BBA - Molecular Cell Research (v.1833, #3)

Protein kinase CK2 is a pleiotropic enzyme, which is implicated in the regulation of numerous biological processes. It seems to regulate the various functions by binding to other proteins and by phosphorylation of many different substrates. Here, we identified the activating transcription factor 4 (ATF4), an essential component of the ER stress signaling, as a new binding partner and a new substrate of CK2 in vitro and in vivo. Bifluorescence complementation analysis (BiFC) revealed that CK2α and ATF4 associate in the nucleus. By using mutants of ATF4 we identified serine 215 as the main CK2 phosphorylation site. The ATF4 S215A mutant turned out to be more stable than the wild-type form. We further noticed that an inhibition of CK2 caused an increased transcription of the ATF4 gene. Analyses of the transcription factor activity revealed an impaired activity of the CK2 phosphorylation mutant of ATF4. Thus, we show that (i) ATF4 is a binding partner of CK2α (ii) ATF4 is a substrate of CK2, (iii) the phosphorylation of ATF4 by CK2 influences the stability of ATF4, (iv) the transcription of ATF4 is regulated by CK2 and (v) the transcription factor activity of ATF4 is regulated by the CK2 phosphorylation of ATF4. Thus, CK2 plays an essential role in the regulation of the ER-stress induced signaling pathway.Display Omitted► ATF4 is a new binding partner of protein kinase CK2. ► CK2 phosphorylates ATF4 at serine 215. ► Phosphorylation of ATF4 by CK2 reduces the stability of ATF4. ► Inhibition of CK2 kinase activity results in an elevated transcription of ATF4. ► CK2 phosphorylation of ATF4 stimulates its transcription factor activity.
Keywords: Phosphorylation; Protein kinase; Transcription; Transcription factor; Protein stability;

A role for sequestosome 1/p62 in mitochondrial dynamics, import and genome integrity by M. Lamar Seibenhener; Yifeng Du; Maria-Theresa Diaz-Meco; Jorge Moscat; Michael C. Wooten; Marie W. Wooten (452-459).
As a signaling scaffold, p62/sequestosome (p62/SQSTM1) plays important roles in cell signaling and degradation of misfolded proteins. While localization of p62 to mitochondria has been reported, a description of its function once there, remains unclear. Here, we report that p62 is localized to mitochondria in non-stressed situations and demonstrate that deficiency in p62 exacerbates defects in mitochondrial membrane potential and energetics leading to mitochondrial dysfunction. We report on the relationship between mitochondrial protein import and p62. In a p62 null background, mitochondrial import of the mitochondrial transcription factor TFAM is disrupted. When p62 is returned, mitochondrial function is restored to more normal levels. We identify for the first time that p62 localization plays a role in regulating mitochondrial morphology, genome integrity and mitochondrial import of a key transcription factor. We present evidence that these responses to the presence of p62 extend beyond the protein's immediate influence on membrane potential.► p62 is localized to the mitochondria where it plays a physiological role. ► p62 affects both mitochondrial morphology and function. ► p62 is involved in maintenance of mitochondrial genome stability. ► p62 regulates the import of TFAM to mitochondria. ► mtDNA biogenesis is affected by p62 beyond regulation provided by Δψm.
Keywords: Mitochondrial integrity; Oxidative stress; p62; TFAM; Mitochondrial import;

Induction of a trophoblast-like phenotype by hydralazine in the p19 embryonic carcinoma cell line by Cliona M. O'Driscoll; Jonathan B. Coulter; Joseph P. Bressler (460-467).
Chemicals that affect cellular differentiation through epigenetic mechanisms have potential utility in treating a wide range of diseases. Hydralazine decreases DNA methylation in some cell types but its effect on differentiation has not been well explored. After five days of exposure to hydralazine, P19 embryocarcinoma cells displayed a giant cell morphology and were binucleate, indicative of a trophoblast-like morphology. Other trophoblast-like properties included the intermediary filament Troma-1/cytokeratin 8 and the transcription factor Tead4. A decrease in CpG methylation at three sites in the TEAD4 promoter and the B1 repeated sequence was observed. Knocking down expression of Tead4 with siRNA blocked the increase in Troma-1/cytokeratin 8 and over expression of Tead4 induced the expression of Troma-1/cytokeratin 8. Cells treated for 5 days with hydralazine were no longer capable of undergoing retinoic acid-mediated neuronal differentiation. An irreversible loss of the pluripotent transcription factor Oct-4 was observed following hydralazine exposure. In summary, hydralazine induces P19 cells to assume a trophoblast-like phenotype by upregulating Tead4 expression through a mechanism involving DNA demethylation.► Hydralazine decreases DNA methylation in P19 cells. ► Hydralazine induces P19 cells to express trophoblast-like properties. ► Tead 4 is required to express trophoblast-like properties.
Keywords: Hydralazine; Trophoblast; Tead4; P19 cell; DNA methylation;

A divalent interaction between HPS1 and HPS4 is required for the formation of the biogenesis of lysosome-related organelle complex-3 (BLOC-3) by Carmelo Carmona-Rivera; Dimitre R. Simeonov; Nicholas D. Cardillo; William A. Gahl; Carmen L. Cadilla (468-478).
Hermansky–Pudlak syndrome (HPS) is a group of rare autosomal recessive disorders characterized by oculocutaneous albinism, a bleeding tendency, and sporadic pulmonary fibrosis, granulomatous colitis or infections. Nine HPS-causing genes have been identified in humans. HPS-1 is the most severe subtype with a prevalence of ~ 1/1800 in northwest Puerto Rico due to a founder mutation in the HPS1 gene. Mutations in HPS genes affect the biogenesis of lysosome-related organelles such as melanosomes in melanocytes and platelet dense granules. Two of these genes (HPS1 and HPS4) encode the HPS1 and HPS4 proteins, which assemble to form a complex known as Biogenesis of Lysosome-related Organelle Complex 3 (BLOC-3). We report the identification of the interacting regions in HPS1 and HPS4 required for the formation of this complex. Two regions in HPS1, spanning amino acids 1–249 and 506–700 are required for binding to HPS4; the middle portion of HPS1 (residues 250–505) is not required for this interaction. Further interaction studies showed that the N-termini of HPS1 and HPS4 interact with each other and that a discrete region of HPS4 (residues 340–528) interacts with both the N- and C-termini of the HPS1 protein. Several missense mutations found in HPS-1 patients did not affect interaction with HPS4, but some mutations involving regions interacting with HPS4 caused instability of HPS1. These observations extend our understanding of BLOC-3 assembly and represent an important first step in the identification of domains responsible for the biogenesis of lysosome-related organelles.► An HPS1 isoform encoded by a transcript lacking exon 9 interacts with HPS4 ► The central region of HPS4 protein interacts dually, with either the N- or C-terminus of HPS1 ► Interacting regions of HPS1 and HPS4 exist as soluble and membrane-associated forms ► Missense mutations within interacting regions of HPS1 alter its stability ► Membrane association of HPS1 and HPS4 depends upon specific regions of these proteins
Keywords: HPS; Hermansky–Pudlak syndrome; Oculocutaneous albinism; Bleeding; BLOC-3; Lysosome-related organelle;

Poly(ADP-ribose) polymerase inhibition prevents reactive oxygen species induced inhibition of aldehyde dehydrogenase2 activity by Shu-jian Wei; Jun-hui Xing; Bai-lu Wang; Li Xue; Jia-li Wang; Rui Li; Wei-dong Qin; Juan Wang; Xu-ping Wang; Ming-xiang Zhang; Yu-guo Chen (479-486).
Lipid peroxidation plays a critical role in cardiovascular diseases. Aldehydes are the major end products of lipid peroxidation and can be metabolized into less reactive chemical species by aldehyde dehydrogenase 2 (ALDH2). However, ALDH2 dehydrogenase activity can be affected by many factors including reactive oxygen species. To elucidate how reactive oxygen species inhibit ALDH2 dehydrogenase activity, we stimulated human aortic endothelial cells (HAECs) with oxidized low-density lipoproteins (ox-LDL) and performed a myocardial ischemia–reperfusion model. Ox-LDL treatment and ischemia–reperfusion injury inhibited ALDH2 dehydrogenase activity. Poly(ADP-ribose) polymerase (PARP) was activated by ox-LDL stimulation and ischemia–reperfusion injury and PARP inhibition partly restored ALDH2 dehydrogenase activity in ox-LDL treated HAECs and ischemia–reperfusion rat hearts. SIRT3 was upregulated by ox-LDL stimulation and ischemia–reperfusion injury and downregulated by PARP inhibition. Using siRNA to knock down SIRT3, we demonstrated that SIRT3 mediated deacetylation decreased ALDH2 dehydrogenase activity and PARP inhibition partly restored ALDH2 dehydrogenase activity through preventing SIRT3 expression and subsequently preserving ALDH2 acetylation.► Oxidized low density lipoprotein (ox-LDL) was proven to inhibit aldehyde dehydrogenase2 (ALDH2) activity in a time- and concentration-dependent manner. ► We report that poly(ADP-ribose) polymerase (PARP) is involved in the inhibition of ALDH2 activity by reactive oxygen species. ► We found the role of PARP on ALDH2 dehydrogenase activity might be mediated by SIRT3 expression.
Keywords: poly(ADP-ribose) polymerase; ROS; Aldehyde dehydrogenase 2; SIRT3;

