Translation and emerging functions of non-coding RNAs in inflammation and immunity.

Regulatory non-coding RNAs (ncRNAs) including small non-coding RNAs (sRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs) have gained considerable attention in the last few years. This is mainly due to their condition- and tissue-specific expression and their various modes of action, which suggests them as promising biomarkers and therapeutic targets. One important mechanism of ncRNAs to regulate gene expression is through translation of short open reading frames (sORFs). These sORFs can be located in lncRNAs, in non-translated regions of mRNAs where upstream ORFs (uORFs) represent the majority, or in circRNAs. Regulation of their translation can function as a quick way to adapt protein production to changing cellular or environmental cues, and can either depend solely on the initiation and elongation of translation, or on the roles of the produced functional peptides. Due to the experimental challenges to pinpoint translation events and to detect the produced peptides, translational regulation through regulatory RNAs is not well studied yet. In the case of circRNAs, they have only recently started to be recognized as regulatory molecules instead of mere artifacts of RNA biosynthesis. Of the many roles described for regulatory ncRNAs, we will focus here on their regulation during inflammation and in immunity.

Impact of DJ-1 and Helix 8 on the Proteome and Degradome of Neuron-Like Cells.

DJ-1 is an abundant and ubiquitous component of cellular proteomes. DJ-1 supposedly exerts a wide variety of molecular functions, ranging from enzymatic activities as a deglycase, protease, and esterase to chaperone functions. However, a consensus perspective on its molecular function in the cellular context has not yet been reached. Structurally, the C-terminal helix 8 of DJ-1 has been proposed to constitute a propeptide whose proteolytic removal transforms a DJ-1 zymogen to an active hydrolase with potential proteolytic activity. To better understand the cell-contextual functionality of DJ-1 and the role of helix 8, we employed post-mitotically differentiated, neuron-like SH-SY5Y neuroblastoma cells with stable over-expression of full length DJ-1 or DJ-1 lacking helix 8 (ΔH8), either with a native catalytically active site (C106) or an inactive site (C106A active site mutation). Global proteome comparison of cells over-expressing DJ-1 ΔH8 with native or mutated active site cysteine indicated a strong impact on mitochondrial biology. N-terminomic profiling however did not highlight direct protease substrate candidates for DJ-1 ΔH8, but linked DJ-1 to elevated levels of activated lysosomal proteases, albeit presumably in an indirect manner. Finally, we show that DJ-1 ΔH8 loses the deglycation activity of full length DJ-1. Our study further establishes DJ-1 as deglycation enzyme. Helix 8 is essential for the deglycation activity but dispensable for the impact on lysosomal and mitochondrial biology; further illustrating the pleiotropic nature of DJ-1.

Arabidopsis proteomics: a simple and standardizable workflow for quantitative proteome characterization.

Arabidopsis is the model plant of choice for large-scale proteome analyses, because its genome is well annotated, essentially free of sequencing errors, and relatively small with little redundancy. Furthermore, most Arabidopsis organs are susceptible to standard protein solubilization protocols making protein extraction relatively simple. Many different facets of functional plant proteomics were established with Arabidopsis such as mapping the subcellular proteomes of organelles, proteo-genomic peptide mapping, and numerous studies on the dynamic changes in protein modification and protein abundances. As most standard proteomics technologies are now routinely applied, research interest is increasingly shifting towards the reverse genetic characterization of gene function at the proteome level, i.e., by profiling the quantitative proteome of wild type in comparison with mutant plant tissue. We report here a simple, standardizable protocol for the large-scale comparative quantitative proteome characterization of different Arabidopsis organs based on normalized spectral counting and suggest a statistical framework for data interpretation. Based on existing organellar proteome maps, proteins can be assigned to organelles, thus allowing the identification of organelle-specific responses.

Jasmonate controls polypeptide patterning in undamaged tissue in wounded Arabidopsis leaves.

