BBA - Molecular Cell Research (v.1864, #1)

Constitutive activation of T cells by γ2-herpesviral GPCR through the interaction with cellular CXCR4 by Eun-Kyung Kwon; Chan-Ki Min; Yuri Kim; Jae-Won Lee; Abdimadiyeva Aigerim; Sebastian Schmidt; Hyun-Jun Nam; Seong Kyu Han; Kuglae Kim; Jeong Seok Cha; Hoyoung Kim; Sanguk Kim; Hyun-Soo Cho; Myung-Sik Choi; Nam-Hyuk Cho (1-11).
Members of the herpesviral family use multiple strategies to hijack infected host cells and exploit cellular signaling for their pathogenesis and latent infection. Among the most intriguing weapons in the arsenal of pathogenic herpesviruses are the constitutively active virally-encoded G protein-coupled receptors (vGPCRs). Even though vGPCRs contribute to viral pathogenesis such as immune evasion and proliferative disorders, the molecular details of how vGPCRs continuously activate cellular signaling are largely unknown. Here, we report that the vGPCR of Herpesvirus saimiri (HVS), an oncogenic γ2-herpesvirus, constitutively activates T cells via a heteromeric interaction with cellular CXCR4. Constitutive T cell activation also occurs with expression of the vGPCR of Kaposi's sarcoma-associated herpesvirus (KSHV), but not the vGPCR of Epstein-Barr virus. Expression of HVS vGPCR down-regulated the surface expression of CXCR4 but did not induce the degradation of the chemokine receptor, suggesting that vGPCR/CXCR4 signaling continues in cytosolic compartments. The physical association of vGPCR with CXCR4 was demonstrated by proximity ligation assay as well as immunoprecipitation. Interestingly, the constitutive activation of T cells by HVS vGPCR is independent of proximal T cell receptor (TCR) signaling molecules, such as TCRβ, Lck, and ZAP70, whereas CXCR4 silencing by shRNA abolished T cell activation by vGPCRs of HVS and KSHV. Furthermore, previously identified inactive vGPCR mutants failed to interact with CXCR4. These findings on the positive cooperativity of vGPCR with cellular CXCR4 in T cell activation extend our current understanding of the molecular mechanisms of vGPCR function and highlight the importance of heteromerization for GPCR activity.
Keywords: γ-Herpesvirus; Viral G protein-coupled receptor; T cells; CXCR4;

Involvement of caveolin-1 in low shear stress-induced breast cancer cell motility and adhesion: Roles of FAK/Src and ROCK/p-MLC pathways by Niya Xiong; Shun Li; Kai Tang; Hongxia Bai; Yueting Peng; Hong Yang; Chunhui Wu; Yiyao Liu (12-22).
Tumor cells translocating to distant sites are subjected to hemodynamic shear forces during their passage in the blood vessels. Low shear stress (LSS) plays a critical role in the regulation of various aspects of tumor cells functions, including motility and adhesion. Beyond its structural role, caveolin-1 (Cav-1), the important component of caveolae, represents a modulator of several cancer-associated functions as tumor progression and metastasis. However, the role of Cav-1 in regulating tumor cells response to shear stress remains poorly explored. Here, we characterized the role of LSS and Cav-1 in mediating cell motility and adhesion on human breast carcinoma MDA-MB-231 cells. We first showed that LSS exposure promoted cell polarity and focal adhesion (FA) dynamics, thus indicating elevated cell migration. Silencing of Cav-1 leaded to a significantly lower formation of stress fibers. However, LSS exposure was able to rescue it via the alteration of actin-associated proteins expression, including ROCK, p-MLC, cofilin and filamin A. Time-lapse migration assay indicated that Cav-1 expression fostered MDA-MB-231 cells motility and LSS triggered cells to rapidly generate new lamellipodia. Furthermore, Cav-1 and LSS significantly influenced cell adhesion. Taken together, our findings provide insights into mechanisms underlying LSS triggered events mediated by downstream Cav-1, including FAK/Src and ROCK/p-MLC pathways, involved in the reorganization of the cytoskeleton, cell motility, FA dynamics and breast cancer cell adhesion.
Keywords: Low shear stress; Caveolin-1; Cell motility; Focal adhesion; ROCK; MLC;

