Encapsulation of myoglobin with a mesoporous silicate results in new capabilities

A metmyoglobin (Fe3+), an oxidized form of myoglobin (Fe2+), was confined in nanospaces of about 4 nm in diameter in mesoporous silica (FSM; folded-sheet mesoporous material), forming a metmyoglobin (Fe3+)-FSM nanoconjugate. The spectral characteristics of metmyoglobin (Fe3+)- and myoglobin (Fe2+)-FSM show an absorption curve quite similar to that of native metmyoglobin, indicating that myoglobin retains its higher-order structure in the pores of FSM. The metmyoglobin (Fe3+)-FSM conjugate had not only a peroxidase-like activity in the presence of hydrogen peroxide (a hydrogen acceptor) and 2,2-azino-bis(3-ethylbenzothiazoline)-6-sulfomic acid (ABTS) or guaiacol (a hydrogen donor) but also an advanced molecular recognition ability enabling it to distinguish between ABTS and guaiacol. Furthermore, the metmyoglobin (Fe3+)-FSM showed the peroxidase-like activity even in an organic media using benzoyl peroxide as the hydrogen acceptor and leucocrystal violet as the hydrogen donor. The simple immobilization of metmyoglobin (Fe3+) into FSM results in enhanced catalytic activity in organic media compared to that of native metmyoglobin (Fe3+).     

Hemin (Fe(3+))-- and heme (Fe(2+))--smectite conjugates as a model of hemoprotein based on spectrophotometry

Hemin (Fe(3+)) was adsorbed onto synthetic smectite (clay mineral) in acetone to form a hemin-smectite conjugate. The hemin-smectite conjugate became soluble in water to form a transparent colloidal solution with a dark brown color. Its absorption spectrum in water showed a sharp Soret band at 398 nm with the molar extinction coefficient as epsilon(398nm) = 11.6 x 10(4) M(-1) cm(-1), which is in good agreement with epsilon(398nm) = (12.2 +/- 3) x 10(4) M(-1) cm(-1) of monomeric hematin (1). Hemin (Fe(3+))-smectite conjugate had a peroxidase-like activity in the presence of hydrogen peroxide (a hydrogen acceptor) and guaiacol (a hydrogen donor) in aqueous solution and its activity was higher than that of hematin. Hemin (Fe(3+))-smectite conjugate in water was reduced by adding sodium dithionite to form a heme (Fe(2+))-smectite conjugate which is also a transparent colloidal solution in water. Its absorption spectrum in aqueous solution was surprisingly in close agreement with that of oxyhemoglobin. Its peak positions of alpha, beta, and Soret bands were located in only a 9--3 nm shift to shorter wavelengths in comparison with those of oxyhemoglobin. Therefore, heme (Fe(2+))-smectite conjugate was bound to O(2) to form O(2)-heme (Fe(2+))-smectite conjugate. The addition of carbon monoxide, CO, to O(2)-heme (Fe(2+))-smectite conjugate caused the formation of CO-heme (Fe(2+))-smectite conjugate with a similar absorption spectrum of carboxyhemoglobin (HbCO) accompanied by shifting 8--10 nm to shorter wavelength. Therefore, the transformation of O(2)-heme (Fe(2+))-smectite conjugate to CO-heme (Fe(2+))-smectite conjugate was accompanied by shifting of 7, 4, and 3 nm to shorter wavelengths in the alpha, beta, and Soret bands respectively, which are similar to the spectral change from oxyhemoglobin to carboxyhemoglobin. Also the ratio (1:1.6) of the molar extinction coefficient of Soret band of O(2)-heme (Fe(2+))-smectite conjugate and CO-heme (Fe(2+))-smectite conjugate was surprisingly agreement with ratio (1:1.5) of oxyhemoglobin and carboxyhemoglobin. The phenomenon shown above was unexpectedly found during the course of study of bioconjugate of a bioactive substance, hemin (Fe(3+)) or heme (Fe(2+)), and a clay mineral, smectite, in place of the protein of globin in hemoglobin.     

