Vicinal Dithiol-containing Proteins Research Articles

Chemoselective Fluorogenic Bioconjugation of Vicinal Dithiol-Containing Proteins for Live Cellular Imaging via Small Molecular Conjugate Acceptors.

To develop small molecular fluorogenic tools for the chemoselective labeling of vicinal dithiol-containing proteins (VDPs) in live cells is important for studying intracellular redox homeostasis. With this research, we developed small molecule-based fluorescent probes, achieving selective labeling of VDPs through thiol-thiol substitutions on bisvinylogous thioester conjugated acceptors (IDAs). Initially, IDAs demonstrated its ability to bridge vicinal cysteine-sulfhydryls on a peptide as a mimic. Then, the peptide complex could be decoupled to recover the original peptide-SH in the presence of dithiothreitol. Furthermore, fluorometric signal amplification of the fluorescent probes occurred with high sensitivity, low limit of detection, and selectivity toward vicinal dithiols on reduced bovine serum albumin, as an example of real world VDPs. More importantly, the probes were utilized successfully for labeling of endogenous VDPs at different redox states in live cells. Thus, the bisvinylogous thioester-based receptor as a functional probe represents a new platform for uncovering the function of VDPs in live cells.

In vivo precision imaging of vicinal-dithiol-containing proteins by a FRET molecular probe sensitive to protein environment

We here present a Förster resonance energy transfer (FRET)-based and environment-sensitive fluorescent probe VG-1 for vicinal-dithiol-containing proteins (VDPs). VG-1 uniquely contains two sites sensitive to the protein environment (SPE), thus it shows weak fluorescence in both blue and green channels (a low FRET efficiency) in solution. After specifically binding with VDPs, its fluorescence in the green channel increases, while that in the blue channel disappears, achieving the specific detection of VDPs. The obvious signal changes in fluorescence may be attributed to that the increased rigidity of the molecular skeletons causes the enhanced FRET efficiency. The probe also achieved the cell super-resolution imaging of VDPs and the confocal imaging of VDPs in zebrafish.

Decrease of Protein Vicinal Dithiols in Parkinsonism Disclosed by a Monoarsenical Fluorescent Probe.

Vicinal dithiol-containing proteins (VDPs) play an important role in maintaining the structures and functions of proteins mainly through the conversion between dithiols and disulfide bonds. The content of VDPs also reflects the redox status of an organism. To specifically and expediently detect VDPs, we developed a turn-on monoarsenical fluorescent probe (NEP) based on the intramolecular charge transfer mechanism. Naphthalimide was chosen as a fluorophore and linked with the receptor moiety (cyclic dithiarsolane) via carbamate segment. In the presence of VDPs, NEP displays a strong green fluorescence signal produced by the cyclic dithiarsolane cleavage and subsequent intramolecular cyclization to liberate the fluorophore. Furthermore, NEP exhibits high selectivity toward VDPs over other protein thiols and low molecular weight thiols. The favorable properties of NEP enable it readily to detect VDPs in live cells and in vivo. In addition, a remarkable decrease of VDPs in parkinsonism was disclosed for the first time, highlighting that regulating VDPs level has a therapeutic potential for parkinsonism.

Dual-functional fluorescent molecular rotor for endoplasmic reticulum microviscosity imaging during reticulophagy.

The microviscosity change associated with reticulophagy is an important component for studying endoplasmic reticulum (ER) stress disorders. Here, a BODIPY-arsenicate conjugate 1-based fluorescent molecular rotor was designed to covalently bind vicinal dithiol-containing proteins in the ER, exhibiting a bifunction of reticulophagy initiation and microviscosity evaluation. Therefore, we could quantify the local viscosity changes during reticulophagy based on the fluorescence lifetime changes of probe 1.

Far-Red Fluorescent Probe for Imaging of Vicinal Dithiol-Containing Proteins in Living Cells Based on a pKa Shift Mechanism.

