Human METTL18 is a histidine-specific methyltransferase that targets RPL3 and affects ribosome biogenesis and function.

Protein methylation occurs primarily on lysine and arginine, but also on some other residues, such as histidine. METTL18 is the last uncharacterized member of a group of human methyltransferases (MTases) that mainly exert lysine methylation, and here we set out to elucidate its function. We found METTL18 to be a nuclear protein that contains a functional nuclear localization signal and accumulates in nucleoli. Recombinant METTL18 methylated a single protein in nuclear extracts and in isolated ribosomes from METTL18 knockout (KO) cells, identified as 60S ribosomal protein L3 (RPL3). We also performed an RPL3 interactomics screen and identified METTL18 as the most significantly enriched MTase. We found that His-245 in RPL3 carries a 3-methylhistidine (3MH; τ-methylhistidine) modification, which was absent in METTL18 KO cells. In addition, both recombinant and endogenous METTL18 were found to be automethylated at His-154, thus further corroborating METTL18 as a histidine-specific MTase. Finally, METTL18 KO cells displayed altered pre-rRNA processing, decreased polysome formation and codon-specific changes in mRNA translation, indicating that METTL18-mediated methylation of RPL3 is important for optimal ribosome biogenesis and function. In conclusion, we have here established METTL18 as the second human histidine-specific protein MTase, and demonstrated its functional relevance.

Proton Compared to X-Irradiation Induces Different Protein Profiles in Oral Cancer Cells and Their Derived Extracellular Vesicles.

Extracellular vesicles (EVs) are membrane-bound particles released from cells, and their cargo can alter the function of recipient cells. EVs from X-irradiated cells have been shown to play a likely role in non-targeted effects. However, EVs derived from proton irradiated cells have not yet been studied. We aimed to investigate the proteome of EVs and their cell of origin after proton or X-irradiation. The EVs were derived from a human oral squamous cell carcinoma (OSCC) cell line exposed to 0, 4, or 8 Gy from either protons or X-rays. The EVs and irradiated OSCC cells underwent liquid chromatography-mass spectrometry for protein identification. Interestingly, we found different protein profiles both in the EVs and in the OSCC cells after proton irradiation compared to X-irradiation. In the EVs, we found that protons cause a downregulation of proteins involved in cell growth and DNA damage response compared to X-rays. In the OSCC cells, proton and X-irradiation induced dissimilar cell death pathways and distinct DNA damage repair systems. These results are of potential importance for understanding how non-targeted effects in normal tissue can be limited and for future implementation of proton therapy in the clinic.

Targeting prohibitins at the cell surface prevents Th17-mediated autoimmunity.

T helper (Th)17 cells represent a unique subset of CD4+ T cells and are vital for clearance of extracellular pathogens including bacteria and fungi. However, Th17 cells are also involved in orchestrating autoimmunity. By employing quantitative surface proteomics, we found that the evolutionarily conserved prohibitins (PHB1/2) are highly expressed on the surface of both murine and human Th17 cells. Increased expression of PHBs at the cell surface contributed to enhanced CRAF/MAPK activation in Th17 cells. Targeting surface-expressed PHBs on Th17 cells with ligands such as Vi polysaccharide (Typhim vaccine) inhibited CRAF-MAPK pathway, reduced interleukin (IL)-17 expression and ameliorated disease pathology with an increase in FOXP3+-expressing Tregs in an animal model for multiple sclerosis (MS). Interestingly, we detected a CD4+ T cell population with high PHB1 surface expression in blood samples from MS patients in comparison with age- and sex-matched healthy subjects. Our observations suggest a pivotal role for the PHB-CRAF-MAPK signalling axis in regulating the polarization and pathogenicity of Th17 cells and unveil druggable targets in autoimmune disorders such as MS.

"Organ-in-a-Column" Coupled On-line with Liquid Chromatography-Mass Spectrometry.

