Ferric heme as a CO/NO sensor in the nuclear receptor Rev-Erbß by coupling gas binding to electron transfer.

Rev-Erbß is a nuclear receptor that couples circadian rhythm, metabolism, and inflammation. Heme binding to the protein modulates its function as a repressor, its stability, its ability to bind other proteins, and its activity in gas sensing. Rev-Erbß binds Fe3+-heme more tightly than Fe2+-heme, suggesting its activities may be regulated by the heme redox state. Yet, this critical role of heme redox chemistry in defining the protein's resting state and function is unknown. We demonstrate by electrochemical and whole-cell electron paramagnetic resonance experiments that Rev-Erbß exists in the Fe3+ form within the cell allowing the protein to be heme replete even at low concentrations of labile heme in the nucleus. However, being in the Fe3+ redox state contradicts Rev-Erb's known function as a gas sensor, which dogma asserts must be Fe2+ This paper explains why the resting Fe3+ state is congruent both with heme binding and cellular gas sensing. We show that the binding of CO/NO elicits a striking increase in the redox potential of the Fe3+/Fe2+ couple, characteristic of an EC mechanism in which the unfavorable Electrochemical reduction of heme is coupled to the highly favorable Chemical reaction of gas binding, making the reduction spontaneous. Thus, Fe3+-Rev-Erbß remains heme-loaded, crucial for its repressor activity, and undergoes reduction when diatomic gases are present. This work has broad implications for proteins in which ligand-triggered redox changes cause conformational changes influencing its function or interprotein interactions (e.g., between NCoR1 and Rev-Erbß). This study opens up the possibility of CO/NO-mediated regulation of the circadian rhythm through redox changes in Rev-Erbß.

Oxygen and Conformation Dependent Protein Oxidation and Aggregation by Porphyrins in Hepatocytes and Light-Exposed Cells.

BACKGROUND & AIMS: Porphyrias are caused by porphyrin accumulation resulting from defects in the heme biosynthetic pathway that typically lead to photosensitivity and possible end-stage liver disease with an increased risk of hepatocellular carcinoma. Our aims were to study the mechanism of porphyrin-induced cell damage and protein aggregation, including liver injury, where light exposure is absent. METHODS: Porphyria was induced in vivo in mice using 3,5-diethoxycarbonyl-1,4-dihydrocollidine or in vitro by exposing human liver Huh7 cells and keratinocytes, or their lysates, to protoporphyrin-IX, other porphyrins, or to δ-aminolevulinic acid plus deferoxamine. The livers, cultured cells, or porphyrin exposed purified proteins were analyzed for protein aggregation and oxidation using immunoblotting, mass spectrometry, and electron paramagnetic resonance spectroscopy. Consequences on cell-cycle progression were assessed. RESULTS: Porphyrin-mediated protein aggregation required porphyrin-photosensitized singlet oxygen and porphyrin carboxylate side-chain deprotonation, and occurred with site-selective native protein methionine oxidation. Noncovalent interaction of protoporphyrin-IX with oxidized proteins led to protein aggregation that was reversed by incubation with acidified n-butanol or high-salt buffer. Phototoxicity and the ensuing proteotoxicity, mimicking porphyria photosensitivity conditions, were validated in cultured keratinocytes. Protoporphyrin-IX inhibited proteasome function by aggregating several proteasomal subunits, and caused cell growth arrest and aggregation of key cell proliferation proteins. Light-independent synergy of protein aggregation was observed when porphyrin was applied together with glucose oxidase as a secondary peroxide source. CONCLUSIONS: Photo-excitable porphyrins with deprotonated carboxylates mediate protein aggregation. Porphyrin-mediated proteotoxicity in the absence of light, as in the liver, requires porphyrin accumulation coupled with a second tissue oxidative injury. These findings provide a potential mechanism for internal organ damage and photosensitivity in porphyrias.

Redox Regulation of Heme Oxygenase-2 and the Transcription Factor, Rev-Erb, Through Heme Regulatory Motifs.