Vicinal dithiol-containing proteins (VDPs) play fundamental roles in intracellular redox homeostasis and are responsible for many diseases. In this work, we report a far-red fluorescence turn-on probe MCAs for VDPs exploiting the pKa shift of the imine functionality of the probe. MCAs is composed of a merocyanine Schiff base as the fluorescent reporter and a cyclic 1,3,2-dithiarsenolane as the specific ligand for VDPs. The imine pKa of MCAs is 4.8, and it exists predominantly in the Schiff base (SB) form at physiological pH. Due to the absence of a resonating positive charge, it absorbs at a relatively short wavelength and is essentially nonfluorescent. Upon selective binding to reduced bovine serum albumin (rBSA, selected as the model protein), MCAs was brought from aqueous media to the binding pockets of the protein, causing a large increase in pKa value of MCAs (pKa = 7.1). As a result, an increase in the protonated Schiff base (PSB) form of MCAs was observed at the physiological pH conditions, which in turn leads to a bathochromically shifted chromophore (λabs = 634 nm) and a significant increase in fluorescence intensity (λem = 657 nm) simultaneously. Furthermore, molecular dynamics simulations indicate that the salt bridges formed between the iminium in MCAs and the residues D72 and D517 in rBSA resist the dissociation of proton from the probe, thus inducing an increase of the pKa value. The proposed probe shows excellent sensitivity and specificity toward VDPs over other proteins and biologically relevant species and has been successfully applied for imaging of VDPs in living cells. We believe that the present pKa shift switching strategy may facilitate the development of new fluorescent probes that are useful for a wide range of applications.

Mitochondrion-Targeting, Environment-Sensitive Red Fluorescent Probe for Highly Sensitive Detection and Imaging of Vicinal Dithiol-Containing Proteins.

Mitochondrial vicinal dithiol-containing proteins (VDPs) are key regulators in cellular redox homeostasis and useful markers for diagnostics of redox-dependent diseases. Current probes fail to target mitochondrial VDPs and show limited sensitivity and response rate. We develop a novel fluorescent probe using an engineered benzoxadiazole fluorophore that allows selective targeting of mitochondria and exhibits highly sensitive environment responsiveness. This probe is almost nonfluorescent in aqueous media, while delivering intense fluorescence upon binding to VDPs via a cyclic dithiaarsane ligand. The fluorescence probe is shown to have rapid response within 30 s and high sensitivity for detecting reduced bovine serum albumin (rBSA) in the concentration range from 0 to 0.1 μM with a detection limit of 2 nM. To our knowledge, this is the first fluorescence probe for VDPs which exhibits deep red emission, instantaneous response, high turn-on fluorescence ratio, and specific mitochondrial localization. It may provide a new tool for in situ monitoring mitochondrial VDPs.

Ratiometric Fluorescent Probe for Vicinal Dithiol-Containing Proteins in Living Cells Designed via Modulating the Intramolecular Charge Transfer-Twisted Intramolecular Charge Transfer Conversion Process.

Vicinal dithiol-containing proteins (VDPs) play a significant role in maintaining the cellular redox homeostasis and are implicated in many diseases. To provide new chemical tools for VDPs imaging, we report here a ratiometric fluorescent probe CAsH2 for VDPs using 7-diethylaminiocoumarin as the fluorescent reporter and cyclic 1,3,2-dithiarsenolane as the specific ligand. CAsH2 shows peculiar dual fluorescence emission from the excited intramolecular charge transfer (ICT) and twisted intramolecular charge transfer (TICT) states in aqueous media. However, upon selective binding of protein vicinal dithiols to the trivalent arsenical of CAsH2, the probe was brought from the polar water media into the hydrophobic protein domain, causing the excited state ICT to TICT conversion to be restricted; as a result, an increase from the ICT emission band and a decrease from the TICT emission band were observed simultaneously. The designed probe shows high selectivity toward VDPs over other proteins and biological thiols. Preliminary experiments show that CAsH2 can be used for the ratiometric imaging of endogenous VDPs in living cells. So far as we know, this is a rare example of the ratiometric fluorescent probe designed via modulating the ICT-TICT conversion process, which provides a new way to construct various protein-specific ratiometric fluorescent probes.

Development of a red fluorescent light-up probe for highly selective and sensitive detection of vicinal dithiol-containing proteins in living cells.

