Effects of different sources of copper on Ctr1, ATP7A,ATP7B,MT and DMT1 protein and gene expression in Caco-2 cells

Copper sulfate (CuSO₄), micron copper oxide (micron CuO) and nano copper oxide (nano CuO) at different concentrations were, respectively, added to culture media containing Caco-2 cells and their effects on Ctr1, ATP7A/7B, MT and DMT1 gene expression and protein expression were investigated and compared. The results showed that nano CuO promoted mRNA expression of Ctr1 in Caco-2 cells, and the difference was significant compared with micron CuO and CuSO₄. Nano CuO was more effective in promoting the expression of Ctr1 protein than CuSO₄ and micron CuO at the same concentration. Nano CuO at a concentration of 62.5 μM increased the mRNA expression levels of ATP7A and ATP7B, and the difference was significant compared with CuSO₄. The addition of CuSO₄ and nano CuO to the culture media promoted the expression of ATP7B proteins. CuSO₄ at a concentration of 125 μM increased the mRNA expression level of MT in Caco-2 cells, and the difference was significant compared with nano CuO and micron CuO. Nano CuO at a concentration of 62.5 μM inhibited the mRNA expression of DMT1, and the difference was significant compared with CuSO₄ and micron CuO. Thus, the effects of CuSO₄, micron CuO and nano CuO on the expression of copper transport proteins and the genes encoding these proteins differed considerably. Nano CuO has a different uptake and transport mechanism in Caco-2 cells to those of CuSO₄ and micron CuO.     

CCR7和B7-2在抗原负载树突状细胞诱导特异性CTL抗肿瘤效应中的表达和意义

目的:研究CCR7(趋化因子受体7)和B7-2(白细胞分化抗原86)与抗原负载树突状细胞(dentritic cell,DC)诱导特异性CTL(细胞毒性T淋巴细胞)抗肿瘤效应的关系.方法:分离和培养DC,制备B16黑色素瘤细胞抗原,进行共培养,即为抗原负载的DC,建立B16黑色素瘤小鼠模型,于肿瘤周围皮下注射抗原负载的DC.应用原位杂交和免疫组织化学方法检测CCR7和B7-2的表达情况.结果:原位杂交和免疫组织化学染色显示,CCR7和B7-2阳性细胞主要分布于肿瘤周围组织,随着注射抗原负载DC时间的进展,CCR7和B7-2呈强阳性表达.结论:CCR7和B7-2的表达与抗原负载树突状细胞诱导特异性CTL抗肿瘤效应有关.     

Phosphorylation of protein Phosphatase-1 isoforms by cdc2-cyclin B in vitro

Protein Phosphatase-1 is phosphorylated in vitro by cdc2-cyclin B (Villa-Moruzzi, FEBS Lett 304: 211-215, 1992). In the present study we show that all the three Phosphatase-1 isoforms, , 1, , are phosphorylated by cdc2-cyclin B. Phosphorylation is specific for this kinase and involves a C-terminal Thr. This site is most likely Thr 320 in a (shown by others to be phosphorylated also by cdc2-cyclin A). Such Thr is conserved in 1, and in the testis-specific 2, and is the only Thr that fits the cdc2-consensus sequence in the C-terminal region. Phosphorylation of Phosphatase-1 purified from skeletal muscle, which is a mixture of the a, 1 and isoforms, is up to 0.4 mol/mol and induces 30-35% enzyme inactivation. Following tryptic proteolysis each isoform yields a distinct phosphopeptide map. This is in agreement with the different sequences of the isoforms in the C-terminal regions and may be useful to distinguish the isoforms lts suggest that all the Phosphatase-1 isoforms may be potentially regulated at M-phase.     

人B7—1和B7—2cDNA的克隆及鉴定

目的：为探索性构建全新型重组人B7－PE40绿脓杆菌外毒素融合蛋白以长期诱导免疫耐受,本研究从急性B淋巴细胞白血病细胞株Raji中隆N－末端分别缺失34和16个氨基酸的人B7－1和B7－2基因胞外区,并构建含此基因的重组质粒。方法：根据B7－1和B7－2基因序列设计合成可增B7－1和B7－2cDNA的特异性引物,用RT－PCR的方法从Raji细胞总RNA中扩增B7－1和B7－2cDNA,并克隆至pGEM－T载体中,经酶切鉴定后再进行序列分析。结果和结论：从Raji细胞中扩增出预期 的624和675bp的B7－1和B7－2cDNA,将其克隆至pGEM-T载体中,分别经EcoRI/HindⅢ和BamHI/SphI双酶切电泳和序列分析确证,为进一步构建人B7－PE40外毒素融合蛋白奠定了基础。     