Intersectin scaffold proteins and their role in cell signaling and endocytosis by Erika Herrero-Garcia; John P. O'Bryan (23-30).
Intersectins (ITSNs) are a family of multi-domain proteins involved in regulation of diverse cellular pathways. These scaffold proteins are well known for regulating endocytosis but also play important roles in cell signaling pathways including kinase regulation and Ras activation. ITSNs participate in several human cancers, such as neuroblastomas and glioblastomas, while their downregulation is associated with lung injury. Alterations in ITSN expression have been found in neurodegenerative diseases such as Down Syndrome and Alzheimer's disease. Binding proteins for ITSNs include endocytic regulatory factors, cytoskeleton related proteins (i.e. actin or dynamin), signaling proteins as well as herpes virus proteins. This review will summarize recent studies on ITSNs, highlighting the importance of these scaffold proteins in the aforementioned processes.
Keywords: Intersectin; Scaffold; Endocytosis; Actin; Cancer; Neurons;

The SAM domain inhibits EphA2 interactions in the plasma membrane by Deo R. Singh; Fozia Ahmed; Michael D. Paul; Manasee Gedam; Elena B. Pasquale; Kalina Hristova (31-38).
All members of the Eph receptor family of tyrosine kinases contain a SAM domain near the C terminus, which has been proposed to play a role in receptor homotypic interactions and/or interactions with binding partners. The SAM domain of EphA2 is known to be important for receptor function, but its contribution to EphA2 lateral interactions in the plasma membrane has not been determined. Here we use a FRET-based approach to directly measure the effect of the SAM domain on the stability of EphA2 dimers on the cell surface in the absence of ligand binding. We also investigate the functional consequences of EphA2 SAM domain deletion. Surprisingly, we find that the EphA2 SAM domain inhibits receptor dimerization and decreases EphA2 tyrosine phosphorylation. This role is dramatically different from the role of the SAM domain of the related EphA3 receptor, which we previously found to stabilize EphA3 dimers and increase EphA3 tyrosine phosphorylation in cells in the absence of ligand. Thus, the EphA2 SAM domain likely contributes to a unique mode of EphA2 interaction that leads to distinct signaling outputs.
Keywords: Receptor tyrosine kinase; EphA2; SAM domain; Interactions;

Saccharomyces cerevisiae Hsp31p is a DJ-1/ThiJ/PfpI family protein that was previously shown to be important for survival in the stationary phase of growth and under oxidative stress. Recently, it was identified as a chaperone or as glutathione-independent glyoxalase. To elucidate the role played by this protein in budding yeast cells, we investigated its involvement in the protection against diverse environmental stresses. Our study revealed that HSP31 gene expression is controlled by multiple transcription factors, including Yap1p, Cad1p, Msn2p, Msn4p, Haa1p and Hsf1p. These transcription factors mediate the HSP31 promoter responses to oxidative, osmotic and thermal stresses, to potentially toxic products of glycolysis, such as methylglyoxal and acetic acid, and to the diauxic shift. We also demonstrated that the absence of the HSP31 gene sensitizes cells to these stressors. Overproduction of Hsp31p and its homologue Hsp32p rescued the sensitivity of glo1Δ cells to methylglyoxal. Hsp31p also reversed the increased sensitivity of the ald6Δ strain to acetic acid. Since Hsp31p glyoxalase III coexists in S. cerevisiae cells with thousand-fold more potent glyoxalase I/II system, its biological purpose requires substantiation. We postulate that S. cerevisiae Hsp31p may have broader substrate specificity than previously proposed and is able to eliminate various toxic products of glycolysis. Alternatively, Hsp31p might be effective under high concentration of exogenous methylglyoxal present in some natural environmental niches populated by budding yeast, when glyoxalase I/II system capacity is saturated.
Keywords: Fermentation; Saccharomyces cerevisiae; Environmental stress; Ethanol; Methylglyoxal; Acetic acid;

