Mechanisms of hepatotoxicity associated with the monocyclic ß-lactam antibiotic BAL30072.

BAL30072 is a new monocyclic ß-lactam antibiotic under development which provides a therapeutic option for the treatment of severe infections caused by multi-drug-resistant Gram-negative bacteria. Despite the absence of liver toxicity in preclinical studies in rats and marmosets and in single dose clinical studies in humans, increased transaminase activities were observed in healthy subjects in multiple-dose clinical studies. We, therefore, initiated a comprehensive program to find out the mechanisms leading to hepatocellular injury using HepG2 cells (human hepatocellular carcinoma cell line), HepaRG cells (inducible hepatocytes derived from a human hepatic progenitor cell line), and human liver microtissue preparations. Our investigations demonstrated a concentration- and time-dependent reduction of the ATP content of BAL30072-treated HepG2 cells and liver microtissues. BAL30072 impaired oxygen consumption by HepG2 cells at clinically relevant concentrations, inhibited complexes II and III of the mitochondrial electron transport chain, increased the production of reactive oxygen species (ROS), and reduced the mitochondrial membrane potential. Furthermore, BAL 30072 impaired mitochondrial fatty acid metabolism, inhibited glycolysis, and was associated with hepatocyte apoptosis. Co-administration of N-acetyl-L-cysteine partially protected hepatocytes from BAL30072-mediated toxicity, underscoring the role of oxidative damage in the observed hepatocellular toxicity. In conclusion, BAL30072 is toxic for liver mitochondria and inhibits glycolysis at clinically relevant concentrations. Impaired hepatic mitochondrial function and inhibition of glycolysis can explain liver injury observed in human subjects receiving long-term treatment with this compound.

Utility of spherical human liver microtissues for prediction of clinical drug-induced liver injury.

Drug-induced liver injury (DILI) continues to be a major source of clinical attrition, precautionary warnings, and post-market withdrawal of drugs. Accordingly, there is a need for more predictive tools to assess hepatotoxicity risk in drug discovery. Three-dimensional (3D) spheroid hepatic cultures have emerged as promising tools to assess mechanisms of hepatotoxicity, as they demonstrate enhanced liver phenotype, metabolic activity, and stability in culture not attainable with conventional two-dimensional hepatic models. Increased sensitivity of these models to drug-induced cytotoxicity has been demonstrated with relatively small panels of hepatotoxicants. However, a comprehensive evaluation of these models is lacking. Here, the predictive value of 3D human liver microtissues (hLiMT) to identify known hepatotoxicants using a panel of 110 drugs with and without clinical DILI has been assessed in comparison to plated two-dimensional primary human hepatocytes (PHH). Compounds were treated long-term (14 days) in hLiMT and acutely (2 days) in PHH to assess drug-induced cytotoxicity over an 8-point concentration range to generate IC50 values. Regardless of comparing IC50 values or exposure-corrected margin of safety values, hLiMT demonstrated increased sensitivity in identifying known hepatotoxicants than PHH, while specificity was consistent across both assays. In addition, hLiMT out performed PHH in correctly classifying hepatotoxicants from different pharmacological classes of molecules. The hLiMT demonstrated sufficient capability to warrant exploratory liver injury biomarker investigation (miR-122, HMGB1, α-GST) in the cell-culture media. Taken together, this study represents the most comprehensive evaluation of 3D spheroid hepatic cultures up to now and supports their utility for hepatotoxicity risk assessment in drug discovery.

Extending the limits of quantitative proteome profiling with data-independent acquisition and application to acetaminophen-treated three-dimensional liver microtissues.