Effective immobilization of subunit protein in mesoporous silica modified with ethanol

Ethoxylated FSM-type mesoporous silica (folded-sheet mesoporous material) with a pore diameter of 6.2 nm (FSM6.2) remarkably enhances rigidly of the structure in aqueous solutions. The esterified material could be used successfully as an adsorbent to accommodate subunit protein, methemoglobin (Fe(3+)). Furthermore, methemoglobin (Fe(3+)) in the pores of ethoxy-FSM is maintained a peroxidase activity similar to the native, indicating methemoglobin retains its fore subunit structure in the pores of FSM6.2.     

Catalase encapsulated in mesoporous silica and its performance

Catalase was confined in a nanospace of about 7 nm in diameter in mesoporous silica (FSM; folded-sheet mesoporous material), forming a catalase-FSM conjugate. That the enzyme was encapsulated in the pores of mesoporous materials was proven by the direct visualization technique. The catalase-FSM showed not only an activity similar to that of the native catalase in the decomposition of H₂O₂ but also much higher stability: specifically, the catalase immobilized in FSM still retained its original activity after being applied 50 times to the decomposition, whereas the native lost its activity after 20 times recycling. Furthermore, the catalase-FSM showed catalase activity even in an organic media in which the native catalase had almost no catalytic activity.     

The ensemble of hetero-proteins in inorganic nanochannels

The assembly and proper alignment of two heterofluorescent proteins (sGFP and DsRed) in the mesoporous channels of ethanol-treated FSM6.2 (a folded-sheet mesoporous material with a pore diameter of 6.2 nm) was confirmed using a fluorescence resonance energy transfer (FRET) technique. The sGFP-DsRed-FSM6.2 conjugate showed a large decrease in the emission of donor (sGFP) fluorescence, indicating that the conjugate functions as an energy transfer system through the combination of the two heteroproteins, due to the successful encapsulation of the sGFP-DsRed pairs in the mesopores. Fluorescence spectral analysis demonstrated that the proteins were highly dispersed and homogeneously encapsulated in the mesopores of FSM6.2, even at high concentration, although they spontaneously aggregated and showed a red shift in solution at the concentration corresponding to that in the conjugate. Furthermore, an increase in the amount of sGFP and DsRed adsorbed to the pores of FSM6.2 led to a decrease in the distance between these proteins, resulting in enhancement of FRET efficiency.     

Hemin induces active chloride secretion in Caco-2 cells

Enterocytes maintain fluid-electrolyte homeostasis by keeping a tight barrier and regulating ion channels. Carbon monoxide (CO), a product of heme degradation, modulates electrolyte transport in kidney and lung epithelium, but its role in regulating intestinal fluid-electrolyte homeostasis has not been studied. The major source of endogenous CO formation comes from the degradation of heme via heme oxygenase. We hypothesized that heme activates electrolyte transport in intestinal epithelial cells. Basolateral hemin treatment increased baseline Caco-2 cell short-circuit currents (I(sc)) twofold (control = 1.96 +/- 0.14 microA/cm(2) vs. hemin = 4.07 +/- 0.16 microA/cm(2), P < 0.01); apical hemin had no effect. Hemin-induced I(sc) was caused by Cl- secretion because it was inhibited in Cl- -free medium, with ouabain, 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), or DIDS. Apical electrogenic Na+ channel inhibitor benzamil had no effect on hemin-induced I(sc). Hemin did not alter the ability of Caco-2 cells to respond maximally to forskolin, but a soluble guanylate cyclase inhibitor, [1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ) inhibited the effects of hemin. A CO-releasing molecule, tricarbonyldichlororuthenium II, induced active Cl- secretion that was also inhibited with ODQ. We conclude that hemin induces active Cl- secretion in Caco-2 cells via a cGMP-dependent pathway. These effects are probably the consequence of CO formation. Heme and CO may be important regulators of intestinal fluid-electrolyte homeostasis.     

Hemin control of heme biosynthesis in mouse Friend virus-transformed erythroleukemia cells in culture.

