BBA - Molecular Cell Research (v.1773, #6)

Nuclear immobilization of DsRed1 tagged proteins: A novel tool for studying DNA–protein interactions? by Anastasios E. Damdimopoulos; Jan-Åke Gustafsson; Giannis Spyrou (687-690).
DsRed1 is a red fluorescent protein that can be used as a fusion partner with other proteins to determine their subcellular localization, similarly to the popular green fluorescent proteins (GFP). Here, we report that fusion of DsRed1 to estrogen receptor α (ERα) renders the transcription factor immobile within the nucleus. Furthermore, we show that the immobilization is dependent on DNA interaction and that the binding to the DNA can be direct as well as indirect for DsRed to immobilize with its fusion partners. This observation could provide a new tool to be used for the identification of target genes containing low affinity binding sites for several transcription factors including ERα. In addition, it could be employed for studies on protein–DNA interactions as well as protein–protein interactions during protein complex formation on chromatin in the event of transcription initiation and regulation.
Keywords: Estrogen receptor; DNA interactions; DsRed; Nuclear protein mobility; Chromatin;

There is growing interest in the potential use of 5-HT1A receptor agonists as neuroprotective agents in stroke and tramautic brain injury. However, a new study using a recombinant 5-HT1A receptor cell line suggests that these agonists may promote as well as inhibit apoptotic responses. Because heterologously expressed receptors may couple promiscuously to inappropriate signal transduction pathways, the results should be interpreted with caution.
Keywords: Apoptosis; Neuroprotection; Signal transduction; Extracellular-regulated kinase (Erk); c-jun-N-terminal kinase (JNK); Promiscuous coupling; G protein coupled receptors (GPCRs); Heterologous expression;

SUMO on the road to neurodegeneration by Véronique Dorval; Paul E. Fraser (694-706).
Sumoylation is a post-translational modification by which small ubiquitin-like modifiers (SUMO) are covalently conjugated to target proteins. This reversible pathway provides a rapid and efficient way to modulate the subcellular localization, activity and stability of a wide variety of substrates. Similar to its well-known cousin ubiquitin, SUMO co-localize with the neuronal inclusions associated with several neurodegenerative diseases, including multiple system atrophy, Huntington's disease and other related polyglutamine disorders. The identification of huntingtin, ataxin-1, tau and α-synuclein as SUMO substrates further supports the involvement of sumoylation in the pathogenesis of this family of neurological diseases. In addition to direct targeting of these constituent proteins, sumoylation also impacts other disease pathways such as oxidative stress, protein aggregation and proteasome-mediated degradation. This review highlights the recent advances in understanding the contributions of SUMO to neurodegeneration and the underlying pathogenic mechanisms of these diseases.
Keywords: SUMO; Neurodegenerative diseases; Proteasome; Ubiquitin; Neuronal inclusions;

Role of extracellular charged amino acids in the yeast α-factor receptor by Anshika Bajaj; Sara M. Connelly; Austin U. Gehret; Fred Naider; Mark E. Dumont (707-717).
The yeast pheromone receptor, Ste2p, is a G protein coupled receptor that initiates cellular responses to α-mating pheromone, a 13 residue peptide that carries a net positive charge at physiological pH. We have examined the role of extracellular charged groups on the receptor in response to the pheromone. Substitutions of Asn or Ala for one extracellular residue, Asp275, affected both pheromone binding and signaling, suggesting that this position interacts directly with ligand. The other seven extracellular acidic residues could be individually replaced by polar residues with no detectable effects on receptor function. However, substitution of Ala for each of these seven residues resulted in impairment of signaling without affecting pheromone binding, implying that the polar nature of these residues promotes receptor activation. In contrast, substitution of Ala for each of the six positively charged residues at the extracellular surface of Ste2p did not affect signaling.
Keywords: G protein coupled receptor; Saccharomyces cerevisiae; STE2; Pheromone; Electrostatic; Pheromone receptor; Yeast; Ligand binding; α-factor;

Plasmin decreases the BH3-only protein BimEL via the ERK1/2 signaling pathway in hepatocytes by Naoyuki Kawao; Kiyotaka Okada; Syuhei Kawata; Chikako Okamoto; Mitsuhiro Tsuritani; Shigeru Ueshima; Osamu Matsuo (718-727).
Since the signal transduction mechanisms responsible for liver regeneration mediated by the plasminogen/plasmin system remain largely undetermined, we have investigated whether plasmin regulates the pro-apoptotic protein BimEL in primary hepatocytes. Plasmin bound to hepatocytes in part via its lysine binding sites (LBS). Plasmin also triggered phosphorylation of ERK1/2 without cell detachment. The plasmin-induced phosphorylation of ERK1/2 was inhibited by the LBS inhibitor epsilon-aminocaproic acid (EACA), the serine protease inhibitor aprotinin, and the MEK inhibitor PD98059. DFP-inactivated plasmin failed to phosphorylate ERK1/2. Plasmin temporally decreased the starvation-induced expression of BimEL and activation of caspase-3 via the ERK1/2 signaling pathway, resulting in an enhancement of cell survival. The amount of mRNA for Bim increased 1 day after the injection of CCl4 in livers of plasminogen knockout (Plg-KO) and the wild-type (WT) mice. The increase in BimEL protein persisted for at least 7 days post-injection in livers of Plg-KO mice, whereas WT mice showed an increase in BimEL protein 1 day after the injection. Plg-KO and WT mice showed notable phosphorylation of ERK1/2 7 and 3 days after the injection of CCl4, respectively. Our data suggest that the plasminogen/plasmin system could decrease BimEL expression via the ERK1/2 signaling pathway during liver regeneration.
Keywords: Plasmin(ogen); Bim; ERK1/2; Hepatocytes; Signal transduction;

