Induction of ER stress protects gastric cancer cells against apoptosis induced by cisplatin and doxorubicin through activation of p38 MAPK

Porphyromonas gingivalis acquires heme through an outer-membrane heme transporter HmuR and heme-binding hemophore-like lipoprotein HmuY. Here, we compare binding of iron(III) mesoporphyrin IX (mesoheme) and iron(III) deuteroporphyrin IX (deuteroheme) to HmuY with that of iron(III) protoporphyrin IX (protoheme) and protoporphyrin IX (PPIX) using spectroscopic methods. In contrast to PPIX, mesoheme and deuteroheme enter the HmuY heme cavity and are coordinated by His134 and His166 residues in a fully analogous way to protoheme binding. However, in the case of deuteroheme two forms of HmuY–iron porphyrin complex were observed differing by a 180° rotation of porphyrin about the α-γ-meso-carbon axis. Since the use of porphyrins either as active photosensitizers or in combination with antibiotics may have therapeutic value for controlling bacterial growth in vivo, it is important to compare the binding of heme derivatives to HmuY.     

Frataxin and mitochondrial carrier proteins, Mrs3p and Mrs4p, cooperate in providing iron for heme synthesis

Frataxin is a conserved mitochondrial protein implicated in cellular iron metabolism. Deletion of the yeast frataxin homolog (YFH1) was combined with deletions of MRS3 and MRS4, mitochondrial carrier proteins implicated in iron homeostasis. As previously reported, the Deltayfh1 mutant accumulated iron in mitochondria, whereas the triple mutant (DeltaDeltaDelta) did not. When wild-type, Deltamrs3/4, Deltayfh1, and DeltaDeltaDelta strains were incubated anaerobically, all strains were devoid of heme and protected from iron and oxygen toxicity. The cultures were then shifted to air for a short time (4-5 h) or a longer time (15 h), and the evolving mutant phenotypes were analyzed (heme-dependent growth, total heme, cytochromes, heme proteins, and iron levels). A picture emerges from these data of defective heme formation in the mutants, with a markedly more severe defect in the DeltaDeltaDelta than in the individual Deltamrs3/4 or Deltayfh1 mutants (a "synthetic" defect in the genetic sense). The defect(s) in heme formation could be traced to lack of iron. Using a real time assay of heme biosynthesis, porphyrin precursor and iron were presented to permeabilized cells, and the appearance and disappearance of fluorescent porphyrins were followed. The Mrs3/4p carriers were required for rapid iron transport into mitochondria for heme synthesis, whereas there was also evidence for an alternative slower system. A different role for Yfh1p was observed under conditions of low mitochondrial iron and aerobic growth (revealed in the DeltaDeltaDelta), acting to protect bioavailable iron within mitochondria and to facilitate its use for heme synthesis.     

A direct and simultaneous detection of zinc protoporphyrin IX, free protoporphyrin IX, and fluorescent heme degradation product in red blood cell hemolysates

Fluorescence emission of free protoporphyrin IX (PPIX, em. approximately 626 nm), zinc protoporphyrin IX (ZPP, em. approximately 594 nm) and fluorescent heme degradation product (FHDP, em. approximately 466 nm) are identified and simultaneously detected in mouse and human red cell hemolysates, when excited at 365 nm. A novel method is established for comparing relative FHDP, PPIX and ZPP levels in hemolysates without performing red cell porphyrin extractions. The ZPP fluorescence directly measured in hemolysates (F(365/594)) correlates with the ZPP fluorescence obtained from acetone/water extraction (R(2) = 0.9515, P < 0.0001). The relative total porphyrin (ZPP and PPIX) fluorescence obtained from direct hemolysate fluorescence measurements also correlates with red blood cell total porphyrins determined by ethyl acetate extraction (Piomelli extraction, R(2) = 0.88, P < 0.0001). These fluorescent species serves as biomarkers for alterations in Hb synthesis and Hb stability.     

