Protoporphyrin IX regulates peripheral benzodiazepine receptor associated protein 7 (PAP7) and divalent metal transporter 1 (DMT1) in K562 cells

BackgroundProtoporphyrin IX (PP IX), the immediate precursor to heme, combines with ferrous iron to make this product. The effects of exogenous PP IX on iron metabolism remain to be elucidated. Peripheral-type benzodiazepine receptor (PBR) is implicated in the transport of coproporphyrinogen into the mitochondria for conversion to PP IX. We have demonstrated that PBR-Associated Protein 7 (PAP7) bound to the Iron Responsive Element (IRE) isoform of divalent metal transporter 1 (DMT1). PP IX and PAP7 are ligands for PBR, thus, we hypothesized that PAP7 interact with PP IX via PBR.MethodsWe have examined in K562 cells, which can be induced to undergo erythroid differentiation by PP IX and hemin, the effects of PP IX on the expression of PAP7 and other proteins involved in cellular iron metabolism, transferrin receptor 1 (TfR1), DMT1, ferritin heavy chain (FTH), c-Myc and C/EBPα by western blot and quantitative real time PCR analyses.ResultsPP IX significantly decreased mRNA levels of DMT1 (IRE) and (non-IRE) from 4 h. PP IX markedly decreased protein levels of C/EBPα, PAP7 and DMT1. In contrast, hemin, which like PP IX also induces K562 cell differentiation, had no effect on PAP7 or DMT1 expression.ConclusionWe hypothesize that PP IX binds to PBR displacing PAP7 protein, which is then degraded, decreasing the interaction of PAP7 with DMT1 (IRE) and resulting in increased turnover of DMT1.General significanceThese results suggest that exogenous PP IX disrupts iron metabolism by decreasing the protein expression levels of PAP7, DMT1 and C/EBPα.     

Evaluation of renal cell carcinoma vaccines targeting carbonic anhydrase IX using heat shock protein 110

Carbonic anhydrase IX (CA9) is a renal cell carcinoma (RCC)-specific tumor protein that is targeted using heat shock protein 110 (hsp110). The chaperoning ability of hsp110 can be utilized to form a complex with CA9 (hsp110 + CA9) in vitro, which can be administered as a highly concentrated tumor vaccine. In a tumor prevention model, hsp110 + CA9 prevented the growth of RENCA tumors in BALB/c mice, and produced IFN-γ response measured using ELISPOT and an antibody response measured using ELISA. To test a second vaccine strategy, hsp110 complexed to a previously described CA9 peptide prevented tumor growth and produced a very weak IFN-γ response, but no antibody response. A plasmid vector containing grp170, a member of the hsp110 family, linked to CA9 did not produce an antitumor response and produced no IFN-γ response or antibodies. In a model of metastatic RCC, RENCA cells were injected intradermally prior to vaccination. Hsp110 + CA9 decreased tumor growth compared to control vaccinations. These studies suggest that recombinant hsp110 complexed to CA9 should be evaluated for treatment of RCC.     

基于碳酸酐酶IX的肿瘤成像和治疗研究进展

碳酸酐酶IX (carbonic anhydrase IX, CAIX)是一种在乏氧肿瘤细胞表面特异性过表达的跨膜蛋白,具有调节肿瘤细胞内外酸碱度的功能,与肿瘤增殖、侵袭和转移息息相关。因此,CAIX是一个很有潜力的肿瘤成像和治疗靶点。本文详细阐述了基于CAIX的肿瘤成像、治疗和诊疗一体化的研究进展,并对CAIX作为抗肿瘤靶点的应用前景进行了展望。     

Two-photon irradiation of an intracellular singlet oxygen photosensitizer: Achieving localized sub-cellular excitation in spatially-resolved experiments

Abstract

The response of a given cell to spatially-resolved sub-cellular irradiation of a singlet oxygen photosensitizer (protoporphyrin IX, PpIX) using a focused laser was assessed. In these experiments, incident light was scattered over a volume greater than that defined by the dimensions of the laser beam as a consequence of the inherent inhomogeneity of the cell. Upon irradiation at a wavelength readily absorbed by PpIX in a one-photon transition, this scattering of light eliminated any advantage accrued to the use of focused irradiation. However, upon irradiation at a longer wavelength where PpIX can only absorb light under non-linear two-photon conditions, meaningful intracellular resolution was achieved in the small spatial domain where the light intensity was high enough for absorption to occur.     

Light control of porphyrin accumulation in acifluorfen-methyl-treated Lemna pausicostata

In Lemna pausicostata Hegelm. 6746, light is required for sufficient acifluorfenmethyl (AFM) stimulation of protoporphyrin IX (Proto IX) accumulation to cause significant herbicidal action. In darkness, AFM causes Proto IX levels to increase for about 2 h, after which Proto IX content is stable at levels significantly lower than those accumulated in light. In darkness, sucrose cannot increase levels of AFM-induced Proto IX. However, addition of δ-aminolevulinic acid (ALA) increases Proto IX levels in AFM-treated plants in darkness, demonstrating that the herbicide blocks the porphyrin pathway in darkness as it does in the light. Thus, Proto IX accumulation in darkness appears to be limited by ALA availability. This is supported by the finding that dioxoheptanoic acid caused more ALA to accumulate in light than in darkness. Heme is a feedback inhibitor of ALA synthesis, and heme synthesis is inhibited by AFM. However, total extractable heme levels were reduced by AFM by about the same amount in both light and darkness. Exogenously supplied hemin reduced AFM-caused Proto IX accumulation and herbicidal damage in the light and also reduced Proto IX accumulation caused by AFM or AFM plus ALA in darkness. AFM-stimulated Proto IX accumulation was inversely proportional to the log of the photon flux density between 5 and 500 μmol in m⁻² s⁻¹. Reduced effects of higher photon fluxes on AFM-stimulated Proto IX accumulation are probably due to both increased photobleaching of Proto IX and reduced porphyrin synthesis because of herbicidal damage. AFM-stimulated Proto IX accumulation in darkness could not be demonstrated to be under phytochrome control, but it appeared to be under the negative influence of protochlorophyllide levels.     

