A mechanism for oxidative damage repair at gene regulatory elements.

Oxidative genome damage is an unavoidable consequence of cellular metabolism. It arises at gene regulatory elements by epigenetic demethylation during transcriptional activation1,2. Here we show that promoters are protected from oxidative damage via a process mediated by the nuclear mitotic apparatus protein NuMA (also known as NUMA1). NuMA exhibits genomic occupancy approximately 100 bp around transcription start sites. It binds the initiating form of RNA polymerase II, pause-release factors and single-strand break repair (SSBR) components such as TDP1. The binding is increased on chromatin following oxidative damage, and TDP1 enrichment at damaged chromatin is facilitated by NuMA. Depletion of NuMA increases oxidative damage at promoters. NuMA promotes transcription by limiting the polyADP-ribosylation of RNA polymerase II, increasing its availability and release from pausing at promoters. Metabolic labelling of nascent RNA identifies genes that depend on NuMA for transcription including immediate-early response genes. Complementation of NuMA-deficient cells with a mutant that mediates binding to SSBR, or a mitotic separation-of-function mutant, restores SSBR defects. These findings underscore the importance of oxidative DNA damage repair at gene regulatory elements and describe a process that fulfils this function.

Integration of JAK/STAT receptor-ligand trafficking, signalling and gene expression in Drosophila melanogaster cells.

The JAK/STAT pathway is an essential signalling cascade required for multiple processes during development and for adult homeostasis. A key question in understanding this pathway is how it is regulated in different cell contexts. Here, we have examined how endocytic processing contributes to signalling by the single cytokine receptor in Drosophila melanogaster cells, Domeless. We identify an evolutionarily conserved di-leucine (di-Leu) motif that is required for Domeless internalisation and show that endocytosis is required for activation of a subset of Domeless targets. Our data indicate that endocytosis both qualitatively and quantitatively regulates Domeless signalling. STAT92E, the single STAT transcription factor in Drosophila, appears to be the target of endocytic regulation, and our studies show that phosphorylation of STAT92E on Tyr704, although necessary, is not always sufficient for target transcription. Finally, we identify a conserved residue, Thr702, which is essential for Tyr704 phosphorylation. Taken together, our findings identify previously unknown aspects of JAK/STAT pathway regulation likely to play key roles in the spatial and temporal regulation of signalling in vivo.

Detection of busulfan adducts on proteins.

RATIONALE: Busulfan is a bifunctional alkyl sulfonate antineoplastic drug. This alkylating agent was described as forming covalent adducts on proteins. However, only limited data are available regarding the interaction of busulfan with proteins. Mass spectrometry and bioinformatics were used to identify busulfan adducts on human serum albumin and hemoglobin. METHODS: Albumin and hemoglobin were incubated with busulfan or control compounds, digested with trypsin and analyzed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) on a Thermo Fisher LTQ Orbitrap Velos Pro. MS data were used to generate spectral libraries of non-modified peptides and an open modification search was performed to identify potential adduct mass shifts and possible modification sites. Results were confirmed by a second database search including identified mass shifts and by visual inspection of annotated tandem mass spectra of adduct-carrying peptides. RESULTS: Five structures of busulfan adducts were detected and a chemical structure could be attributed to four of them. Two were primary adducts corresponding to busulfan monoalkylation and alkylation of two amino acid residues by a single busulfan molecule. Two others corresponded to secondary adducts generated during sample processing. Adducts were mainly detected on Asp, Glu, and His residues. These findings were confirmed by subsequent database searches and experiments with synthetic peptides. CONCLUSIONS: The combination of in vitro incubation of proteins with the drug of interest or control compounds, high-resolution mass spectrometry, and open modification search allowed confirmation of the direct interaction of busulfan with proteins and characterization of the resulting adducts. Our results also showed that careful analysis of the data is required to detect experimental artifacts. Copyright © 2016 John Wiley & Sons, Ltd.

Role of proinflammatory CD68(+) mannose receptor(-) macrophages in peroxiredoxin-1 expression and in abdominal aortic aneurysms in humans.

OBJECTIVE: Abdominal aortic aneurysms (AAAs), dilations of the infrarenal aorta, are characterized by inflammation and oxidative stress. We previously showed increased levels of peroxiredoxin-1 (PRDX-1) in macrophages cultured from AAA patients. The purpose of the study was to determine which subpopulation of macrophages is present in AAAs and is involved in upregulation of PRDX-1 in aneurysmal disease. METHODS AND RESULTS: This study used immunohistochemistry with antibodies against CD68 and mannose receptor (MR) to determine the subtype of macrophages in AAA tissue samples (n=33); laser capture microdissection to isolate each subtype; and quantitative-reverse transcriptase-polymerase chain reaction, Western blot, and ELISA to assess PRDX-1 mRNA and PRDX-1protein levels in both types. Proinflammatory CD68(+)MR(-) macrophages predominated in adventitial tissue, whereas the intraluminal thrombus contained CD68(+)MR(+) macrophages. The presence of lipids and iron-containing deposits confirmed their phagocytic phenotype. Laser capture microdissection-isolated CD68(+)MR(-) and CD68(+)MR(+) macrophages, characterized by quantitative-reverse transcriptase-polymerase chain reaction (TNF, IL1B, MRC1, and CCL18) and Western blot (stabilin and hemoglobin), validated the microdissected subtypes. PRDX-1 expression was colocalized with CD68(+)MR(-) macrophages. PRDX-1 mRNA and PRDX-1 protein were both more abundant in CD68(+)MR(-) than CD68(+)MR(+) macrophages in AAA. CONCLUSIONS: These findings suggest that the proteins or mRNAs expressed by the proinflammatory CD68(+)MR(-) macrophages may contribute to aneurysmal pathology.

