Modification of cysteine residues by cyclopentenone prostaglandins: Interplay with redox regulation of protein function

40 p.-5 fig.-1 tab.Cyclopentenone prostaglandins (cyPG) are endogenous lipid mediators involved in the resolution of inflammation and the regulation of cell proliferation and cellular redox status. Upon exogenous administration they have shown beneficial effects in models of inflammation and tissue injury, as well as potential antitumoral actions, which have raised a considerable interest in their study for the development of therapeutic tools. Due to their electrophilic nature, the best-known mechanism of action of these mediators is the covalent modification of proteins at cysteine residues through Michael addition. Identification of cyPG targets through proteomic approaches, including MS/MS analysis to pinpoint the modified residues, is proving critical to characterize their mechanisms of action. Among the targets of cyPG are proinflammatory transcription factors, proteins involved in cell defense, such as the regulator of the antioxidant response Keap1 and detoxifying enzymes like GST, and key signaling proteins like Ras proteins. Moreover, cyPG may interact with redox-active small molecules, such as glutathione and hydrogen sulfide. Much has been learned about cyPG in the past few years and this knowledge has also contributed to clarify both pharmacological actions and signaling mechanisms of these and other electrophilic lipids. Given the fact that many cyPG targets are involved in or are targets for redox regulation, there is a complex interplay with redox-induced modifications. Here we address the modification of protein cysteine residues by cyPG elucidated by proteomic studies, paying special attention to the interplay with redox signaling.Work in the authors’ laboratory is supported by grants from MINECO SAF2009-11642 and SAF2012-36519, RETIC RIRAAF RD07/0064/0007 and RD12/0013/0008 from ISCIII and COST action CM1001. CLO is the recipient of a FPI fellowship from MINECO.Peer reviewe

The redox-responsive roles of intermediate filaments in cellular stress detection, integration and mitigation

Intermediate filaments are critical for cell and tissue homeostasis and for stress responses. Cytoplasmic intermediate filaments form versatile and dynamic assemblies that interconnect cellular organelles, participate in signaling and protect cells and tissues against stress. Here we have focused on their involvement in redox signaling and oxidative stress, which arises in numerous pathophysiological situations. We pay special attention to type III intermediate filaments, mainly vimentin, because it provides a physical interface for redox signaling, stress responses and mechanosensing. Vimentin possesses a single cysteine residue that is a target for multiple oxidants and electrophiles. This conserved residue fine tunes vimentin assembly, response to oxidative stress and crosstalk with other cellular structures. Here we integrate evidence from the intermediate filament and redox biology fields to propose intermediate filaments as redox sentinel networks of the cell. To support this, we appraise how vimentin detects and orchestrates cellular responses to oxidative and electrophilic stress

Betaine homocysteine S-methyltransferase emerges as a new player of the nuclear methionine cycle

The paradigm of a cytoplasmic methionine cycle synthesizing/eliminating metabolites that are transported into/out of the nucleus as required has been challenged by detection of significant nuclear levels of several enzymes of this pathway. Here, we show betaine homocysteine S-methyltransferase (BHMT), an enzyme that exerts a dual function in maintenance of methionine levels and osmoregulation, as a new component of the nuclear branch of the cycle. In most tissues, low expression of Bhmt coincides with a preferential nuclear localization of the protein. Conversely, the liver, with very high Bhmt expression levels, presents a main cytoplasmic localization. Nuclear BHMT is an active homotetramer in normal liver, although the total enzyme activity in this fraction is markedly lower than in the cytosol. N-terminal basic residues play a role in cytoplasmic retention and the ratio of glutathione species regulates nucleocytoplasmic distribution. The oxidative stress associated with D-galactosamine (Gal) or buthionine sulfoximine (BSO) treatments induces BHMT nuclear translocation, an effect that is prevented by administration of N-acetylcysteine (NAC) and glutathione ethyl ester (EGSH), respectively. Unexpectedly, the hepatic nuclear accumulation induced by Gal associates with reduced nuclear BHMT activity and a trend towards increased protein homocysteinylation. Overall, our results support the involvement of BHMT in nuclear homocysteine remethylation, although moonlighting roles unrelated to its enzymatic activity in this compartment cannot be excluded.This work was supported by grants of the Ministerio de Economía y Competitividad (BFU2008-00666 and BFU2009-08977 to MAP; SAF2012-36519 and SAF2015-68590R to DPS) and Instituto de Salud Carlos III (RETIC RIRAAF RD12/0013/0008 and ARADYAL RD16/0006/0021 to DPS).Peer reviewe

