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IntroductionPost translational modification of proteins plays a significant role in immune recognition. In particular the modification of arginine to citrulline which is mediated by PAD enzymes is increased during cellular stress (autophagy) which permits the presentation of modified epitopes upon MHC class II molecules for recognition by CD4 T cells. Citrullination also occurs in tumour cells as a result of continuous environmental stresses and increased autophagy. We have shown in animal models the efficient stimulation of citrullinated epitope specific CD4 T cells resulting in dramatic elimination/regression of tumours. The ER chaperone glucose-regulated protein 78 (GRP78) is known to also be required for stress-induced autophagy and is directly linked to autophagosome formation. GRP78 is known to be highly expressed by many tumour types. In this study we investigate the potential of targeting citrullinated GRP78 for cancer therapy.MethodsA citrullinated GRP78 specific antibody was used to assess citrullinated GRP78 expression in murine and human tumour cells by flow cytometry. Five peptides were selected and used to vaccinate HLA transgenic mice and immune responses were characterised by ex vivo cytokine ELISpot assay. T cell repertoire in humans was assessed through proliferation assays and cytokine ELISpot assay. Citrullinated peptide was identified in murine B16 melanoma by mass spectrometry and the peptide vaccine was assessed for tumour therapy in a mouse melanoma model.ResultsWe show the identification CD4 T cell responses to one citrullinated GRP78 epitope that are restricted through HLA DP*0401 and HLA-DR*0101 alleles. This peptide is detected by mass spectrometry in B16 melanoma grown in vivo and citrulline specific CD4 responses to two peptides spanning this epitope mediate efficient therapy of established B16 melanoma tumours in HHDII/DP4 (p

ABSTRACT Import and oxidative folding of proteins in the mitochondrial intermembrane space differ among eukaryotic lineages. While opisthokonts such as yeast rely on the receptor and oxidoreductase Mia40 in combination with the Mia40:cytochrome c oxidoreductase Erv, kinetoplastid parasites and other Excavata/Discoba lack Mia40 but have a functional Erv homologue. Whether excavate Erv homologues rely on a Mia40 replacement or directly interact with imported protein substrates remains controversial. Here, we used the CRISPR-Cas9 system to generate a set of tagged and untagged homozygous mutants of LTERV from the kinetoplastid model parasite Leishmania tarentolae. Modifications of the shuttle cysteine motif of LtErv were lethal, whereas replacement of clamp residue Cys17 or removal of the kinetoplastida-specific second (KISS) domain had no impact on parasite viability under standard growth conditions. However, removal of the KISS domain rendered parasites sensitive to heat stress and led to the accumulation of homodimeric and mixed LtErv disulfides. We therefore determined and compared the redox interactomes of tagged wild-type LtErv and LtErvΔKISS using stable isotope labeling by amino acids in cell culture (SILAC) and quantitative mass spectrometry. While the Mia40-replacement candidate Mic20 and all but one typical substrate with twin Cx3/9C-motifs were absent in both redox interactomes, we identified a small set of alternative potential interaction partners with putative redox-active cysteine residues. In summary, our study reveals parasite-specific intracellular structure-function relationships and redox interactomes of LtErv with implications for current hypotheses on mitochondrial protein import in nonopisthokonts. IMPORTANCE The discovery of the redox proteins Mia40/CHCHD4 and Erv1/ALR, as well as the elucidation of their relevance for oxidative protein folding in the mitochondrial intermembrane space of yeast and mammals, founded a new research topic in redox biology and mitochondrial protein import. The lack of Mia40/CHCHD4 in protist lineages raises fundamental and controversial questions regarding the conservation and evolution of this essential pathway. Do protist Erv homologues act alone, or do they use the candidate Mic20 or another protein as a Mia40 replacement? Furthermore, we previously showed that Erv homologues in L. tarentolae and the human pathogen L. infantum are not only essential but also differ structurally and mechanistically from yeast and human Erv1/ALR. Here, we analyzed the relevance of such structural differences in vivo and determined the first redox interactomes of a nonopisthokont Erv homologue. Our data challenge recent hypotheses on mitochondrial protein import in nonopisthokonts.

