Measurement and meaning of cellular thiol:disufhide redox status

The functional group of cysteine is a thiol group (SH) that, due to its chemical reactivity, is able to undergo a wide array of modifications each with the potential to confer a different property or function to the molecule harboring this residue. Most of these modifications involve the reversible oxidation of the thiol to sulfenic acid (SOH), and disulfide, including intra- and intermolecular disulfides between polypeptides and glutathione (glutathionylation). The reversibility of these oxidations allows thiol groups to serve as versatile chemical and structural transducing elements in several low molecular mass metabolites and proteins. A plethora of cellular functions such as DNA and protein synthesis, protein secretion, cytoskeleton architecture, differentiation, apoptosis, and anti-oxidant defense, are recognized to be modulated, at certain stage, by thiol–disulfide exchange mechanisms of redox active thiol groups. All organisms are equipped with enzymatic systems composed by NADPH-dependent reductases, redoxins, and peroxidases that provide kinetic control of global thiol-redox homeostasis as well as target selectivity. These redox systems are distributed in different subcellular compartments and are not in equilibrium with each other. In consequence, measuring cellular thiol–disulfide status represents a challenge for studies aimed to obtain dynamic and spatio-temporal resolution. This review provides a summary of the methods and tools available to quantify the thiol redox status of cells.     

Vinyl sulfone functionalization: a feasible approach for the study of the lectin-carbohydrate interactions

Carbohydrate-mediated molecular recognition is involved in many biological aspects such as cellular adhesion, immune response, blood coagulation, inflammation, and infection. Considering the crucial importance of such biological events in which proteins are normally involved, synthetic saccharide-based systems have emerged as powerful tools for the understanding of protein-carbohydrate interactions. As a new approach to create saccharide-based systems, a set of representative monosaccharides (D-mannose, D-glucose, N-acetyl-D-glucosamine, and L-fucose) and disaccharides (lactose, maltose, and melibiose) were derivatized at their anomeric carbon with a vinyl sulfone group spanned by an ethylthio linker. This vinyl sulfone functionalization is demonstrated to be a general strategy for the covalent linkage of a saccharide in mild conditions via Michael-type additions with the amine and thiol groups from functionalized supports and those naturally present in biomolecules. The introduction of the ethylthio linker between the biorecognizable element (i.e., saccharide) and the reactive group (i.e., vinyl sulfone) was found to preserve the functionality of the former. The capability of the vinyl sulfone saccharides for the study of lectin-carbohydrate interactions was demonstrated by (i) immobilizing them on both amine-functionalized supports (glass slides and microwell plates) and polylysine-coated glass slides to create sugar arrays that selectively bind lectins (ii) coupling to model proteins to yield neoglycoproteins that are recognized by lectins and (iii) using vinyl sulfone saccharides as tags to allow the detection of the labeled biomolecule by HRP-lectins. The above results were further put tothe test with a real case: detection of carbohydrate binding proteins present in rice ( Oryza sativa ).     

Oxidation state of tissue thiol groups and content of protein carbonyl groups in chickens with inherited muscular dystrophy.

Indirect evidence suggests that oxidative stress may play a role in the pathogenesis of inherited muscular dystrophy, but the significance and precise extent of this contribution is poorly understood. Compared with normal muscle, significantly higher contents of glutathione, glutathione disulphide, protein-glutathione mixed disulphides and protein carbonyl groups, and significantly lower contents of free protein thiol groups, were found in pectoralis major muscle of genetically dystrophic chickens (the muscle affected by this disease) at 4 weeks of age. Other tissues did not show such marked disease-related differences. Interestingly, the protein pool in normal, but not dystrophic, pectoralis major muscle was relatively less oxidized in relation to the glutathione pool as compared with other tissues studied. The mechanisms by which this unique relationship between the thiol pools is maintained remain unknown. Although the physiological consequences of the increased content of protein carbonyl groups and the altered thiol pools in dystrophic muscle are not clear, the changes evident at such a young age are consistent with the occurrence of oxidative stress and may reflect significant damage to cellular proteins in this disease.     