Statistical differences between amperometric traces recorded from chromaffin cells using K+ and Ba2+ secretagogues support the assertion that readily releasable pool (RRP) and reserve pool (RP) vesicles can be probed with pool-specific secretagogues. Release from the RRP was evoked by K+ while release from the RP was evoked by Ba2+. Similar temperature-dependent changes in spike area and half-width for both pools suggest that the content of RRP and RP vesicles is similar and packaged in the same way. Differences between the vesicle pools were revealed in the temperature dependence of spike frequency. While the burst spike frequency of the RRP, which is comprised of pre-docked and primed vesicles, increased 2.8% per °C, the RP spike frequency increased 12% per °C. This difference is attributed to a temperature-dependent mobilization of the RP. Furthermore, the RP exhibited more foot events at room temperature than the RRP but this difference was not apparent at 37 °C. This trend suggests that RP vesicle membranes have a compromised surface tension compared to RRP vesicles. Collectively, the changes of release characteristics with temperature reveal distinctions between the RRP and the RP.
Keywords: Exocytosis; Amperometry; Carbon-fiber microelectrode; Catecholamine; Vesicle pools;

The amyloid beta-precursor protein (APP) and the Notch receptor are both type 1 integral transmembrane proteins, and both are cleaved by presenilin-dependent gamma-secretase activity. In this study, we have demonstrated that the Notch intracellular domain (Notch1-IC) suppresses APP-intracellular domain (AICD)-mediated ROS generation and cell death after being processed by gamma secretase. Notch1-IC physically interacts with AICD, Fe65, and Tip60, thereby disrupting the association of the AICD–Fe65–Tip60 trimeric transcription activator complex in AICD signaling. AICD–Fe65–Tip60 mediated reactive oxygen species generation was found to be suppressed by Notch1-IC. Furthermore, AICD–Fe65–Tip60 was shown to mediate cell death in human neuroblastoma cells, and the overexpression of Notch1-IC inhibited cell death induced by AICD–Fe65–Tip60. Collectively, our findings indicate that Notch1-IC plays the role of a negative regulator in AICD signaling via the disruption of the AICD–Fe65–Tip60 trimeric complex.

The FN13 peptide inhibits human tumor cells invasion through the modulation of αvβ3 integrins organization and the inactivation of ILK pathway by Nicoletta Zoppi; Marco Ritelli; Alessandro Salvi; Marina Colombi; Sergio Barlati (747-763).
We report the effect of the stable expression of a 13 amino acid human fibronectin (FN) peptide (FN13) on the organization of the FN extracellular matrix (ECM) and of FN integrin receptors (FNRs), in relationship with the inhibition of cellular invasion, in three FN-ECM defective human tumor-derived cell lines: SK-Hep1C3, hepatoma, ACN, neuroblastoma, and SK-OV-3, ovary carcinoma. All these cell lines stably expressing the FN13 peptide, organized an FN-ECM, disorganized αvβ1 integrins and inactivated the ILK pathway, with the loss of secretion of MMP-9. This was associated with the inhibition of cell invasion in Matrigel matrix only in SK-Hep1C3 and ACN, but not in SK-OV-3 cells. Analysis of the integrin receptors organization showed that the FN13 expressing cells SK-Hep1C3 and ACN organized αvβ3 integrins, whereas SK-OV-3 organized αvβ5 dimers. The functional block of αvβ5 integrins, with an inactivating anti-αvβ5 antibody, led to the induction of αvβ3 integrins also in SK-OV-3 cells, and to the inhibition of cell invasion. These data show that in the human tumor cells studied FN13 inhibits the in vitro invasion through the dissociation of αvβ1 dimers, leading to ILK pathway inactivation, only when the organization of αvβ3 integrins is induced in the plasma membrane.
Keywords: FN13 peptide; αvβ3 integrin; Tumor cells invasion; Fibronectin; Extracellular matrix; ILK;

KCNQ channels are involved in the regulatory volume decrease response in primary neonatal rat cardiomyocytes by Kirstine Calloe; Morten Schak Nielsen; Morten Grunnet; Nicole Schmitt; Nanna Koschmieder Jorgensen (764-773).
Cardiomyocytes may experience significant cell swelling during ischemia and reperfusion. Such changes in cardiomyocyte volume have been shown to affect the electrical properties of the heart, possibly leading to cardiac arrhythmia. In the present study the regulatory volume decrease (RVD) response of neonatal rat cardiomyocytes was studied in intact single cells attached to coverslips, i.e. with an intact cytoskeleton. The potential contribution of KCNQ (Kv7) channels to the RVD response and the possible involvement of the F-actin cytoskeleton were investigated. The rate of RVD was significantly inhibited in the presence of the KCNQ channel blocker XE-991 (10 and 100 μM). Electrophysiological experiments confirmed the presence of an XE-991 sensitive current and Western blotting analysis revealed that KCNQ1 channel protein was present in the neonatal rat cardiomyocytes. Hypoosmotic cell swelling changes the structure of the F-actin cytoskeleton, leading to a more rounded cell shape, less pronounced F-actin stress fibers and patches of actin. In the presence of cytochalasin D (1 μM), a potent inhibitor of actin polymerization, the RVD response was strongly reduced, confirming a possible role for an intact F-actin cytoskeleton in linking cell swelling to activation of ion transport in neonatal rat cardiomyocytes.
Keywords: KCNQ1; Kv7.1; Cell swelling; Ischemia; Arrhythmia;