SIGNIFICANCE: Heme binds to and serves as a cofactor for a myriad of proteins that are involved in diverse biological processes. Hemoproteins also exhibit varying modes of heme binding, suggesting that the protein environment contributes to the functional versatility of this prosthetic group. The subject of this review is a subset of hemoproteins that contain at least one heme regulatory motif (HRM), which is a short sequence containing a Cys-Pro core that, in many cases, binds heme with the Cys acting as an axial ligand. Recent Advances: As more details about HRM-containing proteins are uncovered, some underlying commonalities are emerging, including a role in regulating protein stability. Further, the cysteines of some HRMs have been shown to form disulfide bonds. Because the cysteines must be in the reduced, dithiol form to act as a heme axial ligand, heme binds at these sites in a redox-regulated manner, as demonstrated for heme oxygenase-2 (HO2) and Rev-erbß. CRITICAL ISSUES: HRM-containing proteins have wide variations in heme affinity, utilize different axial ligand schemes, and exhibit differences in the ability to act as a redox sensor-all while having a wide variety of biological functions. Here, we highlight HO2 and Rev-erbß to illustrate the similarities and differences between two hemoproteins that contain HRMs acting as redox sensors. FUTURE DIRECTIONS: HRMs acting as redox sensors may be applicable to other HRM-containing proteins as many contain multiple HRMs and/or other cysteine residues, which may become more evident as the functional significance of HRMs is probed in additional proteins.

The heme-regulatory motif of nuclear receptor Rev-erbß is a key mediator of heme and redox signaling in circadian rhythm maintenance and metabolism.

Rev-erbß is a heme-responsive transcription factor that regulates genes involved in circadian rhythm maintenance and metabolism, effectively bridging these critical cellular processes. Heme binding to Rev-erbß indirectly facilitates its interaction with the nuclear receptor co-repressor (NCoR1), resulting in repression of Rev-erbß target genes. Fe3+-heme binds in a 6-coordinate complex with axial His and Cys ligands, the latter provided by a heme-regulatory motif (HRM). Rev-erbß was thought to be a heme sensor based on a weak Kd value for the Rev-erbß·heme complex of 2 µm determined with isothermal titration calorimetry. However, our group demonstrated with UV-visible difference titrations that the Kd value is in the low nanomolar range, and the Fe3+-heme off-rate is on the order of 10-6 s-1 making Rev-erbß ineffective as a sensor of Fe3+-heme. In this study, we dissected the kinetics of heme binding to Rev-erbß and provided a Kd for Fe3+-heme of ∼0.1 nm Loss of the HRM axial thiolate via redox processes, including oxidation to a disulfide with a neighboring cysteine or dissociation upon reduction of Fe3+- to Fe2+-heme, decreased binding affinity by >20-fold. Furthermore, as measured in a co-immunoprecipitation assay, substitution of the His or Cys heme ligands in Rev-erbß was accompanied by a significant loss of NCoR1 binding. These results demonstrate the importance of the Rev-erbß HRM in regulating interactions with heme and NCoR1 and advance our understanding of how signaling through HRMs affects the major cellular processes of circadian rhythm maintenance and metabolism.

High Affinity Heme Binding to a Heme Regulatory Motif on the Nuclear Receptor Rev-erbß Leads to Its Degradation and Indirectly Regulates Its Interaction with Nuclear Receptor Corepressor.