Protein kinase C epsilon (PKCε) contributes to multiple signaling pathways affecting human disease. The function of PKCε requires it to undergo changes in subcellular distribution in response to signaling events. While the mechanisms underlying this translocation are incompletely understood, it involves the receptor for activated C kinase protein (RACK2/β′-COP). This receptor also functions as a vesicle coat protein in the secretory pathway where it is regulated by the small GTP-binding protein ADP-ribosylation factor, ARF1. We inhibited ARF1 activation to test the requirement for RACK2/β′-COP in PKCε localization in NIH3T3 fibroblasts. We found that steady-state localization of PKCε at the Golgi complex requires ARF1-regulated RACK2/β′-COP function. By contrast, we did not observe any defects in phorbol ester-induced translocation when ARF1 was inhibited. We also found that PKCε bound to isolated membranes through two distinct mechanisms. One mechanism was dependent upon RACK2/β′-COP while a second was RACK2/β′-COP-independent and stimulated by phorbol esters. Finally, we show that RACK2/β′-COP affects the subcellular distribution of a constitutively active form of PKCε, in a manner similar to what we observed for wild-type PKCε. Together, our data support a role for RACK2/β′-COP in the steady-state localization of PKCε at the Golgi apparatus, which may be independent of its role during PKCε translocation to the cell surface.► Localization of PKCε and RACK2/β′-COP at the Golgi apparatus required ARF1 activity. ► Phorbol-ester-induced translocation of PKCε was independent of ARF1 activation. ► PKCε bound to membranes using RACK2/β′-COP-dependent and -independent mechanisms.
Keywords: Protein kinase C; Coatomer; ADP-ribosylation factor-1 (ARF1); Receptor for activated C kinase-2 (RACK2); Golgi apparatus; Phorbol ester;

Mitochondrial metabolite carriers are hydrophobic proteins which catalyze the flux of several charged or hydrophilic substrates across the inner membrane of mitochondria. These proteins, like most mitochondrial proteins, are nuclear encoded and after their synthesis in the cytosol are transported into the inner mitochondrial membrane.Most metabolite carriers, differently from other nuclear encoded mitochondrial proteins, are synthesized without a cleavable presequence and contain several, poorly characterized, internal targeting signals. However, an interesting aspect is the presence of a positively charged N-terminal presequence in a limited number of mitochondrial metabolite carriers.Over the last few years the molecular mechanisms of import of metabolite carrier proteins into mitochondria have been thoroughly investigated. This review summarizes the present knowledge and discusses recent advances on the import and sorting of mitochondrial metabolite carriers.► Summarizes the present knowledge on mitochondrial carrier biogenesis ► Reports the most recent achievements regarding the biogenesis of mitochondrial metabolite carriers ► Discusses recent advances on the import and sorting of mitochondrial metabolite carrier
Keywords: Mitochondria biogenesis; Protein import; Mitochondrial carrier; Precursor protein; TOM complex; TIM complex;

Maturation of autophagosomes and endosomes: A key role for Rab7 by Juha M.T. Hyttinen; Minna Niittykoski; Antero Salminen; Kai Kaarniranta (503-510).
Macroautophagy is an important route in cellular maintenance, in the breakdown and reuse of intracellular materials. It is closely related to endocytosis, the means by which the cell can absorb extracellular material, as both macroautophagy and endocytosis have converging steps and common participating molecules. The point where autophagosomes and endosomes fuse with lysosomes to permit for the final degradation of their contents is important. One of the most substantial molecules in the maturation of autophagosomes/endosomes is Rab7, a member of small GTPases. Rab7 designates the maturation of endosomes and also autophagosomes, directing the trafficking of cargos along microtubules, and finally, participating in the fusion step with lysosomes. Rab7 is an effective multifunctional regulator of autophagy and endocytosis. Since many aggregation-based diseases, e.g. age-related macular degeneration of the eye (AMD) and Alzheimer's disease are due of malfunctioning in the autophagic process, the management of Rab7 activity might hold potential as a therapeutic target against these diseases.► Autophagy and endosomal degradation processes do converge and also contain common molecules ► Rab7 protein acts in a critical point of maturation process of both autophagosomes and endosomes ► Rab7 takes part in the transportation of autophagosomes and endosomes towards lysosomal degradation ► The action of Rab7 in autophagy and endocytosis is highly regulated ► The control of Rab7 may provide a means to control diseases in which autophagy or endocytosis is disturbed.
Keywords: Rab7; Autophagy; Endocytosis; Trafficking; Maturation;

Cardiac glycoside ouabain induces activation of ATF-1 and StAR expression by interacting with the α4 isoform of the sodium pump in Sertoli cells by Raimund Dietze; Lutz Konrad; Mazen Shihan; Ulrike Kirch; Georgios Scheiner-Bobis (511-519).
Sertoli cells express α1 and α4 isoforms of the catalytic subunit of Na+,K+-ATPase (sodium pump). Our recent findings demonstrated that interactions of the α4 isoform with cardiotonic steroids (CTS) like ouabain induce signaling cascades that resemble the so-called non-classical testosterone pathway characterized by activation of the c-Src/c-Raf/Erk1/2/CREB signaling cascade. Here we investigate a possible physiological significance of the activated cascade. The results obtained in the current investigation show that the ouabain-induced signaling cascade also leads to the activation of the CREB-related activating transcription factor 1 (ATF-1) in the Sertoli cell line 93RS2 in a concentration- and time-dependent manner, as demonstrated by detection of ATF-1 phosphorylated on Ser63 in western blots. The ouabain-activated ATF-1 protein was found to localize to the cell nuclei. The sodium pump α4 isoform mediates this activation, as it is ablated when cells are incubated with siRNA to the α4 isoform. Ouabain also leads to increased expression of steroidogenic acute regulator (StAR) protein, which has been shown to be a downstream consequence of CREB/ATF-1 activation. Taking into consideration that CTS are most likely produced endogenously, the demonstrated induction of StAR expression by ouabain establishes a link between CTS, the α4 isoform of the sodium pump, and steroidogenesis crucial for male fertility and reproduction.► The cardiotonic steroid (CTS) ouabain induces in Sertoli cells a signaling cascade. ► This happens by its interaction with the α4 isoform of Na+,K+-ATPase. ► It induces phosphorylation of the activating transforming factor 1 (ATF-1). ► As a consequence, activated ATF-1 stimulates the expression of StAR protein. ► Interactions between endogenous CTS and α4 might be crucial for male fertility.
Keywords: Ouabain; Sertoli cell; ATF-1; StAR; Alpha4 isoform; Sodium pump;

Protons stabilize the closed conformation of gain-of-function mutants of the TRPV1 channel by Stepana Boukalova; Jan Teisinger; Viktorie Vlachova (520-528).
The vanilloid transient receptor potential channel TRPV1 is a molecular integrator of noxious stimuli, including capsaicin, heat and protons. Despite clear similarities between the overall architecture of TRPV1 and voltage-dependent potassium (Kv) channels, the extent of conservation in the molecular logic for gating is unknown. In Kv channels, a small contact surface between S1 and the pore-helix is required for channel functioning. To explore the function of S1 in TRPV1, we used tryptophan-scanning mutagenesis and characterized the responses to capsaicin and protons. Wild-type-like currents were generated in 9 out of 17 mutants; three mutants (M445W, A452W, R455W) were non-functional. The conservative mutation R455K in the extracellular extent of S1 slowed down capsaicin-induced activation and prevented normal channel closure. This mutant was neither activated nor potentiated by protons, on the contrary, protons promoted a rapid deactivation of its currents. Similar phenotypes were found in two other gain-of-function mutants and also in the pore-helix mutant T633A, known to uncouple proton activation. We propose that the S1 domain contains a functionally important region that may be specifically involved in TRPV1 channel gating, and thus be important for the energetic coupling between S1–S4 sensor activation and gate opening. Analogous to Kv channels, the S1-pore interface might serve to stabilize conformations associated with TRPV1 channel gating.► Low pH stabilizes the closed conformations of specific activating mutations of TRPV1 ► The S1 region plays an important role in TRPV1 channel gating ► TRPV1 and potassium channels might share a similar gating mechanism involving S1 ► The S1-pore interface stabilizes conformations associated with TRPV1 gating
Keywords: Vanilloid receptor subtype 1; Transient receptor potential; Gating; Tryptophan-scanning; Mutagenesis;

