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。     

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.     

锌转运蛋白基因研究进展

锌作为一种重要的微量元素参与了植物体内广泛的生理和生化过程,本文详细介绍了涉及Zn^2＋吸收转运的ZIP基因家族（ZRT／IRT相关蛋白）和CDF（Cation　diffusion　facilitator）家族。ZIP家族转运蛋白主要负责将Zn^2＋等二价阳离子跨膜转运进细胞内,以完成细胞内多种生理生化反应。CDF家族转运蛋白主要负责将过量Zn^2＋运出细胞,或者将细胞内过量Zn^2＋进行区室化隔离,降低Zn^2＋对细胞的危害作用。ZIP家族转运蛋白和CDF家族转运蛋白的相互协调使得Zn^2＋在细胞和有机体水平上维持着稳态,进而为细胞内各种生理生化反应的进行供一种保障机制。     

Neuronal zinc regulation and the prion protein

Zinc, the most abundant trace metal in the brain, has numerous functions in health and disease. It is released into the synaptic cleft alongside glutamate and this connection between zinc and glutamatergic neurotransmission allows the ion to modulate overall excitability of the brain and influence synaptic plasticity. To maintain healthy synapses, extracellular zinc levels need to be tightly regulated. We recently reported that the cellular prion protein (PrP^C) can directly influence neuronal zinc concentrations by promoting zinc uptake via AMPA receptors. The octapeptide repeat region of PrP^C is involved in zinc sensing or scavenging and the AMPA receptor provides the channel for transport of the metal across the membrane, facilitated by a direct interaction between the N-terminal polybasic region of PrP^C and AMPA receptors. PrP^C has been evolutionarily linked to the Zrt/Irt-like protein (ZIP) metal ion transport family with the C-terminus of PrP^C sharing sequence similarities with the N-terminal extracellular domains of ZIP 5, 6 and 10. By incorporating the properties of ZIP transporters (both zinc sensing and zinc transport) into two existing neuronal proteins, (PrP^C as zinc sensor, AMPA receptor as zinc transporter), neuronal cells are enhancing their biological efficiency and functionality.     

Cloning and functional identification of two members of the ZIP (Zrt, Irt-like protein) gene family in rice (Oryza sativa L.)

Two ZIP (Zrt, Irt-like Protein) cDNAs were isolated from rice (Oryza sativa L.) by RT-PCR approach, and named as OsZIP7a and OsZIP8 respectively. The predicted proteins of OsZIP7a and OsZIP8 consist of 384 and 390 amino acid residues respectively, and display high similarity to other plant ZIP proteins. Each protein contains eight transmembrane (TM) domains and a highly conserved ZIP signature motif, with a histidine-rich region in the variable region between TM domains III and IV. By semi-quantitative RT-PCR approach, it was found that the expression of OsZIP7a was significantly induced in rice roots by iron-deficiency, while that of OsZIP8 induced in both rice roots and shoots by zinc-deficiency. When expressed in yeast cells, OsZIP7a and OsZIP8 could complement an iron-uptake-deficient yeast mutant and a zinc-uptake-deficient yeast mutant respectively. It suggested that the OsZIP7a and OsZIP8 might encode an iron and a zinc transporter protein in rice respectively. Xia Yang and Ji Huang are contributed equally to this work.     

酵母和植物的锌转运系统及其调控

锌是所有生物体必需的微量元素之一,是多种蛋白的辅酶并参与催化生物体内的一些重要生化反应。生物体为了维持细胞内适当的锌浓度以保证其正常功能而进化出了复杂的锌转运及调控系统。本文主要论述酵母和植物中的锌转运系统及其调控,以及锌吸收的分子标记和QTL位点分析。     

Genomic analysis and expression pattern of <Emphasis Type="Italic">OsZIP1, OsZIP3</Emphasis>, and <Emphasis Type="Italic">OsZIP4</Emphasis> in two rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) genotypes with different zinc efficiency

The ZRT-and IRT-like proteins (ZIP) comprise a large family of transition metal transporters in plants that have diverse functions to transport zinc, iron, copper, etc. Here, we provided a complete overview of this gene family in rice (Oryza sativa L.). Based on the hidden Markov model and BLAST analysis, a total of 17 ZIP-coding genes were identified and further studied by semi-quantitative RT-PCR analysis. Sequence analysis revealed 17 putative genes distributed randomly on eight chromosomes. Although most of the predicted proteins had typical characteristics of the ZIP protein family, the extent of their sequence similarity varied considerably. The expression patterns of OsZIP1, OsZIP3, and OsZIP4, which encode Zn²⁺ transporters in rice, were studied in the Zn-efficient and Zn-inefficient rice genotypes (IR8192 and Erjiufeng) by semi-quantitative RT-PCR analysis of roots, shoots, and panicle from the plants grown under Zn deficiency and normal conditions. OsZIP1 was expressed only in the roots and very weakly if at all in the panicles, while the other two genes were expressed in all parts of plants under study. The Zn-deficient conditions up-regulated the expression of OsZIP1, OsZIP3, and OsZIP4 in the roots and that of OsZIP4 in the shoots of both genotypes, indicating that all these genes may participate in rice zinc nutrition. Furthermore, the expression of OsZIP3 and OsZIP4 was found to be much stronger in the roots of IR8192 than those of Erjiufeng, which suggests that these genes may contribute to high Zn efficiency in rice. The expression patterns and the roles of other OsZIPs are also discussed on the basis of the phylogenetic tree of ZIP proteins and RT-PCR analysis of the two rice genotypes with different zinc efficiency.     

