The Reactions of H2_{2}O2_{2} 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 H$_{2}$O$_{2}$ 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

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 com- posed of a set of numerous protein fragments cleaved C-terminally to V, I, A, T, or C residues, significantly over-represented in AC. A peptide set linked by a common VIATC cleavage consensus was the only prominent can- cer-specific proteolytic fingerprint detected. This se- quence consensus indicated neutrophil elastase as a source of the fingerprint. We also found that a large frac- tion 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 pep- tides that are possible markers of tumor-infiltrating neu- trophil 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 anal- ysis of large global proteomic analysis data sets as op- posed to protein-wise analysis, in which outlier differen- tial peptides are usually neglected

Di-Tyrosine Cross-link Decreases the Collisional Cross-Section of Aβ Peptide Dimers and Trimers in the Gas Phase – an Ion Mobility Study

Oligomeric forms of Aβ peptide are most likely the main synaptotoxic and neurotoxic agent in Alzheimer’s disease. Toxicity of various Aβ 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 Aβ 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

Molecular models of cross-link Aβ oligomer.

<p>Molecular models of the compact form of Aβ hexamer cross-linked by di- (A) or tri-Tyr (B) bond.</p

Spectra for covalently stabilized DIM⁹⁺.

<p>Fragments of mass spectra collected before (right panel) and after 90 min of reaction (left panel). Shown is the fragment of spectra in which a new signal, absent before reaction, was observed. This signal corresponded to 9+ charged dimer at 963 m/z.</p

Fragments of IMS-MS spectra for different charged dimer.

<p>Selected regions of ion mobility separated mass spectra (IMS-MS) covering dimeric signals in 2D rendition (colored panels, drift time at horizontal axis and m/z at vertical axis). Lower panels show the corresponding ion mobility drift time profiles, i.e., either projections of the signal group on the drift time axis or cross-sections at a different m/z value, as described below. Panels showing isotopic envelopes are cross-sections at indicated (blue arrows) drift time values correspond to a given oligomeric form, for instance DIM⁸⁺ denoting a non-covalently stabilized dimer charged 8+, whereas cDIM⁸⁺ is a covalently stabilized dimer charged 8+, etc. Spectra are shown before reaction with H₂O₂/HRP (<b>A,C,E,G</b>) and at 90 min of reaction <b>(B,D,F,H</b>). (<b>A,B</b>) 1082–1085 m/z region with overlapping MON⁴⁺ and DIM⁸⁺ signals present. The two drift time profiles are collected at m/z values indicated by black arrows, showing a new isotopic envelope after reaction (<b>B</b>), corresponding to cDIM⁸⁺, characterized by a smaller m/z and drift time (7.17 ms) longer than DIM⁸⁺/MON⁴⁺ envelope at 6.28 ms. (<b>C,D</b>) 1236–1240 m/z region, showing signals of the two structurally alternate (at 7.61 and 8.16 ms) DIM⁷⁺ forms before reaction (<b>C</b>), after incubation (<b>D</b>) accompanied by a strong new signal of cDIM⁷⁺ at smaller m/z and shorter drift time (6.95 ms). (E,F) 1442–1446 m/z region with MON³⁺ and DIM⁶⁺ signals present. The two drift time profiles are collected at m/z values indicated by black arrows. (<b>G,H</b>) 1731–1735 m/z region before reaction (<b>G</b>) showing signals of at least four structurally alternate DIM⁵⁺ forms, with no new forms after incubation (<b>H</b>) but with equilibrium shifted towards the most compact of the four forms present before reaction. Experiment was repeated at least three times. See also <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100200#pone.0100200.s002" target="_blank">Figure S2</a>.</b></p

Oligomer evolution scheme.

<p>Oligomer evolution scheme showing an on-pathway scenario, leading to fibrils, and off-pathway scenario.</p

Experimental values of molecular masses (M_B, M_A), IMS drift times (t_D) and collisional cross-section (Ω) for dimer and trimer.

<p>Molecular masses (M_B, M_A), drift times (t_D) and collisional cross-section (Ω) values corresponding to dimeric (upper table) and trimeric (lower table) forms detected before (columns 2–5) and after (columns 6–10) reaction with HRP and H₂O₂. Species are denoted by their charge state z (column 1) assigned by the analysis of the isotopic envelope peak spacing. This allowed a direct calculation of the molecular mass of the species (column 3 and 7) from the average m/z (columns 2 and 6). HRP/H₂O₂ reaction results in di-Tyr and tri-Tyr covalent cross-link formation with concomitant molecular mass loss of 2 Da per each bond formed. This is illustrated by the observed loss of the mass of the oligomeric form after reaction (column 10). The presented results are mean values from two independent experiments. The maximum difference for m/z (columns 2 and 6) was ±0.025, corresponding to the largest difference of mass values of ±0.175 Da (columns 3 and 7). The largest difference in drift time values was 0.11 for dim⁸⁺ signal (dt 6.28) which corresponds to the difference of CCS values of 1.1%. In all other cases the discrepancy between two replicate measurements was lower than 0.9%.</p