Comparative Proteomic Analysis of Toxoplasma gondii RH Wild-Type and Four SRS29B (SAG1) Knock-Out Clones Reveals Significant Differences between Individual Strains.

In T. gondii, as well as in other model organisms, gene knock-out using CRISPR-Cas9 is a suitable tool to identify the role of specific genes. The general consensus implies that only the gene of interest is affected by the knock-out. Is this really the case? In a previous study, we generated knock-out (KO) clones of TgRH88_077450 (SRS29B; SAG1) which differed in the numbers of the integrated dihydrofolate-reductase-thymidylate-synthase (MDHFR-TS) drug-selectable marker. Clones 18 and 33 had a single insertion of MDHFR-TS within SRS29B. Clone 6 was disrupted by the insertion of a short unrelated DNA-sequence, but the marker was integrated elsewhere. In clone 30, the marker was inserted into SRS29B, and several other MDHFR-TS copies were found in the genome. KO and wild-type (WT) tachyzoites had similar shapes, dimensions, and vitality. This prompted us to investigate the impact of genetic engineering on the overall proteome patterns of the four clones as compared to the respective WT. Comparative shotgun proteomics of the five strains was performed. Overall, 3236 proteins were identified. Principal component analysis of the proteomes revealed five distinct clusters corresponding to the five strains by both iTop3 and iLFQ algorithms. Detailed analysis of the differentially expressed proteins revealed that the target of the KO, srs29B, was lacking in all KO clones. In addition to this protein, 20 other proteins were differentially expressed between KO clones and WT or between different KO clones. The protein exhibiting the highest variation between the five strains was srs36D encoded by TgRH_016110. The deregulated expression of SRS36D was further validated by quantitative PCR. Moreover, the transcript levels of three other selected SRS genes, namely SRS36B, SRS46, and SRS57, exhibited significant differences between individual strains. These results indicate that knocking out a given gene may affect the expression of other genes. Therefore, care must be taken when specific phenotypes are regarded as a direct consequence of the KO of a given gene.

Computed tomography-based radiomics decodes prognostic and molecular differences in interstitial lung disease related to systemic sclerosis.

BACKGROUND: Radiomic features calculated from routine medical images show great potential for personalised medicine in cancer. Patients with systemic sclerosis (SSc), a rare, multiorgan autoimmune disorder, have a similarly poor prognosis due to interstitial lung disease (ILD). Here, our objectives were to explore computed tomography (CT)-based high-dimensional image analysis ("radiomics") for disease characterisation, risk stratification and relaying information on lung pathophysiology in SSc-ILD. METHODS: We investigated two independent, prospectively followed SSc-ILD cohorts (Zurich, derivation cohort, n=90; Oslo, validation cohort, n=66). For every subject, we defined 1355 robust radiomic features from standard-of-care CT images. We performed unsupervised clustering to identify and characterise imaging-based patient clusters. A clinically applicable prognostic quantitative radiomic risk score (qRISSc) for progression-free survival (PFS) was derived from radiomic profiles using supervised analysis. The biological basis of qRISSc was assessed in a cross-species approach by correlation with lung proteomic, histological and gene expression data derived from mice with bleomycin-induced lung fibrosis. RESULTS: Radiomic profiling identified two clinically and prognostically distinct SSc-ILD patient clusters. To evaluate the clinical applicability, we derived and externally validated a binary, quantitative radiomic risk score (qRISSc) composed of 26 features that accurately predicted PFS and significantly improved upon clinical risk stratification parameters in multivariable Cox regression analyses in the pooled cohorts. A high qRISSc score, which identifies patients at risk for progression, was reverse translatable from human to experimental ILD and correlated with fibrotic pathway activation. CONCLUSIONS: Radiomics-based risk stratification using routine CT images provides complementary phenotypic, clinical and prognostic information significantly impacting clinical decision making in SSc-ILD.

Effect of Sample Transportation on the Proteome of Human Circulating Blood Extracellular Vesicles.