Rev-erbα and Rev-erbß are heme-binding nuclear receptors (NR) that repress the transcription of genes involved in regulating metabolism, inflammation, and the circadian clock. Previous gene expression and co-immunoprecipitation studies led to a model in which heme binding to Rev-erbα recruits nuclear receptor corepressor 1 (NCoR1) into an active repressor complex. However, in contradiction, biochemical and crystallographic studies have shown that heme decreases the affinity of the ligand-binding domain of Rev-erb NRs for NCoR1 peptides. One explanation for this discrepancy is that the ligand-binding domain and NCoR1 peptides used for in vitro studies cannot replicate the key features of the full-length proteins used in cellular studies. However, the combined in vitro and cellular results described here demonstrate that heme does not directly promote interactions between full-length Rev-erbß (FLRev-erbß) and an NCoR1 construct encompassing all three NR interaction domains. NCoR1 tightly binds both apo- and heme-replete FLRev-erbß·DNA complexes; furthermore, heme, at high concentrations, destabilizes the FLRev-erbß·NCoR1 complex. The interaction between FLRev-erbß and NCoR1 as well as Rev-erbß repression at the Bmal1 promoter appear to be modulated by another cellular factor(s), at least one of which is related to the ubiquitin-proteasome pathway. Our studies suggest that heme is involved in regulating the degradation of Rev-erbß in a manner consistent with its role in circadian rhythm maintenance. Finally, the very slow rate constant (10(-6) s(-1)) of heme dissociation from Rev-erbß rules out a prior proposal that Rev-erbß acts as an intracellular heme sensor.

Modulation of nuclear receptor function by cellular redox poise.

Nuclear receptors (NRs) are ligand-responsive transcription factors involved in diverse cellular processes ranging from metabolism to circadian rhythms. This review focuses on NRs that contain redox-active thiol groups, a common feature within the superfamily. We will begin by describing NRs, how they regulate various cellular processes and how binding ligands, corepressors and/or coactivators modulate their activity. We will then describe the general area of redox regulation, especially as it pertains to thiol-disulfide interconversion and the cellular systems that respond to and govern this redox equilibrium. Lastly, we will discuss specific examples of NRs whose activities are regulated by redox-active thiols. Glucocorticoid, estrogen, and the heme-responsive receptor, Rev-erb, will be described in the most detail as they exhibit archetypal redox regulatory mechanisms.

Apoprotein isolation and activation, and vibrational structure of the Helicobacter mustelae iron urease.

The micro aerophilic pathogen Helicobacter mustelae synthesizes an oxygen-labile, iron-containing urease (UreA2B2) in addition to its standard nickel-containing enzyme (UreAB). An apoprotein form of the iron urease was prepared from ureA2B2-expressing recombinant Escherichia coli cells that were grown in minimal medium. Temperature-dependent circular dichroism measurements of holoprotein and apoprotein demonstrate an enhancement of thermal stability associated with the UreA2B2 metallocenter. In parallel to the situation reported for nickel activation of the standard urease apoprotein, incubation of UreA2B2 apoprotein with ferrous ions and bicarbonate generated urease activity in a portion of the nascent active sites. In addition, ferrous ions were shown to be capable of reductively activating the oxidized metallocenter. Resonance Raman spectra of the inactive, aerobically-purified UreA2B2 holoprotein exhibit vibrations at 495cm(-1) and 784cm(-1), consistent with ν(s) and ν(as) modes of an Fe(III)OFe(III) center; these modes undergo downshifts upon binding of urea and were unaffected by changes in pH. The low-frequency mode also exhibits an isotopic shift from 497 to 476cm(-1) upon (16)O/(18)O bulk water isotope substitution. Expression of subunits of the conventional nickel-containing Klebsiella aerogenes urease in cells grown in rich medium without nickel resulted in iron incorporation into a portion of the protein. The inactive iron-loaded species exhibited a UV-visible spectrum similar to oxidized UreA2B2 and was capable of being reductively activated under anoxic conditions. Results from these studies more clearly define the formation and unique properties of the iron urease metallocenter.

Function of UreB in Klebsiella aerogenes urease.

