神经母细胞瘤SK-N-SH细胞内DHRS4L2基因一种新选择性剪接亚型S4的克隆和亚细胞定位

目的 神经母细胞瘤SK-N-SH细胞系脱氢酶/还原酶(SDR家族)成员4类2[dehydrogenase/reductase(SDR family)member 4 like 2,DHRS4L2]基因的一种新的选择性剪接亚型克隆、生物信息学分析及其亚细胞定位.方法 以SK-N-SH细胞cDNA为模板,PCR扩增DHRS4基因簇Ea1转录本.将PCR产物A-T克隆至pGEMT-Easy质粒,对质粒进行DNA Sanger测序.将测序所得序列用NCBI ORF finder分析其编码区,用Motif Scan分析预测蛋白氨基酸序列.用Clustal Omega进行蛋白序列比对分析.将新亚型完整编码框cDNA以及删除偶核定位信号的编码框分别插入pEGFP-C1质粒,所得质粒和空质粒分别转染SK-N-SH细胞,在荧光显微镜下观察转染表达蛋白亚细胞定位.结果 用RT-PCR和Sanger测序方法发现,SK-N-SH表达DHRS4L2 Ea1转录本,未检测到其表达脱氢酶/还原酶(SDR家族)成员4类1[dehydrogenase/reductase(SDR family)member 4 like 1,DHRS4L1]的Ea2转录本.DHRS4L2 Ea1表达一个新的选择性剪接亚型DHRS4L2-S4(KU141377),由AY616183基础上在Ea1与E2外显子之间插入新外显子Ej形成,外显子Ej含有新亚型翻译起始密码子ATG.转录本KU141377预测蛋白羧基端具有偶核定位信号(bipartite nuclear localization signal,NLS),提示其可能定位于细胞核.绿色荧光蛋白融合蛋白实验显示,在SK-N-SH细胞该蛋白定位于细胞核.该蛋白还含有一个甘氨酸密集区(glycine-rich region)和阿片样生长因子受体重复(opioid growth factor receptor repeat)序列.结论 研究发现SK-N-SH细胞表达的一种DHRS4L2新选择性剪接亚型KU141377,其预测编码蛋白含有细胞偶核定位信号,融合荧光蛋白实验显示该新亚型定位于细胞核,这为后续研究DHRS4L2在神经母细胞瘤中的潜在功能奠定基础.     

A C-terminal di-leucine is required for localization of the Menkes protein in the trans-Golgi network

The human X-linked recessive disorder of copper metabolism, Menkes disease, is caused by a defect in the MNK ( ATP7A ) gene which encodes a transmembrane copper-transporting P-type ATPase (MNK). MNK is an important component of the mammalian copper transport pathway, and previous studies in cultured cells have localized MNK to the final compartment of the Golgi apparatus, the trans -Golgi network (TGN). At this location, MNK is predicted to supply copper to copper-dependent enzymes as they migrate through the secretory pathway. However, under conditions of elevated extracellular copper, the MNK protein undergoes a rapid relocalization to the plasma membrane where it functions in the efflux of copper from cells. In this study, three di-leucine motifs and a cluster of four acidic amino acids within the C-terminal region of MNK were investigated as candidate signals necessary for steady-state TGN localization. In vitro mutagenesis of the human MNK cDNA and immunofluorescence detection of mutant forms of MNK expressed in cultured cells demonstrated that the di-leucine, L1487L1488, was essential for localization of MNK within the TGN, but not for copper efflux. We suggest that this di-leucine motif is a putative endocytic targeting motif necessary for the retrieval of MNK from the plasma membrane to the TGN. Our data, along with the recent demonstration that the third transmembrane region of MNK functions as a TGN targeting signal, suggests that MNK localization to the TGN may be a two-step process involving TGN retention via the transmembrane region, and recycling to this compartment from the plasma membrane via the L1487L1488 motif.      