分子佐剂C3d上调Raji细胞协同刺激分子B7-1和B7-2的表达(简报)

我们在以往研究中,引入选择性增强体液免疫效应的新型分子佐剂C3d,成功构建了重组避孕疫苗hCGB-C3d3,通过免疫Th2型优势的 BALB/c小鼠和,Th1型优势的C57BL/6小鼠,显示分子佐剂C3d在不同品系小鼠均使免疫效应从Th1型细胞免疫向Th2型体液免疫偏倚。     

Copper-transporting ATPases ATP7A and ATP7B: cousins, not twins

Copper plays an essential role in human physiology and is indispensable for normal growth and development. Enzymes that are involved in connective tissue formation, neurotransmitter biosynthesis, iron transport, and others essential physiological processes require copper as a cofactor to mediate their reactions. The biosynthetic incorporation of copper into these enzymes takes places within the secretory pathway and is critically dependent on the activity of copper-transporting ATPases ATP7A or ATP7B. In addition, ATP7A and ATP7B regulate intracellular copper concentration by removing excess copper from the cell. These two transporters belong to the family of P₁-type ATPases, share significant sequence similarity, utilize the same general mechanism for their function, and show partial colocalization in some cells. However, the distinct biochemical characteristics and dissimilar trafficking properties of ATP7A and ATP7B in cells, in which they are co-expressed, indicate that specific functions of these two copper-transporting ATPases are not identical. Immuno-detection studies in cells and tissues have begun to suggest specific roles for ATP7A and ATP7B. These experiments also revealed technical challenges associated with quantitative detection of copper-transporting ATPases in tissues, as illustrated here by comparing the results of ATP7A and ATP7B immunodetection in mouse cerebellum. This work was supported by the National Institute of Health grants PO1 GM 067166–01 and DK R01 DK071865 to S.L.     

Cryptic association of B7‐2 molecules and its implication for clustering

T‐cell co‐stimulation through CD28/CTLA4:B7‐1/B7‐2 axis is one of the extensively studied pathways that resulted in the discovery of several FDA‐approved drugs for autoimmunity and cancer. However, many aspects of the signaling mechanism remain elusive, including oligomeric association and clustering of B7‐2 on the cell surface. Here, we describe the structure of the IgV domain of B7‐2 and its cryptic association into 1D arrays that appear to represent the pre‐signaling state of B7‐2 on the cell membrane. Super‐resolution microscopy experiments on heterologous cells expressing B7‐2 and B7‐1 suggest, B7‐2 form relatively elongated and larger clusters compared to B7‐1. The sequence and structural comparison of other B7 family members, B7‐1:CTLA4 and B7‐2:CTLA‐4 complex structures, support our view that the observed B7‐2 1D zipper array is physiologically important. This observed 1D zipper‐like array also provides an explanation for its clustering, and upright orientation on the cell surface, and avoidance of spurious signaling.     

Trafficking of the copper-ATPases, ATP7A and ATP7B: role in copper homeostasis

Copper is essential for human health and copper imbalance is a key factor in the aetiology and pathology of several neurodegenerative diseases. The copper-transporting P-type ATPases, ATP7A and ATP7B are key molecules required for the regulation and maintenance of mammalian copper homeostasis. Their absence or malfunction leads to the genetically inherited disorders, Menkes and Wilson diseases, respectively. These proteins have a dual role in cells, namely to provide copper to essential cuproenzymes and to mediate the excretion of excess intracellular copper. A unique feature of ATP7A and ATP7B that is integral to these functions is their ability to sense and respond to intracellular copper levels, the latter manifested through their copper-regulated trafficking from the transGolgi network to the appropriate cellular membrane domain (basolateral or apical, respectively) to eliminate excess copper from the cell. Research over the last decade has yielded significant insight into the enzymatic properties and cell biology of the copper-ATPases. With recent advances in elucidating their localization and trafficking in human and animal tissues in response to physiological stimuli, we are progressing rapidly towards an integrated understanding of their physiological significance at the level of the whole animal. This knowledge in turn is helping to clarify the biochemical and cellular basis not only for the phenotypes conferred by individual Menkes and Wilson disease patient mutations, but also for the clinical variability of phenotypes associated with each of these diseases. Importantly, this information is also providing a rational basis for the applicability and appropriateness of certain diagnostic markers and therapeutic regimes. This overview will provide an update on the current state of our understanding of the localization and trafficking properties of the copper-ATPases in cells and tissues, the molecular signals and posttranslational interactions that govern their trafficking activities, and the cellular basis for the clinical phenotypes associated with disease-causing mutations.     

