Stable Isoform Research Articles

Update on biochemical properties of recombinant Pseudomonas diminuta phosphotriesterase

Phosphotriesterase from Pseudomonas diminuta (PTE; EC 3.1.8.1) hydrolyzes organophosphate insecticides and chemical warfare agents. The two zinc cations in the active center can be substituted. Co2+-containing PTE is the most efficient but least stable isoform. Gel filtration showed that PTE is monomeric at the submicromolar concentrations used in kinetic assays. The analysis of the recombinant enzyme by X-ray fluorescence spectrometry and CCT-ICP-MS, confirms that recombinant Zn-PTE contains only Zn2+ whereas Co-PTE has Zn2+ and Co2+ in equimolar amount, with Co2+ most likely in the reported labile β-site. We noted that recombinant PTE is unstable at low concentrations and must be stabilized by a protein environment. We tested the effect of excess of various metal cofactors on PTE-catalyzed hydrolysis of paraoxon. We notably observed that ZnCl2 induces a non-competitive partial inhibition of Zn2+- and Co2+-PTE at pH 8.5 (apparent Ki=155 μM and 52 μM, respectively). Inhibition results from interactions with colloidal Zn(OH)2 formed in alkaline buffer that alters the catalytic machinery. NiCl2 caused a similar effect at higher concentrations (apparent Ki=3 mM). We observed that mutating His123, a surface residue close to an alleged allosteric site, dramatically altered the bacterial expression yield of Co2+-PTE, Ki for Zn(OH)2 inhibition, kcat (up to 60 fold) for paraoxon hydrolysis, but not KM. Issues addressed in this work are important for future biotechnological developments of PTE as a detoxifying enzyme.

Enhancing the thermal stability of mitochondrial cytochrome b5 by introducing a structural motif characteristic of the less stable microsomal isoform

Outer mitochondrial membrane cytochrome b5 (OM b5) is the most thermostable cytochrome b5 isoform presently known. Herein, we show that OM b5 thermal stability is substantially enhanced by swapping an apparently invariant motif in its heme-independent folding core with the corresponding motif characteristic of its less stable evolutionary relative, microsomal cytochrome b5 (Mc b5). The motif swap involved replacing two residues, Arg15 with His and Glu20 with Ser, thereby introducing a Glu11-His15-Ser20 H-bonding triad on the protein surface along with a His15/Trp22 pi-stacking interaction. The ferric and ferrous forms of the OM b5 R15H/E20S double mutant have thermal denaturation midpoints (Tm values) of approximately 93 degrees C and approximately 104 degrees C, respectively. A 15 degrees C increase in apoprotein Tm plays a key role in the holoprotein thermal stability enhancement, and is achieved by one of the most common natural mechanisms for stabilization of thermophilic versus mesophilic proteins: raising the unfolding free energy along the entire stability curve.

Comments on Classical and Atypical Scrapie in Domestic Sheep and Goats

Scrapie is a neurodegenerative disease in domestic sheep and goats. Scrapie has a long incubation period that can vary from two to five years. The etiology is an extremely stable isoform (PrP8c) of the normal cellular prion protein (PrPc). The accumulation of this protein in the brain is thought to result in the spongiform encephalopathy that characterizes the disease. Scrapie has been reported in the United Kingdom, Ireland, most European countries, Canada, the United States, India and Japan, but not in Australia and New Zealand. Clinical signs are mostly neurological and diagnosis is usually made with immunohistochemical staining of the brainstem and retropharyngeal lymph nodes. Genetics plays a significant role in the susceptibility and incubation period of the disease and is used in control strategies. The primary method of prevention is maintenance of a closed ewe flock, purchase of sheep from uninfected farms enrolled in an approved flock certification program, or purchase of genetically resistant stock. Following diagnosis of scrapie in a flock, depopulation of genetically susceptible sheep is the only effective control measure at this time. If a sheep or goat flock is diagnosed with atypical scrapie the herd is depopulated.