Binding Specificity of the Porphyromonas gingivalis Heme and Hemoglobin Receptor HmuR, Gingipain K, and Gingipain R1 for Heme, Porphyrins, and Metalloporphyrins

Previous genetic and biochemical studies have confirmed that hemoglobin and hemin utilization in Porphyromonas gingivalis is mediated by the outer membrane hemoglobin and heme receptor HmuR, as well as gingipain K (Kgp), a lysine-specific cysteine protease, and gingipain R1 (HRgpA), one of two arginine-specific cysteine proteases. In this study we report on the binding specificity of the recombinant P. gingivalis HmuR protein and native gingipains for hemoglobin, hemin, various porphyrins, and metalloporphyrins as assessed by spectrophotometric assays, by affinity chromatography, and by enzyme-linked immunosorbent assay. Protoporphyrin, mesoporphyrin, deuteroporphyrin, hematoporphyrin, and some of their iron, copper, and zinc derivatives were examined to evaluate the role of both the central metal ion and the peripheral substituents on binding to recombinant HmuR and soluble gingipains. Scatchard analysis of hemin binding to Escherichia coli cells expressing recombinant membrane-associated six-His-tagged HmuR yielded a linear plot with a binding affinity of 2.4 x 10(-5) M. Recombinant E. coli cells bound the iron, copper, and zinc derivatives of protoporphyrin IX (PPIX) with similar affinities, and approximately four times more tightly than PPIX itself, which suggests that the active site of HmuR contains a histidine that binds the metal ion in the porphyrin ring. Furthermore, we found that recombinant HmuR prefers the ethyl and vinyl side chains of the PPIX molecule to either the larger hydroxyethyl or smaller hydrogen side chains. Kgp and HRgpA were demonstrated to bind various porphyrins and metalloporphyrins with affinities similar to those for hemin, indicating that the binding of Kgp and HRgpA to these porphyrins does not require a metal within the porphyrin ring. We did not detect the binding of RgpB, the arginine-specific cysteine protease that lacks a C-terminal hemagglutinin domain, to hemoglobin, porphyrins, or metalloporphyrins. Kgp and HRgpA, but not RgpB, were demonstrated to bind directly to soluble recombinant six-His-tagged HmuR. Several possible mechanisms for the cooperation between outer membrane receptor HmuR and proteases Kgp and HRgpA in hemin and hemoglobin binding and utilization are discussed.     

Abcb10 Role in Heme Biosynthesis In Vivo: Abcb10 Knockout in Mice Causes Anemia with Protoporphyrin IX and Iron Accumulation

Abcb10, member 10 of the ABC transporter family, is reportedly a part of a complex in the mitochondrial inner membrane with mitoferrin-1 (Slc25a37) and ferrochelatase (Fech) and is responsible for heme biosynthesis in utero. However, it is unclear whether loss of Abcb10 causes pathological changes in adult mice. Here, we show that Abcb10^−/− mice lack heme biosynthesis and erythropoiesis abilities and die in midgestation. Moreover, we generated Abcb10^F/−; Mx1-Cre mice, with Abcb10 in hematopoietic cells deleted, which showed accumulation of protoporphyrin IX and maturation arrest in reticulocytes. Electron microscopy images of Abcb10^−/− hematopoietic cells showed a marked increase of iron deposits at the mitochondria. These results suggest a critical role for Abcb10 in heme biosynthesis and provide new insights into the pathogenesis of erythropoietic protoporphyria and sideroblastic anemia.     

Effect of decreased ferrochelatase activity on iron and porphyrin content in mitochondria of mice with porphyria induced by griseofulvin