Identifying protein partners of CLN8, an ER-resident protein involved in neuronal ceroid lipofuscinosis by Rosa Passantino; Caterina Cascio; Irene Deidda; Giacoma Galizzi; Domenica Russo; Gianpiero Spedale; Patrizia Guarneri (529-540).
Neuronal ceroid lipofuscinoses (NCLs) are a genetically heterogeneous group of neurodegenerative diseases characterized by cognitive and motor decline, epilepsy, visual loss and by lysosomal autofluorescent inclusions. Two distinct clinical phenotypes, the progressive epilepsy with mental retardation (EPMR) and a late-infantile variant of NCLs (CLN8-vLINCL) are associated with mutations in the CLN8 gene that encodes a transmembrane protein predominantly located to the endoplasmic reticulum (ER). To gain insight into the function of CLN8 protein, we employed the split-ubiquitin membrane-based yeast two-hybrid (MYTH) system, which detects protein-protein interactions in a membrane environment, using the full-length human CLN8 as bait and a human brain cDNA library as prey. We identified several potential protein partners of CLN8 and especially referred to VAPA, c14orf1/hERG28, STX8, GATE16, BNIP3 and BNIP3L proteins that are associated with biologically relevant processes such as synthesis and transport of lipids, vesicular/membrane trafficking, autophagy/mitophagy and apoptosis. Interactions of CLN8 with VAPA and GATE16 were further validated by co-immunoprecipitation and co-localization assays in mammalian cells. Using a new C-terminal-oriented CLN8 antibody, CLN8-VAPA interaction was also confirmed by co-staining in close spatial proximity within different CNS tissues. The results of this study shed light on potential interactome networks of CLN8 and provide a powerful starting point for understanding protein function(s) and molecular aspects of diseases associated with CLN8 deficiency.► CLN8p–protein interactions identified by a split-ubiquitin MYTH library screen ► Putative partners identified: VAPA, c14orf1/hERG28, STX8, GATE16, BNIP3 and BNIP3L. ► Co-immunoprecipitation and co-localization assays support some interactions. ► These partners predict roles of CLN8 in four main biological processes. ► Lipid synthesis/transport, vesicular trafficking, autophagy/mitophagy, apoptosis
Keywords: vLINCL; EPMR; CLN8; Split-ubiquitin yeast two hybrid; Protein interaction;

Anti-mitochondrial therapy in human breast cancer multi-cellular spheroids by Edna Ayerim Mandujano-Tinoco; Juan Carlos Gallardo-Pérez; Alvaro Marín-Hernández; Rafael Moreno-Sánchez; Sara Rodríguez-Enríquez (541-551).
During multi-cellular tumor spheroid growth, oxygen and nutrient gradients develop inducing specific genetic and metabolic changes in the proliferative and quiescent cellular layers. An integral analysis of proteomics, metabolomics, kinetomics and fluxomics revealed that both proliferative- (PRL) and quiescent-enriched (QS) cellular layers of mature breast tumor MCF-7 multi-cellular spheroids maintained similar glycolytic rates (3–5 nmol/min/106 cells), correlating with similar GLUT1, GLUT3, PFK-1, and HKII contents, and HK and LDH activities. Enhanced glycolytic fluxes in both cell layer fractions also correlated with higher HIF-1α content, compared to MCF-7 monolayer cultures. On the contrary, the contents of the mitochondrial proteins GA-K, ND1, COXIV, PDH-E1α, 2-OGDH, SDH and F1-ATP synthase (20 times) and the oxidative phosphorylation (OxPhos) flux (2-times) were higher in PRL vs. QS. Enhanced mitochondrial metabolism in the PRL layers correlated with an increase in the oncogenes h-Ras and c-Myc, and transcription factors p32 and PGC-1α, which are involved in the OxPhos activation. On the other hand, the lower mitochondrial function in QS was associated with an increase in Beclin, LC3B, Bnip3 and LAMP protein levels, indicating active mitophagy and lysosome biosynthesis processes. Although a substantial increase in glycolysis was developed, OxPhos was the predominant ATP supplier in both QS and PRL layers. Therefore, targeted anti-mitochondrial therapy by using oligomycin (IC50  = 11 nM), Casiopeina II-gly (IC50  = 40 nM) or Mitoves (IC50  = 7 nM) was effective to arrest MCF-7 spheroid growth without apparent effect on normal epithelial breast tissue at similar doses; canonical anti-neoplastic drugs such as cisplatin and tamoxifen were significantly less potent.Display Omitted► Proliferative and quiescent breast cancer micro-regions depend on mitochondrial ATP. ► Anti-mitochondrial therapy at nanomolar doses arrests breast tumor spheroid growth. ► Glycolysis and chemotherapy inhibitors do not affect breast tumor spheroid growth. ► Anti-mitochondrial therapy does not modify energy metabolism of normal breast spheroids. ► Anti-mitochondrial drugs prevent, and revert, breast tumor spheroid growth.
Keywords: Breast cancer cell; Glycolysis; Multi-cellular tumor spheroids; Oxidative phosphorylation; Anti-mitochondrial therapy;

Role of somatomedin-B-like domains on ENPP1 inhibition of insulin signaling by Claudia Dimatteo; Antonella Marucci; Antonio Palazzo; Carmela Cisternino; René Massimiliano Marsano; Vincenzo Trischitta; Rosa Di Paola (552-558).
The exact mechanism by which ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) inhibits insulin signaling is not known. ENPP1 contains two somatomedin-B-like domains (i.e. SMB 1 and 2) involved in ENPP1 dimerization in animal cells. The aim of the present study was to investigate if these domains modulate ENPP1 inhibitory activity on insulin signaling in human insulin target cells (HepG2). ENPP1 (ENPP1–3′myc), ENPP1 deleted of SMB 1 (ENPP1-ΔI-3′myc) or of SMB 2 (ENPP1-ΔII-3′myc) domain were cloned in frame with myc tag in mammalian expression vector pRK5. Plasmids were transiently transfected in human liver HepG2 cells. ENPP1 inhibitory activity on insulin signaling, dimerization and protein–protein interaction with insulin receptor (IR), reported to mediate the modulation of ENPP1 inhibitory activity, were studied. As compared to untransfected cells, a progressive increase of ENPP1 inhibitory activity on insulin-induced IR β-subunit autophosphorylation and on Akt-S473 phosphorylation was observed in ENPP1–3′myc, ENPP1-ΔI-3′myc and ENPP1-ΔII-3′myc cells. Under non reducing conditions a 260 kDa homodimer, indicating ENPP1 dimerization, was observed. The ratio of non reduced (260 kDa) to reduced (130 kDa) ENPP1 was significantly decreased by two thirds in ENPP1-ΔII-3′myc vs. ENPP1–3′myc but not in ENPP1-ΔI-3′myc. A similar ENPP1/IR interaction was detectable by co-immunoprecipitation in ENPP1–3′myc, ENPP1-ΔI-3′myc and ENPP1-ΔII-3′myc cells. In conclusion, SMB 1 and SMB 2 are negative modulators of ENPP1 inhibitory activity on insulin signaling. For SMB 2 such effect might be mediated by a positive role on protein dimerization.► ENPP1 dimerization occurs in human insulin target cells. ► SMB 2, but not SMB 1, is instrumental for ENPP1 dimerization. ► SMB 1 and SMB 2 are inhibitory domains of ENPP1 modulation of IR signaling.
Keywords: ENPP1; Somatomedin B like domain; Insulin action;

Ubiquitin C-terminal Hydrolase 37, a novel predictor for hepatocellular carcinoma recurrence, promotes cell migration and invasion via interacting and deubiquitinating PRP19 by Ying Fang; Da Fu; Wenqing Tang; Yu Cai; Duan Ma; Huijun Wang; Ruyi Xue; Taotao Liu; Xiaowu Huang; Ling Dong; Hao Wu; Xizhong Shen (559-572).
Ubiquitin C-terminal hydrolase 37 (UCH37) plays a crucial role in numerous biological processes and is also involved in oncogenesis. In this study, clinicopathologic data showed that UCH37 was over-expressed in hepatocellular carcinoma (HCC) cancerous tissues and was a significant predictor for time to recurrence (TTR). In vitro, we discovered that UCH37 could promote cell migration and invasion. Subsequently, we utilized Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) to identify differentially expressed proteins in UCH37 over-expressing cells compared with the control cells, and found that PRP19, an essential RNA splicing factor, was up-regulated. The relationship between UCH37, PRP19 and the capability of cell migration and invasion was further confirmed. Collectively, this study demonstrated that UCH37 could promote cell migration and invasion in HCC cell lines through interacting and deubiquitinating PRP19, and suggested that UCH37 could be a novel predictor for HCC recurrence after curative resection.Display Omitted► UCH37 was over-expressed in HCC tissues and was an independent predictor for TTR. ► iTRAQ was used to identify differentially expressed proteins. ► UCH37 could interact and deubiquitinate PRP19, an essential RNA splicing factor. ► UCH37 could promote cell migration and invasion via PRP19.
Keywords: UCH37; PRP19; Migration; Invasion; HCC;