Chronic sub-lethal oxidative stress by spermine oxidase overactivity induces continuous DNA repair and hypersensitivity to radiation exposure by M. Bianchi; A. Bellini; M. Cervelli; P. Degan; L. Marcocci; F. Martini; M. Scatteia; P. Mariottini; R. Amendola (774-783).
In the aging process and in most degenerative diseases, the oxidant by-products of cellular metabolism lead to oxidative stress. Oxidative stress plays an important role in switching from cell proliferation to its opposite outcome, cell death. The metabolic pathways in charge of the interconversion and degradation of the polyamines are responsible for oxidant by-products. In the past few years, spermine metabolism has been found closely related to DNA oxidation and apoptosis. Moreover, that the ectopical expression of murine spermine oxidase induced DNA damage in the neuroblastoma cell line, and this was uncoupled with any increase in cell mortality, thus suggests an activation of DNA repair. In this work, we provide new evidence showing that only spermine oxidase overactivity can deliver sub-lethal chronic DNA damage and repair without affecting transcriptional and enzymatic levels of the PA key regulatory enzymes ODC and SSAT. Chronic sub-lethal DNA damage is below the cell cycle arrest induction threshold, but is able to activate apurinic/apyrimidinic endonuclease protein (APE1) and γH2AX. Of therapeutic interest, the chronic sub-lethal DNA damage and activation of the repair processes are in turn responsible for inducing hypersensitivity after exposure to radiation with no induction of adaptive response to damage.
Keywords: Oxidative stress; DNA damage; DNA repair; Radiation; Spermine oxidase;

Rab-regulated interaction of early endosomes with lipid droplets by Pingsheng Liu; René Bartz; John K. Zehmer; Yun-shu Ying; Meifang Zhu; Ginette Serrero; Richard G.W. Anderson (784-793).
Recent studies indicate that lipid droplets isolated from a variety of different cells are rich in proteins known to regulate membrane traffic. Among these proteins are multiple Rab GTPases. Rabs are GTP switches that regulate intracellular membrane traffic through an ability to control membrane–membrane docking as well as vesicle motility. Here we present evidence that the multiple Rabs associated with droplets have a function in regulating membrane traffic. Droplet Rabs are removed by Rab GDP-dissociation inhibitor (RabGDI) in a GDP-dependent reaction, and are recruited to Rab-depleted droplets from cytosol in a GTP-dependent reaction. Rabs also control the recruitment of the early endosome (EE) marker EEA1 from cytosol. We use an in vitro reconstitution assay to show that transferrin receptor positive EEs bind to the droplet in a GTP/Rab-dependent reaction that appears not to lead to membrane fusion. This docking reaction is insensitive to ATPγs but is blocked by ATP. Finally, we show that when GTP bound active or GDP bound inactive Rab5 is targeted to the droplet, the active form recruits EEA1. We conclude that the Rabs associated with droplets may be capable of regulating the transient interaction of specific membrane systems, probably to transport lipids between membrane compartments.
Keywords: Membrane traffic; Endosome; Rabs; Adiposomes; Lipid droplets;

The intestinal epithelium provides a barrier to the transport of harmful luminal molecules into the systemic circulation. A dysfunctional epithelial barrier is closely associated with the pathogenesis of a variety of intestinal and systemic disorders. We investigated here the effects of nitric oxide (NO) and hydrogen peroxide (H2O2) on the barrier function of a human intestinal epithelial cell line, Caco-2. When treated with H2O2, Caco-2 cell monolayers grown on permeable supports exhibited several remarkable features of barrier dysfunction as follows: a decrease in transepithelial electrical resistance, an increase in paracellular permeability to dextran, and a disruption of the intercellular junctional localization of the scaffolding protein ZO-1. In addition, an induction of tyrosine phosphorylation of numerous cellular proteins including ZO-1, E-cadherin, and β-catenin, components of tight and adherens junctions, was observed. On the other hand, combined treatment of Caco-2 monolayers with H2O2 and an NO donor (NOC5 or NOC12) relieved the damage to the barrier function and suppressed the protein tyrosine phosphorylation induced by H2O2 alone. These results suggest that NO protects the barrier function of intestinal epithelia from oxidative stress by modulating some intracellular signaling pathways of protein tyrosine phosphorylation in epithelial cells.
Keywords: Intestinal epithelial cell; Tight junction; Nitric oxide; Hydrogen peroxide; Protein tyrosine phosphorylation; Caco-2 cell;

Nuclear import of human sexual regulator DMRT1 is mediated by importin-β by Ming Ying; Bo Chen; Yihao Tian; Yu Hou; Qin Li; Xuan Shang; Jinhua Sun; Hanhua Cheng; Rongjia Zhou (804-813).
Human DMRT1 (Doublesex-Mab3-Related Transcription factor 1) encodes a male-specific transcriptional regulator with a conserved zinc-finger-like DNA-binding domain, so called DM domain, which is similar to male sexual regulatory genes doublesex of Drosophila and mab-3 of Caenorhabditis elegans. As a key transcription factor critical to sex determination and differentiation, however, human DMRT1 nuclear import mechanism remains unknown. We have identified a functional nuclear localization signal (NLS) located between the two intertwined zinc-binding sites of the DM domain. Site-directed mutagenesis indicates that K92 and R93 within the DM domain are critical for DMRT1 nuclear localization. Analysis of deletion mutants shows that importin-β1 binds directly to DMRT1 via the DM domain, mediating its nuclear import. Co-immunoprecipitation analysis confirms the interaction of mouse Dmrt1 in Sertoli cells with importin-β1 in vivo. In addition, in vitro docking or nuclear transport assay in digitonin-permeabilized cells shows that DMRT1 is docked at the nuclear pore complex (NPC) or accumulated in the nucleus when importin-β1, but not importin-α1 added. Furthermore, transduction of anti-importin-β1 antibody into live Sertoli cells effectively inhibits DMRT1 nuclear import. These results suggest that zinc finger domain of DMRT1 functions as a nuclear localization signal and DMRT1 is transported into the nucleus in an importinβ1-mediated manner. Thus, effective nuclear import of DMRT1 and its interaction with importin-β1 insure the nuclear retention of the DMRT1 and further exertion of its influence on downstream targets in the cascade of sexual development.
Keywords: Transcription factor; Nuclear localization; Importin; DM domain;

