peptide-cleaved (red arrow), PEXEL-cleaved (blue arrow), and GFP-only (food vacuole) species are shown. Densitometry of each band per lane (colored traces) is shown. Uncleaved (Un, black), signal peptide-cleaved (SP, red), and PEXEL-cleaved (PEX, blue) bands are shown as percentage of total intensity per lane. Signal peptide-cleaved PfEMP3-GFP can be seen (red arrow, trace). (F) The experiment in (E) was performed after 916 treatment (20 µM for 5 h). Uncleaved protein was most abundant. Note low intensity of bands indicated by red and blue arrows and delay in their appearance compared to (E). (G) Densitometry showing the ratio of PEXEL-cleaved to -uncleaved PfEMP3-GFP with or without 916 treatment (determined using 15 min lanes in (E) and (F)).</p

A PEXEL-mimetic inhibitor of PMV.

<p>(A) Nomenclature for each residue in the PEXEL substrate (circles) and each pocket of the PMV active site (semicircles) with respect to the cleavage site (arrow). (B) Compound structures in this study include PMV inhibitor WEHI-916 and control analogs WEHI-024 and WEHI-025. (C) Structural model of PfPMV bound to WEHI-916. Residues forming the S₃ site, Try177, Glu179, and Glu215, form interactions with the guanidine side chain of WEHI-916. The Leu side chain of WEHI-916 packs tightly against the side-chain groups of Ile116, Tyr177, and Val227 in the S₁ site. The statine hydroxyl forms hydrogen bonds with the two catalytic aspartate residues Asp118 and Asp365. (D) Inhibition of PfPMV and PvPMV by WEHI-916 (blue) and weak activities of WEHI-024 (red) and WEHI-025 (brown). The grey box summarizes compound activity against BACE-1 and Cathepsin D (CathD) and lack of toxicity against human HEpG2 cells and Human Foreskin Fibroblasts (HFF).</p

PMV inhibition impairs protein export, PfEMP1 display, and cytoadherence.

<p>(A) (Top) Immunofluorescent micrographs show Hyp8-GFP is exported and localizes within puncta in the infected erythrocyte. (Middle) Hyp8-HA localizes within SBP1-containing MCs. (Right) Immunoelectron microscopy shows Hyp8-HA localization at MCs. Scale bar is 100 nm. (B) Maximum intensity projection micrographs showing export of Hyp8-GFP to MCs and secretion of EXP2 to the parasitophorous vacuole membrane following treatment with DMSO or 916 (50 µM). Puncta of nonexported GFP within the parasite and vacuole is shown (arrows). (C) GFP intensity in MCs (outside EXP2 signal) and within the parasite and parasitophorous vacuole (inside EXP2 signal) was quantified following drug treatment (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001897#s4" target="_blank">Materials and Methods</a>) and is presented as a ratio: exported = ratio outside/inside EXP2. The number of infected cells counted (<i>n</i>) is shown. (D) GFP within the parasite and parasitophorous vacuole (inside EXP2 signal) was quantified across treatments and is presented as a ratio: nonexported = ratio inside/outside EXP2. (E) EXP2 intensity in the parasitophorous vacuole membrane (red) was quantified across treatments and is presented as average (threshold) signal. (F) The number of GFP-positive MCs per infected erythrocyte was quantified across treatments. Error bars represent the mean ±SEM, and <i>p</i> values were determined by ANOVA in (C–F). (G) Surface-exposed PfEMP1 (VAR2CSA) on infected erythrocytes was measured following inhibitor treatment by FACS using monoclonal human PAM1.4 serum <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001897#pbio.1001897-Barfod1" target="_blank">[30]</a>. Geometric mean fluorescence of >100,000 cells per condition are shown relative to no treatment. Parasites received inhibitor at 15 µM for 23 h (15 µM) or 50 µM for 12 h followed by a reduction to 15 µM for 11 h (50>15 µM). Shown is a single representative of duplicate experiments. Raw FACS data are presented in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001897#pbio.1001897.s007" target="_blank">Figure S7</a>. (H) Adhesion of infected red blood cells (iRBCs) to chondroitin sulfate A (CSA) under static conditions. Adherent iRBCs were counted in ten 0.28 mm² fields of view per sample from triplicate samples and are shown as number of iRBCs per mm². Shown is a single representative of duplicate experiments. Data represent mean ±SEM. 95% confidence intervals are shown (grey dashed lines). 916 significantly reduced adhesion to CSA <i>p</i><.0001.</p