Monopyrroles in porphyria,psychosis and lead exposure

• 1.1. It has been shown that the monopyrrole other than porphobilinogen excreted in excess in acute porphyria is 3-ethyl-5-hydroxy-4,5-dimethyl-Δ³-pyrrolin-2-one (hydroxyhaemopyrrole lactam). A gas liquid chromatographic method has been developed for measurement of the concentrations of this compound. Hydroxyhaemopyrrole lactam was measured in the urine of patients with porphyria, psychiatric in-patients and subjects with industrial lead exposure. Concentrations were found to be raised in porphyria, but no correlation could be found between the concentrations of this compound and current clinical condition. In psychiatric patients, there was again significant elevation of hydroxyhaemopyrrole lactam, although no such rise could be found in lead workers. The studies in the lead workers and porphyria patients would suggest that there is an association between the concentrations of hydroxyhae-mopyrrole lactam in urine and porphobilinogen excretion.
• 2.2. The effects of the monopyrroles; hydroxyhaemopyrrole lactam; haemopyrrole; hydroxykryptopyr-role lactam; kryptopyrrole phyllopyrrole and ethyl-3-acetyl-2,4-dimethyl-pyrrole-5-carboxylate (EADC), have been examined on porphyrin metabolism in the rat. All significantly raised urinary porphyrin excretion and hepatic porphyrin concentrations. Hydroxyhaemopyrrole lactam caused an increase in the excretion of porphyrins associated with an increase in the activity of the rate-limiting enzyme of haem biosynthesis, δ-aminolaevulinic acid synthase. These results suggest some association between such pyrrole production and the biochemical changes found in acute porphyria.

     

Urinary excretion of purines,purine nucleosides,and pseudouridine in immunodeficient children

Studies have been carried out using ion-exchange analysis for determination of urinary purines, pyrimidines, and nucleosides in children with immunodeficiency disorders. Using cation and anion-exchange techniques, the following compounds of urine have been quantitatively determined: deoxyadenosine, adenosine, adenine, pseudouridine, 7-methylguanine, N²,N²-dimethylguanosine, hypoxanthine, and xanthine. Excretion levels of these compounds did not differ significantly from normal values in six children with various immunodeficiency diseases, excluding severe combined immunodeficiency. However, of the seven children studied with severe combined immunodeficiency disease (normal adenosine deaminase), four showed increased excretion levels for one or more of the compounds studied. A germ-free child with severe combined immunodeficiency had lower excretion levels than the mean normal value for most of these same compounds. The possibility is considered that the increased excretion levels noted may be a consequence of repeated episodes of infection in most children with severe combined immunodeficiency.     

Cytochrome P-450 heme and the regulation of hepatic heme oxygenase activity

The degradation of cytochrome P-450 heme in the liver has been studied by a new approach. In rats, hepatic heme was labeled by administration of a tracer pulse of [5-¹⁴C]δ-aminolevulinic acid (ALA), and its degradation was analyzed in terms of labeled carbon monoxide (¹⁴CO) excretion, which is a specific degradation product of the labeled heme. Within minutes after administration of [5-¹⁴C]ALA, 14CO was detectable and increased after 2 h to an “early peak,” reflecting the elimination of labeled heme from a rapidly turning over pool in the liver. Beyond the early peak, the rate of ¹⁴CO production decreased in a log-linear manner, consistent with the degradation of heme in stable hepatic hemoproteins. From the rate at which ¹⁴CO production declined during this phase, from the predominant labeling of cytochrome P-450 heme by the administered [5-¹⁴C]ALA and from the known turnover characteristics of this hemoprotein in the liver, it could be inferred that production of ¹⁴CO—between 16 and 30 h after administration of labeled ALA—largely reflected degradation of cytochrome P-450 heme. This approach, which permits serial measurements in a single animal, was used to study the effect on cytochrome P-450 heme of administered heme or endotoxin, both of which are potent stimulators of hepatic heme oxygenase activity. Both of these substances caused marked acceleration of the degradation of cytochrome P-450 heme, the effect occurring over the same dose range as that for stimulation of hepatic heme oxygenase. The findings suggest that stimulation of this enzyme activity in the liver is closely related to the rate of degradation of cytochrome P-450 heme.     