Circulating extracellular vesicles (cEV) are released by many kinds of cells and play an important role in cellular communication, signaling, inflammation modulation, coagulation, and tumor growth. cEV are of growing interest, not only as biomarkers, but also as potential treatment targets. However, very little is known about the effect of transporting biological samples from the clinical ward to the diagnostic laboratory, notably on the protein composition. Pneumatic tube systems (PTS) and human carriers (C) are both routinely used for transport, subjecting the samples to different ranges of mechanical forces. We therefore investigated qualitatively and quantitatively the effect of transport by C and PTS on the human cEV proteome and particle size distribution. We found that samples transported by PTS were subjected to intense, irregular, and multidirectional shocks, while those that were transported by C mostly underwent oscillations at a ground frequency of approximately 4 Hz. PTS resulted in the broadening of nanoparticle size distribution in platelet-free (PFP) but not in platelet-poor plasma (PPP). Cell-type specific cEV-associated protein abundances remained largely unaffected by the transport type. Since residual material of lymphocytes, monocytes, and platelets seemed to dominate cEV proteomes in PPP, it was concluded that PFP should be preferred for any further analyses. Differential expression showed that the impact of the transport method on cEV-associated protein composition was heterogeneous and likely donor-specific. Correlation analysis was nonetheless able to detect that vibration dose, shocks, and imparted energy were associated with different terms depending on the transport, namely in C with cytoskeleton-regulated cell organization activity, and in PTS with a release of extracellular vesicles, mainly from organelle origin, and specifically from mitochondrial structures. Feature selection algorithm identified proteins which, when considered together with the correlated protein-protein interaction network, could be viewed as surrogates of network clusters.

Common Molecular Targets of a Quinolone Based Bumped Kinase Inhibitor in Neospora caninum and Danio rerio.

Neospora caninum is an apicomplexan parasite closely related to Toxoplasma gondii, and causes abortions, stillbirths and/or fetal malformations in livestock. Target-based drug development has led to the synthesis of calcium-dependent protein kinase 1 inhibitors, collectively named bumped kinase inhibitors (BKIs). Previous studies have shown that several BKIs have excellent efficacy against neosporosis in vitro and in vivo. However, several members of this class of compounds impair fertility in pregnant mouse models and cause embryonic malformation in a zebrafish (Danio rerio) model. Similar to the first-generation antiprotozoal drug quinine, some BKIs have a quinoline core structure. To identify common targets in both organisms, we performed differential affinity chromatography with cell-free extracts from N. caninum tachyzoites and D. rerio embryos using the 5-aminopyrazole-4-carboxamide (AC) compound BKI-1748 and quinine columns coupled to epoxy-activated sepharose followed by mass spectrometry. BKI-binding proteins of interest were identified in eluates from columns coupled to BKI-1748, or in eluates from BKI-1748 as well as quinine columns. In N. caninum, 12 proteins were bound specifically to BKI-1748 alone, and 105 proteins, including NcCDPK1, were bound to both BKI-1748 and quinine. For D. rerio, the corresponding numbers were 13 and 98 binding proteins, respectively. In both organisms, a majority of BKI-1748 binding proteins was involved in RNA binding and modification, in particular, splicing. Moreover, both datasets contained proteins involved in DNA binding or modification and key steps of intermediate metabolism. These results suggest that BKI-1748 interacts with not only specific targets in apicomplexans, such as CDPK1, but also with targets in other eukaryotes, which are involved in common, essential pathways.

Inference of kinase-signaling networks in human myeloid cell line models by Phosphoproteomics using kinase activity enrichment analysis (KAEA).

