Intracellular Cysteines Research Articles

Palmitoylation of the P2X7 receptor, an ATP‐gated channel, controls its expression and association with lipid rafts

The P2X7 receptor (P2X7R) is an ATP-gated cationic channel expressed by hematopoietic, epithelial, and neuronal cells. Prolonged ATP exposure leads to the formation of a nonselective pore, which can result in cell death. We show that P2X7R is associated with detergent-resistant membranes (DRMs) in both transfected human embryonic kidney (HEK) cells and primary macrophages independently from ATP binding. The DRM association requires the posttranslational modification of P2X7R by palmitic acid. Treatment of cells with the palmitic acid analog 2-bromopalmitate as well as mutations of cysteine to alanine residues abolished P2X7R palmitoylation. Substitution of the 17 intracellular cysteines of P2X7R revealed that 4 regions of the carboxyl terminus domain are involved in palmitoylation. Palmitoylation-defective P2X7R mutants showed a dramatic decrease in cell surface expression because of their retention in the endoplasmic reticulum and proteolytic degradation. Taken together, our data demonstrate that P2X7R palmitoylation plays a critical role in its association with the lipid microdomains of the plasma membrane and in the regulation of its half-life.

Intracellular cysteines within channel subunits modulate ENaC activity

Intracellular cysteines within channel subunits modulate ENaC activity

S-acylation regulates Kv1.5 channel surface expression

The number of ion channels expressed on the cell surface shapes the complex electrical response of excitable cells. An imbalance in the ratio of inward and outward conducting channels is unfavorable and often detrimental. For example, over- or underexpression of voltage-gated K(+) (Kv) channels can be cytotoxic and in some cases lead to disease. In this study, we demonstrated a novel role for S-acylation in Kv1.5 cell surface expression. In transfected fibroblasts, biochemical evidence showed that Kv1.5 is posttranslationally modified on both the NH(2) and COOH termini via hydroxylamine-sensitive thioester bonds. Pharmacological inhibition of S-acylation, but not myristoylation, significantly decreased Kv1.5 expression and resulted in accumulation of channel protein in intracellular compartments and targeting for degradation. Channel protein degradation was rescued by treatment with proteasome inhibitors. Time course experiments revealed that S-acylation occurred in the biosynthetic pathway of nascent channel protein and showed that newly synthesized Kv1.5 protein, but not protein expressed on the cell surface, is sensitive to inhibitors of thioacylation. Sensitivity to inhibitors of S-acylation was governed by COOH-terminal, but not NH(2)-terminal, cysteines. Surprisingly, although intracellular cysteines were required for S-acylation, mutation of these residues resulted in an increase in Kv1.5 cell surface channel expression, suggesting that screening of free cysteines by fatty acylation is an important regulatory step in the quality control pathway. Together, these results show that S-acylation can regulate steady-state expression of Kv1.5.

The anticancer activity of the fungal metabolite terrecyclic acid A is associated with modulation of multiple cellular stress response pathways

Tumors are dependent on cellular stress responses, in particular the heat shock response, for survival in their hypoxic, acidotic, and nutrient-deprived microenvironments. Using cell-based reporter assays, we have identified terrecyclic acid A (TCA) from Aspergillus terreus, a fungus inhabiting the rhizosphere of Opuntia versicolor of the Sonoran desert, as a small-molecule inducer of the heat shock response that shows anticancer activity. Further characterization suggested that TCA also affects oxidative and inflammatory cellular stress response pathways. The presence of an alpha-methylene ketone moiety suggested that TCA may form adducts with sulfhydryl groups of proteins. Reaction with labile intracellular cysteines was supported by our finding that the glutathione precursor N-acetyl-cysteine protected tumor cells from the cytotoxic effects of TCA whereas the glutathione-depleting agent buthionine sulfoximine enhanced its activity. Related sesquiterpenes have been shown to increase levels of reactive oxygen species (ROS) and to inhibit nuclear factor kappaB (NF-kappaB) transcriptional activity. To assess whether TCA could have similar activities, we used a ROS-sensitive dye and flow cytometry to show that TCA does indeed increase ROS levels in 3LL cells. When tested in cells carrying NF-kappaB reporter constructs, TCA also exhibited concentration-dependent inhibition of cytokine-induced NF-kappaB transcriptional activity. These findings suggest that TCA modulates multiple stress pathways-the oxidative, heat shock, and inflammatory responses-in tumor cells that promote their survival. Small-molecule natural products such as TCA may serve as useful probes for understanding the relationships between these pathways, potentially providing leads for the design of novel and effective anticancer drugs.

