Chemical Modifications of Globular Proteins Phototriggered by an Endogenous Photosensitizer.

The main goal of the present work was to investigate the damages photoinduced by pterin (Ptr), an endogenous photosensitizer present in human skin under pathological conditions, on a globular protein such as ubiquitin (Ub). Particular attention has been paid on the formation of covalent adducts between Ptr and the protein that can behave as photoantigen and provoke an immune system response. Here, a multifaceted approach including UV-visible spectrophotometry, fluorescence spectroscopy, electrophoresis, size exclusion chromatography, and mass spectrometry is used to establish the Ub changes triggered by UV-A irradiation in the presence of Ptr. Under anaerobic conditions, the only reaction corresponds to the formation of a covalently bound Ptr-Ub adduct that retains the spectroscopic properties of the free photosensitizer. A more complex scheme is observed in air-equilibrated solutions with the occurrence of three different processes, that is, formation of a Ptr-Ub adduct, dimerization, and fragmentation of the protein.

Long-lived coherences: improved dispersion in the frequency domain using continuous-wave and reduced-power windowed sustaining irradiation.

Long-lived coherences (LLC's) are detectable magnetisation modes with favourable relaxation times that translate as sharp resonances upon Fourier transform. The frequency domain of LLC's was previously limited to the range of J-couplings within pairs of homonuclear spins. LLC evolution at high magnetic fields needs to be sustained by radio-frequency irradiation. We show that LLC-based spectral dispersion can be extended beyond the J-couplings domain using adapted carrier offsets and introduce a new reduced-power sustaining method to preserve LLC's within the required range of offsets. Spectral resolution is enhanced as the natively narrow lines of LLC's are further dispersed, making them potential probes for the study of biomolecules featuring strong resonance overlap and for media where NMR spectroscopy is commonly hindered by line broadening.

Effects of combined gamma-irradiation and metal (Al+Cd) exposures in Atlantic salmon (Salmo salar L.).

These experiments were designed to investigate transcriptional effects in Atlantic salmon (Salmo salar) after exposure in vivo to ionizing gamma radiation combined with subtoxic levels of aluminum (Al) and cadmium (Cd). Juvenile fish (35 g) in freshwater with or without Al and Cd (255 microg Al/L + 6 microg Cd/L) were exposed to a 75 mGy dose of gamma-irradiation, and induced responses were compared to those of controls. The transcriptional levels of eight genes encoding proteins known to respond to stress in fish were quantified in liver of fish exposed for 5 h to gamma radiation, to Al and Cd or to the combination of Al, Cd and gamma radiation. The studied genes were caspase 3B, caspase 6A, caspase 7, p53 (apoptosis), glutathione reductase (GR), phospholipid hydroperoxide glutathione peroxidase (GSH-Px), (oxidative stress), metallothionein (MT-A) (metal stress) and ubiquitin (Ubi) (protein degradation). The results showed that gamma-irradiation alone induced significant upregulation of caspase 6A, GR, GSH-Px, MT-A and Ubi compared to the control group, while 5 h exposure to Al+Cd alone did not induce any of the studied genes compared to the control. No significant upregulation of the series of investigated genes could be observed in fish exposed to gamma-irradiation in combination with Al+Cl. In conclusion, the results suggest that the presence of Al+Cd in the water counteracted the gamma-irradiation effect by modifying the transcription of genes encoding proteins involved in the defense mechanisms against free radicals in the cells.

Histone H3 and H4 ubiquitylation by the CUL4-DDB-ROC1 ubiquitin ligase facilitates cellular response to DNA damage.

Posttranslational histone modifications play important roles in transcription and other chromatin-based processes. Compared to acetylation, methylation, and phosphorylation, very little is known about the function of histone ubiquitylation. Here, we report the purification and functional characterization of a histone H3 and H4 ubiquitin ligase complex, CUL4-DDB-ROC1. We demonstrate that CUL4-DDB-ROC1-mediated H3 and H4 ubiquitylation occurs both in vitro and in vivo. Importantly, CUL4-DDB-ROC1-mediated H3 and H4 ubiquitylation is regulated by UV irradiation. Reduction of histone H3 and H4 ubiquitylation by knockdown of CUL4A impairs recruitment of the repair protein XPC to the damaged foci and inhibits the repair process. Biochemical studies indicate that CUL4-DDB-ROC1-mediated histone ubiquitylation weakens the interaction between histones and DNA and facilitates the recruitment of repair proteins to damaged DNA. Thus, our studies uncover CUL4-DDB-ROC1 as a histone ubiquitin ligase and demonstrate that histone H3 and H4 ubiquitylation participates in the cellular response to DNA damage.