PRDM16/MEL1: A novel Smad binding protein expressed in murine embryonic orofacial tissue by Dennis R. Warner; Kristin H. Horn; Lisa Mudd; Cynthia L. Webb; Robert M. Greene; M. Michele Pisano (814-820).
TGFβ signaling regulates central cellular processes such as proliferation and extracellular matrix production during development of the orofacial region. Extracellular TGFβ binds to cell surface receptors to activate the nucleocytoplasmic Smad proteins that, along with other transcription factors and cofactors, bind specific DNA sequences in the promoters of target genes to regulate their expression. To determine the identity of Smad binding proteins that regulate TGFβ signaling in developing murine orofacial tissue, a yeast two-hybrid screening approach was employed. The PR-domain containing protein, PRDM16/MEL1 was identified as a novel Smad binding protein. The interaction between PRDM16/MEL1 and Smad 3 was confirmed by GST pull-down assays. The expression of PRDM16/MEL1 was detected in developing orofacial tissue by both Northern blot and in situ hybridization. PRDM16/MEL1 was constitutively expressed in orofacial tissue on E12.5–E14.5 as well as other embryonic tissues such as heart, brain, liver, and limb buds. Taken together, these results demonstrate that PRDM16/MEL1 is a Smad binding protein that may be important for development of orofacial structures through modulation of the TGFβ signaling pathway.
Keywords: TGFβ; Smad; PRDM16; MEL1; Orofacial;

MAD2 expression and its significance in mitotic checkpoint control in testicular germ cell tumour by Maggie K.-L. Fung; Hiu-Wing Cheung; Hing-Lok Wong; Hiu-Fung Yuen; Ming-Tat Ling; Kowk-Wah Chan; Yong-Chuan Wong; Annie L.-M. Cheung; Xianghong Wang (821-832).
Chromosomal instability (CIN) is a common characteristic in testicular germ cell tumour (TGCT). A functional mitotic checkpoint control is important for accurate chromosome segregation during mitosis. Mitotic arrest deficient 2 (MAD2) is a key component of this checkpoint and inactivation of MAD2 is correlated with checkpoint impairment. The aim of this study was to investigate the function of mitotic checkpoint control in TGCT cells and to study its association with MAD2 expression using 8 TGCT cell lines as well as 23 TGCT tissue samples. We found that in response to microtubule disruption, 6 of 8 TGCT cell lines (75%) failed to arrest in mitosis demonstrated by the decreased mitotic index and aberrant expression of mitosis regulators, indicating that mitotic checkpoint defect is a common event in TGCT cells. This loss of mitotic checkpoint control was correlated with reduced MAD2 protein expression in TGCT cell lines implicating that downregulation of MAD2 may play a critical role in an impaired mitotic checkpoint control in these cells. In addition, immunohistochemistry studies on 23 seminomas and 12 normal testis tissues demonstrated that nuclear expression of MAD2 was much lower in seminomas (p  < 0.0001) but cytoplasmic MAD2 expression was higher in seminomas (p  = 0.06) than normal samples. Our results suggest that aberrant MAD2 expression may play an essential role in a defective mitotic checkpoint in TGCT cells, which may contribute to CIN commonly observed in TGCT tumours.
Keywords: Testicular germ cell tumour; MAD2; Mitotic checkpoint;

EGFR phosphorylation-dependent formation of cell–cell contacts by Ras/Erks cascade inhibition by Eun-Sil Kang; Min-A Oh; Sin-Ae Lee; Tae Young Kim; Sung-Hoon Kim; Noriko Gotoh; Yong-Nyun Kim; Jung Weon Lee (833-843).
Cell–cell contacts play important roles in the homeostasis of normal epithelium and in the steps of metastasis of tumor cells, although signaling mechanisms to regulate cell–cell contacts are unclear. In this study, we observed that phenotype of no cell–cell contacts in rat intestinal epithelial cell subline (RIE1-Sca) correlated with increased Erk1/2 signaling activity, compared to that of parental RIE1 cells growing in colonies. Furthermore, cell–cell contacts between RIE1-Sca cells were reformed by treatment with a specific MEK inhibitor (U0126), with translocation of ZO1 and β-catenin to cell–cell contacts, without changes of their expression levels. U0126 treatment also increased EGFR phosphorylation in a ligand-independent manner. Pretreatment with EGFR kinase inhibitor abolished U0126 treatment-mediated EGFR phosphorylation, and expression of dominant negative H-Ras N17 allowed EGFR phosphorylation and cell–cell contacts even without U0126 treatment. Furthermore, the expression of a nonphosphorylatable EGFR Y5F mutant abolished U0126-mediated cell–cell contacts. U0126 treatment also caused less efficient wound healing by keeping monolayer integrity intact, compared to control untreated cells. This U0126-mediated reduction in wound healing was further altered either by pretreatment of EGFR kinase inhibitor or expression of H-Ras N17 or EGFR Y5F. Taken together, this study supports a unique mechanism of cell–cell contact formation through MEK/Erks inhibition-mediated EGFR phosphorylation.
Keywords: Erk; EGFR; Cell–cell contacts; wound healing;

Polycomb group protein RING1B is a direct substrate of Caspases-3 and -9 by Chung Kai Wong; Zhengming Chen; Ka Lun So; De Li; Peng Li (844-852).
Both Caspase-3 and Caspase-9 play critical roles in the execution of mitochondria-mediated apoptosis. Caspase-9 binds to Apaf-1 in the presence of cytochrome c and dATP/ATP, and is activated by self-cleavage. Caspase-3 is activated by cleavage of caspase-8 and caspase-9. Over hundred direct caspase-3 substrates are identified whereas only few direct caspase-9 substrates are known. Here, we demonstrate that Ring1B, a component of polycomb protein complex that plays important roles in modulating chromatin structures, is a direct substrate of active caspase-3 and caspase-9 both in vitro and in vivo. The specific cleavage sites for caspase-3 and caspase-9 were mapped to Asp175 and Asp208, respectively. Importantly, cleavage of Ring1B by active caspases-3 and caspase-9 triggers the redistribution of Ring1B, from exclusive nuclear localization to even distribution throughout the entire cell. The transcriptional repression activity of Ring1B was also disrupted by caspase cleavage. Our data suggest that caspases-3 and caspase-9 play novel roles in transcription by regulating polycomb protein function through direct cleaving of Ring1B.
Keywords: Ring1B; Polycomb group protein; Caspase-3; Caspase-9; Nuclear localization; Transcriptional repression;