Novel membrane traffic steps regulate the exocytosis of the Menkes disease ATPase

The Menkes disease protein (ATP7A or MNK) is a P-type transmembrane ATPase that regulates translocation of cytosolic copper ions across intracellular membranes of compartments along the secretory pathway. In this study, we show that endogenous MNK in cultured cell lines is localized to the distal Golgi apparatus and translocates to the plasma membrane in response to exogenous copper ions. This transport event is not blocked by expression of a dominant-negative mutant protein kinase D, an enzyme implicated in regulating constitutive trafficking from the trans-Golgi network (TGN) to the plasma membrane, whereas constitutive transport of CD4 is inhibited. In contrast, protein kinase A inhibitors block copper-stimulated MNK delivery to the plasma membrane. Expression of constitutively active Rho GTPases such as Cdc42, Rac1 and RhoA reveals a requirement for Cdc42 in the trafficking of MNK, to the cell surface. Furthermore, overexpression of WASp inhibits anterograde transport of MNK, further supporting regulation by the Cdc42 GTPase. These findings define a novel step in TGN-to-plasma membrane traffic required to export MNK to the cell surface.     

Immunocytochemical localization of the Menkes copper transport protein (ATP7A) to the trans-Golgi network

We have generated polyclonal antibodies against the amino-terminal third of the Menkes protein (ATP7A; MNK) by immunizing rabbits with a histidine-tagged MNK fusion construct containing metal-binding domains 1-4. The purified antibodies were used in Western analysis of cell lysates and in indirect immunofluorescence experiments on cultured cells. On Western blots, the antibodies recognized the approximately 165 kDa MNK protein in CHO cells and human fibroblasts. No MNK signal could be detected in fibroblasts from a patient with Menkes disease or in Hep3B hepatocellular carcinoma cells, confirming the specificity of the antibodies. Immunocytochemical analysis of CHO cells and human fibroblasts showed a distinct perinuclear signal corresponding to the pattern of the Golgi complex. This staining pattern was similar to that of alpha-mannosidase II which is a known resident enzyme of the Golgi complex. Using brefeldin A, a fungal inhibitor of protein secretion, we further demonstrated that the MNK protein is localized to the trans- Golgi network. This data provides direct evidence for a subcellular localization of the MNK protein which is similar to the proposed vacuolar localization of Ccc2p, the yeast homolog of MNK and WND (ATP7B), the Wilson disease gene product. In light of the proposed role of MNK both in subcellular copper trafficking and in copper efflux, these data suggest a model for how these two processes are linked and represent an important step in the functional analysis of the MNK protein.      

Intracellular localization and loss of copper responsiveness of Mnk, the murine homologue of the Menkes protein, in cells from blotchy (Mo blo) and brindled (Mo br) mouse mutants.

Menkes disease is an X-linked copper deficiency disorder that results from mutations in the ATP7A ( MNK ) gene. A wide range of disease-causing mutations within ATP7A have been described, which lead to a diversity of phenotypes exhibited by Menkes patients. The mottled locus ( Mo, Atp7a, Mnk ) represents the murine homologue of the ATP7A gene, and the mottled mutants exhibit a diversity of phenotypes similar to that observed among Menkes patients. Therefore, these mutants are valuable models for studying Menkes disease. Two of the mottled mutants are brindled and blotchy and their phenotypes resemble classical Menkes disease and occipital horn syndrome (OHS) in humans, respectively. That is, the brindled mutant and patients with classical Menkes disease are severely copper deficient and have profound neurological problems, while OHS patients and the blotchy mouse have a much milder phenotype with predominantly connective tissue defects. In this study, in an attempt to understand the basis for the brindled and blotchy phenotypes, the copper transport characteristics and intracellular distribution of the Mnk protein were assessed in cultured cells from these mutants. The results demonstrated that the abnormal copper metabolism of brindled and blotchy cells may be related to a number of factors, which include the amount of Mnk protein, the intracellular location of the protein and the ability of Mnk to redistribute in elevated copper. The data also provide evidence for a relationship between the copper transport function and copper-dependent trafficking of Mnk.     