BACKGROUND: Despite the introduction of targeted therapies, most patients with myeloid malignancies will not be cured and progress. Genomics is useful to elucidate the mutational landscape but remains limited in the prediction of therapeutic outcome and identification of targets for resistance. Dysregulation of phosphorylation-based signaling pathways is a hallmark of cancer, and therefore, kinase-inhibitors are playing an increasingly important role as targeted treatments. Untargeted phosphoproteomics analysis pipelines have been published but show limitations in inferring kinase-activities and identifying potential biomarkers of response and resistance. METHODS: We developed a phosphoproteomics workflow based on titanium dioxide phosphopeptide enrichment with subsequent analysis by liquid chromatography tandem mass spectrometry (LC-MS). We applied a novel Kinase-Activity Enrichment Analysis (KAEA) pipeline on differential phosphoproteomics profiles, which is based on the recently published SetRank enrichment algorithm  with reduced false positive rates. Kinase activities were inferred by this algorithm using an extensive reference database comprising five experimentally validated kinase-substrate meta-databases complemented with the NetworKIN in-silico prediction tool. For the proof of concept, we used human myeloid cell lines (K562, NB4, THP1, OCI-AML3, MOLM13 and MV4-11) with known oncogenic drivers and exposed them to clinically established kinase-inhibitors. RESULTS: Biologically meaningful over- and under-active kinases were identified by KAEA in the unperturbed human myeloid cell lines (K562, NB4, THP1, OCI-AML3 and MOLM13). To increase the inhibition signal of the driving oncogenic kinases, we exposed the K562 (BCR-ABL1) and MOLM13/MV4-11 (FLT3-ITD) cell lines to either Nilotinib or Midostaurin kinase inhibitors, respectively. We observed correct detection of expected direct (ABL, KIT, SRC) and indirect (MAPK) targets of Nilotinib in K562 as well as indirect (PRKC, MAPK, AKT, RPS6K) targets of Midostaurin in MOLM13/MV4-11, respectively. Moreover, our pipeline was able to characterize unexplored kinase-activities within the corresponding signaling networks. CONCLUSIONS: We developed and validated a novel KAEA pipeline for the analysis of differential phosphoproteomics MS profiling data. We provide translational researchers with an improved instrument to characterize the biological behavior of kinases in response or resistance to targeted treatment. Further investigations are warranted to determine the utility of KAEA to characterize mechanisms of disease progression and treatment failure using primary patient samples.

Cellular and Molecular Targets of Nucleotide-Tagged Trithiolato-Bridged Arene Ruthenium Complexes in the Protozoan Parasites Toxoplasma gondii and Trypanosoma brucei.

Toxoplasma gondii is an apicomplexan parasite that infects and proliferates within many different types of host cells and infects virtually all warm-blooded animals and humans. Trypanosoma brucei is an extracellular kinetoplastid that causes human African trypanosomiasis and Nagana disease in cattle, primarily in rural sub-Saharan Africa. Current treatments against both parasites have limitations, e.g., suboptimal efficacy and adverse side effects. Here, we investigate the potential cellular and molecular targets of a trithiolato-bridged arene ruthenium complex conjugated to 9-(2-hydroxyethyl)-adenine (1), which inhibits both parasites with IC50s below 10-7 M. Proteins that bind to 1 were identified using differential affinity chromatography (DAC) followed by shotgun-mass spectrometry. A trithiolato-bridged ruthenium complex decorated with hypoxanthine (2) and 2-hydroxyethyl-adenine (3) were included as controls. Transmission electron microscopy (TEM) revealed distinct ultrastructural modifications in the mitochondrion induced by (1) but not by (2) and (3) in both species. DAC revealed 128 proteins in T. gondii and 46 proteins in T. brucei specifically binding to 1 but not 2 or 3. In T. gondii, the most abundant was a protein with unknown function annotated as YOU2. This protein is a homolog to the human mitochondrial inner membrane translocase subunit Tim10. In T. brucei, the most abundant proteins binding specifically to 1 were mitochondrial ATP-synthase subunits. Exposure of T. brucei bloodstream forms to 1 resulted in rapid breakdown of the ATP-synthase complex. Moreover, both datasets contained proteins involved in key steps of metabolism and nucleic acid binding proteins.

Production of neutralizing antibodies against the secreted Clostridium chauvoei toxin A (CctA) upon blackleg vaccination.

Clostridium chauvoei is the etiologic agent of blackleg in cattle, inducing fever, severe myonecrosis, oedemic lesions and ultimately death of infected animals. The pathogen often results in such rapid death that antibiotic therapy is futile and thus vaccination is the only efficient strategy in order to control the disease. The ß-barrel pore forming leucocidin Clostridium chauvoei toxin A (CctA) is one of the best characterised toxins of C. chauvoei and has been shown to be an important virulence factor. It has been reported to induce protective immunity and is conserved across C. chauvoei strains collected from diverse geographical locations for more than 50 years. The aim of this study was to identify the location of the CctA toxin during liquid culture fermentation and to use CctA to develop an in vitro assay to replace the current guinea pig challenge assay for vaccine potency in standard batch release procedures. We report that CctA is fully secreted in C. chauvoei culture and show that it is found abundantly in the supernatant of liquid cultures. Sera from cattle vaccinated with a commercial blackleg vaccine revealed strong haemolysin-neutralizing activity against recombinant CctA which reached titres of 1000 times 28 days post-vaccination. Similarly, guinea pig sera from an official potency control test reached titres of 600 times 14 days post-vaccination. In contrast, ELISA was not able to specifically measure anti-CctA antibodies in cattle serum due to strong cross-reactions with antibodies against other proteins present pre-vaccination. We conclude that haemolysin-neutralizing antibodies are a valuable measurement for protective immunity against blackleg and have the potential to be a suitable replacement of the guinea pig challenge potency test, which would forego the unnecessary challenge of laboratory animals.

