Intersubunit Disulfide Bond Research Articles

Arrangement and Mobility of the Voltage Sensor Domain in Prokaryotic Voltage-gated Sodium Channels

Prokaryotic voltage-gated sodium channels (Na(V)s) form homotetramers with each subunit contributing six transmembrane α-helices (S1-S6). Helices S5 and S6 form the ion-conducting pore, and helices S1-S4 function as the voltage sensor with helix S4 thought to be the essential element for voltage-dependent activation. Although the crystal structures have provided insight into voltage-gated K channels (K(V)s), revealing a characteristic domain arrangement in which the voltage sensor domain of one subunit is close to the pore domain of an adjacent subunit in the tetramer, the structural and functional information on Na(V)s remains limited. Here, we show that the domain arrangement in NaChBac, a firstly cloned prokaryotic Na(V), is similar to that in K(V)s. Cysteine substitutions of three residues in helix S4, Q107C, T110C, and R113C, effectively induced intersubunit disulfide bond formation with a cysteine introduced in helix S5, M164C, of the adjacent subunit. In addition, substituting two acidic residues with lysine, E43K and D60K, shifted the activation of the channel to more positive membrane potentials and consistently shifted the preferentially formed disulfide bond from T110C/M164C to Q107C/M164C. Because Gln-107 is located closer to the extracellular side of helix S4 than Thr-110, this finding suggests that the functional shift in the voltage dependence of activation is related to a restriction of the position of helix S4 in the lipid bilayer. The domain arrangement and vertical mobility of helix S4 in NaChBac indicate that the structure and the mechanism of voltage-dependent activation in prokaryotic Na(V)s are similar to those in canonical K(V)s.

Opening the Pore of ASIC1a

Acid-sensing ion channels (ASICs) are voltage-independent proton-gated ion channels expressed throughout neurons of the mammalian central and peripheral nervous systems. These channels are organized as homo- or hetero-trimers. Each subunit has two transmembrane segments (TMs) connected by a large extracellular region with the N- and C- termini on the intracellular side. The underlying mechanism coupling proton binding in the extracellular region to pore opening is unknown. To define structural rearrangements associated with gating and desensitization in the outer vestibule of the pore of ASIC1a, we explored the reactivity toward methanethiosulfonate (MTS) reagents of channels bearing Cys substitution in individual residues of the tract I426-Q436. Mutant channels were expressed in Xenopus oocytes and the activity was studied with the two-electrode voltage clamp technique. We found that positively-charged MTS reagents trigger pore opening of G428C in the absence of extracellular acidification. Scanning mutagenesis and double-mutant cycle analysis indicated that the covalently modified side chain of Cys at position 428 interacts with Tyr424 in the region preceding TM2. A Tyr424 to Phe substitution disrupted proton-dependent gating, although pore opening was triggered by MTSET. Our results suggest that the region preceding TM2 provides functional coupling between the extracellular proton-binding sites and the ion pore. Strikingly, Y424C-G428C monomers were associated by inter-subunit disulfide bonds and were insensitive to MTSET. Conformational restrictions on the outer vestibule of the pore arising from these inter-subunit 424-428 disulfide bonds do not prevent the channel from cycling through the resting, open and desensitized states. Our studies suggest that the opening of the ion conductive pathway involves coordinated rotation of the TM2 helices.

Higher Order Structure of TMEM16a/Anoctamin-1 as Compared to Other Chloride-Conducting Channels