The formation of N-alkylprotoporphyrin IX and destruction of cytochrome P-450 in the liver of rats after treatment with 3,5-diethoxycarbonyl-1,4-dihydrocollidine and its 4-ethyl analog

The effects of two porphyrogenic agents, 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) and 3,5-diethoxycarbonyl-2,6-dimethyl-4-ethyl-1,4-dihydropyridine (DDEP), have been studied in rats. The administration of these compounds leads to the formation and accumulation in the liver of N-methylprotoporphyrin IX and N-ethylprotoporphyrin IX, respectively. In each case, the alkyl group of the porphyrin is derived from the 4-alkyl group of the porphyrogenic chemical. Each N-alkylporphyrin is a potent inhibitor of protoheme ferrolyase (EC 4.99.1.1) (ferrochelatase) activity. N-Methylprotoporphyrin IX is somewhat more potent than N-ethylprotoporphyrin IX as an inhibitor of ferrochelatase activity in vitro. However, more N-ethylprotoporphyrin IX accumulates in rat liver than does the N-methyl analog. Since alkylporphyrins are formed during the catabolism of heme (or hemoprotein), the effects of DDC and DDEP on hepatic microsomal cytochrome P-450 were also studied. Whereas DDC treatment led to only a slight decrease in cytochrome P-450 levels (25%), DDEP administration led to a marked decrease (75%) in the total cytochrome P-450 level. In phenobarbital- and 3-methylcholanthrene-treated rats, DDC administration did not alter the hepatic microsomal cytochrome P-450 content, while administration of DDEP to either phenobarbital-treated or 3-methylcholanthrene-treated rats led to marked reduction of levels in cytochrome P-450. Although the N-methylprotoporphyrin IX level was not increased following DDC administration to either phenobarbital- or 3-methylcholanthrene-treated rats, there was a marked increase in N-ethylprotoporphyrin IX accumulation in both phenobarbital- and 3-methylcholanthrene-treated rats after the administration of DDEP. These results suggest that DDC and DDEP react with different forms of rat hepatic microsomal cytochrome P-450.     

Identification of porphyrin present in apo-cytochrome c oxidase of copper-deficient yeast cells

Heme a was not detected either in mitochondria isolated from copper-deficient yeast or in the intact cells. Nevertheless, the intracellular concentration of free porphyrins indicated that the pathway of porphyrin and heme synthesis was not impaired in copper-deficient cells. The immunoprecipitated apo-oxidase from copper-deficient cells revealed an absorption spectrum with maxima at 645, 592, 559, 519 and 423 nm, similar to that of purified porphyrin a. When solubilized mitochondria from [³H]leucine and δ-amino[¹⁴C]levulinic acid-labeled copper-deficient yeast cells were incubated with rabbit antiserum against cytochrome c oxidase, a precipitate was obtained. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of this immunoprecipitate showed [³H]leucine associated with six bands and δ-amino[¹⁴C]levulinic acid resolved in a single band. HCl fractionation of copper-deficient mitochondria labeled with δ-amino[¹⁴C]levulinic acid showed a high specific radioactivity in the fraction extracted by 20% HCl, a solvent which extracts porphyrin a. Thinlayer chromatography of the radioactivity found in 20% HCl showed an R_F value identical to that of purified porphyrin a. When δ-amino[³H]levulinic acid-labeled, copper-deficient yeast cells are grown in copper-supplemented medium, the porphyrin a accumulated in copper-deficient cells wa converted into heme a, and this conversion was prevented by cycloheximidine.These observations suggest that porphyrin a is present in the apo-oxidase of copper-deficient cells, but that the conversion to heme a does not occur. This conversion reaction appears to be a point in the biosynthetic pathway of cytochrome c oxidase which is blocked by copper deficieny.     

