Evidence for oligomeric forms of transducins alpha subunit: formation of intermolecular alpha-alpha disulfide linkages

Transducin, the retinal G-protein, is a heterotrimeric protein composed of alpha, beta and gamma subunits. Intermolecular disulfide linkages between the alpha-subunits of transducin molecules are spontaneously formed when the purified G-protein is placed in a non-reducing buffer system. The beta and gamma subunits do not participate in the intermolecular disulfide bridge formation. The alpha-alpha subunit disulfide bonds result in the inhibition of transducin activation by bleached rhodopsin which is restored by reducing the disulfides with dithiothreitol. The trapping of oligomers by disulfide bond formation provides physical evidence for specific intermolecular interactions between alpha-subunits of transducin.     

Human cofilin forms oligomers exhibiting actin bundling activity.

Human cofilin possesses the tendency for self-association, as indicated by the rapid formation of dimers and oligomers when reacted with water-soluble carbodiimide, Ellman's reagent, or glutathione disulfide. Intermolecular disulfide bonds involve Cys(39) and probably Cys(147) of two adjacent cofilin units. The disulfide-linked dimers and oligomers exhibit a biological activity distinct from the monomer. While monomeric cofilin decreased viscosity and light-scattering of F-actin solutions, dimers and oligomers caused an increase in viscosity and light scattering. Electron microscopy revealed that cofilin oligomers induce the formation of highly ordered actin bundles with occasionally blunt ends similar to actin-cofilin rods observed in cells under oxidative stress. Bundling activity of the disulfide-linked oligomers could be completely reversed into severing activity by dithiothreitol. Formation of cofilin oligomers occurred also in the presence of actin at pH 8, but not at pH 6.6, and was significantly enhanced in the presence of phosphatidylinositol 4,5-bisphosphate. Our data are consistent with the idea that cofilin exists in two forms in vivo also: as monomers exhibiting the known severing activity and as oligomers exhibiting actin bundling activity. However, stabilization of cofilin oligomers in cytoplasm is probably achieved not by disulfide bonds but by a local increase in cofilin concentration and/or binding of regulatory proteins.     

Disulfide-mediated oligomerization of Peripherin/Rds and Rom-1 in photoreceptor disk membranes. Implications for photoreceptor outer segment morphogenesis and degeneration

Peripherin/Rds is a tetraspanning membrane protein that has been implicated in photoreceptor outer segment morphogenesis and inherited retinal degenerative diseases. Together with the structurally related protein, Rom-1, it forms a complex along the rims of rod and cone disc membranes. We have compared the oligomeric structure of these proteins from nonreduced and dithiothreitol reduced membranes by velocity sedimentation, SDS-gel electrophoresis, immunoaffinity chromatography, and chemical cross-linking. Under reducing conditions peripherin/Rds and Rom-1 existed as homomeric and heteromeric core complexes devoid of intermolecular disulfide bonds. Under nonreducing conditions core complexes associated through intermolecular disulfide bonds to form oligomers. One intermediate-size oligomer contained monomers and disulfide-linked dimers of peripherin/Rds and Rom-1, while larger oligomers consisted only of disulfide-linked peripherin/Rds dimers when analyzed on nonreducing SDS gels. Consistent with this result, disc membranes contained twice as much peripherin/Rds as Rom-1. Peripherin/Rds individually expressed in COS-1 cells also formed disulfide-linked oligomers bridged through Cys-150 residues, whereas Rom-1 showed little tendency to form oligomers. These results indicate that peripherin/Rds and Rom-1 associate noncovalently to form multisubunit core complexes. Peripherin/Rds containing core complexes interact through specific intermolecular disulfide bonds to form oligomers which may play a crucial role in photoreceptor disc morphogenesis and retinal degenerative diseases.     

