Dynamic posttranslational modifications of cytoskeletal proteins unveil hot spots under nitroxidative stress.

The cytoskeleton is a supramolecular structure consisting of interacting protein networks that support cell dynamics in essential processes such as migration and division, as well as in responses to stress. Fast cytoskeletal remodeling is achieved with the participation of regulatory proteins and posttranslational modifications (PTMs). Redox-related PTMs are emerging as critical players in cytoskeletal regulation. Here we used a cellular model of mild nitroxidative stress in which a peroxynitrite donor induced transient changes in the organization of three key cytoskeletal proteins, i.e., vimentin, actin and tubulin. Nitroxidative stress-induced reconfiguration of intermediate filaments, microtubules and actin structures were further correlated with their PTM profiles and dynamics of the PTM landscape. Using high-resolution mass spectrometry, 62 different PTMs were identified and relatively quantified in vimentin, actin and tubulin, including 12 enzymatic, 13 oxidative and 2 nitric oxide-derived modifications as well as 35 modifications by carbonylated lipid peroxidation products, thus evidencing the occurrence of a chain reaction with formation of numerous reactive species and activation of multiple signaling pathways. Our results unveil the presence of certain modifications under basal conditions and their modulation in response to stress in a target-, residue- and reactive species-dependent manner. Thus, some modifications accumulated during the experiment whereas others varied transiently. Moreover, we identified protein PTM "hot spots", such as the single cysteine residue of vimentin, which was detected in seven modified forms, thus, supporting its role in PTM crosstalk and redox sensing. Finally, identification of novel PTMs in these proteins paves the way for unveiling new cytoskeleton regulatory mechanisms.

Single molecule turnover of fluorescent ATP by myosin and actomyosin unveil elusive enzymatic mechanisms.

Benefits of single molecule studies of biomolecules include the need for minimal amounts of material and the potential to reveal phenomena hidden in ensembles. However, results from recent single molecule studies of fluorescent ATP turnover by myosin are difficult to reconcile with ensemble studies. We found that key reasons are complexities due to dye photophysics and fluorescent contaminants. After eliminating these, through surface cleaning and use of triple state quenchers and redox agents, the distributions of ATP binding dwell times on myosin are best described by 2 to 3 exponential processes, with and without actin, and with and without the inhibitor para-aminoblebbistatin. Two processes are attributable to ATP turnover by myosin and actomyosin respectively, whereas the remaining process (rate constant 0.2-0.5 s-1) is consistent with non-specific ATP binding to myosin, possibly accelerating ATP transport to the active site. Finally, our study of actin-activated myosin ATP turnover without sliding between actin and myosin reveals heterogeneity in the ATP turnover kinetics consistent with models of isometric contraction.

Myosin and gelsolin cooperate in actin filament severing and actomyosin motor activity.

Actin is a major intracellular protein with key functions in cellular motility, signaling, and structural rearrangements. Its dynamic behavior, such as polymerization and depolymerization of actin filaments in response to intracellular and extracellular cues, is regulated by an abundance of actin binding proteins. Out of these, gelsolin is one of the most potent for filament severing. However, myosin motor activity also fragments actin filaments through motor-induced forces, suggesting that these two proteins could cooperate to regulate filament dynamics and motility. To test this idea, we used an in vitro motility assay, where actin filaments are propelled by surface-adsorbed heavy meromyosin (HMM) motor fragments. This allows studies of both motility and filament dynamics using isolated proteins. Gelsolin, at both nanomolar and micromolar Ca2+ concentration, appreciably enhanced actin filament severing caused by HMM-induced forces at 1 mM MgATP, an effect that was increased at higher HMM motor density. This finding is consistent with cooperativity between actin filament severing by myosin-induced forces and by gelsolin. We also observed reduced sliding velocity of the HMM-propelled filaments in the presence of gelsolin, providing further support of myosin-gelsolin cooperativity. Total internal reflection fluorescence microscopy-based single molecule studies corroborated that the velocity reduction was a direct effect of gelsolin binding to the filament and revealed different filament severing pattern of stationary and HMM propelled filaments. Overall, the results corroborate cooperative effects between gelsolin-induced alterations in the actin filaments and changes due to myosin motor activity leading to enhanced F-actin severing of possible physiological relevance.

Cross-talk between lipid and protein carbonylation in a dynamic cardiomyocyte model of mild nitroxidative stress.

Reactive oxygen and nitrogen species (ROS/RNS) play an important role in the regulation of cardiac function. Increase in ROS/RNS concentration results in lipid and protein oxidation and is often associated with onset and/or progression of many cardiovascular disorders. However, interplay between lipid and protein modifications has not been simultaneously studied in detail so far. Biomolecule carbonylation is one of the most common biomarkers of oxidative stress. Using a dynamic model of nitroxidative stress we demonstrated rapid changes in biomolecule carbonylation in rat cardiomyocytes. Levels of carbonylated species increased as early as 15min upon treatment with the peroxynitrite donor, 3-morpholinosydnonimine (SIN-1), and decreased to values close to control after 16h. Total (lipids+proteins) vs. protein-specific carbonylation showed different dynamics, with a significant increase in protein-bound carbonyls at later time points. Treatment with SIN-1 in combination with inhibitors of proteasomal and autophagy/lysosomal degradation pathways allowed confirmation of a significant role of the proteasome in the degradation of carbonylated proteins, whereas lipid carbonylation increased in the presence of autophagy/lysosomal inhibitors. Electrophilic aldehydes and ketones formed by lipid peroxidation were identified and relatively quantified using LC-MS/MS. Molecular identity of reactive species was used for data-driven analysis of their protein targets. Combination of different enrichment strategies with LC-MS/MS analysis allowed identification of more than 167 unique proteins with 332 sites modified by electrophilic lipid peroxidation products. Gene ontology analysis of modified proteins demonstrated enrichment of several functional categories including proteins involved in cytoskeleton, extracellular matrix, ion channels and their regulation. Using calcium mobilization assays, the effect of nitroxidative stress on the activity of several ion channels was further confirmed.