Urease from Klebsiella aerogenes is composed of three subunits (UreA-UreB-UreC) that assemble into a (UreABC)(3) quaternary structure. UreC harbors the dinuclear nickel active site, whereas the functions of UreA and UreB remain unknown. UreD and UreF accessory proteins previously were suggested to reposition UreB and increase the level of exposure of the nascent urease active site, thus facilitating metallocenter assembly. In this study, cells were engineered to separately produce (UreAC)(3) or UreB, and the purified proteins were characterized. Monomeric UreB spontaneously binds to the trimeric heterodimer of UreA and UreC to form (UreABC*)(3) apoprotein, as shown by gel filtration chromatography, integration of electrophoretic gel band intensities, and mass spectrometry. Similar to the authentic urease apoprotein, the active enzyme is produced by incubation of (UreABC*)(3) with Ni(2+) and bicarbonate. Conversely, UreBΔ1-19, lacking the 19-residue potential hinge and tether to UreC, does not form a complex with (UreAC)(3) and yields negligible levels of the active enzyme when incubated under activation conditions with (UreAC)(3). Comparison of activities and nickel contents for (UreAC)(3), (UreABC*)(3), and (UreABC)(3) samples treated with Ni(2+) and bicarbonate and then desalted indicates that UreB facilitates efficient incorporation of the metal into the active site and protects the bound metal from chelation. Amylose resin pull-down studies reveal that MBP-UreD (a fusion of maltose binding protein with UreD) forms complexes with (UreABC)(3), (UreAC)(3), and UreB in vivo, but not in vitro. By contrast, MBP-UreD does not form an in vivo complex with UreBΔ1-19. The soluble MBP-UreD-UreF-UreG complex binds in vitro to (UreABC)(3), but not to (UreAC)(3) or UreB. Together, these data demonstrate that UreB facilitates the interaction of urease with accessory proteins during metallocenter assembly, with the N-terminal hinge and tether region being specifically required for this process. In addition to its role in urease activation, UreB enhances the stability of UreC against proteolytic cleavage.

Iron-containing urease in a pathogenic bacterium.

Helicobacter mustelae, a gastric pathogen of ferrets, synthesizes a distinct iron-dependent urease in addition to its archetypical nickel-containing enzyme. The iron-urease is oxygen-labile, with the inactive protein exhibiting a methemerythrin-like electronic spectrum. Significantly, incubation of the oxidized protein with dithionite under anaerobic conditions leads to restoration of activity and bleaching of the spectrum. Structural analysis of the oxidized species reveals a dinuclear iron metallocenter bridged by a lysine carbamate, closely resembling the traditional nickel-urease active site. Although the iron-urease is less active than the nickel-enzyme, its activity allows H. mustelae to survive the carnivore's low-nickel gastric environment.

Efficacy and safety study of tazarotene cream 0.1% for the treatment of brittle nail syndrome.

Brittle nail syndrome refers to nails that exhibit surface roughness, raggedness, and peeling. It is a common problem, with a higher prevalence among elderly patients. The goal of this study was to determine if tazarotene cream 0.1% ameliorates the signs and symptoms of brittle nails. In this open-label, single-center trial, participants applied tazarotene cream to the nails twice daily for 24 weeks. Signs and symptoms were rated by the investigators and by the participants during treatment and 12 weeks after discontinuation. Twenty participants were enrolled in the study; 1 participant withdrew prior to the 4-week followup visit. Of the 18 participants available for analysis (1 participant was excluded because baseline photographs were not available) for the primary end point of improvement in the physician global improvement assessment (PGIA), all 18 participants achieved improvement of the target nails at week 12 as well as 16 participants (88.9%) at week 24. All 18 participants had improvement in the PGIA score 12 weeks posttreatment at week 36. The physician global assessment (PGA) improved for 14 of 19 participants (73.7%) at both weeks 12 and 24; at week 24, 4 of 19 participants had achieved a PGA score of none. At week 36, 17 of 19 participants (89.5%) agreed that their nails had improved overall. Only 1 participant (5.3%) reported mild local irritation. This study demonstrated that tazarotene improves some of the changes noted in conjunction with brittle nail syndrome with minimal to no irritation.

Characterization of the Klebsiella aerogenes urease accessory protein UreD in fusion with the maltose binding protein.