Biochemical and biophysical characterization of a novel plant protein disulfide isomerase

We recently isolated a protein disulfide isomerase (PDI) from the Rubiaceae (coffee family) plant Oldenlandia affinis (OaPDI) and demonstrated that it facilitates the production of disulfide-knotted defense proteins called cyclotides. PDIs are major folding catalysts in the eukaryotic ER where they are responsible for formation, breakage, or shuffling of disulfide bonds in substrate polypeptides and are important chaperones in the secretory pathway. Here, we report the first detailed analysis of the oligomerization behavior of a plant PDI, based on characterization of OaPDI using various biochemical and biophysical techniques, including size-exclusion chromatography, NMR spectroscopy, surface plasmon resonance and atomic force microscopy. In solution at low concentration OaPDI comprises mainly monomers, but fractions of dimers and/or higher-order oligomers were observed at increased conditions, raising the possibility that dimerization and/or oligomerization could be a mechanism to adapt to the various-sized polypeptide substrates of PDI. Unlike mammalian PDIs, oligomerization of the plant PDI is not driven by the formation of intermolecular disulfide bonds, but by noncovalent interactions. The information derived in this study advances our understanding of the oligomerization behavior of OaPDI in particular but is potentially of broader interest for understanding the mechanism and role of oligomerization, and hence the catalytic and physiological mechanism, of the ubiquitous folding catalyst PDI.     

Stress-Activated Protein Kinase/c-Jun N-Terminal Kinase Phosphorylates τ Protein

Abstract: A proportion of the neuronal microtubule-associated protein (MAP) τ is highly phosphorylated in foetal and adult brain, whereas the majority of τ in the neurofibrillary tangles of Alzheimer's patients is hyperphosphorylated; many of the phosphorylation sites are serines or threonines followed by prolines. Several kinases phosphorylate τ at such sites in vitro. We have now shown that purified recombinant stress-activated protein kinase/c-Jun N-terminal kinase, a proline-directed kinase of the MAP kinase extended family, phosphorylates recombinant τ in vitro on threonine and serine residues. Western blots using antibodies to phosphorylation-dependent τ epitopes demonstrated that phosphorylation occurs in both of the main phosphorylated regions of τ protein. Unlike glycogen synthase kinase-3, the c-Jun N-terminal kinase readily phosphorylates Thr²⁰⁵ and Ser⁴²², which are more highly phosphorylated in Alzheimer τ than in foetal or adult τ. Glycogen synthase kinase-3 may preferentially phosphorylate the sites found physiologically, in foetal and to a smaller extent in adult τ, whereas stress-activated/c-Jun N-terminal kinase and/or other members of the extended MAP kinase family may be responsible for pathological proline-directed phosphorylations. Inflammatory processes in Alzheimer brain might therefore contribute directly to the pathological formation of the hyperphosphorylated τ found in neurofibrillary tangles.     

Conserved Cysteine Residues Provide a Protein-Protein Interaction Surface in Dual Oxidase (DUOX) Proteins

Intramolecular disulfide bond formation is promoted in oxidizing extracellular and endoplasmic reticulum compartments and often contributes to protein stability and function. DUOX1 and DUOX2 are distinguished from other members of the NOX protein family by the presence of a unique extracellular N-terminal region. These peroxidase-like domains lack the conserved cysteines that confer structural stability to mammalian peroxidases. Sequence-based structure predictions suggest that the thiol groups present are solvent-exposed on a single protein surface and are too distant to support intramolecular disulfide bond formation. To investigate the role of these thiol residues, we introduced four individual cysteine to glycine mutations in the peroxidase-like domains of both human DUOXs and purified the recombinant proteins. The mutations caused little change in the stabilities of the monomeric proteins, supporting the hypothesis that the thiol residues are solvent-exposed and not involved in disulfide bonds that are critical for structural integrity. However, the ability of the isolated hDUOX1 peroxidase-like domain to dimerize was altered, suggesting a role for these cysteines in protein-protein interactions that could facilitate homodimerization of the peroxidase-like domain or, in the full-length protein, heterodimeric interactions with a maturation protein. When full-length hDUOX1 was expressed in HEK293 cells, the mutations resulted in decreased H₂O₂ production that correlated with a decreased amount of the enzyme localized to the membrane surface rather than with a loss of activity or with a failure to synthesize the mutant proteins. These results support a role for the cysteine residues in intermolecular disulfide bond formation with the DUOX maturation factor DUOXA1.     