Profiling and relative quantification of multiply nitrated and oxidized fatty acids.

The levels of nitro fatty acids (NO2-FA), such as nitroarachidonic, nitrolinoleic, nitrooleic, and dinitrooleic acids, are elevated under various inflammatory conditions, and this results in different anti-inflammatory effects. However, other multiply nitrated and nitro-oxidized FAs have not been studied so far. Owing to the low concentrations in vivo, NO2-FA analytics usually relies on targeted gas chromatography-tandem mass spectrometry (MS/MS) or liquid chromatography-MS/MS, and thus require standard compounds for method development. To overcome this limitation and increase the number and diversity of analytes, we performed in-depth mass spectrometry (MS) profiling of nitration products formed in vitro by incubating fatty acids with NO2BF4, and ONOO(-). The modified fatty acids were used to develop a highly specific and sensitive multiple reaction monitoring LC-MS method for relative quantification of 42 different nitrated and oxidized species representing three different groups: singly nitrated, multiply nitrated, and nitro-oxidized fatty acids. The method was validated in in vitro nitration kinetic studies and in a cellular model of nitrosative stress. NO2-FA were quantified in lipid extracts from 3-morpholinosydnonimine-treated rat primary cardiomyocytes after 15, 30, and 70 min from stress onset. The relatively high levels of dinitrooleic, nitroarachidonic, hydroxynitrodocosapenataenoic, nitrodocosahexaenoic, hydroxynitrodocosahexaenoic, and dinitrodocosahexaenoic acids confirm the presence of multiply nitrated and nitro-oxidized fatty acids in biological systems for the first time. Thus, in vitro nitration was successfully used to establish a targeted LC-MS/MS method that was applied to complex biological samples for quantifying diverse NO2-FA. Graphical Abstract Schematic representation of study design which combined in vitro nitration of different fatty acids, MS/MS characterization and optimization of MRM method for relative quantification, which was applied to follow dynamic of fatty acid nitration in cellular model of SIN-1 treated cardiomyoctes.

Fluorescence labeling of carbonylated lipids and proteins in cells using coumarin-hydrazide.

Carbonylation is a generic term which refers to reactive carbonyl groups present in biomolecules due to oxidative reactions induced by reactive oxygen species. Carbonylated proteins, lipids and nucleic acids have been intensively studied and often associated with onset or progression of oxidative stress related disorders. In order to reveal underlying carbonylation pathways and biological relevance, it is crucial to study their intracellular formation and spatial distribution. Carbonylated species are usually identified and quantified in cell lysates and body fluids after derivatization using specific chemical probes. However, spatial cellular and tissue distribution have been less often investigated. Here, we report coumarin-hydrazide, a fluorescent chemical probe for time- and cost-efficient labeling of cellular carbonyls followed by fluorescence microscopy to evaluate their intracellular formation both in time and space. The specificity of coumarin-hydrazide was confirmed in time- and dose-dependent experiments using human primary fibroblasts stressed with paraquat and compared with conventional DNPH-based immunocytochemistry. Both techniques stained carbonylated species accumulated in cytoplasm with strong perinuclear clustering. Using a complimentary array of analytical methods specificity of coumarin-hydrazide probe towards both protein- and lipid-bound carbonyls has been shown. Additionally, co-distribution of carbonylated species and oxidized phospholipids was demonstrated.

Protein and lipid carbonylation in cellular model of nitrosative stress: mass spectrometry, biochemistry and microscopy study.

Within the wide range of oxidative modifications, "carbonylation" formed by the incorporation of aldehyde/keto groups, is commonly studied due to its role in cell physiology and as prospective biomarkers for numerous disorders. Despite close biochemical and physiological links between protein and lipid carbonylation, these two types of modifications are rarely addressed simultaneously in a single study. In nitrosative stress cell model we investigated levels of protein and lipid carbonylation and addressed the main modified species by combining LC-MS, biochemical, and microscopy studies. The influence of nitrosative stress on carbonylation of proteins and lipids was investigated for primary cardiomyocytes treated with SIN-1 for different time intervals. Lipid carbonylation was quantified by RPC-ESI-MS/MS. The results demonstrate dynamic generation, degradation and adduct formations of 25 different species including alkanals, alkenals, alkadienals, alkatrienals and oxo-carboxylic acids. Several new PL-bound aldehydes were present exclusively after a long incubation period. Carbonylated proteins were identified after aldehyde reactive probe derivatization, affinity enrichment and RPC-ESI-MS/MS. More than 200 proteins were identified and evaluated by systems biology to deduce the biological significance of the protein modifications. The protein carbonylation degree was verified using oxyblot and correlated with changes in 20S/26S proteasome activities. Furthermore, a new fluorescence microscopy based technique to stain carbonylated biomolecules was developed and compared with conventional DNPH-based immunocytochemistry. Subcellular localization of carbonylated species was investigated using mitochondrial and ER-specific co-localization experiments. Thus, the combination of lipidomics, proteomics, biochemical techniques, and microscopy imaging revealed a complex molecular pattern of "carbonylation stress" in the studied nitrosative stress cell model.