The data-independent acquisition (DIA) approach has recently been introduced as a novel mass spectrometric method that promises to combine the high content aspect of shotgun proteomics with the reproducibility and precision of selected reaction monitoring. Here, we evaluate, whether SWATH-MS type DIA effectively translates into a better protein profiling as compared with the established shotgun proteomics. We implemented a novel DIA method on the widely used Orbitrap platform and used retention-time-normalized (iRT) spectral libraries for targeted data extraction using Spectronaut. We call this combination hyper reaction monitoring (HRM). Using a controlled sample set, we show that HRM outperformed shotgun proteomics both in the number of consistently identified peptides across multiple measurements and quantification of differentially abundant proteins. The reproducibility of HRM in peptide detection was above 98%, resulting in quasi complete data sets compared with 49% of shotgun proteomics. Utilizing HRM, we profiled acetaminophen (APAP)(1)-treated three-dimensional human liver microtissues. An early onset of relevant proteome changes was revealed at subtoxic doses of APAP. Further, we detected and quantified for the first time human NAPQI-protein adducts that might be relevant for the toxicity of APAP. The adducts were identified on four mitochondrial oxidative stress related proteins (GATM, PARK7, PRDX6, and VDAC2) and two other proteins (ANXA2 and FTCD). Our findings imply that DIA should be the preferred method for quantitative protein profiling.

Differential basolateral-apical distribution of scavenger receptor, class B, type I in cultured cells and the liver.

The high-density lipoprotein (HDL) receptor, scavenger receptor class B, type I (SR-BI), mediates selective cholesteryl ester uptake into the liver, which finally results in cholesterol secretion into the bile. Despite several reports, the distribution of hepatic SR-BI between the sinusoidal and canalicular membranes is still under debate. We present immunohistological data using specific markers showing that the bulk of SR-BI is present in sinusoidal membranes and, to a lesser extent, in canalicular membranes in murine and human liver sections. In addition, SR-BI was detected in preparations of rat liver canalicular membranes. We also compared the in vivo findings to HepG2 cells, a widely used in vitro hepatocyte model. Interestingly, SR-BI was enriched in bile canalicular-like (BC-like) structures in polarized HepG2 cells, which were cultivated either conventionally to form a monolayer or in Matrigel to form three-dimensional structures. Fluorescently labeled HDL was transported into close proximity of BC-like structures, whereas HDL labeled with the fluorescent cholesterol analog BODIPY-cholesterol was clearly detected within these structures. Importantly, similarly to human and mouse liver, SR-BI was localized in basolateral membranes in three-dimensional liver microtissues from primary human liver cells. Our results demonstrate that SR-BI is highly enriched in sinusoidal membranes and is also found in canalicular membranes. There was no significant basolateral-apical redistribution of hepatic SR-BI in fasting and refeeding experiments in mice. Furthermore, in vitro studies in polarized HepG2 cells showed explicit differences as SR-BI was highly enriched in BC-like structures. These structures are, however, functional and accumulated HDL-derived cholesterol. Thus, biological relevant model systems should be employed when investigating SR-BI distribution in vitro.

Hepatic toxicology following single and multiple exposure of engineered nanomaterials utilising a novel primary human 3D liver microtissue model.

BACKGROUND: The liver has a crucial role in metabolic homeostasis as well as being the principal detoxification centre of the body, removing xenobiotics and waste products which could potentially include some nanomaterials (NM). With the ever increasing public and occupational exposure associated with accumulative production of nanomaterials, there is an urgent need to consider the possibility of detrimental health consequences of engineered NM exposure. It has been shown that exposure via inhalation, intratracheal instillation or ingestion can result in NM translocation to the liver. Traditional in vitro or ex vivo hepatic nanotoxicology models are often limiting and/or troublesome (i.e. reduced metabolism enzymes, lacking important cell populations, unstable with very high variability, etc.). METHODS: In order to rectify these issues and for the very first time we have utilised a 3D human liver microtissue model to investigate the toxicological effects associated with a single or multiple exposure of a panel of engineered NMs (Ag, ZnO, MWCNT and a positively charged TiO2). RESULTS: Here we demonstrate that the repeated exposure of the NMs is more damaging to the liver tissue as in comparison to a single exposure with the adverse effects more significant following treatment with the Ag and ZnO as compared with the TiO2 and MWCNT NMs (in terms of cytotoxicity, cytokine secretion, lipid peroxidation and genotoxicity). CONCLUSIONS: Overall, this study demonstrates that the human microtissue model utilised herein is an excellent candidate for replacement of traditional in vitro single cell hepatic models and further progression of liver nanotoxicology.