Pannexin1 as mediator of inflammation and cell death by Sara Crespo Yanguas; Joost Willebrords; Scott R. Johnstone; Michaël Maes; Elke Decrock; Marijke De Bock; Luc Leybaert; Bruno Cogliati; Mathieu Vinken (51-61).
Pannexins form channels at the plasma membrane surface that establish a pathway for communication between the cytosol of individual cells and their extracellular environment. By doing so, pannexin signaling dictates several physiological functions, but equally underlies a number of pathological processes. Indeed, pannexin channels drive inflammation by assisting in the activation of inflammasomes, the release of pro-inflammatory cytokines, and the activation and migration of leukocytes. Furthermore, these cellular pores facilitate cell death, including apoptosis, pyroptosis and autophagy. The present paper reviews the roles of pannexin channels in inflammation and cell death. In a first part, a state-of-the-art overview of pannexin channel structure, regulation and function is provided. In a second part, the mechanisms behind their involvement in inflammation and cell death are discussed.
Keywords: Pannexin; Inflammation; Apoptosis; Pyroptosis; Autophagy;

The cochaperone BAG3 coordinates protein synthesis and autophagy under mechanical strain through spatial regulation of mTORC1 by Barbara Kathage; Sebastian Gehlert; Anna Ulbricht; Laura Lüdecke; Victor E. Tapia; Zacharias Orfanos; Daniela Wenzel; Wilhelm Bloch; Rudolf Volkmer; Bernd K. Fleischmann; Dieter O. Fürst; Jörg Höhfeld (62-75).
The cochaperone BAG3 is a central protein homeostasis factor in mechanically strained mammalian cells. It mediates the degradation of unfolded and damaged forms of the actin-crosslinker filamin through chaperone-assisted selective autophagy (CASA). In addition, BAG3 stimulates filamin transcription in order to compensate autophagic disposal and to maintain the actin cytoskeleton under strain. Here we demonstrate that BAG3 coordinates protein synthesis and autophagy through spatial regulation of the mammalian target of rapamycin complex 1 (mTORC1). The cochaperone utilizes its WW domain to contact a proline-rich motif in the tuberous sclerosis protein TSC1 that functions as an mTORC1 inhibitor in association with TSC2. Interaction with BAG3 results in a recruitment of TSC complexes to actin stress fibers, where the complexes act on a subpopulation of mTOR-positive vesicles associated with the cytoskeleton. Local inhibition of mTORC1 is essential to initiate autophagy at sites of filamin unfolding and damage. At the same time, BAG3-mediated sequestration of TSC1/TSC2 relieves mTORC1 inhibition in the remaining cytoplasm, which stimulates protein translation. In human muscle, an exercise-induced association of TSC1 with the cytoskeleton coincides with mTORC1 activation in the cytoplasm. The spatial regulation of mTORC1 exerted by BAG3 apparently provides the basis for a simultaneous induction of autophagy and protein synthesis to maintain the proteome under mechanical strain.Display Omitted
Keywords: Autophagy; Chaperones; Protein synthesis; Signaling; Proteostasis; Mechanical strain;

The Sal-like 4 - integrin α6β1 network promotes cell migration for metastasis via activation of focal adhesion dynamics in basal-like breast cancer cells by Junji Itou; Sunao Tanaka; Wenzhao Li; Atsuo Iida; Atsuko Sehara-Fujisawa; Fumiaki Sato; Masakazu Toi (76-88).
During metastasis, cancer cell migration is enhanced. However, the mechanisms underlying this process remain elusive. Here, we addressed this issue by functionally analyzing the transcription factor Sal-like 4 (SALL4) in basal-like breast cancer cells. Loss-of-function studies of SALL4 showed that this transcription factor is required for the spindle-shaped morphology and the enhanced migration of cancer cells. SALL4 also up-regulated integrin gene expression. The impaired cell migration observed in SALL4 knockdown cells was restored by overexpression of integrin α6 and β1. In addition, we clarified that integrin α6 and β1 formed a heterodimer. At the molecular level, loss of the SALL4 - integrin α6β1 network lost focal adhesion dynamics, which impairs cell migration. Over-activation of Rho is known to inhibit focal adhesion dynamics. We observed that SALL4 knockdown cells exhibited over-activation of Rho. Aberrant Rho activation was suppressed by integrin α6β1 expression, and pharmacological inhibition of Rho activity restored cell migration in SALL4 knockdown cells. These results indicated that the SALL4 - integrin α6β1 network promotes cell migration via modulation of Rho activity. Moreover, our zebrafish metastasis assays demonstrated that this gene network enhances cell migration in vivo. Our findings identify a potential new therapeutic target for the prevention of metastasis, and provide an improved understanding of cancer cell migration.
Keywords: Breast cancer; Cell migration; Focal adhesion dynamics; Integrin; SALL4;