Detection of oxidative stress-induced carbonylation in live mammalian cells

Oxidative stress is often associated with etiology and/or progression of disease conditions, such as cancer, neurodegenerative diseases, and diabetes. At the cellular level, oxidative stress induces carbonylation of biomolecules such as lipids, proteins, and DNA. The presence of carbonyl-containing biomolecules as a hallmark of these diseases provides a suitable target for diagnostic detection. Here, a simple, robust method for detecting cellular aldehydes and ketones in live cells using a fluorophore is presented. A hydrazine-functionalized synthetic fluorophore serves as an efficient nucleophile that rapidly reacts with reactive carbonyls in the cellular milieu. The product thus formed exhibits a wavelength shift in the emission maximum accompanied by an increase in emission intensity. The photochemical characteristics of the fluorophore enable the identification of the fluorophore-conjugated cellular biomolecules in the presence of unreacted dye, eliminating the need for removal of excess fluorophore. Moreover, this fluorophore is found to be nontoxic and is thus appropriate for live cell analysis. Utility of the probe is demonstrated in two cell lines, PC3 and A549. Carbonylation resulting from serum starvation and hydrogen peroxide-induced stress is detected in both cell lines using fluorescence microscopy and a fluorescence plate reader. The fluorescent signal originates from carbonylated proteins and lipids but not from oxidized DNA, and the majority of the fluorescence signal (>60%) is attributed to fluorophore-conjugated lipid oxidation products. This method should be useful for detecting cellular carbonylation in a high-content assay or high-throughput assay format.     

Ablation of the mammalian methionine sulfoxide reductase A affects the expression level of cysteine deoxygenase

Methionine sulfoxide reductases (Msrs) are able to reduce methionine sulfoxide to methionine both in proteins and free amino acids. By their action it is possible to regulate the function of specific proteins and the cellular antioxidant defense against oxidative damage. Similarly, cysteine deoxygenase (CDO) may be involved in the regulation of protein function and antioxidant defense mechanisms by its ability to oxidized cysteine residues. The two enzymes' involvement in sulfur amino-acids metabolism seems to be connected. Lack of methionine sulfoxide reductase A (MsrA) in liver of MsrA-/- led to a significant drop in the cellular level of thiol groups and lowered the CDO level of expression. Moreover, following selenium deficient diet (applied to decrease the expression levels of selenoproteins like MsrB), the latter effect was maintained while the basal levels of thiol decreased in both mouse strains. We suggest that both enzymes are working in coordination to balance cellular antioxidant defense.     

Nitrosative capacity of macrophages is dependent on nitric-oxide synthase induction signals

Nitrosative stress can occur when reactive nitric oxide (NO) species compromise the function of biomolecules via formation of NO adducts on critical amine and thiol residues. The capacity of inducible nitric-oxide synthase (iNOS) to generate nitrosative stress was investigated in the murine macrophage line ANA-1. Sequential activation with the cytokines IFN-gamma and either tumor necrosis factor-alpha or interleukin-1beta resulted in the induction of iNOS and production of nitrite (20 nM/min) but failed to elicit nitrosation of extracellular 2,3-diaminonapthalene. Stimulation with IFN-gamma and bacterial lipopolysaccharide increased the relative level of iNOS protein and nitrite production of ANA-1 cells 2-fold; however, a substantial level of NO in the media was also observed, and nitrosation of 2,3-diaminonapthalene was increased greater than 30-fold. Selective scavenger compounds suggested that the salient nitrosating mechanism was the NO/O(2) reaction leading to N(2)O(3) formation. These data mimicked the pattern observed with a 5 microM concentration of the synthetic NO donor (Z)-1-[N-ammoniopropyl)-N-(n-propyl)amino]diazen-1-ium -1,2-diolate (PAPA/NO). The NO profiles derived from iNOS can be distinct and depend on the inductive signal cascades. The diverse consequences of NO production in macrophages may reside in the cellular mechanisms that control the ability of iNOS to form N(2)O(3) and elicit nitrosative stress.     