Hemin treatment of mouse Friend virus-transformed cells in cultured caused a dose-dependent increase in hemoglobin synthesis. By the addition of radioactively labeled hemin and by the analysis of the radioactive heme in hemoglobin, only 60 to 70% of heme in the newly synthesized hemoglobin was accounted for by the exogenously added hemin. In keeping with this finding, hemin treatment increased the activity of two enzymes in the heme biosynthetic activity, i.e. delta-aminolevulinate (ALA) dehydratase and uroporphyrinogen-I (URO) synthase in these cells. Incorporation of [2(-14C)]glycine, [14C]ALA, and 59Fe into heme was also significantly increased in the cells treated with hemin, suggesting that essentially all enzyme activities in the heme biosynethetic pathway were increased after hemin treatment. These results indicate that heme in the newly synthesized hemoglobin in hemin-treated Friend cells derives both from hemin added to the culture and from heme synthesized intracellularly. In addition, these results suggest that the stimulation of heme biosynthesis by hemin in Friend virus-transformed cells is in contrast to the hemin repression of heme biosynthesis in liver cells.     

Neurotrophic activity of neudesin, a novel extracellular heme-binding protein, is dependent on the binding of heme to its cytochrome b5-like heme/steroid-binding domain

Neudesin is a secreted protein with neurotrophic activity in neurons and undifferentiated neural cells. We report here that neudesin is an extracellular heme-binding protein and that its neurotrophic activity is dependent on the binding of heme to its cytochrome b(5)-like heme/steroid-binding domain. At first, we found that at least a portion of the purified recombinant neudesin appeared to bind hemin because the purified neudesin solution was tinged with green and had a sharp absorbance peak at 402 nm. The addition of exogenous hemin extensively increased the amount of hemin-bound neudesin. In contrast, neudesinDeltaHBD, a mutant lacking the heme-binding domain, could not bind hemin. The neurotrophic activity of the recombinant neudesin that bound exogenous hemin (neudesin-hemin) was significantly greater than that of the recombinant neudesin in either primary cultured neurons or Neuro2a cells, suggesting that the activity of neudesin depends on hemin. The neurotrophic activity of neudesin was enhanced by the binding of Fe(III)-protoporphyrin IX, but neither Fe(II)-protoporphyrin IX nor protoporphyrin IX alone. The inhibition of endogenous neudesin by RNA interference significantly decreased cell survival in Neuro2a cells. This indicates that endogenous neudesin possibly contains hemin. The experiment with anti-neudesin antibody suggested that the endogenous neudesin detected in the culture medium of Neuro2a cells was associated with hemin because it was not retained on a heme-affinity column at all. Neudesin is the first extracellular heme-binding protein that shows signal transducing activity by itself. The present findings may shed new light on the function of extracellular heme-binding proteins.     

Bacillus anthracis IsdG, a heme-degrading monooxygenase

Bacillus anthracis, the causative agent of anthrax, utilizes hemin and hemoglobin for growth in culture, suggesting that these host molecules serve as sources for the nutrient iron during bacterial infection. Bioinformatic analyses of the B. anthracis genome revealed genes with similarity to the iron-regulated surface determinant (isd) system responsible for heme uptake in Staphylococcus aureus. We show that the protein product of one of these genes, isdG, binds hemin in a manner resembling the heme binding of known heme oxygenases. Formation of IsdG:hemin complexes in the presence of a suitable electron donor, e.g., ascorbate or cytochrome P450 reductase, promotes catalytic degradation of hemin to biliverdin with concomitant release of iron. IsdG is required for B. anthracis utilization of hemin as a sole iron source, and it is also necessary for bacterial protection against heme-mediated toxicity. These data suggest that IsdG functions as a heme-degrading monooxygenase in B. anthracis.     

