Thiol-reactive Reagents Research Articles

Biotinylation of K16 in histone H4 causes chromatin condensation

Histone modifications play crucial roles in the control of gene activity, nuclear architecture, and genomic stability. Nucleosomes are the basic units of chromatin in eukaryotes, consisting of an octamer of histone core proteins wrapped by ~147 bp of DNA. Biotinylation of lysines (K) in histones H3 and H4 by holocarboxylase synthetase (HLCS) is a rare epigenetic repression mark, e.g., H4K16bio. Here, we tested the hypothesis that H4K16bio contributes toward chromatin condensation and, therefore, gene repression. We generated mutant recombinant histone H4 in which K16 has been replaced with cysteine (H4K16C) for subsequent chemical biotinylation with a thiol reactive reagent (H4K16Cbio). Nucleosomes were assembled by salt dialysis, using recombinant histones and the Widom 601 DNA sequence. DNA binding was analyzed by atomic force microscopy. Core octamers containing H4K16Cbio bound ~29% more DNA compared with octamers containing H4K16 or H4K16C (controls), suggesting biotin‐dependent condensation of chromatin. We conclude that biotinylation of H4 increases the nucleosomal affinity for DNA. We propose that histone biotinylation contributes to gene repression, in addition to the emerging role of HLCS as a member of a multiprotein gene repression complex in human chromatin.Grant Funding Source: ARD, and NIH DK063945, DK077816, and DK082476.

Lysine 210 Regulates Voltage-Dependent Gating of the Anion-Proton Exchanger CLC-5

The ClC-type Cl-/H+ exchanger ClC-5 displays complex voltage-dependent gating that regulates the number of active transporters and is modified by the concentration of internal and external transport substrates. So far, the molecular basis of ClC transporter gating is insufficiently understood. One residue, a conserved amino acid facing the anion permeation pathway at the external side of the protein, is known to be a crucial determinant of this process. Adjacent to this so-called gating glutamate (E211), there is a positive amino acid (K210) that is also highly conserved among the ClC family. Studies of the corresponding residue in ClC-1 (Fahlke et al. (1997) Nature 390, 529-532) demonstrated the importance of this residue for anion selectivity. Because of the well established connection between anion permeation and voltage-dependent gating in ClC proteins, this residue might also shape the voltage dependence of ClC-5. To test this hypothesis, we introduced the point mutation K210C in the non-conducting E268Q ClC-5. Because the gating process, observed at positive voltages is associated with prominent gating currents, we assessed the effects of K210C by measuring the voltage dependence of the nonlinear capacitances of ClC-5. The charge neutralization K210C shifted the voltage dependence of the bell shaped capacitance curve of ClC-5 by ∼25 mV to the left, which indicates that this residue is part of the voltage sensing machinery of the transporter. Surprisingly, the modification of K210C with the thiol-reactive reagent MTSET that restores the positive charge at this position induced additional 40 mV towards hyperpolarized potentials. Therefore, not only the positive charge of the lysine K210 but also the size and the pK of its side chain contribute to shaping the voltage dependence of ClC-5.

Identification and Quantification of S-Nitrosylation by Cysteine Reactive Tandem Mass Tag Switch Assay

Redox-switches are critical cysteine thiols that are modified in response to changes in the cell's environment conferring a functional effect. S-nitrosylation (SNO) is emerging as an important modulator of these regulatory switches; however, much remains unknown about the nature of these specific cysteine residues and how oxidative signals are interpreted. Because of their labile nature, SNO-modifications are routinely detected using the biotin switch assay. Here, a new isotope coded cysteine thiol-reactive multiplex reagent, cysTMT(6), is used in place of biotin, for the specific detection of SNO-modifications and determination of individual protein thiol-reactivity. S-nitrosylation was measured in human pulmonary arterial endothelia cells in vitro and in vivo using the cysTMT(6) quantitative switch assay coupled with mass spectrometry. Cell lysates were treated with S-nitrosoglutathione and used to identify 220 SNO-modified cysteines on 179 proteins. Using this approach it was possible to discriminate potential artifacts including instances of reduced protein disulfide bonds (6) and S-glutathionylation (5) as well as diminished ambiguity in site assignment. Quantitative analysis over a range of NO-donor concentrations (2, 10, 20 μm; GSNO) revealed a continuum of reactivity to SNO-modification. Cysteine response was validated in living cells, demonstrating a greater number of less sensitive cysteine residues are modified with increasing oxidative stimuli. Of note, the majority of available cysteines were found to be unmodified in the current treatment suggesting significant additional capacity for oxidative modifications. These results indicate a possible mechanism for the cell to gauge the magnitude of oxidative stimuli through the progressive and specific accumulation of modified redox-switches.

