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

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

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

Targeted and non-targeted proteomics to characterize the parasite proteins of Echinococcus multilocularis metacestodes.

The larval stage of the cestode Echinococcus multilocularis is the causative agent of alveolar echinococcosis. To investigate the biology of these stages and to test novel compounds, metacestode cultures represent a suitable in vitro model system. These metacestodes are vesicles surrounded by an envelope formed by the vesicle tissue (VT), which is formed by the laminated and germinal layer, and filled with vesicle fluid (VF). We analyzed the proteome of VF and VT by liquid chromatography tandem mass spectrometry (LC-MS/MS) and identified a total of 2,954 parasite proteins. The most abundant protein in VT was the expressed conserved protein encoded by EmuJ_000412500, followed by the antigen B subunit AgB8/3a encoded by EmuJ_000381500 and Endophilin B1 (protein p29). In VF, the pattern was different and dominated by AgB subunits. The most abundant protein was the AgB8/3a subunit followed by three other AgB subunits. In total, the AgB subunits detected in VF represented 62.1% of the parasite proteins. In culture media (CM), 63 E. multilocularis proteins were detected, of which AgB subunits made up 93.7% of the detected parasite proteins. All AgB subunits detected in VF (encoded by EmuJ_000381100-700, corresponding to AgB8/2, AgB8/1, AgB8/4, AgB8/3a, AgB8/3b, and AgB8/3c) were also found in CM, except the subunit encoded by EmuJ_000381800 (AgB8/5) that was very rare in VF and not detected in CM. The relative abundance of the AgB subunits in VF and CM followed the same pattern. In VT, only the subunits EmuJ_000381500 (AgB8/3a) and EmuJ_000381200 (AgB8/1) were detected among the 20 most abundant proteins. To see whether this pattern was specific to VF from in vitro cultured metacestodes, we analyzed the proteome of VF from metacestodes grown in a mouse model. Here, the AgB subunits encoded by EmuJ_000381100-700 constituted the most abundant proteins, namely, 81.9% of total protein, with the same order of abundance as in vitro. Immunofluorescence on metacestodes showed that AgB is co-localized to calcareous corpuscles of E. multilocularis. Using targeted proteomics with HA-tagged EmuJ_000381200 (AgB8/1) and EmuJ_000381100 (AgB8/2), we could show that uptake of AgB subunits from CM into VF occurs within hours

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

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, mono- cytes, 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. Correla- tion 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

Multi-laboratory experiment PME11 for the standardization of phosphoproteome analysis

PhosphoproteomeFosfoproteomaFosfoproteomaGlobal 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.ProteoRed, PRB3 is supported by grant PT17/0019/0001, of the PE I+D+i 2013-2016, funded by ISCIII and ERDF

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

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

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

40S hnRNP particles are a novel class of nuclear biomolecular condensates.

Heterogenous nuclear ribonucleoproteins (hnRNPs) are abundant proteins implicated in various steps of RNA processing that assemble on nuclear RNA into larger complexes termed 40S hnRNP particles. Despite their initial discovery 55 years ago, our understanding of these intriguing macromolecular assemblies remains limited. Here, we report the biochemical purification of native 40S hnRNP particles and the determination of their complete protein composition by label-free quantitative mass spectrometry, identifying A-group and C-group hnRNPs as the major protein constituents. Isolated 40S hnRNP particles dissociate upon RNA digestion and can be reconstituted in vitro on defined RNAs in the presence of the individual protein components, demonstrating a scaffolding role for RNA in nucleating particle formation. Finally, we revealed their nanometer scale, condensate-like nature, promoted by intrinsically disordered regions of A-group hnRNPs. Collectively, we identify nuclear 40S hnRNP particles as novel dynamic biomolecular condensates