The molecular identity of Ca-activated chloride channels (CaCCs) has been unknown until recently, when three independent studies using different strategies identified TMEM16A as a protein with the classic CaCC properties. Due to the lack of sequence homology to other channel proteins, the possible oligomeric structure of TMEM16A cannot be inferred by analogy. Here, we used blue native PAGE (BN-PAGE) and chemical cross-linking combined with BN-PAGE and SDS-PAGE to determine the quaternary structure of the mouse TMEM16A(a) and TMEM16A(ac). As positive controls, we co-analyzed chloride-conducting channels of known oligomeric structure such as the glycine-gated homopentameric α1 glycine receptor (GlyR) and the voltage-gated homodimeric chloride channel hCLC-1. All constructs carried a hexahistidyl tag to allow for one-step affinity purification. Two-electrode voltage clamp recordings revealed that N- or C-terminal His-tagging did not affect the TMEM16A(a)-mediated ion currents in Xenopus laevis oocytes. The proteins were purified on a Ni-NTA affinity resin under non-denaturing conditions using digitonin as a mild detergent from both X. laevis oocytes and HEK293 cells. The non-denatured TMEM16A(a) and TMEM16A(ac) migrated in the BN-PAGE gel as a homodimer, as judged by comparison with a concatenated TMEM16A(a) homodimer, the α1 GlyR, and the CLC-1. Cross-linking with glutaraldehyde corroborated the homodimeric TMEM16A(a) structure. The homodimeric TMEM16A(a) assembly dissociated into protomers following denaturation with SDS, and non-reducing SDS-PAGE provided no evidence for inter-subunit disulfide bonds. Based on the identical homodimeric assembly of both the ER-resident and the plasma membrane-bound TMEM16A(a) protein, the non-covalent homodimerization must have already occurred in the ER. This observation classifies the TMEM16A(a) channel as a permanent or obligate oligomer, (i.e. a protein that occurs only in the oligomeric state). Together, our data show that the CaCC member TMEM16A shares an obligate homodimeric architecture with the hCLC-1 channel.

TMEM16A(a)/anoctamin-1 Shares a Homodimeric Architecture with CLC Chloride Channels

TMEM16A/anoctamin-1 has been identified as a protein with the classic properties of a Ca2+-activated chloride channel. Here, we used blue native polyacrylamide gel electrophoresis (BN-PAGE) and chemical cross-linking to assess the quaternary structure of the mouse TMEM16A(a) and TMEM16A(ac) splice variants as well as a genetically concatenated TMEM16A(a) homodimer. The constructs carried hexahistidyl (His) tags to allow for their purification using a nondenaturing metal affinity resin. Neither His-tagging nor head-to-tail concatenation of two copies of TMEM16A(a) noticeably affected Ca2+-induced measured macroscopic Cl− currents compared with the wild-type TMEM16A(a) channel. The digitonin-solubilized, nondenatured TMEM16A(a) protein migrated in the BN-PAGE gel as a homodimer, as judged by comparison with the concatenated TMEM16A(a) homodimer and channel proteins of known oligomeric structures (e.g. the voltage-gated Cl− channel CLC-1). Cross-linking with glutaraldehyde corroborated the homodimeric structure of TMEM16A(a). The TMEM16A(a) homodimer detected in Xenopus laevis oocytes and HEK 293 cells dissociated into monomers following denaturation with SDS, and reducing versus nonreducing SDS-PAGE provided no evidence for the presence of intersubunit disulfide bonds. Together, our data demonstrate that the Ca2+-activated chloride channel member TMEM16A shares an obligate homodimeric architecture with the hCLC-1 channel.

Thermal Stability of RNA Phage Virus-Like Particles Displaying Foreign Peptides

BackgroundTo be useful for genetic display of foreign peptides a viral coat protein must tolerate peptide insertions without major disruption of subunit folding and capsid assembly. The folding of the coat protein of RNA phage MS2 does not normally tolerate insertions in its AB-loop, but an engineered single-chain dimer readily accepts them as long as they are restricted to one of its two halves.ResultsHere we characterize the effects of peptide insertions on the thermal stabilities of MS2 virus-like particles (VLPs) displaying a variety of different peptides in one AB-loop of the coat protein single-chain dimer. These particles typically denature at temperatures around 5-10°C lower than unmodified VLPs. Even so, they are generally stable up to about 50°C. VLPs of the related RNA phage PP7 are cross-linked with intersubunit disulfide bonds and are therefore significantly more stable. An AB-loop insertion also reduces the stability of PP7 VLPs, but they only begin to denature above about 70°C.ConclusionsVLPs assembled from MS2 single-chain dimer coat proteins with peptide insertions in one of their AB-loops are somewhat less stable than the wild-type particle, but still resist heating up to about 50°C. Because they possess disulfide cross-links, PP7-derived VLPs provide an alternate platform with even higher stability.