Extracellular Heme Peroxidases in Actinomycetes: a Case of Mistaken Identity

Actinomycetes secrete into their surroundings a suite of enzymes involved in the biodegradation of plant lignocellulose; these have been reported to include both hydrolytic and oxidative enzymes, including peroxidases. Reports of secreted peroxidases have been based upon observations of peroxidase-like activity associated with fractions that exhibit optical spectra reminiscent of heme peroxidases, such as the lignin peroxidases of wood-rotting fungi. Here we show that the appearance of the secreted pseudoperoxidase of the thermophilic actinomycete Thermomonospora fusca BD25 is also associated with the appearance of a heme-like spectrum. The species responsible for this spectrum is a metalloporphyrin; however, we show that this metalloporphyrin is not heme but zinc coproporphyrin. The same porphyrin was found in the growth medium of the actinomycete Streptomyces viridosporus T7A. We therefore propose that earlier reports of heme peroxidases secreted by actinomycetes were due to the incorrect assignment of optical spectra to heme groups rather than to non-iron-containing porphyrins and that lignin-degrading heme peroxidases are not secreted by actinomycetes. The porphyrin, an excretory product, is degraded during peroxidase assays. The low levels of secreted peroxidase activity are associated with a nonheme protein fraction previously shown to contain copper. We suggest that the role of the secreted copper-containing protein may be to bind and detoxify metals that can cause inhibition of heme biosynthesis and thus stimulate porphyrin excretion.     

Stimulation of porphyrin accumulation in chick embryo liver cell cultures by partially purified erythroprotein

Step III and Step IV erythropoieten derived from sheep plasma stimulated the accumulation of porphyrins in cultured chick embryo liver cells. Increased porphyrin accumulation occurred within hepatocytes. It was not accompanied by increased hemoglobin formation. Stimulation of porphyrin accumulation was inhibited by hematin but was unimpaired by heating erythropoietin to 60°C for 10 min or preincubating it with trypsin. A more highly purified preparation of erythropoietin from human urine had no effect on porphyrin accumulation. The data indicate that a component in partially purified sheep erythropoietin can increase levels of a heme precursor in non-erythroid tissue. The participation of such a component should be considered when interpreting biochemical effects observed with crude erythropoietin preparations, other than ⁵⁹Fe incorporation into red cells or heme.     

Induction of hepatic heme oxygenase activity by bromobenzene

Hepatic heme oxygenase, an enzyme which converts heme to carbon monoxide and bile pigment in vitro, is inducible by heme but also by large “toxic” doses of such nonheme substances as hormones, endotoxin, and heavy metal ions. When we gave rats a single hepatotoxic dose of allyl alcohol, ethionine, acetaminophen, furosemide, or endotoxin, hepatic heme oxygenase activity rose modestly (two- to fivefold) after 20 h. In contrast, administration of bromobenzene (5 mmol/kg) induced heme oxygenase in the liver an average of 15-fold after 20 h but was without effect on the enzyme in the kidney or spleen. The change in heme oxygenase was accompanied by a loss in cytochrome P-450 concentration and, in rats labeled with 5-δ-amino[¹⁴C]levulinic acid, an increased rate of degradation of hepatic [¹⁴C]heme to ¹⁴CO. Induction of heme oxygenase by bromobenzene was blocked by cycloheximide, an inhibitor of protein synthesis, but not by actinomycin D, an inhibitor of RNA synthesis. This suggests that bromobenzene stimulates de novo enzyme synthesis at the step of translation. Subtoxic doses of bromobenzene (less than 1 mmol/kg) gave proportionately greater induction of heme oxygenase. Furthermore, induction of the enzyme remained unaffected when bromobenzene hepatotoxicity was blocked by pretreatment of rats with SKF-525A, 3-methylcholanthrene, or cysteine (which supplements liver sulfhydryl content), or when hepatotoxicity was enhanced by pretreatment with phenobarbital or with diethylmaleate (which depletes hepatic glutathione). These data suggest that with induction of heme oxygenase by bromobenzene, neither liver cell necrosis nor alteration in hepatic sulfhydryl metabolism is indispensible. The latter characteristic differs from induction of the enzyme by metal ions in which depletion of sulfhydryl-containing constituents has been thought to be essential. We conclude that bromobenzene is a novel inducer of heme oxygenase activity in the liver, differing from other nonheme substances in potency and specificity for the liver, and in utilizing mechanism(s) which require neither production of hepatotoxicity, depletion of hepatic glutathione, nor sensitivity to actinomycin D.     

