Proteomics Analysis of Monocyte-Derived Hepatocyte-Like Cells Identifies Integrin Beta 3 as a Specific Biomarker for Drug-Induced Liver Injury by Diclofenac.

Idiosyncratic drug-induced liver injury (iDILI) is a major cause of acute liver failure resulting in liver transplantation or death. Prediction and diagnosis of iDILI remain a great challenge, as current models provide unsatisfying results in terms of sensitivity, specificity, and prognostic value. The absence of appropriate tools for iDILI detection also impairs the development of reliable biomarkers. Here, we report on a new method for identification of drug-specific biomarkers. We combined the advantages of monocyte-derived hepatocyte-like (MH) cells, able to mimic individual characteristics, with those of a novel mass spectrometry-based proteomics technology to assess potential biomarkers for Diclofenac-induced DILI. We found over 2,700 proteins differentially regulated in MH cells derived from individual patients. Herefrom, we identified integrin beta 3 (ITGB3) to be specifically upregulated in Diclofenac-treated MH cells from Diclofenac-DILI patients compared to control groups. Finally, we validated ITGB3 by flow cytometry analysis of whole blood and histological staining of liver biopsies derived from patients diagnosed with Diclofenac-DILI. In summary, our results show that biomarker candidates can be identified by proteomics analysis of MH cells. Application of this method to a broader range of drugs in the future will exploit its full potential for the development of drug-specific biomarkers. Data are available via ProteomeXchange with identifier PXD008918.

Genetic diagnosis of Mendelian disorders via RNA sequencing.

Across a variety of Mendelian disorders, ∼50-75% of patients do not receive a genetic diagnosis by exome sequencing indicating disease-causing variants in non-coding regions. Although genome sequencing in principle reveals all genetic variants, their sizeable number and poorer annotation make prioritization challenging. Here, we demonstrate the power of transcriptome sequencing to molecularly diagnose 10% (5 of 48) of mitochondriopathy patients and identify candidate genes for the remainder. We find a median of one aberrantly expressed gene, five aberrant splicing events and six mono-allelically expressed rare variants in patient-derived fibroblasts and establish disease-causing roles for each kind. Private exons often arise from cryptic splice sites providing an important clue for variant prioritization. One such event is found in the complex I assembly factor TIMMDC1 establishing a novel disease-associated gene. In conclusion, our study expands the diagnostic tools for detecting non-exonic variants and provides examples of intronic loss-of-function variants with pathological relevance.

Circular non-coding RNA ANRIL modulates ribosomal RNA maturation and atherosclerosis in humans.

Circular RNAs (circRNAs) are broadly expressed in eukaryotic cells, but their molecular mechanism in human disease remains obscure. Here we show that circular antisense non-coding RNA in the INK4 locus (circANRIL), which is transcribed at a locus of atherosclerotic cardiovascular disease on chromosome 9p21, confers atheroprotection by controlling ribosomal RNA (rRNA) maturation and modulating pathways of atherogenesis. CircANRIL binds to pescadillo homologue 1 (PES1), an essential 60S-preribosomal assembly factor, thereby impairing exonuclease-mediated pre-rRNA processing and ribosome biogenesis in vascular smooth muscle cells and macrophages. As a consequence, circANRIL induces nucleolar stress and p53 activation, resulting in the induction of apoptosis and inhibition of proliferation, which are key cell functions in atherosclerosis. Collectively, these findings identify circANRIL as a prototype of a circRNA regulating ribosome biogenesis and conferring atheroprotection, thereby showing that circularization of long non-coding RNAs may alter RNA function and protect from human disease.

Plasma Proteome Profiling to Assess Human Health and Disease.