Robust Label-free, Quantitative Profiling of Circulating Plasma Microparticle (MP) Associated Proteins.

Cells of the vascular system release spherical vesicles, called microparticles, in the size range of 0.1-1 µm induced by a variety of stress factors resulting in variable concentrations between health and disease. Furthermore, microparticles have intercellular communication/signaling properties and interfere with inflammation and coagulation pathways. Today's most used analytical technology for microparticle characterization, flow cytometry, is lacking sensitivity and specificity, which might have led to the publication of contradicting results in the past.We propose the use of nano-liquid chromatography two-stage mass spectrometry as a nonbiased tool for quantitative MP proteome analysis.For this, we developed an improved microparticle isolation protocol and quantified the microparticle protein composition of twelve healthy volunteers with a label-free, data-dependent and independent proteomics approach on a quadrupole orbitrap instrument.Using aliquots of 250 µl platelet-free plasma from one individual donor, we achieved excellent reproducibility with an interassay coefficient of variation of 2.7 ± 1.7% (mean ± 1 standard deviation) on individual peptide intensities across 27 acquisitions performed over a period of 3.5 months. We show that the microparticle proteome between twelve healthy volunteers were remarkably similar, and that it is clearly distinguishable from whole cell and platelet lysates. We propose the use of the proteome profile shown in this work as a quality criterion for microparticle purity in proteomics studies. Furthermore, one freeze thaw cycle damaged the microparticle integrity, articulated by a loss of cytoplasm proteins, encompassing a specific set of proteins involved in regulating dynamic structures of the cytoskeleton, and thrombin activation leading to MP clotting. On the other hand, plasma membrane protein composition was unaffected. Finally, we show that multiplexed data-independent acquisition can be used for relative quantification of target proteins using Skyline software. Mass spectrometry data are available via ProteomeXchange (identifier PXD003935) and panoramaweb.org (https://panoramaweb.org/labkey/N1OHMk.url).

Vitamin E and vitamin E acetate absorption from self-assembly systems under pancreas insufficiency conditions.

We determined the bioavailability of vitamin E from self-assembly structures in patients with diagnosed chronic pancreas insufficiency. Vitamin E solubilized in dispersed inverted bicontinuous cubic phase and in micellar formulation was delivered directly to the small intestine by tube-feeding. A cross-over study with randomization of 6 subjects and 2 treatments including a combined dose of 18 mg (27 IU) of vitamin E (RRR-[5,7-methyl-((2)H6)]-α-tocopherol) and 27 mg (27 IU) vitamin E acetate (RRR-[5-methyl-(2)H3]-α-tocopheryl acetate) was applied over a time period of 1 h. Plasma samples were collected for 56 h and analyzed by liquid chromatography-mass spectrometry. Appearance of labeled tocopherols originating from the treatment started at 25 h and reached Cmax (0.6-4.6 µM depending on subject) in the 7-9 h window. From the Tmax onwards, both forms of tocopherols diminished slowly to 30-50% of their maxima within 56 h. Strong inter-individual variation was observed in the plasma appearance curves (relative standard deviation varied between 38-45%). No significant discrimination was found between the absorption of free or acetylated forms of deuterated α-tocopherol confirming that application of acetylated α-tocopherol provides the same bioavailability as free α-tocopherol. This observation is valid in both dispersed inverted bicontinuous cubic phase and micellar formulations. Furthermore, since the area-under-the-curve values from cubic phase and from micellar formulations are similar, the cubic phase formulation could represent an alternative delivery system for lipophilic micronutrients in conditions or studies where polysorbate-based micelles cannot be generated.

The Aeromonas salmonicida subsp. salmonicida exoproteome: global analysis, moonlighting proteins and putative antigens for vaccination against furunculosis.