The content of iron and protoporphyrin in liver mitochondria from mice with porphyria induced by griseofulvin was measured. The amount of porphyrin was 0.0076 +/- 0.0043, 4.11 +/- 0.58 and 22.2 +/- 6.8 nmol/mg protein (n = 5) in mitochondria from control animals and animals treated with griseofulvin for 3 days and 4-5 weeks, respectively. The energy coupling of the mitochondria was greatly diminished after 4-5 weeks of treatment, and the ferrochelatase activity was inhibited 80-90%, compared to that of control animals. Mitochondrial preparations isolated by differential centrifugation were contaminated with iron-containing lysosomes which could be removed by Percoll density-gradient centrifugation. In purified mitochondrial preparations no change in the amount of non-heme iron was found after griseofulvin feeding, representing 3.36 +/- 0.15, 3.97 +/- 0.40 and 3.59 +/- 0.23 nmol/mg protein for control animals, 3 days- and 4-5 weeks-treated animals, respectively (n = 4). A mitochondrial iron pool previously identified in rat liver mitochondria and shown to be available for heme synthesis in vitro (Tanger?s, A. (1985) Biochim. Biophys. Acta 843, 199-207) was also present in mitochondria from mice. The magnitude of this iron pool, as well as its availability for heme synthesis, was not changed after treatment of the animals with griseofulvin. The fact that porphyrin, but not iron, accumulated in the mitochondria when ferrochelatase was inhibited is discussed with regard to our understanding of the process of heme synthesis and its regulation.     

Identification of the Mitochondrial Heme Metabolism Complex

Heme is an essential cofactor for most organisms and all metazoans. While the individual enzymes involved in synthesis and utilization of heme are fairly well known, less is known about the intracellular trafficking of porphyrins and heme, or regulation of heme biosynthesis via protein complexes. To better understand this process we have undertaken a study of macromolecular assemblies associated with heme synthesis. Herein we have utilized mass spectrometry with coimmunoprecipitation of tagged enzymes of the heme biosynthetic pathway in a developing erythroid cell culture model to identify putative protein partners. The validity of these data obtained in the tagged protein system is confirmed by normal porphyrin/heme production by the engineered cells. Data obtained are consistent with the presence of a mitochondrial heme metabolism complex which minimally consists of ferrochelatase, protoporphyrinogen oxidase and aminolevulinic acid synthase-2. Additional proteins involved in iron and intermediary metabolism as well as mitochondrial transporters were identified as potential partners in this complex. The data are consistent with the known location of protein components and support a model of transient protein-protein interactions within a dynamic protein complex.     

Mitochondrial Translocator Protein (TSPO) Function Is Not Essential for Heme Biosynthesis

Function of the mammalian translocator protein (TSPO; previously known as the peripheral benzodiazepine receptor) remains unclear because its presumed role in steroidogenesis and mitochondrial permeability transition established using pharmacological methods has been refuted in recent genetic studies. Protoporphyrin IX (PPIX) is considered a conserved endogenous ligand for TSPO. In bacteria, TSPO was identified to regulate tetrapyrrole metabolism and chemical catalysis of PPIX in the presence of light, and in vertebrates, TSPO function has been linked to porphyrin transport and heme biosynthesis. Positive correlation between high TSPO expression in cancer cells and susceptibility to photodynamic therapy based on their increased ability to convert the precursor 5-aminolevulinic acid (ALA) to PPIX appeared to reinforce this mechanism. In this study, we used TSPO knock-out (Tspo^−/−) mice, primary cells, and different tumor cell lines to examine the role of TSPO in erythropoiesis, heme levels, PPIX biosynthesis, phototoxic cell death, and mitochondrial bioenergetic homeostasis. In contrast to expectations, our results demonstrate that TSPO deficiency does not adversely affect erythropoiesis, heme biosynthesis, bioconversion of ALA to PPIX, and porphyrin-mediated phototoxic cell death. TSPO expression levels in cancer cells do not correlate with their ability to convert ALA to PPIX. In fibroblasts, we observed that TSPO deficiency decreased the oxygen consumption rate and mitochondrial membrane potential (ΔΨm) indicative of a cellular metabolic shift, without a negative impact on porphyrin biosynthetic capability. Based on these findings, we conclude that mammalian TSPO does not have a critical physiological function related to PPIX and heme biosynthesis.     