Functionally Active T1-T1 Interfaces Revealed by the Accessibility of Intracellular Thiolate Groups in Kv4 Channels

Gating of voltage-dependent K+ channels involves movements of membrane-spanning regions that control the opening of the pore. Much less is known, however, about the contributions of large intracellular channel domains to the conformational changes that underlie gating. Here, we investigated the functional role of intracellular regions in Kv4 channels by probing relevant cysteines with thiol-specific reagents. We find that reagent application to the intracellular side of inside-out patches results in time-dependent irreversible inhibition of Kv4.1 and Kv4.3 currents. In the absence or presence of Kv4-specific auxiliary subunits, mutational and electrophysiological analyses showed that none of the 14 intracellular cysteines is essential for channel gating. C110, C131, and C132 in the intersubunit interface of the tetramerization domain (T1) are targets responsible for the irreversible inhibition by a methanethiosulfonate derivative (MTSET). This result is surprising because structural studies of Kv4-T1 crystals predicted protection of the targeted thiolate groups by constitutive high-affinity Zn2+ coordination. Also, added Zn2+ or a potent Zn2+ chelator (TPEN) does not significantly modulate the accessibility of MTSET to C110, C131, or C132; and furthermore, when the three critical cysteines remained as possible targets, the MTSET modification rate of the activated state is ∼200-fold faster than that of the resting state. Biochemical experiments confirmed the chemical modification of the intact α-subunit and the purified tetrameric T1 domain by MTS reagents. These results conclusively demonstrate that the T1–T1 interface of Kv4 channels is functionally active and dynamic, and that critical reactive thiolate groups in this interface may not be protected by Zn2+ binding.

Accessibility of Cysteines in the Native Bovine Rod cGMP-Gated Channel

Cyclic nucleotide-gated channels of photoreceptors and olfactory sensory neurons are tetramers consisting of A and B subunits. Here, the accessibility of the cysteines of the bovine rod cyclic nucleotide-gated channel is examined as a function of ligand binding. N-Ethylmaleimide-modified cysteines of both subunits were identified by mass spectrometry after trypsin digestion. In the absence of ligand, the intracellular carboxy-terminal cysteines of both subunits were accessible to N-ethylmaleimide. Activation of the channel abolished the accessibility of Cys505 of the A subunit and Cys1104 of the B subunit, with both being conserved cysteines of the cyclic nucleotide-binding sites. The cysteine of the pore loop of the B subunit was also found to be modified by this reagent in the absence of ligand. The total number of accessible cysteines of each subunit was determined by mass shifting upon modification with polyethylene glycol maleimide. In the absence of cyclic nucleotides, this hydrophilic reagent only weakly labeled cysteines of the A subunit but readily labeled at least three cysteines of the B subunit. Ligand binding exposed two cysteines of the A subunit and one cysteine of the B subunit to chemical modification. Double-modification experiments suggest that some of these cysteines are in or close to membrane-spanning domains. However, these cysteines could not yet be identified. Together, the cysteine accessibility of the native rod cyclic nucleotide-gated channel varies markedly upon ligand binding, thus indicating major structural rearrangements, which are of functional importance for channel activation.

CD81 Associates with 14-3-3 in a Redox-regulated Palmitoylation-dependent Manner

As a member of the tetraspanin superfamily of proteins, CD81 has been linked to a number of biologic functions including cellular proliferation, differentiation, activation, and degranulation. As a co-receptor for hepatitis C virus, and a requirement for hepatocytes for infectivity of human Plasmodium falciparum and rodent P. yoelii sporozoite infectivity, CD81 may also play a vital role in pathology. Despite the importance of CD81 in multiple cellular functions, the molecular mechanism of action of CD81 in these processes has remained elusive. Here we report an association between CD81 and the epsilon isoform of 14-3-3, a serine/threonine-binding intracellular signaling protein. Furthermore, we provide evidence that in human, this association is influenced by the palmitoylation state of the CD81 cytoplasmic tails. We have generated a series of CD81 cysteine mutants to identify palmitoylated intracellular motifs of CD81, and reveal palmitoylation on the N- and C-terminal tails as well as the intracellular loop between transmembrane domains 2 and 3. One of these mutants lacks all five of its intracellular cysteines and therefore cannot be palmitoylated. This unpalmitoylated version of CD81 shows constitutive association with 14-3-3. Interestingly, we find that under oxidative conditions, CD81 palmitoylation is inhibited and that condition correlates with the association of CD81 and 14-3-3. These finding suggest that CD81 signaling events could be mediated by 14-3-3 adapter proteins, and these signals may be dependent on cellular redox.

Peroxynitrite inactivates the human dopamine transporter by modification of cysteine 342: potential mechanism of neurotoxicity in dopamine neurons.