BACKGROUND: Aeromonas salmonicida subsp. salmonicida, the etiologic agent of furunculosis, is a major pathogen of fisheries worldwide. Despite the identification of several virulence factors the pathogenesis is still poorly understood. We have used high-throughput proteomics to display the differences between in vitro secretome of A. salmonicida wild-type (wt, hypervirulent, JF5054) and T3SS-deficient (isogenic ΔascV, extremely low-virulent, JF2747) strains in exponential (GP) and stationary (SP) phases of growth. RESULTS: Among the different experimental conditions we obtained semi-quantitative values for a total of 2136 A. salmonicida proteins. Proteins of specific A. salmonicida species were proportionally less detected than proteins common to the Aeromonas genus or those shared with other Aeromonas species, suggesting that in vitro growth did not induce the expression of these genes. Four detected proteins which are unidentified in the genome of reference strains of A. salmonicida were homologous to components of the conjugative T4SS of A. hydrophila pRA1 plasmid. Polypeptides of three proteins which are specific to the 01-B526 strain were also discovered. In supernatants (SNs), the number of detected proteins was higher in SP (326 for wt vs 329 for mutant) than in GP (275 for wt vs 263 for mutant). In pellets, the number of identified proteins (a total of 1536) was approximately the same between GP and SP. Numerous highly conserved cytoplasmic proteins were present in A. salmonicida SNs (mainly EF-Tu, EF-G, EF-P, EF-Ts, TypA, AlaS, ribosomal proteins, HtpG, DnaK, peptidyl-prolyl cis-trans isomerases, GAPDH, Enolase, FbaA, TpiA, Pgk, TktA, AckA, AcnB, Mdh, AhpC, Tpx, SodB and PNPase), and several evidences support the theory that their extracellular localization was not the result of cell lysis. According to the Cluster of Orthologous Groups classification, 29% of excreted proteins in A. salmonicida SNs were currently poorly characterized. CONCLUSIONS: In this part of our work we elucidated the whole in vitro exoproteome of hypervirulent A. salmonicida subsp. salmonicida and showed the secretion of several highly conserved cytoplasmic proteins with putative moonlighting functions and roles in virulence. All together, our results offer new information about the pathogenesis of furunculosis and point out potential candidates for vaccine development.

The Aeromonas salmonicida subsp. salmonicida exoproteome: determination of the complete repertoire of Type-Three Secretion System effectors and identification of other virulence factors.

BACKGROUND: Aeromonas salmonicida subsp. salmonicida, the etiologic agent of furunculosis, is a major pathogen of fisheries worldwide. Several virulence factors have been described, but the type-three secretion system (T3SS) is recognized as having a major effect on virulence by injecting effectors directly into fish cells. In this study we used high-throughput proteomics to display the differences between in vitro secretome of A. salmonicida wild-type (wt, hypervirulent, JF2267) and T3SS-deficient (isogenic ΔascV, extremely low-virulent, JF2747) strains in exponential and stationary phases of growth. RESULTS: Results confirmed the secretion of effectors AopH, AexT, AopP and AopO via T3SS, and for the first time demonstrated the impact of T3SS in secretion of Ati2, AopN and ExsE that are known as effectors in other pathogens. Translocators, needle subunits, Ati1, and AscX were also secreted in supernatants (SNs) dependent on T3SS. AopH, Ati2, AexT, AopB and AopD were in the top seven most abundant excreted proteins. EF-G, EF-Tu, DnaK, HtpG, PNPase, PepN and MdeA were moderately secreted in wt SNs and predicted to be putative T3 effectors by bioinformatics. Pta and ASA_P5G088 were increased in wt SNs and T3-associated in other bacteria. Ten conserved cytoplasmic proteins were more abundant in wt SNs than in the ΔascV mutant, but without any clear association to a secretion system. T1-secreted proteins were predominantly found in wt SNs: OmpAI, OmpK40, DegQ, insulinase ASA_0716, hypothetical ASA_0852 and ASA_3619. Presence of T3SS components in pellets was clearly decreased by ascV deletion, while no impact was observed on T1- and T2SS. Our results demonstrated that the ΔascV mutant strain excreted well-described (VapA, AerA, AerB, GCAT, Pla1, PlaC, TagA, Ahe2, GbpA and enolase) and yet uncharacterized potential toxins, adhesins and enzymes as much as or even more than the wt strain. Other putative important virulence factors were not detected. CONCLUSIONS: We demonstrated the whole in vitro secretome and T3SS repertoire of hypervirulent A. salmonicida. Several toxins, adhesins and enzymes that are not part of the T3SS secretome were secreted to a higher extent in the extremely low-virulent ΔascV mutant. All together, our results show the high importance of an intact T3SS to initiate the furunculosis and offer new information about the pathogenesis.