Self-association of the histidine kinase CheA as studied by pulsed dipolar ESR spectroscopy

Biologically important protein complexes often involve molecular interactions that are low affinity or transient. We apply pulsed dipolar electron spin resonance spectroscopy and site-directed spin labeling in what to our knowledge is a new approach to study aggregation and to identify regions on protein surfaces that participate in weak, but specific molecular interactions. As a test case, we have probed the self-association of the chemotaxis kinase CheA, which forms signaling clusters with chemoreceptors and the coupling protein CheW at the poles of bacterial cells. By measuring the intermolecular dipolar interactions sensed by spin-labels distributed over the protein surface, we show that the soluble CheA kinase aggregates to a small extent through interactions mediated by its regulatory (P5) domain. Direct dipolar distance measurements confirm that a hydrophobic surface at the periphery of P5 subdomain 2 associates CheA dimers in solution. This result is further supported by differential disulfide cross-linking from engineered cysteine reporter sites. We suggest that the periphery of P5 is an interaction site on CheA for other similar hydrophobic surfaces and plays an important role in structuring the signaling particle.     

Lamin dimers. Presence in the nuclear lamina of surf clam oocytes and release during nuclear envelope breakdown

The nuclear lamina of surf clam oocytes contains dimers of 67-kDa lamin which are stabilized by both noncovalent interactions and disulfide bonds. The latter can be reduced but re-form when the reducing agent is removed. The cysteine residues involved in these disulfide bonds are inaccessible to alkylating agents unless the protein is unfolded in urea. During nuclear envelope breakdown the lamin is released as a mixture of oligomers in which disulfide-stabilized dimers are associated noncovalently with lamin monomers. Concurrent with solubilization, both dimers and monomers are phosphorylated to a similar extent, indicating that the interactions which maintain these complexes are not destabilized by lamin phosphorylation. Our results suggest the existence of two types of interactions between the lamin molecules in the polymer, which react differently to phosphorylation during nuclear envelope breakdown.     

τ Protein Kinase II Is Involved in the Regulation of the Normal Phosphorylation State of τ Protein

Abstract: To study the phosphorylation state of τ in vivo, we have prepared antisera by immunizing rabbits with synthetic phosphopeptides containing phosphoamino acids at specific sites that are potential targets for τ protein kinase II. Immunoblot experiments using these antisera demonstrated that τ in microtubule-associated proteins is phosphorylated at Ser¹⁴⁴ and at Ser³¹⁵. Almost all τ variants separated on two-dimensional gel electrophoresis were phosphorylated at Ser¹⁴⁴ and nearly one-half of them at Ser³¹⁵. Phosphorylation at Ser¹⁴⁴ and at Thr¹⁴⁷ of τ isolated from heat-stable brain extracts was shown to be developmentally regulated, with the highest level of phosphorylation found at postnatal week 1. In vitro phosphorylation of τ by τ protein kinase I, a kinase responsible for abnormal phosphorylation of τ found in paired helical filaments of patients with Alzheimer's disease, was enhanced by prior phosphorylation of τ by τ protein kinase II. Thus, we suggest that τ protein kinase II is indirectly involved, at least in part, in the regulation of the phosphorylation state of τ in neuronal cells.     