NADPH-dependent thioredoxin reductase and 2-Cys peroxiredoxins are needed for the protection of Mg-protoporphyrin monomethyl ester cyclase

The chloroplast-localized NADPH-dependent thioredoxin reductase (NTRC) has been found to be able to reduce hydrogen peroxide scavenging 2-Cys peroxiredoxins. We show that the Arabidopsis ntrc mutant is perturbed in chlorophyll biosynthesis and accumulate intermediates preceding protochlorophyllide formation. A specific involvement of NTRC during biosynthesis of protochlorophyllide is indicated from in vitro aerobic cyclase assays in which the conversion of Mg-protoporhyrin monomethyl ester into protochlorophyllide is stimulated by addition of the NTRC/2-Cys peroxiredoxin system. These findings support the hypothesis that this NADPH-dependent hydrogen peroxide scavenging system is particularly important during periods with limited reducing power from photosynthesis, e.g. under chloroplast biogenesis.     

Allosteric Inhibition as a New Mode of Action for BAY 1213790, a Neutralizing Antibody Targeting the Activated Form of Coagulation Factor XI

Factor XI (FXI), the zymogen of activated FXI (FXIa), is an attractive target for novel anticoagulants because FXI inhibition offers the potential to reduce thrombosis risk while minimizing the risk of bleeding. BAY 1213790, a novel anti-FXIa antibody, was generated using phage display technology. Crystal structure analysis of the FXIa–BAY 1213790 complex demonstrated that the tyrosine-rich complementarity-determining region 3 loop of the heavy chain of BAY 1213790 penetrated deepest into the FXIa binding epitope, forming a network of favorable interactions including a direct hydrogen bond from Tyr102 to the Gln451 sidechain (2.9 Å). The newly discovered binding epitope caused a structural rearrangement of the FXIa active site, revealing a novel allosteric mechanism of FXIa inhibition by BAY 1213790. BAY 1213790 specifically inhibited FXIa with a binding affinity of 2.4 nM, and in human plasma, prolonged activated partial thromboplastin time and inhibited thrombin generation in a concentration-dependent manner.     

利用烟草表达人凝血IX因子(hFIX)的研究

血友病B是凝血IX因子(hFIX)缺乏所导致的一种出血性疾病,通过输血和hFIX浓缩剂进行治疗疗效显著,但存在治疗费用高和安全隐患,因而获得安全、廉价的人凝血IX因子对血友病B治疗具有重要意义。植物系统表达外源蛋白在生产成本和安全性方面具有优势。为此,构建含人凝血IX因子基因(hFIX,2.8kb)植物双元表达载体p35s2300∷gus∷noster,用农杆菌介导法转化烟草“百日红”,通过PCR和Southernblot分析证实获得4株独立转基因植株,hFIX在转基因烟草基因组中的拷贝数为1～4个;RTPCR和ELISA检测结果表明,hFIX在转录和翻译水平已成功表达,hFIX在转基因烟草叶片中的表达量为2.5～8.8ng/g·FW,并具有免疫活性。为利用植物系统表达hFIX的后续研究作了必要准备,也为利用植物系统表达其他药用蛋白研究提供了一些理论和实验参考。     

Ambient Light Promotes Selective Subcellular Proteotoxicity after Endogenous and Exogenous Porphyrinogenic Stress

Hepatic accumulation of protoporphyrin-IX (PP-IX) in erythropoietic protoporphyria (EPP) or X-linked-dominant protoporphyria (XLP) cause liver damage. Hepatocyte nuclear lamin aggregation is a sensitive marker for PP-IX-mediated liver injury. We tested the hypothesis that extracellular or intracellular protoporphyria cause damage to different subcellular compartments, in a light-triggered manner. Three hepatoma cell lines (HepG2, Hepa-1, and Huh-7) were treated with exogenous PP-IX (mimicking XLP extrahepatic protoporphyria) or with the iron chelator deferoxamine and the porphyrin precursor 5-aminolevulinic acid (ALA) (mimicking intracellular protoporphyrin accumulation in EPP). Exogenous PP-IX accumulated predominantly in the nuclear fraction and caused nuclear shape deformation and cytoplasmic vacuoles containing electron-dense particles, whereas ALA+deferoxamine treatment resulted in higher PP-IX in the cytoplasmic fraction. Protein aggregation in the nuclear and cytoplasmic fractions paralleled PP-IX levels and, in cell culture, the effects were exclusively ambient light-mediated. PP-IX and ALA caused proteasomal inhibition, whereas endoplasmic reticulum protein aggregation was more prominent in ALA-treated cells. The enhanced ALA-related toxicity is likely due to generation of additional porphyrin intermediates including uroporphyrin and coproporphyrin, based on HPLC analysis of cell lysates and the culture medium, as well as cell-free experiments with uroporphyrin/coproporphyrin. Mouse livers from drug-induced porphyria phenocopied the in vitro findings, and mass spectrometry of liver proteins isolated in light/dark conditions showed diminished (as compared with light-harvested) but detectable aggregation under dark-harvested conditions. Therefore, PP-IX leads to endoplasmic reticulum stress and proteasome inhibition in a manner that depends on the source of porphyrin buildup and light exposure. Porphyrin-mediated selective protein aggregation provides a potential mechanism for porphyria-associated tissue injury.