Stimulation of ubiquitin-proteasome pathway through the expression of amidohydrolase for N-terminal asparagine (Ntan1) in cultured rat hippocampal neurons exposed to static magnetism.

In order to elucidate mechanisms underlying modulation by static magnetism of the cellular functionality and/or integrity in the brain, we screened genes responsive to brief magnetism in cultured rat hippocampal neurons using differential display analysis. We have for the first time cloned and identified Ntan1 (amidohydrolase for N-terminal asparagine) as a magnetism responsive gene in rat brain. Ntan1 is an essential component of a protein degradation signal, which is a destabilizing N-terminal residue of a protein, in the N-end rule. In situ hybridization histochemistry revealed abundant expression of Ntan1 mRNA in hippocampal neurons in vivo. Northern blot analysis showed that Ntan1 mRNA was increased about three-fold after 3 h in response to brief magnetism. Brief magnetism also increased the transcriptional activity of Ntan1 promoter by luciferase reporter assay. Brief magnetism induced degradation of microtubule-associated protein 2 (MAP2) without affecting cell morphology and viability, which was prevented by a selective inhibitor of 26S proteasome in hippocampal neurons. Overexpression of Ntan1 using recombinant Ntan1 adenovirus vector resulted in a marked decrease in the MAP2 protein expression in hippocampal neurons. Our results suggest that brief magnetism leads to the induction of Ntan1 responsible for MAP2 protein degradation through ubiquitin-proteasome pathway in rat hippocampal neurons.

Effective novel dissociation methods for intact protein: heat-assisted nozzle-skimmer collisionally induced dissociation and infrared multiphoton dissociation using a Fourier transform ion cyclotron resonance mass spectrometer equipped with a micrometal electrospray ionization emitter.

Heating of a nano-electrospray ionization (nanoESI) source can improve the dissociation efficiency of collisionally induced dissociation (CID) methods, such as nozzle-skimmer CID (NS-CID) and infrared multiphoton dissociation (IRMPD), for large biomolecule fragmentation. A metal nanoESI emitter was used due to its resistance to heating above 250 degrees C. This novel method for the dissociation of large biomolecular ions is termed "heat-assisted NS-CID" (HANS-CID) or "heat-assisted IRMPD" (HA-IRMPD). Multiple charged nonreduced protein ions (8.6 Da ubiquitin, 14 kDa lysozyme, and 67 kDa bovine serum albumin) were directly dissociated by HANS-CID and HA-IRMPD to effectively yield fragment ions that could be assigned. The fragment ions of ubiquitin by HANS-CID can be analyzed by tandem mass spectrometry (MS/MS) using sustained off-resonance irradiation CID (SORI-CID) and IRMPD. In addition, a native large protein, immunoglobulin G (IgG, 150 kDa), was efficiently dissociated by HA-IRMPD. The product ions that were obtained reflected the domain structure of IgG. However, these product ions of IgG and lysozyme were not dissociated by MS/MS using the same heating energetic methods such as IRMPD and SORI-CID.

Unfolding of ubiquitin studied by picosecond time-resolved fluorescence of the tyrosine residue.