Gastrointestinal hormones stimulate growth of Foregut Neuroendocrine Tumors by transactivating the EGF receptor by Alessia Di Florio; Veronica Sancho; Paola Moreno; Gianfranco Delle Fave; Robert T. Jensen (573-582).
Foregut neuroendocrine tumors [NETs] usually pursuit a benign course, but some show aggressive behavior. The treatment of patients with advanced NETs is marginally effective and new approaches are needed. In other tumors, transactivation of the EGF receptor (EGFR) by growth factors, gastrointestinal (GI) hormones and lipids can stimulate growth, which has led to new treatments. Recent studies show a direct correlation between NET malignancy and EGFR expression, EGFR inhibition decreases basal NET growth and an autocrine growth effect exerted by GI hormones, for some NETs. To determine if GI hormones can stimulate NET growth by inducing transactivation of EGFR, we examined the ability of EGF, TGFα and various GI hormones to stimulate growth of the human foregut carcinoid,BON, the somatostatinoma QGP-1 and the rat islet tumor,Rin-14B-cell lines. The EGFR tyrosine-kinase inhibitor, AG1478 strongly inhibited EGF and the GI hormones stimulated cell growth, both in BON and QGP-1 cells. In all the three neuroendocrine cell lines studied, we found EGF, TGFα and the other growth-stimulating GI hormones increased Tyr1068 EGFR phosphorylation. In BON cells, both the GI hormones neurotensin and a bombesin analogue caused a time- and dose-dependent increase in EGFR phosphorylation, which was strongly inhibited by AG1478. Moreover, we found this stimulated phosphorylation was dependent on Src kinases, PKCs, matrix metalloproteinase activation and the generation of reactive oxygen species. These results raise the possibility that disruption of this signaling cascade by either EGFR inhibition alone or combined with receptor antagonists may be a novel therapeutic approach for treatment of foregut NETs/PETs.► In BON and QGP cells, various GI hormones/neurotransmitters stimulated growth (bradykinin, Bombesin, PACAP, Neurotensin). ► NET growth by the GI hormones neurotransmitters was inhibited by AG1478, an EGFR inhibitor. ► Each of the GI hormones/neurotransmitters stimulated EGFR transactivation with Y1068 tyrosine phosphorylation of the EGFR. ► EGFR transactivation required activation of Src kinases, PKC and matrix metalloproteinases. ► EGFR transactivation required activation of reactive oxygen species.
Keywords: Neuroendocrine tumors; Transactivation; Gastrointestinal hormones; Cell growth; Pancreatic endocrine tumors;

Stress-induced nuclear import of apoptosis signal-regulating kinase 1 is mediated by karyopherin α2/β1 heterodimer by Emmanuel Sturchler; Daniel Feurstein; Weimin Chen; Patricia McDonald; Derek Duckett (583-592).
The apoptosis signal-regulating kinase 1 (ASK1) is activated in response to a wide variety of extracellular stressors. Consequently, dysregulation of ASK1 is associated with multiple pathologies. Here, we show that ASK1 translocates from the cytoplasm to the nucleus in HEK293 cells and human cardiomyocytes in response to hydrogen peroxide (H2O2) or angiotensin respectively. Immunoprecipitation and mass spectrometry experiments reveal that ASK1 physically interacts with the karyopherin α2/β1 heterodimer in response to stress and genetic knockdown experiments confirm that this association mediates H2O2-induced ASK1 nuclear translocation. In addition, we have identified a nuclear localization signal (NLS)-like motif within the primary amino acid sequence of ASK1 composed of two clusters of basic amino acids separated by an intervening 16 amino acid spacer, KR[ACANDLLVDEFLKVSS]KKKK. Mutation of the downstream lysine cluster markedly reduces the H2O2-induced ASK1-karyopherin α2/β1 interaction and inhibits ASK1 nuclear translocation. Furthermore, we demonstrate that nuclear ASK1 is active and participates in H2O2-induced ASK1-mediated cell death. Collectively, our findings have identified a functional interaction between ASK1 and the karyopherin α2/β1 heterodimer and have also revealed a novel mechanism by which nuclear trafficking regulates the apoptotic function of ASK1 in response to stress.► ASK1 translocates from the cytoplasm to the nucleus in response to stress. ► Karyopherin α2/β1 heterodimer physically interacts with ASK1 via a newly identified NLS motif, facilitating ASK1 nuclear entry. ► In response to stress, active nuclear ASK1 participates in cytotoxicity. ► Stress-induced ASK1 nuclear import is a novel mechanism for controlling ASK1 function.
Keywords: Apoptosis signal-regulated kinase 1; Karyopherin α2/β1; Nuclear localization signal; Nuclear translocation;

Lack of Sir2 increases acetate consumption and decreases extracellular pro-aging factors by Nadia Casatta; Alessandra Porro; Ivan Orlandi; Luca Brambilla; Marina Vai (593-601).
Yeast chronological aging is regarded as a model for aging of mammalian post-mitotic cells. It refers to changes occurring in stationary phase cells over a relatively long period of time. How long these cells can survive in such a non-dividing state defines the chronological lifespan. Several factors influence cell survival including two well known normal by-products of yeast glucose fermentation such as ethanol and acetic acid. In fact, the presence in the growth medium of these C2 compounds has been shown to limit the chronological lifespan. In the chronological aging paradigm, a pro-aging role has also emerged for the deacetylase Sir2, the founding member of the Sirtuin family, whose loss of function increases the depletion of extracellular ethanol by an unknown mechanism. Here, we show that lack of Sir2 strongly influences carbon metabolism. In particular, we point out a more efficient acetate utilization which in turn may have a stimulatory effect on ethanol catabolism. This correlates with an enhanced glyoxylate/gluconeogenic flux which is fuelled by the acetyl-CoA produced from the acetate activation. Thus, when growth relies on a respiratory metabolism such as that on ethanol or acetate, SIR2 inactivation favors growth. Moreover, in the chronological aging paradigm, the increase in the acetate metabolism implies that sir2Δ cells avoid acetic acid accumulation in the medium and deplete ethanol faster; consequently pro-aging extracellular signals are reduced. In addition, an enhanced gluconeogenesis allows replenishment of intracellular glucose stores which may be useful for better long-term cell survival.► Lack of Sir2 affects positively acetate metabolism. ► Chronologically aging sir2Δ cells avoid extracellular acetic acid accumulation. ► SIR2 inactivation favors growth on ethanol and acetate. ► Lack of Sir2 increases gluconeogenesis.
Keywords: Chronological aging; Sir2; Acetate metabolism; Acetyl-CoA; Ethanol; Gluconeogenesis;

Palmitoylation of TNF alpha is involved in the regulation of TNF receptor 1 signalling by Marjorie Poggi; Imène Kara; Jean-Michel Brunel; Jean-François Landrier; Roland Govers; Bernadette Bonardo; Regina Fluhrer; Christian Haass; Marie-Christine Alessi; Franck Peiretti (602-612).
The pleiotropic pro-inflammatory cytokine tumour necrosis factor alpha (TNF) is synthesised as a transmembrane protein that is subject to palmitoylation. In this study, the roles of this acylation on TNF-mediated biological effects were investigated. We found that the lipid raft partitioning of TNF is regulated by its palmitoylation. Furthermore, we demonstrated that this palmitoylation process interferes with the cleavage/degradation of TNF intracellular fragments but is not involved in the regulation of its ectodomain shedding. Moreover, we found that the palmitoylation of TNF hinders the binding of soluble TNF to TNFR1 and regulates the integration/retention of TNFR1 into lipid rafts. Finally, we demonstrate that the transmembrane forms of wild-type and palmitoylation-defective TNF interact differently with TNFR1 and regulate NFκB activity, Erk1/2 phosphorylation and interleukin-6 synthesis differently, strongly suggesting that palmitoylation of TNF is involved in the regulation of TNFR1 signalling. An evidence for the physiological intervention of this regulation is provided by the fact that, in macrophages, the binding of endogenous soluble TNF to TNFR1 is enhanced by inhibition of palmitoylation. Therefore, our data introduce the new concept that palmitoylation of TNF is one of the means by which TNF-producing cells regulate their sensitivity to soluble TNF.► Palmitoylation of transmembrane TNF promotes its lipid raft integration. ► Palmitoylation of TNF is required for the processing of its intracellular fragments. ► Palmitoylation of TNF modulates the binding of soluble TNF to TNFR1. ► Palmitoylation of transmembrane TNF alters its interaction with TNFR1. ► Palmitoylation of transmembrane TNF modulates cellular responses to soluble TNF.
Keywords: TNF; TNFR1; Lipid rafts; Palmitoylation; Signalling;