Generation of a Stable Activated Thrombin Activable Fibrinolysis Inhibitor Variant

Activated thrombin activable fibrinolysis inhibitor (TAFIa), generated upon activation of TAFI, exerts an antifibrinolytic effect. TAFIa is a thermolabile enzyme, inactivated through a conformational change. The objective of the current study was to generate a stable variant of human TAFIa. Using a site-directed as well as a random mutagenesis approach to generate a library of TAFI mutants, we identified two mutations that increase TAFIa stability, i.e. a Ser305 to Cys and a Thr329 to Ile mutation, respectively. Combining these mutations in TAFI-Ala147-Ile325, the most stable isoform of TAFIa (half-life of 9.4 +/- 0.4 min), revealed a TAFIa half-life of 70 +/- 3.1 min (i.e. an 11-fold increase versus 6.3 +/- 0.3 min for TAFIa-Ala147-Thr325, the most frequently occurring isoform of TAFI in humans) at 37 degrees C. Moreover, clot lysis (induced by tissue plasminogen activator) experiments in which TAFI-Ala147-Cys305-Ile325-Ile329 was added to TAFI-depleted plasma revealed a 50% clot lysis time of 313 +/- 77 min (i.e. a 3.0-fold increase versus 117 +/- 10 min for TAFI-Ala147-Thr325). The availability of a more stable TAFIa variant will facilitate the search for inhibitors and allow further structural analysis to elucidate the mechanisms of the instability of TAFIa.

Acute and repeated cocaine induces alterations in FosB/ΔFosB expression in the paraventricular nucleus of the hypothalamus

Apart from activation of the brain reward system, cocaine administration influences the activity of the hypothalamo–pituitary–adrenal (HPA) axis by affecting CRH neurons in the paraventricular nucleus of the hypothalamus (PVN). In order to find a molecular mechanism of cocaine-evoked effects in the PVN, in the present study, we investigated the impact of cocaine on the expression of FosB/ΔFosB transcription factors in the PVN. Using an immunohistochemical method, we found that acute cocaine treatment (25 mg/kg) induced a relatively long-lasting (at least 72 h) expression of FosB/ΔFosB in the PVN, whereas repeated cocaine administration (25 mg/kg, once daily for 5 consecutive days) caused accumulation of FosB/ΔFosB in the PVN. The latter observation was further confirmed by the Western blot technique which revealed that repeated exposure to cocaine specifically increased the expression of a stable isoform of ΔFosB (35 kDa). Using a double-labeling immunofluorescent method, it was established that FosB/ΔFosB proteins induced by repeated cocaine treatment were present in a small population of CRF-immunoreactive neurons of the PVN. Furthermore, it was found that pretreatment with the specific antagonist of dopamine D1-like receptors SCH 23390 (1 mg/kg) attenuated the expression and accumulation of FosB/ΔFosB in the PVN, evoked by repeated cocaine administration. Although functional consequences of the above effects for the process of addiction remain to be established, the obtained results indicate that cocaine administration can produce relatively long-lasting changes in the expression of FosB/ΔFosB transcription factors in PVN neurons (in some populations of CRF-immunoreactive neurons, among others) and that dopamine D1-like receptors are involved in the above effects. Finally, it is proposed that the long-lasting expression as well as the accumulation of ΔFosB in the PVN may constitute a molecular basis underlying adaptive changes occurring in the HPA axis after relatively high doses of cocaine.

Detection of a short CCR5 messenger RNA isoform in human spermatozoa.

It has recently been reported that the Regulated upon Activation of Normal T-cells Expressed and Secreted (RANTES) chemokine may exhibit a chemotactic effect on sperm. The RANTES chemokine acts on target cells by binding to the CCR5 receptor, which is present on the surface of various cells. Spermatozoa contain a complex repertoire of messenger RNAs (mRNAs) that may provide an insight into past events of spermatogenesis. The type and amount of CCR5 chemokine receptor transcript were investigated in spermatozoa that were isolated by the swim-up method from semen samples of men with normozoospermia. Using reverse transcription and real-time quantitative polymerase chain reaction (RQ-PCR) analysis, we found that the CCR5 mRNA isoform in human spermatozoa consists of exons 3 and 4, and is shorter than the transcript in leukocytes. This CCR5 transcript may represent a more stable mRNA isoform; one that is used to biosynthesize the CCR5 receptor in spermatogenesis or the early stages of embryonic development.

