The Reactions of H2O2 and GSNO with the Zinc Finger Motif of XPA. Not A Regulatory Mechanism, But No Synergy with Cadmium Toxicity.

Tetrathiolate zinc fingers are potential targets of oxidative assault under cellular stress conditions. We used the synthetic 37-residue peptide representing the tetrathiolate zinc finger domain of the DNA repair protein XPA, acetyl-DYVICEECGKEFMSYLMNHFDLPTCDNCRDADDKHK-amide (XPAzf) as a working model to study the reaction of its Zn(II) complex (ZnXPAzf) with hydrogen peroxide and S-nitrosoglutathione (GSNO), as oxidative and nitrosative stress agents, respectively. We also used the Cd(II) substituted XPAzf (CdXPAzf) to assess the situation of cadmium assault, which is accompanied by oxidative stress. Using electrospray mass spectrometry (ESI-MS), HPLC, and UV-vis and circular dichroism spectroscopies we demonstrated that even very low levels of H2O2 and GSNO invariably cause irreversible thiol oxidation and concomitant Zn(II) release from ZnXPAzf. In contrast, CdXPAzf was more resistant to oxidation, demonstrating the absence of synergy between cadmium and oxidative stresses. Our results indicate that GSNO cannot act as a reversible modifier of XPA, and rather has a deleterious effect on DNA repair.

Comprehensive list of SUMO targets in Caenorhabditis elegans and its implication for evolutionary conservation of SUMO signaling.

Post-translational modification by small ubiquitin-related modifier (SUMO) is a key regulator of cell physiology, modulating protein-protein and protein-DNA interactions. Recently, SUMO modifications were postulated to be involved in response to various stress stimuli. We aimed to identify the near complete set of proteins modified by SUMO and the dynamics of the modification in stress conditions in the higher eukaryote, Caenorhabditis elegans. We identified 874 proteins modified by SUMO in the worm. We have analyzed the SUMO modification in stress conditions including heat shock, DNA damage, arsenite induced cellular stress, ER and osmotic stress. In all these conditions the global levels of SUMOylation was significantly increased. These results show the evolutionary conservation of SUMO modifications in reaction to stress. Our analysis showed that SUMO targets are highly conserved throughout species. By comparing the SUMO targets among species, we approximated the total number of proteins modified in a given proteome to be at least 15-20%. We developed a web server designed for convenient prediction of potential SUMO modification based on experimental evidences in other species.

A Strong Neutrophil Elastase Proteolytic Fingerprint Marks the Carcinoma Tumor Proteome.

Proteolytic cascades are deeply involved in critical stages of cancer progression. During the course of peptide-wise analysis of shotgun proteomic data sets representative of colon adenocarcinoma (AC) and ulcerative colitis (UC), we detected a cancer-specific proteolytic fingerprint composed of a set of numerous protein fragments cleaved C-terminally to V, I, A, T, or C residues, significantly overrepresented in AC. A peptide set linked by a common VIATC cleavage consensus was the only prominent cancer-specific proteolytic fingerprint detected. This sequence consensus indicated neutrophil elastase as a source of the fingerprint. We also found that a large fraction of affected proteins are RNA processing proteins associated with the nuclear fraction and mostly cleaved within their functionally important RNA-binding domains. Thus, we detected a new class of cancer-specific peptides that are possible markers of tumor-infiltrating neutrophil activity, which often correlates with the clinical outcome. Data are available via ProteomeXchange with identifiers: PXD005274 (Data set 1) and PXD004249 (Data set 2). Our results indicate the value of peptide-wise analysis of large global proteomic analysis data sets as opposed to protein-wise analysis, in which outlier differential peptides are usually neglected.

HD2C histone deacetylase and a SWI/SNF chromatin remodelling complex interact and both are involved in mediating the heat stress response in Arabidopsis.

Studies in yeast and animals have revealed that histone deacetylases (HDACs) often act as components of multiprotein complexes, including chromatin remodelling complexes (CRCs). However, interactions between HDACs and CRCs in plants have yet to be demonstrated. Here, we present evidence for the interaction between Arabidopsis HD2C deacetylase and a BRM-containing SWI/SNF CRC. Moreover, we reveal a novel function of HD2C as a regulator of the heat stress response. HD2C transcript levels were strongly induced in plants subjected to heat treatment, and the expression of selected heat-responsive genes was up-regulated in heat-stressed hd2c mutant, suggesting that HD2C acts to down-regulate heat-activated genes. In keeping with the HDAC activity of HD2C, the altered expression of HD2C-regulated genes coincided in most cases with increased histone acetylation at their loci. Microarray transcriptome analysis of hd2c and brm mutants identified a subset of commonly regulated heat-responsive genes, and the effect of the brm hd2c double mutation on the expression of these genes was non-additive. Moreover, heat-treated 3-week-old hd2c, brm and brm hd2c mutants displayed similar rates of growth retardation. Taken together, our findings suggest that HD2C and BRM act in a common genetic pathway to regulate the Arabidopsis heat stress response.