AKT-dependent phosphorylation of the SAM domain induces oligomerization and activation of the scaffold protein CNK1 by Adrian Fischer; Wilfried Weber; Bettina Warscheid; Gerald Radziwill (89-100).
Scaffold proteins are hubs for the coordination of intracellular signaling networks. The scaffold protein CNK1 promotes several signal transduction pathway. Here we demonstrate that sterile motif alpha (SAM) domain-dependent oligomerization of CNK1 stimulates CNK1-mediated signaling in growth factor-stimulated cells. We identified Ser22 located within the SAM domain as AKT-dependent phosphorylation site triggering CNK1 oligomerization. Oligomeric CNK1 increased the affinity for active AKT indicating a positive AKT feedback mechanism. A CNK1 mutant lacking the SAM domain and the phosphorylation-defective mutant CNK1S22A antagonizes oligomerization and prevents CNK1-driven cell proliferation and matrix metalloproteinase 14 promoter activation. The phosphomimetic mutant CNK1S22D constitutively oligomerizes and stimulates CNK1 downstream signaling. Searching the COSMIC database revealed Ser22 as putative target for oncogenic activation of CNK1. Like the phosphomimetic mutant CNK1S22D, the oncogenic mutant CNK1S22F forms clusters in serum-starved cells comparable to clusters of CNK1 in growth factor-stimulated cells. CNK1 clusters induced by activating Ser22 mutants correlate with enhanced cell invasion and binding to and activation of ADP ribosylation factor 1 associated with tumor formation. Mutational analysis indicate that EGF-triggered phosphorylation of Thr8 within the SAM domain prevents AKT binding and antagonizes CNK1-mediated AKT signaling. Our findings reveal SAM domain-dependent oligomerization by AKT as switch for CNK1 activation.Display Omitted
Keywords: Signaling; Scaffold protein; Oligomerization; Oncogene;

Metformin impairs systemic bile acid homeostasis through regulating SIRT1 protein levels by Qi Chen; Xiaoying Yang; Huabing Zhang; Xingxing Kong; Lu Yao; Xiaona Cui; Yongkang Zou; Fude Fang; Jichun Yang; Yongsheng Chang (101-112).
Metformin is widely used to treat hyperglycemia. However, metformin treatment may induce intrahepatic cholestasis and liver injury in a few patients with type II diabetes through an unknown mechanism. Here we show that metformin decreases SIRT1 protein levels in primary hepatocytes and liver. Both metformin-treated wild-type C57 mice and hepatic SIRT1-mutant mice had increased hepatic and serum bile acid levels. However, metformin failed to change systemic bile acid levels in hepatic SIRT1-mutant mice. Molecular mechanism study indicates that SIRT1 directly interacts with and deacetylates Foxa2 to inhibit its transcriptional activity on expression of genes involved in bile acids synthesis and transport. Hepatic SIRT1 mutation elevates Foxa2 acetylation levels, which promotes Foxa2 binding to and activating genes involved in bile acids metabolism, impairing hepatic and systemic bile acid homeostasis. Our data clearly suggest that hepatic SIRT1 mediates metformin effects on systemic bile acid metabolism and modulation of SIRT1 activity in liver may be an attractive approach for treatment of bile acid-related diseases such as cholestasis.
Keywords: Bile acid metabolism; Deacetylate; Foxa2; Metformin; SIRT1;

RIP3 antagonizes a TSC2-mediated pro-survival pathway in glioblastoma cell death by Gregory Fettweis; Emmanuel Di Valentin; Laurent L'homme; Cédric Lassence; Franck Dequiedt; Marianne Fillet; Isabelle Coupienne; Jacques Piette (113-124).
Glioblastomas are the deadliest type of brain cancer and are frequently associated with poor prognosis and a high degree of recurrence despite removal by surgical resection and treatment by chemo- and radio-therapy. Photodynamic therapy (PDT) is a treatment well known to induce mainly necrotic and apoptotic cell death in solid tumors. 5-Aminolevulinic acid (5-ALA)-based PDT was recently shown to sensitize human glioblastoma cells (LN-18) to a RIP3 (Receptor Interacting Protein 3)-dependent cell death which is counter-acted by activation of autophagy. These promising results led us to investigate the pathways involved in cell death and survival mechanisms occurring in glioblastoma following PDT. In the present study, we describe a new TSC2 (Tuberous Sclerosis 2)-dependent survival pathway implicating MK2 (MAPKAPK2) kinase and 14-3-3 proteins which conducts to the activation of a pro-survival autophagy. Moreover, we characterized a new RIP3/TSC2 complex where RIP3 is suggested to promote cell death by targeting TSC2-dependent survival pathway. These results highlight (i) a new role of TSC2 to protect glioblastoma against PDT-induced cell death and (ii) TSC2 and 14-3-3 as new RIP3 partners.
Keywords: Photodynamic therapy; Glioblastoma; Autophagy regulation; Programmed necrosis; MAP kinases;