Breast cancer resistance protein (Bcrp1/Abcg2) is expressed in the harderian gland and mediates transport of conjugated protoporphyrin IX

Proper regulation of intracellular levels of protoporphyrin IX (PPIX), the direct precursor of heme, is important for cell survival. A deficiency in ferrochelatase, which mediates the final step in heme biosynthesis, leads to erythropoietic protoporphyria (EPP), a photosensitivity syndrome caused by the accumulation of PPIX in the skin. We have previously shown that mice with a deficiency in the ABC transporter Bcrp1/Abcg2 display a novel type of protoporphyria. This protoporphyria is mild compared with ferrochelatase-dependent EPP, and in itself not sufficient to cause phototoxicity, but it might exacerbate the consequences of other porphyrias. In this study, we identified the mouse harderian gland as a novel expression site of Bcrp1. Because of its pronounced role in porphyrin secretion, the harderian gland presents a useful tool to study the mechanism of Bcrp1-related protoporphyria and transport of porphyrins. Bcrp1^–/– harderian gland displayed a highly increased accumulation of PPIX glycoconjugates, and a similar shift was seen in Bcrp1^–/– liver. Tear- and hepatobiliary excretion data suggest that Bcrp1 controls intracellular levels of PPIX by mediating high affinity transport of its glycoconjugates and possibly low-affinity transport of unconjugated PPIX. This mechanism may allow cells to prevent or reduce cytotoxicity of PPIX under excess conditions, without spillage under physiological conditions where PPIX is needed. ATP binding cassette transporter; knockout mice; protoporphyria     

Porphyrin accumulation in mitochondria is mediated by 2-oxoglutarate carrier

Heme (Fe-protoporphyrin IX), an endogenous porphyrin derivative, is an essential molecule in living aerobic organisms and plays a role in a variety of physiological processes such as oxygen transport, respiration, and signal transduction. For the biosynthesis of heme or the mitochondrial heme proteins, heme or its biosynthetic precursor porphyrin must be transported into mitochondria from cytosol. The mechanism of porphyrin accumulation in the mitochondrial inner membrane is unclear. In the present study, we analyzed the mechanism of mitochondrial translocation of porphyrin derivatives. We showed that palladium meso-tetra(4-carboxyphenyl)porphyrin (PdTCPP), a phosphorescent porphyrin derivative, accumulated in the mitochondria of several cell lines. Using affinity latex beads, we showed that 2-oxoglutarate carrier (OGC), the mitochondrial transporter of 2-oxoglutarate, bound to PdTCPP, and in vitro PdTCPP inhibited 2-oxoglutarate uptake into mitochondria in a competitive manner (Ki = 15 microM). Interestingly, all types of porphyrin derivatives examined in this study competitively inhibited 2-oxoglutarate uptake into mitochondria, including protoporphyrin IX, coproporphyrin III, and hemin. Furthermore, mitochondrial accumulation of porphyrins was inhibited by 2-oxoglutarate or OGC inhibitor. These results suggested that porphyrin accumulation in mitochondria is mediated by OGC and that porphyrins are able to competitively inhibit 2-oxoglutarate uptake into mitochondria. This is the first report of a putative mechanism for accumulation of porphyrins in the mitochondrial inner membrane.     

Effect of decreased ferrochelatase activity on iron and porphyrin content in mitochondrai of mice with porphyria induced by griseofulvin

The content of iron and protoporphyrin in liver mitochondria from mice with porphyria induced by griseofulvin was measured. The amount of porphyrin was 0.0076 ± 0.0043, 4.11 ± 0.58 and 22.2 ± 6.8 nmol/mg protein (n = 5) in mitochondria from control animals and animals treated with griseofulvin for 3 days and 4–5 weeks, respectively. The energy coupling of the mitochondia was greatly diminished after 4–5 weeks of treatment, and the ferrochelatase activity was inhibited 80–90%, compared to that of control animals. Mitochondrial preparations isolated by differential centrifugation were contaminated with iron-containing lysosomes which could be removed by Percoll density-gradient centrifugation. In purified mitochondrial preparations no change in the amount of non-heme iron was found after griseofulvin feeding, representing 3.36±0.15, 3.97±0.40 and 3.59±0.23 nmol/mg protein for control animals, 3 days- and 4–5 weeks-treated animals, respectively (n = 4). A mitochondrial iron pool previously identified in rat liver mitochondria and shown to be available for heme synthesis in vitro (Tangerås, A. (1985)) Biochim. Biophys. Acta 843 199–207) was also present in mitochondria from mice. The magnitude of this iron pool, as well as its availability for heme synthesis, was not changed after treatment of the animals with griseofulvin. The fact that porphyrin, but not iron, accumulated in the mitochondria when ferrochelatase was inhibited is discussed with regard to our understanding of the process of heme synthesis and its regulation.     