An Ethnographic Study of Patient Life Experience in Early-Stage Parkinson's Disease in the United States and Germany.

INTRODUCTION: Existing qualitative research on early-stage Parkinson's disease draws on patients' reported disease experience, aiming to capture the symptoms and impacts most relevant to patients living with the disease. As a complement to this research, the present study investigated the patient experience of early-stage Parkinson's disease from a holistic, ethnographic perspective. We explored the attitudes, beliefs, and social structures that shape how people understand and adapt to life with early-stage Parkinson's disease. METHODS: Researchers interviewed 30 people with early-stage Parkinson's disease, 10 relatives, and 10 neurologists and movement disorder specialists in the USA and Germany. Many of these interviews took place in-person, allowing researchers to spend time in participants' homes and witness their daily lives. A multidisciplinary team of social scientists, clinical researchers, and patient organization representatives led the mixed-methods study design and analysis. In-depth ethnographic interviews yielded qualitative insights, with a quantitative survey following to assess their prevalence in a larger sample of 150 patients. RESULTS: In addition to developing a patient life experience pathway of early-stage Parkinson's disease, we identified five key thematic findings that provide insight into how the clinical features of the disease become meaningful to patients on the context of their daily lives, family relations, and subjective well-being: (1) People with early-stage Parkinson's disease start coming to terms with their disease before receiving a medical diagnosis; (2) Acceptance is not a finalized achievement, but a cyclical process; (3) People with early-stage Parkinson's disease "live in the moment" to make the future more manageable; (4) Slowing disease progression is an important goal driving the actions of people with early-stage Parkinson's; and (5) People with early-stage Parkinson's disease value information that is grounded in lived experience and relevant to their stage of disease progression. CONCLUSION: This holistic, ethnographic approach to patient life experience provided five key thematic findings that complement insights from qualitative and quantitative datasets on early-stage Parkinson's disease. An enhanced understanding of how early-stage Parkinson's symptoms impact patients' health-related quality of life and their broader social lives can help us better understand how patients make decisions about their usage of healthcare services and therapies.

This study aimed to understand the experience of people living with early-stage Parkinson's. In addition to looking at how symptoms impact people's daily lives, this research examined how people think about and give meaning to early-stage Parkinson's. The first step was to conduct interviews with people with early-stage Parkinson's, their relatives, and doctors. These interviews covered topics such as how people with early-stage Parkinson's are eventually diagnosed, where they go for information, and how they approach the future. In the second step recordings and transcripts of the interviews were analyzed in detail. The ideas and themes that emerged from analysis were used to create a picture of how people experience early-stage Parkinson's as part of their broader lives. Researchers identified five key insights: (1) people often begin to come to terms with Parkinson's before being diagnosed; (2) accepting Parkinson's is an ongoing process; (3) people with early-stage Parkinson's value living in the moment; (4) people with early-stage Parkinson's see slowing the worsening of the disease as an important goal; and (5) learning from the first-hand experience of others can be more valuable than scientific information. Ultimately, this research shows that understanding how early-stage Parkinson's fits into people's everyday lives can help researchers, doctors, and patient organizations provide more effective support and care.

PARP1 ADP-ribosylates lysine residues of the core histone tails.