Protein trafficking at the crossroads to mitochondria by Michal Wasilewski; Katarzyna Chojnacka; Agnieszka Chacinska (125-137).
Mitochondria are central power stations in the cell, which additionally serve as metabolic hubs for a plethora of anabolic and catabolic processes. The sustained function of mitochondria requires the precisely controlled biogenesis and expression coordination of proteins that originate from the nuclear and mitochondrial genomes. Accuracy of targeting, transport and assembly of mitochondrial proteins is also needed to avoid deleterious effects on protein homeostasis in the cell. Checkpoints of mitochondrial protein transport can serve as signals that provide information about the functional status of the organelles. In this review, we summarize recent advances in our understanding of mitochondrial protein transport and discuss examples that involve communication with the nucleus and cytosol.Display Omitted
Keywords: Mitochondrial protein biogenesis; Protein transport; Retrograde signaling; UPRmt; UPRam;

Consequences of acute oxidative stress in Leishmania amazonensis: From telomere shortening to the selection of the fittest parasites by Marcelo Santos da Silva; Marcela Segatto; Raphael Souza Pavani; Fernanda Gutierrez-Rodrigues; Vanderson da Silva Bispo; Marisa Helena Gennari de Medeiros; Rodrigo Tocantins Calado; Maria Carolina Elias; Maria Isabel Nogueira Cano (138-150).
Leishmaniasis is a spectrum of diseases caused by parasites of the genus Leishmania that affects millions of people around the world. During infection, the parasites use different strategies to survive the host's defenses, including overcoming exposure to reactive oxidant species (ROS), responsible for causing damage to lipids, proteins and DNA. This damage especially affects telomeres, which frequently results in genome instability, senescence and cell death. Telomeres are the physical ends of the chromosomes composed of repetitive DNA coupled with proteins, whose function is to protect the chromosomes termini and avoid end-fusion and nucleolytic degradation. In this work, we induced acute oxidative stress in promastigote forms of Leishmania amazonensis by treating parasites with 2 mM hydrogen peroxide (H2O2) for 1 h, which was able to increase intracellular ROS levels. In addition, oxidative stress induced DNA damage, as confirmed by 8-oxodGuo quantification and TUNEL assays and the dissociation of LaRPA-1 from the 3′ G-overhang, leading to telomere shortening. Moreover, LaRPA-1 was observed to interact with newly formed C-rich single-stranded telomeric DNA, probably as a consequence of the DNA damage response. Nonetheless, acute oxidative stress caused the death of some of the L. amazonensis population and induced cell cycle arrest at the G2/M phase in survivor parasites, which were able to continue proliferating and replicating DNA and became more resistant to oxidative stress. Taken together, these results suggest that adaptation occurs through the selection of the fittest parasites in terms of repairing oxidative DNA damage at telomeres and maintaining genome stability in a stressful environment.Display Omitted
Keywords: Leishmania amazonensis; Oxidative stress; Telomere shortening; LaRPA-1; DNA repair;