p38α phosphorylates serine 258 within the cytoplasmic domain of tissue factor and prevents its incorporation into cell-derived microparticles by Camille Ettelaie; Azza M. ElKeeb; Anthony Maraveyas; Mary Elizabeth W. Collier (613-621).
We previously showed that the phosphorylation of Ser253 within the cytoplasmic domain of human tissue factor (TF) initiates the incorporation and release of this protein into cell-derived microparticles. Furthermore, subsequent phosphorylation of Ser258 terminates this process. However, the identity of the kinase responsible for the phosphorylation of Ser258 and mode of action of this enzyme remain unknown. In this study, p38α was identified as the proline-directed kinase capable of phosphorylating Ser258 specifically, and without any detectable activity towards Ser253. Furthermore, using synthetic peptides, it was shown that the Km for the reaction decreased by approximately 10 fold on substitution of Ser253 with phospho-Ser253. Either inhibition of p38 using SB202190 or knockdown of p38α expression in coronary artery endothelial cells overexpressing wild-type TF, resulted in decreased phosphorylation of Ser258, following activation of cells with PAR2-agonist peptide (PAR2-AP). In agreement with our previous data, inhibition of phosphorylation of this residue maintained the release of TF. Activation of PAR2 in cells transfected to overexpress TF, resulted in two separate peaks of p38 activity at approximately 40 and 120 min post-activation. Furthermore, overexpression of Ala253-substituted TF enhanced the second p38 activation peak. However, the second peak was absent in cells devoid of TF or in cells overexpressing the Asp253-substituted TF. Our data clearly identifies p38α as a kinase capable of phosphorylating Ser258 within the cytoplasmic domain of TF. Moreover, it appears that the presence of TF within the cells regulates the late activation of p38 and consequently the termination of TF release into microparticles.Display Omitted► The kinase that phosphorylates Ser258 within TF is identified as p38α ► The reaction rate increases by 10 fold on previous phosphorylation of Ser253 in TF ► This arises as a result of exposure of Ser258 following phosphorylation of Ser253 ► The activation of p38 may occur by the retention of TF within activated cells ► A mechanism for the regulation of the release of TF into microparticles is proposed
Keywords: Tissue factor; Cytoplasmic domain; Serine-phosphorylation; p38 kinase; Microparticle;

Destabilization of fructose 1,6-bisphosphatase–Z-line interactions is a mechanism of glyconeogenesis down-regulation in vivo by Agnieszka Gizak; Jakub Mazurek; Marta Wozniak; Ewa Maciaszczyk-Dziubinska; Dariusz Rakus (622-628).
Although it is well known that insulin controls the synthesis of glycogen from non-carbohydrates by down-regulating expression of several glyconeogenic enzymes, a mechanism of short-term inhibition of glyconeogenesis remains unknown. In recent years, we have shown that in skeletal muscle, fructose 1,6-bisphosphatase (FBPase) is a part of the hypothetical glyconeogenic complex located on sarcomeric Z-line. Here, we show that inhibition of glycogen synthase kinase-3 causes disruption of the FBPase–Z-line interactions and reduction of muscle glycogen content in vivo. The normal, striated pattern of muscle FBPase localization is also disturbed by insulin treatment but preserved when insulin is applied together with Akt inhibitor. We suggest that destabilization of FBPase–Z-line interaction is a universal cellular mechanism of glyconeogenesis down-regulation, allowing for preferential utilization of glucose for insulin-stimulated muscle glycogen synthesis.► GSK-3 inhibition results in destabilization of FBPase–Z-line interaction in muscle. ► Disruption of FBPase–Z-line binding results in a fast inhibition of glyconeogenesis. ► This allows for preferential usage of glucose to insulin-induced glycogen synthesis. ► GSK-3 inhibition leads to translocation of FBPase to mitochondria in vivo.
Keywords: FBPase; Glyconeogenesis; GSK3; Insulin; Striated muscle;

The COOH-terminus of TM4SF5 in hepatoma cell lines regulates c-Src to form invasive protrusions via EGFR Tyr845 phosphorylation by Oisun Jung; Yoon-Ju Choi; Tae Kyoung Kwak; Minkyung Kang; Mi-Sook Lee; Jihye Ryu; Hye-Jin Kim; Jung Weon Lee (629-642).
Transmembrane 4 L six family member 5 (TM4SF5) enhances cell migration and invasion, although how TM4SF5 mechanistically mediates these effects remains unknown. In the study, during efforts to understand TM4SF5-mediated signal transduction, TM4SF5 was shown to bind c-Src and thus hepatoma cell lines expressing TM4SF5 were analyzed for the significance of the interaction in cell invasion. The C-terminus of TM4SF5 bound both inactive c-Src that might be sequestered to certain cellular areas and active c-Src that might form invasive protrusions. Wildtype (WT) TM4SF5 expression enhanced migration and invasive protrusion formation in a c-Src-dependent manner, compared with TM4SF5-null control hepatoma cell lines. However, tailless TM4SF5ΔC cells were more efficient than WT TM4SF5 cells, suggesting a negative regulatory role by the C-terminus. TM4SF5 WT- or TM4SF5ΔC-mediated formation of invasive protrusions was dependent or independent on serum or epidermal growth factor treatment, respectively, although they both were dependent on c-Src. The c-Src activity of TM4SF5 WT- or TM4SF5ΔC-expressing cells correlated with enhanced Tyr845 phosphorylation of epidermal growth factor receptor. Y845F EGFR mutation abolished the TM4SF5-mediated invasive protrusions, but not c-Src phosphorylation. Our findings demonstrate that TM4SF5 modulates c-Src activity during TM4SF5-mediated invasion through a TM4SF5/c-Src/EGFR signaling pathway, differentially along the leading protrusive edges of an invasive cancer cell.► The C-terminus of TM4SF5 binds c-Src family kinases in preference to the inactive form. ► TM4SF5 requires a signaling link from c-Src to Tyr845 EGFR for invasive protrusion. ► Tailless TM4SF5 enhances c-Src/EGFR-dependent invasion even without EGF treatment. ► The C-terminus of TM4SF5 may play a negative regulatory role in hepatocyte invasion.
Keywords: EGFR; Hepatocarcinoma; Invasion; Src family kinase; TM4SF5;

Store-operated Ca2 + entry in proliferating and retinoic acid-differentiated N- and S-type neuroblastoma cells by Natalie Bell; Victoria Hann; Christopher P.F. Redfern; Timothy R. Cheek (643-651).
Neuroblastoma cell lines are heterogeneous, comprised of at least three distinct cell phenotypes; neuroblastic N-type cells, non-neuronal substrate-adherent S-type cells and intermediate I-type cells. N- and S-type cell populations were enriched from the parental SH-SY5Y neuroblastoma cell line and induced to differentiate by the addition of retinoic acid (RA), a drug used in the treatment of neuroblastoma. N- and S-type cells were identified based on their differential expression of β-tubulin III, vimentin and Bcl-2. Store-operated Ca2 + entry (SOCE) was then measured in proliferating and differentiated N- and S-type cell populations and the expression of STIM1, Orai1 and TRPC1, three proteins reported to play a key role in SOCE, was determined. In N-type cells the RA-induced switch from proliferation to differentiation was accompanied by a down-regulation in SOCE. STIM1 and Orai1 expression became down-regulated in differentiated cells, consistent with their respective roles as ER Ca2 + sensor and store-operated Ca2 + channel (SOC). TRPC1 became up-regulated suggesting that TRPC1 is not involved in SOCE, at least in differentiated N-type cells. In S-type cells SOCE remained active following the RA-induced switch from proliferation to differentiation and the expression of STIM1 and Orai1 remained unchanged. TRPC1 was not expressed in S-type cells. Our results indicate that differentiation of neuronal cells is associated with a remodelling of SOCE. Therapeutic targeting of SOCE proteins could potentially be a means of promoting neuronal differentiation in the treatment of neuroblastoma.► SOCE is down-regulated in differentiated N-type cells. ► STIM1 and Orai1 expression is down-regulated in differentiated N-type cells. ► TRPC1 expression is up-regulated in differentiated N-type cells. ► SOCE proteins could provide a therapeutic target in the treatment of neuroblastoma.
Keywords: Store-operated Ca2 + entry; Differentiation; STIM1; Orai1; TRPC1; Neuroblastoma;