Copper-independent lysosomal localisation of the Wilson disease protein ATP7B

In hepatocytes, the Wilson disease protein ATP7B resides on the trans-Golgi network (TGN) and traffics to peripheral lysosomes to export excess intracellular copper through lysosomal exocytosis. We found that in basal copper or even upon copper chelation, a significant amount of ATP7B persists in the endolysosomal compartment of hepatocytes but not in non-hepatic cells. These ATP7B-harbouring lysosomes lie in close proximity of ~10 nm to the TGN. ATP7B constitutively distributes itself between the sub-domain of the TGN with a lower pH and the TGN-proximal lysosomal compartments. The presence of ATP7B on TGN-lysosome colocalising sites upon Golgi disruption suggested a possible exchange of ATP7B directly between the TGN and its proximal lysosomes. Manipulating lysosomal positioning significantly alters the localisation of ATP7B in the cell. Contrary to previous understanding, we found that upon copper chelation in a copper-replete hepatocyte, ATP7B is not retrieved back to TGN from peripheral lysosomes; rather, ATP7B recycles to these TGN-proximal lysosomes to initiate the next cycle of copper transport. We report a hitherto unknown copper-independent lysosomal localisation of ATP7B and the importance of TGN-proximal lysosomes but not TGN as the terminal acceptor organelle of ATP7B in its retrograde pathway.     

Ontogeny of a novel pituitary protein (7B2) in the human fetal intestine

The developmental profile of the concentration of a novel pituitary protein (7B2) was studied immunochemically in the human gastrointestinal tract from 12 weeks of gestation to 4 months after birth and was compared to the distribution in the adult. 7B2-like immunoreactivity (IR-7B2) was detected in all segments studied, but no gross changes were seen through fetal life. At term higher concentrations of IR-7B2 were found in the duodenum and the antrum, which is similar to the distribution of adult man. Gel permeation chromatography revealed that the main peak of 7B2 immunoreactivity in the fetal intestinal extract eluted with a Kav of 0.3. Similar elution profiles were also observed in extracts of human adult intestine.     

ATP7B和PCNA在大鼠、小鼠空肠及肾的表达

随机选取6只SD大鼠(Rattus norvegicus)和7只昆明小鼠(Mus musculus),用免疫组织化学单标和双标法检测其空肠及肾ATP7B与PCNA的表达,并分析表达的相关性。结果发现,对于大鼠及小鼠,ATP7B主要表达于小肠腺与空肠上皮的纹状缘、近腔面和近基底部,肾小管与集合管;PCNA在空肠腺及小肠绒毛中轴的结缔组织中表达,在肾小管、集合管及肾小球的少数细胞表达;ATP7B与PCNA虽在空肠上皮、肠腺、肾小管和集合管有共表达现象,但二者在大鼠与小鼠空肠及肾的免疫反应阳性物积分光密度间均无显著相关性(P0.05)。提示ATP7B与PCNA在正常大鼠与小鼠空肠及肾的表达相似,ATP7B的表达与组织增殖活跃程度间的相关性不明显。     

Insight into the correlation between lag time and aggregation rate in the kinetics of protein aggregation

Under favorable conditions, many proteins can assemble into macroscopically large aggregates such as the amyloid fibrils that are associated with Alzheimer's, Parkinson's, and other neurological and systemic diseases. The overall process of protein aggregation is characterized by initial lag time during which no detectable aggregation occurs in the solution and by maximal aggregation rate at which the dissolved protein converts into aggregates. In this study, the correlation between the lag time and the maximal rate of protein aggregation is analyzed. It is found that the product of these two quantities depends on a single numerical parameter, the kinetic index of the curve quantifying the time evolution of the fraction of protein aggregated. As this index depends relatively little on the conditions and/or system studied, our finding provides insight into why for many experiments the values of the product of the lag time and the maximal aggregation rate are often equal or quite close to each other. It is shown how the kinetic index is related to a basic kinetic parameter of a recently proposed theory of protein aggregation. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Interplay between protein homeostasis networks in protein aggregation and proteotoxicity