Proteins in the circulatory system mirror an individual's physiology. In daily clinical practice, protein levels are generally determined using single-protein immunoassays. High-throughput, quantitative analysis using mass-spectrometry-based proteomics of blood, plasma, and serum would be advantageous but is challenging because of the high dynamic range of protein abundances. Here, we introduce a rapid and robust "plasma proteome profiling" pipeline. This single-run shotgun proteomic workflow does not require protein depletion and enables quantitative analysis of hundreds of plasma proteomes from 1 µl single finger pricks with 20 min gradients. The apolipoprotein family, inflammatory markers such as C-reactive protein, gender-related proteins, and >40 FDA-approved biomarkers are reproducibly quantified (CV <20% with label-free quantification). Furthermore, we functionally interpret a 1,000-protein, quantitative plasma proteome obtained by simple peptide pre-fractionation. Plasma proteome profiling delivers an informative portrait of a person's health state, and we envision its large-scale use in biomedicine.

Ultra-deep and quantitative saliva proteome reveals dynamics of the oral microbiome.

BACKGROUND: The oral cavity is home to one of the most diverse microbial communities of the human body and a major entry portal for pathogens. Its homeostasis is maintained by saliva, which fulfills key functions including lubrication of food, pre-digestion, and bacterial defense. Consequently, disruptions in saliva secretion and changes in the oral microbiome contribute to conditions such as tooth decay and respiratory tract infections. Here we set out to quantitatively map the saliva proteome in great depth with a rapid and in-depth mass spectrometry-based proteomics workflow. METHODS: We used recent improvements in mass spectrometry (MS)-based proteomics to develop a rapid workflow for mapping the saliva proteome quantitatively and at great depth. Standard clinical cotton swabs were used to collect saliva form eight healthy individuals at two different time points, allowing us to study inter-individual differences and interday changes of the saliva proteome. To accurately identify microbial proteins, we developed a method called "split by taxonomy id" that prevents peptides shared by humans and bacteria or between different bacterial phyla to contribute to protein identification. RESULTS: Microgram protein amounts retrieved from cotton swabs resulted in more than 3700 quantified human proteins in 100-min gradients or 5500 proteins after simple fractionation. Remarkably, our measurements also quantified more than 2000 microbial proteins from 50 bacterial genera. Co-analysis of the proteomics results with next-generation sequencing data from the Human Microbiome Project as well as a comparison to MALDI-TOF mass spectrometry on microbial cultures revealed strong agreement. The oral microbiome differs between individuals and changes drastically upon eating and tooth brushing. CONCLUSION: Rapid shotgun and robust technology can now simultaneously characterize the human and microbiome contributions to the proteome of a body fluid and is therefore a valuable complement to genomic studies. This opens new frontiers for the study of host-pathogen interactions and clinical saliva diagnostics.

Versatile and Efficient Site-Specific Protein Functionalization by Tubulin Tyrosine Ligase.

A novel chemoenzymatic approach for simple and fast site-specific protein labeling is reported. Recombinant tubulin tyrosine ligase (TTL) was repurposed to attach various unnatural tyrosine derivatives as small bioorthogonal handles to proteins containing a short tubulin-derived recognition sequence (Tub-tag). This novel strategy enables a broad range of high-yielding and fast chemoselective C-terminal protein modifications on isolated proteins or in cell lysates for applications in biochemistry, cell biology, and beyond, as demonstrated by the site-specific labeling of nanobodies, GFP, and ubiquitin.

DNA methylation requires a DNMT1 ubiquitin interacting motif (UIM) and histone ubiquitination.

DNMT1 is recruited by PCNA and UHRF1 to maintain DNA methylation after replication. UHRF1 recognizes hemimethylated DNA substrates via the SRA domain, but also repressive H3K9me3 histone marks with its TTD. With systematic mutagenesis and functional assays, we could show that chromatin binding further involved UHRF1 PHD binding to unmodified H3R2. These complementation assays clearly demonstrated that the ubiquitin ligase activity of the UHRF1 RING domain is required for maintenance DNA methylation. Mass spectrometry of UHRF1-deficient cells revealed H3K18 as a novel ubiquitination target of UHRF1 in mammalian cells. With bioinformatics and mutational analyses, we identified a ubiquitin interacting motif (UIM) in the N-terminal regulatory domain of DNMT1 that binds to ubiquitinated H3 tails and is essential for DNA methylation in vivo. H3 ubiquitination and subsequent DNA methylation required UHRF1 PHD binding to H3R2. These results show the manifold regulatory mechanisms controlling DNMT1 activity that require the reading and writing of epigenetic marks by UHRF1 and illustrate the multifaceted interplay between DNA and histone modifications. The identification and functional characterization of the DNMT1 UIM suggests a novel regulatory principle and we speculate that histone H2AK119 ubiquitination might also lead to UIM-dependent recruitment of DNMT1 and DNA methylation beyond classic maintenance.