A Plasma Membrane Wound Proteome: REVERSIBLE EXTERNALIZATION OF INTRACELLULAR PROTEINS FOLLOWING REPARABLE MECHANICAL DAMAGE

Cells in mechanically active tissues undergo constant plasma membrane damage that must be repaired to allow survival. To identify wound-associated proteins, a cell-impermeant, thiol-reactive biotinylation reagent was used to label and subsequently isolate intracellular proteins that become exposed on the surface of cultured cells after plasma membrane damage induced by scraping from substratum or crushing with glass beads. Scrape-damaged cells survived injury and were capable of forming viable colonies. Proteins that were exposed to the cell surface were degraded or internalized a few seconds to several minutes after damage, except for vimentin, which was detectable on the cell surface for at least an hour after injury. Seven major biotinylated protein bands were identified on SDS-PAGE gels. Mass spectrometric studies identified cytoskeletal proteins (caldesmon-1 and vimentin), endoplasmic reticulum proteins (ERp57, ERp5, and HSP47), and nuclear proteins (lamin C, heterogeneous nuclear ribonucleoprotein F, and nucleophosmin-1) as major proteins exposed after injury. Although caldesmon was a major wound-associated protein in calpain small subunit knock-out fibroblasts, it was rapidly degraded in wild-type cells, probably by calpains. Lamin C exposure after wounding was most likely the consequence of nuclear envelope damage. These studies document major intracellular proteins associated with the cell surface of reversibly damaged somatic cells. The studies also show that externalization of some proteins reported to have physiologic or pathologic roles on the cell surface can occur in cells undergoing plasma membrane damage and subsequent repair.

Antitumor Activity of Ribonuclease Multimers Created by Site-Specific Covalent Tethering

Site-specific cross-linking can generate homogeneous multimeric proteins of defined valency. Pancreatic-type ribonucleases are an especially attractive target, as their natural dimers can enter mammalian cells, evade the cytosolic ribonuclease inhibitor (RI), and exert their toxic ribonucleolytic activity. Here, we report on the use of eight distinct thiol-reactive cross-linking reagents to produce dimeric and trimeric conjugates of four pancreatic-type ribonucleases. Both the site of conjugation and, to a lesser extent, the propinquity of the monomers within the conjugate modulate affinity for RI, and hence cytotoxicity. Still, the cytotoxicity of the multimers is confounded in vitro by their increased hydrodynamic radius, which attenuates cytosolic entry. A monomeric RI-evasive variant of bovine pancreatic ribonuclease (RNase A) inhibits the growth of human prostate and lung tumors in mice. An RI-evasive trimeric conjugate inhibits tumor growth at a lower dose and with less frequent administration than does the monomer. This effect is attributable to an enhanced persistence of the trimers in circulation. On a molecular basis, the trimer is ∼300-fold more efficacious and as well tolerated as erlotinib, which is in clinical use for the treatment of lung cancer. These data encourage the development of mammalian ribonucleases for the treatment of human cancers.

Pore-opening mechanism in trimeric P2X receptor channels

The opening of ion channels in response to ligand binding, voltage or membrane stretch underlies electrical and chemical signalling throughout biology. Two structural classes of pore-opening mechanisms have been established, including bending of pore-lining helices in the case of tetrameric cation channels, or tilting of such helices in mechanosensitive channels. In this paper, we explore how the structure of the pore changes during opening in P2X receptors by measuring the modification of introduced cysteine residues in transmembrane helices by thiol-reactive reagents, and by engineering metal bridges. Our results are consistent with the X-ray structure of the closed state, and demonstrate that expansion of the gate region in the external pore is accompanied by a significant narrowing of the inner pore, indicating that pore-forming helices straighten on ATP binding to open the channel. This unique pore-opening mechanism has fundamental implications for the role of subunit interfaces in the gating mechanism of P2X receptors and points to a role of the internal pore in ion permeation.