The chromatin-associated enzyme PARP1 has previously been suggested to ADP-ribosylate histones, but the specific ADP-ribose acceptor sites have remained enigmatic. Here, we show that PARP1 covalently ADP-ribosylates the amino-terminal histone tails of all core histones. Using biochemical tools and novel electron transfer dissociation mass spectrometric protocols, we identify for the first time K13 of H2A, K30 of H2B, K27 and K37 of H3, as well as K16 of H4 as ADP-ribose acceptor sites. Multiple explicit water molecular dynamics simulations of the H4 tail peptide into the catalytic cleft of PARP1 indicate that two stable intermolecular salt bridges hold the peptide in an orientation that allows K16 ADP-ribosylation. Consistent with a functional cross-talk between ADP-ribosylation and other histone tail modifications, acetylation of H4K16 inhibits ADP-ribosylation by PARP1. Taken together, our computational and experimental results provide strong evidence that PARP1 modifies important regulatory lysines of the core histone tails.

Molecular mechanism of poly(ADP-ribosyl)ation by PARP1 and identification of lysine residues as ADP-ribose acceptor sites.

Poly(ADP-ribose) polymerase 1 (PARP1) synthesizes poly(ADP-ribose) (PAR) using nicotinamide adenine dinucleotide (NAD) as a substrate. Despite intensive research on the cellular functions of PARP1, the molecular mechanism of PAR formation has not been comprehensively understood. In this study, we elucidate the molecular mechanisms of poly(ADP-ribosyl)ation and identify PAR acceptor sites. Generation of different chimera proteins revealed that the amino-terminal domains of PARP1, PARP2 and PARP3 cooperate tightly with their corresponding catalytic domains. The DNA-dependent interaction between the amino-terminal DNA-binding domain and the catalytic domain of PARP1 increased V(max) and decreased the K(m) for NAD. Furthermore, we show that glutamic acid residues in the auto-modification domain of PARP1 are not required for PAR formation. Instead, we identify individual lysine residues as acceptor sites for ADP-ribosylation. Together, our findings provide novel mechanistic insights into PAR synthesis with significant relevance for the different biological functions of PARP family members.

Sumoylation of poly(ADP-ribose) polymerase 1 inhibits its acetylation and restrains transcriptional coactivator function.

Poly(ADP-ribose) polymerase 1 (PARP1) is a chromatin-associated nuclear protein and functions as a molecular stress sensor. At the cellular level, PARP1 has been implicated in a wide range of processes, such as maintenance of genome stability, cell death, and transcription. PARP1 functions as a transcriptional coactivator of nuclear factor kappaB (NF-kappaB) and hypoxia inducible factor 1 (HIF1). In proteomic studies, PARP1 was found to be modified by small ubiquitin-like modifiers (SUMOs). Here, we characterize PARP1 as a substrate for modification by SUMO1 and SUMO3, both in vitro and in vivo. PARP1 is sumoylated at the single lysine residue K486 within its automodification domain. Interestingly, modification of PARP1 with SUMO does not affect its ADP-ribosylation activity but completely abrogates p300-mediated acetylation of PARP1, revealing an intriguing crosstalk of sumoylation and acetylation on PARP1. Genetic complementation of PARP1-depleted cells with wild-type and sumoylation-deficient PARP1 revealed that SUMO modification of PARP1 restrains its transcriptional coactivator function and subsequently reduces gene expression of distinct PARP1-regulated target genes.

Physarum nitric oxide synthases: genomic structures and enzymology of recombinant proteins.

Physarum polycephalum expresses two closely related, calcium-independent NOSs (nitric oxide synthases). In our previous work, we showed that both NOSs are induced during starvation and apparently play a functional role in sporulation. In the present study, we characterized the genomic structures of both Physarum NOSs, expressed both enzymes recombinantly in bacteria and characterized their biochemical properties. Whereas the overall genomic organization of Physarum NOS genes is comparable with various animal NOSs, none of the exon-intron boundaries are conserved. Recombinant expression of clones with various N-termini identified N-terminal amino acids essential for enzyme activity, but not required for haem binding or dimerization, and suggests the usage of non-AUG start codons for Physarum NOSs. Biochemical characterization of the two Physarum isoenzymes revealed different affinities for L-arginine, FMN and 6R-5,6,7,8-tetrahydro-L-biopterin.