Lucidone Promotes the Cutaneous Wound Healing Process via Activation of the PI3K/AKT, Wnt/β-catenin and NF-κB Signaling Pathways by Hsin-Ling Yang; Yu-Cheng Tsai; Mallikarjuna Korivi; Chia-Ting Chang; You-Cheng Hseu (151-168).
Lucidone, which comprises a naturally occurring cyclopentenedione, has been investigated for its in vitro and in vivo wound healing properties, and the underlying molecular signaling cascades in the wound healing mechanism have been elucidated. We demonstrated the cell −/dose-specific responses of lucidone (0.5–8 μM) on proliferation and migration/invasion of keratinocyte HaCaT and fibroblast Hs68 cells. In keratinocytes, lucidone-induced nuclear translocation of β-catenin was accompanied by increased transcriptional target genes, including c-Myc and cyclin-D1, through GSK3β-dependent pathway. Correspondingly, lucidone promoted the cell-cycle by increasing PCNA/CDK4 and decreasing p21/p27 expressions. Lucidone induced EMT through the downregulation of epithelial (E-cadherin/occludin) and upregulation of mesenchymal (vimentin/Twist/Snail) marker proteins. Activated MMP-9/− 2 and uPA/uPAR as well as suppressed TIMP-1/− 2 and PAI-1 expressions by lucidone may promote the migration/invasion of keratinocytes. Notably, lucidone activated NF-κB signaling via IKK-mediated-IκB degradation, and its inhibition abolished MMP-9 activation and keratinocyte migration. Inhibition of PI3K/AKT signaling impaired the lucidone-induced proliferation/migration with corresponding suppression of β-catenin/c-Myc/cyclin-D1 and NF-κB/MMP-9 expressions. Results indicate that lucidone-induced PI3K/AKT signaling anchored the β-catenin/NF-κB-mediated healing mechanism. β-catenin knockdown substantially diminished lucidone-induced keratinocyte migration. Furthermore, lucidone increased endothelial cell proliferation/migration and triggered angiogenesis (MMP-9/uPA/ICAM-1). In macrophages, lucidone-activated NF-κB-mediated inflammation (COX-2/iNOS/NO) and VEGF, which may contribute to the growth of keratinocytes/fibroblasts and endothelial cells. Punched wounds on mice were rapidly healed with the topical application of lucidone (5 mM) compared with control ointment-treated mice. Taken together, lucidone accelerates wound healing through the cooperation of keratinocyte/fibroblast/endothelial cell growth and migration and macrophage inflammation via PI3K/AKT, Wnt/β-catenin and NF-κB signaling cascade activation.
Keywords: Wound healing; lucidone; Wnt/β-catenin; NF-κB; E-cadherin; MMP;

Sirtuin 5 protects mitochondria from fragmentation and degradation during starvation by Hala Guedouari; Tanya Daigle; Luca Scorrano; Etienne Hebert-Chatelain (169-176).
During starvation, intra-mitochondrial sirtuins, NAD+ sensitive deacylating enzymes that modulate metabolic homeostasis and survival, directly adjust mitochondrial function to nutrient availability; concomitantly, mitochondria elongate to escape autophagic degradation. However, whether sirtuins also impinge on mitochondrial dynamics is still uncharacterized. Here we show that the mitochondrial Sirtuin 5 (Sirt5) is essential for starvation induced mitochondrial elongation. Deletion of Sirt5 in mouse embryonic fibroblasts increased levels of mitochondrial dynamics of 51 kDa protein and mitochondrial fission protein 1, leading to mitochondrial accumulation of the pro-fission dynamin related protein 1 and to mitochondrial fragmentation. During starvation, Sirt5 deletion blunted mitochondrial elongation, resulting in increased mitophagy. Our results indicate that starvation induced mitochondrial elongation and evasion from autophagic degradation requires the energy sensor Sirt5.
Keywords: Sirtuin 5; Mitochondrial fragmentation; Mitochondrial degradation;

ATM-ROS-iNOS axis regulates nitric oxide mediated cellular senescence by Meisam Bagheri; Raji R. Nair; Krishna Kumar Singh; Deepak Kumar Saini (177-190).
Cellular senescence is an outcome of the accumulation of DNA damage which induces the growth arrest in cells. Physiologically, it is presumed to be mediated by accumulation of reactive oxygen species (ROS). Here, we show that another free radical, nitric oxide (NO) produced during inflammation or present as an environmental pollutant can also induce cellular senescence. In primary cells and various immortalized cell lines, exposure to chronic NO, through external addition or internally generated by iNOS expression, leads to the activation of DNA damage response and causes cellular senescence. The phenotype generated by NO includes robust growth arrest, increase in the levels of the DNA damage foci, ROS, SAβ-gal staining, and inflammatory cytokines like IL-6 and IL-8, all hallmarks of cellular senescence similar to replicative senescence. Mechanistically, inhibitor and knockdown analysis revealed that NO mediates senescence through ATM kinase activation and the viability of cells is dependent on both ROS and ATM kinase involving the ATM-ROS-iNOS axis. Overall, we demonstrate that nitric oxide mediates cellular senescence through a novel free radical dependent genotoxic stress pathway.Display Omitted
Keywords: Cellular senescence; Free radicals; ATM kinase; iNOS; Nitric oxide; DNA damage response;