Peroxynitrite (ONOO(-)) has been implicated as a causative factor in dopamine neuronal damage resulting from exposure to methamphetamine and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and it may be involved in the etiology of Parkinson's Disease. ONOO(-) causes a concentration-dependent and irreversible reduction in dopamine uptake by EM4 cells stably expressing the human dopamine transporter (hDAT). The effect of ONOO(-) is manifested as a reduction in V(max). Cysteine, dithiothreitol, glutathione, and N-acetyl-cysteine, reagents that interact directly with ONOO(-), prevent this inhibition, whereas a scavenger of hydroxyl radical (dimethylsulfoxide), hydrogen peroxide (catalase), and superoxide (superoxide dismutase) did not. Dopamine in the extracellular medium protects the hDAT from ONOO(-), whereas intracellular dopamine does not. Parachloromercuribenzoic acid and 2-aminoethyl methanethiosulfonate (MTSEA), which share with ONOO(-) the ability to modify cysteine sulfhydryls, also inhibit hDAT function. ONOO(-) treatment lowers cysteine-specific labeling of the hDAT by MTSEA-biotin, suggesting that ONOO(-) reacts with one or more cysteines in hDAT. A mutant of hDAT (X7C) in which all intracellular and extracellular loop cysteines were mutated was resistant to inhibition by ONOO(-). Sensitivity to ONOO(-) was restored in mutants of hDAT in which reduced cysteines were present only in the first (C135) and third (C342) intracellular loops (CD-DAT), or in which C342 alone had been reintroduced into X7C (X7C-M342C). These results indicate that the hDAT is inhibited by ONOO(-) through oxidation of cysteine 342. Our studies also substantiate the possibility that drugs known to decrease DAT function in vivo (e.g., methamphetamine and MPTP) may exert their effects through ONOO(-)-mediated oxidative stress.

Peroxynitrite Inactivates the Human Dopamine Transporter by Modification of Cysteine 342: Potential Mechanism of Neurotoxicity in Dopamine Neurons

Peroxynitrite (ONOO⁻) has been implicated as a causative factor in dopamine neuronal damage resulting from exposure to methamphetamine and1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and it may be involved in the etiology of Parkinson9s Disease. ONOO⁻ causes a concentration-dependent and irreversible reduction in dopamine uptake by EM4 cells stably expressing the human dopamine transporter (hDAT). The effect of ONOO⁻ is manifested as a reduction in<i>V</i>_max. Cysteine, dithiothreitol, glutathione, and <i>N</i>-acetyl-cysteine, reagents that interact directly with ONOO⁻, prevent this inhibition, whereas a scavenger of hydroxyl radical (dimethylsulfoxide), hydrogen peroxide (catalase), and superoxide (superoxide dismutase) did not. Dopamine in the extracellular medium protects the hDAT from ONOO⁻, whereas intracellular dopamine does not. Parachloromercuribenzoic acid and 2-aminoethyl methanethiosulfonate (MTSEA), which share with ONOO⁻ the ability to modify cysteine sulfhydryls, also inhibit hDAT function. ONOO⁻ treatment lowers cysteine-specific labeling of the hDAT by MTSEA-biotin, suggesting that ONOO⁻ reacts with one or more cysteines in hDAT. A mutant of hDAT (X7C) in which all intracellular and extracellular loop cysteines were mutated was resistant to inhibition by ONOO⁻. Sensitivity to ONOO⁻ was restored in mutants of hDAT in which reduced cysteines were present only in the first (C135) and third (C342) intracellular loops (CD-DAT), or in which C342 alone had been reintroduced into X7C (X7C-M342C). These results indicate that the hDAT is inhibited by ONOO⁻ through oxidation of cysteine 342. Our studies also substantiate the possibility that drugs known to decrease DAT function <i>in vivo</i> (e.g., methamphetamine and MPTP) may exert their effects through ONOO⁻-mediated oxidative stress.

Intracellular Cysteines of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Modulate Channel Gating

The cystic fibrosis transmembrane conductance regulator (CFTR), a member of the ATP-binding cassette superfamily, is a cAMP-activated chloride channel. CFTR contains two transmembrane domains (TMDs), two nucleotide-binding domains (NBDs), and a regulatory (R) domain. We found that whole-cell CFTR-dependent Cl- currents in Xenopus laevis oocytes were sensitive to HgCl₂, suggesting that modification of endogenous cysteines alters channel activity. To understand better this phenomenon, site-directed mutagenesis was employed to generate both individual cysteine replacements and a version of the molecule with no cysteines in the hydrophobic sector. Each mutant displayed a forskolin/IBMX-activated Cl^- conductance similar to wild type, indicating that none of the cysteines located within the TMDs is essential. Subsequent single-channel analysis of inside-out patches excised from HEK293 cells expressing either cysteine-less or wild-type CFTR showed that intracellular application of a membrane impermeant sulphydryl reagent, p-chloromercuribenzosulfonate (PCMBS), significantly reduced open probability without affecting ion selectivity or conductance. The cysteine-less molecule also acquired a voltage-dependent sensitivity to extracellular PCMBS not observed in the wild type, perhaps due to a more flexible conformation that allowed PCMBS access to the intracellular surface. Together, these experiments suggest that endogenous intracellular cysteines, located primarily within the NBDs and/or R domain, influence channel gating.