Inhibition of the CaaX proteases Rce1p and Ste24p by peptidyl (acyloxy)methyl ketones by Stephen B. Porter; Emily R. Hildebrandt; Sarah R. Breevoort; David Z. Mokry; Timothy M. Dore; Walter K. Schmidt (853-862).
The CaaX proteases Rce1p and Ste24p can independently promote a proteolytic step required for the maturation of certain isoprenylated proteins. Although functionally related, Rce1p and Ste24p are unrelated in primary sequence. They have distinct enzymatic properties, which are reflected in part by their distinct inhibitor profiles. Moreover, Rce1p has an undefined catalytic mechanism, whereas Ste24p is an established zinc-dependent metalloprotease. This study demonstrates that both enzymes are inhibited by peptidyl (acyloxy)methyl ketones (AOMKs), making these compounds the first documented dual specificity inhibitors of the CaaX proteases. Further investigation of AOMK-mediated inhibition reveals that varying the peptidyl moiety can significantly alter the inhibitory properties of AOMKs toward Rce1p and Ste24p and that these enzymes display subtle differences in sensitivity to AOMKs. This observation suggests that this compound class could potentially be engineered to be selective for either of the CaaX proteases. We also demonstrate that the reported sensitivity of Rce1p to TPCK is substrate-dependent, which significantly alters the interpretation of certain reports having used TPCK sensitivity for mechanistic classification of Rce1p. Finally, we show that an AOMK inhibits the isoprenylcysteine carboxyl methyltransferase Ste14p. In sum, our observations raise important considerations regarding the specificity of agents targeting enzymes involved in the maturation of isoprenylated proteins, some of which are being developed as anti-cancer therapeutic agents.
Keywords: Ras; Protease; CaaX protein; Post-translational modification; Isoprenylation; (acyloxy)methyl ketone;

Human 1A6/DRIM, the homolog of yeast Utp20, functions in the 18S rRNA processing by You Wang; Jiangying Liu; Hong Zhao; Wenqing Lü; Jun Zhao; Lin Yang; Ning Li; Xiaojuan Du; Yang Ke (863-868).
1A6/DRIM is a nucleolar protein with a nucleolar targeting sequence in its 3′-terminus. Bioinformatic analysis indicated that human 1A6/DRIM shares 23% identity and 43% similarity with yeast Utp20, which has been reported as a component of U3 snoRNA protein complex and has been implicated in 18S rRNA processing. In the present study, we found, by utilizing RT-PCR with RNA extracted from anti-1A6/DRIM immunoprecipitates and Northern blotting, that 1A6/DRIM is associated with U3 snoRNA. Pulse-chase labeling assays showed that silencing of 1A6/DRIM expression in HeLa cells resulted in a delayed 18S rRNA processing. Furthermore, immunoprecipitations revealed that 1A6/DRIM was also associated with fibrillarin, another U3 RNP component in HeLa cells. These results indicate that 1A6/DRIM is involved in 18S rRNA processing and is the bona fide mammalian Utp20.
Keywords: 1A6/DRIM; UTP20; 18S rRNA processing; U3 snoRNA;

Switch activation of PI-PLC downstream signals in activated macrophages with wortmannin by Der-Zen Liu; Hong-Jen Liang; Chien-Ho Chen; Shyr-Yi Lin; Wen-Bin Zhong; Feng-Ming Ho; Wen-Chi Hou; Jui-Lien Lo; Yuan-Soon Ho; Pei-Jung Lin; Ling-Fang Hung; Yu-Chih Liang (869-879).
Phosphatidylinositol (4,5)-bisphosphate (PtdIns(4,5)P2) has been known to serve as a substrate for phosphatidylinositol 3-kinase (PI3K) and phosphoinositide-specific phospholipase C (PI-PLC), which can produce PtdIns(3,4,5)P3 and inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and diacylglycerol (DAG), respectively. In this study, we elucidated the role of PI-PLC during the LPS-activated mouse macrophages RAW264.7 treated with PI3K inhibitor wortmannin. First, wortmannin treatment enhanced Ins(1,4,5)P3 production and iNOS expression in LPS-activated macrophages. Inhibition of PI3K by p85 siRNA also showed an enhancement of iNOS expression. On the other hand, overexpression of PI3K by ras-p110 expression plasmid significantly decreased iNOS expression in LPS-activated macrophages. In addition, overexpression of wild-type or dominant-negative Akt expression plasmid did not affect the iNOS expression in LPS-activated macrophages. Second, treatment of PI-PLC inhibitor U73122 reversed the enhancement of iNOS expression, the increase of phosphorylation level of ERK, JNK and p38, and the increase of AP-1-dependent gene expression in wortmannin-treated and LPS-activated macrophages. However, NF-κB activity determined by EMSA assay and reporter plasmid assay did not change during LPS-activated macrophages with or without wortmannin. We propose that the inhibition of PI3K by wortmannin in mouse macrophages enhances the PI-PLC downstream signals, and subsequently increases the LPS induction of iNOS expression independently of Akt pathway.
Keywords: Phosphatidylinositol 3-kinase; Phosphoinositide-specific phospholipase C; Inducible nitric oxide synthase; Wortmannin; Lipopolysaccharide;