Vicinal dithiol-containing proteins (VDPs) play a key role in cellular redox homeostasis and are responsible for many diseases. Here, we develop a red fluorescent light-up probe FAsH for the highly selective and sensitive detection of VDPs using the environment-sensitive 2-(4-dimethylaminophenyl)-4-(2-carboxyphenyl)-7-diethylamino-1-benzopyrylium (F1) as the fluorescent reporter and cyclic dithiaarsane as the targeting unit. FAsH is almost nonfluorescent in aqueous solution. However, it exhibits intense fluorescence emission upon binding to reduced bovine serum albumin (rBSA, selected as the model protein). The fluorescence intensity of FAsH is directly proportional to the concentration of rBSA over the range of 0.06-0.9 μM, with a detection limit (3δ) of 0.015 μM. Importantly, the fast kinetics of binding between FAsH and VDPs (∼2.5 min) enables the dynamic tracing of VDPs in biological systems. Preliminary experiments show that FAsH can be used for the no-wash imaging of endogenous VDPs in living cells. In addition, our study shows that F1 presents both high environment-sensitivity and good fluorescence properties, and is promising for the development of no-wash fluorescent light-up probes for target-specific proteins in living cells.

Sensitive detection of tumor cells by a new cytosensor with 3D-MWCNTs array based on vicinal-dithiol-containing proteins (VDPs)

Vicinal-dithiol-containing Proteins (VDPs) are overexpressed in cancer cells and become a potential biomarker for aggressive tumors, the synthesized 2-p-aminophenyl-1, 3, 2-dithiarsenolane (VTA2) is proved to be a highly selective ligand for vicinal dithiols of VDPs in cells. In this report, we developed a new cytosensor based on three-dimensional (3D)-like VTA2-conjugated multiwalled carbon nanotubes (VTA2@MWCNTs) array modified indium tin oxide (ITO) electrode for sensitive and selective detection of VDPs-overexpression tumor cells. The layer-by-layer assembling and cellular detection performances of the 3D-VTA2@MWCNTs-based cytosensor were investigated by atomic force microscopy (AFM) and electrochemical methods including cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV). Attributed to signal amplification and targeting recognition of the 3D-structured MWCNTs@VTA2, the fabricated cytosensor showed high specificity and sensitivity to the detection of VDPs-overexpression HL-60 cells ranging from 2.7×102 to 2.7×107cellsmL−1 with a low detection limit of ca. 90cellsmL−1. Furthermore, the captured cancer cells on the cytosensor also could be directly visualized by optical microscopy technology. This proposed 3D-MWCNTs array-based cytosensing strategy provides a simple, sensitive approach for non-invasive cell detection, presenting potential applications in cancer diagnosis.

Selective and ratiometric fluorescent trapping and quantification of protein vicinal dithiols and in situ dynamic tracing in living cells.

Protein vicinal dithiols play fundamental roles in intracellular redox homeostasis due to their involvement in protein synthesis and function through the reversible vicinal dithiol oxidation to disulfide. To provide quantitative information about the global distribution and dynamic changes of protein vicinal dithiols in living cells, we have designed and synthesized a ratiometric fluorescent probe (VTAF) for trapping of vicinal dithiol-containing proteins (VDPs) in living cells. VTAF exhibits a ratiometric fluorescence signal upon single excitation, which enables self-calibration of the fluorescence signal and quantification of endogenous vicinal dithiols of VDPs. Its potential for in situ dynamic tracing of changes of protein vicinal dithiols under different cellular redox conditions was exemplified. VTAF facilitated the direct observation of subcellular distribution of endogenous VDPs via ratiometric fluorescence imaging and colocalization assay. And the results suggested that there are abundant VDPs in mitochondria. Moreover, some redox-sensitive VDPs are also present on cell surface which can respond to redox stimulus. This ratiometric fluorescence technique presents an important extension to previous fluorescence intensity-based probes for trapping and quantifying protein vicinal dithiols in living cells, as well as its visible dynamic tracing of VDPs.

Versatile Probes for the Selective Detection of Vicinal‐Dithiol‐Containing Proteins: Design, Synthesis, and Application in Living Cells

Endogenous vicinal-dithiol-containing proteins (VDPs) that have two thiol groups close to each other in space play a significant importance in maintaining the cellular redox microenvironment. Approaches to identify VDPs mainly rely on monitoring the different concentration of monothiol and total thiol groups or on indirect labeling of vicinal thiols by using p-aminophenylarsenoxide (PAO). Our previous work has reported the direct labeling of VDPs with a highly selective receptor PAO analogue, which could realize fluorescence detection of VDPs directly in living cells. Herein, we developed a conjugated approach to expand detectable tags to nitrobenzoxadiazole (NBD), fluorescein, naphthalimide, and biotin for the synthesis of a series of probes. Different linkers have also been introduced toward conjugation of VTA2 with these functional tags. These synthesized flexible probes with various features will offer new tools for the potential identification and visualization of vicinal dithiols existing in different regions of VDPs in living cells. These probes are convenient tools for proteomics studies of various disease-related VDPs and for the discovery of new drug targets.