Assembly of the Klebsiella aerogenes urease metallocenter requires four accessory proteins, UreD, UreE, UreF, and UreG, to effectively deliver and incorporate two Ni2+ ions into the nascent active site of the urease apoprotein (UreABC). Each accessory protein has been purified and characterized with the exception of UreD due to its insolubility when it is overproduced in recombinant cells. In this study, a translational fusion was made between the maltose binding protein (MBP) and UreD, with the resulting MBP-UreD found to be soluble in Escherichia coli cell extracts and able to complement a DeltaureD-urease cluster in this host microorganism. MBP-UreD was purified as a large multimer (> 670 kDa) that bound approximately 2.5 Ni2+ ions (K(d) of approximately 50 microM, where K(d) is the dissociation constant) per UreD protomer according to equilibrium dialysis measurements. Zn2+ directly competes with 10-fold higher affinity (approximately 4 Zn2+ ions per protomer; K(d) of 5 microM) for the Ni2+ binding sites. MBP pulldown experiments demonstrated that the UreD domain of MBP-UreD formed in vivo complexes with UreF, UreG, UreF plus UreG, or UreABC when these proteins were overproduced in the same E. coli cells. In addition, a UreABC-(MBP-UreD)-UreFG complex was observed in cells producing all urease components. Comparative in vitro binding experiments with purified proteins demonstrated an approximate 1:1 binding ratio between the UreD domain of MBP-UreD and the UreF domain of the UreEF fusion, only weak or transient interaction between MBP-UreD and UreG, and no binding with UreABC. These studies are the first to describe the properties of purified UreD, and they extend our understanding of its binding partners both in vitro and in the cell.

Purification and characterization of the folate catabolic enzyme p-aminobenzoyl-glutamate hydrolase from Escherichia coli.

The abg locus of the Escherichia coli chromosome includes three genes encoding proteins (AbgA, AbgB, and AbgT) that enable uptake and utilization of the folate breakdown product, p-aminobenzoyl-glutamate (PABA-GLU). We report on the purification and characterization of the p-aminobenzoyl-glutamate hydrolase (PGH) holoenzyme encoded by abgA and abgB. One-step purification was accomplished using a plasmid carrying abgAB with a hexahistidine tag on the carboxyl terminus of AbgB and subsequent metal affinity chromatography (MAC). Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed two subunits (approximately 53-kDa and approximately 47-kDa proteins) of the expected masses of AbgB and AbgA; N-terminal sequencing confirmed the subunit identification, and amino acid analysis yielded a 1:1 ratio of the subunits. Size exclusion chromatography coupled with light-scattering analysis of purified PGH revealed a predominant molecular mass of 206 kDa and a minor component of 400 to 500 kDa. Both peaks contained PGH activity, and SDS-PAGE revealed that fractions containing activity were composed of both AbgA and AbgB. MAC-purified PGH was highly stimulated by manganese chloride. Kinetic analysis of MAC-purified PGH revealed a K(m) value for PABA-GLU of 60 +/- 0.08 microM and a specific activity of 63,300 +/- 600 nmol min(-1) mg(-1). Folic acid and a variety of dipeptides served as poor substrates of PGH. This locus of the E. coli chromosome may encode a portion of a folate catabolism pathway.

Interplay of metal ions and urease.

Urease, the first enzyme to be crystallized, contains a dinuclear nickel metallocenter that catalyzes the decomposition of urea to produce ammonia, a reaction of great agricultural and medical importance. Several mechanisms of urease catalysis have been proposed on the basis of enzyme crystal structures, model complexes, and computational efforts, but the precise steps in catalysis and the requirement of nickel versus other metals remain unclear. Purified bacterial urease is partially activated via incubation with carbon dioxide plus nickel ions; however, in vitro activation also has been achieved with manganese and cobalt. In vivo activation of most ureases requires accessory proteins that function as nickel metallochaperones and GTP-dependent molecular chaperones or play other roles in the maturation process. In addition, some microorganisms control their levels of urease by metal ion-dependent regulatory mechanisms.

Response of Sinorhizobium meliloti to elevated concentrations of cadmium and zinc.

Whole-genome transcriptional profiling was used to identify genes in Sinorhizobium meliloti 1021 that are differentially expressed during exposure to elevated concentrations of cadmium and zinc. Mutant strains with insertions in metal-regulated genes and in genes encoding putative metal efflux pumps were analyzed for their metal sensitivities, revealing a crucial role for the SMc04128-encoded P-type ATPase in the defense of S. meliloti against cadmium and zinc stress.