The photophysics of the single tyrosine in bovine ubiquitin (UBQ) was studied by picosecond time-resolved fluorescence spectroscopy, as a function of pH and along thermal and chemical unfolding, with the following results: First, at room temperature (25 degrees C) and below pH 1.5, native UBQ shows single-exponential decays. From pH 2 to 7, triple-exponential decays were observed and the three decay times were attributed to the presence of tyrosine, a tyrosine-carboxylate hydrogen-bonded complex, and excited-state tyrosinate. Second, at pH 1.5, the water-exposed tyrosine of either thermally or chemically unfolded UBQ decays as a sum of two exponentials. The double-exponential decays were interpreted and analyzed in terms of excited-state intramolecular electron transfer from the phenol to the amide moiety, occurring in one of the three rotamers of tyrosine in UBQ. The values of the rate constants indicate the presence of different unfolded states and an increase in the mobility of the tyrosine residue during unfolding. Finally, from the pre-exponential coefficients of the fluorescence decays, the unfolding equilibrium constants (KU) were calculated, as a function of temperature or denaturant concentration. Despite the presence of different unfolded states, both thermal and chemical unfolding data of UBQ could be fitted to a two-state model. The thermodynamic parameters Tm = 54.6 degrees C, DeltaHTm = 56.5 kcal/mol, and DeltaCp = 890 cal/mol//K, were determined from the unfolding equilibrium constants calculated accordingly, and compared to values obtained by differential scanning calorimetry also under the assumption of a two-state transition, Tm = 57.0 degrees C, DeltaHm= 51.4 kcal/mol, and DeltaCp = 730 cal/mol//K.

The role of the ubiquitin/proteasome system in cellular responses to radiation.

In the last few years, the ubiquitin(Ub)/proteasome system has become increasingly recognized as a controller of numerous physiological processes, including signal transduction, DNA repair, chromosome maintenance, transcriptional activation, cell cycle progression, cell survival, and certain immune cell functions. This is in addition to its more established roles in the removal of misfolded, damaged, and effete proteins. This review examines the role of the Ub/proteasome system in processes underlying the classical effects of irradiation on cells, such as radiation-induced gene expression, DNA repair and chromosome instability, oxidative damage, cell cycle arrest, and cell death. Furthermore, recent evidence suggests that the proteasome is a redox-sensitive target for ionizing radiation and other oxidative stress signals. In other words, the Ub/proteasome system may not simply be a passive player in radiation-induced responses, but may modulate them. The extent of the modulation will be influenced by the functional and structural diversity that is expressed by the system. Cell types vary in the Ub/proteasome structures they possess and the level at which they function, and this changes as they go from the normal to the cancerous condition. Cancer-related functional changes within the Ub/proteasome system may therefore present unique targets for cancer therapy, especially when targeting agents are used in combination with radio- or chemotherapy. The peptide boronic acid compound PS-341, which was designed to inhibit proteasome chymotryptic activity, is in clinical trials for the treatment of solid and hematogenous tumors. It has shown some efficacy on its own and in combination with chemotherapy. Preclinical studies have shown that PS-341 will also potentiate the cytotoxic effects of radiation therapy. In addition, other drugs in common clinical use have been shown to affect proteasome function, and their activities may be valuably reconsidered from this perspective.

Secondary and tertiary structures of gaseous protein ions characterized by electron capture dissociation mass spectrometry and photofragment spectroscopy.

Over the last decade a variety of MS measurements, such as HD exchange, collision cross sections, and electron capture dissociation (ECD), have been used to characterize protein folding in the gas phase, in the absence of solvent. To the extensive data already available on ubiquitin, here photofragmentation of its ECD-reduced (M + nH)(n-1)+* ions shows that only the 6+ to 9+, not the 10+ to 13+ ions, have tertiary noncovalent bonding; this is indicated as hydrogen bonding by the 3,050-3,775 cm(-1) photofragment spectrum. ECD spectra and HD exchange of the 13+ ions are consistent with an all alpha-helical secondary structure, with the 11+ and 10+ ions sufficiently destabilized to denature small bend regions near the helix termini. In the 8+ and 9+ ions these terminal helical regions are folded over to be antiparallel and noncovalently bonded to part of the central helix, whereas this overlap is extended in the 7+, 6+, and, presumably, 5+ ions to form a highly stable three-helix bundle. Thermal denaturing of the 7+ to 9+ conformers both peels and slides back the outer helices from the central one, but for the 6+ conformer, this instead extends the protein ends away to shrink the three-helix bundle. Thus removal of H2O from a native protein negates hydrophobic interactions, preferentially stabilizes the alpha-helical secondary structure with direct solvation of additional protons, and increases tertiary interhelix dipole-dipole and hydrogen bonding.