Evidence for VirB4-Mediated Dislocation of Membrane-Integrated VirB2 Pilin during Biogenesis of the Agrobacterium VirB/VirD4 Type IV Secretion System

Agrobacterium VirB2 pilin is required for assembly of the VirB/VirD4 type IV secretion system (T4SS). The propilin is processed by signal sequence cleavage and covalent linkage of the N and C termini, and the cyclized pilin integrates into the inner membrane (IM) as a pool for assembly of the secretion channel and T pilus. Here, by use of the substituted cysteine accessibility method (SCAM), we defined the VirB2 IM topology and then identified distinct contributions of the T4SS ATPase subunits to the pilin structural organization. Labeling patterns of Cys-substituted pilins exposed to the membrane-impermeative, thiol-reactive reagent 3-(N-maleimidopropionyl)biocytin (MPB) supported a topology model in which two hydrophobic stretches comprise transmembrane domains, an intervening hydrophilic loop (residues 90 to 94) is cytoplasmic, and the hydrophilic N and C termini joined at residues 48 and 121 form a periplasmic loop. Interestingly, the VirB4 ATPase, but not a Walker A nucleoside triphosphate (NTP) binding motif mutant, induced (i) MPB labeling of Cys94, a residue that in the absence of the ATPase is located in the cytoplasmic loop, and (ii) release of pilin from the IM upon osmotic shock. These findings, coupled with evidence for VirB2-VirB4 complex formation by coimmunoprecipitation, support a model in which VirB4 functions as a dislocation motor to extract pilins from the IM during T4SS biogenesis. The VirB11 ATPase functioned together with VirB4 to induce a structural change in the pilin that was detectable by MPB labeling, suggestive of a role for VirB11 as a modulator of VirB4 dislocase activity.

Structure-Function Studies of the SLC17 Transporter Sialin Identify Crucial Residues and Substrate-induced Conformational Changes

Salla disease and infantile sialic acid storage disorder are human diseases caused by loss of function of sialin, a lysosomal transporter that mediates H(+)-coupled symport of acidic sugars N-acetylneuraminic acid and glucuronic acid out of lysosomes. Along with the closely related vesicular glutamate transporters, sialin belongs to the SLC17 transporter family. Despite their critical role in health and disease, these proteins remain poorly understood both structurally and mechanistically. Here, we use substituted cysteine accessibility screening and radiotracer flux assays to evaluate experimentally a computationally generated three-dimensional structure model of sialin. According to this model, sialin consists of 12 transmembrane helices (TMs) with an overall architecture similar to that of the distantly related glycerol 3-phosphate transporter GlpT. We show that TM4 in sialin lines a large aqueous cavity that forms a part of the substrate permeation pathway and demonstrate substrate-induced alterations in accessibility of substituted cysteine residues in TM4. In addition, we demonstrate that one mutant, F179C, has a dramatically different effect on the apparent affinity and transport rate for N-acetylneuraminic acid and glucuronic acid, suggesting that it may be directly involved in substrate recognition and/or translocation. These findings offer a basis for further defining the transport mechanism of sialin and other SLC17 family members.

Contribution of exofacial thiol groups in the reducing activity of Lactococcus lactis

Lactococcus lactis can decrease the redox potential at pH 7 (E(h7)) from 200 to -200 mV in oxygen free Man-Rogosa-Sharpe media. Neither the consumption of oxidizing compounds or the release of reducing compounds during lactic acid fermentation were involved in the decrease in E(h7) by the bacteria. Thiol groups located on the bacterial cell surface appear to be the main components that are able to establish a greater exchange current between the Pt electrode and the bacteria. After the final E(h7) (-200 mV) was reached, only thiol-reactive reagents could restore the initial E(h7) value. Inhibition of the proton motive force showed no effect on maintaining the final E(h7) value. These results suggest that maintaining the exofacial thiol (-SH) groups in a reduced state does not depend on an active mechanism. Thiol groups appear to be displayed by membrane proteins or cell wall-bound proteins and may participate in protecting cells against oxidative stress.