Hypoxia alters the adhesive properties of lymphatic endothelial cells. A transcriptional and functional study by M. Irigoyen; E. Ansó; E. Martínez; M. Garayoa; J.J. Martínez-Irujo; A. Rouzaut (880-890).
Recent advances in our understanding of the molecular biology of lymphatic endothelial cells have revealed that these vessels, besides their known function in tissue homeostasis and immunity, constitute conduits for the tumor cells to metastasize. One of the factors that contribute to tumor spread is the acquisition of an angiogenic phenotype as a response to the onset of tumor hypoxia. To our knowledge, little is known about the effects of low oxygen levels on the lymphatic vasculature. Therefore, we used cDNA microarrays to study the transcriptional changes occurring in hypoxia exposed lymphatic endothelial cells. Our analysis was then complemented by functional assays showing that these cells responded with increased attachment to the extracellular matrix, delayed proliferation and production of reactive oxygen species. Differential expression of genes involved in these processes such as NADPH oxidase 4, the tissue inhibitor of metalloproteinase 3, and TGFβ induced protein I, was found. Hypoxia was also found to increase mRNA levels of the cytokine CXCL-12 and its receptor CXCR4. Moreover, adhesion experiments revealed that hypoxia increased the binding of non-small cell lung carcinoma cells to this endothelium in a CXCR4 dependent way. We thus illustrate the response of lymphatic endothelial cells to hypoxia and suggest targets to study tumor metastasis through these vessels.
Keywords: Lymphatic; Hypoxia; Adhesion; Endothelium; Gene Expression;

Identification and mapping of protein kinase A binding sites in the costameric protein myospryn by Joseph G. Reynolds; Sarah A. McCalmon; Thomas Tomczyk; Francisco J. Naya (891-902).
Recently we identified a novel target gene of MEF2A named myospryn that encodes a large, muscle-specific, costamere-restricted α-actinin binding protein. Myospryn belongs to the tripartite motif (TRIM) superfamily of proteins and was independently identified as a dysbindin-interacting protein. Dysbindin is associated with α-dystrobrevin, a component of the dystrophin–glycoprotein complex (DGC) in muscle. Apart from these initial findings little else is known regarding the potential function of myospryn in striated muscle. Here we reveal that myospryn is an anchoring protein for protein kinase A (PKA) (or AKAP) whose closest homolog is AKAP12, also known as gravin/AKAP250/SSeCKS. We demonstrate that myospryn co-localizes with RIIα, a type II regulatory subunit of PKA, at the peripheral Z-disc/costameric region in striated muscle. Myospryn interacts with RIIα and this scaffolding function has been evolutionarily conserved as the zebrafish ortholog also interacts with PKA. Moreover, myospryn serves as a substrate for PKA. These findings point to localized PKA signaling at the muscle costamere.
Keywords: MEF2 target; Muscle-specific; Tripartite motif; Scaffolding protein; Costamere; Protein kinase A;

Genistein, a soy isoflavone with anti-tumor properties, has both estrogenic and non-estrogenic activities. Genistein sensitive/estrogen receptor negative (ER-) MDA-MB-231 cells and genistein resistant/ER + MCF-7 cells are frequently cited as examples of differential responses to genistein due to different ER status. Other factors that may affect genistein response, however, are largely unknown. Based on our finding that MCF-7 is caspase-3 deficient, we examined whether caspase-3 status plays a role in the differential responses between the two cell lines. We demonstrate that reconstitution of caspase-3 significantly sensitizes MCF-7 cells to genistein. Specific knockdown of caspase-3 in MDA-MB-231 cells renders the cells resistant to genistein. We also found that caspases-4 and -10 were downregulated in MCF-7 cells. Reconstitution of caspase-10 in MCF-7 cells, however, resulted in little sensitization. Moreover, we show that caspase-3 downregulation is very common in breast cancer cell lines and tumor tissues. Taken together, our data indicate that caspase-3 is a critical determinant of cellular response to genistein, which may have important implications in studying soy/genistein-mediated anti-tumor activities.
Keywords: Genistein; Caspase-3; Apoptosis; Breast cancer; MCF-7; MDA-MB-231;

Relocalization of the polypyrimidine tract-binding protein during PKA-induced neurite growth by Shumei Ma; Guodong Liu; Yuan Sun; Jiuyong Xie (912-923).
Neurite RNA binding proteins are important for neurite growth, a process critical for neuronal development and regeneration after injury. It has been known that many RNA binding proteins undergo nucleocytoplasmic shuttling but how their nucleocytoplasmic distributions are regulated during neurite growth has not been well explored. Here we found that the polypyrimidine tract binding protein (PTB) was exported from the nucleus and accumulated at growing neurite terminals upon activation of the PKA pathway in PC12 cells in a PKA-target Ser16-dependent manner. RNA interference (RNAi) of PTB significantly disrupted the neurite growth. We then examined the role of cytoplasmic PTB in relation to mRNAs involved in neurite growth. We found that PTB was preferentially associated with the β-actin mRNA transcripts in cytoplasmic fractions. RNAi of PTB reduced neurite accumulation of the endogenous actin proteins. It is thus likely that, during PKA-induced neurite growth, PTB is relocalized through Ser16 phosphorylation to the cytoplasm where it is associated with β-actin mRNA and is critical for the mRNA localization to neurites.
Keywords: Polypyrimidine tract binding protein; PTB; PKA; Nucleocytoplasmic transport; Neurite growth; Phosphorylation; mRNA localization;