Facile synthesis of peptide–porphyrin conjugates: Towards artificial catalase

A facile synthetic method for peptide–porphyrin conjugates containing four peptide units on one porphyrin was developed using chemoselective reactions. The key building blocks, 5,10,15,20-tetrakis(3-azidophenyl)porphyrin 1 and 5,10,15,20-tetrakis(5-azido-3-pyridyl)porphyrin 2, were efficiently synthesized and used as substrates for two well-known chemoselective reactions, traceless Staudinger ligation and copper-catalyzed azide alkyne cycloaddition (so-called click chemistry). Both reactions gave the desired compounds, and click chemistry was superior for our purpose. To confirm the value of the established methodology, nine peptide–porphyrin conjugates were synthesized, and their catalase- and peroxidase-like activity in water was evaluated. Our synthetic strategy is expected to be valuable for the preparation of artificial heme protein models.     

Biochemical characterization of porphobilinogen deaminase-deficient mice during phenobarbital induction of heme synthesis and the effect of enzyme replacement

Acute intermittent porphyria (AIP) is a genetic disorder caused by a deficiency of porphobilinogen deaminase (PBGD), the 3rd enzyme in heme synthesis. It is clinically characterized by acute attacks of neuropsychiatric symptoms and biochemically by increased urinary excretion of the porphyrin precursors porphobilinogen (PBG) and 5-aminolevulinic acid (ALA). A mouse model that is partially deficient in PBGD and biochemically mimics AIP after induction of the hepatic ALA synthase by phenobarbital was used in this study to identify the site of formation of the presumably toxic porphyrin precursors and study the effect of enzyme-replacement therapy by using recombinant human PBGD (rhPBGD). After 4 d of phenobarbital administration, high levels of PBG and ALA were found in liver, kidney, plasma, and urine of the PBGD-deficient mice. The administration of rhPBGD intravenously or subcutaneously after a 4-d phenobarbital induction was shown to lower the PBG level in plasma in a dose-dependent manner with maximal effect seen after 30 min and 2 h, respectively. Injection of rhPBGD subcutaneously twice daily during a 4-d phenobarbital induction reduced urinary PBG excretion to 25% of the levels found in PBGD-deficient mice administered with only phenobarbital. This study points to the liver as the main producer of PBG and ALA in the phenobarbital-induced PBGD-deficient mice and demonstrates efficient removal of accumulated PBG in plasma and urine by enzyme-replacement therapy.     

Multiple regulatory steps in erythroid heme biosynthesis

Current models for regulation of heme synthesis during erythropoiesis propose that the first enzyme of the pathway, 5-aminolevulinate synthase (ALAS), is the rate-limiting enzyme. We have examined cellular porphyrin excretion in differentiating murine erythroleukemia cells to determine in situ rate-limiting steps in heme biosynthesis. The data demonstrate that low levels of coproporphyrin and protoporphyrin accumulate in the culture medium under normal growth conditions and that during erythroid differentiation the level of excretion of coproporphyrin increases approximately 100-fold. Iron supplementation lowered, but did not eliminate, porphyrin accumulation. While ALAS induction is necessary for increased heme synthesis, these data indicate that other enzymes, in particular coproporphyrinogen oxidase, represent down-stream rate-limiting steps.     