Poly(ADP-ribose) polymerase 1 promotes tumor cell survival by coactivating hypoxia-inducible factor-1-dependent gene expression.

Hypoxia-inducible factor 1 (HIF-1) is the key transcription factor regulating hypoxia-dependent gene expression. Lack of oxygen stabilizes HIF-1, which in turn modulates the gene expression pattern to adapt cells to the hypoxic environment. Activation of HIF-1 is also detected in most solid tumors and supports tumor growth through the expression of target genes that are involved in processes like cell proliferation, energy metabolism, and oxygen delivery. Poly(ADP-ribose) polymerase 1 (PARP1) is a chromatin-associated protein, which was shown to regulate transcription. Here we report that chronic myelogenous leukemia cells expressing small interfering RNA against PARP1, which were injected into wild-type mice expressing PARP1, showed tumor growth with increased levels of necrosis, limited vascularization, and reduced expression of GLUT-1. Of note, PARP1-deficient cells showed a reduced HIF-1 transcriptional activation that was dependent on PARP1 enzymatic activity. PARP1 neither influenced binding of HIF-1 to its hypoxic response element nor changed HIF-1alpha protein levels in hypoxic cells. However, PARP1 formed a complex with HIF-1alpha through direct protein interaction and coactivated HIF-1alpha-dependent gene expression. These findings provide convincing evidence that wild-type mice expressing PARP1 cannot compensate for the loss of PARP1 in tumor cells and strengthen the importance of the role of PARP1 as a transcriptional coactivator of HIF-1-dependent gene expression during tumor progression.

TET enzymes control antibody production and shape the mutational landscape in germinal centre B cells.

Upon activation by antigen, B cells form germinal centres where they clonally expand and introduce affinity-enhancing mutations into their B-cell receptor genes. Somatic mutagenesis and class switch recombination (CSR) in germinal centre B cells are initiated by the activation-induced cytidine deaminase (AID). Upon germinal centre exit, B cells differentiate into antibody-secreting plasma cells. Germinal centre maintenance and terminal fate choice require transcriptional reprogramming that associates with a substantial reconfiguration of DNA methylation patterns. Here we examine the role of ten-eleven-translocation (TET) proteins, enzymes that facilitate DNA demethylation and promote a permissive chromatin state by oxidizing 5-methylcytosine, in antibody-mediated immunity. Using a conditional gene ablation strategy, we show that TET2 and TET3 guide the transition of germinal centre B cells to antibody-secreting plasma cells. Optimal AID expression requires TET function, and TET2 and TET3 double-deficient germinal centre B cells show defects in CSR. However, TET2/TET3 double-deficiency does not prevent the generation and selection of high-affinity germinal centre B cells. Rather, combined TET2 and TET3 loss-of-function in germinal centre B cells favours C-to-T and G-to-A transition mutagenesis, a finding that may be of significance for understanding the aetiology of B-cell lymphomas evolving in conditions of reduced TET function.

Transcriptomic, Proteomic, and Functional Long-Term Characterization of Multicellular Three-Dimensional Human Liver Microtissues.

Three-Dimensional (3D) liver microtissues, specifically prepared from primary human hepatocytes (PHH) in coculture with nonparenchymal cells (NPCs), have been shown to be a valuable tool for in vitro toxicology. However, a lack of thorough characterization on a functional, transcriptomic, and proteomic level of such models during long-term cultivation is evident. By integrating multiple omics technologies, we provide in this study an in-depth long-term characterization of 3D microtissues composed of PHH from three different donors cocultured with primary NPCs. The 3D human liver microtissues (hLiMTs) exhibited stable adenosine triphosphate (ATP) content and albumin secretion over 5 weeks. Histological analysis indicated a healthy liver tissue with polarized expression of bile salt export pump (BSEP) and multidrug resistance protein 2 (MRP2) in a structure reminiscent of bile canaliculi. The 3D microtissues exhibited stable basal and inducible cytochrome P450 activities up to 5 weeks in culture. Analysis of 40,716 transcripts using RNA arrays revealed distinct similarities to native human liver gene expression. Long-term culture showed a stable phenotype up to 5 weeks, with differences in liver gene expression primarily attributed to individual donors. Proteomic profiling of 2200 unique proteins by label-free LC-MS/MS revealed a relatively stable protein expression where only 7.3% were up- or downregulated more than twofold from day 7 to 35 in culture. Taken together, these results suggest that hLiMTs represent a responsive and physiologically relevant in vitro liver model that maintains stable function over 5 weeks and is therefore well suited for repeated-dose toxicity testing.