Unusual compartmentalization of CTCF and other transcription factors in the course of terminal erythroid differentiation by Omar L. Kantidze; Olga V. Iarovaia; Elena S. Philonenko; Irina I. Yakutenko; Sergey V. Razin (924-933).
It is demonstrated that in chicken embryonic and mature erythrocyte nuclei the distribution of a versatile transcription factor CTCF differs drastically from its distribution in nuclei of proliferating erythroid and non-erythroid cells. In the latter case CTCF was distributed throughout the whole nucleus volume, being concentrated in many small compartments (punctuate nuclear staining). In contrast, in embryonic and mature erythrocytes CTCF was concentrated in a limited number of large compartments. These large CTCF-containing compartments were not observed in other cells. Occasionally, but not in all cells, some of these compartments were localized close to nucleoli but did not colocalize with them. In mature erythrocytes a clear exclusion of CTCF-containing compartments from the chromatin domain was observed. This exclusion correlated with a tight association of CTCF with the nuclear matrix. Concentration in relatively large compartments and exclusion from the chromatin domain in nuclei of mature erythrocytes were also observed for RNA polymerase II and several transcription factors. The data are discussed in the context of a hypothesis postulating that relocalization of different components of the transcriptional machinery from the chromatin domain into the interchromatin compartment is an important step of the terminal inactivation of chicken erythrocyte nuclei.
Keywords: CTCF; Interchromatin domain; Erythrocytes; Nuclear compartments;

Difference in nucleocytoplasmic shuttling sequences of rat and human constitutive active/androstane receptor by Yuichiro Kanno; Motoyoshi Suzuki; Yukari Miyazaki; Midori Matsuzaki; Takayuki Nakahama; Kouichi Kurose; Jun-ichi Sawada; Yoshio Inouye (934-944).
Fluorescence recovery after photobleaching (FRAP) in spontaneous multinuclear cells shows that both rat and human constitutive active/androstane receptors (CARs) are shuttling proteins with both nuclear localization signals (NLSs) and nuclear export signals (NESs). We previously identified two NLSs in rat CAR: NLS1 in the hinge region (residues 100–108) and NLS2 in the ligand-binding domain (residues 111–320). In the present study, we compared the intracellular localization signals between rat and human CARs. There was a marked difference in their intracellular localization in COS-7 cells because, unlike rat CAR, human CAR does not contain NLS1 due to an amino acid change at position 106. A CRM1-dependent leucine-rich NES, which is sensitive to an inhibitory effect of leptomycin B, was found in the cytoplasmic retention region previously identified within the ligand-binding domain of rat CAR (residues 220–258). We found that human CAR instead has a NES in the ligand-binding domain between residues 170 and 220. Also, we detected CRM1-independent C-terminal NESs between residues 317–358 of rat and human CARs. Removal of NLS1 by N-terminal truncation and mutation of xenochemical response signal caused rat CAR to localize in the cytoplasm of COS-7 cells, which we suspect is due to the masking of NLS2.
Keywords: Constitutive androstane receptor; Nuclear localization signal; Nuclear export signal; Nucleocytoplasmic shuttling;

Aqueous peroxyl radical exposure to THP-1 cells causes glutathione loss followed by protein oxidation and cell death without increased caspase-3 activity by Marion Kappler; Andrew B. Gerry; Erin Brown; Linzi Reid; David S. Leake; Steven P. Gieseg (945-953).
Protein oxidation within cells exposed to oxidative free radicals has been reported to occur in an uninhibited manner with both hydroxyl and peroxyl radicals. In contrast, THP-1 cells exposed to peroxyl radicals (ROO) generated by thermo decomposition of the azo compound AAPH showed a distinct lag phase of at least 6 h, during which time no protein oxidation or cell death was observed. Glutathione appears to be the source of the lag phase as cellular levels were observed to rapidly decrease during this period. Removal of glutathione with buthionine sulfoxamine eliminated the lag phase. At the end of the lag phase there was a rapid loss of cellular MTT reducing activity and the appearance of large numbers of propidium iodide/annexin-V staining necrotic cells with only 10% of the cells appearing apoptotic (annexin-V staining only). Cytochrome c was released into the cytoplasm after 12 h of incubation but no increase in caspase-3 activity was found at any time points. We propose that the rapid loss of glutathione caused by the AAPH peroxyl radicals resulted in the loss of caspase activity and the initiation of protein oxidation. The lack of caspase-3 activity appears to have caused the cells to undergo necrosis in response to protein oxidation and other cellular damage.
Keywords: AAPH; THP-1; Macrophage; Protein-oxidation; Glutathione; Apoptosis; Cell-death; Caspase;

Modulation of apolipoprotein D expression and translocation under specific stress conditions by Sonia Do Carmo; Louis-Charles Levros; Eric Rassart (954-969).
Apolipoprotein D is a lipocalin, primarily associated with high density lipoproteins in human plasma. Its expression is induced in several pathological and stressful conditions including growth arrest suggesting that it could act as a nonspecific stress protein. A survey of cellular stresses shows those causing an extended growth arrest, as hydrogen peroxide and UV light increase apoD expression. Alternatively, lipopolysaccharide (LPS), a pro-inflammatory agonist showed a time- and dose-dependent effect on apoD expression that correlates with an increase in proliferation. At the promoter level, NF-kB, AP-1 and APRE-3 proved to be the elements implicated in the LPS response. Colocalization of apoDh–GFP fusion constructs with DNA and Golgi markers, immunocytochemistry of the endogenous protein and cell fractionation showed that both serum starvation and LPS treatment caused a displacement of apoD localization. In normal conditions, apoD is mainly perinuclear but it accumulates in cytoplasm and nucleus under these stress conditions. Since nuclear apoD appears derived from the secreted protein, it may act as an extracellular ligand transporter as well as a transcriptional regulator depending on its location. This role of apoD inside the cell is not only dependent of endogenous apoD but may also be provided by exogenous apoD entering the cell.
Keywords: Apolipoprotein D; Growth arrest; Inflammation; Lipocalin; Nuclear translocation;