Hemin rescues adrenodoxin, heme a and cytochrome oxidase activity in frataxin-deficient oligodendroglioma cells

Mutations in the frataxin gene cause neurodegeneration and demyelination in Friedreich's ataxia. We showed earlier that frataxin deficiency causes primary iron-sulfur cluster defects, and later causes defects in heme and cytochrome c hemoprotein levels. Iron-sulfur (Fe/S) clusters are required in two enzymes of heme biosynthesis in humans i.e. in ferrochelatase and adrenodoxin. However, decreases in ferrochelatase activity have not been observed in frataxin-deficient HeLa cells or patient lymphoblasts. We knocked down frataxin in oligodendroglioma cells using siRNA, which produced significant defects in the activity of the Fe/S cluster enzymes adrenodoxin and aconitase, the adrenodoxin product heme a, and cytochrome oxidase, for which heme a serves as a prosthetic group. Exogenous hemin produced a significant rescue of adrenodoxin, aconitase, heme a levels and cytochrome oxidase activity. Thus hemin rescues iron-sulfur cluster defects that are the result of frataxin-deficiency, perhaps as a consequence of increasing the pool of bioavailable iron, and thus should be more fully tested for beneficial effects in Friedreich's ataxia models.     

Non-heme induction of heme oxygenase-1 does not alter cellular iron metabolism

The catabolism of heme is carried out by members of the heme oxygenase (HO) family. The products of heme catabolism by HO-1 are ferrous iron, biliverdin (subsequently converted to bilirubin), and carbon monoxide. In addition to its function in the recycling of hemoglobin iron, this microsomal enzyme has been shown to protect cells in various stress models. Implicit in the reports of HO-1 cytoprotection to date are its effects on the cellular handling of heme/iron. However, the limited amount of uncommitted heme in non-erythroid cells brings to question the source of substrate for this enzyme in non-hemolytic circumstances. In the present study, HO-1 was induced by either sodium arsenite (reactive oxygen species producer) or hemin or overexpressed in the murine macrophage-like cell line, RAW 264.7. Both of the inducers elicited an increase in active HO-1; however, only hemin exposure caused an increase in the synthesis rate of the iron storage protein, ferritin. This effect of hemin was the direct result of the liberation of iron from heme by HO. Cells stably overexpressing HO-1, although protected from oxidative stress, did not display elevated basal ferritin synthesis. However, these cells did exhibit an increase in ferritin synthesis, compared with untransfected controls, in response to hemin treatment, suggesting that heme levels, and not HO-1, limit cellular heme catabolism. Our results suggest that the protection of cells from oxidative insult afforded by HO-1 is not due to the catabolism of significant amounts of cellular heme as thought previously.     

Expression and characterization of cyanobacterium heme oxygenase, a key enzyme in the phycobilin synthesis. Properties of the heme complex of recombinant active enzyme.

An efficient bacterial expression system of cyanobacterium Synechocystis sp. PCC 6803 heme oxygenase gene, ho-1, has been constructed, using a synthetic gene. A soluble protein was expressed at high levels and was highly purified, for the first time. The protein binds equimolar free hemin to catabolize the bound hemin to ferric-biliverdin IX alpha in the presence of oxygen and reducing equivalents, showing the heme oxygenase activity. During the reaction, verdoheme intermediate is formed with the evolution of carbon monoxide. Though both ascorbate and NADPH-cytochrome P450 reductase serve as an electron donor, the heme catabolism assisted by ascorbate is considerably slow and the reaction with NADPH-cytochrome P450 reductase is greatly retarded after the oxy-heme complex formation. The optical absorption spectra of the heme-enzyme complexes are similar to those of the known heme oxygenase complexes but have some distinct features, exhibiting the Soret band slightly blue-shifted and relatively strong CT bands of the high-spin component in the ferric form spectrum. The heme-enzyme complex shows the acid-base transition, where two alkaline species are generated. EPR of the nitrosyl heme complex has established the nitrogenous proximal ligand, presumably histidine 17 and the obtained EPR parameters are discriminated from those of the rat heme oxygenase-1 complex. The spectroscopic characters as well as the catabolic activities strongly suggest that, in spite of very high conservation of the primary structure, the heme pocket structure of Synechocystis heme oxygenase isoform-1 is different from that of rat heme oxygenase isoform-1, rather resembling that of bacterial heme oxygenase, H mu O.     