Proteomic characterization of Toxoplasma gondii ME49 derived strains resistant to the artemisinin derivatives artemiside and artemisone implies potential mode of action independent of ROS formation.

The sesquiterpene lactone artemisinin and its amino-artemisinin derivatives artemiside (GC008) and artemisone (GC003) are potent antimalarials. The mode of action of artemisinins against Plasmodium sp is popularly ascribed to 'activation' of the peroxide group by heme-Fe(II) or labile Fe(II) to generate C-radicals that alkylate parasite proteins. An alternative postulate is that artemisinins elicit formation of reactive oxygen species by interfering with flavin disulfide reductases resposible for maintaining intraparasitic redox homeostasis. However, in contradistinction to the heme-activation mechanism, the amino-artemisinins are effective in vitro against non-heme-degrading apicomplexan parasites including T. gondii, with IC 50 values of 50-70 nM, and induce distinct ultrastructural alterations. However, T. gondii strains readily adapted to increased concentrations (2.5 µM) of these two compounds within few days. Thus, T. gondii strains that were resistant against artemisone and artemiside were generated by treating the T. gondii reference strain ME49 with stepwise increasing amounts of these compounds, yielding the artemisone resistant strain GC003R and the artemiside resistant strain GC008R. Differential analyses of the proteomes of these resistant strains compared to the wildtype ME49 revealed that 215 proteins were significantly downregulated in artemisone resistant tachyzoites and only 8 proteins in artemiside resistant tachyzoites as compared to their wildtype. Two proteins, namely a hypothetical protein encoded by ORF TGME49_236950, and the rhoptry neck protein RON2 encoded by ORF TGME49_300100 were downregulated in both resistant strains. Interestingly, eight proteins involved in ROS scavenging including catalase and superoxide dismutase were amongst the differentially downregulated proteins in the artemisone-resistant strain. In parallel, ROS formation was significantly enhanced in isolated tachyzoites from the artemisone resistant strain and - to a lesser extent - in tachyzoites from the artemiside resistant strain as compared to wildtype tachyzoites. These findings suggest that amino-artemisinin derivatives display a mechanism of action in T. gondii distinct from Plasmodium.

Renal FGF23 signaling depends on redox protein Memo1 and promotes orthovanadate-sensitive protein phosphotyrosyl phosphatase activity.

Memo1 deletion in mice causes premature aging and an unbalanced metabolism partially resembling Fgf23 and Klotho loss-of-function animals. We report a role for Memo's redox function in renal FGF23-Klotho signaling using mice with postnatally induced Memo deficiency in the whole body (cKO). Memo cKO mice showed impaired FGF23-driven renal ERK phosphorylation and transcriptional responses. FGF23 actions involved activation of oxidation-sensitive protein phosphotyrosyl phosphatases in the kidney. Redox proteomics revealed excessive thiols of Rho-GDP dissociation inhibitor 1 (Rho-GDI1) in Memo cKO, and we detected a functional interaction between Memo's redox function and oxidation at Rho-GDI1 Cys79. In isolated cellular systems, Rho-GDI1 did not directly affect FGF23-driven cell signaling, but we detected disturbed Rho-GDI1 dependent small Rho-GTPase protein abundance and activity in the kidney of Memo cKO mice. Collectively, this study reveals previously unknown layers in the regulation of renal FGF23 signaling and connects Memo with the network of small Rho-GTPases.

Proteome remodelling during development from blood to insect-form Trypanosoma brucei quantified by SILAC and mass spectrometry.

BACKGROUND: Trypanosoma brucei is the causative agent of human African sleeping sickness and Nagana in cattle. In addition to being an important pathogen T. brucei has developed into a model system in cell biology. RESULTS: Using Stable Isotope Labelling of Amino acids in Cell culture (SILAC) in combination with mass spectrometry we determined the abundance of >1600 proteins in the long slender (LS), short stumpy (SS) mammalian bloodstream form stages relative to the procyclic (PC) insect-form stage. In total we identified 2645 proteins, corresponding to ~30% of the total proteome and for the first time present a comprehensive overview of relative protein levels in three life stages of the parasite. CONCLUSIONS: We can show the extent of pre-adaptation in the SS cells, especially at the level of the mitochondrial proteome. The comparison to a previously published report on monomorphic in vitro grown bloodstream and procyclic T. brucei indicates a loss of stringent regulation particularly of mitochondrial proteins in these cells when compared to the pleomorphic in vivo situation. In order to better understand the different levels of gene expression regulation in this organism we compared mRNA steady state abundance with the relative protein abundance-changes and detected moderate but significant correlation indicating that trypanosomes possess a significant repertoire of translational and posttranslational mechanisms to regulate protein abundance.