Identification of Two Forms of Qβ Replicase with Different Thermal Stabilities but Identical RNA Replication Activity

The enzyme Qβ replicase is an RNA-dependent RNA polymerase, which plays a central role in infection by the simple single-stranded RNA virus bacteriophage Qβ. This enzyme has been used in a number of applications because of its unique activity in amplifying RNA from an RNA template. Determination of the thermal stability of Qβ replicase is important to gain an understanding of its function and potential applications, but data reported to date have been contradictory. Here, we provide evidence that these previous inconsistencies were due to the heterogeneous forms of the replicase with different stabilities. We purified two forms of replicase expressed in Escherichia coli, which differed in their thermal stability but showed identical RNA replication activity. Furthermore, we found that the replicase undergoes conversion between these forms due to oxidation, and the Cys-533 residue in the catalytic β subunit and Cys-82 residue in the EF-Tu subunit of the replicase are essential prerequisites for this conversion to occur. These results strongly suggest that the thermal stable replicase contains the intersubunit disulfide bond between these cysteines. The established strategies for isolating and purifying a thermally stable replicase should increase the usefulness of Qβ replicase in various applications, and the data regarding thermal stability obtained in this study may yield insight into the precise mechanism of infection by bacteriophage Qβ.

Capillary electrophoresis for analysis of microheterogeneous glutelin subunits in rice (Oryza sativa L.)

Glutelin, the major storage protein of rice seed, consists of microheterogenous subunits and partially exists in a macromolecular form that is polymerized by intersubunit disulfide bonds. In order to analyze the glutelin subunits using high-throughput CE, we first identified a sample preparation procedure suitable for CE. The polymerized glutelin treated with a reductant could not dissociate into its constituent monomer subunits when it was dissolved in an acidic solution. However, the glutelin dissociated into its subunits and component α and β polypeptides when it was denatured and reduced by an appropriate amount of urea and 2-mercaptoethanol at a specific incubation time and temperature. The molecular species of the completely dissociated α and β polypeptides were identified and quantitatively analyzed by CE using glutelin mutants. The CE analysis also demonstrated that the actual subunit variation in terms of the charge and/or size of the β polypeptides is much smaller than predicted when compared with that of α polypeptides, even under denaturing and reducing condition. Thus, the combined analytical system described here will be useful for basic and applied research, such as the kinetic characterization of higher-order structure and the quantitative evaluation of glutelin in a large number of diverse rice varieties.

Subunit 간의 disulfide 결합 형성에 의한 Mycobacterium smegmatis DevS histidine kinase의 불활성화

The DevSR two-component system is a major regulatory system involved in redox sensing in Mycobacterium smegmatis. The DevSR system consists of the DevS histidine kinase and its cognate DevR response regulator. When exposed to hypoxic conditions, the DevS histidine kinase is activated to phosphorylate the DevR response regulator, leading to the transcriptional activation of the DevR regulation. The ligand-binding state of the heme embedded in the N-terminal GAF domain of DevS determines the kinase activity of DevS. In this study, we demonstrated that the redox-responsive cysteine (C547) in the C-terminal kinase domain is involved in the redox-dependent control of DevS kinase activity. The formation of an intersubunit disulfide bond between the C547 residues in the presence of O₂ led to inactivation of DevS kinase activity. The reduction of the oxidized DevS with reductants such as β-mercaptoethanol and dithiothreitol resulted in the restoration of DevS kinase activity. It was demonstrated in vivo by complementation test that the substitution of C547 to alanine partially impaired the sensory function of DevS in M. smegmatis.

Heterologous quaternary structure of CXCL12 and its relationship to the CC chemokine family