Identification of additional proteins in differential proteomics using protein interaction networks.

In this study, we developed a novel computational approach based on protein-protein interaction networks to identify a list of proteins that might have remained undetected in differential proteomic profiling experiments. We tested our computational approach on two sets of human smooth muscle cell protein extracts that were affected differently by DNase I treatment. Differential proteomic analysis by saturation DIGE resulted in the identification of 41 human proteins. The application of our approach to these 41 input proteins consisted of four steps: (i) Compilation of a human protein-protein interaction network from public databases; (ii) calculation of interaction scores based on functional similarity; (iii) determination of a set of candidate proteins that are needed to efficiently and confidently connect the 41 input proteins; and (iv) ranking of the resulting 25 candidate proteins. Two of the three highest-ranked proteins, beta-arrestin 1, and beta-arrestin 2, were experimentally tested, revealing that their abundance levels in human smooth muscle cell samples were indeed affected by DNase I treatment. These proteins had not been detected during the experimental proteomic analysis. Our study suggests that our computational approach may represent a simple, universal, and cost-effective means to identify additional proteins that remain elusive for current 2D gel-based proteomic profiling techniques.

Quantitative mass spectrometry analysis of intact hemoglobin A2 by precursor ion isolation and detection.

Precise and accurate quantification of proteins is essential in clinical laboratories. Here, we present a mass spectrometry (MS)-based method for the quantification of intact proteins in an ion trap mass spectrometer. The developed method is based on the isolation and detection of precursor ions for the quantification of the corresponding signals. The method was applied for the quantification of hemoglobin (Hb) A2, a marker used for the diagnosis of a ß-thalassemia trait. The α and δ globin chains, corresponding to total Hb and HbA2, respectively, were isolated in the ion trap at specific charge states and ejected without activation. Areas of the corresponding isolated precursor ions were used to calculate the δ to α ratio. Three series of quantifications were performed on 7 different days. The standard curve fitted linearly (R(2) = 0.9982) and allowed quantification of HbA2 over a concentration range from 3% to 18% of total Hb. Analytical imprecision ranged from 3.5% to 5.3%, which is enough to determine if the HbA2 level is below 3.5% or above 3.7%. In conclusion, our method reaches precision requirements that would be acceptable for the quantitative measurement of diagnostic proteins, such as HbA2, in clinical laboratories.

PGFinder, a novel analysis pipeline for the consistent, reproducible, and high-resolution structural analysis of bacterial peptidoglycans.

Many software solutions are available for proteomics and glycomics studies, but none are ideal for the structural analysis of peptidoglycan (PG), the essential and major component of bacterial cell envelopes. It icomprises glycan chains and peptide stems, both containing unusual amino acids and sugars. This has forced the field to rely on manual analysis approaches, which are time-consuming, labour-intensive, and prone to error. The lack of automated tools has hampered the ability to perform high-throughput analyses and prevented the adoption of a standard methodology. Here, we describe a novel tool called PGFinder for the analysis of PG structure and demonstrate that it represents a powerful tool to quantify PG fragments and discover novel structural features. Our analysis workflow, which relies on open-access tools, is a breakthrough towards a consistent and reproducible analysis of bacterial PGs. It represents a significant advance towards peptidoglycomics as a full-fledged discipline.

Resveratrol addition to Chinese hamster ovary cell culture media: The effect on cell growth, monoclonal antibody synthesis, and its chemical modification.

The effect of the addition of resveratrol to cell culture media during the production of monoclonal antibodies was investigated. Treatments of Chinese hamster ovary (CHO) cells expressing immunoglobulin G (IgG) with 25 and 50 µM resveratrol showed that resveratrol was capable of slowing cell growth while almost doubling cell-specific productivity to 4.7 ± 0.6 pg IgG/cell·day, resulting in up to a 1.37-fold increase of the final IgG titer. A resveratrol concentration of 50 µM slowed the progression through the cell cycle temporarily by trapping cells in the S-phase. Cation exchange chromatography showed no significant difference in the composition of acidic or basic IgG species and size exclusion chromatography indicated no change in fragmentation or aggregation of the recombinant IgG in the treatment groups. Resveratrol could be used as a chemical additive to CHO media where it would enhance IgG productivity and provide a degree of protection against hydroxyl and superoxide free radicals, expanding the range of options for process improvement available to monoclonal antibody manufacturers.