26] Phenylarsine oxide affinity chromatography to identify proteins involved in redox regulation: Dithiol-disulfide equilibrium in serine/threonine phosphatase calcineurin

Publisher Summary This chapter discusses affinity chromatography using an immobilized phenylarsine oxide derivative coupled to Sepharose for the purification of vicinal dithiolcontaining proteins. This methodology uses a phenylarsine oxide (PAO) resin to selectively bind, elute, and identify proteins that might be involved in redox regulation and to test a possible disulfide formation as a regulatory mechanism in oxidative stress. Because such conditions are found in activated polymorphonuclear leukocytes (PMN), a cytosolic fraction from PMN is used to isolate potential target proteins. This way the isolation and identification of six proteins with affinity for PAO can be achieved: thioredoxin (Trx), calcineurin (CAN), glutathione S-transferase (GST), calgranulin, L-plastin, and cofilin. From these candidates, a more detailed characterization of the interaction of PAO and oxidants with the serine-threonine phosphatase CaN (also known as protein phosphatase 2B) can be explored, which strongly suggests a dithiol-disulfide equilibrium in the enzyme as a mechanism for redox regulation.

Translation of chloroplast psbA mRNA is regulated by signals initiated by both photosystems II and I.

Light controls the translation of several mRNAs in fully developed chloroplasts via at least two regulatory pathways. In the first, the light signal is transduced as a thiol-mediated signal that modulates translation in parallel to light intensity. The second light-controlled pathway, termed priming, is a prerequisite to the thiol-mediated regulatory pathway. Light regulation is rapid and requires intrachloroplast photoreceptor(s). To delineate the signaling pathways controlling each of these regulatory events, we assayed the effect of photosynthetic inhibitors and electron donors on the translation of chloroplastic psbA mRNA. We show that the thiol-mediated signal is generated by photosystem I and transduced by vicinal dithiol-containing proteins. We also found that the priming signal probably initiates on reduction of plastoquinone. These findings suggest that translation of chloroplast psbA mRNA is controlled by both linear photosynthetic electron transport, exerted by the reduction of the ferredoxin-thioredoxin system, and the relative activities of photosystems I and II, signaled by the redox state of the plastoquinone pool. These data underscore the function of the light-capturing reactions of photosynthesis as chloroplast photoreceptors.

Mechanism of dehydroascorbic acid reduction by glutaredoxin (thioltransferase): [formula omitted] Rushad Daruwala, Mark Levine. NIDDK, NIH, Bethesda, MD 20892

This chapter discusses the conditions that permit the use of p-nitrophenyl disulfide (PNPD) to measure the disulfide reductase capacity of cells. This reductive capacity reflects the reaction of PNPD with the cellular thiols and measures the potential of the cell to regenerate glutathione (GSH) by means of glutathione reductase (GSSG-R). There are two main reactions involved in the capacity of the cell to reduce PNPD. The first reaction is the direct reduction by NADPH-thioredoxin reductase and GSH-dependent thioltransferase or glutaredoxin. The second reaction is the indirect reduction involving (1) the PNPD conversion of GSH to GSSG followed by the NADPH-GSSG-reductase-catalyzed regeneration of GSH and (2) the conversion of monothiol proteins to mixed disulfides and of vicinal dithiol-containing proteins to disulfides. The addition of PNPD to cells results in the inhibition of phosphotyrosine phosphatases by the formation of both mixed disulfide- and disulfide-containing proteins. The activity of these enzymes is regenerated by treatment with dithiothreitol (DTT). This result suggests that PNPD can be used to inhibit phosphotyrosine phosphatases transiently and to study the enzymes involved in the cellular reduction of mixed disulfide- and disulfide-containing proteins to regenerate the activity of these important regulatory proteins.