Nitric oxide evolution and perception

Various experimental data indicate signalling roles for nitric oxide (NO) in processes such as xylogenesis, programmed cell death, pathogen defence, flowering, stomatal closure, and gravitropism. However, it still remains unclear how NO is synthesized. Nitric oxide synthase-like activity has been measured in various plant extracts, NO can be generated from nitrite via nitrate reductase and other mechanisms of NO generation are also likely to exist. NO removal mechanisms, for example, by reaction with haemoglobins, have also been identified. NO is a gas emitted by plants, with the rate of evolution increasing under conditions such as pathogen challenge or hypoxia. However, exactly how NO evolution relates to its bioactivity in planta remains to be established. NO has both aqueous and lipid solubility, but is relatively reactive and easily oxidized to other nitrogen oxides. It reacts with superoxide to form peroxynitrite, with other cellular components such as transition metals and haem-containing proteins and with thiol groups to form S-nitrosothiols. Thus, diffusion of NO within the plant may be relatively restricted and there might exist 'NO hot-spots' depending on the sites of NO generation and the local biochemical micro-environment. Alternatively, it is possible that NO is transported as chemical precursors such as nitrite or as nitrosothiols that might function as NO reservoirs. Cellular perception of NO may occur through its reaction with biologically active molecules that could function as 'NO-sensors'. These might include either haem-containing proteins such as guanylyl cyclase which generates the second messenger cGMP or other proteins containing exposed reactive thiol groups. Protein S-nitrosylation alters protein conformation, is reversible and thus, is likely to be of biological significance.     

Synthesis and Characterization of Dual-Functionalized Core-Shell Fluorescent Microspheres for Bioconjugation and Cellular Delivery

The efficient transport of micron-sized beads into cells, via a non-endocytosis mediated mechanism, has only recently been described. As such there is considerable scope for optimization and exploitation of this procedure to enable imaging and sensing applications to be realized. Herein, we report the design, synthesis and characterization of fluorescent microsphere-based cellular delivery agents that can also carry biological cargoes. These core-shell polymer microspheres possess two distinct chemical environments; the core is hydrophobic and can be labeled with fluorescent dye, to permit visual tracking of the microsphere during and after cellular delivery, whilst the outer shell renders the external surfaces of the microspheres hydrophilic, thus facilitating both bioconjugation and cellular compatibility. Cross-linked core particles were prepared in a dispersion polymerization reaction employing styrene, divinylbenzene and a thiol-functionalized co-monomer. These core particles were then shelled in a seeded emulsion polymerization reaction, employing styrene, divinylbenzene and methacrylic acid, to generate orthogonally functionalized core-shell microspheres which were internally labeled via the core thiol moieties through reaction with a thiol reactive dye (DY630-maleimide). Following internal labeling, bioconjugation of green fluorescent protein (GFP) to their carboxyl-functionalized surfaces was successfully accomplished using standard coupling protocols. The resultant dual-labeled microspheres were visualized by both of the fully resolvable fluorescence emissions of their cores (DY630) and shells (GFP). In vitro cellular uptake of these microspheres by HeLa cells was demonstrated conventionally by fluorescence-based flow cytometry, whilst MTT assays demonstrated that 92% of HeLa cells remained viable after uptake. Due to their size and surface functionalities, these far-red-labeled microspheres are ideal candidates for in vitro, cellular delivery of proteins.     

Protective mechanisms against peptide and protein peroxides generated by singlet oxygen

Reaction of certain amino acids, peptides, and proteins with singlet oxygen yields substrate-derived peroxides. Recent studies have shown that these species are formed within intact cells and can inactivate key cellular enzymes. This study examines potential mechanisms by which cells might remove or detoxify such peroxides. It is shown that catalase, horseradish peroxidase, and Cu/Zn superoxide dismutase do not react rapidly with these peroxides. Oxymyoglobin and oxyhemoglobin, but not the met (Fe3+) forms of these proteins, react with peptide but not protein, peroxides with oxidation of the heme iron. Glutathione peroxidase, in the presence of reduced glutathione (GSH) rapidly removes peptide, but not protein, peroxides, consistent with substrate size being a key factor. Protein thiols, GSH, other low-molecular-weight thiols, and the seleno-compound ebselen react, in a nonstoichiometric manner, with both peptide and protein peroxides. Cell lysate studies show that thiol consumption and peroxide removal occur in parallel; the stoichiometry of these reactions suggests that thiol groups are the major direct, or indirect, reductants for these species. Ascorbic acid and some derivatives can remove both the parent peroxides and radicals derived from them, whereas methionine and the synthetic phenolic antioxidants Probucol and BHT show little activity. These studies show that cells do not have efficient enzymatic defenses against protein peroxides, with only thiols and ascorbic acid able to remove these materials; the slow removal of these species is consistent with protein peroxides playing a role in cellular dysfunction resulting from oxidative stress.     