The misfolding and aggregation of disease proteins is characteristic of numerous neurodegenerative diseases. Particular neuronal populations are more vulnerable to proteotoxicity while others are more apt to tolerate the misfolding and aggregation of disease proteins. Thus, the cellular environment must play a significant role in determining whether disease proteins are converted into toxic or benign forms. The endomembrane network of eukaryotes divides the cell into different subcellular compartments that possess distinct sets of molecular chaperones and protein interaction networks. Chaperones act as agonists and antagonists of disease protein aggregation to prevent the accumulation of toxic intermediates in the aggregation pathway. Interacting partners can also modulate the conformation and localization of disease proteins and thereby influence proteotoxicity. Thus, interplay between these protein homeostasis network components can modulate the self‐association of disease proteins and determine whether they elicit a toxic or benign outcome. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 229–236, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Presence of the Novel Pituitary Protein „7B2” in Bovine Chromaffin Granules: Possible Co-Release of 7B2 and Catecholamine as Induced by Nicotine

We observed the presence of the novel pituitary protein "7B2" and its release in the bovine adrenal medulla. The 7B2 concentration (mean +/- SEM) in extracts of the bovine adrenal medulla was 952 +/- 155 pg/mg tissue (n = 6). 7B2 was distributed in the chromaffin granule fraction prepared from the bovine adrenal medulla and was released by high K+ and/or nicotine from cultured cells of the bovine adrenal medulla. Co-release of 7B2 with catecholamine induced by nicotine from the cultured bovine chromaffin cells was also observed. In an analysis of the bovine adrenal medulla chromaffin granule fraction on gel permeation chromatography, there was a major peak with an apparent molecular weight of 45,000, whereas a major peak with an apparent molecular weight of 20,000 was found in that on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On reverse-phase HPLC, a major peak with a retention time of 35 min was observed in the bovine chromaffin granule fraction and in the bovine anterior pituitary extract. These findings indicate that 7B2 is a secretory protein in the bovine adrenal medulla. The possibility that 7B2 might be released with catecholamine, possibly in response to stress, warrants investigation.     

乙型肝炎病毒X蛋白对细胞凋亡的影响存在剂量依赖性

乙型肝炎病毒(HBV)X蛋白(HBx)与HBV相关肝细胞肝癌(HCC)的发生和发展密切相关.深入研究HBx在HCC形成中的作用将为探索HBV致癌机制提供重要依据.HBx是多功能蛋白,其对细胞凋亡的影响至今仍存在分歧.许多研究表明,HBx既有促进细胞凋亡又有抑制细胞凋亡的功能,但原因不清楚.本研究中,将表达HBx的质粒短...     

Functional characterization of new mutations in Wilson disease gene (ATP7B) using the yeast model

The Wilson disease gene, a copper transporting ATPase (Atp7b), is responsible for the sequestration of Cu into secretory vesicles, and this function is exhibited by the orthologous Ccc2p in the yeast. In this study, we aimed to characterize clinically relevant new mutations of human ATP7B (p.T788I, p.V1036I and p.R1038G-fsX83) in yeast lacking the CCC2 gene. Expression of human wild type ATP7B gene in ccc2Δ mutant yeast restored the growth deficiency and copper transport activity; however, expression of the mutant forms did not restore the copper transport functions and only partially supported the cell growth. Our data support that p.T788I, p.V1036I and p.R1038G-fsX83 mutations cause functional deficiency in ATP7B functions and suggest that these residues are important for normal ATP7B function.     

Protection from cytosolic prion protein toxicity by modulation of protein translocation

Failure to promptly dispose of undesirable proteins is associated with numerous diseases. In the case of cellular prion protein (PrP), inhibition of the proteasome pathway can generate a highly aggregation-prone, cytotoxic form of PrP implicated in neurodegeneration. However, the predominant mechanisms that result in delivery of PrP, ordinarily targeted to the secretory pathway, to cytosolic proteasomes have been unclear. By accurately measuring the in vivo fidelity of protein translocation into the endoplasmic reticulum (ER), we reveal a slight inefficiency in PrP signal sequence function that generates proteasomally degraded cytosolic PrP. Attenuating this source of cytosolic PrP completely eliminates the dependence on proteasomes for PrP degradation. This allows cells to tolerate both higher expression levels and decreased proteasomal capacity without succumbing to the adverse consequences of misfolded PrP. Thus, the generation of potentially toxic cytosolic PrP is controlled primarily during its initial translocation into the ER. These results suggest that a substantial proportion of the cell's constitutive proteasomal burden may consist of proteins that, like PrP, fail to cotranslationally enter the secretory pathway with high fidelity.     