Interchain disulfide bonds promote protein cross-linking during protein folding

We provide evidence that in vitro protein cross-linking can be accomplished in three concerted steps: (i) a change in protein conformation; (ii) formation of interchain disulfide bonds; and (iii) formation of interchain isopeptide cross-links. Oxidative refolding and thermal unfolding of ribonuclease A, lysozyme, and protein disulfide isomerase led to the formation of cross-linked dimers/oligomers as revealed by SDS-polyacrylamide gel electrophoresis. Chemical modification of free amino groups in these proteins or unfolding at pH < 7.0 resulted in a loss of interchain isopeptide cross-linking without affecting interchain disulfide bond cross-linking. Furthermore, preformed interchain disulfide bonds were pivotal for promoting subsequent interchain isopeptide cross-links; no dimers/oligomers were detected when the refolding and unfolding solution contained the reducing agent dithiothreitol. Similarly, the Cys326Ser point mutation in protein disulfide isomerase abrogated its ability to cross-link into homodimers. Heterogeneous proteins become cross-linked following the formation of heteromolecular interchain disulfide bonds during thermal unfolding of a mixture of of ribonuclease A and lysozyme. The absence of glutathione and glutathione disulfide during the unfolding process attenuated both the interchain disulfide bond cross-links and interchain isopeptide cross-links. No dimers/oligomers were detected when the thermal unfolding temperature was lower than the midpoint of thermal denaturation temperature.     

Reducer driven baric denaturation and oligomerisation of whey proteins

The influence of high pressure on alpha-lactalbumin (ALA)/beta-lactoglobulin (BLG) mixtures of various compositions was studied at pH 8.5 by gel-permeation chromatography and sodium dodecyl sulphate (SDS) gel electrophoresis without 2-mercaptoethanol. High-molecular protein disulfide oligomers formed after denaturation by the pressure of 10 kbar (1000 MPa) if the weight fraction of BLG (W(BLG)(0)) in the protein mixture exceeded 0.2. The maximum yield of these oligomers of order 80-85% is observed at W(BLG)(0)>/=0.4. Conversions of both proteins in the oligomers are roughly the same. The estimates of the oligomerisation yield obtained by the gel-permeation chromatography and SDS gel electrophoresis agree well. This indicates that the formation of intermolecular disulfide bonds is necessary for the oligomerisation. Thus, the oligomerisation of pressure denatured ALA and BLG is driven by the thiol<-->disulfide exchange rendered possible by the vigorous baric denaturation and the exposure of the free thiol group of BLG, which acts as an initiator of disulfide bridges scrambling.     

Dissociation of intermolecular disulfide bonds in P22 tailspike protein intermediates in the presence of SDS

Each chain of the native trimeric P22 tailspike protein has eight cysteines that are reduced and buried in its hydrophobic core. However, disulfide bonds have been observed in the folding pathway and they are believed to play a critical role in the registration of the three chains. Interestingly, in the presence of sodium dodecyl sulfate (SDS) only monomeric chains, rather than disulfide-linked oligomers, have been observed from a mixture of folding intermediates. Here we show that when the oligomeric folding intermediates were separated from the monomer by native gel electrophoresis, the reduction of intermolecular disulfide bonds did not occur in the subsequent second-dimension SDS-gel electrophoresis. This result suggests that when tailspike monomer is present in free solution with SDS, the partially unfolded tailspike monomer can facilitate the reduction of disulfide bonds in the tailspike oligomers.     

Disulfide Scrambling Describes the Oligomer Formation of Superoxide Dismutase (SOD1) Proteins in the Familial Form of Amyotrophic Lateral Sclerosis

Dominant mutations in Cu,Zn-superoxide dismutase (SOD1) are a cause of a familial form of amyotrophic lateral sclerosis. Wild-type SOD1 forms a highly conserved intra-molecular disulfide bond, whereas pathological SOD1 proteins are cross-linked via intermolecular disulfide bonds and form insoluble oligomers. A thiol-disulfide status in SOD1 will thus play a regulatory role in determining its folding/misfolding pathways; however, it remains unknown how pathogenic mutations in SOD1 affect the thiol-disulfide status to facilitate the protein misfolding. Here, we show that the structural destabilization of SOD1 scrambles a disulfide bond among four Cys residues in an SOD1 molecule. The disulfide scrambling produces SOD1 monomers with distinct electrophoretic mobility and also reproduces the formation of disulfide-linked oligomers. We have also found that the familial form of amyotrophic lateral sclerosis-causing mutations facilitate the disulfide scrambling in SOD1. Based upon our results, therefore, scrambling of the conserved disulfide bond will be a key event to cause the pathological changes in disease-associated mutant SOD1 proteins.     

