Redox control of 20S proteasome gating.

UNLABELLED: The proteasome is the primary contributor in intracellular proteolysis. Oxidized or unstructured proteins can be degraded via a ubiquitin- and ATP-independent process by the free 20S proteasome (20SPT). The mechanism by which these proteins enter the catalytic chamber is not understood thus far, although the 20SPT gating conformation is considered to be an important barrier to allowing proteins free entrance. We have previously shown that S-glutathiolation of the 20SPT is a post-translational modification affecting the proteasomal activities. AIMS: The goal of this work was to investigate the mechanism that regulates 20SPT activity, which includes the identification of the Cys residues prone to S-glutathiolation. RESULTS: Modulation of 20SPT activity by proteasome gating is at least partially due to the S-glutathiolation of specific Cys residues. The gate was open when the 20SPT was S-glutathiolated, whereas following treatment with high concentrations of dithiothreitol, the gate was closed. S-glutathiolated 20SPT was more effective at degrading both oxidized and partially unfolded proteins than its reduced form. Only 2 out of 28 Cys were observed to be S-glutathiolated in the proteasomal α5 subunit of yeast cells grown to the stationary phase in glucose-containing medium. INNOVATION: We demonstrate a redox post-translational regulatory mechanism controlling 20SPT activity. CONCLUSION: S-glutathiolation is a post-translational modification that triggers gate opening and thereby activates the proteolytic activities of free 20SPT. This process appears to be an important regulatory mechanism to intensify the removal of oxidized or unstructured proteins in stressful situations by a process independent of ubiquitination and ATP consumption. Antioxid. Redox Signal. 16, 1183-1194.

Fragmentation features of intermolecular cross-linked peptides using N-hydroxy- succinimide esters by MALDI- and ESI-MS/MS for use in structural proteomics.

The use of mass spectrometry coupled with chemical cross-linking of proteins has become one of the most useful tools for proteins structure and interactions studies. One of the challenges in these studies is the identification of the cross-linked peptides. The interpretation of the MS/MS data generated in cross-linking experiments using N-hydroxy succinimide esters is not trivial once a new amide bond is formed allowing new fragmentation pathways, unlike linear peptides. Intermolecular cross-linked peptides occur when two different peptides are connected by the cross-linker and they yield information on the spatial proximity of different domains (within a protein) or proteins (within a complex). In this article, we report a detailed fragmentation study of intermolecular cross-linked peptides, generated from a set of synthetic peptides, using both ESI and MALDI to generate the precursor ions. The fragmentation features observed here can be helpful in the interpretation and identification of cross-linked peptides present in cross-linking experiments and be further implemented in search engine's algorithms.

Traveling-wave ion mobility mass spectrometry analysis of isomeric modified peptides arising from chemical cross-linking.

Traveling-wave ion mobility (TWIM) coupled to mass spectrometry (MS) has emerged as a powerful tool for structural and conformational analysis of proteins and peptides, allowing the analysis of isomeric peptides (or proteins) with the same sequence but modified at different residues. This work demonstrates the use of the novel TWIM-MS technique to separate isomeric peptide ions derived from chemical cross-linking experiments, which enables the acquisition of distinct product ion spectra for each isomer, clearly indicating modification on different sites. Experiments were performed with four synthetic peptides, for which variable degrees of mobility separation were achieved. In cases of partially overlapping mobility arrival time distributions (ATDs), extracting the ATDs of fragment ions belonging to each individual isomer allowed their separation into two distinct ATDs. Accumulation over regions from the specific ATDs generates the product ion spectrum of each isomer, or a spectrum highly enriched in their fragments. The population of both modified peptide isomers was correlated with the intrinsic reactivities of different Lys residues from reactions conducted at different pH conditions.

A ditryptophan cross-link is responsible for the covalent dimerization of human superoxide dismutase 1 during its bicarbonate-dependent peroxidase activity.

Unlike intermolecular disulfide bonds, other protein cross-links arising from oxidative modifications cannot be reversed and are presumably more toxic to cells because they may accumulate and induce protein aggregation. However, most of these irreversible protein cross-links remain poorly characterized. For instance, the antioxidant enzyme human superoxide dismutase 1 (hSod1) has been reported to undergo non-disulfide covalent dimerization and further oligomerization during its bicarbonate-dependent peroxidase activity. The dimerization was shown to be dependent on the oxidation of the single, solvent-exposed Trp(32) residue of hSod1, but the covalent dimer was not isolated nor was its structure determined. In this work, the hSod1 covalent dimer was isolated, digested with trypsin in H(2)O and H(2)(18)O, and analyzed by UV-Vis spectroscopy and mass spectrometry (MS). The results demonstrate that the covalent dimer consists of two hSod1 subunits cross-linked by a ditryptophan, which contains a bond between C3 and N1 of the respective Trp(32) residues. We further demonstrate that the cross-link cleaves under usual MS/MS conditions leading to apparently unmodified Trp(32), partially hinders proteolysis, and provides a mechanism to explain the formation of hSod1 covalent trimers and tetramers. This characterization of the covalent hSod1 dimer identifies a novel oxidative modification of protein Trp residues and provides clues for studying its occurrence in vivo.

Collision-induced dissociation of Lys-Lys intramolecular crosslinked peptides.

The use of chemical crosslinking is an attractive tool that presents many advantages in the application of mass spectrometry to structural biology. The correct assignment of crosslinked peptides, however, is still a challenge because of the lack of detailed fragmentation studies on resultant species. In this work, the fragmentation patterns of intramolecular crosslinked peptides with disuccinimidyl suberate (DSS) has been devised by using a set of versatile, model peptides that resemble species found in crosslinking experiments with proteins. These peptides contain an acetylated N-terminus followed by a random sequence of residues containing two lysine residues separated by an arginine. After the crosslinking reaction, controlled trypsin digestion yields both intra- and intermolecular crosslinked peptides. In the present study we analyzed the fragmentation of matrix-assisted laser desorption/ionization-generated peptides crosslinked with DSS in which both lysines are found in the same peptide. Fragmentation starts in the linear moiety of the peptide, yielding regular b and y ions. Once it reaches the cyclic portion of the molecule, fragmentation was observed to occur either at the following peptide bond or at the peptide crosslinker amide bond. If the peptide crosslinker bond is cleaved, it fragments as a regular modified peptide, in which the DSS backbone remains attached to the first lysine. This fragmentation pattern resembles the fragmentation of modified peptides and may be identified by common automated search engines using DSS as a modification. If, on the other hand, fragmentation happens at the peptide bond itself, rearrangement of the last crosslinked lysine is observed and a product ion containing the crosslinker backbone and lysine (m/z 222) is formed. The detailed identification of fragment ions can help the development of softwares devoted to the MS/MS data analysis of crosslinked peptides.