Escherichia coli abg genes enable uptake and cleavage of the folate catabolite p-aminobenzoyl-glutamate.

Escherichia coli AbgT was first identified as a structural protein enabling the growth of p-aminobenzoate auxotrophs on exogenous p-aminobenzoyl-glutamate (M. J. Hussein, J. M. Green, and B. P. Nichols, J. Bacteriol. 180:6260-6268, 1998). The abg region includes abgA, abgB, abgT, and ogt; these genes may be regulated by AbgR, a divergently transcribed LysR-type protein. Wild-type cells transformed with a high-copy-number plasmid encoding abgT demonstrate saturable uptake of p-aminobenzoyl-glutamate (K(T)=123 microM); control cells expressing vector demonstrate negligible uptake. The addition of metabolic poisons inhibited uptake of p-aminobenzoyl-glutamate, consistent with this process requiring energy. p-Aminobenzoyl-glutamate taken in by cells expressing large amounts of AbgT alone is not rapidly metabolized to a form that is trapped in the cell, as the addition of nonradioactive p-aminobenzoyl-glutamate to these cells results in a rapid loss of intracellular label. The addition of nonradioactive p-aminobenzoate has no effect. The abgA, abgB, and abgAB genes were cloned into the medium-copy-number plasmid pACYC184; p-aminobenzoate auxotrophs transformed with the clone encoding abgAB demonstrated enhanced ability to grow on low levels of p-aminobenzoyl-glutamate. When transformed with complementary plasmids encoding high-copy levels of abgT and medium-copy levels of abgAB, p-aminobenzoate auxotrophs grew on 50 nM p-aminobenzoyl-glutamate. Our data are consistent with a model of p-aminobenzoyl-glutamate utilization in which AbgT catalyzes transport of p-aminobenzoyl-glutamate, followed by cleavage to p-aminobenzoate by a protein composed of subunits encoded by abgA and abgB. While endogenous expression of these genes is very low under the conditions in which we performed our experiments, these genes may be induced by AbgR bound to an unknown molecule. The true physiological role of this region may be related to some molecule similar to p-aminobenzoyl-glutamate, such as a dipeptide.

A randomized, double-blind comparison of two topical anesthetic formulations prior to electrodesiccation of dermatosis papulosa nigra.

BACKGROUND: Liposomal lidocaine 4% (L.M.X.4 cream, Ferndale Laboratories Inc., Ferndale, MI, USA) has been proposed as a more rapidly acting topical anesthetic than the eutectic mixture of lidocaine 2.5% and prilocaine 2.5% (EMLA cream, AstraZeneca LP, Wilmington, DE, USA) for venipuncture and laser procedures. However, their anesthetic efficacy has not been previously compared for electrosurgical destruction of superficial skin lesions. OBJECTIVE: To test the hypothesis that L.M.X.4 and EMLA differ in anesthetic efficacy when applied under occlusion for 30 minutes prior to electrodesiccation of papules of dermatosis papulosa nigra. METHODS: Forty adults were randomly assigned to treatment with either agent for 30 minutes under Tegaderm. The study drug was administered for an additional 30 minutes if the electrodesiccation of the first few papules was too painful. RESULTS: One subject treated with EMLA versus none treated with L.M.X.4 experienced complete anesthesia after a single 30-minute application. Nineteen of 20 (95%) subjects treated with EMLA versus 18 of 20 (90%) subjects treated with L.M.X.4 required only a single application (p = .49). Pain scores after the initial 30-minute application (scale: 0 = none to 10 = very severe) were EMLA 3.3 +/- 2.2 (mean +/- SD) versus L.M.X. 4 2.9 +/- 2.0 (p = .46). CONCLUSION: EMLA and L.M.X.4 provide comparable levels of anesthesia after a single 30-minute application under occlusion prior to electrodesiccation of superficial skin lesions.