Protoporphyrin IX accumulation disrupts mitochondrial dynamics and function in ABCG2-deficient hepatocytes

Targeted inhibition of multidrug ABCG2 transporter is believed to improve cancer therapeutics. However, the consequences of ABCG2 inhibition have not been systematically evaluated since ABCG2 is expressed in several organs including the liver. Here, we demonstrate that ABCG2-deficient hepatocytes have increased amounts of fragmental mitochondria accompanied by disruption of mitochondrial dynamics and functions. This disruption was due to ABCG2 knockout elevating intracellular protoporphyrin IX, which led to upregulation of DRP-1-mediated mitochondrial fission. The finding that ABCG2 deficiency can generate dysfunctional mitochondria in hepatocytes raises concerns regarding the systematic use of ABCG2 inhibitor in cancer patients.     

In Vitro Comparison of Hypericin and 5-Aminolevulinic Acid-Derived Protoporphyrin IX for Photodynamic Inactivation of Medulloblastoma Cells

Background

Hypericin (HYP) is a naturally occurring photosensitizer. Cellular uptake and photodynamic inactivation after incubation with this photosensitizer have neither been examined in medulloblastoma cells in vitro, nor compared with 5-aminolevulinic acid-derived protoporphyrin IX (5-ALA-derived PpIX).

Methods

In 3 medulloblastoma cell lines (D283 Med, Daoy, and D341 Med) the time- and concentration-dependent intracellular accumulation of HYP and 5-ALA-derived PpIX was analyzed by fluorescence microscopy (FM) and FACS. Photocytotoxicity was measured after illumination at 595 nm (HYP) and 635 nm (5-ALA-derived PpIX) in D283 Med cells and compared to U373 MG glioma cells.

Results

All medulloblastoma cell lines exhibited concentration- and time-dependent uptake of HYP. Incubation with HYP up to 10 µM resulted in a rapid increase in fluorescence intensity, which peaked between 2 and 4 hours. 5-ALA-derived PpIX accumulation increased in D283 Med cells by 22% over baseline after 5-ALA incubation up to 1.2 mM. Photocytotoxicity of 5-ALA-derived PpIX was higher in D283 Med medulloblastoma compared to U373MG glioma. The [lethal dose (light dose that is required to reduce cell survival to 50% of control)] of 5-ALA-derived PpIX was 3.8 J/cm² in D283 Med cells versus 5.7 J/cm2 in U373MG glioma cells. Photocytotoxicity of HYP in D283 Med cells was determined at 2.5 µM after an incubation time of 2 h and an illumination wavelength of 595 nm. The value was 0.47 J/cm².

Conclusion

By its 5-fold increase in fluorescence over autofluorescence levels HYP has excellent properties for tumor visualization in medulloblastomas. The high photocytotoxicity of HYP, compared to 5-ALA-derived PpIX, is convincingly demonstrated by its 8- to 13-fold lower . Therefore HYP might be a promising molecule for intraoperative visualization and photodynamic treatment of medulloblastomas.     

Expression of Murine 5-Aminolevulinate Synthase Variants Causes Protoporphyrin IX Accumulation and Light-Induced Mammalian Cell Death

5-Aminolevulinate synthase (ALAS; EC 2.3.1.37) catalyzes the first committed step of heme biosynthesis in animals. The erythroid-specific ALAS isozyme (ALAS2) is negatively regulated by heme at the level of mitochondrial import and, in its mature form, certain mutations of the murine ALAS2 active site loop result in increased production of protoporphyrin IX (PPIX), the precursor for heme. Importantly, generation of PPIX is a crucial component in the widely used photodynamic therapies (PDT) of cancer and other dysplasias. ALAS2 variants that cause high levels of PPIX accumulation provide a new means of targeted, and potentially enhanced, photosensitization. In order to assess the prospective utility of ALAS2 variants in PPIX production for PDT, K562 human erythroleukemia cells and HeLa human cervical carcinoma cells were transfected with expression plasmids for ALAS2 variants with greater enzymatic activity than the wild-type enzyme. The levels of accumulated PPIX in ALAS2-expressing cells were analyzed using flow cytometry with fluorescence detection. Further, cells expressing ALAS2 variants were subjected to white light treatments (21–22 kLux) for 10 minutes after which cell viability was determined. Transfection of HeLa cells with expression plasmids for murine ALAS2 variants, specifically for those with mutated mitochondrial presequences and a mutation in the active site loop, caused significant cellular accumulation of PPIX, particularly in the membrane. Light treatments revealed that ALAS2 expression results in an increase in cell death in comparison to aminolevulinic acid (ALA) treatment producing a similar amount of PPIX. The delivery of stable and highly active ALAS2 variants has the potential to expand and improve upon current PDT regimes.     