Dissociation of ethanol and saccharin preference in fosB knockout mice

The Fos family of transcription factors may play a key role in various forms of brain plasticity. Among different genes coding Fos proteins is the fosB gene. Protein products of the fosB gene are thought to be critically involved in neural adaptations produced by chronic treatment with drugs of abuse. fosB gene transcription leads to accumulation of full-length FosB as well as its truncated form, Δ fosB. Stable isoforms of Δ fosB called chronic FRAs accumulate in the brain after chronic administration of various drugs of abuse. The purpose of the present study was to evaluate the role of the fosB gene in two-bottle choice ethanol self-administration. For this aim, ethanol (2–8% v/v) intake and preference was assessed in fosB mutant ( n=17) and wild-type (WT) mice ( n=16). For comparison, consumption of saccharin (0.05–0.8% w/v) and quinine (15–960 μM) solutions was assessed in the same animals. Ethanol preference in both groups varied from around 50% for the lowest to 20% for the highest ethanol concentration. Neither ethanol intake (g/kg) nor preference differed between the two genotypes. In contrast, saccharin preference, but not intake, was higher in the fosB mutants. Only slight and inconsistent between-group differences were observed in terms of quinine preference. The present results suggest that permanent elimination of fosB gene products does not alter ethanol intake but may enhance preference for sweet solutions in mice.

Inducible, brain region-specific expression of a dominant negative mutant of c-Jun in transgenic mice decreases sensitivity to cocaine

Administration of cocaine induces the Fos family of transcription factors in the striatum, including the nucleus accumbens (NAc), a brain region important for the rewarding effects of addictive drugs. Several Fos proteins are induced acutely by cocaine, with stable isoforms of ΔFosB predominating after chronic drug administration. However, it has been difficult to study the functional consequences of these Fos responses in vivo. Fos proteins heterodimerize with members of the Jun family to form active AP-1 transcription factor complexes. In the present study, we took advantage of this property and generated transgenic mice, using the tetracycline gene regulation system, that support the inducible, brain region-specific expression of a dominant negative mutant form of c-Jun (Δc-Jun), which can antagonize the actions of Fos proteins. Expression of Δc-Jun in the striatum and certain other brain regions of adult mice decreases their development of cocaine-induced conditioned place preference, suggesting reduced sensitivity to the rewarding effects of cocaine. In contrast, Δc-Jun expression had no effect on cocaine-induced locomotor activity or sensitization. However, expression of Δc-Jun in adult mice blocked the ability of chronic cocaine administration to induce three known targets for AP-1 in the NAc: the AMPA glutamate receptor subunit GluR2, the cyclin-dependent protein kinase Cdk5, and the transcription factor nuclear factor-κB (NFκB), without affecting several other proteins examined for comparison. Taken together, these results provide further support for an important role of AP-1-mediated transcription in some of the behavioral and molecular mechanisms underlying cocaine addiction.

Cytosolic isoform-like structure of domain II is important for the stability of ascorbate peroxidase of Galdieria partita under ascorbate-depleted conditions.

Chloroplastic ascorbate peroxidases (APXs), enzymes involved in scavenging system of reactive oxygen species, are rapidly inactivated by attack of hydrogen peroxide if the electron donor ascorbate is not available. In contrast, another APX isoform localized in cytosol is relatively stable. The mechanism of the different stability among APX isoforms remains unknown. We isolated a clone of the cDNA encoding a stable isoform of ascorbate peroxidase (APX-B) of Galdieria partita, an acido- and thermophilic red alga. The recombinant protein of APX-B produced in Escherichia coli retained its activity when it was incubated in the medium containing 10 mM of ascorbate for 3 hr, while recombinant stromal APX of spinach chloroplasts lost its activity within 1 hr due to the depletion of ascorbate. Interestingly, APX-B was a hybrid between the cytosolic and chloroplastic APXs in its primary structure; the N-terminal half (domain I) relatively similar to that of domain I of the chloroplastic isoform of higher plants and the C-terminal half (domain II) similar to that of domain II of the cytosolic isoform. A chimeric APX protein that had a domain I of APX-B fused to a domain II of stromal APX of spinach was created. It showed rapid inactivation when ascorbate was depleted. These results indicate that cytosolic APX-like domain II is important for its stability of Galdieria APX-B and the stability or instability of APX isoforms is due to their structures of domain II, rather than domain I.

