Redox-sensitive sulfenic acid modification regulates surface expression of the cardiovascular voltage-gated potassium channel Kv1.5.

RATIONALE: Kv1.5 (KCNA5) is expressed in the heart, where it underlies the I(Kur) current that controls atrial repolarization, and in the pulmonary vasculature, where it regulates vessel contractility in response to changes in oxygen tension. Atrial fibrillation and hypoxic pulmonary hypertension are characterized by downregulation of Kv1.5 protein expression, as well as with oxidative stress. Formation of sulfenic acid on cysteine residues of proteins is an important, dynamic mechanism for protein regulation under oxidative stress. Kv1.5 is widely reported to be redox-sensitive, and the channel possesses 6 potentially redox-sensitive intracellular cysteines. We therefore hypothesized that sulfenic acid modification of the channel itself may regulate Kv1.5 in response to oxidative stress. OBJECTIVE: To investigate how oxidative stress, via redox-sensitive modification of the channel with sulfenic acid, regulates trafficking and expression of Kv1.5. METHODS AND RESULTS: Labeling studies with the sulfenic acid-specific probe DAz and horseradish peroxidase-streptavidin Western blotting demonstrated a global increase in sulfenic acid-modified proteins in human patients with atrial fibrillation, as well as sulfenic acid modification to Kv1.5 in the heart. Further studies showed that Kv1.5 is modified with sulfenic acid on a single COOH-terminal cysteine (C581), and the level of sulfenic acid increases in response to oxidant exposure. Using live-cell immunofluorescence and whole-cell voltage-clamping, we found that modification of this cysteine is necessary and sufficient to reduce channel surface expression, promote its internalization, and block channel recycling back to the cell surface. Moreover, Western blotting demonstrated that sulfenic acid modification is a trigger for channel degradation under prolonged oxidative stress. CONCLUSIONS: Sulfenic acid modification to proteins, which is elevated in diseased human heart, regulates Kv1.5 channel surface expression and stability under oxidative stress and diverts channel from a recycling pathway to degradation. This provides a molecular mechanism linking oxidative stress and downregulation of channel expression observed in cardiovascular diseases.

Fluorescent coumarin thiols measure biological redox couples.

In this report we present a new chemical probe, 3-HTC, that can reversibly and ratiometrically measure the thiol-disulfide equilibrium of biological systems. 3-HTC is composed of a coumarin that has a thiolate directly conjugated to its extended aromatic π system while formation of a disulfide attenuates this conjugation. The fluorescence and absorption properties of 3-HTC are therefore very sensitive to the redox state of its thiol. 3-HTC reacts reversibly with thiols and disulfides enabling its use to measure dynamic GSH/GSSH ratios in vitro as well as to monitor the reversible redox status of whole cell lysates.

Mining the thiol proteome for sulfenic acid modifications reveals new targets for oxidation in cells.

Oxidation of cysteine to sulfenic acid has emerged as a biologically relevant post-translational modification with particular importance in redox-mediated signal transduction; however, the identity of modified proteins remains largely unknown. We recently reported DAz-1, a cell-permeable chemical probe capable of detecting sulfenic acid modified proteins directly in living cells. Here we describe DAz-2, an analogue of DAz-1 that exhibits significantly improved potency in vitro and in cells. Application of this new probe for global analysis of the sulfenome in a tumor cell line identifies most known sulfenic acid modified proteins: 14 in total, plus more than 175 new candidates, with further testing confirming oxidation in several candidates. The newly identified proteins have roles in signal transduction, DNA repair, metabolism, protein synthesis, redox homeostasis, nuclear transport, vesicle trafficking, and ER quality control. Cross-comparison of these results with those from disulfide, S-glutathionylation, and S-nitrosylation proteomes reveals moderate overlap, suggesting fundamental differences in the chemical and biological basis for target specificity. The combination of selective chemical enrichment and live-cell compatibility makes DAz-2 a powerful new tool with the potential to reveal new regulatory mechanisms in signaling pathways and identify new therapeutic targets.

Substituent effects on the sensitivity of a quinoline photoremovable protecting group to one- and two-photon excitation.