Site-specifically 89Zr-labeled monoclonal antibodies for ImmunoPET

Three thiol reactive reagents were developed for the chemoselective conjugation of desferrioxamine (Df) to a monoclonal antibody via engineered cysteine residues (thio-trastuzumab). The in vitro stability and in vivo imaging properties of site-specifically radiolabeled (89)Zr-Df-thio-trastuzumab conjugates were investigated. The amino group of desferrioxamine B was acylated by bromoacetyl bromide, N-hydroxysuccinimidyl iodoacetate, or N-hydroxysuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate to obtain thiol reactive reagents bromoacetyl-desferrioxamine (Df-Bac), iodoacetyl-desferrioxamine (Df-Iac) and maleimidocyclohexyl-desferrioxamine (Df-Chx-Mal), respectively. Df-Bac and Df-Iac alkylated the free thiol groups of thio-trastuzumab by nucleophilic substitution forming Df-Ac-thio-trastuzumab, while the maleimide reagent Df-Chx-Mal reacted via Michael addition to provide Df-Chx-Mal-thio-trastuzumab. The conjugates were radiolabeled with (89)Zr and evaluated for serum stability, and their positron emission tomography (PET) imaging properties were investigated in a BT474M1 (HER2-positive) breast tumor mouse model. The chemoselective reagents were obtained in 14% (Df-Bac), 53% (Df-Iac) and 45% (Df-Chx-Mal) yields. Site-specific conjugation of Df-Chx-Mal to thio-trastuzumab was complete within 1 h at pH 7.5, while Df-Iac and Df-Bac respectively required 2 and 5 h at pH 9. Each Df modified thio-trastuzumab was chelated with (89)Zr in yields exceeding 75%. (89)Zr-Df-Ac-thio-trastuzumab and (89)Zr-Df-Chx-Mal-thio-trastuzumab were stable in mouse serum and exhibited comparable PET imaging capabilities in a BT474M1 (HER2-positive) breast cancer model reaching 20-25 %ID/g of tumor uptake and a tumor to blood ratio of 6.1-7.1. The new reagents demonstrated good reactivity with engineered thiol groups of trastuzumab and very good chelation properties with (89)Zr. The site-specifically (89)Zr-labeled thio-antibodies were stable in serum and showed PET imaging properties comparable to lysine conjugates.

Dissecting the mechanism of Ca2+‐triggered membrane fusion: Probing protein function using thiol reactivity

1. Ca(2+)-triggered membrane fusion involves the coordinated actions of both lipids and proteins, but the specific mechanisms remain poorly understood. The urchin cortical vesicle model is a stage-specific native preparation fully enabling the directly coupled functional-molecular analyses necessary to identify critical components of fast triggered membrane fusion. 2. Recent work on lipidic components has established a direct role for cholesterol in the fusion mechanism via local contribution of negative curvature to readily enable the formation of transient lipidic fusion intermediates. In addition, cholesterol- and sphingomyelin-enriched domains regulate the efficiency of fusion by focally organizing other components to ensure an optimized response to the triggering Ca(2+) transient. 3. There is less known about the identity of proteins involved in the Ca(2+)-triggering steps of membrane fusion. Thiol reagents can be used as unbiased tools to probe protein functions. Comparisons of several thiol-reactive reagents have identified different effects on Ca(2+) sensitivity and the extent of fusion, suggesting that there are at least two distinct thiol sites that participate in the fusion mechanism: one that regulates the efficiency of Ca(2+) sensing/triggering and one that may function during the membrane merger event itself. 4. To identify the proteins that regulate Ca(2+) sensitivity, the fluorescent thiol reagent Lucifer yellow iodoacetamide was used to potentiate fusion and simultaneously tag the proteins involved. Ongoing work involves the isolation of cholesterol-enriched membrane fractions to reduce the complexity of the labelled proteome, narrowing the number of candidate proteins.