DeltaFosB: a molecular switch underlying long-term neural plasticity.

Numerous chronic perturbations have been shown to induce highly stable isoforms of the transcription factor deltaFosB in the brain in a region-specific manner. This review examines the functional consequences of the induction of deltaFosB in particular neuronal populations as well as its possible role in behavioral abnormalities such as drug addiction and movement disorders.

Expression of the transcription factor deltaFosB in the brain controls sensitivity to cocaine.

Acute exposure to cocaine transiently induces several Fos family transcription factors in the nucleus accumbens, a region of the brain that is important for addiction. In contrast, chronic exposure to cocaine does not induce these proteins, but instead causes the persistent expression of highly stable isoforms of deltaFosB. deltaFosB is also induced in the nucleus accumbens by repeated exposure to other drugs of abuse, including amphetamine, morphine, nicotine and phencyclidine. The sustained accumulation of deltaFosB in the nucleus accumbens indicates that this transcription factor may mediate some of the persistent neural and behavioural plasticity that accompanies chronic drug exposure. Using transgenic mice in which deltaFosB can be induced in adults in the subset of nucleus accumbens neurons in which cocaine induces the protein, we show that deltaFosB expression increases the responsiveness of an animal to the rewarding and locomotor-activating effects of cocaine. These effects of deltaFosB appear to be mediated partly by induction of the AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole) glutamate receptor subunit GluR2 in the nucleus accumbens. These results support a model in which deltaFosB, by altering gene expression, enhances sensitivity to cocaine and may thereby contribute to cocaine addiction.

15] Gel electrofocusing method for studying protein S-nitrosylation

Publisher Summary This chapter discusses the use of chemical modification reactions that adapt the method for the determination of protein S-thiolation, protein S-nitrosylation, and even irreversible damage (oxidation) to reactive sulfhydryls on a specific protein. Oxidative modifications of protein sulfhydryls can increase the negative charge on a protein—that is, either S-glutathiolation or sulfhydryl oxidation to a cysteic acid. Other oxidative modifications such as S-cysteylation or Snitrosylation produce charge-silent modifications. This method produces stable isoforms where S-glutathiolated and cysteic acid containing protein species have a net negative charge. S-nitrosylated proteins may also be analyzed by electrofocusing with little concern for decomposition. The electrofocusing method can be extended to the study of protein sulfhydryl modification in complex mixtures of proteins. The method can also be extended to proteins that are not completely water-soluble by including detergents in the electrophoresis gel. However, it must be emphasized that protein denaturants expose sulfhydryls normally buried within the protein structure. With no added detergent, the most reactive and accessible protein sulfhydryls are easily protected from artifactual modification during processing by the alkylating agents used to stop the reaction.

The Cyclophilin-like Domain Mediates the Association of Ran-Binding Protein 2 with Subunits of the 19 S Regulatory Complex of the Proteasome

The combination of the Ran-binding domain 4 and cyclophilin domains of Ran-binding protein 2 selectively associate with a subset of G protein-coupled receptors, red/green opsins, upon cis-trans prolyl isomerase-dependent and direct modification of opsin followed by association of the modified opsin isoform to Ran-binding domain 4. This effect enhances in vivo the production of functional receptor and generates an opsin isoform with no propensity to self-aggregate in vitro. We now show that another domain of Ran-binding protein 2, cyclophilin-like domain, specifically associates with the 112-kDa subunit, P112, and other subunits of the 19 S regulatory complex of the 26 S proteasome in the neuroretina. This association possibly mediates Ran-binding protein 2 limited proteolysis into a smaller and stable isoform. Also, the interaction of Ran-binding protein 2 with P112 regulatory subunit of the 26 S proteasome involves still another protein, a putative kinesin-like protein. Our results indicate that Ran-binding protein 2 is a key component of a macro-assembly complex selectively linking protein biogenesis with the proteasome pathway and, thus, with potential implications for the presentation of misfolded and ubiquitin-like modified proteins to this proteolytic machinery.

Identification of a novel alternatively spliced mRNA of murine pulmonary surfactant protein B.