TRF2 recruits ORC through TRFH domain dimerization by Mitsunori Higa; Tatsunori Kushiyama; Seiichiro Kurashige; Daisuke Kohmon; Kouki Enokitani; Satoko Iwahori; Nozomi Sugimoto; Kazumasa Yoshida; Masatoshi Fujita (191-201).
Telomeres are specialized chromatin structures that prevent the degradation and instability of the ends of linear chromosomes. While telomerase maintains long stretches of the telomeric repeat, the majority of telomeric DNA is duplicated by conventional DNA replication. A fundamental step in eukaryotic DNA replication involves chromatin binding of the origin recognition complex (ORC). In human cells, telomeric repeat binding factor 2 (TRF2) is thought to play a role in the recruitment of ORC onto telomeres. To better understand the mechanism of TRF2-mediated ORC recruitment, we utilized a lacO-LacI protein tethering system in U2OS cells and found that ectopically targeted TRF2, but not TRF1, can recruit ORC onto the lacO array. We further found that the TRF homology (TRFH) dimerization domain of TRF2, but not its mutant defective in dimerization, is sufficient for ORC and minichromosome maintenance (MCM) recruitment. Mutations impairing the dimerization also compromised ORC recruitment by full-length TRF2. Similar results were obtained using immunoprecipitation and GST pull-down assays. Together, these results suggest that dimerized TRF2 recruits ORC and stimulates pre-replication complex (pre-RC) formation at telomeres through the TRFH domain.Display Omitted
Keywords: DNA replication; Telomere; MCM; ORC; TRF2; Pre-replication complex;

The Bacillus subtilis TatAdCd system exhibits an extreme level of substrate selectivity by Kelly M. Frain; Alexander S. Jones; Ronald Schoner; Kelly L. Walker; Colin Robinson (202-208).
The Tat system preferentially transports correctly folded proteins across the bacterial membrane although little is known of the proofreading mechanism. Most research has focused on TatABC systems from Gram-negative bacteria, especially Escherichia coli, and much less is known of the TatAC-type systems from Gram-positive organisms. We have previously shown that the Bacillus subtilis TatAdCd system is functional in an E. coli tat null background and able to transport TorA-GFP and native TorA (TMAO reductase); here, we examined its ability to transport other proteins bearing a TorA signal sequence. We show that whereas E. coli TatABC transports a wide range of biotherapeutics including human growth hormone, interferon α2b, a VH domain protein and 2 different scFvs, TatAdCd transports the scFvs but completely rejects the other proteins. The system also rejects two native E. coli substrates, NrfC and FhuD. Moreover, we have shown that TatABC will transport a wide range of folded scFv variants with the surface altered to incorporate multiple salt bridges, charged residues (5 glutamate, lysine or arginine), or hydrophobic residues (up to 6 leucines). In contrast, TatAdCd completely rejects many of these variants including those with 5 or 6 added Leu residues. The combined data show that the TatABC and TatAdCd systems have very different substrate selectivities, with the TatAdCd system displaying an extreme level of selectivity when compared to the E. coli system. The data also provide a preliminary suggestion that TatAdCd may not tolerate substrates that contain surface domains with a level of hydrophobicity above a certain threshold.
Keywords: Tat; Twin-arginine; Signal peptide; TatAdCd; Bacillus subtilis;