44] Labeling of protein vicinal dithiols: Role of protein-S2 to protein-(SH)2 conversion in metabolic regulation and oxidative stress

Publisher Summary Native proteins with cysteine-sulfhydryls in proximity that can undergo reversible dithiol/disulfide conversions are here referred to as vicinal dithiol-containing proteins (VDPs). The dithiols of interest are present at the protein surface, allowing interaction with substrates, with oxidants, and with proteins catalyzing disulfide reduction. VDPs include enzymes whose active sites contain redox-active dithiols, such as nucleotide and oxidized glutathione (GSSG) reductases, thioredoxin reductase-thioredoxin, and glutaredoxin. Additionally, proteins that bind to DNA including transcription regulatory proteins with zinc fingers and most polymerases are VDPs. Also included are a series of enzymes in which the dithiol/disulfide conversion serves as an allosteric regulator of the enzymatic activity. Hormone or steroid receptors, such as the triiodothyronine nuclear receptor and the cytoplasmic glucocorticoid receptor have a dithiol that regulates hormone or steroid binding to the receptor and subsequent binding of the receptor to DNA. Another distinctive property of VDPs is their high affinity for trivalent arsenicals. This interaction is stable in the absence of other dithiols. On addition of small dithiols, such as 1,2-dithioglycerol (BAL), dithiothreitol, or lipoic acid, however, the binding of the arsenical to the dithiol protein can be reversed.

Arsenical-Based Affinity Chromatography of Vicinal Dithiol-Containing Proteins: Purification of L1210 Leukemia Cytoplasmic Proteins and the Recombinant Rat c-erb Aβ 1 T 3 Receptor

Proteins containing vicinal dithiols were purified by affinity chromatography using Sepharose 4B linked to aminohexanoyl-4-aminophenylarsineoxide (As-Sepharose). The protein vicinal dithiols form stable dithioarsine derivatives with the arsine oxide moieties of the gel. The adsorbed proteins were eluted, at physiological pH, by buffers containing β-mercaptoethanol or dithiothreitol. The dithiol proteins were identified by their specific labeling with N-iodoacetyl-3-[ 125I]-iodotyrosine. Cytoplasmic thiol proteins of L1210 murine leukemia lymphoblasts were separated into three classes by interaction with As-Sepharose. Proteins that did not bind to the gel consisted of monothiol proteins; proteins eluted by β-mercaptoethanol include vicinal dithiol-containing proteins with low affinity for the arsine oxide. DL-dithiothreitol (DTT) elutes a large group of vicinal dithiol-containing proteins with high affinity for the arsine groups. Gradient elution allowed characterization of the relative affinities of dithiol proteins for the As-Sepharose. A one-step purification of the L-triiodothyronine recombinant rat c-erb Aβ 1 T 3 receptor synthesized in yeast required pretreatment with DTT for binding to As-Sepharose and resulted in a 62-fold increase in specific activity. The procedure allows purification of proteins inhibited by phenylarsine oxide such as phosphotyrosine phosphatases, proteins that are subject to redox regulation, and dithiol proteins that are targets of oxidative stress.

Uptake and binding of radiolabelled phenylarsine oxide in 3T3-L1 adipocytes

Phenylarsine oxide (PAO), a trivalent arsenical, has been shown to inhibit insulin-stimulated glucose transport in 3T3-L1 adipocytes, implicating vicinal dithiols in signal transmission [Frost & Lane (1985) J. Biol. Chem. 260, 2646-2652]. To assist in the direct identification of a PAO-binding protein which might be involved in this process, we have synthesized [3H]acetylaminophenylarsine oxide [( 3H]APAO) from the amino derivative of phenylarsine oxide (NPAO). To assess the inhibitory effect of the product, a dual-labelling experiment was performed which showed that [3H]APAO inhibited insulin-stimulated 2-deoxy[1-14C]glucose transport in 3T3-L1 adipocytes with a Ki of 21 microM, identical with that of the parent compound, NPAO. Further characterization revealed that over a wide concentration range, uptake of the labelled arsine oxide was linear. Although the dithiol reagent 2,3-dimercaptopropanol (DMP) reversed PAO-induced inhibition of transport, it had no effect on the uptake of [3H]APAO. In a simple fractionation experiment approx. 50% of the radioactivity was associated with the cytosolic fraction and 50% with the total membrane fraction. Identification of radiolabelled proteins by non-reducing SDS/PAGE revealed fraction-specific binding, although many proteins were observed. Covalent modification was time-dependent and could be reversed by addition of DMP. These data further support a role for vicinal dithiols in insulin-stimulated glucose transport. Additionally, the probe described may offer a new means with which to identify the inhibitory protein or, more globally, to investigate mechanisms of action of vicinal dithiol-containing proteins.