Organoids, i.e., laboratory-grown organ models developed from stem cells, are emerging tools for studying organ physiology, disease modeling, and drug development. On-line analysis of organoids with mass spectrometry would provide analytical versatility and automation. To achieve these features with robust hardware, we have loaded liquid chromatography column housings with induced pluripotent stem cell (iPSC) derived liver organoids and coupled the "organ-in-a-column" units on-line with liquid chromatography-mass spectrometry (LC-MS). Liver organoids were coloaded with glass beads to achieve an even distribution of organoids throughout the column while preventing clogging. The liver organoids were interrogated "on column" with heroin, followed by on-line monitoring of the drug's phase 1 metabolism. Enzymatic metabolism of heroin produced in the "organ-in-a-column" units was detected and monitored using a triple quadrupole MS instrument, serving as a proof-of-concept for on-line coupling of liver organoids and mass spectrometry. Taken together, the technology allows direct integration of liver organoids with LC-MS, allowing selective and automated tracking of drug metabolism over time.

FSAP Protects against Histone-Mediated Increase in Endothelial Permeability In Vitro.

Factor-VII-activating protease (FSAP) is involved in the regulation of hemostasis and inflammation. Extracellular histones play a role in inflammation and the conversion of latent pro-FSAP into active FSAP. FSAP has been shown to regulate endothelial permeability, but the mechanisms are not clear. Here, we have investigated the effects of FSAP on endothelial permeability in vitro. A mixture of histones from calf thymus stimulated permeability, and the wild-type (WT) serine protease domain (SPD) of FSAP blocked this effect. WT-SPD-FSAP did not influence permeability on its own, nor that stimulated by thrombin or vascular endothelial growth factor (VEGF)-A165. Histones induced a large-scale rearrangement of the junction proteins VE-cadherin and zona occludens-1 from a clear junctional distribution to a diffuse pattern. The presence of WT-SPD-FSAP inhibited these changes. Permeability changes by histones were blocked by both TLR-2 and TLR4 blocking antibodies. Histones upregulated the expression of TLR-2, but not TLR-4, in HUVEC cells, and WT-SPD-FSAP abolished the upregulation of TLR-2 expression. An inactive variant, Marburg I (MI)-SPD-FSAP, did not have any of these effects. The inhibition of histone-mediated permeability may be an important function of FSAP with relevance to sepsis, trauma, and stroke and the need to be investigated further in in vivo experiments.

Proteomic Profiling of Saliva and Tears in Radiated Head and Neck Cancer Patients as Compared to Primary Sjögren's Syndrome Patients.

Patients with head and neck cancer (HNC) and patients with primary Sjögren's syndrome (pSS) may exhibit similar symptoms of dry mouth and dry eyes, as a result of radiotherapy (RT) or a consequence of disease progression. To identify the proteins that may serve as promising disease biomarkers, we analysed saliva and tears from 29 radiated HNC patients and 21 healthy controls, and saliva from 14 pSS patients by mass spectrometry-based proteomics. The study revealed several upregulated, and in some instances overlapping, proteins in the two patient groups. Histone H1.4 and neutrophil collagenase were upregulated in whole saliva of both patient groups, while caspase-14, histone H4, and protein S100-A9 were upregulated in HNC saliva only. In HCN tear fluid, the most highly upregulated protein was mucin-like protein 1. These overexpressed proteins in saliva and tears play central roles in inflammation, host cell injury, activation of reactive oxygen species, and tissue repair. In conclusion, the similarities and differences in overexpressed proteins detected in saliva from HNC and pSS patients may contribute to the overall understanding of the different pathophysiological mechanisms inducing dry mouth. Thus, the recurring proteins identified could possibly serve as future promising biomarkers.

Protease activated receptors (PAR)-1 and -2 mediate cellular effects of factor VII activating protease (FSAP).

Factor VII activating protease (FSAP) is a circulating serine protease implicated in thrombosis, atherosclerosis, stroke, and cancer. Using an overexpression strategy, we have systematically investigated the role of protease activated receptors (PAR)-1, -2, -3, and -4 on FSAP-mediated signaling in HEK293T and A549 cells. Cleavage of PAR-reporter constructs and MAPK phosphorylation was used to monitor receptor activation. FSAP cleaved PAR-2 and to a lesser degree PAR-1, but not PAR-3 or PAR-4 in both cell types. Robust MAPK activation in response to FSAP was observed after PAR-2, but not PAR-1 overexpression in HEK293T. Recombinant serine protease domain of wild type FSAP, but not the Marburg I isoform of FSAP, could reproduce the effects of plasma purified FSAP. Canonical cleavage of both PARs was suggested by mass spectrometric analysis of synthetic peptide substrates from the N-terminus of PARs and site directed mutagenesis studies. Surprisingly, knockdown of endogenous PAR-1, but not PAR-2, prevented the apoptosis-inhibitory effect of FSAP, suggesting that PAR1 is nevertheless a direct or indirect target in some cell types. This molecular characterization of PAR-1 and -2 as cellular receptors of FSAP will help to define the actions of FSAP in the context of cancer and vascular biology.