Oligomerization of H(+)-pyrophosphatase and its structural and functional consequences

The H(+)-pyrophosphatase (H(+)-PPase) consists of a single polypeptide, containing 16 or 17 transmembrane domains. To determine the higher order oligomeric state of Streptomyces coelicolor H(+)-PPase, we constructed a series of cysteine substitution mutants and expressed them in Escherichia coli. Firstly, we analyzed the formation of disulfide bonds, promoted by copper, in mutants with single cysteine substitutions. 28 of 39 mutants formed disulfide bonds, including S545C, a substitution at the periplasmic side. The formation of intermolecular disulfide bonds suppressed the enzyme activity of several, where the substituted residues were located in the cytosol. Creating disulfide links in the cytosol may interfere with the enzyme's catalytic function. Secondly, we prepared double mutants by introducing second cysteine substitutions into the S545C mutant. These double-cysteine mutants produced cross-linked complexes, estimated to be at least tetramers and possibly hexamers. Thirdly, we co-expressed epitope-tagged, wild type, and inactive mutant H(+)-PPases in E. coli and confirmed the formation of oligomers by co-purifying one subunit using the epitope tag used to label the other. The enzyme activity of these oligomers was markedly suppressed. We propose that H(+)-PPase is present as an oligomer made up of at least two or three sets of dimers.     

Oligomerization and Phosphorylation Dependent Regulation of ArgBP2 Adaptive Capabilities and Associated Functions

ArgBP2 (Arg-Binding Protein 2/SORBS2) is an adaptor protein involved in cytoskeleton associated signal transduction, thereby regulating cell migration and adhesion. These features are associated with its antitumoral role in pancreatic cancer cells. Tyrosine phosphorylation of ArgBP2, mediated by c-Abl kinase and counterbalanced by PTP-PEST phosphatase, regulates many of its interactions. However, the exact mechanisms of action and of regulation of ArgBP2 remain largely unknown. We found that ArgBP2 has the capacity to form oligomers which are destabilized by tyrosine phosphorylation. We could show that ArgBP2 oligomerization involves the binding of one of its SH3 domains to a specific proline rich cluster. ArgBP2 self-association increases its binding to some of its molecular partners and decreased its affinity for others. Hence, the phosphorylation/oligomerization state of ArgBP2 directly regulates its functions by modulating its adaptive capabilities. Importantly, using a human pancreatic cancer cell model (MiaPaCa-2 cells), we could validate that this property of ArgBP2 is critical for its cytoskeleton associated functions. In conclusions, we describe a new mechanism of regulation of ArgBP2 where tyrosine phosphorylation of the protein interfere with a SH3 mediated self-interaction, thereby controlling its panel of interacting partners and related functions.     

Phosphorylation of τ In Situ: Inhibition of Calcium-Dependent Proteolysis

Abstract: In this study, the in situ phosphorylation and subsequent calcium-activated proteolysis of τ protein were examined in human neuroblastoma (LA-N-5) cells, which were differentiated into a neuronal phenotype. The phosphorylation of τ was increased by treating the cells with forskolin and rolipram, which elevate cyclic AMP levels, by treating with the phosphatase inhibitor okadaic acid, or by treating with a combination of both treatments. Phosphorylated τ migrated slightly slower on sodium dodecyl sulfate-polyacrylamide gels than τ from untreated cells. Immunostaining with the phosphate-sensitive monoclonal antibody Tau-1 was also decreased in cells treated with okadaic acid, indicating an increase in the phosphorylation of specific Ser-Pro motifs within the molecule. Calcium-dependent, in situ proteolysis of τ protein was induced by treating the cells with the calcium ionophore A23187. τ protein was proteolyzed to a significantly lesser extent in cells treated with forskolin and rolipram, okadaic acid, or both than in cells in which phosphorylation was not increased. Partially purified τ protein from cells treated with a combination of forskolin, rolipram, and okadaic acid was also more resistant to proteolysis by calpain in vitro compared with τ isolated from control cells. These data suggest a possible role for phosphorylation in the regulation of τ metabolism and in pathological conditions in which the balance between protein kinases and phosphatases is disrupted.     