Histone H1 Variants in Arabidopsis Are Subject to Numerous Post-Translational Modifications, Both Conserved and Previously Unknown in Histones, Suggesting Complex Functions of H1 in Plants.

Linker histones (H1s) are conserved and ubiquitous structural components of eukaryotic chromatin. Multiple non-allelic variants of H1, which differ in their DNA/nucleosome binding properties, co-exist in animal and plant cells and have been implicated in the control of genetic programs during development and differentiation. Studies in mammals and Drosophila have revealed diverse post-translational modifications of H1s, most of which are of unknown function. So far, it is not known how this pattern compares with that of H1s from other major lineages of multicellular Eukaryotes. Here, we show that the two main H1variants of a model flowering plant Arabidopsis thaliana are subject to a rich and diverse array of post-translational modifications. The distribution of these modifications in the H1 molecule, especially in its globular domain (GH1), resembles that occurring in mammalian H1s, suggesting that their functional significance is likely to be conserved. While the majority of modifications detected in Arabidopsis H1s, including phosphorylation, acetylation, mono- and dimethylation, formylation, crotonylation and propionylation, have also been reported in H1s of other species, some others have not been previously identified in histones.

Di-tyrosine cross-link decreases the collisional cross-section of aß peptide dimers and trimers in the gas phase: an ion mobility study.

Oligomeric forms of Aß peptide are most likely the main synaptotoxic and neurotoxic agent in Alzheimer's disease. Toxicity of various Aß oligomeric forms has been confirmed in vivo and also in vitro. However, in vitro preparations were found to be orders of magnitude less toxic than oligomers obtained from in vivo sources. This difference can be explained by the presence of a covalent cross-link, which would stabilize the oligomer. In the present work, we have characterized the structural properties of Aß dimers and trimers stabilized by di- and tri-tyrosine cross-links. Using ion mobility mass spectrometry we have compared the collisional cross-section of non-cross-linked and cross-linked species. We have found that the presence of cross-links does not generate new unique forms but rather shifts the equilibrium towards more compact oligomer types that can also be detected for non-cross-linked peptide. In consequence, more extended forms, probable precursors of off-pathway oligomeric species, become relatively destabilized in cross-linked oligomers and the pathway of oligomer evolution becomes redirected towards fibrillar structures.

Reaction of the XPA zinc finger with S-nitrosoglutathione.

S-Nitrosoglutathione (GSNO) is an intracellular redox signaling molecule, also implicated in nitrosative stress. GSNO actions include modifications of Cys thiols in proteins. In this study, we focused on a GSNO reaction with a Cys4 zinc finger (ZF) sequence of human protein XPA, crucial to the nucleotide excision repair pathway of DNA repair. By using a corresponding synthetic 37-residue peptide acetyl-DYVICEECGKEFMDSYLMNHFDLPTCDNCRDADDKHK-amide (XPAzf) and combining the detection of noncovalent and covalent complexes by ESI-MS with zinc release monitored by the zinc-sensitive chromophore 4-(2-pyridylazo)resorcinol (PAR), we demonstrated that the reaction of XPAzf with GSNO yielded S-nitrosylated intermediates, intrapeptide disulfides, and mixed glutathione disulfides. The reaction started with the formation of a complex of GSNO with ZnXPAzf followed by thiol transnitrosylation reactions and the final formation of disulfides. The results obtained suggest that at low levels/transient exposures, GSNO may act as a reversible regulator of Cys4 ZF activity, whereas transnitrosylation by GSNO, occurring at prolonged exposures, may cause deleterious effects to the functions of Cys 4 ZF proteins. In the case of XPA, this may lead to DNA repair inhibition.

Quantitative electrospray ionization mass spectrometry of zinc finger oxidation: the reaction of XPA zinc finger with H(2)O(2).

Oxidation plays an important role in the functioning of zinc fingers (ZFs). Electrospray ionization mass spectrometry (ESI-MS) is a very useful technique to study products of ZF oxidation, but its application has been limited largely to qualitative analysis of reaction products. On the other hand, ESI-MS has been applied successfully on several occasions to determine binding constants in metalloproteins. We used a synthetic 37-residue peptide acetyl-DYVICEECGKEFMDSYLMNHFDLPTCDNCRDADDKHK-amide (XPAzf), which corresponds to the Cys4 ZF sequence of human nucleotide excision repair protein XPA, to find out whether ESI-MS might be used quantitatively to study ZF reaction kinetics. For this purpose, we studied oxidation of the Zn(II) complex of XPAzf (ZnXPAzf) by H(2)O(2) using three techniques in parallel: high-performance liquid chromatography (HPLC) of covalent reaction products, 4-(2-pyridylazo)-resorcinol monosodium salt (PAR)-based spectrophotometric zinc release assay, and ESI-MS. Single and double intrapeptide disulfides were detected by ESI-MS to be the sole reaction products. All three techniques yielded independently the same reaction rate, thereby demonstrating that ESI-MS may indeed be used in quantitative kinetic studies of ZF reactions. The comparison of experimental information demonstrated that the formation of the Cys5-Cys8 single disulfide was responsible for zinc release.