Chemical probes for profiling fatty acid-associated proteins in living cells

Chemical probes appended with reactive electrophiles afford powerful tools for profiling discrete protein families in living cells. Herein, we have synthesized cell-permeable chemical probes that target fatty acid-associated proteins. These fatty acid-based chemical probes contain acyloxymethylketone or fluorophosphonate functional groups and an alkyne click chemistry tag for visualization of covalently modified proteins by in-gel fluorescence scanning. Our fatty acid-based chemical probe affords new tools to evaluate the activity/expression of lipid-associated proteins that should facilitate their functional characterization and inhibitor discovery.     

Chemical and genetic probes for analysis of protein palmitoylation

Reversible protein palmitoylation is one of the most important posttranslational modifications that has been implicated in the regulation of protein signaling, trafficking, localizing and enzymatic activities in cells and tissues. In order to achieve a precise understanding of mechanisms and functions of protein palmitoylation as well as its roles in physiological processes and disease progression, it is necessary to develop techniques that can qualitatively and quantitatively monitor the dynamic protein palmitoylation in vivo and in vitro. This review will highlight recent advances in both chemical and genetic encoded probes that have been developed for accurate analysis of protein palmitoylation, including identification and quantification of acyl moieties and palmitoylated proteins, localization of amino acid residues on which acyl moieties are attached, and imaging of cellular distributions of palmitoylated proteins. The role of major techniques of fluorescence microscopy and mass spectrometry in facilitating the analysis of protein palmitoylation will also be explored.     

Chemical probes for histone-modifying enzymes

The histone-modifying enzymes that catalyze reversible lysine acetylation and methylation are central to the epigenetic regulation of chromatin remodeling. From the early discovery of histone deacetylase inhibitors to the more recent identification of histone demethylase blockers, chemical approaches offer increasingly sophisticated tools for the investigation of the structure and function of these lysine-modifying enzymes. This review summarizes progress to date on compounds identified from screens or by design that can modulate the activity of classical histone deacetylases, sirtuins, histone acetyltransferases, histone methyltransferases and histone demethylases. We highlight applications of compounds to mechanistic and functional studies involving these enzymes and discuss future challenges regarding target specificity and general utility.     

Cytokinin inhibits the proteasome-mediated degradation of carbonylated proteins in Arabidopsis leaves

Under normal conditions, plants contain numerous carbonylated proteins, which are thought to be indicative of oxidative stress damage. Conditions that promote formation of reactive oxygen species (ROS) enhance protein carbonylation, and protein degradation is required to reverse the damage. However, it is not clear how the degradation of carbonylated proteins is controlled in planta. In this report, we show that detached Arabidopsis leaves rapidly and selectively degrade carbonylated proteins when kept in the dark. The loss of carbonylated proteins corresponded to a loss of soluble protein and accumulation of free amino acids. Degradation of carbonylated proteins and the loss of soluble protein was blocked by MG132 but not 3-methyladenine, suggesting that the 26S proteasome pathway rather than the autophagic pathway was involved. Consistent with this, rpn10 and rpn12 mutants, which are defective in proteasome function, had increased (rather than decreased) levels of carbonylated proteins when detached in the dark. Feeding metabolites (amino acids and sucrose) to detached leaves of wild-type Arabidopsis in the dark had little or no effect on the loss of carbonylated proteins, whereas providing soybean xylem sap via the transpiration stream effectively prevented degradation. The effect of xylem sap was mimicked by feeding 10 muM kinetin. We postulate that disruption of cytokinin flux to detached leaves triggers the selective degradation of carbonylated proteins via the proteasome pathway. The results may have implications for the control of protein mobilization in response to changes in N availability.     

Studies of phosphorescent probes for proteins

1. Certain capabilities and limitations of using bound phosphorescent chromophores to study protein structure were investigated. Carbonic anhydrase inhibitors with three different arrangements of singlet and triplet energy levels relative to those of tryptophan were used to determine their ability to transfer triplet energy. 2. Ligands representing each of the three spectroscopic energy level arrangements were found to exhibit triplet-triplet energy transfer with a tryptophan residue at the active site of carbonic anhydrase. This greatly increases the number of ligands which may be useful as phosphorescent probes. 3. The efficiency of energy transfer occurs to varying degrees depending upon the inhibitor. This is a potential source of data for determining the position of the ligand in the binding site.     

