真核生物锌转运体及其活性的调控

真核生物的锌内稳态是由其众多特异转运体协同转运来实现的。有两个锌转运体家族ZIP和CDF被相继发现。ZIP家族的主要功能是摄取锌,而CDF家族成员主要参与锌的外排及锌在细胞内的区室化以达到解毒或贮存的目的。锌可在转录水平和翻译水平调控两类转运体的活性以维持锌在细胞和生物体水平的内稳态。     

Copper and zinc cause delivery of the prion protein from the plasma membrane to a subset of early endosomes and the Golgi

The cellular isoform of prion protein (PrPC) is a plasma membrane glycoprotein whose conformational conversion into PrPSc is the central molecular event in the propagation of infectious prions. However, the physiological function of PrPC has remained uncertain. The finding that PrPC binds copper ions with low micromolar affinity, coupled with several other observations, has led to the proposal that the protein plays a role in copper homeostasis. Using biochemical techniques, we had shown previously that copper ions rapidly and reversibly stimulate endocytosis of PrPC from the cell surface. In this report, we employ immunofluorescence microscopy to further investigate the specificity and kinetics of metal effects on PrPC trafficking and to identify the intracellular compartments to which internalized PrPC is delivered in response to copper and zinc. We find that both of these metals stimulate redistribution of surface PrPC to a subset of transferrin-containing early endosomes as well as to Golgi compartments. These results are consistent with models in which PrPC plays a role in the cellular uptake or efflux of transition metals.     

Correlation between ZIP2 messenger RNA expression and zinc level in rat lateral prostate

Zinc content in rat lateral prostate (LP) is higher compared with the other tissues, but the zinc retention system in the prostate remains unclear. In the present study, we examined the expression of ZRT, and IRT-like protein (ZIP) family transporter in rat prostate. The zinc level in rat LP was higher compared with the ventral (VP) and dorsal prostate (DP). The predicted ZIP2 mRNA was really expressed in LP at a high level. The expression was decreased in LP from castrated rats, associated with a decrease in zinc level, and these changes were prevented by testosterone replacement. Moreover, ZIP2 expression levels in LP positively correlated with the zinc levels. These findings strongly suggest that ZIP2 is involved in zinc homeostasis of rat prostate.     

Plant-based FRET biosensor discriminates environmental zinc levels

Heavy metal accumulation in the environment poses great risks to flora and fauna. However, monitoring sites prone to accumulation poses scale and economic challenges. In this study, we present and test a method for monitoring these sites using fluorescent resonance energy transfer (FRET) change in response to zinc (Zn) accumulation in plants as a proxy for environmental health. We modified a plant Zn transport protein by adding flanking fluorescent proteins (FPs) and deploying the construct into two different species. In Arabidopsis thaliana, FRET was monitored by a confocal microscope and had a 1.4-fold increase in intensity as the metal concentration increased. This led to a 16.7% overall error-rate when discriminating between a control (1μm Zn) and high (10mm Zn) treatment after 96h. The second host plant (Populus tremula×Populu salba) also had greater FRET values (1.3-fold increase) when exposed to the higher concentration of Zn, while overall error-rates were greater at 22.4%. These results indicate that as plants accumulate Zn, protein conformational changes occur in response to Zn causing differing interaction between FPs. This results in greater FRET values when exposed to greater amounts of Zn and monitored with appropriate light sources and filters. We also demonstrate how this construct can be moved into different host plants effectively including one tree species. This chimeric protein potentially offers a method for monitoring large areas of land for Zn accumulation, is transferable among species, and could be modified to monitor other specific heavy metals that pose environmental risks.     

Aberrant metal binding by prion protein in human prion disease

Human prion diseases are characterized by the conversion of the normal prion protein (PrP(C)) into a pathogenic isomer (PrP(Sc)). Distinct PrP(Sc) conformers are associated with different subtypes of prion diseases. PrP(C) binds copper and has antioxidation activity. Changes in metal-ion occupancy can lead to significant decline of the antioxidation activity and changes in conformation of the protein. We studied the trace element status of brains from patients with sporadic Creutzfeldt-Jakob disease (sCJD). We found a decrease of up to 50% of copper and an increase in manganese of approximately 10-fold in the brain tissues from sCJD subjects. We have also studied the metal occupancy of PrP in sCJD patients. We observed striking elevation of manganese and, to a lesser extent, of zinc accompanied by significant reduction of copper bound to purified PrP in all sCJD variants, determined by the PrP genotype and PrP(Sc) type, combined. Both zinc and manganese were undetectable in PrP(C) preparations from controls. Copper and manganese changes were pronounced in sCJD subjects homozygous for methionine at codon 129 and carrying PrP(Sc) type-1. Anti-oxidation activity of purified PrP was dramatically reduced by up to 85% in the sCJD variants, and correlated with increased in oxidative stress markers in sCJD brains. These results suggest that altered metal-ion occupancy of PrP plays a pivotal role in the pathogenesis of prion diseases. Since the metal changes differed in each sCJD variants, they may contribute to the diversity of PrP(Sc) and disease phenotype in sCJD. Finally, this study also presented two potential approaches in the diagnosis of CJD; the significant increase in brain manganese makes it potentially detectable by MRI, and the binding of manganese by PrP in sCJD might represent a novel diagnostic marker.     