Involvement of the Carboxyl-Terminal Region of the Yeast Peroxisomal Half ABC Transporter Pxa2p in Its Interaction with Pxa1p and in Transporter Function

Background

The peroxisome is a single membrane-bound organelle in eukaryotic cells involved in lipid metabolism, including β-oxidation of fatty acids. The human genetic disorder X-linked adrenoleukodystrophy (X-ALD) is caused by mutations in the ABCD1 gene (encoding ALDP, a peroxisomal half ATP-binding cassette [ABC] transporter). This disease is characterized by defective peroxisomal β-oxidation and a large accumulation of very long-chain fatty acids in brain white matter, adrenal cortex, and testis. ALDP forms a homodimer proposed to be the functional transporter, whereas the peroxisomal transporter in yeast is a heterodimer comprising two half ABC transporters, Pxa1p and Pxa2p, both orthologs of human ALDP. While the carboxyl-terminal domain of ALDP is engaged in dimerization, it remains unknown whether the same region is involved in the interaction between Pxa1p and Pxa2p.

Methods/Principal Findings

Using a yeast two-hybrid assay, we found that the carboxyl-terminal region (CT) of Pxa2p, but not of Pxa1p, is required for their interaction. Further analysis indicated that the central part of the CT (designated CT₂) of Pxa2p was indispensable for its interaction with the carboxyl terminally truncated Pxa1_NBD. An interaction between the CT of Pxa2p and Pxa1_NBD was not detected, but could be identified in the presence of Pxa2_NBD-CT₁. A single mutation of two conserved residues (aligned with X-ALD-associated mutations at the same positions in ALDP) in the CT₂ of the Pxa2_NBD-CT protein impaired its interaction with Pxa1_NBD or Pxa1_NBD-CT, resulting in a mutant protein that exhibited a proteinase K digestion profile different from that of the wild-type protein. Functional analysis of these mutant proteins on oleate plates indicated that they were defective in transporter function.

Conclusions/Significance

The CT of Pxa2p is involved in its interaction with Pxa1p and in transporter function. This concept may be applied to human ALDP studies, helping to establish the pathological mechanism for CT-related X-ALD disease.     

Growth Cycle-Dependent Overproduction and Accumulation of Protoporphyrin IX in Tetrahymena: Effect of Heavy Metals1

Cells of the ciliate Tetrahymena pyriformis GL overproduce and accumulate massive quantities of the heme intermediate, protoporphyrin IX. Protoporphyrin is localized intracellularly in discrete membranous compartments. The amount of porphyrin stored in the cell changes dramatically as cells progress through the growth cycle. Porphyrin overproduction is stimulated by δ-aminolevulinic acid, but only during the mid-stationary phase. Overproduction of protoporphyrin IX apparently results from an increase, late in the growth cycle, of activities subsequent to δ-aminolevulinic acid synthetase. Feedback inhibition in the pathway by accumulated protoporphyrin IX does not occur. The presence of Co²⁺ completely inhibits accumulation of protoporphyrin IX in a manner reversed by δ-aminolevulinic acid. Sn⁴⁺ stimulates protoporphyrin IX accumulation in the culture.     