X-ray crystallographic studies reveal that CXCL12 is able to form multiple dimer types, a traditional CXC dimer and a 'CC-like' form. Phylogenetic analysis of all known human chemokines demonstrates CXCL12 is more closely related to the CC chemokine class than other CXC chemokines. These observations indicate that CXCL12 contains genomic and structural elements characteristic of both CXC and CC chemokines.Chemokines are members of a superfamily of proteins involved in the migration of cells to the proper anatomical position during embryonic development or in response to infection or stress during an immune response. There are two major (CC and CXC) and two minor (CX3C and XC) families based on the sequence around the first conserved cysteine. The topology of all structures is essentially identical with a flexible N-terminal region of 3-8 amino acids, a 10-20 residue N-terminal loop, a short 3{sub 10}-helix, three {beta}-strands, and a {alpha}-helix. The major consequence of the subtle difference between the families occurs at the oligomeric level. Monomers of the CC, CXC, and CX3C families form dimers in a family-specific manner. The XCL1 chemokine is a monomer that can interconvert between two folded states. All chemokines activate GPCRs according to family-specificity, however there are a fewmore » examples of chemokines crossing the family boundary to function as antagonists. A two-stage mechanism for chemokine activation of GPCRs has been proposed. The N-terminal region of the receptor interacts with the chemokine, followed by receptor activation by the chemokine N-terminal region. Monomeric chemokines have been demonstrated to be the active form for receptor function. There are numerous examples of both chemokines and their receptors forming dimers. While family-specific dimerization may be an attractive explanation for why specific chemokines only activate GPCRs within their own family, the role of dimers in the function of chemokines has not been resolved. Given that CXCL12 is in the CXC family, the CXC dimer is considered the physiologic dimer in all previous studies based on crystallographic evidence. NMR and mutational studies agree with the CXC dimer form in solution. The CXC form of the dimer is seen in recent structures of CXCL12 bound to a heparin disaccharide and several CXCR4 peptides. In one case, crystals of the CXC-type dimer were soaked in a heparin disaccharide solution to determine the interactions between this dimer and bound disaccharide. In another case, in order to overcome NMR chemical shift line broadening when CXCR4 peptides are added, a 'locked' dimer was constructed by introducing a cysteine mutant that linked subunits as a CXC dimer through an inter-subunit disulfide bond. The solution structures of the locked CXC dimer with CXCR4 peptides were determined. The locked CXC dimer retained Ca{sup 2+} mobilization yet lost chemotaxis activity, presumably because the monomer is the active form. In addition to existing as a monomer and CXC dimer, CXCL12 is now demonstrated to have the capacity to form CC type dimers in the presence of a CXCR4 peptide.« less

Oxidant Regulated Intersubunit Disulfide Bond Formation between ASIC1a Subunits

The acid‐sensing ion channel‐1a (ASIC1a) is composed of three subunits and is activated by a fall in extracellular pH. It plays an important role in diseases associated with a reduced pH and production of oxidants. Previous work showed that oxidants reduce ASIC1a currents. However, the effects on channel structure and composition are unknown. We found that ASIC1a formed intersubunit disulfide bonds and the oxidant H2O2 increased this link between subunits. Cys495 in the ASIC1a C‐terminus was particularly important for intersubunit disulfide bond formation, although other C‐terminal cysteines contributed. Intersubunit disulfide bonds produced some ASIC1a complexes larger than trimers. Intersubunit disulfide bond formation also reduced the proportion of ASIC1a located on the cell surface and contributed to the H2O2‐induced decrease in H+‐gated current. These results indicate that channel function is controlled by disulfide bond formation between intracellular residues on distinct ASIC1a subunits. They also suggest a mechanism by which the redox state can dynamically regulate membrane protein activity by forming intracellular bridges.

Insulin‐mediated redox regulation of cyclic AMP‐dependent protein kinase (PKA) in rat adipocytes

Lipolysis and lipogenesis are two highly regulated pathways. Catecholamines promote lipolysis via cAMP, whereas insulin stimulates lipogenesis. H2O2 mimics insulin action inhibiting lipolysis, and insulin stimulates NADPH oxidase in adipocytes and transiently generates H2O2. Therefore, the possibility that H2O2 might regulate cAMP‐dependent protein kinase (PKA) was explored.Results show that H2O2 generated by insulin in rat adipocytes impaired cAMP‐mediated amplification cascade of lipolysis. Micromolar concentrations of H2O2 added before cAMP suppressed cAMP activation of both type IIß or type IIα cyclic AMP‐dependent protein kinase (PKA) holoenzyme. However, H2O2 was ineffective on reconstituted PKA holoenzyme preincubated with cAMP, or if added to the catalytic αsubunit alone. The same results were obtained if the catalytic αsubunit was substituted by its C199A mutant. H2O2 inhibition of PKA activation remained after H2O2 elimination but was reverted with thioredoxin. Electrophoresis of holoenzyme in SDS gels showed separation of catalytic and regulatory subunits after cAMP incubation but a single band after H2O2 incubation. These data strongly suggest that H2O2 promotes the formation of an intersubunit disulfide bond, impairing PKA activation. Phylogenetic analysis showed that Cys‐97 is conserved only in type II (but not type I) regulatory subunits. Supported by DGAPA‐UNAM grant IN208308