3-hydroxypyridine chromophores are endogenous sensitizers of photooxidative stress in human skin cells

Photocarcinogenesis and photoaging are established consequences of chronic exposure of human skin to solar irradiation. Accumulating evidence supports a causative involvement of UVA irradiation in skin photo-damage. UVA photodamage has been attributed to photosensitization by endogenous skin chromophores leading to the formation of reactive oxygen species and organic free radicals as key mediators of cellular photooxidative stress. In this study, 3-hydroxypyridine derivatives contained in human skin have been identified as a novel class of potential endogenous photosensitizers. A structure-activity relationship study of skin cell photosensitization by endogenous pyridinium derivatives (pyridinoline, desmosine, pyridoxine, pyridoxamine, pyridoxal, pyridoxal-5'-phosphate) and various synthetic hydroxypyridine isomers identified 3-hydroxypyridine and N-alkyl-3-hydroxypyridinium cation as minimum phototoxic chromophores sufficient to effect skin cell sensitization toward UVB and UVA, respectively. Photosensitization of cultured human skin keratinocytes (HaCaT) and fibroblasts (CF3) by endogenous and synthetic 3-hydroxypyridine derivatives led to a dose-dependent inhibition of proliferation, cell cycle arrest in G2/M, and induction of apoptosis, all of which were reversible by thiol antioxidant intervention. Enhancement of UVA-induced intracellular peroxide formation and p38 mitogen-activated protein kinase-dependent stress signaling suggest a photooxidative mechanism of skin cell photosensitization by 3-hydroxypyridine derivatives. 3-hydroxypyridine derivatives were potent photosensitizers of macromolecular damage, effecting protein (RNase A) photocross-linking and peptide (melittin) photooxidation with incorporation of molecular oxygen. Based on these results, we conclude that 3-hydroxypyridine derivatives comprising a wide range of skin biomolecules, such as enzymatic collagen cross-links, B6 vitamers, and probably advanced glycation end products in chronologically aged skin constitute a novel class of UVA photosensitizers, capable of skin photooxidative damage.     

α-Amino acid containing degradable polymers as functional biomaterials: rational design, synthetic pathway, and biomedical applications

Currently, biomedical engineering is rapidly expanding, especially in the areas of drug delivery, gene transfer, tissue engineering, and regenerative medicine. A prerequisite for further development is the design and synthesis of novel multifunctional biomaterials that are biocompatible and biologically active, are biodegradable with a controlled degradation rate, and have tunable mechanical properties. In the past decades, different types of α-amino acid-containing degradable polymers have been actively developed with the aim to obtain biomimicking functional biomaterials. The use of α-amino acids as building units for degradable polymers may offer several advantages: (i) imparting chemical functionality, such as hydroxyl, amine, carboxyl, and thiol groups, which not only results in improved hydrophilicity and possible interactions with proteins and genes, but also facilitates further modification with bioactive molecules (e.g., drugs or biological cues); (ii) possibly improving materials biological properties, including cell-materials interactions (e.g., cell adhesion, migration) and degradability; (iii) enhancing thermal and mechanical properties; and (iv) providing metabolizable building units/blocks. In this paper, recent developments in the field of α-amino acid-containing degradable polymers are reviewed. First, synthetic approaches to prepare α-amino acid-containing degradable polymers will be discussed. Subsequently, the biomedical applications of these polymers in areas such as drug delivery, gene delivery and tissue engineering will be reviewed. Finally, the future perspectives of α-amino acid-containing degradable polymers will be evaluated.     

