Protective Alzheimer's disease-associated APP A673T variant predominantly decreases sAPPß levels in cerebrospinal fluid and 2D/3D cell culture models.

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 (Aß) 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 APPß (sAPPß) and Aß42 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 Aß, 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 sAPPß levels. Concomitantly, the levels of sAPPα were increased, while decreased levels of CTFß and Aß42 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.

Citrullinated glucose-regulated protein 78 is a candidate target for melanoma immunotherapy.

Introduction: Post 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. Methods: A 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. Results: We 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<0.0001) transgenic mouse model. Finally, we demonstrate the existence of a repertoire of responses to the citrullinated GRP78 peptide in healthy individuals (p=0.0023) with 13/17 (76%) individuals showing a response to this peptide. Conclusion: We propose that citrullinated GRP78 is a candidate tumour antigen and vaccination against citrullinated GRP78 may provide a promising tumour therapy approach.

Comparative Assessment of Quantification Methods for Tumor Tissue Phosphoproteomics.

With increasing sensitivity and accuracy in mass spectrometry, the tumor phosphoproteome is getting into reach. However, the selection of quantitation techniques best-suited to the biomedical question and diagnostic requirements remains a trial and error decision as no study has directly compared their performance for tumor tissue phosphoproteomics. We compared label-free quantification (LFQ), spike-in-SILAC (stable isotope labeling by amino acids in cell culture), and tandem mass tag (TMT) isobaric tandem mass tags technology for quantitative phosphosite profiling in tumor tissue. Compared to the classic SILAC method, spike-in-SILAC is not limited to cell culture analysis, making it suitable for quantitative analysis of tumor tissue samples. TMT offered the lowest accuracy and the highest precision and robustness toward different phosphosite abundances and matrices. Spike-in-SILAC offered the best compromise between these features but suffered from a low phosphosite coverage. LFQ offered the lowest precision but the highest number of identifications. Both spike-in-SILAC and LFQ presented susceptibility to matrix effects. Match between run (MBR)-based analysis enhanced the phosphosite coverage across technical replicates in LFQ and spike-in-SILAC but further reduced the precision and robustness of quantification. The choice of quantitative methodology is critical for both study design such as sample size in sample groups and quantified phosphosites and comparison of published cancer phosphoproteomes. Using ovarian cancer tissue as an example, our study builds a resource for the design and analysis of quantitative phosphoproteomic studies in cancer research and diagnostics.

Tryptophan metabolism is inversely regulated in the tumor and blood of patients with glioblastoma.

Tryptophan (Trp)-catabolic enzymes (TCEs) produce metabolites that activate the aryl hydrocarbon receptor (AHR) and promote tumor progression and immunosuppression in glioblastoma. As therapies targeting TCEs or AHR become available, a better understanding of Trp metabolism is required. Methods: The combination of LC-MS/MS with chemical isobaric labeling enabled the simultaneous quantitative comparison of Trp and its amino group-bearing metabolites in multiple samples. We applied this method to the sera of a cohort of 43 recurrent glioblastoma patients and 43 age- and sex-matched healthy controls. Tumor volumes were measured in MRI data using an artificial neural network-based approach. MALDI MSI visualized Trp and its direct metabolite N-formylkynurenine (FK) in glioblastoma tissue. Analysis of scRNA-seq data was used to detect the presence of Trp metabolism and AHR activity in different cell types in glioblastoma. Results: Compared to healthy controls, glioblastoma patients showed decreased serum Trp levels. Surprisingly, the levels of Trp metabolites were also reduced. The decrease became smaller with more enzymatic steps between Trp and its metabolites, suggesting that Trp availability controls the levels of its systemic metabolites. High tumor volume associated with low systemic metabolite levels and low systemic kynurenine levels associated with worse overall survival. MALDI MSI demonstrated heterogeneity of Trp catabolism across glioblastoma tissues. Analysis of scRNA-seq data revealed that genes involved in Trp metabolism were expressed in almost all the cell types in glioblastoma and that most cell types, in particular macrophages and T cells, exhibited AHR activation. Moreover, high AHR activity associated with reduced overall survival in the glioblastoma TCGA dataset. Conclusion: The novel techniques we developed could support the identification of patients that may benefit from therapies targeting TCEs or AHR activation.

In Vivo Structure-Function Analysis and Redox Interactomes of Leishmania tarentolae Erv.

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.

Combined Tissue-Fluid Proteomics to Unravel Phenotypic Variability in Amyotrophic Lateral Sclerosis.