Oxidant regulated inter-subunit disulfide bond formation between ASIC1a subunits

The acid-sensing ion channel-1a (ASIC1a) is composed of 3 subunits and is activated by a decrease in extracellular pH. It plays an important role in diseases associated with a reduced pH and production of oxidants. Previous work showed that oxidants reduce ASIC1a currents. However, the effects on channel structure and composition are unknown. We found that ASIC1a formed inter-subunit disulfide bonds and the oxidant H(2)O(2) increased this link between subunits. Cys-495 in the ASIC1a C terminus was particularly important for inter-subunit disulfide bond formation, although other C-terminal cysteines contributed. Inter-subunit disulfide bonds also produced some ASIC1a complexes larger than trimers. Inter-subunit disulfide bond formation reduced the proportion of ASIC1a located on the cell surface and contributed to the H(2)O(2)-induced decrease in H(+)-gated current. These results indicate that channel function is controlled by disulfide bond formation between intracellular residues on distinct ASIC1a subunits. They also suggest a mechanism by which the redox state can dynamically regulate membrane protein activity by forming intracellular bridges.

Human pancreas-specific protein disulfide isomerase homolog (PDIp) is redox-regulated through formation of an inter-subunit disulfide bond

Protein disulfide isomerase (PDI) and its homologs are catalysts of the formation of disulfide bonds in secretory proteins, and they also serve as molecular chaperones. In the present study, we investigated the redox-mediated regulation of the structures and functions of human pancreas-specific PDI homolog (PDIp). We found that formation of an inter-subunit disulfide bond in the recombinant human PDIp can alter not only its structure, but also its functions. PDIp exists predominantly as monomer under reducing conditions, but the dimeric form is significantly increased following the removal of the reducing agent, due to the formation of an inter-subunit disulfide bond. The oxidized PDIp (with an inter-subunit disulfide bond) appears to expose more hydrophobic patches and is more sensitive to protease digestion compared to the reduced form. Along with these structural changes, the oxidized PDIp also exhibits an enhanced chaperone activity. The formation of the inter-subunit disulfide bond in PDIp is mainly contributed by its non-active cysteine residue (cysteine-4), which is only present in human and primate PDIp, but not in rodent PDIp. In addition, we observed that the formation of the inter-subunit disulfide bond in PDIp is redox-dependent and is favored under oxidizing conditions, and that PDIp can function as a chaperone to form stable complexes with various non-native cellular proteins, particularly under oxidizing conditions. In light of these observations, it is concluded that the structures and functions of human PDIp are redox-regulated through formation of an inter-subunit disulfide bond between two cysteine-4 residues.

Membrane-damaging activity with A chain and B chain of β-bungarotoxin

β-Bungarotoxin (β-Bgt) consists of A chain (a phospholipase A 2 subunit) and B chain, cross-linked by an intersubunit disulfide bridge. In contrast to a marginal activity noted with β-Bgt, recombinant A1 chain and B1 chain markedly induced release of calcein from phospholipid vesicles. Reduction of intersubunit disulfide bond by dithiothreitol or glutathione enhanced membrane-damaging activity of β-Bgt. Moreover, phospholipid-binding capability of recombinant A1 and B1 chains was higher than that of β-Bgt. In contrast to β-Bgt, A1 and B1 chains preferably bound lipids with a preference for anionic over zwitterionic phospholipids. Removal of positively charged residues lying on the interface between A chain and B chain resulted in abolishment of membrane-permeabilizing activity of B chain. Taken together, our data indicate that both A and B chains possess the capability to induce vesicle leakage, and reduction of interchain disulfide bond markedly releases this ability from intact β-Bgt molecule.