Analogs of farnesylcysteine induce apoptosis in HL-60 cells

AbstractS-Farnesyl-thioacetic acid (FTA), a competitive inhibitor of isoprenylated protein methyltransferase, potently suppressed the growth of HL-60 cells and induced apoptosis, as evidenced by the development of increased annexin-V binding, decreased binding of DNA dyes and internucleosomal DNA degradation. FTA did not impair the membrane association of ras proteins, conversely, it brought about a decrease in the proportion of ras present in the cytosolic fraction. Farnesylated molecules which are weak inhibitors of the methyltransferase also induced DNA laddering and reduced the proportion of cytosolic ras. These findings suggest that neither inhibition of isoprenylated protein methylation nor impairment of ras membrane association are essential for apoptosis induced by farnesylcysteine analogs

Drawbacks of dialysis procedures for removal of EDTA

9 p.-3 fig.Ethylenediaminetetraacetic acid (EDTA) is a chelating agent commonly used in protein purification, both to eliminate contaminating divalent cations and to inhibit protease activity. For a number of subsequent applications EDTA needs to be exhaustively removed. Most purification methods rely in extensive dialysis and/or gel filtration in order to exchange or remove protein buffer components, including metal chelators. We report here that dialysis protocols,even as extensive as those typically employed for protein refolding, may not effectively remove EDTA, which is reduced only by approximately two-fold and it also persists after spin-column gel filtration, as determined by NMR and by colorimetric methods. Remarkably, the most efficient removal was achieved by ultrafiltration, after which EDTA became virtually undetectable. These results highlight a potentially widespread source of experimental variability affecting free divalent cation concentrations in protein applications.This work has been funded by the European Union's Horizon 2020 research and innovation programme under the Marie Sklowdowska-Curie grant agreement number 675132 (http://cordis.europa.eu/project/rcn/198275_en.html), and by grants from the Spanish Ministerio de Economía y Competitividad (MINECO/FEDER, http://www.mineco.gob.es/ portal/site/mineco/idi) SAF2015-68590R to DPS and CTQ2015-64597-C2-2-P to FJC.Peer reviewe

Photosensitivity to triflusal: formation of a photoadduct with ubiquitin demonstrated by photophysical and proteomic techniques

8 p.-7 fig.1 tab.Triflusal is a platelet aggregation inhibitor chemically related to acetylsalicylic acid, which is used for the prevention and/or treatment of vascular thromboembolisms, which acts as a prodrug. Actually, after oral administration it is absorbed primarily in the small intestine, binds to plasma proteins (99%) and is rapidly biotransformed in the liver into its deacetylated active metabolite 2-hydroxy-4-trifluoromethylbenzoic acid (HTB). In healthy humans, the half-life of triflusal is ca. 0.5 h, whereas for HTB it is ca. 35 h. From a pharmacological point of view, it is interesting to note that HTB is itself highly active as a platelet anti-aggregant agent. Indeed, studies on the clinical profile of both drug and metabolite have shown no significant differences between them. It has been evidenced that HTB displays ability to induce photoallergy in humans. This phenomenon involves a cell-mediated immune response, which is initiated by covalent binding of a light-activated photosensitizer (or a species derived therefrom) to a protein. In this context, small proteins like ubiquitin could be appropriate models for investigating covalent binding by means of MS/MS and peptide fingerprint analysis. In previous work, it was shown that HTB forms covalent photoadducts with isolated lysine. Interestingly, ubiquitin contains seven lysine residues that could be modified by a similar reaction.With this background, the aim of the present work is to explore adduct formation between the triflusal metabolite and ubiquitin as model protein upon sunlight irradiation, combining proteomic and photophysical (fluorescence and laser flash photolysis) techniques. Photophysical and proteomic analysis demonstrates monoadduct formation as the major outcome of the reaction. Interestingly, addition can take place at any of the +-amino groups of the lysine residues of the protein and involves replacement of the trifluoromethyl moiety with a new amide function. This process can in principle occur with other trifluoroaromatic compounds and may be responsible for the appearance of undesired photoallergic side effects.Financial supportfromtheGeneralitatValenciana(Prometeo Program), the Spanish Government (MINECOCTQ2015-70164- P toVL-Vand SAF2012-36519 to DP-S) and the Carlos III Institute of Health(Grant RIRAAF,RETIC Sprogram, RD12/0013/0009 to MM and RD12/0013/0008 to DP-S,and Miguel Servet Contract CP11/00154forIA) is gratefully acknowledged.Peer reviewe

Gemelli-obturator complex in the deep gluteal space: an anatomic and dynamic study