The lack of biomarkers for early diagnosis, clinical stratification and to monitor treatment response has hampered the development of new therapies for amyotrophic lateral sclerosis (ALS), a clinically heterogeneous neurodegenerative disorder with a variable site of disease initiation and rate of progression. To identify new biomarkers and therapeutic targets, two separate proteomic workflows were applied to study the immunological response and the plasma/brain proteome in phenotypic variants of ALS. Conventional multiplex (TMT) proteomic analysis of peripheral blood mononuclear cells (PBMCs) was performed alongside a recently introduced method to profile neuronal-derived proteins in plasma using brain tissue-enhanced isobaric tagging (TMTcalibrator). The combined proteomic analysis allowed the detection of regulated proteins linked to ALS pathogenesis (RNA-binding protein FUS, superoxide dismutase Cu-Zn and neurofilaments light polypeptide) alongside newly identified candidate biomarkers (myosin-9, fructose-bisphosphate aldolase and plectin). In line with the proteomic results, orthogonal immunodetection showed changes in neurofilaments and ApoE in bulbar versus limb onset fast progressing ALS. Functional analysis of significantly regulated features showed enrichment of pathways involved in regulation of the immune response, Rho family GTPases, semaphorin and integrin signalling. Our cross-phenotype investigation of PBMCs and plasma/brain proteins provides a more sensitive biomarker exploratory platform than conventional case-control studies in a single matrix. The reported regulated proteins may represent novel biomarker candidates and potentially druggable targets.

Post-translational modifications in Alzheimer's disease and the potential for new biomarkers.

The ability to detect and diagnose Alzheimer's disease (AD) early is an ever pressing issue, and the development of markers of disease progression that are able to distinguish AD patients from normal aging and patients with alternative forms of dementia, is at the center of the issue. Protein markers of disease, or biomarkers, can be used not only to monitor the progression of AD, but also allow identification of patients suitable for potential therapy, and the response to therapy to be monitored. Cerebrospinal fluid protein biomarkers are important in this early AD diagnosis, and three such biomarkers have been extensively studied and are reviewed here. In addition, post translational protein modifications of proteins important in AD pathology are also discussed. If additional biomarkers can be identified and thoroughly understood, potential therapeutic agents can be better designed, and the effects of therapeutic intervention on disease progression can be monitored.

Quantification of pancreatic cancer proteome and phosphorylome: indicates molecular events likely contributing to cancer and activity of drug targets.

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Differential genomic and proteomic profiling of glioblastoma cells exposed to terpyridineplatinum(II) complexes.

Terpyridineplatinum(II) complexes (TPCs) efficiently inhibit the proliferation of glioblastoma cells in vitro and have been tested successfully in a rodent glioblastoma model. Apart from intercalation with DNA, the major mechanism of action of TPCs is a very potent and specific interaction with the human selenoprotein thioredoxin reductase (TrxR). TrxR plays a crucial role in cellular redox homeostasis and protection against oxidative damage. In many malignant cells the thioredoxin system is upregulated, promoting tumor growth and progression. Thus, the thioredoxin system has been proposed to be an attractive target for cancer therapy. This study gives the first comprehensive overview of the effects of TPCs on the transcriptome and proteome of glioblastoma cells. We reveal that under TPC treatment, mechanisms countersteering TrxR inhibition are activated in parallel to DNA-damage-responsive pathways. TPC pressure results in long-term compensatory upregulation of TrxR expression. In parallel, p53 is activated, leading to a range of regulations typical for cell-cycle-arrested cells such as upregulation of CDKN1A, induction of GADD45, inhibition of eIF5A maturation, and reduced phosphorylation of stathmin. We also show that TPCs induce endoplasmic reticulum stress, as they activate the unfolded protein response. This profiling study provides a thorough insight into the spectrum of cellular events resulting from specific TrxR inhibition and characterizes the TPC mode of action.

Identification of proteins targeted by the thioredoxin superfamily in Plasmodium falciparum.

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.

Glutathione- and thioredoxin-related enzymes are modulated by sulfur-containing chemopreventive agents.

We studied the effects of sulfur-containing chemopreventive agents, including allyl sulfides and isothiocyanates, on human redox networks. Isothiocyanates inhibited isolated redox-active enzymes in a time- and dose-dependent manner. As shown for the most active compound, benzyl isothiocyanate (BITC), on thioredoxin reductase, the inhibition has an initial competitive part (Ki=6.1+/-1.0 microM) followed by a time-dependent irreversible inhibition (k2=72.8+/-25.5 M(-1) s(-1)). Also, glutathione reductase and glutathione S-transferase were irreversibly modified by BITC. Sulforaphane led to irreversible inhibition of the studied redox enzymes, but with 5-10 times lower k2 values. In contrast, allyl sulfides had only moderate effects on the tested enzymes. However, diallyl disulfide was found to react directly with reduced glutathione (k2=100 M(-2) s(-1)). This reaction might contribute to enhanced oxidative stress and the induction of the selenoprotein glutathione peroxidase as determined on activity and transcript levels. All chemopreventive agents tested induced transcript levels of genes associated with cell cycle arrest and apoptosis. This upregulation was accompanied by a dose-dependent decrease in cell number. Our data indicate that modulation of cellular redox networks is likely to contribute to the effects of sulfur-containing chemopreventive agents.