Dynamic Recruitment of Human CD2 into Lipid Rafts: LINKAGE TO T CELL SIGNAL TRANSDUCTION

CD2 mediates T cell adhesion via its ectodomain and signal transduction utilizing its 117-amino acid cytoplasmic tail. Here we show that a significant fraction of human CD2 molecules is inducibly recruited into lipid rafts upon CD2 cross-linking by a specific pair of mitogenic anti-CD2 monoclonal antibodies (anti-T11(2) + anti-T11(3)) or during cellular conjugate formation by CD58, the physiologic ligand expressed on antigen-presenting cells. Translocation to lipid microdomains is independent of the T cell receptor (TCR) and, unlike inducible TCR-raft association, requires no tyrosine phosphorylation. Structural integrity of rafts is necessary for CD2-stimulated elevation of intracellular free calcium and tyrosine phosphorylation of cellular substrates. Whereas murine CD2 contains two membrane-proximal intracellular cysteines, partitioning CD2 into cholesterol-rich lipid rafts constitutively, human CD2 has no cytoplasmic cysteines. Mapping studies using CD2 point mutation, deletion, and chimeric molecules suggest that conformational change in the CD2 ectodomain participates in inducible raft association and excludes the membrane-proximal N-linked glycans, the transmembrane segment, and the CD2 cytoplasmic region (residues 8-117) as necessary for translocation. Translocation of CD2 into lipid rafts may reorganize the membrane into an activation-ready state prior to TCR engagement by a peptide associated with a major histocompatibility complex molecule, accounting for synergistic T cell stimulation by CD2 and the TCR.

PH-dependent Gating of ROMK (Kir1.1) Channels Involves Conformational Changes in Both N and C Termini

ROMK channels (Kir1.1) are members of the superfamily of inward rectifier potassium channels (Kir) and represent the channels underlying K+ secretion in the kidney. As their native counterparts, Kir1.1 channels are gated by intracellular pH, with acidification leading to channel closure. Although a lysine residue (Lys80) close to the first hydrophobic segment M1 has been identified as the pH sensor, little is known about how opening and closing of the channel is accomplished. Here we investigate the gating process of Kir1.1 channels exploiting their state-dependent modification by water-soluble oxidants and sulfhydryl reagents. Mutagenesis of all intracellular cysteines either alone or in combination revealed two residues targeted by these reagents, one in the N terminus (Cys49) and one in the C terminus (Cys308) of the channel protein. Both sites reacted with the thiol reagents only in the closed state and not in the open state. These results indicate that pH-dependent gating of Kir1.1 channels involves movement of protein domains in both N and C termini of the Kir1.1 protein.

Cocaine alters the accessibility of endogenous cysteines in putative extracellular and intracellular loops of the human dopamine transporter.

Cocaine and other psychostimulants act by blocking the dopamine transporter. Binding of the cocaine analog, [3H]2-beta-carbomethoxy-3-beta-(4-fluorophenyl) tropane (CFT) to the dopamine transporter is sensitive to polar sulfhydryl-specific derivatives of methanethiosulfonate (MTS). These reagents preferentially react with water-accessible, reduced cysteines. The human dopamine transporter has 13 cysteines. Their topology is not completely determined. We sought to identify those cysteine residues the modification of which affects CFT binding and to determine the topology of these reactive cysteines. We mutated each of the cysteines, one at a time and in various combinations, to residues that preserved binding and transport, and we tested the sensitivity of each of the mutant transporters to the reagents. One construct, X5C, had five mutated cysteines (C90A, C135A, C306A, C319F, and C342A). Using a membrane preparation in which both extracellular and intracellular cysteines could be accessible, we found that CFT binding in X5C, as compared with wild-type transporter, was two orders of magnitude less sensitive to MTS ethylammonium (MTSEA). The wild-type cysteines were substituted back into X5C, one at a time, and these constructs were tested in cells and in membranes. Cys-90 and Cys-306 appear to be extracellular, and Cys-135 and Cys-342 appear to be intracellular. Each of these residues is predicted to be in extramembranous loops. The binding of cocaine increases the rate of reaction of MTSEA and MTS ethyltrimethylammonium with the extracellular Cys-90 and therefore acts by inducing a conformational change. Cocaine decreases the rate of reaction of MTSEA with Cys-135 and Cys-342, acting either directly or indirectly on these intracellular residues.