Identification of four novel mutations in classical Menkes disease and successful prenatal DNA diagnosis

Menkes disease is an X-linked recessive disorder of the copper metabolism and affected males suffer a systemic copper deficiency due to malabsorption and defective distribution of dietary copper. It is caused by a defect in the Menkes (ATP7A) gene, which encodes a transmembrane copper-transporting P-type ATPase. A variety of mutations were reported; however, only a few mutations were reported in Asian patients. We identified four novel mutations and one known mutation in five Korean patients. Arg646Ter in exon 8, a novel mutation transmitted from his carrier mother, was identified in one patient. Prenatal DNA diagnosis on an unaffected fetus in this carrier mother was successfully accomplished. An additional three novel mutations, Leu706Arg in exon 9, Gly1118Asp in exon 17, and Gly1255Arg in exon 19, were identified. Splicing mutation was not identified. Menkes disease in Korean patients appears to be caused by heterogeneous mutations with different spectrums from Caucasian patients.     

The Menkes copper transporter is required for the activation of tyrosinase

Menkes disease is an X-linked recessive copper deficiency disorder caused by mutations in the ATP7A (MNK) gene. The MNK gene encodes a copper-transporting P-type ATPase, MNK, which is localized predominantly in the trans-Golgi network (TGN). The MNK protein relocates to the plasma membrane in cells exposed to elevated copper where it functions in copper efflux. A role for MNK at the TGN in mammalian cells has not been demonstrated. In this study, we investigated whether the MNK protein is required for the activity of tyrosinase, a copper-dependent enzyme involved in melanogenesis that is synthesized within the secretory pathway. We demonstrate that recombinant tyrosinase expressed in immortalized Menkes fibroblast cell lines was inactive, whereas in normal fibroblasts known to express MNK protein there was substantial tyrosinase activity. Co-expression of the Menkes protein and tyrosinase from plasmid constructs in Menkes fibroblasts led to the activation of tyrosinase and melanogenesis. This MNK-dependent activation of tyrosinase was impaired by the chelation of copper in the medium of cells and after mutation of the invariant phosphorylation site at aspartic acid residue 1044 of MNK. Collectively, these findings suggest that the MNK protein transports copper into the secretory pathway of mammalian cells to activate copper-dependent enzymes and reveal a second copper transport role for MNK in mammalian cells. These findings describe a single cell-based system that allows both the copper transport and trafficking functions of MNK to be studied. This study also contributes to our understanding of the molecular basis of pigmentation in mammalian cells.     

Characterization of the Menkes protein copper-binding domains and their role in copper-induced protein relocalization.

Menkes disease is a fatal X-linked disorder of copper metabolism. The gene defective in Menkes disease (ATP7A) encodes a copper transporting P-type ATPase (MNK or ATP7A) with six copper-binding domains at its N-terminus. MNK is normally localized to the trans -Golgi network in cultured cells, but relocates to the plasma membrane in the presence of elevated extracellular copper. In this study, the role of the six copper-binding domains on copper-induced redistribution is investigated. In a recombinant clone, when all the wild-type copper-binding motifs are mutated from GMXCXXC to GMXSXXS and the cells grown in medium containing elevated copper, relocalization of the recombinant protein to the plasma membrane was not observed. Using the same assay with any one of the six copper-binding domains intact, MNK moves to the plasma membrane in a way indistinguishable from the wild-type protein. Therefore, the copper-binding domains are vital for MNK trafficking and only a single domain is sufficient for this redistribution to occur.     

The Wilson disease protein ATP7B resides in the late endosomes with Rab7 and the Niemann-Pick C1 protein

Wilson disease is a genetic disorder characterized by the accumulation of copper in the body due to a defect of biliary copper excretion. Although the Wilson disease gene has been cloned, the cellular localization of the gene product (ATP7B) has not been fully clarified. Therefore, the precise physiological action of ATP7B is still unknown. We examined the distribution of ATP7B using an anti-ATP7B antibody, green fluorescent protein (GFP)-ATP7B (GFP-ATP7B) and ATP7B-DsRed in various cultured cells. Intracellular organelles were visualized by fluorescence microscopy. The distribution of ATP7B was compared with that of Rab7 and Niemann-Pick C1 (NPC1), proteins that localize in the late endosomes. U18666A, which induces the NPC phenotype, was used to modulate the intracellular vesicle traffic. GFP-ATP7B colocalized with various late endosome markers including Rab7 and NPC1 but not with Golgi or lysosome markers. U18666A induced the formation of late endosome-lysosome hybrid organelles, with GFP-ATP7B localized with NPC1 in these structures. We have confirmed that ATP7B is a late endosome-associated membrane protein. ATP7B appears to translocate copper from the cytosol to the late endosomal lumen, thus participating in biliary copper excretion via lysosomes. Thus, defective copper ATPase activity of ATP7B in the late endosomes appears to be the main defect of Wilson disease.     