A new thioether-ligated iron porphyrin as a model of a protonated form of P450 active site

Thioether-ligated iron porphyrin (complex 1) was synthesized as a model of the protonated form of P450 to explore the possible involvement of the protonated form in the catalytic cycle, and ether-ligated iron porphyrin (complex 2) was also synthesized for comparison. The thioether and ether ligands enhanced heterolytic O-O bond cleavage of peroxy acid-iron porphyrin complex even in highly hydrophobic media without the assistance of acid or base, using mCPPAA as an oxidant. Competitive oxidation of cyclooctane/cyclooctene catalyzed by iron porphyrins showed that complexes 1 and 2 are less effective than heme thiolate (P450 and a synthetic heme thiolate (SR complex)) in oxidizing alkane. The possibility that thiol-ligated heme, which is a protonated form of heme thiolate, is not involved in the active intermediate structure of P450 is indicated by this result. This is the first report concerning the oxidizing ability of a thioether-ligated iron porphyrin.     

Effects of starvation for heme on the synthesis of porphyrins in Escherichia coli

A study is described of the regulation of porphyrin synthesis in Escherichia coli using a heme-permeable, hemH deletion mutant, designated VS212. This strain utilizes only exogenous hemin that is supplied in the medium and accumulates porphyrins since the final step in the synthesis of heme is genetically blocked. It is possible, therefore, to monitor the rate of synthesis of heme by examining the accumulation of porphyrins. Using this system, we found that the rate of production of porphyrins depended on the availability of heme. The lower the concentration of hemin in the medium, the higher the level of porphyrins that accumulated. We next examined the mechanism responsible for the activation of porphyrin synthesis upon starvation for heme. The main activation occurred at the step that leads to the synthesis of 5-aminolevulinic acid (ALA). Starvation for heme induced the expression of a hemA-lacZ fusion gene, as previously reported, but an activation pathway that is independent of the hemA promoter was also identified. We found that starvation for heme caused the stringent response, and such starvation promoted the synthesis of porphyrins without having any effect on the expression of the hemA-lacZ fusion gene. We suggest a model for the regulation of porphyrin synthesis whereby the synthesis of porphyrins is coordinated with that of proteins.     

In situ assessment of porphyrin photosensitizers in Propionibacterium acnes

Porphyrins are known to be efficient photosensitizer molecules and the combined action of light and porphyrins in Propionibacterium acnes have a lethal action on the cells. Identification and quantification of in situ porphyrins in P. acnes have been done using an integrating sphere connected to an ordinary absorption spectrophotometer, and the amounts of porphyrins in the cells were quantified by measuring scattering free absorption spectra of the cell suspensions. The concentration of porphyrins in P. acnes cells were increased in either of two ways; by the addition of delta-aminolevulinic acid (ALA), which lead to the formation of coproporphyrin III under the incubation conditions used in these experiments, or by the addition of protoporphyrin IX (PPIX) to the cell suspension. In the latter case, PPIX molecules are taken up by the cells in a membrane-mediated uptake mechanism, and accumulate in the cells either on a monomeric or a particular aggregate form. The fraction of porphyrins on aggregate form increased with increasing PPIX additions. In the case of ALA induced porphyrin production, only monomeric porphyrins were stored in the cells. In both cases, the cells have a limited binding capacity of monomeric porphyrins, which is estimated to be 3 x 10(5) molecules/cell, or one porphyrin molecule to every 100st lipid molecule in the cell membrane.     