Intraplastidic Localization of the Enzymes That Convert delta-Aminolevulinic Acid to Protoporphyrin IX in Etiolated Cucumber Cotyledons

The intraplastidic localization of the enzymes that catalyze the conversion of δ-aminolevulinic acid (ALA) to protoporphyrin IX (Proto) is a controversial issue. While some researchers assign a stromal location for these enzymes, others favor a membranebound one. Etiochloroplasts were isolated from etiolated cucumber cotyledons (Cucumis sativus, L.) by differential centrifugation and were purified further by Percoll density gradient centrifugation. Purified plastids were highly intact, and contamination by other subcellular organelles was reduced five- to ninefold in comparison to crude plastid preparations. Most of the ALA to Proto conversion activity was found in the plastids. On a unit protein basis, the ALA to Proto conversion activity of isolated mitochondria was about 2% that of the purified plastids, and could be accounted for by contamination of the mitochondrial preparation by plastids. Lysis of the purified plastids by osmotic shock followed by high speed centrifugation, yielded two subplastidic fractions: a soluble clear stromal fraction and a pelleted yellowish one. The stromal fraction contained about 11% of the plastidic ALA to Proto conversion activity while the membrane fraction contained the remaining 89%. The stromal ALA to Proto conversion activity was in the range of stroma contamination by subplastidic membrane material. Complete solubilization of the ALA to Proto activity was achieved by high speed shearing and cavitation, in the absence of detergents. Solubilization of the ALA to Proto conversion activity was accompanied by release of about 30% of the membrane-bound protochlorophyllide. It is proposed that the enzymes that convert ALA to Proto are loosely associated with the plastid membranes and may be solubilized without the use of detergents. It is not clear at this stage whether the enzymes are associated with the outer or inner plastid membranes and whether they form a multienzyme complex or not.     

Chloroplast Biogenesis: XXIV. Intrachloroplastic Localization of the Biosynthesis and Accumulation of Protoporphyrin IX, Magnesium-Protoporphyrin Monoester, and Longer Wavelength Metalloporphyrins during Greening

The intraplastidic localization of the endogenous metabolic pools from protoporphyrin to protochlorophyll was determined in Cucumis sativus. The endogenous protoporphyrin, Mg-protoporphyrin monoester + longer wavelength metalloporphyrins, protochlorophyllide and protochlorophyllide ester were membrane-bound. Protoporphyrin was synthesized in the stroma and subsequently became associated with the membranes. The membrane-associated protoporphyrin was then converted into Mg-protoporphyrin monoester + longer wavelength metalloporphyrins by membrane-bound enzymes. Although lysed plastids were capable of converting exogenous δ-aminolevulinic acid to protochlorophyllide, the net synthesis of protochlorophyllide from exogenous δ-aminolevulinic acid was lost upon segregating the lysed plastids into stromal and membrane fractions and then recombining the stromal and membrane fraction prior to incubation. The segregated membrane fraction was still capable of converting protoporphyrin into Mg-protoporphyrin monoester + longer wavelength metalloporphyrins in the presence or absence of the stromal fraction. These results indicated that although the reactions from protoporphyrin to Mg-protoporphyrin monoester and longer wavelength metalloporphyrins could survive a considerable degree of plastid disruption, the reactions from Mg-protoporphyrin monoester and longer wavelength metalloporphyrins to protochlorophyllide were more sensitive to structural disorganization.     

GxySBA ABC Transporter of Agrobacterium tumefaciens and Its Role in Sugar Utilization and vir Gene Expression