Menkes protein localization in rat parotid acinar cells

The aim was to study the subcellular localization of the Menkes protein (MNK; ATP7A) in the rat parotid acinar cell. MNK protein is a copper transporting P-type ATPase whose absence or dysfunction causes a fatal neurodegenerative disorder, MNK disease. Rat parotid glands were fixed and low-temperature embedded in Lowicryl K4M resin, and ultrathin sections were prepared for immunocytochemical analysis. Immunolocalization of MNK was demonstrated mainly over the trans Golgi network (TGN) area. Immature and mature secretory granules were also labelled, indicating that MNK protein could be involved here in copper secretion from acinar cells into saliva, consistent with a proposed cariostatic role for copper.     

Functional analysis and intracellular localization of the human menkes protein (MNK) stably expressed from a cDNA construct in Chinese hamster ovary cells (CHO-K1)

The Menkes protein (MNK or ATP7A) is an important component of the mammalian copper transport pathway and is defective in Menkes disease, a fatal X-linked disorder of copper transport. To study the structure and function of this protein and to elucidate its role in cellular copper homeostasis, a cDNA construct encoding the full-length MNK protein was cloned into a mammalian expression vector under the control of the CMV promoter. Transfection of this plasmid construct into CHO-K1 cells yielded clones that expressed MNK at varying levels. Detailed characterization of four clones showed that an increase in MNK protein expression led to a corresponding increase in the level of copper resistance of the cells. Subcellular localization studies showed that in the parental CHO-K1 and the transfected cell lines, MNK was located in a post-Golgi compartment which, based on immunogold electron microscopic analyses, most likely represented the trans -Golgi network (TGN). When the extracellular copper concentration was increased, MNK in the clones as well as in CHO-K1 cells was redistributed to the cytoplasm and plasma membrane, but returned to the TGN under basal, low copper conditions. This report presents the first ultrastructural evidence for the association of MNK with vesicles within the cell and with the TGN and plasma membrane. It also demonstrates the stable expression of a functional MNK protein from a cDNA construct in mammalian cells, as well as the copper-induced redistribution of MNK in a cell line (CHO-K1) that was not selected for copper resistance or overexpression of MNK.      

Effect of the toxic milk mutation (tx) on the function and intracellular localization of Wnd, the murine homologue of the Wilson copper ATPase

Wilson disease is an autosomal recessive copper transport disorder resulting from defective biliary excretion of copper and subsequent hepatic copper accumulation and liver failure if not treated. The disease is caused by mutations in the ATP7B (WND) gene, which is expressed predominantly in the liver and encodes a copper-transporting P-type ATPase that is structurally and functionally similar to the Menkes protein (MNK), which is defective in the X-linked copper transport disorder Menkes disease. The toxic milk (tx) mouse has a clinical phenotype similar to Wilson disease patients and, recently, the tx mutation within the murine WND homologue (WND:) of this mouse was identified, establishing it as an animal model for Wilson disease. In this study, cDNA constructs encoding the wild-type (Wnd-wt) and mutant (Wnd-tx) Wilson proteins (Wnd) were generated and expressed in Chinese hamster ovary (CHO) cells. The tx mutation disrupted the copper-induced relocalization of Wnd in CHO cells and abrogated Wnd-mediated copper resistance of transfected CHO cells. In addition, co-localization experiments demonstrated that while Wnd and MNK are located in the trans-Golgi network in basal copper conditions, with elevated copper, these proteins are sorted to different destinations within the same cell. Ultrastructural studies showed that with elevated copper levels, Wnd accumulated in large multi-vesicular structures resembling late endosomes that may represent a novel compartment for copper transport. The data presented provide further support for a relationship between copper transport activity and the copper-induced relocalization response of mammalian copper ATPases, and an explanation at a molecular level for the observed phenotype of tx mice.