Acetylation of spermidine in polyamine catabolism

Treatment with thioacetamide (150 mg/kg)_ was used to enhance polyamine metabolism in rat liver. The increased uptake and catabolism of [¹⁴C]spermine and the changes of putrescine, spermidine and spermine concentrations indicated enhanced polyamine turnover rates. The increase of hepatic putrescine concentration was accompanied by an increase of monoacetylputrescine and N¹-monoacetylspermidine concentration. In control animals, the latter compound was below detection levels. Thioacetamide treatment also enhanced putrescine excretion, which again was concomitant with an increased excretion of N¹-acetylspermidine.The close time-dependent correlation between induced putrescine formation and enhanced formation of N¹-acetylsperimidine at a time when liver spermidine and spermine concentrations are not changed, favors the notion that acetylation is an essential step in polyamine degradation and elimination. The increase of polyamine oxidase and decrease of acetylpolyamine deacetylase activities in the liver of thioacetamide-treated rats is in line with an increased polyamine turnover, but these enzymes. although essential, are not rate-limiting in the catabolic reactions.     

Renal cytochrome P-450's-electrophoretic and electron paramagnetic resonance studies.

The cytochrome P-450's of the microsomal mixed function oxidase systems from the rabbit renal cortex, outer medulla, inner medulla, and the liver were compared. Sodium dodecyl sulfate-(SDS) gel electrophoresis and electron paramagnetic resonance (EPR) studies detected cytochrome P-450 proteins in the liver, renal cortex, and outer medulla but not the inner medulla of normal animals. Two cytochrome P-450 peptides, which had molecular weights of 54,500 and 58,900 and which comigrated with known hepatic cytochrome P-450's on SDS gels, were identified in the cortex and outer medulla. Treatment of animals with 3-methylcholanthrene (MC) enhanced the 54,500 and 58,900 peptides in the liver and cortex but produced little change in outer medulla. MC treatment induced faint cytochrome P-450 bands in the inner medulla. The EPR studies detected low spin heme iron absorption lines at g = 2.42, 2.26, and 1.92 in liver, cortex, and outer medulla from untreated animals. The amplitude of the low spin absorption lines was increased by ethanol, a reverse type I compound, and reduced by chloroform, a type I compound, in these tissues. MC treatment increased the amplitude of the heme absorption lines in these tissues, and it induced a barely detectable heme spectrum in the inner medulla. Differences in exogenous substrate binding between hepatic and renal microsomes from MC-treated animals were detected by EPR and optical difference spectroscopy. Acetone, 1-butanol, and 2-propanol gave evidence of binding to the hepatic cytochrome P-450's but no evidence of binding to renal cortical microsomes. These results, along with previous enzymatic studies, suggest that the liver and each area of the kidney contain different substrate specificities and pathways for the metabolism of organic compounds.     

Protective effect of heme oxygenase induction in ethinylestradiol‐induced cholestasis