Thioredoxin networks in the malarial parasite Plasmodium falciparum.

The intraerythrocytic protozoan parasite Plasmodium falciparum is responsible for more than 500 million clinical cases of tropical malaria annually. Although exposed to high fluxes of reactive oxygen species, Plasmodium lacks the antioxidant enzymes catalase and glutathione peroxidase. Thus, the parasite depends on the antioxidant capacity of its host cell and its own peroxidases. These are fuelled by the thioredoxin system and are considered to represent the major defense line against peroxides. Five peroxidases that act in different compartments have been described in P. falciparum. They include two typical 2-Cys peroxiredoxins (Prx), a 1-Cys Prx, the so-called antioxidant protein (AOP), which is a further Prx acting on the basis of a 1-Cys mechanism, and a glutathione peroxidase-like thioredoxin peroxidase. Because of their central function in redox regulation and antioxidant defense, some of these proteins might represent highly interesting targets for structure-based drug development. In this article we summarize the present knowledge on the thioredoxin and peroxiredoxin metabolism in malaria parasitized red blood cells. We furthermore report novel data on the biochemical and kinetic characterization of different thioredoxins, of AOP, and of the classic 1-Cys peroxiredoxin of P. falciparum.

Antiglioma activity of 2,2':6',2"-terpyridineplatinum(II) complexes in a rat model--effects on cellular redox metabolism.

The mammalian thioredoxin system, comprising the selenoenzyme thioredoxin reductase (TrxR) and the 12-kDa protein thioredoxin (Trx), is implicated in thiol-mediated antioxidant defense and redox regulatory processes including transcriptional control, DNA synthesis, and apoptosis. Cell proliferation supported by the thioredoxin system can be suppressed by TrxR inhibition. In this study, we assessed the effects of the potent hTrxR inhibitors 4-mercaptopyridine (4'-chloro-2,2':6',2"-terpyridine)platinum nitrate (I(23)2N) and 2-mercaptopyridine (4'-chloro-2,2':6',2"-terpyridine)platinum nitrate (I(25)2N) on glioblastoma in a rat model. These compounds show no or little cross-resistance with cisplatin and are thus of great clinical interest. Triple intravenous application of 25-35 mg/kg of the compounds led to a significant decrease of tumor growth as determined by magnetic resonance imaging. Metabolic as well as redox parameters in the blood of the animals were not altered. However, TrxR activity was significantly decreased in the tumor tissue, and redox parameters-including glutathione concentrations, total antioxidant status, and the activities of different antioxidant enzymes-showed tissue-specific variations. As indicated by different apoptotic markers, the antitumor activity of I(23)2N is not mediated by the induction of programmed cell death but rather by hTrxR inhibition and DNA intercalation leading to cell cycle arrest.

Mechanistic studies on a novel, highly potent gold-phosphole inhibitor of human glutathione reductase.

The homodimeric flavoprotein glutathione reductase (GR) is a central player of cellular redox metabolism, connecting NADPH to the large pool of redox-active thiols. In this work, the inhibition of human GR by a novel gold-phosphole inhibitor (GoPI) has been studied in vitro. Two modes of inhibition are observed, reversible inhibition that is competitive with GSSG followed by irreversible inhibition. When approximately 1 nm GoPI is incubated with NADPH-reduced GR (1.4 nm) the enzyme becomes 50% inhibited. This appears to be the most potent stable inhibitor of human GR to date. Analyzing the monophasic oxidative half-reaction of reduced GR with GSSG at pH 6.9 revealed a K(d)((app)) for GSSG of 63 microm, and a k((obs)max) of 106 s(-1) at 4 degrees C. The reversible inhibition by the gold-phosphole complex [{1-phenyl-2,5-di(2-pyridyl)phosphole}AuCl] involves formation of a complex at the GSSG-binding site of GR (K(d) = 0.46 microm) followed by nucleophilic attack of an active site cysteine residue that leads to covalent modification and complete inactivation of the enzyme. Data from titration spectra, molecular modeling, stopped-flow, and steady-state kinetics support this theory. In addition, covalent binding of the inhibitor to human GR was demonstrated by mass spectrometry. The extraordinary properties of the compound and its derivatives might be exploited for cell biological studies or medical applications, e.g. as an anti-tumor or antiparasitic drug. Preliminary experiments with glioblastoma cells cultured in vitro indicate an anti-proliferative effect of the inhibitor in the lower micromolar range.