Prediction of Safety Margin and Optimization of Dosing Protocol for a Novel Antibiotic using Quantitative Systems Pharmacology Modeling.

Elevations of liver enzymes have been observed in clinical trials with BAL30072, a novel antibiotic. In vitro assays have identified potential mechanisms for the observed hepatotoxicity, including electron transport chain (ETC) inhibition and reactive oxygen species (ROS) generation. DILIsym, a quantitative systems pharmacology (QSP) model of drug-induced liver injury, has been used to predict the likelihood that each mechanism explains the observed toxicity. DILIsym was also used to predict the safety margin for a novel BAL30072 dosing scheme; it was predicted to be low. DILIsym was then used to recommend potential modifications to this dosing scheme; weight-adjusted dosing and a requirement to assay plasma alanine aminotransferase (ALT) daily and stop dosing as soon as ALT increases were observed improved the predicted safety margin of BAL30072 and decreased the predicted likelihood of severe injury. This research demonstrates a potential application for QSP modeling in improving the safety profile of candidate drugs.

Pro-fibrotic compounds induce stellate cell activation, ECM-remodelling and Nrf2 activation in a human 3D-multicellular model of liver fibrosis.

BACKGROUND & AIMS: Currently most liver fibrosis research is performed in vivo, since suitable alternative in vitro systems which are able to recapitulate the cellular events leading to liver fibrosis are lacking. Here we aimed at generating a system containing cells representing the three key players of liver fibrosis (hepatocyte, Kupffer cells and stellate cells) and assess their response to pro-fibrotic compounds such as TGF-ß1, methotrexate (MTX) and thioacetamide (TAA). METHODS: Human cell lines representing hepatocytes (HepaRG), Kupffer cell (THP-1 macrophages) and stellate cells (hTERT-HSC) were co-cultured using the InSphero hanging drop technology to generate scaffold-free 3D microtissues, that were treated with pro-fibrotic compounds (TGF-ß1, MTX, TAA) for up to 14 days. The response of the microtissues was evaluated by determining the expression of cytokines (TNF-α, TGF-ß1 and IL6), the deposition and secretion of ECM proteins and induction of gene expression of fibrosis biomarkers (e.g. αSMA). Induction of Nrf2 and Keap1, as key player of defence mechanism, was also evaluated. RESULTS: We could demonstrate that the multicellular 3D microtissue cultures could be maintained in a non-activated status, based on the low expression levels of activation markers. Macrophages were activated by stimulation with LPS and hTERT-HSC showed activation by TGF-ß1. In addition, MTX and TAA elicited a fibrotic phenotype, as assessed by gene-expression and protein-deposition of ECM proteins such as collagens and fibronectin. An involvement of the antioxidant pathway upon stimulation with pro-fibrotic compounds was also observed. CONCLUSION: Here, for the first time, we demonstrate the in vitro recapitulation of key molecular and cellular events leading to liver fibrosis: hepatocellular injury, antioxidant defence response, activation of Kupffer cells and activation of HSC leading to deposition of ECM.

Hepatic 3D spheroid models for the detection and study of compounds with cholestatic liability.