Differential Affinity Chromatography Coupled to Mass Spectrometry: A Suitable Tool to Identify Common Binding Proteins of a Broad-Range Antimicrobial Peptide Derived from Leucinostatin.

Leucinostatins are antimicrobial peptides with a broad range of activities against infectious agents as well as mammalian cells. The leucinostatin-derivative peptide ZHAWOC_6027 (peptide 6027) was tested in vitro and in vivo for activity against the intracellular apicomplexan parasite Toxoplasma gondii. While highly efficacious in vitro (EC50 = 2 nM), subcutaneous application of peptide 6027 (3 mg/kg/day for 5 days) in mice experimentally infected with T. gondii oocysts exacerbated the infection, caused mild clinical signs and elevated cerebral parasite load. Peptide 6027 also impaired the proliferation and viability of mouse splenocytes, most notably LPS-stimulated B cells, in vitro. To identify common potential targets in Toxoplasma and murine splenocytes, we performed differential affinity chromatography (DAC) with cell-free extracts from T. gondii tachyzoites and mouse spleens using peptide 6027 or an ineffective analogue (peptide 21,358) coupled to N-hydroxy-succinimide sepharose, followed by mass spectrometry. Proteins specifically binding to peptide 6027 were identified in eluates from the peptide 6027 column but not in peptide 21,358 nor the mock column eluates. In T. gondii eluates, 269 proteins binding specifically to peptide 6027 were identified, while in eluates from mouse spleen extracts 645 proteins specifically binding to this peptide were detected. Both datasets contained proteins involved in mitochondrial energy metabolism and in protein processing and secretion. These results suggest that peptide 6027 interacts with common targets in eukaryotes involved in essential pathways. Since this methodology can be applied to various compounds as well as target cell lines or organs, DAC combined with mass spectrometry and proteomic analysis should be considered a smart and 3R-relevant way to identify drug targets in pathogens and hosts, thereby eliminating compounds with potential side effects before performing tedious and costly safety and efficacy assessments in animals or humans.

Multi-laboratory experiment PME11 for the standardization of phosphoproteome analysis.

Global analysis of protein phosphorylation by mass spectrometry proteomic techniques has emerged in the last decades as a powerful tool in biological and biomedical research. However, there are several factors that make the global study of the phosphoproteome more challenging than measuring non-modified proteins. The low stoichiometry of the phosphorylated species and the need to retrieve residue specific information require particular attention on sample preparation, data acquisition and processing to ensure reproducibility, qualitative and quantitative robustness and ample phosphoproteome coverage in phosphoproteomic workflows. Aiming to investigate the effect of different variables in the performance of proteome wide phosphoprotein analysis protocols, ProteoRed-ISCIII and EuPA launched the Proteomics Multicentric Experiment 11 (PME11). A reference sample consisting of a yeast protein extract spiked in with different amounts of a phosphomix standard (Sigma/Merck) was distributed to 31 laboratories around the globe. Thirty-six datasets from 23 laboratories were analyzed. Our results indicate the suitability of the PME11 reference sample to benchmark and optimize phosphoproteomics strategies, weighing the influence of different factors, as well as to rank intra and inter laboratory performance.

Partially shielded enzymes capable of processing large protein substrates.

We report the first method of enzyme protection enabling the production of partially shielded enzymes capable of processing substrates as large as proteins. We show that partially shielded sortase retains its transpeptidase activity and can perform bioconjugation reactions on antibodies. Moreover, a partially shielded trypsin is shown to outperform its soluble counterpart in terms of proteolytic kinetics. Remarkably, partial enzyme shielding results in a drastic increase in temporal stability of the enzyme.

Neospora caninum: Differential Proteome of Multinucleated Complexes Induced by the Bumped Kinase Inhibitor BKI-1294.