FKBP52 is involved in the regulation of SOCE channels in the human platelets and MEG 01 cells by Esther López; Alejandro Berna-Erro; Ginés M. Salido; Juan A. Rosado; Pedro C. Redondo (652-662).
Immunophilins are FK506-binding proteins that have been involved in the regulation of calcium homeostasis, either by modulating Ca2 + channels located in the plasma membrane or in the rough endoplasmic reticulum (RE). We have investigated whether immunophilins would participate in the regulation of stored-operated Ca2 + entry (SOCE) in human platelets and MEG 01. Both cell types were loaded with fura-2 for determining cytosolic calcium concentration changes ([Ca2 +]c), or stimulated and fixed to evaluate the protein interaction profile by performing immunoprecipitation and western blotting. We have found that incubation of platelets with FK506 increases Ca2 + mobilization. Thapsigargin (TG)-evoked, Thr-evoked SOCE and TG-evoked Mn2 + entry resulted in significant reduction by treatment of platelets with immunophilin antagonists. We confirmed by immunoprecipitation that immunophilins interact with transient receptor potential channel 1 (TRPC1) and Orai1 in human platelets. FK506 and rapamycin reduced the association between TRPC1 and Orai1 with FK506 binding protein (52) (FKBP52) in human platelets, and between TRPC1 and the type II IP3R, which association is known to be crucial for the maintenance of SOCE in human platelets. FKBP52 role in SOCE activation was confirmed by silencing FKBP52 using SiRNA FKBP52 in MEG 01 as demonstrated by single cell configuration imaging technique. TRPC1 silencing and depletion of cell of TRPC1 and FKBP52 simultaneously, impair activation of SOCE evoked by TG in MEG 01. Finally, in MEG 01 incubated with FK506 we observed a reduction in TRPC1/FKBP52 coupling, and similarly, FKBP52 silencing reduced the association between IP3R type II and TRPC1 during SOCE. All together, these results demonstrate that immunophilins participate in the regulation of SOCE in human platelets.► FK506 modifies SOCE and TRPC1/type II IP3R coupling in platelets and MEG 01. ► Association between FKBP52/TRPC1 and FKBP52/Orai 1 is impaired by FK506 in platelets. ► FKBP52 silencing using siRNA reduces SOCE and coupling between TRPC1/type II IP3R. ► TRPC1 silencing reduces SOCE in MEG 01 thus reinforcing the role of TRPC1 in SOCE.
Keywords: FKBP52; FK506; shTRPC1; Orai1; siFKBP52;

Histone deacetylase inhibitor induction of epithelial–mesenchymal transitions via up-regulation of Snail facilitates cancer progression by Guan-Min Jiang; Hong-Sheng Wang; Fan Zhang; Kun-Shui Zhang; Zong-Cai Liu; Rui Fang; Hao Wang; Shao-Hui Cai; Jun Du (663-671).
Histone deacetylase inhibitors (HDACIs) are now emerging as a new class of anticancer drugs. Some of them have been used in clinical treatment for tumors, most impressively in the hematological tumors. But their single-agent activities in epithelial-derived tumors are limited. The mechanisms of these actions of HDACIs are not yet well understood. In this study, it was found for the first time that HDACIs were able to induce epithelial–mesenchymal transitions (EMT) which is believed to trigger tumor cell invasion and metastasis. We show that HDACIs induce fibroblast-like morphology, up-regulate Snail and Vimentin and down-regulate E-cadherin in epithelial cell-derived tumor cell lines. It demonstrates that HDACI treatment enhances further Snail acetylation and reduces its ubiquitylation, and induces Snail transcription as well as Snail nuclear translocation in CNE2 cells. Snail knockdown by siRNAs prevents the change in cell morphology and Vimentin up-regulation in response to HDACIs. The results suggested that Snail plays an important role in the HDACI-induced EMT. It is very crucial for a better understanding of clinical therapeutical failure of HDACIs in the patients with epithelial cell-derived cancers. Therefore, our results indicate that more attention should be paid to the cancer treatment using HDACIs due to the fact that it will enhance the spread risks of cancer cells to facilitate cancer progression and it is very important to select appropriate drugs for different tumors.► We found that HDACIs induced EMT in cancer cells to facilitate cancer progression. ► In CNE2, LoVo, HepG2, HDACIs up-regulate Snail and Vimentin and down-regulate E-cadherin. ► We provided that Snail plays an important role in the HDACI-induced EMT. ► It shows that more attention should be paid to the cancer treatment using HDACIs.
Keywords: Epithelial–mesenchymal transitions; Histone deacetylase inhibitors; Snail; Tumor metastasis; Vimentin;

UCP2 inhibition triggers ROS-dependent nuclear translocation of GAPDH and autophagic cell death in pancreatic adenocarcinoma cells by Ilaria Dando; Claudia Fiorini; Elisa Dalla Pozza; Chiara Padroni; Chiara Costanzo; Marta Palmieri; Massimo Donadelli (672-679).
Mitochondrial uncoupling protein 2 (UCP2) can moderate oxidative stress by favoring the influx of protons into the mitochondrial matrix, thus reducing electron leakage from respiratory chain and mitochondrial superoxide production. Here, we demonstrate that UCP2 inhibition by genipin or UCP2 siRNA strongly increases reactive oxygen species (ROS) production inhibiting pancreatic adenocarcinoma cell growth. We also show that UCP2 inhibition triggers ROS-dependent nuclear translocation of the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH), formation of autophagosomes, and the expression of the autophagy marker LC3-II. Consistently, UCP2 over-expression significantly reduces basal autophagy confirming the anti-autophagic role of UCP2. Furthermore, we demonstrate that autophagy induced by UCP2 inhibition determines a ROS-dependent cell death, as indicated by the apoptosis decrease in the presence of the autophagy inhibitors chloroquine (CQ) or 3-methyladenine (3-MA), or the radical scavenger NAC. Intriguingly, the autophagy induced by genipin is able to potentiate the autophagic cell death triggered by gemcitabine, the standard chemotherapeutic drug for pancreatic adenocarcinoma, supporting the development of an anti-cancer therapy based on UCP2 inhibition associated to standard chemotherapy. Our results demonstrate for the first time that UCP2 plays a role in autophagy regulation bringing new insights into mitochondrial uncoupling protein field.► UCP2 inhibition stimulates ROS-dependent nuclear translocation of GAPDH. ► UCP2 inhibition induces ROS-dependent autophagy. ► Autophagy induced by UCP2 inhibition has an antiproliferative role. ► Genipin enhances autophagy induced by chemotherapy.
Keywords: UCP2; Autophagy; ROS; GAPDH; Cancer; Genipin;

ADAM17 cleaves CD16b (FcγRIIIb) in human neutrophils by Yue Wang; Jianming Wu; Robert Newton; Nooshin S. Bahaie; Chunmei Long; Bruce Walcheck (680-685).
CD16b (FcγRIIIb) is exclusively expressed by human neutrophils and binds IgG in immune complexes. Cell surface CD16b undergoes efficient ectodomain shedding upon neutrophil activation and apoptosis. Indeed, soluble CD16b is present at high levels in the plasma of healthy individuals, which appears to be maintained by the daily turnover of apoptotic neutrophils. At this time, the principal protease responsible for CD16b shedding is not known. We show that CD16b plasma levels were significantly decreased in patients administered a selective inhibitor targeting the metalloproteases ADAM10 and ADAM17. Additional analysis with inhibitors selective for ADAM10 or ADAM17 revealed that only inhibition of ADAM17 significantly blocked the cleavage of CD16b following neutrophil activation and apoptosis. CD16b shedding by ADAM17 was further demonstrated using a unique ADAM17 function-blocking mAb and a cell-based ADAM17 reconstitution assay. Unlike human CD16, however, mouse CD16 did not undergo efficient ectodomain shedding upon neutrophil stimulation or apoptosis, indicating that this mechanism cannot be modeled in normal mice. Taken together, our findings are the first to directly demonstrate that ADAM17 cleaves CD16 in human leukocytes.Display Omitted► Plasma CD16 in humans is generated by ADAM protease activity. ► ADAM17 cleaves CD16a and CD16b in activated NK cells and neutrophils, respectively. ► CD16b undergoes ectodomain shedding in apoptotic neutrophils by ADAM17. ► Unlike human CD16, mouse CD16 is not efficiently regulated by ectodomain shedding.
Keywords: Neutrophil; Metalloprotease; ADAM17; Apoptosis; Fc receptor;