Cell free expression and functional reconstitution of eukaryotic drug transporters

Polyspecific organic cation and anion transporters of the SLC22 protein family are critically involved in absorption and excretion of drugs. To elucidate transport mechanisms, functional and biophysical characterization of purified transporters is required and tertiary structures must be determined. Here, we synthesized rat organic cation transporters OCT1 and OCT2 and rat organic anion transporter OAT1 in a cell free system in the absence of detergent. We solubilized the precipitates with 2% 1-myristoyl-2-hydroxy- sn-glycero-3-[phospho- rac-(1-glycerol)] (LMPG), purified the transporters in the presence of 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) or octyl glucoside, and reconstituted them into proteoliposomes. From 1 mL reaction vessels 0.13-0.36 mg of transporter proteins was purified. Thus, from five to ten 1 mL reaction vessels sufficient protein for crystallization was obtained. In the presence of 1% LMPG and 0.5% CHAPS, OCT1 and OAT1 formed homo-oligomers but no hetero-oligomers. After reconstitution of OCT1, OCT2, and OAT1 into proteoliposomes, similar Michaelis-Menten K m values were measured for uptake of 1-methyl-4-phenylpyridinium and p-aminohippurate (PAH (-)) by the organic cation and anion transporters, respectively, as after expression of the transporters in cells. Using the reconstituted system, evidence was obtained that OAT1 operates as obligatory and electroneutral PAH (-)/dicarboxylate antiporter and contains a low-affinity chloride binding site that stimulates turnover. PAH (-) uptake was observed only with alpha-ketoglutarate (KG (2-)) on the trans side, and trans-KG (2-) increased the PAH (-) concentration in voltage-clamped proteoliposomes transiently above equilibrium. The V max of PAH (-)/KG (2-) antiport was increased by Cl (-) in a manner independent of gradients, and PAH (-)/KG (2-) antiport was independent of membrane potential in the absence or presence of Cl (-).     

Nitrate transporters and peptide transporters

In higher plants, two types of nitrate transporters, NRT1 and NRT2, have been identified. In Arabidopsis, there are 53 NRT1 genes and 7 NRT2 genes. NRT2 are high-affinity nitrate transporters, while most members of the NRT1 family are low-affinity nitrate transporters. The exception is CHL1 (AtNRT1.1), which is a dual-affinity nitrate transporter, its mode of action being switched by phosphorylation and dephosphorylation of threonine 101. Two of the NRT1 genes, CHL1 and AtNRT1.2, and two of the NRT2 genes, AtNRT2.1 and AtNRT2.2, are known to be involved in nitrate uptake. In addition, AtNRT1.4 is required for petiole nitrate storage. On the other hand, some members of the NRT1 family are dipeptide transporters, called PTRs, which transport a broad spectrum of di/tripeptides. In barley, HvPTR1, expressed in the plasma membrane of scutellar epithelial cells, is involved in mobilizing peptides, produced by hydrolysis of endosperm storage protein, to the developing embryo. In higher plants, there is another family of peptide transporters, called oligopeptide transporters (OPTs), which transport tetra/pentapeptides. In addition, some OPTs transport GSH, GSSH, GSH conjugates, phytochelatins, and metals.     

葡萄中蔗糖转运蛋白家族的序列分析及功能推测

以葡萄中的蔗糖转运蛋白为主要研究对象,结合了功能研究较为深入的拟南芥和水稻蔗糖转运蛋白家族序列,分析讨论了这些基因在启动子区域顺式作用元件的异同,以及这些差异可能对mRNA的转录带来的影响;同时,根据蔗糖转运蛋白氨基酸序列对其家族进行了分类,分析了不同亚类蔗糖转运蛋白基因结构的特点;最后还对蔗糖转运蛋白家族中氨基酸的保守性进行了分析。这些分析将为后续蔗糖转运蛋白功能基因组学的研究以及通过基因工程技术精确调节植物代谢提供一定的依据。     

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.     

Choline Transporters in Human Lung Adenocarcinoma： Expression and Functional Implications

Choline is an essential nutrient for cell survival and proliferation, however, the expression and function of choline transporters have not been well identified in cancer. In this study, we detected the mRNA and protein expression of organic cation transporter OCT3, carnitine/cation transporters OCTN 1 and OCTN2, and choline transporter-like protein CTL1 in human lung adenocarcinoma cell lines A549, H 1299 and SPC-A-1. Their expression pattern was further confirmed in 25 human primary adenocarcinoma tissues. The choline uptake in these cell lines was significantly blocked by CTL1 inhibitor, but only partially inhibited by OCT or OCTN inhibitors. The efficacy of these inhibitors on cell proliferation is closely correlated with their abilities to block choline transport. Under the native expression of these transporters, the total choline uptake was notably blocked by specific PI3K/AKT inhibitors. These results describe the expression of choline transporters and their relevant function in cell proliferation of human lung adenocarcinoma, thus providing a potential ＂choline-starvation＂ strategy of cancer interference through targeting choline transporters, especially CTL1.