Drug-induced cholestasis (DIC) is poorly understood and its preclinical prediction is mainly limited to assessing the compound's potential to inhibit the bile salt export pump (BSEP). Here, we evaluated two 3D spheroid models, one from primary human hepatocytes (PHH) and one from HepaRG cells, for the detection of compounds with cholestatic liability. By repeatedly co-exposing both models to a set of compounds with different mechanisms of hepatotoxicity and a non-toxic concentrated bile acid (BA) mixture for 8 days we observed a selective synergistic toxicity of compounds known to cause cholestatic or mixed cholestatic/hepatocellular toxicity and the BA mixture compared to exposure to the compounds alone, a phenomenon that was more pronounced after extending the exposure time to 14 days. In contrast, no such synergism was observed after both 8 and 14 days of exposure to the BA mixture for compounds that cause non-cholestatic hepatotoxicity. Mechanisms behind the toxicity of the cholestatic compound chlorpromazine were accurately detected in both spheroid models, including intracellular BA accumulation, inhibition of ABCB11 expression and disruption of the F-actin cytoskeleton. Furthermore, the observed synergistic toxicity of chlorpromazine and BA was associated with increased oxidative stress and modulation of death receptor signalling. Combined, our results demonstrate that the hepatic spheroid models presented here can be used to detect and study compounds with cholestatic liability.

Several posttranslational modifications act in concert to regulate gephyrin scaffolding and GABAergic transmission.

GABAA receptors (GABAARs) mediate the majority of fast inhibitory neurotransmission in the brain via synergistic association with the postsynaptic scaffolding protein gephyrin and its interaction partners. However, unlike their counterparts at glutamatergic synapses, gephyrin and its binding partners lack canonical protein interaction motifs; hence, the molecular basis for gephyrin scaffolding has remained unclear. In this study, we identify and characterize two new posttranslational modifications of gephyrin, SUMOylation and acetylation. We demonstrate that crosstalk between SUMOylation, acetylation and phosphorylation pathways regulates gephyrin scaffolding. Pharmacological intervention of SUMO pathway or transgenic expression of SUMOylation-deficient gephyrin variants rescued gephyrin clustering in CA1 or neocortical neurons of Gabra2-null mice, which otherwise lack gephyrin clusters, indicating that gephyrin SUMO modification is an essential determinant for scaffolding at GABAergic synapses. Together, our results demonstrate that concerted modifications on a protein scaffold by evolutionarily conserved yet functionally diverse signalling pathways facilitate GABAergic transmission.

Histone ADP-ribosylation in DNA repair, replication and transcription.

Most published work on post-translational histone modifications focuses on small covalent alterations such as acetylation, methylation and phosphorylation. By contrast, fewer data are available on the modification of histones by ADP-ribose. Discussion of the biological significance of histone ADP-ribosylation has often been restricted to functions of the modifying enzymes, rather than to histones as ADP-ribose acceptors. In particular, the identification of specific lysine residues as ADP-ribose acceptor sites in histones and the identification of ADP-ribose binding modules raise this modification to a par with acetylation, methylation or phosphorylation. We discuss here the functional aspects of histone ADP-ribosylation and its influence on DNA repair, replication and transcription.

Identification of distinct amino acids as ADP-ribose acceptor sites by mass spectrometry.

ADP-ribosylation is a well-known post-translational protein modification, which regulates a variety of -cellular processes. The proteins able to catalyze mono- or poly ADP-ribosylation of proteins belong to the family of ADP-ribosyltransferases. A variety of nuclear proteins has been described to be ADP-ribosylated, including ARTD1 itself and histone proteins. Despite intensive research during the last 40 years, the acceptor amino acids in ARTD1 or histone proteins could be identified and confirmed only recently by MS/MS and by site-directed mutagenesis. The establishment of a standardized protocol including the specific enrichment of ADP-ribosylated proteins and peptides and subsequent mass spectrometric analysis allows the identification of ADP-ribose acceptor sites of modified proteins and to address the functional contribution of ADP-ribosylation in vitro as well as in vivo.