BACKGROUND: the apicomplexan parasite Neospora caninum causes important reproductive problems in farm animals, most notably in cattle. After infection via oocysts or tissue cysts, rapidly dividing tachyzoites infect various tissues and organs, and in immunocompetent hosts, they differentiate into slowly dividing bradyzoites, which form tissue cysts and constitute a resting stage persisting within infected tissues. Bumped kinase inhibitors (BKIs) of calcium dependent protein kinase 1 are promising drug candidates for the treatment of Neospora infections. BKI-1294 exposure of cell cultures infected with N. caninum tachyzoites results in the formation of massive multinucleated complexes (MNCs) containing numerous newly formed zoites, which remain viable for extended periods of time under drug pressure in vitro. MNC and tachyzoites exhibit considerable antigenic and structural differences. METHODS: Using shotgun mass spectrometry, we compared the proteomes of tachyzoites to BKI-1294 induced MNCs, and analyzed the mRNA expression levels of selected genes in both stages. RESULTS: More than half of the identified proteins are downregulated in MNCs as compared to tachyzoites. Only 12 proteins are upregulated, the majority of them containing SAG1 related sequence (SRS) domains, and some also known to be expressed in bradyzoites Conclusions: MNCs exhibit a proteome different from tachyzoites, share some bradyzoite-like features, but may constitute a third stage, which remains viable and ensures survival under adverse conditions such as drug pressure. We propose the term "baryzoites" for this stage (from Greek ßαρυσ = massive, bulky, heavy, inert).

A Digital Pathology-Based Shotgun-Proteomics Approach to Biomarker Discovery in Colorectal Cancer.

BACKGROUND: Biomarkers in colorectal cancer are scarce, especially for patients with Stage 2 disease. The aim of our study was to identify potential prognostic biomarkers from colorectal cancers using a novel combination of approaches, whereby digital pathology is coupled to shotgun proteomics followed by validation of candidates by immunohistochemistry (IHC) using digital image analysis (DIA). METHODS AND RESULTS: Tissue cores were punched from formalin-fixed paraffin-embedded colorectal cancers from patients with Stage 2 and 3 disease (n = 26, each). Protein extraction and liquid chromatography-mass spectrometry (MS) followed by analysis using three different methods were performed. Fold changes were evaluated. The candidate biomarker was validated by IHC on a series of 413 colorectal cancers from surgically treated patients using a next-generation tissue microarray. DIA was performed by using a pan-cytokeratin serial alignment and quantifying staining within the tumor and normal tissue epithelium. Analysis was done in QuPath and Brightness_Max scores were used for statistical analysis and clinicopathological associations. MS identified 1947 proteins with at least two unique peptides. To reinforce the validity of the biomarker candidates, only proteins showing a significant (P < 0.05) fold-change using all three analysis methods were considered. Eight were identified, and of these, cathepsin B was selected for further validation. DIA revealed strong associations between higher cathepsin B expression and less aggressive tumor features, including tumor node metastasis stage and lymphatic vessel and venous vessel invasion (P < 0.001, all). Cathepsin B was associated with more favorable survival in univariate analysis only. CONCLUSIONS: Our results present a novel approach to biomarker discovery that includes MS and digital pathology. Cathepsin B expression analyzed by DIA within the tumor epithelial compartment was identified as a strong feature of less aggressive tumor behavior and favorable outcome, a finding that should be further investigated on a more functional level.

Mapping the Orthosteric Binding Site of the Human 5-HT3 Receptor Using Photo-cross-linking Antagonists.

The serotonin-gated 5-HT3 receptor is a ligand-gated ion channel. Its location at the synapse in the central and peripheral nervous system has rendered it a prime pharmacological target, for example, for antiemetic drugs that bind with high affinity to the neurotransmitter binding site and prevent the opening of the channel. Advances in structural biology techniques have led to a surge of disclosed three-dimensional receptor structures; however, solving ligand-bound high-resolution 5-HT3 receptor structures has not been achieved to date. Ligand binding poses in the orthosteric binding site have been largely predicted from mutagenesis and docking studies. We report the synthesis of a series of photo-cross-linking compounds whose structures are based on the clinically used antiemetic drug granisetron (Kytril). These displaced [3H]granisetron from the orthosteric binding site with low nanomolar affinities and showed specific photo-cross-linking with the human 5-HT3 receptor. Detailed analysis by protein-MS/MS identified a residue (Met-228) near the tip of binding loop C as the covalent modification site.