The use of catechins in Chinese hamster ovary cell media for the improvement of monoclonal antibody yields and a reduction of acidic species.

Catechin compounds have potential benefits for recombinant monoclonal antibody (Mab) production as chemical additives in cell culture media. In this study, four catechin compounds catechin (Cat), epicatechin (EC), gallocatechin-gallate (GCG), and epigallocatechin-gallate (EGCG) were added to cell culture media (at 50 µM) and their effects on the recombinant Chinese hamster ovary (CHO) cell culture, specific productivity, and Mab quality were assessed. The results indicate that the improvement of specific productivity was linked to cell growth inhibition. All catechins caused cell phase growth arrest by lowering the number of cells in the G1/G0 phase and increasing the cells in the S and G2/M phases. Late addition of the catechin resulted in a significantly higher final IgG concentration. Cat and EC caused an improvement in the final antibody titer of 1.5 ± 0.1 and 1.3 ± 0.1 fold, respectively. Catechins with a galloyl group (GCG and EGCG) arrested cell growth and reduced cell specific productivity at the concentrations tested. The Cat-treated IgG was found to have reduced acidic species with a corresponding increase in the main peak.

Detection of Unknown Chemical Adduct Modifications on Proteins: From Wet to Dry Laboratory.

The detection and characterization of chemical adducts on proteins is of increasing interest. Here, we described a step-by-step procedure to identify unknown chemical adduct modifications on proteins resulting from the interaction with a given reactive compound. The protocol can be divided into two equally important parts: (1) the wet laboratory work, to produce high quality mass spectrometry (MS) data of in vitro modified proteins and (2) the dry laboratory work, to analyze the generated MS data and provide highly confident qualitative and quantitative results on the chemical composition and amino acid localization of adducts. This protocol is applicable to the study of any pharmaceutical or chemical compound forming covalent protein adducts, detectable in LC-MS/MS experiments.

Proteomic profiling of macrophages by 2D electrophoresis.

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The key role of macrophages has been shown in various pathological disorders such as inflammatory, immunological, and infectious diseases. In this protocol, we use primary cultures of human monocyte-derived macrophages that can be differentiated into the M1 (pro-inflammatory) or the M2 (anti-inflammatory) phenotype. This in vitro model is reliable for studying the biological activities of M1 and M2 macrophages and also for a proteomic approach. Proteomic techniques are useful for comparing the phenotype and behaviour of M1 and M2 macrophages during host pathogenicity. 2D gel electrophoresis is a powerful proteomic technique for mapping large numbers of proteins or polypeptides simultaneously. We describe the protocol of 2D electrophoresis using fluorescent dyes, named 2D Differential Gel Electrophoresis (DIGE). The M1 and M2 macrophages proteins are labelled with cyanine dyes before separation by isoelectric focusing, according to their isoelectric point in the first dimension, and their molecular mass, in the second dimension. Separated protein or polypeptidic spots are then used to detect differences in protein or polypeptide expression levels. The proteomic approaches described here allows the investigation of the macrophage protein changes associated with various disorders like host pathogenicity or microbial toxins.

Quantitative mass spectrometry analysis using PAcIFIC for the identification of plasma diagnostic biomarkers for abdominal aortic aneurysm.

BACKGROUND: Abdominal aortic aneurysm (AAA) is characterized by increased aortic vessel wall diameter (>1.5 times normal) and loss of parallelism. This disease is responsible for 1-4% mortality occurring on rupture in males older than 65 years. Due to its asymptomatic nature, proteomic techniques were used to search for diagnostic biomarkers that might allow surgical intervention under nonlife threatening conditions. METHODOLOGY/PRINCIPAL FINDINGS: Pooled human plasma samples of 17 AAA and 17 control patients were depleted of the most abundant proteins and compared using a data-independent shotgun proteomic strategy, Precursor Acquisition Independent From Ion Count (PAcIFIC), combined with spectral counting and isobaric tandem mass tags. Both quantitative methods collectively identified 80 proteins as statistically differentially abundant between AAA and control patients. Among differentially abundant proteins, a subgroup of 19 was selected according to Gene Ontology classification and implication in AAA for verification by Western blot (WB) in the same 34 individual plasma samples that comprised the pools. From the 19 proteins, 12 were detected by WB. Five of them were verified to be differentially up-regulated in individual plasma of AAA patients: adiponectin, extracellular superoxide dismutase, protein AMBP, kallistatin and carboxypeptidase B2. CONCLUSIONS/SIGNIFICANCE: Plasma depletion of high abundance proteins combined with quantitative PAcIFIC analysis offered an efficient and sensitive tool for the screening of new potential biomarkers of AAA. However, WB analysis to verify the 19 PAcIFIC identified proteins of interest proved inconclusive save for five proteins. We discuss these five in terms of their potential relevance as biological markers for use in AAA screening of population at risk.