Biomarkers have the potential to move personalized medicine a step forward for the patients' benefit. Blood is a non-invasive source of biomarkers. The cell-free compartment of the blood has traditionally been used to search for biomarkers and currently used cardiovascular biomarkers are mainly measured in plasma or serum fractions. While most of these biomarkers are proteins, very few belong to the RNA family due to methodological constraints inherent to the assessment of RNAs. Over the past decade, technological developments have overcome these constraints and have allowed the assessment of RNAs in blood cells. Thus, the search for novel cardiovascular biomarkers among RNAs expressed in blood cells has emerged. This review highlights the potential of the transcriptome of blood cells to be used as a reservoir of novel cardiovascular biomarkers. First, the benefit of using biomarkers for personalized medicine is introduced. The focus is on the RNA family of biomarkers, both coding and non-coding. The value of systems-based approaches for biomarker discovery is discussed. Then, the current knowledge of the use of blood cells' transcriptome for biomarker discovery is reviewed. Recent technological developments that have facilitated the study of the transcriptome of blood cells are presented. Further axes of developments are proposed to bring RNA-based biomarkers to clinical application. Lastly, some directions for future work are presented.
Keywords: Biomarker; Cardiovascular disease; RNA; Transcriptomics; Noncoding RNA; Personalized medicine;

Tetraspanin 3: A central endocytic membrane component regulating the expression of ADAM10, presenilin and the amyloid precursor protein by Lisa Seipold; Markus Damme; Johannes Prox; Björn Rabe; Petr Kasparek; Radislav Sedlacek; Hermann Altmeppen; Michael Willem; Barry Boland; Markus Glatzel; Paul Saftig (217-230).
Despite existing knowledge about the role of the A Disintegrin and Metalloproteinase 10 (ADAM10) as the α-secretase involved in the non-amyloidogenic processing of the amyloid precursor protein (APP) and Notch signalling we have only limited information about its regulation. In this study, we have identified ADAM10 interactors using a split ubiquitin yeast two hybrid approach. Tetraspanin 3 (Tspan3), which is highly expressed in the murine brain and elevated in brains of Alzheimer´s disease (AD) patients, was identified and confirmed to bind ADAM10 by co-immunoprecipitation experiments in mammalian cells in complex with APP and the γ-secretase protease presenilin. Tspan3 expression increased the cell surface levels of its interacting partners and was mainly localized in early and late endosomes. In contrast to the previously described ADAM10-binding tetraspanins, Tspan3 did not affect the endoplasmic reticulum to plasma membrane transport of ADAM10. Heterologous Tspan3 expression significantly increased the appearance of carboxy-terminal cleavage products of ADAM10 and APP, whereas N-cadherin ectodomain shedding appeared unaffected. Inhibiting the endocytosis of Tspan3 by mutating a critical cytoplasmic tyrosine-based internalization motif led to increased surface expression of APP and ADAM10. After its downregulation in neuroblastoma cells and in brains of Tspan3-deficient mice, ADAM10 and APP levels appeared unaltered possibly due to a compensatory increase in the expression of Tspans 5 and 7, respectively. In conclusion, our data suggest that Tspan3 acts in concert with other tetraspanins as a stabilizing factor of active ADAM10, APP and the γ-secretase complex at the plasma membrane and within the endocytic pathway.Display Omitted
Keywords: ADAM10; Tetraspanin; APP; Presenilin;

Extreme calorie restriction in yeast retentostats induces uniform non-quiescent growth arrest by Markus M.M. Bisschops; Marijke A.H. Luttik; Anne Doerr; Peter J.T. Verheijen; Frank Bruggeman; Jack T. Pronk; Pascale Daran-Lapujade (231-242).
Non-dividing Saccharomyces cerevisiae cultures are highly relevant for fundamental and applied studies. However, cultivation conditions in which non-dividing cells retain substantial metabolic activity are lacking. Unlike stationary-phase (SP) batch cultures, the current experimental paradigm for non-dividing yeast cultures, cultivation under extreme calorie restriction (ECR) in retentostat enables non-dividing yeast cells to retain substantial metabolic activity and to prevent rapid cellular deterioration. Distribution of F-actin structures and single-cell copy numbers of specific transcripts revealed that cultivation under ECR yields highly homogeneous cultures, in contrast to SP cultures that differentiate into quiescent and non-quiescent subpopulations. Combined with previous physiological studies, these results indicate that yeast cells subjected to ECR survive in an extended G1 phase. This study demonstrates that yeast cells exposed to ECR differ from carbon-starved cells and offer a promising experimental model for studying non-dividing, metabolically active, and robust eukaryotic cells.
Keywords: Extreme calorie restriction; Retentostat; Non-dividing; mRNA FISH; Actin structure; Heterogeneity;