Preventing Voltage-dependent Gating of Anthrax Toxin Channels Using Engineered Disulfides

The channel-forming component of anthrax toxin, (PA63)7, is a heptameric water-soluble protein at neutral pH, but under acidic conditions it spontaneously inserts into lipid bilayers to form a 14-stranded β-barrel ion-conducting channel. This channel plays a vital role in anthrax pathogenesis because it serves as a conduit for the membrane translocation of the two enzymatic components of anthrax toxin, lethal factor and edema factor. Anthrax channels open and close in response to changes in transmembrane voltage, a property shared by several other pore-forming toxins. We have discovered an unexpected phenomenon in cysteine-substituted channels that provides a window into this gating process: their normal voltage-dependent gating can be abolished by reaction with methanethiosulfonate (MTS) reagents or exposure to oxidizing conditions. Remarkably, this perturbation is seen with cysteines substituted at sites all along the ∼100 Å length of the channel's β-barrel. In contrast, reaction with N-ethylmaleimide, a thiol-reactive compound that does not form a mixed disulfide, does not affect gating at any of the sites tested. These findings, coupled with our biochemical detection of dimers, have led us to conclude that MTS reagents are catalyzing the formation of intersubunit disulfide bonds that lock channels in a conducting state, and that voltage gating requires a conformational change that involves the entire β-barrel.

Stabilization of tRNA (m1G37) methyltransferase [TrmD] from Aquifex aeolicus by an intersubunit disulfide bond formation

Recombinant Aquifex aeolicus TrmD protein has a Cys20-Cys20 disulfide bond between its two subunits. This was demonstrated by SDS-polyacrylamide gel analysis of wild-type enzyme and C20S mutant protein (in which the Cys20 residue is substituted by serine), in the absence or presence of various concentrations of dithiothreitol. Analytical gel-filtration chromatography revealed that the C20S mutant protein forms a dimer structure even though it is missing the disulfide bond. Western blotting analysis suggests that the Cys20-Cys20 disulfide bond is formed in native TrmD protein in living A. aeolicus cells. Incubation at 85 degrees C for 20 min caused the precipitation of more than half of the C20S protein, while more than 70% of the wild-type enzyme was soluble at that temperature. This assay clearly demonstrates that the disulfide bond enhances the protein stability at 85 degrees C. A kinetic assay showed that the methyl-transfer activity of the C20S mutant protein was slightly less than that of the wild-type enzyme at 70 degrees C. Comparison of the CD-spectra of wild-type and C20S proteins reveals that some of the alpha-helices in the C20S mutant protein are less tightly packed than those of the wild-type enzyme at 70 degrees C.

Conversion of Bacillus subtilis OhrR from a 1-Cys to a 2-Cys Peroxide Sensor

OhrR proteins can be divided into two groups based on their inactivation mechanism: 1-Cys (represented by Bacillus subtilis OhrR) and 2-Cys (represented by Xanthomonas campestris OhrR). A conserved cysteine residue near the amino terminus is present in both groups of proteins and is initially oxidized to the sulfenic acid. The B. subtilis 1-Cys OhrR protein is subsequently inactivated by formation of a mixed-disulfide bond with low-molecular-weight thiols or by cysteine overoxidation to sulfinic and sulfonic acids. In contrast, the X. campestris 2-Cys OhrR is inactivated when the initially oxidized cysteine sulfenate forms an intersubunit disulfide bond with a second Cys residue from the other subunit of the protein dimer. Here, we demonstrate that the 1-Cys B. subtilis OhrR can be converted into a 2-Cys OhrR by introducing another cysteine residue in either position 120 or position 124. Like the X. campestris OhrR protein, these mutants (G120C and Q124C) are inactivated by intermolecular disulfide bond formation. Analysis of oxidized 2-Cys variants both in vivo and in vitro indicates that intersubunit disulfide bond formation can occur simultaneously at both active sites in the protein dimer. Rapid formation of intersubunit disulfide bonds protects OhrR against irreversible overoxidation in the presence of strong oxidants much more efficiently than do the endogenous low-molecular-weight thiols.

A Novel Mercaptopyruvate Sulfurtransferase Thioredoxin-Dependent Redox-Sensing Molecular Switch: A Mechanism for the Maintenance of Cellular Redox Equilibrium

An intermolecular disulfide bond serves as a thioredoxin-dependent redox-sensing switch for the regulation of the enzymatic activity of 3-mercaptopyruvate sulfurtransferase (MST, EC.2.8.1.2). A cysteine residue on the surface of each subunit was oxidized to form an intersubunit disulfide bond so as to decrease MST activity, and thioredoxin-specific conversion of a dimer to a monomer increased MST activity. Further, a low redox potential sulfenate was reversibly formed at a catalytic site cysteine so as to inhibit MST, and thioredoxin-dependent reduction of the sulfenate restored the MST activity. Concludingly, MST partly contributes to the maintenance of cellular redox homeostasis via exerting control over cysteine catabolism.