Estrogen‐induced cholestasis is characterized by impaired hepatic uptake and biliary bile acids secretion because of changes in hepatocyte transporter expression. The induction of heme oxygenase‐1 (HMOX1), the inducible isozyme in heme catabolism, is mediated via the Bach1/Nrf2 pathway, and protects livers from toxic, oxidative and inflammatory insults. However, its role in cholestasis remains unknown. Here, we investigated the effects of HMOX1 induction by heme on ethinylestradiol‐induced cholestasis and possible underlying mechanisms. Wistar rats were given ethinylestradiol (5 mg/kg s.c.) for 5 days. HMOX1 was induced by heme (15 μmol/kg i.p.) 24 hrs prior to ethinylestradiol. Serum cholestatic markers, hepatocyte and renal membrane transporter expression, and biliary and urinary bile acids excretion were quantified. Ethinylestradiol significantly increased cholestatic markers (P ≤ 0.01), decreased biliary bile acid excretion (39%, P = 0.01), down‐regulated hepatocyte transporters (Ntcp/Oatp1b2/Oatp1a4/Mrp2, P ≤ 0.05), and up‐regulated Mrp3 (348%, P ≤ 0.05). Heme pre‐treatment normalized cholestatic markers, increased biliary bile acid excretion (167%, P ≤ 0.05) and up‐regulated hepatocyte transporter expression. Moreover, heme induced Mrp3 expression in control (319%, P ≤ 0.05) and ethinylestradiol‐treated rats (512%, P ≤ 0.05). In primary rat hepatocytes, Nrf2 silencing completely abolished heme‐induced Mrp3 expression. Additionally, heme significantly increased urinary bile acid clearance via up‐regulation (Mrp2/Mrp4) or down‐regulation (Mrp3) of renal transporters (P ≤ 0.05). We conclude that HMOX1 induction by heme increases hepatocyte transporter expression, subsequently stimulating bile flow in cholestasis. Also, heme stimulates hepatic Mrp3 expression via a Nrf2‐dependent mechanism. Bile acids transported by Mrp3 to the plasma are highly cleared into the urine, resulting in normal plasma bile acid levels. Thus, HMOX1 induction may be a potential therapeutic strategy for the treatment of ethinylestradiol‐induced cholestasis.     

Heme Mediates Cytotoxicity from Artemisinin and Serves as a General Anti-Proliferation Target

Heme (Fe2+ protoporphyrin IX) is an essential molecule that has been implicated the potent antimalarial action of artemisinin and its derivatives, although the source and nature of the heme remain controversial. Artemisinins also exhibit selective cytotoxicity against cancer cells in vitro and in vivo. We demonstrate that intracellular heme is the physiologically relevant mediator of the cytotoxic effects of artemisinins. Increasing intracellular heme synthesis through the addition of aminolevulinic acid, protoporphyrin IX, or transferrin-bound iron increased the cytotoxicity of dihydroartemisinin, while decreasing heme synthesis through the addition of succinyl acetone decreased its cytotoxic activity. A simple and robust high throughput assay was developed to screen chemical compounds that were capable of interacting with heme. A natural products library was screened which identified the compound coralyne, in addition to artemisinin, as a heme interacting compound with heme synthesis dependent cytotoxic activity. These results indicate that cellular heme may serve a general target for the development of both anti-parasitic and anti-cancer therapeutics.     

Design,synthesis, anti-inflammatory activity,and molecular docking studies of perimidine derivatives containing triazole

We report here the design, synthesis, and anti-inflammatory activities of a series of perimidine derivatives containing triazole (5a–s). The chemical structures of the synthesized compounds have been assigned on the basis of IR, ¹H NMR, ¹³C NMR, and HRMS spectral analyses. The anti-inflammatory properties of the synthesized perimidine derivatives were evaluated in a lipopolysaccharide (LPS)-stimulated inflammation model. Among the tested compounds, compound 7-(3-methylbenzyl)-7H-[1,2,4]triazolo[4,3-a]perimidine (hereafter referred to as 5h) and compound 7-(2-fluorobenzyl)-7H-[1,2,4]triazolo[4,3-a]perimidine (hereafter referred to as 5n) caused a reduction in the levels of the pro-inflammatory cytokines—tumor necrosis factor (TNF)-α and interleukin (IL)-6—in RAW264.7 cells. The anti-inflammatory potential of compounds 5h and 5n was also evaluated in vivo in a xylene-induced ear inflammation model. Compound 5n showed the most potent anti-inflammatory activity with an inhibition of 49.26% at a dose of 50 mg/kg. This activity is more potent than that of the reference drug ibuprofen (28.13%), and slightly less than that of indometacin (49.36%). To further elucidate the mechanisms underlying these inhibitory effects, LPS-induced nuclear factor-κB (NF-κB) activation and mitogen-activated protein kinase (MAPK) phosphorylation were studied. The results of western blotting showed that the extract obtained from compound 5n inhibited NF-κB (p65) activation and MAPK (extracellular signal-regulated kinase (ERK) and p38) phosphorylation in a dose-dependent manner. Moreover, the results of a docking study of compound 5n into the COX-2 binding site revealed that its mechanism was possibly similar to that of naproxen, a COX-2 inhibitor. The effect of compound 5n on COX-2 antibody was showed it could significantly inhibit COX-2 activity.     