Effects of starvation for heme on the synthesis of porphyrins in Escherichia coli

A study is described of the regulation of porphyrin synthesis in Escherichia coli using a heme-permeable, hemH deletion mutant, designated VS212. This strain utilizes only exogenous hemin that is supplied in the medium and accumulates porphyrins since the final step in the synthesis of heme is genetically blocked. It is possible, therefore, to monitor the rate of synthesis of heme by examining the accumulation of porphyrins. Using this system, we found that the rate of production of porphyrins depended on the availability of heme. The lower the concentration of hemin in the medium, the higher the level of porphyrins that accumulated. We next examined the mechanism responsible for the activation of porphyrin synthesis upon starvation for heme. The main activation occurred at the step that leads to the synthesis of 5-aminolevulinic acid (ALA). Starvation for heme induced the expression of a hemA-lacZ fusion gene, as previously reported, but an activation pathway that is independent of the hemA promoter was also identified. We found that starvation for heme caused the stringent response, and such starvation promoted the synthesis of porphyrins without having any effect on the expression of the hemA-lacZ fusion gene. We suggest a model for the regulation of porphyrin synthesis whereby the synthesis of porphyrins is coordinated with that of proteins. Received: 28 January 1997 / Accepted: 13 March 1997     

Porphyrin-mediated cell surface heme capture from hemoglobin by Porphyromonas gingivalis

The porphyrin requirements for growth recovery of Porphyromonas gingivalis in heme-depleted cultures are investigated. In addition to physiologically relevant sources of heme, growth recovery is stimulated by a number of noniron porphyrins. These data demonstrate that, as for Haemophilus influenzae, reliance on captured iron and on exogenous porphyrin is manifest as an absolute growth requirement for heme. A number of outer membrane proteins including some gingipains contain the hemoglobin receptor (HA2) domain. In cell surface extracts, polypeptides derived from HA2-containing proteins predominated in hemoglobin binding. The in vitro porphyrin-binding properties of a recombinant HA2 domain were investigated and found to be iron independent. Porphyrins that differ from protoporphyrin IX in only the vinyl aspect of the tetrapyrrole ring show comparable effects in competing with hemoglobin for HA2 and facilitate growth recovery. For some porphyrins which differ from protoporphyrin IX at both propionic acid side chains, the modification is detrimental in both these assays. Correlations of porphyrin competition and growth recovery imply that the HA2 domain acts as a high-affinity hemophore at the cell surface to capture porphyrin from hemoglobin. While some proteins involved with heme capture bind directly to the iron center, the HA2 domain of P. gingivalis recognizes heme by a mechanism that is solely porphyrin mediated.     

In vitro catabolic effect of protoporphyrin IX in human osteoblast-like cells: possible role of the 18 kDa mitochondrial translocator protein

In several pathological conditions, when conversion of Protoporphyrin (PP)IX into heme is impaired, a toxic accumulation of PPIX might occur. PPIX has been found to have affinity to the mitochondrial Translocator Protein 18 kDa. Since it is known that TSPO is abundant in human osteoblast cells, thus we assumed that PPIX can affect cellular functions via interactions with TSPO in these cells. Therefore we aimed to study the metabolic responses of human osteoblast to a high (10^?5M) concentration of PPIX in vitro. We found that in primary culture of human osteoblast-like cells cell numbers decreased following exposure to PPIX(10^?5M). Cellular [¹⁸F]-FDG incorporation, mitochondrial mass, ATP content were suppressed, and ΔΨm collapsed. Lactate dehydrogenase activity was enhanced in culture media, indicating overall cell death, while no increase in apoptotic levels was observed. Cellular proliferation was not affected. Protein expression of TSPO, VDAC 1, and hexokinase 2 decreased, although the synthesis of mRNA for hexokinase 2 increased. Thus, PPIX(10^?5M) has a cytotoxic effect on human osteoblast-like cell in vitro. Since these cells remain viable following exposure to another TSPO ligand, PK 11195 (10^?5M), as observed previously by us, the mode of action of PPIX on osteoblast-like cells is not identical to that of PK 11195. Accordingly pathological accumulation of PPIX may cause necrosis of osteoblasts leading to bone mass loss. We show that this phenomenon is unrelated to iron overload.