LC-MS/MS phospho-proteomics is an essential technology to help unravel the complex molecular events that lead to and propagate cancer. We have developed a global phospho-proteomic workflow to determine activity of signaling pathways and drug targets in pancreatic cancer tissue for clinical application.Peptides resulting from tryptic digestion of proteins extracted from frozen tissue of pancreatic ductal adenocarcinoma and background pancreas (n = 12), were labelled with tandem mass tags (TMT 8-plex), separated by strong cation exchange chromatography, then were analysed by LC-MS/MS directly or first enriched for phosphopeptides using IMAC and TiO2, prior to analysis. In-house, commercial and freeware bioinformatic platforms were used to identify relevant biological events from the complex dataset.Of 2,101 proteins identified, 152 demonstrated significant difference in abundance between tumor and non-tumor tissue. They included proteins that are known to be up-regulated in pancreatic cancer (e.g. Mucin-1), but the majority were new candidate markers such as HIPK1 & MLCK. Of the 6,543 unique phosphopeptides identified (6,284 unique phosphorylation sites), 635 showed significant regulation, particularly those from proteins involved in cell migration (Rho guanine nucleotide exchange factors & MRCKα) and formation of focal adhesions. Activator phosphorylation sites on FYN, AKT1, ERK2, HDAC1 and other drug targets were found to be highly modulated (≥2 fold) in different cases highlighting their predictive power.Here we provided critical information enabling us to identify the common and unique molecular events likely contributing to cancer in each case. Such information may be used to help predict more bespoke therapy suitable for an individual case.

The malarial parasite Plasmodium falciparum possesses a functional thioredoxin and glutathione system comprising the dithiol-containing redox proteins thioredoxin (Trx) and glutaredoxin (Grx), as well as plasmoredoxin (Plrx), which is exclusively found in Plasmodium species. All three proteins belong to the thioredoxin superfamily and share a conserved Cys-X-X-Cys motif at the active site. Only a few of their target proteins, which are likely to be involved in redox reactions, are currently known. The aim of the present study was to extend our knowledge of the Trx-, Grx-, and Plrx-interactome in Plasmodium. Based on the reaction mechanism, we generated active site mutants of Trx and Grx lacking the resolving cysteine residue. These mutants were bound to affinity columns to trap target proteins from P. falciparum cell extracts after formation of intermolecular disulfide bonds. Covalently linked proteins were eluted with dithiothreitol and analyzed by mass spectrometry. For Trx and Grx, we were able to isolate 17 putatively redox-regulated proteins each. Furthermore, the approach was successfully established for Plrx, leading to the identification of 21 potential target proteins. In addition to confirming known interaction partners, we captured potential target proteins involved in various processes including protein biosynthesis, energy metabolism, and signal transduction. The identification of three enzymes involved in S-adenosylmethionine (SAM) metabolism furthermore suggests that redox control is required to balance the metabolic fluxes of SAM between methyl-group transfer reactions and polyamine synthesis. To substantiate our data, the binding of the redoxins to S-adenosyl-L-homocysteine hydrolase and ornithine aminotransferase (OAT) were verified using BIAcore surface plasmon resonance. In enzymatic assays, Trx was furthermore shown to enhance the activity of OAT. Our approach led to the discovery of several putatively redox-regulated proteins, thereby contributing to our understanding of the redox interactome in malarial parasites.

Proteome studies contribute markedly to our understanding of parasite biology, host-parasite interactions, and mechanisms of drug action. For most antimalarial drugs neither mode of action nor mechanisms of resistance development are fully elucidated although this would be important prerequisites for successfully developing urgently required novel antimalarials. Here, we establish a large-scale quantitative proteomic approach to examine protein expression changes in trophozoite stages of the malarial parasite Plasmodium falciparum following chloroquine and artemisinin treatment. For this purpose SIL (stable isotope labeling) using (14)N-isoleucine and (13)C(6),(15)N(1)-isoleucine was optimized to obtain 99% atomic percent enrichment. Proteome fractionation with anion exchange chromatography was used to reduce sample complexity and increase quantitative coverage of protein expression. Tryptic peptides of subfractions were subjected to SCX/RP separation, measured by LC-MS/MS and quantified using the novel software tool Census. In drug treated parasites, we identified a total number of 1,253 proteins, thus increasing the overall number of proteins identified in the trophozoite stage by 30%. A relative quantification was obtained for more than 800 proteins. Under artemisinin and chloroquine treatment 41 and 38 proteins respectively were upregulated (>1.5) whereas 14 and 8 proteins were down-regulated (