Minimal, encapsulated proteomic-sample processing applied to copy-number estimation in eukaryotic cells.

Mass spectrometry (MS)-based proteomics typically employs multistep sample-preparation workflows that are subject to sample contamination and loss. We report an in-StageTip method for performing sample processing, from cell lysis through elution of purified peptides, in a single, enclosed volume. This robust and scalable method largely eliminates contamination or loss. Peptides can be eluted in several fractions or in one step for single-run proteome analysis. In one day, we obtained the largest proteome coverage to date for budding and fission yeast, and found that protein copy numbers in these cells were highly correlated (R(2) = 0.78). Applying the in-StageTip method to quadruplicate measurements of a human cell line, we obtained copy-number estimates for 9,667 human proteins and observed excellent quantitative reproducibility between replicates (R(2) = 0.97). The in-StageTip method is straightforward and generally applicable in biological or clinical applications.

Establishment of a robust single axis of cell polarity by coupling multiple positive feedback loops.

Establishment of cell polarity--or symmetry breaking--relies on local accumulation of polarity regulators. Although simple positive feedback is sufficient to drive symmetry breaking, it is highly sensitive to stochastic fluctuations typical for living cells. Here, by integrating mathematical modelling with quantitative experimental validations, we show that in the yeast Saccharomyces cerevisiae a combination of actin- and guanine nucleotide dissociation inhibitor-dependent recycling of the central polarity regulator Cdc42 is needed to establish robust cell polarity at a single site during yeast budding. The guanine nucleotide dissociation inhibitor pathway consistently generates a single-polarization site, but requires Cdc42 to cycle rapidly between its active and inactive form, and is therefore sensitive to perturbations of the GTPase cycle. Conversely, actin-mediated recycling of Cdc42 induces robust symmetry breaking but cannot restrict polarization to a single site. Our results demonstrate how cells optimize symmetry breaking through coupling between multiple feedback loops.

Fluorescent protein specific Nanotraps to study protein-protein interactions and histone-tail peptide binding.

Fluorescent proteins are widely used to study protein localization and protein dynamics in living cells. Additional information on peptide binding, DNA binding, enzymatic activity, and complex formation can be obtained with various methods including chromatin immunoprecipitation (ChIP) and affinity purification. Here we describe two specific GFP- and RFP binding proteins based on antibody fragments derived from llama single domain antibodies. The binding proteins can be produced in bacteria and coupled to monovalent matrixes generating so-called Nanotraps. Both Nanotraps allow a fast and efficient (one-step) isolation of fluorescent fusion proteins and their interacting factors for biochemical analyses including mass spectroscopy and enzyme activity measurements. Here we provide protocols for precipitation of fluorescent fusion proteins from crude cell extracts to identify and map protein-protein interactions as well as specific histone tail peptide binding in an easy and reliable manner.

Versatile toolbox for high throughput biochemical and functional studies with fluorescent fusion proteins.

Fluorescent fusion proteins are widely used to study protein localization and interaction dynamics in living cells. However, to fully characterize proteins and to understand their function it is crucial to determine biochemical characteristics such as enzymatic activity and binding specificity. Here we demonstrate an easy, reliable and versatile medium/high-throughput method to study biochemical and functional characteristics of fluorescent fusion proteins. Using a new system based on 96-well micro plates comprising an immobilized GFP-binding protein (GFP-mulitTrap), we performed fast and efficient one-step purification of different GFP- and YFP-fusion proteins from crude cell lysate. After immobilization we determined highly reproducible binding ratios of cellular expressed GFP-fusion proteins to histone-tail peptides, DNA or selected RFP-fusion proteins. In particular, we found Cbx1 preferentially binding to di-and trimethylated H3K9 that is abolished by phosphorylation of the adjacent serine. DNA binding assays showed, that the MBD domain of MeCP2 discriminates between fully methylated over unmethylated DNA and protein-protein interactions studies demonstrate, that the PBD domain of Dnmt1 is essential for binding to PCNA. Moreover, using an ELISA-based approach, we detected endogenous PCNA and histone H3 bound at GFP-fusions. In addition, we quantified the level of H3K4me2 on nucleosomes containing different histone variants. In summary, we present an innovative medium/high-throughput approach to analyse binding specificities of fluroescently labeled fusion proteins and to detect endogenous interacting factors in a fast and reliable manner in vitro.