Comparison of LFQ and IPTL for Protein Identification and Relative Quantification.

(1) Background: Mass spectrometry-based quantitative proteome profiling is most commonly performed by label-free quantification (LFQ), stable isotopic labeling with amino acids in cell culture (SILAC), and reporter ion-based isobaric labeling methods (TMT and iTRAQ). Isobaric peptide termini labeling (IPTL) was described as an alternative to these methods and is based on crosswise labeling of both peptide termini and MS2 quantification. High quantification accuracy was assumed for IPTL because multiple quantification points are obtained per identified MS2 spectrum. A direct comparison of IPTL with other quantification methods has not been performed yet because IPTL commonly requires digestion with endoproteinase Lys-C. (2) Methods: To enable tryptic digestion of IPTL samples, a novel labeling for IPTL was developed that combines metabolic labeling (Arg-0/Lys-0 and Arg-d4/Lys-d4, respectively) with crosswise N-terminal dimethylation (d4 and d0, respectively). (3) Results: The comparison of IPTL with LFQ revealed significantly more protein identifications for LFQ above homology ion scores but not above identity ion scores. (4) Conclusions: The quantification accuracy was superior for LFQ despite the many quantification points obtained with IPTL.

Design and Characterization of a New pVII Combinatorial Phage Display Peptide Library for Protease Substrate Mining Using Factor VII Activating Protease (FSAP) as Model.

We describe a novel, easy and efficient combinatorial phage display peptide substrate-mining method to map the substrate specificity of proteases. The peptide library is displayed on the pVII capsid of the M13 bacteriophage, which renders pIII necessary for infectivity and efficient retrieval, in an unmodified state. As capture module, the 3XFLAG was chosen due to its very high binding efficiency to anti-FLAG mAbs and its independency of any post-translational modification. This library was tested with Factor-VII activating protease (WT-FSAP) and its single-nucleotide polymorphism variant Marburg-I (MI)-FSAP. The WT-FSAP results confirmed the previously reported Arg/Lys centered FSAP cleavage site consensus as dominant, as well as reinforcing MI-FSAP as a loss-of-function mutant. Surprisingly, rare substrate clones devoid of basic amino acids were also identified. Indeed one of these peptides was cleaved as free peptide, thus suggesting a broader range of WT-FSAP substrates than previously anticipated.

Predominant cleavage of proteins N-terminal to serines and threonines using scandium(III) triflate.

Proteolytic digestion prior to LC-MS analysis is a key step for the identification of proteins. Digestion of proteins is typically performed with trypsin, but certain proteins or important protein sequence regions might be missed using this endoproteinase. Only few alternative endoproteinases are available and chemical cleavage of proteins is rarely used. Recently, it has been reported that some metal complexes can act as artificial proteases. In particular, the Lewis acid scandium(III) triflate has been shown to catalyze the cleavage of peptide bonds to serine and threonine residues. Therefore, we investigated if this compound can also be used for the cleavage of proteins. For this purpose, several single proteins, the 20S immune-proteasome (17 proteins), and the Universal Proteomics Standard UPS1 (48 proteins) were analyzed by MALDI-MS and/or LC-MS. A high cleavage specificity N-terminal to serine and threonine residues was observed, but also additional peptides with deviating cleavage specificity were found. Scandium(III) triflate can be a useful tool in protein analysis as no other reagent has been reported yet which showed cleavage specificity within proteins to serines and threonines.

Sericin-Induced Melanogenesis in Cultured Retinal Pigment Epithelial Cells Is Associated with Elevated Levels of Hydrogen Peroxide and Inflammatory Proteins.