The mechanism of direct heme transfer from the streptococcal cell surface protein Shp to HtsA of the HtsABC transporter

The heme-binding proteins Shp and HtsA are part of the heme acquisition machinery found in Streptococcus pyogenes. The hexacoordinate heme (Fe(II)-protoporphyrin IX) or hemochrome form of holoShp (hemoShp) is stable in air in Tris-HCl buffer, pH 8.0, binds to apoHtsA with a K(d) of 120 +/- 18 microm, and transfers its heme to apoHtsA with a rate constant of 28 +/- 6s(-1) at 25 degrees C, pH 8.0. The hemoHtsA product then autoxidizes to the hexacoordinate hemin (Fe(III)-protoporphyrin IX) or hemichrome form (hemiHtsA) with an apparent rate constant of 0.017 +/- 0.002 s(-1). HemiShp also rapidly transfers hemin to apoHtsA through a hemiShp.apoHtsA complex (K(d) = 48 +/- 7 microM) at a rate approximately 40,000 times greater than the rate of simple hemin dissociation from hemiShp into solvent (k(transfer) = 43 +/- 3s(-1) versus k(-hemin) = 0.0003 +/- 0.00006 s(-1)). The rate constants for hemin binding to and dissociation from HtsA (k'(hemin) approximately 80 microm(-1) s(-1), k(-hemin) = 0.0026 +/- 0.0002 s(-1)) are 50- and 10-fold greater than the corresponding rate constants for Shp (k(hemin) approximately 1.6 microM(-1) s(-1), k(-hemin) = 0.0003 s(-1)), which implies that HtsA has a more accessible active site. However, the affinity of apoHtsA for hemin (k(hemin) approximately 31,000 microm(-1)) is roughly 5-fold greater than that of apoShp (k(hemin) approximately 5,300 microM(-1)), accounting for the net transfer from Shp to HstA. These results support a direct, rapid, and affinity-driven mechanism of heme and hemin transfer from the cell surface receptor Shp to the ATP-binding cassette transporter system.     

Heme oxygenase-1 and the ischemia-reperfusion injury in the rat heart

Carbon monoxide (CO) is a signaling gas produced intracellularly by heme oxygenase (HO) enzymes using heme as a substrate. During heme breakdown, HO-1 and HO-2 release CO, biliverdin, and Fe(2+). In this study, we investigated the effects of manipulation of the HO-1 system in an in vivo model of focal ischemia-reperfusion (FIR) in the rat heart. Male Wistar albino rats, under general anesthesia and artificial ventilation, underwent thoracotomy, the pericardium was opened, and a silk suture was placed around the left descending coronary artery; ischemia was induced by tightening the suture and was monitored for 30 min. Subsequently, the ligature was released to allow reperfusion lasting for 60 min. The first group of rats was sham operated and injected intraperitoneally (i.p.) with saline. The second group underwent FIR. The third group was treated ip 18 hr before FIR with hemin (4 mg/kg). The fourth group was pretreated ip 24 hr before FIR and 6 hr before hemin with zinc protoporphyrin IX (ZnPP-IX, 50 microg/kg). Specimens of the left ventricle were taken for determination of HO expression and activity, infarct size, malonyldialdehyde (MDA) production, and tissue calcium content. FIR led to a significant increase in the generation of MDA and notably raised tissue calcium levels. Induction of HO-1 by hemin significantly decreased infarct size, incidence of reperfusion arrhythmias, MDA generation, and calcium overload induced by FIR. These effects were prevented by the HO-1 inhibitor ZnPP-IX. The present experiments show that the concerted actions of CO, iron, and biliverdin/bilirubin modulate the FIR-induced myocardial injury.     