The rare A673T variant was the first variant found within the amyloid precursor protein (APP) gene conferring protection against Alzheimer's disease (AD). Thereafter, different studies have discovered that the carriers of the APP A673T variant show reduced levels of amyloid beta (Aβ) in the plasma and better cognitive performance at high age. Here, we analyzed cerebrospinal fluid (CSF) and plasma of APP A673T carriers and control individuals using a mass spectrometry-based proteomics approach to identify differentially regulated targets in an unbiased manner. Furthermore, the APP A673T variant was introduced into 2D and 3D neuronal cell culture models together with the pathogenic APP Swedish and London mutations. Consequently, we now report for the first time the protective effects of the APP A673T variant against AD-related alterations in the CSF, plasma, and brain biopsy samples from the frontal cortex. The CSF levels of soluble APPβ (sAPPβ) and Aβ42 were significantly decreased on average 9–26% among three APP A673T carriers as compared to three well-matched controls not carrying the protective variant. Consistent with these CSF findings, immunohistochemical assessment of cortical biopsy samples from the same APP A673T carriers did not reveal Aβ, phospho-tau, or p62 pathologies. We identified differentially regulated targets involved in protein phosphorylation, inflammation, and mitochondrial function in the CSF and plasma samples of APP A673T carriers. Some of the identified targets showed inverse levels in AD brain tissue with respect to increased AD-associated neurofibrillary pathology. In 2D and 3D neuronal cell culture models expressing APP with the Swedish and London mutations, the introduction of the APP A673T variant resulted in lower sAPPβ levels. Concomitantly, the levels of sAPPα were increased, while decreased levels of CTFβ and Aβ42 were detected in some of these models. Our findings emphasize the important role of APP-derived peptides in the pathogenesis of AD and demonstrate the effectiveness of the protective APP A673T variant to shift APP processing towards the non-amyloidogenic pathway in vitro even in the presence of two pathogenic mutations.

Resonance Raman spectroscopy was applied for investigating the malaria pigment hemozoin, which is an important target structure of antimalarial drugs. Morphology-sensitive low wavenumber modes of hemozoin were selectively enhanced with help of excitation wavelengths at λ= 633 nm and λ= 647 nm. The assignment of the most prominent bands in the Raman spectra at 343 cm−1and 368 cm−1was assisted by DFT calculations of the hemozoin dimer. The mode at 343 cm−1in the Raman spectrum of hemozoin is strongly enhanced with λexc.= 647 nm and is represented by a combined, symmetric doming mode of the two hematin units in the hemozoin dimer. The enhancement of this vibration is stronger in the resonance Raman spectrum of hemozoin compared with less crystalline β-hematin. The selective resonance enhancement of the morphology-sensitive Raman modes of hemozoin is caused by absorption bands in the UV-VIS-NIR spectrum. This absorption spectrum of the crystalline malaria pigment hemozoin shows a strong band at 655 nm. Another broad absorption band at 870 nm is the reason for the strong relative resonance enhancement of the mode at 1372 cm−1in the Raman spectrum of crystalline hemozoin with λexc.= 830 nm. In conclusion, resonance Raman micro-spectroscopy with λexc.= 647 nm was shown to have great potential as an analytical tool to probe the morphology of hematin samples. [ABSTRACT FROM AUTHOR]

Raman microspectroscopy was applied for an insitulocalization of the malaria pigment hemozoin in Plasmodium falciparum-infected erythrocytes. The Raman spectra (exc633 nm) of hemozoin show very intense signals with a very good signal-to-noise ratio. These in situRaman signals of hemozoin were compared to Raman spectra of extracted hemozoin, of the synthetic analogue -hematin, and of hematin and hemin. -Hematin was synthesized according to the acid-catalyzed dehydration of hematin and the anhydrous dehydrohalogenation of hemin which lead to good crystals with lengths of about 5−30 m. The Raman spectra (exc1064 nm) of hemozoin and -hematin show almost identical behaviors, while some low wavenumber modes might be used to distinguish between the morphology of differently synthesized -hematin samples. The intensity pattern of the resonance Raman spectra (exc568 nm) of hemozoin and -hematin differ significantly from those of hematin and hemin. The most striking difference is an additional band at 1655 cm-1which was only observed in the spectra of hemozoin and -hematin and cannot be seen in the spectra of hematin and hemin. Raman spectra of the -hematin dimer were calculated ab initio(DFT) for the first time and used for an assignment of the experimentally derived Raman bands. The calculated atomic displacements provide valuable insight into the most important molecular vibrations of the hemozoin dimer. With help from these DFT calculations, it was possible to assign the Raman band at 1655 cm-1to a mode located at the propionic acid side chain, which links the hemozoin dimers to each other. The Raman band at 1568 cm-1, which has been shown to be influenced by an attachment of the antimalarial drug chloroquine in an earlier study, could be assigned to a CC stretching mode spread across one of the porphyrin rings and is therefore expected to be influenced by a −-stacking to the drug. [ABSTRACT FROM AUTHOR]