We previously demonstrated that the silk protein sericin promotes pigmentation of retinal pigment epithelium (RPE) by activating the NF-κB pathway. Among numerous agents, NF-κB can be activated by hydrogen peroxide. In the present study, we explored possible associations between reactive oxygen species and sericin-induced melanogenesis in RPE. The proteome of human fetal RPE cultured for seven days with or without 1% sericin was analyzed using ingenuity pathway analysis (IPA). The proteomic data was verified by immunofluorescence and immunoblotting. Light microscopy and scanning electron microscopy were used to assess morphology. Dihydroethidium (DHE) and dihydrorhodamine (DHR) assays were used to measure superoxide and hydrogen peroxide species. Expression levels of proteins related to inflammation, differentiation, cell survival and cell adhesion were higher in cells cultured in Dulbecco's Modified Eagle Medium (DMEM) with 1% sericin, whereas cells cultured in DMEM alone showed higher expression levels of proteins associated with Bruch's membrane and cytoskeleton. Despite upregulation of inflammatory proteins, sericin co-cultured RPE yielded significantly higher cell viability compared to cells cultured without sericin. Addition of sericin to culture media significantly increased hydrogen peroxide-levels without significantly affecting superoxide-levels. We suggest that sericin-induced melanogenesis in cultured RPE is associated with elevated levels of superoxide dismutase, hydrogen peroxide and inflammatory proteins.

Apoptosis inhibitor 5 is an endogenous inhibitor of caspase-2.

Caspases are key enzymes responsible for mediating apoptotic cell death. Across species, caspase-2 is the most conserved caspase and stands out due to unique features. Apart from cell death, caspase-2 also regulates autophagy, genomic stability and ageing. Caspase-2 requires dimerization for its activation which is primarily accomplished by recruitment to high molecular weight protein complexes in cells. Here, we demonstrate that apoptosis inhibitor 5 (API5/AAC11) is an endogenous and direct inhibitor of caspase-2. API5 protein directly binds to the caspase recruitment domain (CARD) of caspase-2 and impedes dimerization and activation of caspase-2. Interestingly, recombinant API5 directly inhibits full length but not processed caspase-2. Depletion of endogenous API5 leads to an increase in caspase-2 dimerization and activation. Consistently, loss of API5 sensitizes cells to caspase-2-dependent apoptotic cell death. These results establish API5/AAC-11 as a direct inhibitor of caspase-2 and shed further light onto mechanisms driving the activation of this poorly understood caspase.

Ezrin-anchored PKA phosphorylates serine 369 and 373 on connexin 43 to enhance gap junction assembly, communication, and cell fusion.

A limited number of human cells can fuse to form multinucleated syncytia. In the differentiation of human placenta, mononuclear cytotrophoblasts fuse to form an endocrinologically active, non-proliferative, multinucleated syncytium. This syncytium covers the placenta and manages the exchange of nutrients and gases between maternal and fetal circulation. We recently reported protein kinase A (PKA) to be part of a macromolecular signaling complex with ezrin and gap junction protein connexin 43 (Cx43) that provides cAMP-mediated control of gap junction communication. Here, we examined the associated phosphorylation events. Inhibition of PKA activity resulted in decreased Cx43 phosphorylation, which was associated with reduced trophoblast fusion and differentiation. In vitro studies using peptide arrays, together with mass spectrometry, pointed to serine 369 and 373 of Cx43 as the major PKA phosphorylation sites that increases gap junction assembly at the plasmalemma. A combination of knockdown and reconstitution experiments and gap-fluorescence loss in photobleaching assays with mutant Cx43 containing single or double phosphoserine-mimicking amino acid substitutions in putative PKA phosphorylation sites demonstrated that phosphorylation of S369 and S373 mediated gap junction communication, trophoblast differentiation, and cell fusion.

Sumoylation of Rap1 mediates the recruitment of TFIID to promote transcription of ribosomal protein genes.

