Margaret Dah-Tsyr Chang

Differential Activation of p38 Mitogen-activated Protein Kinase and Extracellular Signal-regulated Protein Kinases Confers Cadmium-induced HSP70 Expression in 9L Rat Brain Tumor Cells

We have reported that treatment with CdCl2 at 40–100 μm induces the heat shock proteins (HSPs) in 9L rat brain tumor cells, during which the activation of heat shock factor (HSF) is essentially involved. By exploiting protein kinase inhibitors, we further analyzed the possible participation of specific protein kinases in the above processes. It was found that induction of HSP70 in cells treated with a high concentration of cadmium (i.e. 100 μm) is preceded by the phosphorylation and activation of p38 mitogen-activated protein kinase (p38MAPK), while that in cells treated with a low concentration (60 μm) is accompanied by the phosphorylation and activation of extracellular-regulated protein kinases 1 and 2 (ERK1/2). In 100 μm cadmium-treated cells, both HSP70 induction and HSF1 activation are eliminated in the presence of SB203580, a specific inhibitor of p38MAPK. By contrast, in 60 μm cadmium-treated cells, the processes are not affected by SB203580 but are significantly suppressed by PD98059, which indirectly inhibits ERK1/2 by acting on MAPK-ERK kinase. Taken together, we demonstrate that p38MAPK and ERK1/2 can be simultaneously or independently activated under different concentrations of cadmium and that the signaling pathways participate in the induction of HSP70 by acting on the inducible phosphorylation of HSF1. We thus provide the first evidence that both p38MAPKand ERK signaling pathways can differentially participate in the activation of HSF1, which leads to the induction of HSP70 by cadmium.

The Flexible and Clustered Lysine Residues of Human Ribonuclease 7 Are Critical for Membrane Permeability and Antimicrobial Activity

The ubiquitous ribonucleases (RNases) play important roles in RNA metabolism, angiogenesis, neurotoxicity, and antitumor or antimicrobial activity. Only the antimicrobial RNases possess high positively charged residues, although their mechanisms of action remain unclear. Here, we report on the role of cationic residues of human RNase7 (hRNase7) in its antimicrobial activity. It exerted antimicrobial activity against bacteria and yeast, even at 4 °C. The bacterial membrane became permeable to the DNA-binding dye SYTOX® Green in only a few minutes after bactericidal RNase treatment. NMR studies showed that the 22 positively charged residues (Lys18 and Arg4) are distributed into three clusters on the surface of hRNase7. The first cluster, K1,K3,K111,K112, was located at the flexible coil near the N terminus, whereas the other two, K32,K35 and K96,R97,K100, were located on rigid secondary structures. Mutagenesis studies showed that the flexible cluster K1,K3,K111,K112, rather than the catalytic residues His15, Lys38, and His123 or other clusters such as K32,K35 and K96,R97,K100, is critical for the bactericidal activity. We suggest that the hRNase7 binds to bacterial membrane and renders the membrane permeable through the flexible and clustered Lys residues K1,K3,K111,K112. The conformation of hRNase7 can be adapted for pore formation or disruption of bacterial membrane even at 4 °C.

HCMV IE2-mediated inhibition of HAT activity downregulates p53 function

Targeting of cellular histone acetyltransferases (HATs) by viral proteins is important in the development of virus‐associated diseases. The immediate‐early 2 protein (IE2) of human cytomegalovirus (HCMV) binds to the tumor suppressor, p53, and inactivates its functions by unknown mechanisms. Here, we show that IE2 binds to the HAT domain of the p53 coactivators, p300 and CREB‐binding protein (CBP), and blocks their acetyltransferase activity on both histones and p53. The minimal HAT inactivation region on IE2 involves the N‐terminal 98 amino acids. The in vivo DNA binding of p53 and local histone acetylation on p53‐dependent promoters are all reduced by IE2, but not by mutant IE2 proteins that lack the HAT inhibition region. Furthermore, the p53 acetylation site mutant, K320/373/382R, retains both DNA binding and promoter transactivation activity in vivo and these effects are repressed by IE2 as well. Together with the finding that only wild‐type IE2 exerts an antiapoptotic effect, our results suggest that HCMV IE2 downregulates p53‐dependent gene activation by inhibiting p300/CBP‐mediated local histone acetylation and that IE2 may have oncogenic activity.