Monosaccharides available in the extracellular milieu of Agrobacterium tumefaciens can be transported into the cytoplasm, or via the periplasmic sugar binding protein, ChvE, play a critical role in controlling virulence gene expression. The ChvE-MmsAB ABC transporter is involved in the utilization of a wide range of monosaccharide substrates but redundant transporters are likely given the ability of a chvE-mmsAB deletion strain to grow, albeit more slowly, in the presence of particular monosaccharides. In this study, a putative ABC transporter encoded by the gxySBA operon is identified and shown to be involved in the utilization of glucose, xylose, fucose, and arabinose, which are also substrates for the ChvE-MmsAB ABC transporter. Significantly, GxySBA is also shown to be the first characterized glucosamine ABC transporter. The divergently transcribed gene gxyR encodes a repressor of the gxySBA operon, the function of which can be relieved by a subset of the transported sugars, including glucose, xylose, and glucosamine, and this substrate-induced expression can be repressed by glycerol. Furthermore, deletion of the transporter can increase the sensitivity of the virulence gene expression system to certain sugars that regulate it. Collectively, the results reveal a remarkably diverse set of substrates for the GxySBA transporter and its contribution to the repression of sugar sensitivity by the virulence-controlling system, thereby facilitating the capacity of the bacterium to distinguish between the soil and plant environments.     

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.     

ABCG5 and ABCG8 require MDR2 for secretion of cholesterol into bile

The major pathway for the removal of cholesterol from the body is via secretion into the bile. Three members of the ATP binding cassette (ABC) family, ABCG5 (G5), ABCG8 (G8), and ABCB4 (MDR2), are required for the efficient biliary export of sterols. Here, we examined the interdependence of these three ABC transporters for biliary sterol secretion. Biliary lipid levels in mice expressing no MDR2 (Mdr2-/- mice) were compared with those of Mdr2-/- mice expressing 14 copies of a human G5 (hG5) and hG8 transgene (Mdr2-/-;hG5G8Tg mice). Mdr2-/- mice had only trace amounts of biliary cholesterol and phospholipids. The Mdr2-/-;hG5G8Tg mice had biliary cholesterol levels as low as those of Mdr2-/- mice. Thus, MDR2 expression is required for G5G8-mediated biliary sterol secretion. To determine whether the reduction in fractional absorption of dietary sterols associated with G5G8 overexpression is secondary to the associated increase in biliary cholesterol, we compared the fractional absorption of sterols in Mdr2-/-;hG5G8Tg and hG5G8Tg animals. Inactivation of MDR2 markedly attenuated the reduction in fractional sterol absorption associated with G5G8 overexpression. These results are consistent with the notion that increased biliary cholesterol secretion contributes to the reduction in fractional sterol absorption associated with G5G8 overexpression.     

光动力诊疗中的5-氨基酮戊酸及酯类衍生物的研究进展

5-氨基酮戊酸(ALA)是体表光动力疗法的一个重要前体药物,通过代谢产物原卟啉Ⅸ(PpⅨ)介导发挥光敏作用。ALA制剂的研发和优化促生了一系列产品和技术,不仅推动了体表光动力疗法的应用,而且ALA介导的PpⅨ荧光还可用于肿瘤的荧光可视化和辅助手术。本文将对光动力诊疗中ALA及其酯类衍生物和PpⅨ的研究进展作一个系统介绍。     

Enantioselective Induction of a Glutathione-S-Transferase,a Glutathione Transporter and an ABC Transporter in Maize by Metolachlor and Its (S)-Isomer

The metabolism of chiral herbicides in plants remains poorly understood. Glutathione conjugation reactions are one of the principal mechanisms that plants utilize to detoxify xenobiotics. The induction by rac- and S-metolachlor of the expression of three genes, ZmGST27, ZmGT1 and ZmMRP1, encoding respectively a glutathione-S-transferase, a glutathione transporter and an ATP-binding cassette (ABC) transporter was studied in maize. The results demonstrate that the inducing effect of rac- and S-metolachlor on the expression of ZmGST27 and ZmGT1 is comparable. However, the inducing effect of rac-metolachlor on ZmMRP1 expression is more pronounced than that of S-metolachlor. Furthermore, vanadate, an ABC transporter inhibitor, could greatly reduce the difference in herbicidal activity between rac- and S-metolachlor. These results suggest that the ABC transporters may preferentially transport conjugates of rac-metolachlor, leading to a faster metabolism of the latter. Through comparing the expression of ZmGST27, ZmMRP1 and ZmGT1 after treatment by rac- and S-metolachlor, we provide novel insights into the metabolic processes of chiral herbicides in plants.     