Transcription factors are abundant Sumo targets, yet the global distribution of Sumo along the chromatin and its physiological relevance in transcription are poorly understood. Using Saccharomyces cerevisiae, we determined the genome-wide localization of Sumo along the chromatin. We discovered that Sumo-enriched genes are almost exclusively involved in translation, such as tRNA genes and ribosomal protein genes (RPGs). Genome-wide expression analysis showed that Sumo positively regulates their transcription. We also discovered that the Sumo consensus motif at RPG promoters is identical to the DNA binding motif of the transcription factor Rap1. We demonstrate that Rap1 is a molecular target of Sumo and that sumoylation of Rap1 is important for cell viability. Furthermore, Rap1 sumoylation promotes recruitment of the basal transcription machinery, and sumoylation of Rap1 cooperates with the target of rapamycin kinase complex 1 (TORC1) pathway to promote RPG transcription. Strikingly, our data reveal that sumoylation of Rap1 functions in a homeostatic feedback loop that sustains RPG transcription during translational stress. Taken together, Sumo regulates the cellular translational capacity by promoting transcription of tRNA genes and RPGs.

Factor VII deficiency: Unveiling the cellular and molecular mechanisms underlying three model alterations of the enzyme catalytic domain.

Activated factor (F) VII is a vitamin K-dependent glycoprotein that initiates blood coagulation upon interaction with tissue factor. FVII deficiency is the most common of the rare congenital bleeding disorders. While the mutational pattern has been extensively characterized, the pathogenic molecular mechanisms of mutations, particularly at the intracellular level, have been poorly defined. Here, we aimed at elucidating the mechanisms underlying altered FVII biosynthesis in the presence of three mutation types in the catalytic domain: a missense change, a microdeletion and a frameshift/elongation, associated with severe or moderate to severe phenotypes. Using CHO-K1 cells transiently transfected with expression vectors containing the wild-type FVII cDNA (FVIIwt) or harboring the p.I289del, p.G420V or p.A354V-p.P464Hfs mutations, we found that the secretion of the FVII mutants was severely decreased compared to FVIIwt. The synthesis rate of the mutants was slower than the FVIIwt and delayed, and no degradation of the FVII mutants by proteasomes, lysosomes or cysteine proteases was observed. Confocal immunofluorescence microscopy studies showed that FVII variants were localized into the endoplasmic reticulum (ER) but were not detectable within the Golgi apparatus. These findings suggested that a common pathogenic mechanism, possibly a defective folding of the mutant proteins, was triggered by the FVII mutations. The misfolded state led to impaired trafficking of these proteins causing ER retention, which would explain the low to very low FVII plasma levels observed in patients carrying these mutations.

Absolute two-point quantification of proteins using dimethylated proteotypic peptides.

Absolute targeted proteomics typically employs known amounts of synthetic stable isotopically labeled peptides which are mixed with the analyte and analysed by LC-MS to determine the concentration of proteins. In order to obtain more data, we evaluated the use of two different stable isotopes of the same peptide as spike-in for absolute quantification. For this purpose, peptide labeling by reductive amination was applied, which is a mild reaction for dimethylation of amine groups with very high yield. Three different forms can be generated with e.g., light and heavy labels for spike-in peptides, and medium label for endogenous peptides. The method was studied with peptides of apolipoprotein A-I, apolipoprotein B-100, and leucine-rich alpha-2-glycoprotein without and with serum. In serum, the endogenous protein concentrations were measured across four orders of magnitude by the two-point quantification method. Less than 20% of coefficient of variation (CV) values and strong correlation with R2 of 0.99 across three analytical replicates was observed. Most importantly, the two-point quantification method allows an internal quality control of the spike-in peptide as strong deviations in ratios calculated between the first and second reference indicate a methodical error. Because of the significant lower costs than synthetically stable isotopically labeled peptides, this approach might be particularly interesting for the absolute quantification of multiple proteins.

CTCF modulates Estrogen Receptor function through specific chromatin and nuclear matrix interactions.

Enhancer regions and transcription start sites of estrogen-target regulated genes are connected by means of Estrogen Receptor long-range chromatin interactions. Yet, the complete molecular mechanisms controlling the transcriptional output of engaged enhancers and subsequent activation of coding genes remain elusive. Here, we report that CTCF binding to enhancer RNAs is enriched when breast cancer cells are stimulated with estrogen. CTCF binding to enhancer regions results in modulation of estrogen-induced gene transcription by preventing Estrogen Receptor chromatin binding and by hindering the formation of additional enhancer-promoter ER looping. Furthermore, the depletion of CTCF facilitates the expression of target genes associated with cell division and increases the rate of breast cancer cell proliferation. We have also uncovered a genomic network connecting loci enriched in cell cycle regulator genes to nuclear lamina that mediates the CTCF function. The nuclear lamina and chromatin interactions are regulated by estrogen-ER. We have observed that the chromatin loops formed when cells are treated with estrogen establish contacts with the nuclear lamina. Once there, the portion of CTCF associated with the nuclear lamina interacts with enhancer regions, limiting the formation of ER loops and the induction of genes present in the loop. Collectively, our results reveal an important, unanticipated interplay between CTCF and nuclear lamina to control the transcription of ER target genes, which has great implications in the rate of growth of breast cancer cells.