Wounding initiates a strong and largely jasmonate-dependent remodelling of the transcriptome in the leaf blades of Arabidopsis (Arabidopsis thaliana). How much control do jasmonates exert on wound-induced protein repatterning in leaves? Replicated shotgun proteomic analyses of 2.5-mm-wide leaf strips adjacent to wounds revealed 106 differentially regulated proteins. Many of these gene products have not emerged as being wound regulated in transcriptomic studies. From experiments using the jasmonic acid (JA)-deficient allene oxide synthase mutant we estimated that approximately 95% of wound-stimulated changes in protein levels were deregulated in the absence of JA. The levels of two tonoplast proteins already implicated in defense response regulation, TWO-PORE CHANNEL1 and the calcium-V-ATPase ACA4 increased on wounding, but their transcripts were not wound inducible. The data suggest new roles for jasmonate in controlling the levels of calcium-regulated pumps and transporters, proteins involved in targeted proteolysis, a putative bacterial virulence factor target, a light-dependent catalyst, and a key redox-controlled enzyme in glutathione synthesis. Extending the latter observation we found that wounding increased the proportion of oxidized glutathione in leaves, but only in plants able to synthesize JA. The oxidizing conditions generated through JA signaling near wounds help to define the cellular environment in which proteome remodelling occurs.

Genome-scale proteomics reveals Arabidopsis thaliana gene models and proteome dynamics.

We have assembled a proteome map for Arabidopsis thaliana from high-density, organ-specific proteome catalogs that we generated for different organs, developmental stages, and undifferentiated cultured cells. We matched 86,456 unique peptides to 13,029 proteins and provide expression evidence for 57 gene models that are not represented in the TAIR7 protein database. Analysis of the proteome identified organ-specific biomarkers and allowed us to compile an organ-specific set of proteotypic peptides for 4105 proteins to facilitate targeted quantitative proteomics surveys. Quantitative information for the identified proteins was used to establish correlations between transcript and protein accumulation in different plant organs. The Arabidopsis proteome map provides information about genome activity and proteome assembly and is available as a resource for plant systems biology.

5-Fluorouracil is efficiently removed from DNA by the base excision and mismatch repair systems.

BACKGROUND & AIMS: 5-Fluorouracil (FU) is one of the mainstays of colon cancer chemotherapy. Although developed as an inhibitor of thymidylate synthase, its cytotoxicity has been linked also to its incorporation into RNA. Surprisingly, although FU is incorporated also into DNA, little is known about its metabolism in this nucleic acid. METHODS: Using extracts of human cells and circular DNA substrates containing a single FU residue either paired with adenine or mispaired with guanine, we studied the enzymology of FU processing. RESULTS: In nicked circular substrates, FU/G mispairs were efficiently repaired by mismatch repair (MMR). In covalently closed circular DNA, which is refractory to MMR, FU/G repair was initiated by either thymine-DNA glycosylase or uracil-DNA glycosylase, whereas FU/A pairs were processed by UNG. Methylated CpG binding domain 4 protein and single-strand selective monofunctional uracil-DNA glycosylase 1 did not detectably contribute to FU removal; however, because these recombinant enzymes process FU/G and FU/A in oligonucleotide substrates, respectively, they too may be involved in FU metabolism in vivo. CONCLUSIONS: The functional redundancy of MMR and DNA glycosylases in FU processing should ensure that the drug is efficiently removed from DNA before it can interfere with essential DNA metabolic processes, such as transcription. However, in FU-treated cells, the nucleotide pools are depleted of thymine. The repair synthesis might thus be inhibited and leave cytotoxic gaps or breaks in DNA. Moreover, FU and/or 5-fluorouracil-2'-deoxyuridine-5'-triphosphate removed from DNA will increase the intracellular concentration of the drug and thus exacerbate its cytotoxicity.

The Mass Distance Fingerprint: a statistical framework for de novo detection of predominant modifications using high-accuracy mass spectrometry.

We describe a statistical measure, Mass Distance Fingerprint, for automatic de novo detection of predominant peptide mass distances, i.e., putative protein modifications. The method's focus is to globally detect mass differences, not to assign peptide sequences or modifications to individual spectra. The Mass Distance Fingerprint is calculated from high accuracy measured peptide masses. For the data sets used in this study, known mass differences are detected at electron mass accuracy or better. The proposed method is novel because it works independently of protein sequence databases and without any prior knowledge about modifications. Both modified and unmodified peptides have to be present in the sample to be detected. The method can be used for automated detection of chemical/post-translational modifications, quality control of experiments and labeling approaches, and to control the modification settings of protein identification tools. The algorithm is implemented as a web application and is distributed as open source software.