An alternatively spliced mRNA of pulmonary surfactant protein B (SP-B) was identified in murine lung. Sequencing analysis revealed a 69 base-pair (bp) deletion at the beginning of exon 7 of SP-B, presumably the result of an alternative splicing event. Reverse transcription-polymerase chain reaction (RT-PCR) of mouse, rat, and rabbit lung RNA revealed the existence of full-length and the 69-bp deleted short form. Ribonuclease protection assay of the SP-B messenger RNA (mRNA) demonstrated expression of both isoforms in five strains of adult and fetal mice with different genetic backgrounds, as well as in rabbit, but not in human. Splice junction sequences in exon 6 and at the exon 7 splice boundary for the two isoforms are similar, including AG doublet identity, but sequence differences do not account for species variation in isoform abundance. The abundance of the short SP-B mRNA isoform was approximately 30% of total SP-B mRNA in mouse and rabbit. Analysis of precursor SP-B protein in mouse lung suggested that the two mRNA species are expressed as stable protein isoforms.

The Role of the α3(VI) Chain in Collagen VI Assembly: EXPRESSION OF AN α3(VI) CHAIN LACKING N-TERMINAL MODULES N10–N7 RESTORES COLLAGEN VI ASSEMBLY, SECRETION, AND MATRIX DEPOSITION IN AN α3(VI)-DEFICIENT CELL LINE

Collagen VI is a microfibrillar protein found in the extracellular matrix of virtually all connective tissues. Three genetically distinct subunits, the alpha1(VI), alpha2(VI), and alpha3(VI) chains, associate intracellularly to form triple-helical monomers, which then assemble into disulfide-bonded dimers and tetramers before secretion. Although sequence considerations suggest that collagen VI monomers composed of all three chains are the most stable isoform, the precise chain composition of collagen VI remains controversial and alternative assemblies containing only alpha1(VI) and alpha2(VI) chains have also been proposed. To address this question directly and study the role of the alpha3(VI) chain in assembly, we have characterized collagen VI biosynthesis and in vitro matrix formation by a human osteosarcoma cell line (SaOS-2) that is deficient in alpha3(VI) production. Northern analysis showed an abundance of alpha1(VI) and alpha2(VI) mRNAs, but no detectable alpha3(VI) mRNA was apparent in SaOS-2 cells. By day 30 of culture, however, small amounts of alpha3(VI) mRNA were detected, although the level of expression was still much less than alpha1(VI) and alpha2(VI). Collagen VI protein was not detected in SaOS-2 medium or cell layer samples until day 30 of culture, demonstrating that despite the abundant synthesis of alpha1(VI) and alpha2(VI), no stable collagen VI protein was produced without expression of alpha3(VI). The alpha1(VI) and alpha2(VI) chains produced in the absence of alpha3(VI) were non-helical and were largely retained intracellularly and degraded. The critical role of the alpha3(VI) chain in collagen VI assembly was directly demonstrated after stable transfection of SaOS-2 cells with an alpha3(VI) cDNA expression construct that lacked 4 of the 10 N-terminal type A subdomains. The transfected alpha3(VI) N6-C5 chains associated with endogenous alpha1(VI) and alpha2(VI) and formed collagen VI dimers and tetramers, which were secreted and deposited into an extensive network in the extracellular matrix. These data demonstrated that alpha3(VI) is essential for the formation of stable collagen VI molecules and subdomains N10-N7 are not required for molecular assembly.

Chronic Fos-Related Antigens: Stable Variants of ΔFosB Induced in Brain by Chronic Treatments