Multiple forms of protein kinase CK2 present in leukemic cells: In vitro study of its origin by proteolysis

Human recombinant CK2 subunits were incubated for different times with the two main cytosolic proteases m-calpain and 20 S proteasome. Both, m-calpain in a calcium dependent manner and the 20 S proteasome, were able to degrade CK2 subunits in vitro. In both cases, CK2 was more resistant to these proteases than CK2. When these proteases were assayed on the reconstituted (22 holoenzyme, a 37 kDa -band, analogous to that observed in AML extracts, was generated which was resistant to further degradation. No degradation was observed when the 26 S proteasome was assayed on free subunits. Studies with CK2 deletion mutants showed that m-calpain and the 20 S proteasome acted on the C-terminus end of CK2. These results pointed to cytosolic proteases as agents involved in the control of the amount of free CK2 subunits within the cell, which becomes evident when CK2 is overexpressed as in AML cells.     

Inactivation of the mitochondrial heat shock protein zim17 leads to aggregation of matrix hsp70s followed by pleiotropic effects on morphology and protein biogenesis

The biogenesis of mitochondrial matrix proteins involves the translocase of the outer membrane, the presequence translocase of the inner membrane and the presequence translocase-associated motor. The mitochondrial heat shock protein 70 (mtHsp70) forms the central core of the motor. Recent studies led to the identification of Zim17, a mitochondrial zinc finger motif protein that interacts with mtHsp70. Different views have been reported on the localization of Zim17 in the mitochondrial inner membrane or matrix. Depletion of Zim17 impairs several critical mitochondrial processes, leading to inhibition of protein import, defects of Fe/S protein biogenesis and aggregation of Hsp70s in the matrix. Additionally, we found that inactivation of Zim17 altered the morphology of mitochondria. These pleiotropic effects raise the question of the specific function of Zim17 in mitochondria. Here, we report that Zim17 is a heat shock protein of the mitochondrial matrix that is loosely associated with the inner membrane. To address the function of Zim17 in organello, we generated a temperature-sensitive mutant allele of the ZIM17 gene in yeast. Upon a short-term shift of the yeast mutant cells to a non-permissive temperature, matrix Hsp70s aggregated while protein import, Fe/S protein activity and mitochondrial morphology were not, or only mildly, affected. Only after a long-term shift to non-permissive temperature, were strong defects in protein import, Fe/S protein activity and mitochondrial morphology observed. These findings suggest that the heat shock protein Zim17 plays a specific role in preventing protein aggregation in the mitochondrial matrix, and that aggregation of Hsp70s causes pleiotropic effects on protein biogenesis and mitochondrial morphology.     

ATP7B expression in human breast epithelial cells is mediated by lactational hormones.

A role for the copper transporter, ATP7B, in secretion of copper from the human breast into milk has previously not been reported, although it is known that the murine ortholog of ATP7B facilitates copper secretion in the mouse mammary gland. We show here that ATP7B is expressed in luminal epithelial cells in both the resting and lactating human breast, where it has a perinuclear localization in resting epithelial cells and a diffuse location in lactating tissue. ATP7B protein was present in a different subset of vesicles from those containing milk proteins and did not overlap with Menkes ATPase, ATP-7A, except in the perinuclear region of cells. In the cultured human mammary line, PMC42-LA, treatment with lactational hormones induced a redistribution of ATP7B from a perinuclear region to a region adjacent, but not coincident with, the apical plasma membrane. Trafficking of ATP7B was copper dependent, suggesting that the hormone-induced redistribution of ATP7A was mediated through an increase in intracellular copper. Radioactive copper ((64)Cu) studies using polarized PMC42-LA cells that overexpressed mAtp7B protein showed that this transporter facilitates copper efflux from the apical surface of the cells. In summary, our results are consistent with an important function of ATP7B in the secretion of copper from the human mammary gland.