Photoremovable protecting groups that can reveal biologically important functional groups through one- and two-photon excitation (1PE and 2PE, respectively) have promise in regulating physiological function in a temporally and spatially restricted manner. Only a few chromophores have sufficient sensitivity to 2PE suitable for use as "caging groups" in physiology experiments. It would be useful to develop structure-property relationships of chromophores, so that chromophores with high two-photon uncaging action cross-sections (delta(u)) can be designed. The 8-bromo-7-hydroxyquinolinyl chromophore (BHQ) releases a variety of functional groups through 1PE and 2PE. Swapping the bromine substituent for a nitro (NHQ), cyano (CyHQ), or chloro (CHQ) or exchanging the hydroxy for dimethylamino (DMAQ and DMAQ-Cl) or sulfhydryl (TQ) significantly alters the photochemical and photophysical properties of the quinoline chromophore. CyHQ-OAc demonstrated a 3-fold increase in sensitivity for acetate release, whereas NHQ-OAc was photochemically insensitive. The quantum efficiencies (Q(u)) of the amino and sulfhydryl derivatives were about an order of magnitude lower than that of BHQ-OAc. All of the chromophores showed diminished sensitivity to 2PE compared to BHQ-OAc, but the CyHQ, DMAQ, and DMAQ-Cl chromophores are sufficiently sensitive for physiological use. The high sensitivity of CyHQ to 1PE will be useful in biological applications requiring short exposure with low light intensity.

Expanding the functional diversity of proteins through cysteine oxidation.

The polarizable sulfur atom in cysteine is subject to numerous post-translational oxidative modifications in the cellular milieu, which regulates a wide variety of biological phenomena such as catalysis, metal binding, protein turnover, and signal transduction. The application of chemical rationale to describe the features of different cysteine oxoforms affords a unique perspective on this rapidly expanding field. Moreover, a chemical framework broadens our understanding of the functional roles that specific cysteine oxidation states can play and facilitates the development of mechanistic proposals, which can be tested in both biochemical and cellular studies.

A chemical approach for detecting sulfenic acid-modified proteins in living cells.

Oxidation of the thiol functional group in cysteine (Cys-SH) to sulfenic (Cys-SOH), sulfinic (Cys-SO2H) and sulfonic acids (Cys-SO3H) is emerging as an important post-translational modification that can activate or deactivate the function of many proteins. Changes in thiol oxidation state have been implicated in a wide variety of cellular processes and correlate with disease states but are difficult to monitor in a physiological setting because of a lack of experimental tools. Here, we describe a method that enables live cell labeling of sulfenic acid-modified proteins. For this approach, we have synthesized the probe DAz-1, which is chemically selective for sulfenic acids and cell permeable. In addition, DAz-1 contains an azide chemical handle that can be selectively detected with phosphine reagents via the Staudinger ligation for identification, enrichment and visualization of modified proteins. Through a combination of biochemical, mass spectrometry and immunoblot approaches we characterize the reactivity of DAz-1 and highlight its utility for detecting protein sulfenic acids directly in mammalian cells. This novel method to isolate and identify sulfenic acid-modified proteins should be of widespread utility for elucidating signaling pathways and regulatory mechanisms that involve oxidation of cysteine residues.

Inhibition of kinesin motor proteins by adociasulfate-2.

Kinesin motor proteins are involved in cell division and intracellular transport of vesicles and organelles, and as such, they play a role in neurological disease, cancer, and developmental disorders. Inhibitors of kinesin would be valuable as probes of cell physiology and as potential therapeutics. Adociasulfate-2 (AS-2) is the only known natural product inhibitor of kinesins, but its mechanism of action is unknown. We utilized kinetic studies, dynamic light scattering, and transmission electron microscopy to investigate the inhibitory action of AS-2. Our data suggest that AS-2 is not a classical 1:1 inhibitor. Instead, a rodlike aggregate that mimics microtubules is complexed with kinesin and inhibits its ATPase activity. An intriguing implication of this hypothesis is that aggregates of a chiral natural product can have interesting and biologically relevant properties. This mode of action might represent one way in which a small molecule can disrupt a protein-protein interaction.