Objective: To investigate the behavior of the sciatic nerve during hip rotation at subgluteal space. Materials and methods: Sonographic examination (high-resolution ultrasound machine at 5.0–14 MHZ) of the gemelli-obturator internus complex following two approaches: (1) a study on cadavers and (2) a study on healthy volunteers. The cadavers were examined in pronation, pelvis-fixed position by forcing internal and external rotations of the hip with the knee in 90° flexion. Healthy volunteers were examined during passive internal and external hip rotation (prone position; lumbar and pelvic regions fixed). Subjects with a history of major trauma, surgery or pathologies affecting the examined regions were excluded. Results: The analysis included eight hemipelvis from six fresh cadavers and 31 healthy volunteers. The anatomical study revealed the presence of connective tissue attaching the sciatic nerve to the structures of the gemellus-obturator system at deep subgluteal space. The amplitude of the nerve curvature during rotating position was significantly greater than during resting position. During passive internal rotation, the sciatic nerve of both cadavers and healthy volunteers transformed from a straight structure to a curved structure tethered at two points as the tendon of the obturator internus contracted downwards. Conversely, external hip rotation caused the nerve to relax. Conclusion: Anatomically, the sciatic nerve is closely related to the gemelli-obturator internus complex. This relationship results in a reproducible dynamic behavior of the sciatic nerve during passive hip rotation, which may contribute to explain the pathological mechanisms of the obturator internal gemellus syndrome.Peer ReviewedPostprint (author's final draft

Proteomics in drug hypersensitivity

21 p.-4 fig.-1 tab.Drug hypersensitivity reactions result from the activation of the immune system by drugs or their metabolites. The clinical presentations of drug hypersensitivity can range from relatively mild local manifestations to severe systemic syndromes that can be life-threatening. As in other allergic reactions, the causes are multifactorial as genetic, metabolic and concomitant factors may influence the occurrence of drug hypersensitivity. Formation of drug protein adducts is considered a key step in drug adverse reactions, and in particular in the immunological recognition in drug hypersensitivity reactions. Nevertheless, non-covalent interactions of drugs with receptors in immune cells or with MHC clefts and/or exposed peptides can also play an important role. In recent years, development of proteomic approaches has allowed the identification and characterization of the protein targets for modification by drugs in vivo and in vitro, the nature of peptides exposed on MHC molecules, the changes in protein levels induced by drug treatment, and the concomitant modifications induced by danger signals, thus providing insight into context factors. Nevertheless, given the complexity and multifactorial nature of drug hypersensitivity reactions, understanding the underlying mechanisms also requires the integration of knowledge from genomic, metabolomic and clinical studies.This work has been supported by grants SAF2012-36519 and SAF2015-68590R from MINECO/FEDER and RETIC RD12/0013/0008 from ISCIII to D.P.-S., and by RETIC RD12/0013/0001 and CP15/00103 from ISCIII, and PI-0699-2011 and PI-0179-2014 from Junta de Andalucía to M.I.M.Peer reviewe

The conserved cysteine residue of type III intermediate filaments serves as a structural element and redox sensor

1 p.H2020 grant 675132(Masstrplan); MINECO/FEDER SAF2015–68590-RPeer reviewe

Vimentin filament organization and stress sensing depend on its single cysteine residue and zinc binding

17 pág., 10 figs.The vimentin filament network plays a key role in cell architecture and signalling, as well as in epithelial–mesenchymal transition. Vimentin C328 is targeted by various oxidative modifications, but its role in vimentin organization is not known. Here we show that C328 is essential for vimentin network reorganization in response to oxidants and electrophiles, and is required for optimal vimentin performance in network expansion, lysosomal distribution and aggresome formation. C328 may fulfil these roles through interaction with zinc. In vitro, micromolar zinc protects vimentin from iodoacetamide modification and elicits vimentin polymerization into optically detectable structures; in cells, zinc closely associates with vimentin and its depletion causes reversible filament disassembly. Finally, zinc transportdeficient human fibroblasts show increased vimentin solubility and susceptibility to disruption, which are restored by zinc supplementation. These results unveil a critical role of C328 in vimentin organization and open new perspectives for the regulation of intermediate filaments by zinc. DOI: 10.1038/ncomms8287 OPEN 1This work was supported by grants SAF2012–36519, MINECO, Spain and RD12/0013/0008, ISCIII to D.P.-S., and CTQ2012–32025, MINECO, y CAM MHIT S2010/BMD-2353 to F.J.C. C.L.O. and B.G. have been recipients of fellowships BES-2010–033718 and BES-2007–15806, respectively (FPI, MINECO). We acknowledge support from COST Action CM1001.Peer reviewe