Increased expression of Fe-chelatase leads to increased metabolic flux into heme and confers protection against photodynamically induced oxidative stress

Fe-chelatase (FeCh, EC 4.99.1.1) inserts Fe²⁺ into protoporphyrin IX (Proto IX) to form heme, which influences the flux through the tetrapyrrole biosynthetic pathway as well as fundamental cellular processes. In transgenic rice (Oryza sativa), the ectopic expression of Bradyrhizobium japonicum FeCh protein in cytosol results in a substantial increase of FeCh activity compared to wild-type (WT) rice and an increasing level of heme. Interestingly, the transgenic rice plants showed resistance to oxidative stress caused not only by the peroxidizing herbicide acifluorfen (AF) as indicated by a reduced formation of leaf necrosis, a lower conductivity, lower malondialdehyde and H₂O₂ contents as well as sustained F_v/F_m compared to WT plants, but also by norflurazon, paraquat, salt, and polyethylene glycol. Moreover, the transgenic plants responded to AF treatment with markedly increasing FeCh activity. The accompanying increases in heme content and heme oxygenase activity demonstrate that increased heme metabolism attenuates effects of oxidative stress caused by accumulating porphyrins. These findings suggest that increases in heme levels and porphyrin scavenging capacity support a detoxification mechanism serving against porphyrin-induced oxidative stress. This study also implicates heme as possibly being a positive signal in plant stress responses.     

Transport of Intact Porphyrin by HpuAB, the Hemoglobin-Haptoglobin Utilization System of Neisseria meningitidis

The meningococcal hemA gene was cloned and used to construct a porphyrin biosynthesis mutant. An analysis of the hemA mutant indicated that meningococci can transport intact porphyrin from heme (Hm), hemoglobin (Hb), and Hb-haptoglobin (Hp). By constructing a HemA⁻ HpuAB⁻ double mutant, we demonstrated that HpuAB is required for the transport of porphyrin from Hb and Hb-Hp.     

Proteasome inhibition and oxidative reactions disrupt cellular homeostasis during heme stress