Structural Determinants of Ion Permeation in Crac Channels

CRAC channels generate Ca2+ signals critical for the activation of immune cells and exhibit an intriguing pore profile distinguished by extremely high Ca2+ selectivity, low Cs+ permeability, and small unitary conductance. To identify the conduction pathway of the transported ions and gain insight into the structural bases of these characteristics, we introduced cysteine residues in the CRAC channel pore subunit, Orai1, and probed their accessibility to various thiol-reactive reagents. Our results indicate that the architecture of the ion conduction pathway is characterized by a flexible outer vestibule formed by the TM1-TM2 loop, which leads to a narrow pore flanked by residues of a helical TM1 segment. Residues in TM3, and specifically, E190, a residue considered important for ion selectivity, are not close to the pore. Moreover, the outer vestibule does not significantly contribute to ion selectivity, implying that Ca2+ selectivity is conferred mainly by E106 in the TM1 segment. The pore is sufficiently narrow along much of its length to permit stable coordination of Cd2+ by several TM1 residues, which likely explains the slow flux of ions within the restrained geometry of the pore. Together, these results reveal new insights into the long-sought structural basis for the unique permeation properties of CRAC channels.

Identifying the Ion Access Pathway to the Transmembrane Pore in P2X Receptors

P2X receptors are trimeric ion channels activated by extracellular ATP. Upon activation, P2X receptors promote inward current of cations to evoke action potentials or trigger calcium mediated signaling that are important for pain sensing, inflammation, and the synaptic transmission. However, the molecular mechanism of how extracellular ions access the transmembrane pore of P2X receptors is unknown. According to the zebrafish P2X4 crystal structure in the closed state, extracellular ions appear to be readily accessible to the pore through three identical fenestrations located right above the membrane leaflet (lateral pathway). In addition, ions may access the pore through a second possible pathway that runs through the central voids along the molecular three-fold axis of symmetry (central pathway). While this pathway is hypothetical, as the constrictions flanking the central voids are too narrow for hydrated ions to pass (∼2.3 Å), agonist binding may expand these constrictions to enable ions to access the transmembrane pore. We have begun to explore the pathway ions use to move through the extracellular domain to enter the pore by inserting cysteine residues into rat P2X2 receptor channels and measuring reaction rates with a range of thiol-reactive reagents and ions. We found that MTSEA-Texas Red (MW=∼750) can access T336 in the transmembrane pore with an apparently fast modification rate when the channel is open. These results suggest a large access pathway exists between the extracellular solution and the transmembrane pore, consistent with the fenestrations observed in the crystal structure. We are currently testing whether reagents of various size may also move through the central pathway.

Structural determinants of ion permeation in CRAC channels

CRAC channels generate Ca(2+) signals critical for the activation of immune cells and exhibit an intriguing pore profile distinguished by extremely high Ca(2+) selectivity, low Cs(+) permeability, and small unitary conductance. To identify the ion conduction pathway and gain insight into the structural bases of these permeation characteristics, we introduced cysteine residues in the CRAC channel pore subunit, Orai1, and probed their accessibility to various thiol-reactive reagents. Our results indicate that the architecture of the ion conduction pathway is characterized by a flexible outer vestibule formed by the TM1-TM2 loop, which leads to a narrow pore flanked by residues of a helical TM1 segment. Residues in TM3, and specifically, E190, a residue considered important for ion selectivity, are not close to the pore. Moreover, the outer vestibule does not significantly contribute to ion selectivity, implying that Ca(2+) selectivity is conferred mainly by E106. The ion conduction pathway is sufficiently narrow along much of its length to permit stable coordination of Cd(2+) by several TM1 residues, which likely explains the slow flux of ions within the restrained geometry of the pore. These results provide a structural framework to understand the unique permeation properties of CRAC channels.

2-Dialkynyl derivatives of (N)-methanocarba nucleosides: 'Clickable' A(3) adenosine receptor-selective agonists.