Regulation of DNA methyltransferase 1 by interactions and modifications.

DNA methylation plays a central role in the epigenetic regulation of gene expression during development and disease. Remarkably, the complex and changing patterns of genomic DNA methylation are established and maintained by only three DNA methyltransferases. Here we focus on DNMT1, the major and ubiquitously expressed DNA methyltransferase in vertebrates, to outline possible regulatory mechanisms. A list of all protein interactions and post-translational modifications reported for DNMT1 clearly shows that DNMT1, and by extension also DNA methylation in general, are functionally linked with several other epigenetic pathways and cellular processes. General themes of these interactions and modifications include the activation, stabilization and recruitment of DNMT1 at specific sites and heterochromatin regions. For a comprehensive understanding of the regulation of DNA methylation it is now necessary to systematically quantify the interactions and modifications of DNMT1, to elucidate their function at the molecular level and to integrate these data at the cellular level.

Cooperative DNA and histone binding by Uhrf2 links the two major repressive epigenetic pathways.

Gene expression is regulated by DNA as well as histone modifications but the crosstalk and mechanistic link between these epigenetic signals are still poorly understood. Here we investigate the multi-domain protein Uhrf2 that is similar to Uhrf1, an essential cofactor of maintenance DNA methylation. Binding assays demonstrate a cooperative interplay of Uhrf2 domains that induces preference for hemimethylated DNA, the substrate of maintenance methylation, and enhances binding to H3K9me3 heterochromatin marks. FRAP analyses revealed that localization and binding dynamics of Uhrf2 in vivo require an intact tandem Tudor domain and depend on H3K9 trimethylation but not on DNA methylation. Besides the cooperative DNA and histone binding that is characteristic for Uhrf2, we also found an opposite expression pattern of uhrf1 and uhrf2 during differentiation. While uhrf1 is mainly expressed in pluripotent stem cells, uhrf2 is upregulated during differentiation and highly expressed in differentiated mouse tissues. Ectopic expression of Uhrf2 in uhrf1(-/-) embryonic stem cells did not restore DNA methylation at major satellites indicating functional differences. We propose that the cooperative interplay of Uhrf2 domains may contribute to a tighter epigenetic control of gene expression in differentiated cells.

The multi-domain protein Np95 connects DNA methylation and histone modification.

DNA methylation and histone modifications play a central role in the epigenetic regulation of gene expression and cell differentiation. Recently, Np95 (also known as UHRF1 or ICBP90) has been found to interact with Dnmt1 and to bind hemimethylated DNA, indicating together with genetic studies a central role in the maintenance of DNA methylation. Using in vitro binding assays we observed a weak preference of Np95 and its SRA (SET- and Ring-associated) domain for hemimethylated CpG sites. However, the binding kinetics of Np95 in living cells was not affected by the complete loss of genomic methylation. Investigating further links with heterochromatin, we could show that Np95 preferentially binds histone H3 N-terminal tails with trimethylated (H3K9me3) but not acetylated lysine 9 via a tandem Tudor domain. This domain contains three highly conserved aromatic amino acids that form an aromatic cage similar to the one binding H3K9me3 in the chromodomain of HP1ss. Mutations targeting the aromatic cage of the Np95 tandem Tudor domain (Y188A and Y191A) abolished specific H3 histone tail binding. These multiple interactions of the multi-domain protein Np95 with hemimethylated DNA and repressive histone marks as well as with DNA and histone methyltransferases integrate the two major epigenetic silencing pathways.