Hemopexin decreases hemin accumulation and catabolism by neural cells

Hemopexin is a serum, CSF, and neuronal protein that is protective after experimental stroke. Its efficacy in the latter has been linked to increased expression and activity of heme oxygenase (HO)-1, suggesting that it facilitates heme degradation and subsequent release of cytoprotective biliverdin and carbon monoxide. In this study, the effect of hemopexin on the rate of hemin breakdown by CNS cells was investigated in established in vitro models. Equimolar hemopexin decreased hemin breakdown, as assessed by gas chromatography, by 60–75% in primary cultures of murine neurons and glia. Extracellular hemopexin reduced cell accumulation of ⁵⁵Fe-hemin by over 90%, while increasing hemin export or extraction from membranes by fourfold. This was associated with significant reduction in HO-1 expression and neuroprotection. In a cell-free system, hemin breakdown by recombinant HO-1 was reduced over 80% by hemopexin; in contrast, albumin and two other heme-binding proteins had no effect. Although hemopexin was detected on immunoblots of cortical lysates from adult mice, hemopexin knockout per se did not alter HO activity in cortical cells treated with hemin. These results demonstrate that hemopexin decreases the accumulation and catabolism of exogenous hemin by neural cells. Its beneficial effect in stroke models is unlikely to be mediated by increased production of cytoprotective heme breakdown products.     

Role of heme oxygenase-1 in hypoxia-reoxygenation: requirement of substrate heme to promote cardioprotection

Heme oxygenase-1 (HO-1) catalyzes the enzymatic degradation of heme to carbon monoxide, bilirubin, and iron. All three products possess biological functions; bilirubin, in particular, is a potent free radical scavenger of which its antioxidant property is enhanced at low oxygen tension. Here, we investigated the effect of severe hypoxia and reoxygenation on HO-1 expression in cardiomyocytes and determined whether HO-1 and its product, bilirubin, have a protective role against reoxygenation damage. Hypoxia caused a time-dependent increase in both HO-1 expression and heme oxygenase activity, which gradually declined during reoxygenation. Reoxygenation of hypoxic cardiomyocytes produced marked injury; however, incubation with hemin or bilirubin during hypoxia considerably reduced the damage at reoxygenation. The protective effect of hemin is attributable to increased availability of substrate for heme oxygenase activity, because hypoxic cardiomyocytes generated very little bilirubin when incubated with medium alone but produced substantial bile pigment in the presence of hemin. Interestingly, incubation with hemin also maintained high heme oxygenase activity levels during the reoxygenation period. Reactive oxygen species generation was enhanced after hypoxia, and hemin and bilirubin were capable once again to attenuate this effect. These results indicate that the HO-1-bilirubin pathway can effectively defend hypoxic cardiomyocytes against reoxygenation injury and highlight the issue of heme availability in the cytoprotective action afforded by HO-1.     

Hydrophobic adsorption and covalent immobilization of Candida antarctica lipase B on mixed-function-grafted silica gel supports for continuous-flow biotransformations

Adsorption onto solid supports has proven to be an easy and effective way to improve the mechanical and catalytic properties of lipases. Covalent binding of lipases onto the support surface enhances the active lifetime of the immobilized biocatalysts. Our study indicates that mesoporous silica gels grafted with various functions are ideal supports for both adsorptive and covalent binding for lipase B from Candida antarctica (CaLB). Adsorption of CaLB on phenyl-functionalized silica gels improved in particular its specific activity, whereas adsorption on aminoalkyl-modified silica gels enabling covalent binding with the proper reagents resulted in only moderate specific activity. In addition, adsorption on silica gels modified by mixtures of phenyl- and aminoalkyl silanes significantly increased the productivity of CaLB. Furthermore, CaLB adsorbed onto a phenyl/aminoalkyl-modified surface and then treated with glutardialdehyde (GDA) as cross-linking agent provided a biocatalyst of enhanced durability. Adsorbed and cross-linked CaLB was resistant to detergent washing that would otherwise physically deactivate adsorbed CaLB preparations. The catalytic properties of our best immobilized CaLB variants, including temperature-dependent behavior were compared between 0 and 70 °C with those of two commercial CaLB biocatalysts in the continuous-flow kinetic resolutions of racemic 1-phenylethanol rac-1a and 1-phenylethanamine rac-1b.     