Chemical probes for tagging mycobacterial lipids

Mycobacteria, which cause tuberculosis and related diseases, possess a diverse set of complex envelope lipids that provide remarkable tolerance to antibiotics and are major virulence factors that drive pathogenesis. Recently, metabolic labeling and bio-orthogonal chemistry have been harnessed to develop chemical probes for tagging specific lipids in live mycobacteria, enabling a range of new basic and translational research avenues. A toolbox of probes has been developed for labeling mycolic acids and their derivatives, including trehalose-, arabinogalactan-, and protein-linked mycolates, as well as newer probes for labeling phthiocerol dimycocerosates (PDIMs) and potentially other envelope lipids. These lipid-centric tools have yielded fresh insights into mycobacterial growth and host interactions, provided new avenues for drug target discovery and characterization, and inspired innovative diagnostic and therapeutic strategies.     

Anti-nitroxide immunoglobulin G: analysis of antibody specificity and their application as probes for spin-labeled proteins

Antibodies have been elicited to the nitroxide spin-label 4-maleimido-2,2,6,6-tetramethyl-piperidinyl-1-oxy conjugated, via protein sulfhydryl groups, to bovine serum albumin. Antibody-hapten cross-reactivity was demonstrated by double immunodiffusion and by a broadening of the nitroxide electron paramagnetic resonance spectrum. The specificity of the antibodies with respect to hapten structure was examined by means of a simple filter binding assay. Under these conditions, antibodies were shown to distinguish between the nitroxide and hydroxylamine derivatives and between spin-labels comprising either five- or six-membered ring structures. In addition, protein-bound nitroxide spin-labels were detected at the nanogram level by immunoblotting. By use of this method, the specificity of the antibody-hapten reaction predicted by the filter binding assay procedure was utilized to differentially detect various types of bound spin-label. Finally, antibodies were used to identify protein-bound nitroxide spin-label of protein fractionated by gel electrophoresis.     

Proteomic profile of carbonylated proteins in rat liver: Discovering possible mechanisms for tetracycline‐induced steatosis

To investigate biochemical mechanisms for the tetracycline‐induced steatosis in rats, targeted proteins of oxidative modification were profiled. The results showed that tetracycline induced lipid accumulation, oxidative stress, and cell viability decline in HepG2 cells only under the circumstances of palmitic acid overload. Tetracycline administration in rats led to significant decrement in blood lipids, while resulted in more than four times increment in intrahepatic triacylglycerol and typical microvesicular steatosis in the livers. The triacylglycerol levels were positively correlated with oxidative stress. Proteomic profiles of carbonylated proteins revealed 26 targeted proteins susceptible to oxidative modification and most of them located in mitochondria. Among them, the long‐chain specific acyl‐CoA dehydrogenase was one of the key enzymes regulating fatty acid β‐oxidation. Oxidative modification of the enzyme in the tetracycline group depressed its enzymatic activity. In conclusion, the increased influx of lipid into the livers is the first hit of tetracycline‐induced microvesicular steatosis. Oxidative stress is an essential part of the second hit, which may arise from the lipid overload and attack a series of functional proteins, aggravating the development of steatosis. The 26 targeted proteins revealed here provide a potential direct link between oxidative stress and tetracycline‐induced steatosis.     

Porphyrinmaleimides: towards thiol probes for cysteine residues in proteins

Porphyrinmaleimides were synthesized and characterized. The thiol-containing amino acid L-cysteine reacted with 58% yield with these porphyrins to form bioconjugate adducts. The new thiol-active reagents were labeled cytoplasmic cysteine 140 and 316 in rhodopsin (Rh), a G protein coupled receptor (GPCR).     

Proteomic identification of mitochondrial carbonylated proteins in two maturation stages of pepper fruits

Pepper fruits in green and red maturation stages were selected to study the protein pattern modified by oxidation measuring carbonylated proteins in isolated mitochondria, together with the accumulation of superoxide radical and hydrogen peroxide in the fruits. MALDI‐TOF/TOF analysis identified as carbonylated proteins in both green and red fruits, formate dehydrogenase, NAD‐dependent isocitrate dehydrogenase, porin, and defensin, pointing to a common regulation by carbonylation of these proteins independently of the maturation stage. However, other proteins such as glycine dehydrogenase P subunit and phosphate transporter were identified as targets of carbonylation only in green fruits, whereas aconitase, ATPase β subunit, prohibitin, orfB protein, and cytochrome C oxidase, were identified only in red fruits. In general, the results suggest that carbonylation of mitochondrial proteins is a PTM that drives the complex ripening process, probably establishing the accumulation and functionality of some mitochondrial proteins in the nonclimacteric pepper fruit.     