Subcellular Localization of the Neuronal Glycine Transporter GLYT2 in Brainstem

The neuronal glycine transporter GLYT2 belongs to the neurotransmitter:sodium:symporter (NSS) family and removes glycine from the synaptic cleft, thereby aiding the termination of the glycinergic signal and achieving the reloading of the presynaptic terminal. The task fulfilled by this transporter is fine tuned by regulating both transport activity and intracellular trafficking. Different stimuli such as neuronal activity or protein kinase C (PKC) activation can control GLYT2 surface levels although the intracellular compartments where GLYT2 resides are largely unknown. Here, by biochemical and immunological techniques in combination with electron and confocal microscopy, we have investigated the subcellular distribution of GLYT2 in rat brainstem tissue, and characterized the vesicles that contain the transporter. GLYT2 is shown to be present in small and larger vesicles that contain the synaptic vesicle protein synaptophysin, the recycling endosome small GTPase Rab11, and in the larger vesicle population, the vesicular inhibitory amino acid transporter VIAAT. Rab5A, the GABA transporter GAT1, synaptotagmin2 and synaptobrevin2 (VAMP2) were not present. Coexpression of a Rab11 dominant negative mutant with recombinant GLYT2 impaired transporter trafficking and glycine transport. Dual immunogold labeling of brainstem synaptosomes showed a very close proximity of GLYT2 and Rab11. Therefore, the intracellular GLYT2 resides in a subset of endosomal membranes and may traffic around several compartments, mainly Rab11-positive endosomes.     

Cloning,Sequence and Functional Analysis of Goat ATP-binding Cassette Transporter G2 (ABCG2)

ATP-binding cassette transporter G2 (ABCG2) gene encodes a protein that has a wide variety of substrates and is responsible for the active secretion of clinically and toxicologically important molecules into milk. Although known in many species, this marks the first time this gene product has been reported in goats. In this study, we cloned and sequenced goat ABCG2 gene complete coding sequence and predicted its putative translated protein structure with implicative functional domains. One six-transmembrane span on C-terminal region and at least one coiled-coil domain on N-terminal were predicted and compared primarily with those of other closely related species. In addition, three conserved cysteines (in positions 595, 606, and 611) were determined toward the C-terminal of goat’s ABCG2. Two known functional motifs were identified in goat’s protein through comparative studies with other species. The goat ABCG2 relative expression profile revealed that the gene expression was a function of lactation stage and parallel to goat lactation curve.     

Functional asymmetry of nucleotide-binding domains in ABCG5 and ABCG8

The ATP-binding cassette half-transporters ABCG5 (G5) and ABCG8 (G8) promote secretion of neutral sterols into bile, a major pathway for elimination of sterols. Mutations in either ABCG5 or ABCG8 cause sitosterolemia, a recessive disorder characterized by impaired biliary and intestinal sterol secretion, sterol accumulation, and premature atherosclerosis. The mechanism by which the G5G8 heterodimer couples ATP hydrolysis to sterol transport is not known. Here we examined the roles of the Walker A, Walker B, and signature motifs in the nucleotide-binding domains (NBD) of G5 and G8 using recombinant adenoviruses to reconstitute biliary sterol transport in G5G8-deficient mice. Mutant forms of each half-transporter were co-expressed with their wild-type partners. Mutations at crucial residues in the Walker A and Walker B domains of G5 prevented biliary sterol secretion, whereas mutations of the corresponding residues in G8 did not. The opposite result was obtained when mutations were introduced into the signature motif; mutations in the signature domain of G8 prevented sterol transport, but substitution of the corresponding residues in G5 did not. Taken together, these findings indicate that the NBDs of G5 and G8 are not functionally equivalent. The integrity of the canonical NBD formed by the Walker A and Walker B motifs of G5 and the signature motif of G8 is essential for G5G8-mediated sterol transport. In contrast, mutations in key residues of the NBD formed by the Walker A and B motifs of G8 and the signature sequence of G5 did not affect sterol secretion.