Quantitative phosphoproteome analysis of cisplatin-induced apoptosis in Jurkat T cells.

Protein phosphorylation is one of the most important post-translational modifications (PTMs) due to its vital role in cellular functions and signaling pathways. Protein phosphorylation is also known to be involved in the regulation of apoptosis. Previously, we have performed a SILAC-based analysis of tyrosine phosphorylated peptides of cisplatin-induced apoptotic Jurkat T cells. Here, we analyzed the global phosphorylation profile by enrichment of serine/threonine/tyrosine phosphorylated peptides using TiO2 beads. More than 7000 phosphopeptides of more than 2500 phosphoproteins were identified in four biological replicates. Using two different normalized collision energy (NCE) values for fragmentation by higher-energy collisional dissociation (HCD) revealed complementary results. HCD with NCE 25 accounted for 31% and NCE 35 for 12% uniquely identified phosphopeptides, whereas 57% were found at both NCEs. Different peptide lengths and amino acid compositions were observed at different NCE. A phosphopeptide database was generated out of the results obtained using the Swiss-Prot protein database in order to find differences in regulation of specific phosphorylated sites within multiphosphorylated proteins. Several members of the MAPK signaling pathway were found to be upregulated in apoptotic compared to control cells. Changes of phosphorylation of the transcription factors JUN and ATF2 during apoptosis was confirmed by Western blotting.

Identification of Alternative Splice Variants Using Unique Tryptic Peptide Sequences for Database Searches.

Alternative splicing is a mechanism in eukaryotes by which different forms of mRNAs are generated from the same gene. Identification of alternative splice variants requires the identification of peptides specific for alternative splice forms. For this purpose, we generated a human database that contains only unique tryptic peptides specific for alternative splice forms from Swiss-Prot entries. Using this database allows an easy access to splice variant-specific peptide sequences that match to MS data. Furthermore, we combined this database without alternative splice variant-1-specific peptides with human Swiss-Prot. This combined database can be used as a general database for searching of LC-MS data. LC-MS data derived from in-solution digests of two different cell lines (LNCaP, HeLa) and phosphoproteomics studies were analyzed using these two databases. Several nonalternative splice variant-1-specific peptides were found in both cell lines, and some of them seemed to be cell-line-specific. Control and apoptotic phosphoproteomes from Jurkat T cells revealed several nonalternative splice variant-1-specific peptides, and some of them showed clear quantitative differences between the two states.

Identification of kinases phosphorylating 13 sites in the nuclear, DNA-binding protein NUCKS.

NUCKS is a vertebrate specific, nuclear and DNA-binding phospho protein. The protein is highly expressed in rapidly dividing cells, and is overexpressed in a number of cancer tissues. The phosphorylation of NUCKS is cell cycle and DNA-damage regulated, but little is known about the responsible kinases. By utilizing in vitro and in vivo phosphorylation assays using isolated NUCKS as well as synthetic NUCKS-derived peptides in combination with mass spectrometry, phosphopeptide mapping, phosphphoamino acid analyses, phosphospecific antibodies and the use of specific kinase inhibitors, we found that NUCKS is phosphorylated on 11 sites by CK2. At least 7 of the CK2 sites are phosphorylated in vivo. We also found that NUCKS is phosphorylated on two sites by ATM kinase and DNA-PK in vitro, and is phosphorylated in vivo by ATM kinase in γ-irradiated cells. All together, we identified three kinases phosphorylating 13 out of 39 in vivo phosphorylated sites in mammalian NUCKS. The identification of CK2 and PIKK kinases as kinases phosphorylating NUCKS in vivo provide further evidence for the involvement of NUCKS in cell cycle control and DNA repair.