Signaling the Signal, Cyclic AMP-dependent Protein Kinase Inhibition by Insulin-formed H2O2 and Reactivation by Thioredoxin

Catecholamines in adipose tissue promote lipolysis via cAMP, whereas insulin stimulates lipogenesis. Here we show that H(2)O(2) generated by insulin in rat adipocytes impaired cAMP-mediated amplification cascade of lipolysis. These micromolar concentrations of H(2)O(2) added before cAMP suppressed cAMP activation of type IIbeta cyclic AMP-dependent protein kinase (PKA) holoenzyme, prevented hormone-sensitive lipase translocation from cytosol to storage droplets, and inhibited lipolysis. Similarly, H(2)O(2) impaired activation of type IIalpha PKA holoenzyme from bovine heart and from that reconstituted with regulatory IIalpha and catalytic alpha subunits. H(2)O(2) was ineffective (a) if these PKA holoenzymes were preincubated with cAMP, (b) if added to the catalytic alpha subunit, which is active independently of cAMP activation, and (c) if the catalytic alpha subunit was substituted by its C199A mutant in the reconstituted holoenzyme. H(2)O(2) inhibition of PKA activation remained after H(2)O(2) elimination by gel filtration but was reverted with dithiothreitol or with thioredoxin reductase plus thioredoxin. Electrophoresis of holoenzyme in SDS gels showed separation of catalytic and regulatory subunits after cAMP incubation but a single band after H(2)O(2) incubation. These data strongly suggest that H(2)O(2) promotes the formation of an intersubunit disulfide bond, impairing cAMP-dependent PKA activation. Phylogenetic analysis showed that Cys-97 is conserved only in type II regulatory subunits and not in type I regulatory subunits; hence, the redox regulation mechanism described is restricted to type II PKA-expressing tissues. In conclusion, phylogenetic analysis results, selective chemical behavior, and the privileged position in holoenzyme lead us to suggest that Cys-97 in regulatory IIalpha or IIbeta subunits is the residue forming the disulfide bond with Cys-199 in the PKA catalytic alpha subunit. A new molecular point for cross-talk among heterologous signal transduction pathways is demonstrated.

The crystal structure of the C45S mutant of annelid Arenicola marina peroxiredoxin 6 supports its assignment to the mechanistically typical 2‐Cys subfamily without any formation of toroid‐shaped decamers

The peroxiredoxins (PRDXs) define a superfamily of thiol-dependent peroxidases able to reduce hydrogen peroxide, alkyl hydroperoxides, and peroxynitrite. Besides their cytoprotective antioxidant function, PRDXs have been implicated in redox signaling and chaperone activity, the latter depending on the formation of decameric high-molecular-weight structures. PRDXs have been mechanistically divided into three major subfamilies, namely typical 2-Cys, atypical 2-Cys, and 1-Cys PRDXs, based on the number and position of cysteines involved in the catalysis. We report the structure of the C45S mutant of annelid worm Arenicola marina PRDX6 in three different crystal forms determined at 1.6, 2.0, and 2.4 A resolution. Although A. marina PRDX6 was cloned during the search of annelid homologs of mammalian 1-Cys PRDX6s, the crystal structures support its assignment to the mechanistically typical 2-Cys PRDX subfamily. The protein is composed of two distinct domains: a C-terminal domain and an N-terminal domain exhibiting a thioredoxin fold. The subunits are associated in dimers compatible with the formation of intersubunit disulfide bonds between the peroxidatic and the resolving cysteine residues in the wild-type enzyme. The packing of two crystal forms is very similar, with pairs of dimers associated as tetramers. The toroid-shaped decamers formed by dimer association and observed in most typical 2-Cys PRDXs is not present. Thus, A. marina PRDX6 presents structural features of typical 2-Cys PRDXs without any formation of toroid-shaped decamers, suggesting that it should function more like a cytoprotective antioxidant enzyme or a modulator of peroxide-dependent cell signaling rather than a molecular chaperone.