We modified a series of (N)-methanocarba nucleoside 5'-uronamides to contain dialkyne groups on an extended adenine C2 substituent, as synthetic intermediates leading to potent and selective A(3) adenosine receptor (AR) agonists. The proximal alkyne was intended to promote receptor recognition, and the distal alkyne reacted with azides to form triazole derivatives (click cycloaddition). Click chemistry was utilized to couple an octadiynyl A(3)AR agonist to azido-containing fluorescent, chemically reactive, biotinylated, and other moieties with retention of selective binding to the A(3)AR. A bifunctional thiol-reactive crosslinking reagent was introduced. The most potent and selective novel compound was a 1-adamantyl derivative (K(i) 6.5nM), although some of the click products had K(i) values in the range of 200-400nM. Other potent, selective derivatives (K(i) at A(3)AR innM) were intended as possible receptor affinity labels: 3-nitro-4-fluorophenyl (10.6), alpha-bromophenacyl (9.6), thiol-reactive isothiazolone (102), and arylisothiocyanate (37.5) derivatives. The maximal functional effects in inhibition of forskolin-stimulated cAMP were measured, indicating that this class of click adducts varied from partial to full A(3)AR agonist compared to other widely used agonists. Thus, this strategy provides a general chemical approach to linking potent and selective A(3)AR agonists to reporter groups of diverse structure and to carrier moieties.

Modulation of Cell Surface Protein Free Thiols; A Potential Novel Mechanism of Action of the Sesquiterpene Lactone Parthenolide in Non-Hodgkin's Lymphoma.

Abstract Abstract 3774 Poster Board III-710 Recent data supports the concept that redox regulation of cell surface protein thiols (i.e., exofacial thiols) effects critical cellular functions. We therefore hypothesized that redox-active anti-cancer therapeutics would modulate exofacial thiols, and that such modulation could be related to cell death mechanisms. To test this hypothesis, we used the sesquiterpene lactone parthenolide, a known anti-cancer agent. Parthenolide treatment results in a dose dependent decrease in mantle cell (MCL) and diffuse large cell lymphoma cell viability. Indeed, parthenolide decreases the level of free (ie. reduced) exofacial thiols on Granta MCL cells as assessed by flow cytometry. Parthenolide specifically modifies free thiols of surface proteins having molecular weights of ∼12 kd and ∼22 kd, as determined using a biotinylated thiol reactive reagent N-(biotinoyl)-N-(iodoacetyl) ethylendiamine (BIAM), which binds to free thiol groups and is detected by streptavidin-peroxidase staining of western blots. We further show that the central redox regulator thioredoxin is one of the surface protein thiol targets modified by parthenolide; specifically; (a) thioredoxin is detected on the Granta cell surface; (b) parthenolide directly interacts with free thiol groups on purified human thioredoxin, and; (c) parthenolide directly modifies Granta membrane associated thioredoxin, as determined with BIAM and neutravidin pull-down. To examine the functional consequences of parthenolide induced surface protein thiol modification, Granta cells were pretreated with the cell impermeable thiol antioxidant glutathione (GSH). Pretreatment with GSH inhibits the parthenolide induced; (a) decrease in exofacial free thiols; (b) modification of surface thioredoxin and; (c) Granta cell death. Pretreatment of Granta cells with GSH also inhibits parthenolide-mediated activation of JNK and inhibition of NFkB, two activities previously associated with the drug's anti-cancer activity. Based on these data, we postulate that at least one component of parthenolide's anti-lymphoma activity derives from its ability to modify the redox state of critical exofacial thiols. Indeed, to our knowledge, our data is the first to suggest that cancer cell exofacial thiols may be novel and important targets for cancer therapy. Supported by an NCI SPORE grant in lymphoma 1P50 CA130805. Disclosures: Bernstein: millenium: Consultancy; genentech: Consultancy, Speakers Bureau; enzon: Consultancy.

PGRL1 Participates in Iron-induced Remodeling of the Photosynthetic Apparatus and in Energy Metabolism in Chlamydomonas reinhardtii

PGRL1 RNA and protein levels are increased in iron-deficient Chlamydomonas reinhardtii cells. In an RNAi strain, which accumulates lower PGRL1 levels in both iron-replete and -starved conditions, the photosynthetic electron transfer rate is decreased, respiratory capacity in iron-sufficient conditions is increased, and the efficiency of cyclic electron transfer under iron-deprivation is diminished. Pgrl1-kd cells exhibit iron deficiency symptoms at higher iron concentrations than wild-type cells, although the cells are not more depleted in cellular iron relative to wild-type cells as measured by mass spectrometry. Thiol-trapping experiments indicate iron-dependent and redox-induced conformational changes in PGRL1 that may provide a link between iron metabolism and the partitioning of photosynthetic electron transfer between linear and cyclic flow. We propose, therefore, that PGRL1 in C. reinhardtii may possess a dual function in the chloroplast; that is, iron sensing and modulation of electron transfer.