朊蛋白的细胞生物学研究

朊蛋白病是人和牛羊等哺乳动物所患的致命性的神经系统变性疾病,它是由机体内正常的朊蛋白改变构象后所引起的疾病。本综述对朊蛋白在细胞生物学领域的认知和理解进行了归纳总结,阐述了正常和异常朊蛋白的翻译、表达、定位、裂解、转化等一系列过程,是对疾病本质的有益探索。     

Redundant roles of four ZIP family members in zinc homeostasis and seed development in Arabidopsis thaliana

Zinc (Zn) is essential for normal plant growth and development. The Zn-regulated transporter, iron-regulated transporter (IRT)-like protein (ZIP) family members are involved in Zn transport and cellular Zn homeostasis throughout the domains of life. In this study, we have characterized four ZIP transporters from Arabidopsis thaliana (IRT3, ZIP4, ZIP6, and ZIP9) to better understand their functional roles. The four ZIP proteins can restore the growth defect of a yeast Zn uptake mutant and are upregulated under Zn deficiency. Single and double mutants show no phenotypes under Zn-sufficient or Zn-limited growth conditions. In contrast, triple and quadruple mutants show impaired growth irrespective of external Zn supply due to reduced Zn translocation from root to shoot. All four ZIP genes are highly expressed during seed development, and siliques from all single and higher-order mutants exhibited an increased number of abnormal seeds and decreased Zn levels in mature seeds relative to wild type. The seed phenotypes could be reversed by supplementing the soil with Zn. Our data demonstrate that IRT3, ZIP4, ZIP6, and ZIP9 function redundantly in maintaining Zn homeostasis and seed development in A. thaliana.     

Sequence similarity and functional relationship among eukaryotic ZIP and CDF transporters

ZIP （ZRT/IRT-like Protein） and CDF （Cation Diffusion Facilitator） are two large metal transporter families mainly transporting zinc into and out of the cytosol. Several ZIP and CDF transporters have been characterized in mammals and various model organisms, such as yeast, nematode, fruit fly, and zebrafish, and many candidate genes have been identified by genome projects. Unexpected functions of ZIP and CDF transporters have been recently reported in some model organisms, leading to major advances in our understanding of the functions of mammalian counterparts. Here, we review the recent information on the sequence similarity and functional relationship among eukaryotic ZIP and CDF transporters obtained from the representative model organisms.     

Molecular characterization of ten zinc (Zn) transporter genes and their regulation to Zn metabolism in freshwater teleost yellow catfish Pelteobagrus fulvidraco