Poly(glycoamidoamine)s for gene delivery. structural effects on cellular internalization, buffering capacity, and gene expression

The study of polymeric nucleic acid delivery vehicles has recently grown because of their promise for many biomedical applications. In an effort to understand how the chemical traits of polymers affect the biological mechanisms of nucleic acid delivery, we have calculated the buffering capacity in the physiological pH range of a series of 10 poly(glycoamidoamine)s with systematic structural variations in the amine stoichiometry (from 1 to 4), carbohydrate moiety (d-glucarate or l-tartarate), and amine spacer (ethylene or butylene) within their repeat units. In addition, we have compared the buffering capacity of these polymeric vectors to their polyplex (polymer-DNA complex) stability, cellular internalization, and gene expression profiles to understand the parameters that are important for increasing gene delivery efficiency. The results indicate that the buffering capacity is not always the primary characteristic that determines the gene delivery efficiency for all the poly(glycoamidoamine)s. We have found that the buffering capacity may affect the gene delivery efficiency only when analogous structures containing the same number of amines but different carbohydrates are compared. We reveal that the cellular internalization is the key step in the gene delivery process with systems containing different amine stoichiometry. Also, increasing the number of methylene groups between the secondary amines increases toxicity to a large degree. This systematic and heuristic approach of studying the correlations between structural variables and gene delivery efficiency will facilitate the development of effective synthetic vectors for specific nucleic acid delivery applications.     

Radioprotectors – the Evergreen Topic

To protect organisms from ionizing radiation (IR), and to reduce morbidity or mortality, various agents, called radioprotectors, have been utilized. Because radiation‐induced cellular damage is attributed primarily to the harmful effects of free radicals, molecules with radical‐scavenging properties are particularly promising as radioprotectors. Early development of such agents focused on thiol synthetic compounds, known as WR protectors, but only amifostine (WR‐2721) has been used in clinical trials as an officially approved radioprotector. Besides thiol compounds, various compounds with different chemical structure were investigated, but an ideal radioprotector has not been found yet. Plants and natural products have been evaluated as promising sources of radioprotectors because of their low toxicity, although they exhibit an inferior protection level compared to synthetic thiol compounds. Active plant constituents seem to exert the radioprotection through antioxidant and free radical‐scavenging activities. Our research established that plants containing polyphenolic compounds (raspberry, blueberry, strawberry, grape, etc.) exhibit antioxidative activities and protect genetic material from IR.     

Medicinal chemistry of plasmid DNA with peptide nucleic acids: A new strategy for gene therapy

In this chapter, we describe an approach using a peptide nucleic acid (PNA) clamp to directly and irreversibly modify plasmid DNA, without affecting either its supercoiled conformation or its ability to be efficiently transcribed. This strategy enables investigators to `functionalize' their gene of interest by direct coupling of ligands (fluorophores, peptide, proteins, sugars or oligonucleotides) to plasmid DNA. This approach provides versatile tools to study the mechanisms of gene delivery and to circumvent some of the main obstacles of synthetic gene delivery systems, such as specific targeting and efficient delivery. The proof-of-principal of PNA-dependent gene chemistry (PDGC) was demonstrated with a fluorescently labeled PNA that allowed generation of a highly fluorescent preparation of plasmid DNA that was functionally and conformationally intact. Fluorescent-PNA/DNA was used to identify critical parameters involved in naked DNA and non-viral gene delivery technology. The greatest potential of PDGC lies in the ability to attach specific ligands (e.g., peptides, proteins) to the plasmid DNA in order to overcome cellular barriers of non-viral gene delivery systems. In this regard, specific examples of ligands coupled to DNA are described and their effect on increasing the efficacy of gene therapy is presented.     

