Simple Fluorescence Assay for Cystine Uptake via the xCT in Cells Using Selenocystine and a Fluorescent Probe.

The cystine/glutamate antiporter (xCT) is a crucial transporter that maintains cellular redox balance by regulating intracellular glutathione synthesis via cystine uptake. However, no robust and simple method to determine the cystine uptake activity of xCT is currently available. We have developed a method to measure the xCT activity via the reaction of selenocysteine and fluorescein O,O'-diacrylate (FOdA). Selenocystine, a cystine analogue, is transported into cells through xCT on the cell membrane. The amount of the transported selenocystine was then determined by a reaction using tris(2-carboxyethyl)phosphine (TCEP) and FOdA in a weak acidic buffer at pH 6. Using this method, the cystine uptake activity of xCT in various cells and the inhibitory efficiency of xCT inhibitors, were evaluated.

ß-Galactosidase-Catalyzed Fluorescent Reporter Labeling of Living Cells for Sensitive Detection of Cell Surface Antigens.

The ability to detect cell surface proteins using fluorescent-dye-labeled antibodies is crucial for the reliable identification of many cell types. However, the different types of cell surface proteins used to identify cells are currently limited in number because they need to be expressed at high levels to exceed background cellular autofluorescence, especially in the shorter-wavelength region. Herein we report on a new method, quinone methide-based catalyzed labeling for signal amplification (CLAMP), in which the fluorescence signal is amplified by an enzymatic reaction that strongly facilitates the detection of cell surface proteins on living cells. We used ß-galactosidase as an amplification enzyme and designed a substrate for it, called MUGF, that contains a fluoromethyl group. Upon removal of the galactosyl group in MUGF by ß-galactosidase labeling of the target cell surface proteins, the resulting product containing the quinone methide group was found to be both cell-membrane-permeable and reactive with intracellular nucleophiles, thereby providing fluorescent adducts. Using this method, we successfully detected several cell surface proteins, including programmed death ligand 1 protein, which is difficult to detect using conventional fluorescent-dye-labeled antibodies.

Protein Redox State Monitoring Studies of Thiol Reactivity.

Protein cysteine thiol status is a major determinant of oxidative stress and oxidant signaling. The -SulfoBiotics- Protein Redox State Monitoring Kit provides a unique opportunity to investigate protein thiol states. This system adds a 15-kDa Protein-SHifter to reduced cysteine residues, and this molecular mass shift can be detected by gel electrophoresis. Even in biological samples, Protein-SHifter Plus allows the thiol states of specific proteins to be studied using Western blotting. Peroxiredoxin 6 (Prx6) is a unique one-cysteine peroxiredoxin that scavenges peroxides by utilizing conserved Cysteine-47. Human Prx6 also contains an additional non-conserved cysteine residue, while rat Prx6 only has the catalytic cysteine. In cultured cells, cysteine residues of Prx6 were found to be predominantly fully reduced. The treatment of human cells with hydrogen peroxide (H2O2) formed Prx6 with one cysteine reduced. Since catalytic cysteine becomes oxidized in rat cells by the same H2O2 treatment and treating denatured human Prx6 with H2O2 results in the oxidation of both cysteines, non-conserved cysteine may not be accessible to H2O2 in human cells. We also found that untreated cells contained Prx6 multimers bound through disulfide bonds. Surprisingly, treating cells with H2O2 eliminated these Prx6 multimers. In contrast, treating cell lysates with H2O2 promoted the formation of Prx6 multimers. Similarly, treating purified preparations of the recombinant cyclic nucleotide-binding domain of the human hyperpolarization-activated cyclic nucleotide-modulated channels with H2O2 promoted the formation of multimers. These studies revealed that the cellular environment defines the susceptibility of protein cysteines to H2O2 and determines whether H2O2 acts as a facilitator or a disrupter of disulfide bonds.

Reactive sulfur species regulate tRNA methylthiolation and contribute to insulin secretion.

The 2-methylthio (ms2) modification at A37 of tRNAs is critical for accurate decoding, and contributes to metabolic homeostasis in mammals. However, the regulatory mechanism of ms2 modification remains largely unknown. Here, we report that cysteine hydropersulfide (CysSSH), a newly identified reactive sulfur species, is involved in ms2 modification in cells. The suppression of intracellular CysSSH production rapidly reduced ms2 modification, which was rescued by the application of an exogenous CysSSH donor. Using a unique and stable isotope-labeled CysSSH donor, we show that CysSSH was capable of specifically transferring its reactive sulfur atom to the cysteine residues of ms2-modifying enzymes as well as ms2 modification. Furthermore, the suppression of CysSSH production impaired insulin secretion and caused glucose intolerance in both a pancreatic ß-cell line and mouse model. These results demonstrate that intracellular CysSSH is a novel sulfur source for ms2 modification, and that it contributes to insulin secretion.

Direct determination of the redox status of cysteine residues in proteins in vivo.

The redox states of proteins in cells are key factors in many cellular processes. To determine the redox status of cysteinyl thiol groups in proteins in vivo, we developed a new maleimide reagent, a photocleavable maleimide-conjugated single stranded DNA (DNA-PCMal). The DNA moiety of DNA-PCMal is easily removed by UV-irradiation, allowing DNA-PCMal to be used in Western blotting applications. Thereby the state of thiol groups in intracellular proteins can be directly evaluated. This new maleimide compound can provide information concerning redox proteins in vivo, which is important for our understanding of redox networks in the cell.

Short PEG-linkers improve the performance of targeted, activatable monoclonal antibody-indocyanine green optical imaging probes.

The ability to switch optical imaging probes from the quenched (off) to the active state (on) has greatly improved target to background ratios. The optimal activation efficiency of an optical probe depends on complete quenching before activation and complete dequenching after activation. For instance, monoclonal antibody-indocyanine green (mAb-ICG) conjugates, which are promising agents for clinical translation, are normally quenched, but can be activated when bound to a cell surface receptor and internalized. However, the small fraction of commonly used ICG derivative (ICG-Sulfo-OSu) can bind noncovalently to its mAb and is, thus, gradually released from the mAb leading to relatively high background signal especially in the liver and the abdomen. In this study, we re-engineered a mAb-ICG conjugate, (Panitumumab-ICG) using bifunctional ICG derivatives (ICG-PEG4-Sulfo-OSu and ICG-PEG8-Sulfo-OSu) with short polyethylene glycol (PEG) linkers. Higher covalent binding (70-86%) was observed using the bifunctional ICG with short PEG linkers resulting in less in vivo noncovalent dissociation. Panitumumab-ICG conjugates with short PEG linkers were able to detect human epidermal growth factor receptor 1 (EGFR)-positive tumors with high tumor-to-background ratios (15.8 and 6.9 for EGFR positive tumor-to-negative tumor and tumor-to-liver ratios, respectively, at 3 d postinjection).