BackgroundZn is an essential trace element for vertebrates, and Zn uptake and transport is related with the ZIP family of Zn transporters. Meantime, Zn also influenced the expression of ZIP family members.MethodsWe cloned and characterized the full-length cDNA sequences of ten Zn transport-relevant genes (ZIP1, ZIP3, ZIP6, ZIP7, ZIP8, ZIP9, ZIP10, ZIP11, ZIP13 and ZIP14) from yellow catfish Pelteobagrus fulvidraco, investigated their mRNA tissue expression. These ZIP mRNA expression was also assessed in the primary hepatocytes and intestinal epithelial cells of yellow catfish in response to three Zn levels (0, 30 μM and 60 μM, respectively).ResultsAll these genes shared the similar domains with the corresponding members in mammals. The mRNA expression of the ten ZIP genes was detected in nine-tested tissues, but variable among these tissues. Flow cytometry analysis and confocal microscopy observation indicated that intracellular free Zn²⁺ concentration in hepatocytes and intestinal epithelial cells increased with increasing Zn incubation concentration at both 24 h and 48 h. Zn incubation differentially influenced mRNA levels of ZIP transporters in the hepatocytes and intestinal epithelial cells, in a time- and cells-dependent manners. In the hepatocytes, at 24 h, compared to the control, Zn addition down-regulated mRNA levels of ZIP1, ZIP3, ZIP6, ZIP7, ZIP8, ZIP9, ZIP11 and ZIP14; however, ZIP10 mRNA levels were lower in 60 μM Zn group than those in the control and 30 μM Zn group. At 48 h, mRNA levels of ZIP1, ZIP6, ZIP7, ZIP9, ZIP10 and ZIP14 declined with increasing Zn incubation concentrations; ZIP3 mRNA levels were the lowest in 60 μM Zn group and showed no significant differences between the control and 30 μM Zn group. In the intestinal epithelial cells, at 24 h, Zn addition down-regulated mRNA levels of ZIP1, ZIP6, ZIP7, ZIP8, ZIP9, ZIP10, ZIP11, ZIP13 and ZIP14; ZIP3 mRNA levels were lower in 60 μM Zn group than those in the control and 30 μM Zn group. At 48 h, Zn addition up-regulated mRNA levels of ZIP6 and ZIP9, but down-regulated mRNA levels of ZIP8, ZIP10 and ZIP13. ZIP7, ZIP11 and ZIP14 mRNA abundances were the lowest in 60 μM Zn group and showed no significant differences between the control and 30 μM Zn group.ConclusionFor the first time, our study characterized ten ZIP family members in yellow catfish, explored their mRNA tissue expression. Their regulation to Zn addition were also investigated in the hepatocytes and intestinal epithelial cells of yellow catfish. Our study revealed the mechanism of cells exposed to Zn addition and provided novel insights for the regulatory mechanism of Zn homeostasis.     

Porcine prion protein amyloid

ABSTRACT

Mammalian prions are composed of misfolded aggregated prion protein (PrP) with amyloid-like features. Prions are zoonotic disease agents that infect a wide variety of mammalian species including humans. Mammals and by-products thereof which are frequently encountered in daily life are most important for human health. It is established that bovine prions (BSE) can infect humans while there is no such evidence for any other prion susceptible species in the human food chain (sheep, goat, elk, deer) and largely prion resistant species (pig) or susceptible and resistant pets (cat and dogs, respectively). PrPs from these species have been characterized using biochemistry, biophysics and neurobiology. Recently we studied PrPs from several mammals in vitro and found evidence for generic amyloidogenicity as well as cross-seeding fibril formation activity of all PrPs on the human PrP sequence regardless if the original species was resistant or susceptible to prion disease. Porcine PrP amyloidogenicity was among the studied. Experimentally inoculated pigs as well as transgenic mouse lines overexpressing porcine PrP have, in the past, been used to investigate the possibility of prion transmission in pigs. The pig is a species with extraordinarily wide use within human daily life with over a billion pigs harvested for human consumption each year. Here we discuss the possibility that the largely prion disease resistant pig can be a clinically silent carrier of replicating prions.     

Expression and regulation of SLC39A family zinc transporters in the developing mouse intestine

Several ZIP genes (SLC39A family of metal transporters) play roles in zinc homeostasis. Herein, the temporal and spatial patterns of expression of the mouse ZIP1, 3, 4, and 5 genes in the developing intestine and the effects of maternal dietary zinc deficiency on these patterns of expression were examined. ZIP1 and ZIP3 genes, conserved members of the ZIP subfamily II, were found to be coexpressed during development. Expression of these genes was detected on day 14 of gestation in smooth muscle and the pseudostratified endoderm. By 5 days post-partum, prominent expression became restricted to muscle and connective stroma. In contrast, expression of ZIP4 and ZIP5 genes, members of the ZIP subfamily called LIV-1, coincided with epithelial morphogenesis. ZIP5 expression was detected on d16 of gestation and localized to the basolateral membranes of the single-layered epithelium. ZIP4 expression was detected on d18 of gestation and localized to the apical membrane of villus epithelial cells. When dams were fed a zinc-deficient diet beginning at parturition, ZIP4 expression in the nursing neonate was greatly induced. In contrast, neonatal ZIP5 expression remained unchanged, but this protein was removed from the basolateral membrane of the enterocyte. These responses to dietary zinc deficiency mimic those found in the adult intestine. These studies reveal cell-type-specific expression of ZIP genes during development of the intestine, and suggest that the mouse intestine can elicit an adaptive response to dietary zinc availability at birth.     