The Nε-amino group of lysine residues can be transiently modified by the addition of an acetyl group. Recognized functions of Nε-lysine acetylation include regulation of activity, molecular stabilization and conformational assembly of a protein. For more than forty years lysine acetylation was thought to occur only in the cytosol and nucleus. Targets included cytoskeletal-associated proteins as well as transcription factors, histone proteins and proteins involved in DNA recombination and repair. However, in 2007 we reported that a type I membrane protein involved in the pathogenesis of Alzheimer's disease was transiently acetylated on the ε amino group of seven lysine residues while transiting along the secretory pathway. Surprisingly, the acetylation occurred in the lumen of the endoplasmic reticulum (ER) forcing us to reconsider old paradigms. Indeed, if lysine acetylation can occur in the lumen of the ER, then all the essential biochemical elements of the reaction must be available in the lumen of the organelle. Follow-up studies revealed the existence of ER-based acetyl-CoA:lysine acetyltransferases as well as a membrane transporter that translocates acetyl-CoA from the cytosol into the ER lumen. Large-scale proteomics showed that the list of substrates of the ER-based acetylation machinery includes both transiting and resident proteins. Finally, genetic studies revealed that this machinery is tightly linked to human diseases. Here, we describe these exciting findings as well as recent biochemical and cellular advances, and discuss possible impact on both human physiology and pathology.► Resident and transiting proteins can undergo Nε-lysine acetylation in the ER lumen. ► Two (or more) acetyl-CoA:lysine acetyltransferases are found in the ER. ► A membrane transporter translocates acetyl-CoA into the ER lumen. ► Influx of acetyl-CoA into the ER controls the induction of ERAD(II). ► Defects in the ER-based acetylation machinery are linked to human diseases.
Keywords: Endoplasmic reticulum; Lysine acetylation; ATase; AT-1; PCSK9/NARC-1; Autophagy/ERAD(II);

A role for β-dystroglycan in the organization and structure of the nucleus in myoblasts by Ivette A. Martínez-Vieyra; Alejandra Vásquez-Limeta; Ricardo González-Ramírez; Sara L. Morales-Lázaro; Mónica Mondragón; Ricardo Mondragón; Arturo Ortega; Steve J. Winder; Bulmaro Cisneros (698-711).
We recently characterized a nuclear import pathway for β-dystroglycan; however, its nuclear role remains unknown. In this study, we demonstrate for the first time, the interaction of β-dystroglycan with distinct proteins from different nuclear compartments, including the nuclear envelope (NE) (emerin and lamins A/C and B1), splicing speckles (SC35), Cajal bodies (p80-coilin), and nucleoli (Nopp140). Electron microscopy analysis revealed that β-dystroglycan localized in the inner nuclear membrane, nucleoplasm, and nucleoli. Interestingly, downregulation of β-dystroglycan resulted in both mislocalization and decreased expression of emerin and lamin B1, but not lamin A/C, as well in disorganization of nucleoli, Cajal bodies, and splicing speckles with the concomitant decrease in the levels of Nopp140, and p80-coilin, but not SC35. Quantitative reverse transcription PCR and cycloheximide-mediated protein arrest assays revealed that β-dystroglycan deficiency did not change mRNA expression of NE proteins emerin and lamin B1 bud did alter their stability, accelerating protein turnover. Furthermore, knockdown of β-dystroglycan disrupted NE-mediated processes including nuclear morphology and centrosome-nucleus linkage, which provides evidence that β-dystroglycan association with NE proteins is biologically relevant. Unexpectedly, β-dystroglycan-depleted cells exhibited multiple centrosomes, a characteristic of cancerous cells. Overall, these findings imply that β-dystroglycan is a nuclear scaffolding protein involved in nuclear organization and NE structure and function, and that might be a contributor to the biogenesis of nuclear envelopathies.► β-dystroglycan interacts with nuclear envelope proteins emerin and lamins. ► β-dystroglycan knockdown mislocalized and decreased levels of emerin and lamin B1. ► β-dystroglycan deficiency impairs spatial organization of nucleoli and Cajal bodies. ► Deficiency of β-dystroglycan alters nuclear envelope-associated functions. ► β-dystroglycan is crucial for nuclear envelope structure and function.
Keywords: Dystroglycan; Emerin; Nuclear lamins; Nuclear envelope; Nucleoli;

Osmotic stress poses one of the most fundamental challenges to living cells. Particularly, the largely inextensible plasma membrane of eukaryotic cells easily ruptures under in-plane tension calling for sophisticated strategies to readily respond to osmotic stress. We describe how epithelial cells react and adapt mechanically to the exposure to hypotonic and hypertonic solutions in the context of a confluent monolayer. Site-specific indentation experiments in conjunction with tether pulling on individual cells have been carried out with an atomic force microscope to reveal spatio-temporal changes in membrane tension and surface area. We found that cells compensate for an increase in lateral tension due to hypoosmotic stress by sacrificing excess of membrane area stored in protrusions and invaginations such as microvilli and caveolae. At mild hypotonic conditions lateral tension increases partly compensated by surface are regulation, i.e. the cell sacrifices some of its membrane reservoirs. A loss of membrane–actin contacts occurs upon exposure to stronger hypotonic solutions giving rise to a drop in lateral tension. Tension release recovers on longer time scales by an increasing endocytosis, which efficiently removes excess membrane from the apical side to restore the initial pre-stress. Hypertonic solutions lead to shrinkage of cells and collapse of the apical membrane onto the cortex. Exposure to distilled water leads to stiffening of cells due to removal of excess surface area and tension increase due to elevated osmotic pressure across the plasma membrane.► The way membrane tension is regulated by adaption of the cell's surface area. ► A new way towards simultaneous determination of membrane tension and surface area. ► Swelling and shrinking induce alteration in tension buffering membrane reservoirs. ► Epithelial cells (MDCKII) adjust their surface area in response to tension changes. ► Lysis of the plasma membrane is prevented by loosening membrane–actin links.
Keywords: Osmotic stress; Epithelial cell; Atomic force microscopy; Membrane tension; Surface area regulation; Microvilli;

Prostate cancer is one of the most commonly occurring malignancies in men, and because existing treatments are not able to manage this neoplasm adequately, novel approaches are needed. Although tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has strong antitumor activity via the induction of apoptotic cell death in a wide range of tumor cell types and has negligible toxicity to most normal cells, some prostate carcinoma cells are resistant to the apoptotic effects of TRAIL. Therefore, combinatorial approaches with TRAIL and different chemotherapeutic agents have been developed to overcome the resistance of cancer cells to TRAIL. Here, we investigated the sensitizing effects of ursolic acid (UA), a pentacyclic triterpenoid found in many plants, on TRAIL-induced prostate cancer cell apoptosis. We found TRAIL-induced prostate cancer cells apoptosis was significantly enhanced by UA, and that UA induced CHOP-dependent DR5 up-regulation. This study shows the use of UA as a sensitizer for TRAIL-induced apoptotic cell death offers a promising means of enhancing the efficacy of TRAIL-based prostate cancer treatments.► Ursolic acid sensitized TRAIL-induced prostate cancer cell apoptosis. ► UA induced CHOP-dependent DR5 up-regulation. ► The use of urosolic acid offers a promising means of enhancing the efficacy of TRAIL-based prostate cancer treatments.
Keywords: Prostate cancer; Ursolic acid; TRAIL; Apoptosis; DR5;

Towards delineation of a developmental α-importome in the mammalian male germline by Yoichi Miyamoto; Mark A. Baker; Penny A. Whiley; Arash Arjomand; Justin Ludeman; Chin Wong; David A. Jans; Kate L. Loveland (731-742).
Nucleocytoplasmic transport mediated by importin proteins is central to many developmental processes, such as precisely regulated germ cell differentiation during spermatogenesis. Here we examine for the first time the dynamic association of importins with cargo during two successive spermatogenic stages: meiotic pachytene spermatocytes and haploid round spermatids of the adult rat testis. Immunoprecipitation followed by mass spectrometry yielded the first non-biased identification of proteins selectively interacting with importin α2, α3 and α4 in each of these cell types. Amongst the 22 novel importin binding proteins identified, 11 contain a predicted classical nuclear localization signal (cNLS) for importin α binding using a new algorithm (Kosugi et al. [22]), although only 6 of these have known nuclear functions. An importin α2-immunoprecipitated protein with a key nuclear role in meiosis, structural maintenance of chromosomes 6 (SMC6), contained a predicted bipartite NLS that was shown to be preferentially recognized by importin α together with importin β1. In contrast, the predicted cNLS of synovial sarcoma, X breakpoint 2 interacting protein (SSX2IP) was found not to confer either nuclear accumulation or direct binding to importin αs, implying that NLS prediction algorithms may identify cryptic importin binding sites or require additional refinement to increase their accuracy. Unbiased identification of importin α binding proteins in cellular differentiation represents a powerful tool to help identify the functional roles of importin αs.► This is the first study to reveal the α-importome in mammalian spermatogenesis. ► Highly purified rat male germ cells were used for an unbiased proteomic approach. ► A total of 22 proteins were identified unequivocally. ► The cNLS Mapper was validated for identifying importin α binding motifs. ► The identified SMC6 NLS confers nuclear transport, generally via importin α/β1
Keywords: Nuclear transport; Importin α; NLS; SMC6; Testis; Spermatogenesis;