Biotinylated indoles as probes for indole-binding proteins

Biotinylated indoles were prepared for application as bifunctional probes for the detection of indole-binding proteins which participate in the life processes of humans, animals, plants, and bacteria. The indole nucleus was functionalized, at ring positions 3, 5, or 6, by attachment of a 2-aminoethyl group, which was then coupled to the carboxyl moiety of biotin, via a spacer composed of 3 or 4 concatenated beta-alanine residues. The constructs thus obtained were able to inhibit tryptophanase activity, similarly to indole in a concentration-dependent manner. They also bound strongly to lysozyme and weakly to bovine and human serum albumins, in accordance with the known affinities of these proteins for indole and 3-(2-aminoethyl)indole (tryptamine). The biotin end of the protein-bound bifunctional probes could then be detected by coupling to (strept)avidin conjugated to alkaline phosphatase or horseradish peroxidase, followed by incubation with substrates which are converted by these enzymes to intensely colored or chemiluminescent products.     

Chemical probes and tandem mass spectrometry: a strategy for the quantitative analysis of proteomes and subproteomes

Quantitative proteome profiling using mass spectrometry and stable isotope dilution is being widely applied for the functional analysis of biological systems and for the detection of clinical, diagnostic or prognostic marker proteins. Because of the enormous complexity of proteomes, their comprehensive analysis is unlikely to be routinely achieved in the near future. However, in recent years, significant progress has been achieved focusing quantitative proteomic analyses on specific protein classes or subproteomes that are rich in biologically or clinically important information. Such projects typically combine the use of chemical probes that are specific for a targeted group of proteins and may contain stable isotope signatures for accurate quantification with automated tandem mass spectrometry and bioinformatics tools for data analysis. In this review, we summarize technical and conceptual advances in quantitative subproteome profiling based on tandem mass spectrometry and chemical probes.     

New fluorescent probes for ligand-binding assays of odorant-binding proteins

Fluorescence-linked binding assays allow determination of dissociation constants at equilibrium and have recently become increasingly popular, thanks to their ease of operation. Currently used probes, such as 1-aminoanthracene and N-phenyl-1-naphthylamine, are excited and emit in the ultraviolet region, but alternative ligands operating in the visible spectrum would be highly desirable for applications in biosensing devices. Based on the two above structures, we have designed and synthesised six new fluorescent probes to be used in ligand-binding assays. The compounds are derivatives of naphatalene, anthracene and fluoranthene and present two aromatic moieties linked by an amine nitrogen. We have measured the emission spectra of the new probes and their binding to three odorant-binding proteins. The probes bind the tested proteins with different affinities, generally with dissociation constants about one order of magnitude lower than the parent compounds. The extended aromatic systems present in the new compounds produced a shift of both excitation and emission peaks at higher wavelength, close or within the visible spectrum, thus facilitating measurements in biosensors for odorants and small organic molecules using optical devices.     

Chemical modification probes accessibility to organic phase: proteins on surfaces are more exposed than in lyophilized powders

Chemical modification of myoglobin and cutinase suspended in n-hexane by acyl chlorides and iodine was monitored by electrospray mass spectrometry. The general rate of modification was always much faster for protein adsorbed to supports (silica or polypropylene) than for lyophilized powders. Modification rates were slower for larger acyl chlorides, particularly with lyophilized powders. About 20% of the protein molecules in lyophilized powders were modified much more quickly than the rest, a fraction consistent with those exposed on the surface of the solid. It appears that access to most of the molecules in lyophilized powders requires a very slow stage of solid-phase diffusion. This has been neglected in previous discussion of mass transfer limitation of lyophilized enzymes in organic media, and would not be revealed by the experimental evidence used to dismiss it. Studies of the effects of particle size and dilution with inactive protein are only sensitive to diffusion in liquid-filled pores, not through the solid phase. Slow solid-phase diffusion is not required for access to most support-adsorbed proteins, which is probably a major contributory factor to their enhanced catalytic efficiency in organic media. Hydration of lyophilized proteins accelerates chemical modification rates, as it does their catalytic activity. The main site of reaction of acyl chlorides in organic media is not amino groups (which are probably ion-paired), but is likely to be hydroxyl groups instead.     

Advances in the study of luminescence probes for proteins

Spectral probes (or labels) have been widely used for the investigation and determination of proteins and have made considerable progress. Traditional luminescence probes include fluorescent derivatizing reagents, fluorescent probes and chemiluminescence probes which continue to develop. Of them, near infrared (NIR) fluorescent probes are especially suitable for the determination of biomolecules including proteins, so their development has been rapid. Novel luminescence probes (such as nanoparticle probes and molecular beacons) and resonance light scattering probes recently appeared in the literature. Preliminary results indicate that they possess great potential for ultrasensitive protein detection. This review summarizes recent developments of the above-mentioned probes for proteins and 195 references are cited.