A systematic investigation of production of synthetic prions from recombinant prion protein

According to the protein-only hypothesis, infectious mammalian prions, which exist as distinct strains with discrete biological properties, consist of multichain assemblies of misfolded cellular prion protein (PrP). A critical test would be to produce prion strains synthetically from defined components. Crucially, high-titre ‘synthetic'' prions could then be used to determine the structural basis of infectivity and strain diversity at the atomic level. While there have been multiple reports of production of prions from bacterially expressed recombinant PrP using various methods, systematic production of high-titre material in a form suitable for structural analysis remains a key goal. Here, we report a novel high-throughput strategy for exploring a matrix of conditions, additives and potential cofactors that might generate high-titre prions from recombinant mouse PrP, with screening for infectivity using a sensitive automated cell-based bioassay. Overall, approximately 20 000 unique conditions were examined. While some resulted in apparently infected cell cultures, this was transient and not reproducible. We also adapted published methods that reported production of synthetic prions from recombinant hamster PrP, but again did not find evidence of significant infectious titre when using recombinant mouse PrP as substrate. Collectively, our findings are consistent with the formation of prion infectivity from recombinant mouse PrP being a rare stochastic event and we conclude that systematic generation of prions from recombinant PrP may only become possible once the detailed structure of authentic ex vivo prions is solved.     

Mutant prion protein-mediated aggregation of normal prion protein in the endoplasmic reticulum: implications for prion propagation and neurotoxicity

Familial prion disorders are believed to result from spontaneous conversion of mutant prion protein (PrPM) to the pathogenic isoform (PrPSc). While most familial cases are heterozygous and thus express the normal (PrPC) and mutant alleles of PrP, the role of PrPC in the pathogenic process is unclear. Plaques from affected cases reveal a heterogeneous picture; in some cases only PrPM is detected, whereas in others both PrPC and PrPM are transformed to PrPSc. To understand if the coaggregation of PrPC is governed by PrP mutations or is a consequence of the cellular compartment of PrPM aggregation, we coexpressed PrPM and PrPC in neuroblastoma cells, the latter tagged with green fluorescent protein (PrPC-GFP) for differentiation. Two PrPM forms (PrP231T, PrP217R/231T) that aggregate spontaneously in the endoplasmic reticulum (ER) were generated for this analysis. We report that PrPC-GFP aggregates when coexpressed with PrP231T or PrP217R/231T, regardless of sequence homology between the interacting forms. Furthermore, intracellular aggregates of PrP231T induce the accumulation of a C-terminal fragment of PrP, most likely derived from a potentially neurotoxic transmembrane form of PrP (CtmPrP) in the ER. These findings have implications for prion pathogenesis in familial prion disorders, especially in cases where transport of PrPM from the ER is blocked by the cellular quality control.     

The protease-sensitive N-terminal polybasic region of prion protein modulates its conversion to the pathogenic prion conformer

Conversion of normal prion protein (PrP^C) to the pathogenic PrP^Sc conformer is central to prion diseases such as Creutzfeldt–Jakob disease and scrapie; however, the detailed mechanism of this conversion remains obscure. To investigate how the N-terminal polybasic region of PrP (NPR) influences the PrP^C-to-PrP^Sc conversion, we analyzed two PrP mutants: ΔN6 (deletion of all six amino acids in NPR) and Met4-1 (replacement of four positively charged amino acids in NPR with methionine). We found that ΔN6 and Met4-1 differentially impacted the binding of recombinant PrP (recPrP) to the negatively charged phospholipid 1-palmitoyl-2-oleoylphosphatidylglycerol, a nonprotein cofactor that facilitates PrP conversion. Both mutant recPrPs were able to form recombinant prion (recPrP^Sc) in vitro, but the convertibility was greatly reduced, with ΔN6 displaying the lowest convertibility. Prion infection assays in mammalian RK13 cells expressing WT or NPR-mutant PrPs confirmed these differences in convertibility, indicating that the NPR affects the conversion of both bacterially expressed recPrP and post-translationally modified PrP in eukaryotic cells. We also found that both WT and mutant recPrP^Sc conformers caused prion disease in WT mice with a 100% attack rate, but the incubation times and neuropathological changes caused by two recPrP^Sc mutants were significantly different from each other and from that of WT recPrP^Sc. Together, our results support that the NPR greatly influences PrP^C-to-PrP^Sc conversion, but it is not essential for the generation of PrP^Sc. Moreover, the significant differences between ΔN6 and Met4-1 suggest that not only charge but also the identity of amino acids in NPR is important to PrP conversion.