A Novel Cell-Penetrating Peptide Derived from Human Eosinophil Cationic Protein

Cell-penetrating peptides (CPPs) are short peptides which can carry various types of molecules into cells; however, although most CPPs rapidly penetrate cells in vitro, their in vivo tissue-targeting specificities are low. Herein, we describe cell-binding, internalization, and targeting characteristics of a newly identified 10-residue CPP, denoted ECP32–41, derived from the core heparin-binding motif of human eosinophil cationic protein (ECP). Besides traditional emphasis on positively charged residues, the presence of cysteine and tryptophan residues was demonstrated to be essential for internalization. ECP32–41 entered Beas-2B and wild-type CHO-K1 cells, but not CHO cells lacking of cell-surface glycosaminoglycans (GAGs), indicating that binding of ECP32–41 to cell-surface GAGs was required for internalization. When cells were cultured with GAGs or pre-treated with GAG-digesting enzymes, significant decreases in ECP32–41 internalization were observed, suggesting that cell-surface GAGs, especially heparan sulfate proteoglycans were necessary for ECP32–41 attachment and penetration. Furthermore, treatment with pharmacological agents identified two forms of energy-dependent endocytosis, lipid-raft endocytosis and macropinocytosis, as the major ECP32–41 internalization routes. ECP32–41 was demonstrated to transport various cargoes including fluorescent chemical, fluorescent protein, and peptidomimetic drug into cultured Beas-2B cells in vitro, and targeted broncho-epithelial and intestinal villi tissues in vivo. Hence this CPP has the potential to serve as a novel vehicle for intracellular delivery of biomolecules or medicines, especially for the treatment of pulmonary or gastrointestinal diseases.

A heparan sulfate-facilitated and raft-dependent macropinocytosis of eosinophil cationic protein.

Eosinophil cationic protein (ECP), a human RNAseA superfamily member, highly implicated in asthma pathology, is toxic to bronchial epithelial cells following its endocytosis. The mechanism by which ECP is internalized into cells is poorly understood. In this study, we show that cell surface‐bound heparan sulfate proteoglycans serve as the major receptor for ECP internalization. Removal of cell surface heparan sulfate by heparinases or reducing glycan sulfation by chlorate markedly decreased ECP binding to human bronchial epithelial Beas‐2B cells. In addition, ECP uptake and associated cytotoxicity were reduced in glycosaminoglycan‐defective cells compared with their wild‐type counterparts. Furthermore, pharmacological treatment combined with siRNA knockdown identified a clathrin‐ and caveolin‐independent endocytic pathway as the major route for ECP internalization. This pathway is regulated by Rac1 and ADP‐ribosylating factor 6 GTPases. It requires cholesterol, actin cytoskeleton rearrangement and phosphatidylinositol‐3‐kinase activities, and is compatible with the characteristics of raft‐dependent macropinocytosis. Thus, our results define the early events of ECP internalization and may have implications for novel therapeutic design for ECP‐associated diseases.

TTK/hMps1 mediates the p53-dependent postmitotic checkpoint by phosphorylating p53 at Thr18.

ABSTRACT Upon prolonged arrest in mitosis, cells undergo adaptation and exit mitosis without cell division. These tetraploid cells are either eliminated by apoptosis or arrested in the subsequent G1 phase in a spindle checkpoint- and p53-dependent manner. p53 has long been known to be activated by spindle poisons, such as nocodazole and Taxol, although the underlying mechanism remains elusive. Here we present evidence that stabilization and activation of p53 by spindle disruption requires the spindle checkpoint kinase TTK/hMps1. TTK/hMps1 phoshorylates the N-terminal domain of p53 at Thr18, and this phosphorylation disrupts the interaction with MDM2 and abrogates MDM2-mediated p53 ubiquitination. Phosphorylation at Thr18 enhances p53-dependent activation of not only p21 but also Lats2, two mediators of the postmitotic checkpoint. Furthermore, a phospho-mimicking substitution at Thr18 (T18D) is more competent than the phospho-deficient mutant (T18A) in rescuing the tetraploid checkpoint defect of p53-depleted cells. Our findings therefore provide a mechanism connecting the spindle checkpoint with p53 in the maintenance of genome stability.

MicroRNA-34a modulates genes involved in cellular motility and oxidative phosphorylation in neural precursors derived from human umbilical cord mesenchymal stem cells

