Identification of a four copper folding intermediate in mammalian copper metallothionein by electrospray ionization mass spectrometry

 Mammalian metallothioneins (MT) are known to maximally bind 12 copper ions in two six-Cu(I) ion clusters. Using electrospray ionization mass spectrometry of MT at pH 4.5, a four-Cu(I) ion cluster was observed intermediate to a fully formed six Cu(I) in a single domain or a fully formed Cu₁₂MT species. The four-Cu(I) cluster was observed in both MT1 and MT3 isoforms. Addition of increasing amounts of Cu(I) to MT at pH 4.5 resulted in prominent ions whoses masses were consistent with apo-MT, Cu₄MT, Cu₆MT, and Cu₁₂MT. The cooperativity of cluster formation was reduced at pH 2.5. Addition of Cu(I) to apo-MT at a reduced pH resulted in a series of ions consistent with Cu₄ to Cu₁₂MT species. However, formation of the tetracopper MT species remained cooperative at low pH, suggesting that this species is very stable. To determine whether the tetracopper cluster was formed in either the α or β domain, domain peptides of MT3 were used. Addition of Cu(I) to the apo β domain resulted in a peak consistent with the formation of a four-Cu(I) cluster. This is consistent with reports that Cu(I) ions bind preferentially to the β domain of MTs. Received: 2 June 1998 / Accepted: 21 August 1998     

The influence of biological and environmental factors on metallothionein concentration in the blood

The concentration of metallothionein (MT), a low-molecular-weight protein, is regulated by many factors, primarily metals (zinc, cadmium, copper), cytokines, glucocorticoides and free radicals. These factors are determined by such aspects of human biology as gender, pregnancy and age, as well as by environmental factors including the use of oral contraceptives and cigarette smoking, all which may affect MT levels in the body.The aim of this study was to investigate the influence of these biological and environmental factors on MT concentrations in erythrocyte lysate and in plasma.MT concentrations were determined by a two-step direct enzyme-linked immunosorbent assay. Evaluation of exposure to cigarette smoking was performed by checking cotinine levels in the plasma of subjects.The studies showed higher MT concentrations in both the erythrocyte lysate and plasma of women when compared to men. Furthermore, pregnancy causes an increase of MT concentration in plasma, while oral contraceptives cause an elevated concentration of MT in erythrocyte lysate. Age impacts plasma MT concentrations in men, whereas it does not affect concentrations of MT in erythrocyte lysate.     

Metallothionein and stress combine to affect multiple organ systems

Metallothioneins (MTs) are a family of low molecular weight, cysteine-rich, metal-binding proteins that have a wide range of functions in cellular homeostasis and immunity. MTs can be induced by a variety of conditions including metals, glucocorticoids, endotoxin, acute phase cytokines, stress, and irradiation. In addition to their important immunomodulatory functions, MTs can protect essential cellular compartments from toxicants, serve as a reservoir of essential heavy metals, and regulate cellular redox potential. Many of the roles of MTs in the neuroinflammation, intestinal inflammation, and stress response have been investigated and were the subject of a session at the 6th International Congress on Stress Proteins in Biology and Medicine in Sheffield, UK. Like the rest of the cell stress response, there are therapeutic opportunities that arise from an understanding of MTs, and these proteins also provide potential insights into the world of the heat shock protein.     

MT2A对脂多糖介导的人肺毛细血管内皮细胞损伤的保护作用

目的:探索不同浓度的金属硫蛋2A(Metallothionein 2A, MT2A)对脂多糖(Lipopolysaccharid, LPS)介导的人肺微血管内皮细胞损伤的保护作用。方法:培养人肺毛细血管内皮细胞株(Human lung microvascular endothelial cells, HPMVECs),经过一定浓度LPS溶液进行刺激后,利用不同浓度的MT2A与对照组共同培养,一段时间后观察炎性介质IL-6、TNF-α释放的量及荧光显微镜观察组HPMVECs骨架形态变化。结果:各组TNF-α浓度均在0 h最低,随之逐渐升高,到6 h达到高峰;从各时间点来看,除0 h各组TNF-α浓度无显著差异外(F=0.717, P=0.549),其余各时间点B1、B2、B3均显著高于A组(均P0.05)。各组中IL-6浓度均在0 h最低,A组在2 h达到高峰,随后逐渐下降;B1组在4 h达到高峰,随之下降;B2、B3组从0 h开始逐渐升高,到6 h达到峰值;从各时间点来看,除0 h各处理因素无显著差异外(F=2.341, P=0.092),其余各时间点B1、B2、B3均低于A组(均P0.05)。A组6 h小时后纤维状肌动蛋白(F-actin)明显解聚,分布明显减少,应力纤维排列紊乱或者消失;B1组、B2组、B3组6 h小时后,与A组相比,F-actin的分布明显较多,应力纤维排列较为整齐。结论:LPS刺激人肺毛细血管内皮细胞有明显损伤效应,加入MT2A后细胞相关炎性因子释放量、细胞骨架损伤情况明显减轻,表明一定浓度的MT2A对LPS介导的肺毛细血管损伤有明显保护作用。     