Dual control of cellular heme levels by extracellular scavenger proteins and degradation by heme oxygenases is essential in diseases associated with increased heme release. During severe hemolysis or rhabdomyolysis, uncontrolled heme exposure can cause acute kidney injury and endothelial cell damage. The toxicity of heme was primarily attributed to its pro-oxidant effects; however additional mechanisms of heme toxicity have not been studied systematically. In addition to redox reactivity, heme may adversely alter cellular functions by binding to essential proteins and impairing their function. We studied inducible heme oxygenase (Hmox1)-deficient mouse embryo fibroblast cell lines as a model to systematically explore adaptive and disruptive responses that were triggered by intracellular heme levels exceeding the homeostatic range. We extensively characterized the proteome phenotype of the cellular heme stress responses by quantitative mass spectrometry of stable isotope-labeled cells that covered more than 2000 individual proteins. The most significant signals specific to heme toxicity were consistent with oxidative stress and impaired protein degradation by the proteasome. This ultimately led to an activation of the response to unfolded proteins. These observations were explained mechanistically by demonstrating binding of heme to the proteasome that was linked to impaired proteasome function. Oxidative heme reactions and proteasome inhibition could be differentiated as synergistic activities of the porphyrin. Based on the present data a novel model of cellular heme toxicity is proposed, whereby proteasome inhibition by heme sustains a cycle of oxidative stress, protein modification, accumulation of damaged proteins and cell death.Free heme can accumulate in hemolytic conditions during rhabdomyolysis and locally in wounded or inflamed tissues.¹ The concentration of free heme in the extracellular space and within cells must be controlled within a narrow homeostatic range to avoid cytotoxicity and tissue damage caused by heme stress.²Extracellular release from hemoproteins, cellular uptake, and intracellular metabolism determine the cumulative exposure of cells and tissues to heme.¹ The hemoglobin (Hb) and heme scavenger proteins haptoglobin and hemopexin restrict the accumulation of free heme within the extracellular space and prevent uncontrolled translocation into susceptible cells.^3,4 Within cells, heme is continuously degraded by heme oxygenases (Hmox).^{5, 6, 7, 8} The heme oxygenase system includes the constitutively expressed Hmox2 and inducible Hmox1 that is induced by acute increases in cellular heme such as during exogenous heme exposure.⁹ Cellular heme toxicity can result if excessive extracellular release exceeds the metabolic heme degradation capacity or if Hmox activity is inadequately low, such as that observed in rare conditions associated with loss-of-function mutations in the Hmox1 gene.¹⁰Several mechanisms of heme-triggered cell damage have been explored previously, with a focus on oxidative processes that can be catalyzed by free heme as well as on the activation of innate immunity receptors by the porphyrin.^{3,11, 12, 13, 14, 15, 16, 17} However, there is limited understanding of the ‘metabolic'' disruption that occurs in cells when intracellular free heme exceeds homeostatic levels and causes toxicity. To identify novel mechanisms of heme-triggered cell damage, we systematically explored heme-driven deviations of the cellular proteome phenotype and their underlying molecular mechanisms.The primary signals that consistently appeared throughout our studies suggested that secondary to oxidative processes, the dysfunction of cellular protein homeostasis was the most important component of heme toxicity. These effects could be traced mechanistically to an inhibitory function of the porphyrin in the principal cellular protein degradation machinery: the proteasome.     

A novel kavalactone derivative protects against H2O2-induced PC12 cell death via Nrf2/ARE activation

Oxidative stress is involved in the pathogenesis of neurodegenerative disorders such as Parkinson’s and Alzheimer’s diseases. Natural kavalactones isolated from Piper methysticum (Piperaceae) are capable of activating the Nrf2/ARE (antioxidant response element) pathway and thus enhancing the expression of phase II antioxidant enzymes such as heme oxygenase-1 (HO-1). In an attempt to identify kavalactone derivatives that are more potent in Nrf2/ARE activation than natural compounds, we synthesized a series of chemically-modified kavalactones and studied their effects on the ARE enhancer activity in rat pheochromocytoma PC12 cells. Among 81 compounds tested, a kavalactone derivative, 2′,6′-dichloro-5-methoxymethyl-5,6-dehydrokawain [(E)-6-(2′,6′-dichlorostyryl)-4-methoxy-5-(methoxymethyl)-2H-pyran-2-one] (1), exhibited the strongest ARE enhancer activity. The ARE activation and HO-1 protein induction by the compound 1 were higher than those by natural kavalactones. The compound did not affect cell viability and induced expression of various phase II enzymes. Nuclear translocation of Nrf2 after treatment with 1 was preceded by phosphorylation of ERK1/2 and p38. The compound transiently increased intracellular ROS levels. Finally, pretreatment with the compound ameliorated H₂O₂-induced cell death, which was associated with increased expression of HO-1. These results suggest that the compound 1 protects against oxidative stress-induced neuronal cell death via a preconditioning effect on the Nrf2/ARE activation.