Thromboxane (TX) A2 plays a central role in hemostasis, regulating platelet activation status and vascular tone. We have recently established that the TPβ isoform of the human TXA2 receptor (TP) undergoes rapid, agonist-induced homologous desensitization of signalling largely through a G protein-coupled receptor kinase (GRK) 2/3-dependent mechanism with a lesser role for protein kinase (PK) C. Herein, we investigated the mechanism of desensitization of signalling by the TPα isoform. TPα undergoes profound agonist-induced desensitization of signalling (intracellular calcium mobilization and inositol 1,4,5 trisphosphate generation) in response to the TXA2 mimetic U46619 but, unlike that of TPβ, this is independent of GRKs. Similar to TPβ, TPα undergoes partial agonist-induced desensitization that occurs through a GF 109203X-sensitive, PKC mechanism where Ser145 within intracellular domain (IC)2 represents the key phospho-target. TPα also undergoes more profound sustained PKC- and PKG-dependent desensitization where Thr337 and Ser331, respectively, within its unique C-tail domain were identified as the phospho-targets. Desensitization was impaired by the nitric oxide synthase (NOS), soluble guanylyl cyclase (sGC) and PKG inhibitors l-NAME, LY 83583 and KT5823, respectively, indicating that homologous desensitization of TPα involves nitric oxide generation and signalling. Consistent with this, U46619 led to rapid phosphorylation/activation of endogenous eNOS. Collectively, data herein suggest a mechanism whereby agonist-induced PKC phosphorylation of Ser145 partially and transiently impairs TPα signalling while PKG- and PKC-phosphorylation at both Ser331 and Thr337, respectively, within its C-tail domain profoundly desensitizes TPα, effectively terminating its signalling. Hence, in addition to the agonist-mediated PKC feedback mechanism, U46619-activation of the NOS/sGC/PKG pathway plays a significant role in inducing homologous desensitization of TPα.
Keywords: Thromboxane receptor; Alpha; Desensitization; Phosphorylation; Nitric oxide; G protein coupled receptor;

Iodine deficiency activates antioxidant genes and causes DNA damage in the thyroid gland of rats and mice by J. Maier; H. van Steeg; C. van Oostrom; R. Paschke; R.E. Weiss; K. Krohn (990-999).
Because thyroid nodules are frequent in areas with iodine deficiency the aim of this study was to characterise molecular events during iodine deficiency that could explain mutagenesis and nodule formation. We therefore studied gene expression of catalytic enzymes prominent for H2O2 detoxification and antioxidative defence, quantified DNA oxidation and damage as well as spontaneous mutation rates (SMR) in mice and rats fed an iodine controlled diet. Antioxidative enzymes such as superoxide dismutase 3, glutathione peroxidase 4 and the peroxiredoxins 3 and 5 showed increased mRNA expression, which indicates increased radical burden that could be the cause of additional oxidized base adducts found in thyroidal genomic DNA in our experiments of iodine deficiency. Furthermore, the uracil content of thyroid DNA was significantly higher in the iodine-deficient compared to the control group. While SMR is very high in the normal thyroid gland it is not changed in experimental iodine deficiency. Our data suggest that iodine restriction causes oxidative stress and DNA modifications. A higher uracil content of the thyroid DNA could be a precondition for C → T transitions often detected as somatic mutations in nodular thyroid tissue. However, the absence of increased SMR would argue for more efficient DNA repair in response to iodine restriction.
Keywords: Iodine Deficiency; Mutagenesis; Comet Assay; DNA Damage;

OCTN2VT, a splice variant of OCTN2, does not transport carnitine because of the retention in the endoplasmic reticulum caused by insertion of 24 amino acids in the first extracellular loop of OCTN2 by Satoshi Maekawa; Daisuke Mori; Tadashi Nishiya; Osamu Takikawa; Takahiro Horinouchi; Arata Nishimoto; Emi Kajita; Soichi Miwa (1000-1006).
A novel organic cation transporter OCTN2 is indispensable for carnitine transport across plasma membrane and subsequent fatty acid metabolism in the mitochondria. Here, we report a novel splice variant of OCTN2 (OCTN2VT), in which a 72-base-pair sequence located in the first intron of OCTN2 gene was spliced between exons 1 and 2 of OCTN2, causing the insertion of 24 amino acids in the first extracellular loop of OCTN2. Despite the similarity between OCTN2 and OCTN2VT regarding primary structure and tissue distribution, their biochemical characteristics were significantly different. OCTN2 was expressed on the plasma membrane with robust N-glycosylation, whereas OCTN2VT was retained in the endoplasmic reticulum (ER) with poor N-glycosylation. In addition, the retention in the ER caused no carnitine uptake into the cells. These results demonstrate that the biochemical and functional characteristics of OCTN2VT are distinct from OCTN2 due to the insertion of 24 amino acids in the first extracellular loop.
Keywords: OCTN2; OCTN2VT; Splicing; Carnitine; N-glycosylation; Endoplasmic reticulum;

MUC1 is involved in trophoblast transendothelial migration by Twanda L. Thirkill; Tim Cao; Michael Stout; Thomas N. Blankenship; Abdul Barakat; Gordon C. Douglas (1007-1014).
The factors that regulate trophoblast invasion of the uterine vasculature are incompletely understood. In this paper we show that macaque trophoblasts express the mucin, MUC1, and that it is involved in trophoblast-endothelial interaction. Immunocytochemistry, Western blotting and RT-PCR analyses confirmed that MUC1 was expressed by isolated early gestation macaque trophoblasts. MUC1 was also detected in endovascular trophoblasts in sections of placental–decidual tissue during early gestation. A blocking antibody against MUC1 reduced trophoblast adhesion to uterine endothelial cells and also blocked trophoblast transendothelial migration. MUC1 is known to bind to Intercellular Adhesion Molecule-1 (ICAM-1) in other systems. Incubation in the presence of a blocking antibody against Intercellular Adhesion Molecule-1 (ICAM-1) or recombinant ICAM-1 modestly, but significantly, reduced transendothelial trophoblast migration. These results are consistent with the idea that MUC1 is involved in trophoblast adhesion to uterine endothelial cells and in trophoblast transendothelial migration.
Keywords: Adhesion; Placenta; Uterus; Mucins;