Iron-dependent binding of bovine milk α-casein with holo-lactoferrin, but not holo-transferrin

Bovine milk α-casein has been identified as an iron- and heme-binding protein. However, the physiological role of its iron-binding remains to be elucidated in more detail. α-Casein was immobilized on CNBr-activated Sepharose 4B beads, and the α-casein agarose beads efficiently bound hemin as well as ferrous ammonium sulfate (Fe(2+)) as compared with control beads. Additionally, α-casein-beads bound bovine holo-lactoferrin (Lf), but not holo-transferrin. Lf caused the release of Fe(2+) which had bound to the α-casein-agarose beads beforehand. These results suggest that bovine α-casein iron-dependently binds holo-bovine Lf more strongly than Fe(2+), and that strong binding between them may play a physiological role in regulating iron homeostasis in the bovine mammary gland.     

Role of PUG1 in inducible porphyrin and heme transport in Saccharomyces cerevisiae

Unlike pathogenic fungi, the budding yeast Saccharomyces cerevisiae is not efficient at using heme as a nutritional source of iron. Here we report that for this yeast, heme uptake is induced under conditions of heme starvation. Heme synthesis requires oxygen, and yeast grown anaerobically exhibited an increased uptake of hemin. Similarly, a strain lacking aminolevulinate synthase exhibited a sixfold increase in hemin uptake when grown without 2-aminolevulinic acid. We used microarray analysis of cells grown under reduced oxygen tension or reduced intracellular heme conditions to identify candidate genes involved in heme uptake. Surprisingly, overexpression of PUG1 (protoporphyrin uptake gene 1) resulted in reduced utilization of exogenous heme by a heme-deficient strain and, conversely, increased the utilization of protoporphyrin IX. Pug1p was localized to the plasma membrane by indirect immunofluorescence and subcellular fractionation. Strains overexpressing PUG1 exhibited decreased accumulation of [(55)Fe]hemin but increased accumulation of protoporphyrin IX compared to the wild-type strain. To measure the effect of PUG1 overexpression on intracellular heme pools, we used a CYC1-lacZ reporter, which is activated in the presence of heme, and we monitored the activity of a heme-containing metalloreductase, Fre1p, expressed from a constitutive promoter. The data from these experiments were consistent with a role for Pug1p in inducible protoporphyrin IX influx and heme efflux.     

Effect of heme and heme oxygenase-1 on vascular endothelial growth factor synthesis and angiogenic potency of human keratinocytes

BACKGROUND: Skin injury leads to the release of heme, a potent prooxidant which is degraded by heme oxygenase-1 (HO-1) to carbon monoxide, iron, and biliverdin, subsequently reduced to bilirubin. Recently the involvement of HO-1 in angiogenesis has been shown; however, the role of heme and HO-1 in wound healing angiogenesis has not been yet investigated. RESULTS: Treatment of HaCaT keratinocytes with hemin (heme chloride) induced HO-1 expression and activity. The effect of heme on vascular endothelial growth factor (VEGF) synthesis is variable: induction is significant after a short, 6 h treatment with heme, while longer stimulation may attenuate its production. The involvement of HO-1 in VEGF synthesis was confirmed by inhibition of VEGF expression by SnPPIX, a blocker of HO activity and by attenuation of HO-1 mRNA expression with specific siRNA. Importantly, induction of HO-1 by hemin was able to overcome the inhibitory effect of high glucose on VEGF synthesis. Moreover, HO-1 expression was also induced in keratinocytes cultured in hypoxia, with concomitant augmentation of VEGF production, which was further potentiated by hemin stimulation. Accordingly, conditioned media from keratinocytes overexpressing HO-1 enhanced endothelial cell proliferation and augmented formation of capillaries in angiogenic assay in vitro. CONCLUSIONS: HO-1 is involved in hemin-induced VEGF expression in HaCaT and may play a role in hypoxic regulation of this protein. HO-1 overexpression may be beneficial in restoring the proper synthesis of VEGF disturbed in diabetic conditions.