Changes in Phosphorylation of τ During Ischemia and Reperfusion in the Rabbit Spinal Cord

Abstract: The microtubule-associated protein τ plays an important role in the dynamics of microtubule assembly necessary for axonal growth and neurite plasticity. Ischemia disrupts the neuronal cytoskeleton both by promoting proteolysis of its components and by affecting kinase and phosphatase activities that alter its assembly. In this study the effect of ischemia and reperfusion on the expression and phosphorylation of τ was examined in a reversible model of spinal cord ischemia in rabbits. τ was found to be dephosphorylated in response to ischemia with a time course that closely matched the production of permanent paraplegia. Dephosphorylation of τ was limited to the caudal lumbar spinal cord. In a similar manner, Ca²⁺/calmodulin-dependent kinase II activity was reduced only in the ischemic region. Thus, dephosphorylation of τ is an early marker of ischemia as is the rapid loss of Ca²⁺/calmodulin-dependent kinase II activity, τ, however, was rephosphorylated rapidly during reperfusion at site(s) that cause a reduction in its electrophoretic mobility regardless of the neurological outcome. Alterations in phosphorylation or degradation of τ may affect microtubule stability, possibly contributing to disruption of axonal transport but also facilitating neurite plasticity in a regenerative response.     

Mapping superoxide dismutase 1 domains of non-native interaction: roles of intra- and intermolecular disulfide bonding in aggregation

Superoxide dismutase 1 (SOD1) proteins harboring mutations linked to familial amyotrophic lateral sclerosis (FALS) uniformly show heightened potential to form high molecular weight structures. Here, we examine the domains of SOD1 that are involved in forming these structures (aggregates) and study the role of intra- and intermolecular disulfide bonds. An analysis of disease mutations identified to date reveals a non-random distribution with predominant occurrence at residues within highly conserved beta-strands or at highly conserved residues in loop domains. Using a cell transfection assay for aggregation, we determined that no single domain in SOD1 is indispensable in the formation of sedimentable aggregates, suggesting multiple potential motifs in the protein mediate non-native interactions. By a cell-free aggregation assay, analysis of transgenic mouse tissues, and mutagenesis approaches, we found evidence that redox conditions may modulate SOD1 aggregation; reduction of the native intramolecular disulfide bonds may predispose SOD1 to unfolding and aggregation, whereas non-native intermolecular disulfide linkages may help stabilize aggregates in vivo. The results suggest a possible mechanism for diversity in the structures formed by different SOD1 mutants, and define a potential contribution of redox conditions to SOD1 aggregation.     

Oligomerization of H-pyrophosphatase and its structural and functional consequences

The H⁺-pyrophosphatase (H⁺-PPase) consists of a single polypeptide, containing 16 or 17 transmembrane domains. To determine the higher order oligomeric state of Streptomyces coelicolor H⁺-PPase, we constructed a series of cysteine substitution mutants and expressed them in Escherichia coli. Firstly, we analyzed the formation of disulfide bonds, promoted by copper, in mutants with single cysteine substitutions. 28 of 39 mutants formed disulfide bonds, including S545C, a substitution at the periplasmic side. The formation of intermolecular disulfide bonds suppressed the enzyme activity of several, where the substituted residues were located in the cytosol. Creating disulfide links in the cytosol may interfere with the enzyme's catalytic function. Secondly, we prepared double mutants by introducing second cysteine substitutions into the S545C mutant. These double-cysteine mutants produced cross-linked complexes, estimated to be at least tetramers and possibly hexamers. Thirdly, we co-expressed epitope-tagged, wild type, and inactive mutant H⁺-PPases in E. coli and confirmed the formation of oligomers by co-purifying one subunit using the epitope tag used to label the other. The enzyme activity of these oligomers was markedly suppressed. We propose that H⁺-PPase is present as an oligomer made up of at least two or three sets of dimers.