3-Aminobenzamide protects primary human keratinocytes from UV-induced cell death by a poly(ADP-ribosyl)ation independent mechanism by Petra Lakatos; Éva Szabó; Csaba Hegedűs; György Haskó; Pál Gergely; Péter Bai; László Virág (743-751).
Poly(ADP-ribosyl)ation (PARylation) is a NAD+-dependent protein modification carried out by PARP [poly(ADP-ribose) polymerase] enzymes. Here we set out to investigate whether PARylation regulates UVB-induced cell death in primary human keratinocytes. We used the benchmark PARP inhibitor 3-aminobenzamide (3AB) and a more potent and specific inhibitor PJ34 and found that UVB (0.05–0.2 J/cm2) induced a dose dependent loss of viability that was prevented by 3AB but not by PJ34. Similarly to PJ34, two other new generation PARP inhibitors also failed to protect keratinocytes from UVB-induced loss of viability. Moreover, silencing PARP-1 in HaCaT human keratinocytes sensitized cells to UVB toxicity but 3AB provided protection to both control HaCaT cells and to PARP-1 silenced cells indicating that the photoprotective effect of 3AB is independent of PARP inhibition. Lower UVB doses (0.0125–0.05 J/cm2) caused inhibition of proliferation of keratinocytes which was prevented by 3AB but augmented by PJ34. UVB-induced keratinocyte death displayed the characteristics of both apoptosis (morphology, caspase activity, DNA fragmentation) and necrosis (morphology, LDH release) with all of these parameters being inhibited by 3AB and apoptotic parameters slightly enhanced by PJ34. UVA also caused apoptotic and necrotic cell death in keratinocytes with 3AB protecting and PJ34 sensitizing cells to UVA-induced toxicity. 3AB prevented UVB-induced mitochondrial membrane depolarization and generation of hydrogen peroxide. In summary, PARylation is a survival mechanism in UV-treated keratinocytes. Moreover, 3-aminobenzamide is photoprotective and acts by a PARP-independent mechanism at a premitochondrial step of phototoxicity.Display Omitted► The weak PARP inhibitor 3-aminobenzamide (3AB) protected cells from phototoxicity. ► 3AB prevented both apoptosis and necrosis. ► Specific PARP inhibitors or PARP-1 silencing sensitized cells to phototoxicity. ► The protective effect of 3AB is not due to PARP inhibition.
Keywords: Ultraviolet radiation; Poly(ADP-ribose) polymerase; 3-Aminobenzamide; Cell death; Apoptosis;

ORAI3 silencing alters cell proliferation and cell cycle progression via c-myc pathway in breast cancer cells by Malika Faouzi; Philippe Kischel; Frédéric Hague; Ahmed Ahidouch; Nazim Benzerdjeb; Henri Sevestre; Reinhold Penner; Halima Ouadid-Ahidouch (752-760).
Members of the Orai family are highly selective calcium ion channels that play an important role in store-operated calcium entry. Among the three known Orai isoforms, Orai3 has gained increased attention, notably for its emerging role in cancer. We recently demonstrated that Orai3 channels are over-expressed in breast cancer (BC) biopsies, and involved specifically in proliferation, cell cycle progression and survival of MCF-7 BC cells. Here, we investigate the downstream signaling mechanisms affected by Orai3 silencing, leading to the subsequent functional impact specifically seen in MCF-7 cancer cells. We report a correlation between Orai3 and c-myc expression in tumor tissues and in the MCF-7 cancer cell line by demonstrating that Orai3 down-regulation reduces both expression and activity of the proto-oncogene c-myc. This is likely mediated through the MAP Kinase pathway, as we observed decreased pERK1/2 levels and cell-cycle arrest in G1 phase after Orai3 silencing. Our results provide strong evidence that the c-myc proto-oncogene is influenced by the store-operated calcium entry channel Orai3 through the MAP kinase pathway. This connection provides new clues in the downstream mechanism linking Orai3 channels and proliferation, cell cycle progression and survival of MCF-7 BC cells.► Protein expression of Orai3 Ca2 + channels and c-myc are correlated in tumor tissues ► Orai3 down-regulation reduces expression and activity of the c-myc proto-oncogene ► Orai3 down-regulation induced cell cycle arrest through the c-myc pathway ► Orai3 regulates c-myc-dependent proliferation and survival in breast cancer cells
Keywords: Orai3; Breast cancer; Cell cycle; Cell proliferation; c-myc pathway;

CacyBP/SIP as a novel modulator of the thin filament by Ewelina Jurewicz; Zofia Ostrowska; Jolanta Jozwiak; Maria Jolanta Redowicz; Wieslawa Lesniak; Joanna Moraczewska; Anna Filipek (761-766).
The CacyBP/SIP protein interacts with several targets, including actin. Since the majority of actin filaments are associated with tropomyosin, in this work we characterized binding of CacyBP/SIP to the actin–tropomyosin complex and examined the effects of CacyBP/SIP on actin filament functions. By using reconstituted filaments composed of actin and AEDANS-labeled tropomyosin, we observed that binding of CacyBP/SIP caused an increase in tropomyosin fluorescence intensity indicating the occurrence of conformational changes within the filament. We also found that CacyBP/SIP bound directly to tropomyosin and that these proteins did not compete with each other for binding to actin. Electron microscopy showed that in the absence of tropomyosin CacyBP/SIP destabilized actin filaments, but tropomyosin reversed this effect. Actin-activated myosin S1 ATPase activity assays, performed using a colorimetric method, indicated that CacyBP/SIP reduced ATPase activity and that the presence of tropomyosin enhanced this inhibitory effect. Thus, our results suggest that CacyBP/SIP, through its interaction with both actin and tropomyosin, regulates the organization and functional properties of the thin filament.► CacyBP/SIP directly binds to tropomyosin. ► CacyBP/SIP has an effect on the organization of the actin–tropomyosin filament. ► CacyBP/SIP inhibits actin-activated myosin S1 ATPase activity. ► CacyBP/SIP might be a novel modulator of the thin filament.
Keywords: CacyBP/SIP; Actin; Tropomyosin; Thin filament; ATPase activity;

Granulocyte maturation determines ability to release chromatin NETs and loss of DNA damage response; these properties are absent in immature AML granulocytes by Emilie Lukášová; Zdeněk Kořistek; Martin Klabusay; Vladan Ondřej; Sergei Grigoryev; Alena Bačíková; Martina Řezáčová; Martin Falk; Jiřina Vávrová; Viera Kohútová; Stanislav Kozubek (767-779).
Terminally-differentiated cells cease to proliferate and acquire specific sets of expressed genes and functions distinguishing them from less differentiated and cancer cells. Mature granulocytes show lobular structure of cell nuclei with highly condensed chromatin in which HP1 proteins are replaced by MNEI. These structural features of chromatin correspond to low level of gene expression and the loss of some important functions as DNA damage repair, shown in this work and, on the other hand, acquisition of a new specific function consisting in the release of chromatin extracellular traps in response to infection by pathogenic microbes. Granulocytic differentiation is incomplete in myeloid leukemia and is manifested by persistence of lower levels of HP1γ and HP1β isoforms. This immaturity is accompanied by acquisition of DDR capacity allowing to these incompletely differentiated multi-lobed neutrophils of AML patients to respond to induction of DSB by γ-irradiation. Immature granulocytes persist frequently in blood of treated AML patients in remission. These granulocytes contrary to mature ones do not release chromatin for NETs after activation with phorbol myristate-12 acetate-13 and do not exert the neutrophil function in immune defence. We suggest therefore the detection of HP1 expression in granulocytes of AML patients as a very sensitive indicator of their maturation and functionality after the treatment. Our results show that the changes in chromatin structure underlie a major transition in functioning of the genome in immature granulocytes. They show further that leukemia stem cells can differentiate ex vivo to mature granulocytes despite carrying the translocation BCR/ABL.► Stem cells with BCR/ABL mutation can differentiate ex vivo to mature granulocytes. ► Mature granulocytes neither repair DNA damage nor express DNA repair proteins. ► Granulocytes of AML patients remain immature and keep active DNA repair. ► Immature granulocytes lose the capacity to release chromatin into extracellular space. ► Incomplete chromatin condensation could serve to monitor therapy and functionality.
Keywords: In vivo and ex vivo blood stem cells differentiation; Immature AML neutrophil; Higher-order chromatin remodeling; Neutrophil extracellular trap (NET); HP1 protein; DNA double-strand break repair;