Structure-Function Studies of Claudin Extracellular Domains by Cysteine-scanning Mutagenesis

Claudins form size- and charge-selective pores in the tight junction that control the paracellular flux of inorganic ions and small molecules. However, the structural basis for ion selectivity of paracellular pores is poorly understood. Here we applied cysteine scanning to map the paracellular pathway of ion permeation across claudin-2-transfected Madin-Darby canine kidney type I cells. Four potential pore-lining amino acid residues in the first extracellular loop were mutated to cysteine and screened for their accessibility to thiol-reactive reagents. All mutants were functional except D65C, which formed dimers by intermolecular disulfide bonding, leading to a loss of charge and size selectivity. This suggests that claudin-2 pores are multimeric and that Asp(65) lies close to a protein-protein interface. Methanethiosulfonate reagents of different size and charge and the organic mercury derivate, p-(chloromercuri)benzenesulfonic acid, significantly decreased paracellular ion permeation across I66C-transfected cells by a mechanism that suggests steric blocking of the pore. The conductance of wild-type claudin-2 and the other cysteine mutants was only weakly affected. The rate of reaction with I66C decreased dramatically with increasing size of the reagent, suggesting that Ile(66) is buried deep within a narrow segment of the pore with its side group facing into the lumen. Furthermore, labeling with N-biotinoylaminoethyl methanethiosulfonate showed that I66C was weakly reactive, whereas Y35C was strongly reactive, suggesting that Tyr(35) is located at the protein surface outside of the pore.

Thioredoxin Redox Western Analysis

Increasing evidence suggests that compartment-specific changes are important in redox signaling and control. Examination of thiol/disulfide redox changes in thioredoxin (Trx) family members including Trx1 in cytoplasm and nucleus and Trx2 in mitochondria should aid in the understanding of compartmentalized redox signaling mechanisms. Methods to quantify redox states of both Trx1 and Trx2 by redox western analysis and to further calculate redox potential using the Nernst equation are described in this unit. The procedures to measure redox states of Trx1 and Trx2 consist of three parts, derivatization, redox western blotting, and calculation of redox potentials. Derivatization of proteins with thiol-reactive reagents prior to redox western blotting prevents artifactual oxidation of protein thiols and allows separation of the reduced forms from oxidized forms of the protein. Consequently, the redox western analysis of Trx provides a convenient tool to estimate the redox state of different compartments and improve the understanding of redox signaling.

Substituted Cysteine Accessibility Method Analysis of Human Concentrative Nucleoside Transporter hCNT3 Reveals a Novel Discontinuous Region of Functional Importance within the CNT Family Motif (G/A)XKX3NEFVA(Y/M/F)

The human SLC28 family of integral membrane CNT (concentrative nucleoside transporter) proteins has three members, hCNT1, hCNT2, and hCNT3. Na(+)-coupled hCNT1 and hCNT2 transport pyrimidine and purine nucleosides, respectively, whereas hCNT3 mediates transport of both pyrimidine and purine nucleosides utilizing Na(+) and/or H(+) electrochemical gradients. These and other eukaryote CNTs are currently defined by a putative 13-transmembrane helix (TM) topology model with an intracellular N terminus and a glycosylated extracellular C terminus. Recent mutagenesis studies, however, have provided evidence supporting an alternative 15-TM membrane architecture. In the absence of CNT crystal structures, valuable information can be gained about residue localization and function using substituted cysteine accessibility method analysis with thiol-reactive reagents, such as p-chloromercuribenzene sulfonate. Using heterologous expression in Xenopus oocytes and the cysteineless hCNT3 protein hCNT3C-, substituted cysteine accessibility method analysis with p-chloromercuribenzene sulfonate was performed on the TM 11-13 region, including bridging extramembranous loops. The results identified residues of functional importance and, consistent with a new revised 15-TM CNT membrane architecture, suggest a novel membrane-associated topology for a region of the protein (TM 11A) that includes the highly conserved CNT family motif (G/A)XKX(3)NEFVA(Y/M/F).