Nitric oxide-induced S-glutathionylation and inactivation of glyceraldehyde-3-phosphate dehydrogenase

S-Nitrosylation of protein thiol groups by nitric oxide (NO) is a widely recognized protein modification. In this study we show that nitrosonium tetrafluoroborate (BF4NO), a NO+ donor, modified the thiol groups of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by S-nitrosylation and caused enzyme inhibition. The resultant protein-S-nitrosothiol was found to be unstable and to decompose spontaneously, thereby restoring enzyme activity. In contrast, the NO-releasing compound S-nitrosoglutathione (GSNO) promoted S-glutathionylation of a thiol group of GAPDH both in vitro and under cellular conditions. The GSH-mixed protein disulfide formed led to a permanent enzyme inhibition, but upon dithiothreitol addition a functional active GAPDH was recovered. This S-glutathionylation is specific for GSNO because GSH itself was unable to produce protein-mixed disulfides. During cellular nitrosative stress, the production of intracellular GSNO might channel signaling responses to form protein-mixed disulfide that can regulate intracellular function.     

Medicinal chemistry of plasmid DNA with peptide nucleic acids: A new strategy for gene therapy

Summary In this chapter, we describe an approach using a peptide nucleic acid (PNA) clamp to directly and irreversibly modify plasmid DNA, without affecting either its supercoiled conformation or its ability to be efficiently transcribed. This strategy enables investigators to 'functionalize' their gene of interest by direct coupling of ligands (fluorophores, peptide, proteins, sugars or oligonucleotides) to plasmid DNA. This approach provides versatile tools to study the mechanisms of gene delivery and to circumvent some of the main obstacles of synthetic gene delivery systems, such as specific targeting and efficient delivery. The proof-of-principal of PNA-dependent gene chemistry (PDGC) was demonstrated with a fluorescently labeled PNA that allowed generation of a highly fluorescent preparation of plasmid DNA that was functionally and conformationally intact. Fluorescent-PNA/DNA was used to identify critical parameters involved in naked DNA and non-viral gene delivery technology. The greatest potential of PDGC lies in the ability to attach specific ligands (e.g., peptides, proteins) to the plasmid DNA in order to overcome cellular barriers of non-viral gene delivery systems. In this regard, specific examples of ligands coupled to DNA are described and their effect on increasing the efficacy of gene therapy is presented.     

Mass spectrometry in studies of protein thiol chemistry and signaling: Opportunities and caveats

Mass spectrometry (MS) has become a powerful and widely utilized tool in the investigation of protein thiol chemistry, biochemistry, and biology. Very early biochemical studies of metabolic enzymes have brought to light the broad spectrum of reactivity profiles that distinguish cysteine thiols with functions in catalysis and protein stability from other cysteine residues in proteins. The development of MS methods for the analysis of proteins using electrospray ionization (ESI) or matrix-assisted laser desorption/ionization (MALDI) coupled with the emergence of high-resolution mass analyzers has been instrumental in advancing studies of thiol modifications, both in single proteins and within the cellular context. This article reviews MS instrumentation and methods of analysis employed in investigations of thiols and their reactivity toward a range of small biomolecules. A selected number of studies are detailed to highlight the advantages brought about by the MS technologies along with the caveats associated with these analyses.     

A biological transporter for the delivery of peptide nucleic acids (PNAs) to the nuclear compartment of living cells

To facilitate nuclear delivery of biomolecules we describe the synthesis of a modular transporter bearing a cellular membrane transport peptide (pAntp) and, as a cargo, a 16-mer peptide nucleic acid (PNA) covalently linked to a nuclear localisation signal (NLS[SV40-T]). Transport peptide and PNA are connected via N-terminal activated cysteine to form cleavable disulphide bonds. Internalization and subsequent delivery of PNA to the nucleus was verified in living and fixed cells by confocal laser scanning microscopy (CLSM) and fluorescence correlation spectroscopy (FCS). Double-labelling experiments indicate the cytoplasmic cleavage of the two modules and the effective nuclear import of the chromophore-tagged cargo. A non-degradable linker between transport module and cargo as well as a construct without NLS did not enable nuclear PNA import under the described experimental conditions. FCS-measurements revealed that most of the PNAs delivered into the cytoplasm by the modular transporter are anchored or encapsulated, indicating that intracellular transport of these compounds is not governed by molecular diffusion. Our results clearly demonstrate efficient compartment-directed transport using a synthetic, non-toxic modular transporter in living cells.