BackgroundMesenchymal stem cell (MSC) found in bone marrow (BM-MSCs) and the Whartons jelly matrix of human umbilical cord (WJ-MSCs) are able to transdifferentiate into neuronal lineage cells both in vitro and in vivo and therefore hold the potential to treat neural disorders such as stroke or Parkinsons disease. In bone marrow MSCs, miR-130a and miR-206 have been show to regulate the synthesis of neurotransmitter substance P in human mesenchymal stem cell-derived neuronal cells. However, how neuronal differentiation is controlled in WJ-MSC remains unclear.MethodsWJ-MSCs were isolated from human umbilical cords. We subjected WJ-MSCs into neurogenesis by a published protocol, and the miRNome patterns of WJ-MSCs and their neuronal progenitors (day 9 after differentiation) were analyzed by the Agilent microRNA microarray.ResultsFive miRNAs were enriched in WJ-MSCs, including miR-345, miR-106a, miR-17-5p, miR-20a and miR-20b. Another 11 miRNAs (miR-206, miR-34a, miR-374, miR-424, miR-100, miR-101, miR-323, miR-368, miR-137, miR-138 and miR-377) were abundantly expressed in transdifferentiated neuronal progenitors. Among these miRNAs, miR-34a and miR-206 were the only 2 miRNAs been linked to BM-MSC neurogenesis. Overexpressing miR-34a in cells suppressed the expression of 136 neuronal progenitor genes, which all possess putative miR-34a binding sites. Gene enrichment analysis according to the Gene Ontology database showed that those 136 genes were associated with cell motility, energy production (including those with oxidative phosphorylation, electron transport and ATP synthesis) and actin cytoskeleton organization, indicating that miR-34a plays a critical role in precursor cell migration. Knocking down endogenous miR-34a expression in WJ-MSCs resulted in the augment of WJ-MSC motility.ConclusionsOur data suggest a critical role of miRNAs in MSC neuronal differentiation, and miR-34a contributes in neuronal precursor motility, which may be crucial for stem cells to home to the target sites they should be.

Characterization of Molecular Interactions between Eosinophil Cationic Protein and Heparin

Eosinophil cationic protein (ECP) is currently used as a biomarker for airway inflammation. It is a heparin-binding ribonuclease released by activated eosinophils. Its cytotoxicity toward cancer cell lines is blocked by heparin. The objective of this study was to locate the heparin binding site of ECP by site-directed mutagenesis and construction of a synthetic peptide derived from this region. Synthetic heparin with ≥5 monosaccharide units showed strong inhibition of ECP binding to the cell surface. Analysis of ECP mt1 (R34A/W35A/R36A/K38A) showed that these charged and aromatic residues were involved in ECP binding to heparin and the cell surface. A potential binding motif is located in the loop L3 region between helix α2 and strand β1, outside the RNA binding domain. The synthetic peptide derived from the loop L3 region displayed strong pentasaccharide binding affinity and blocked ECP binding to cells. In addition, ECP mt1 showed reduced cytotoxicity. Thus, the tight interaction between ECP and heparin acts as the primary step for ECP endocytosis. These results provide new insights into the structure and function of ECP for anti-asthma therapy.

GRP78 and Raf-1 cooperatively confer resistance to endoplasmic reticulum stress-induced apoptosis

The chaperone glucose‐regulated protein, 78/immunoglobulin binding protein (GRP78/Bip), protects cells from cytotoxicity induced by DNA damage or endoplasmic reticulum (ER) stress. In this study, we showed that GRP78 is a major inducible protein in human non‐small cell lung cancer H460 cells treated with ER stress inducers, including A23187 and thapsigargin. AEBSF, an inhibitor of serine protease, diminished GRP78 induction, enhanced mitochondrial permeability, and augmented apoptosis in H460 cells during ER stress. Simultaneously, AEBSF promoted Raf‐1 degradation and suppressed phosphorylation of Raf‐1 at Ser338 and/or Tyr340 during ER stress. Coimmunoprecipitation assays and subcellular fractionations showed that GRP78 associated and colocalized with Raf‐1 on the outer membrane of mitochondria, respectively. While treatment of cells with ER stress inducers inactivated BAD by phosphorylation at Ser75, a Raf‐1 phosphorylation site; AEBSF attenuated phosphorylation of BAD, leading to cytochrome c release from mitochondria. Additionally, overexpression of GRP78 and/or Raf‐1 protected cells from ER stress‐induced apoptosis. Taken together, our results indicate that GRP78 may stabilize Raf‐1 to maintain mitochondrial permeability and thus protect cells from ER stress‐induced apoptosis. J. Cell. Physiol. 215: 627–635, 2008.

Outer Membrane Protein I of Pseudomonas aeruginosa Is a Target of Cationic Antimicrobial Peptide/Protein

Cationic antimicrobial peptides/proteins (AMPs) are important components of the host innate defense mechanisms against invading microorganisms. Here we demonstrate that OprI (outer membrane protein I) of Pseudomonas aeruginosa is responsible for its susceptibility to human ribonuclease 7 (hRNase 7) and α-helical cationic AMPs, instead of surface lipopolysaccharide, which is the initial binding site of cationic AMPs. The antimicrobial activities of hRNase 7 and α-helical cationic AMPs against P. aeruginosa were inhibited by the addition of exogenous OprI or anti-OprI antibody. On modification and internalization of OprI by hRNase 7 into cytosol, the bacterial membrane became permeable to metabolites. The lipoprotein was predicted to consist of an extended loop at the N terminus for hRNase 7/lipopolysaccharide binding, a trimeric α-helix, and a lysine residue at the C terminus for cell wall anchoring. Our findings highlight a novel mechanism of antimicrobial activity and document a previously unexplored target of α-helical cationic AMPs, which may be used for screening drugs to treat antibiotic-resistant bacterial infection.