不同应激因子对小鼠肝脏金属硫蛋白诱导合成的影响

目的筛选出小鼠肝脏金属硫蛋白（MT）合成量最大的诱导方式。方法从时间-效应和剂量-效应两方面研究了重金属元素（Cd）、微量元素（Zn）、重金属与微量元素的组合（Cd＋Zn）、生理因子（饥饿）及创伤因子等五大类组合应激因子、19种诱导方式对小鼠肝脏中MT诱导合成的影响及效果。结果生理因子诱导MT量最小,饥饿诱导小鼠肝脏MT的量随饥饿程度的加重而增加,但各组间差异不显著（P〉0.05）;创伤因子诱导产生MT的量最高,其诱导量随创伤恢复时间的增加而降低,各组之间差异显著（P〈0.01）,本实验诱导峰值（9.0241±0.6441μmol/g）出现在创伤后6 h;重金属元素和微量元素诱导量居中,且两者混合诱导量比单独诱导量之和要大。结论成功筛选出诱导小鼠肝脏MT合成最有效的因子和最佳时间,为进一步大量合成MT及研究其功能等奠定基础。     

Impact of cadmium on aquatic bird Cairina moschata

The impact on palmiped Cairina moschata of two levels of dietary cadmium (Cd) contamination (C1: 1 mg kg⁻¹ and C10: 10 mg kg⁻¹) was investigated on liver gene expression by real-time PCR. Genes involved in mitochondrial metabolism, in antioxidant defences, detoxification and in DNA damage repair were studied. Metallothionein (MT) protein levels and Cd bioaccumulation were also investigated in liver, kidneys and muscle. Male ducks were subjected to three periods of exposure: 10, 20 and 40 days. Cd was mainly bioaccumulated in kidneys first and in liver. The concentrations in liver and kidneys appeared to reach a stable level at 20 days of contamination even if the concentrations in muscle still increased. Cd triggered the enhancement of mitochondrial metabolism, the establishment of antioxidant defences (superoxide dismutase Mn and Cu/Zn; catalase) and of DNA repair from 20 days of contamination. Discrepancies were observed in liver between MT protein levels and MT gene up-regulation. MT gene expression appeared to be a late hour biomarker.     

Prion protein protects against zinc-mediated cytotoxicity by modifying intracellular exchangeable zinc and inducing metallothionein expression

PrP^C contains several octapeptide repeats sequences toward the N-terminus which have binding affinity for divalent metals such as copper, zinc, nickel and manganese. However, the link between PrP^C expression and zinc metabolism remains elusive. Here we studied the relationship between PrP^C and zinc ions intracellular homeostasis using a cell line expressing a doxycycline-inducible PrP^C gene. No significant difference in ⁶⁵Zn²⁺ uptake was observed in cells expressing PrP^C when compared with control cells. However, PrP^C-expressing cells were more resistant to zinc-induced toxicity, suggesting an adaptative mechanism induced by PrP^C. Using zinquin-ethyl-ester, a specific fluorophore for vesicular free zinc, we observed a significant re-localization of intracellular exchangeable zinc in vesicles after PrP^C expression. Finally, we demonstrated that PrP^C expression induces metallothionein (MT) expression, a zinc-upregulated zinc-binding protein. Taken together, these results suggest that PrP^C modifies the intracellular localization of zinc rather than the cellular content and induces MT upregulation. These findings are of major importance since zinc deregulation is implicated in several neurodegenerative disorders. It is postulated that in prion diseases the conversion of PrP^C to PrP^Sc may deregulate zinc homeostasis mediated by metallothionein.     

Chemopreventive effect of vanadium in a rodent model of chemical hepatocarcinogenesis: reflections in oxidative DNA damage, energy-dispersive X-ray fluorescence profile and metallothionein expression

In the present study, we investigated the antitumour efficacy of vanadium in a defined rodent model of experimental hepatocarcinogenesis. Hepatic preneoplasia was induced in male Sprague-Dawley rats with a single, necrogenic, intraperitoneal injection of diethylnitrosamine (DEN) (200 mg/kg body weight) followed by promotion with phenobarbital (PB). The levels of modified DNA bases 8-hydroxy-2′-deoxyguanosine (8-OHdG), a potential marker involved in the initiation of carcinogenesis, were measured by high-performance liquid chromatography, whereas tissue trace element status and expression of metallothionein (MT), a Cu–Zn metalloprotein associated with neoplastic cell growth and subsequent development of premalignant phenotype of the cell, were studied by energy-dispersive X-ray fluorescence spectrometry and enzyme-coupled immunohistochemistry, respectively. There was a significant and steady elevation of modified bases (8-OHdG) along with substantial increase in MT immunoexpression and disturbance in trace element homeostasis following DEN exposure. Supplementation of vanadium at a dose of 0.5 ppm for four consecutive weeks strictly abated the formation of 8-OHdG (P < 0.0001; 81.28%) in preneoplastic rat liver. In a long-term DEN plus PB regimen, vanadium was able to limit in situ MT expression with a concomitant decrease in MT immunoreactivity (P < 0.05). Furthermore, vanadium treatment throughout the study restored hepatic levels of essential trace elements and decreased nodular incidence (58.34%) and nodule multiplicity (P < 0.001; 66.89%) in rats treated with DEN plus PB. Taken together, the study provides evidence in support of the chemopreventive potential of vanadium in limiting neoplastic transformation during the preneoplastic stages of hepatocarcinogenesis in rats.