Fos family transcription factors are believed to play an important role in the transcriptional responses of the brain to a variety of stimuli. Previous studies have described 35 and 37 kDa Fos-like proteins, termed chronic Fos-related antigens (FRAs), that are induced in brain in a region-specific manner in response to several chronic perturbations, including chronic electroconvulsive seizures, psychotropic drug treatments, and lesions. We show in this study that the chronic FRAs are isoforms of deltaFosB, a truncated splice variant of FosB that accumulate in brain after chronic treatments because of their stability. doffaFosB cDNA encodes the expression of 33, 35, and 37 kDa proteins that arise from a single AUG translation start site. The 35 and 37 kDa proteins correspond to the chronic FRAs that are induced in brain by chronic treatments, whereas the 33 kDa protein corresponds to a Fos-like protein that is induced in brain by acute treatments, findings based on migration on one- and two-dimensional Western blots with anti-FRA and anti-FosB antibodies. Using cells in which deltaFosB or FosB expression is under the control of a tetracycline-regulated gene expression system, we show that the 37 kDa deltaFosB protein exhibits a remarkably long half-life, the 35 kDa DeltaFosB protein exhibits an intermediate half-life, and the 33 kDa deltaFosB protein and all FosB-derived proteins exhibit relatively short half-lives. Moreover, we show that the 33 kDa deltaFosB protein is the first to appear after activation of deltaFosB expression. Finally, deltaFosB proteins are shown to possess DNA-binding activity and to exert potent transactivating effects in reporter gene assays. Together, these findings support a scheme wherein deltaFosB, expressed as a 33 kDa protein, is modified to form highly stable isoforms of 35 and 37 kDa. As a result, these stable isoforms gradually accumulate in the brain with repeated treatments to mediate forms of long-lasting neural and behavioral plasticity.

Heterogeneity of the human corticotropin-releasing factor-binding protein.

Human corticotropin-releasing factor (hCRF), secreted by the placenta, principally in the third trimester, is specifically bound in the peripheral circulation to a 37-kDa binding protein (CRF-BP). This complex is cleared from the circulation. We postulate that the protein may be returned to the blood in a form that is immunologically altered and not well recognized by the reported RIAs. We report that a stable isoform can result from temporary denaturation of recombinant CRF-BP by 8 mol/L urea. This isoform, urea-treated binding protein, which can bind CRF, has been found to bind to an antibody raised against a synthetic peptide comprising the first 24 amino acid residues of CRF-BP, but not to a second similar N-terminal antibody, although it was closely matched in titer. Urea-treated binding protein also cross-reacts poorly in the RIA with CRF-BP. It is proposed that as a result of in vivo post-ligand binding events, isoforms may be susceptible to cleavage. After affinity purification, which involves denaturation, recombinant CRF-BP was often found to be cleaved after storage in the presence of protease inhibitors. Here we present evidence for a C-terminally truncated form of the native binding protein in the plasma of subjects suffering from rheumatoid arthritis, which may parallel the in vitro truncation.

Purification and kinetics of phenylpropanoid O-methyltransferase activities from Brassica oleracea

Two phenylpropanoid O-methyltransferase isoforms were purified to homogeneity from young cabbage leaves. They catalyzed the meta-O-methylation of caffeic and 5-hydroxyferulic acids to ferulic and sinapic acids, respectively. Both isoforms I and II exhibited different elution patterns from a Mono Q column, distinct apparent pIs on chromatofocusing, different product ratios, and stability on adenosine–agarose affinity column. On the other hand, both isoforms had similar apparent molecular masses (42 kilodaltons) and a pH optimum of 7.6. They exhibited no requirement for divalent cations and were both irreversibly inhibited by iodoacetate. Substrate interaction kinetics of the more stable isoform I, using the 5-hydroxyferulic acid and S-adenosyl-L-methionine, gave converging lines. Product inhibition studies showed competitive inhibition between S-adenosyl-L-methionine and S-adenosyl-L-homocysteine and non-competitive inhibition between the phenylpropanoid substrate and its methylated product. The kinetic patterns are consistent with an ordered bi bi mechanism, where S-adenosyl-L-methionine is the first substrate to bind and S-adenosyl-L-homocysteine is the last product released.Key words: phenylpropanoid O-methyltransferase, purification, isoforms, adenosine–agarose affinity chromatography, kinectic mechanism.

Posttranslational processing of brain actin.

Abstract: Two short‐lived isoforms of actin, named δ‐ and ε‐actin, have been detected in brain extracts from rats labeled in vivo with [35S]methionine. These two molecular species have PI values slightly more basic than β‐ and γ‐actin, the stable isoforms of the protein found in brain tissue. Under the appropriate incubation conditions δ‐ and ε‐actin, synthesized in vivo, can be converted in vitro into β‐ and γ‐actin. This posttranslational processing of δ‐ and ε‐actin requires acetyl coenzyme A, suggesting that an acetylation step, presumably of the NH2‐terminal end, is involved in the transformation of these proteins into β‐ and γ‐actin.