BBA - Molecular Cell Research (v.1803, #10)

The evolution of protein targeting and translocation systems by Markus T. Bohnsack; Enrico Schleiff (1115-1130).
Cells have evolved increasingly complex membrane systems for compartmentalization and thereby for the regulation of multiple cellular pathways. The existence of such membranes required the evolution of molecular machines that allow and regulate the exchange of material between intracellular compartments or with the exterior. Here, we have summarized the current concepts for the origin and evolution of the targeting and translocation systems required for the specific insertion of transmembrane proteins into their target membranes and for the transport of protein cargos across membranes. The basic pathways developed in prokaryotes were modified and extended to suffice for the much more complex membrane systems found in eukaryotes, allowing not only the identification of basic mechanistic principles, but also phylogenetic studies to elucidate evolutionary relations.►translocating machines of the plasma membrane are evolutionary ancient. ►Chaperone system involved in protein transport evolved independent of the membrane inserted machines. ►Omp85 is the ancient outer membrane protein assembly machine. ►recycling of prokaryotic systems led to sorting and transport machineries in eukaryotes.
Keywords: Evolutionary development; Protein transport; Bacteria; Organelles; Molecular machines; Membranes;

S-resistin inhibits adipocyte differentiation and increases TNFα expression and secretion in 3T3-L1 cells by Carmen M. Fernández; Araceli del Arco; Nilda Gallardo; Lidia Aguado; María Rodriguez; Manuel Ros; Jose M. Carrascosa; Antonio Andrés; Carmen Arribas (1131-1141).
S-resistin is a non-secretable resistin spliced variant described in white adipose tissue from Wistar rats. Since resistin has been implicated in adipogenesis regulation, here we have investigated the possible role of this new isoform in this process. For that, we have studied the adipocyte development in 3T3-L1 pre-adipocyte cell line stably expressing s-resistin and resistin. Both isoforms are able to restrain 3T3-L1 pre-adipocyte differentiation though affecting differently the expression pattern of pro-adipogenic transcription factors such CCAAT/enhancer binding proteins α and β (C/EBPα and C/EBPβ) and peroxisome proliferator-activated receptor gamma (PPARγ), as well of proteins implicated in lipid metabolism such perilipin, fatty acid synthase (FAS), adipocyte lipid binding protein (ALBP/aP2) and carnitine palmitoyltransferase1 (CPT1). Likewise, both resistin isoforms impair insulin-stimulated glucose transport by decreasing glucose transport 4 (GLUT4) expression but to a different degree. In addition, s-resistin expressing 3T3-L1 cells display other remarkable differences. Thus, in these cells, endogenous resistin expression falls down while tumor necrosis factor alpha (TNFα) and interleukine 6 (IL-6) productions are increased along differentiation. These findings indicate that s-resistin isoform also impairs adipocyte differentiation affecting the expression pattern of key pro-adipogenic transcription factors and insulin sensitivity. Additionally, s-resistin may play a role in inflammatory processes.► Resistin isoforms impair adipocyte differentiation and insulin response. ► Both isoforms promote IL-6 and PAI-1 secretions. ► Only s-resistin increases TNFα expression and secretion in 3T3-L1 cells. ► S-resistin production is related with inflammatory pathway.
Keywords: S-resistin; Resistin; Adipogenesis; 3T3-L1; Cell differentiation; TNFα; Inflammation;

Mild hyperoxia limits hTR levels, telomerase activity, and telomere length maintenance in hTERT-transduced bone marrow endothelial cells by Christine E. Napier; Laura A. Veas; Chin-Yi Kan; Lisa M. Taylor; Jun Yuan; Victoria W. Wen; Alexander James; Tracey A. O'Brien; Richard B. Lock; Karen L. MacKenzie (1142-1153).
Reactivation of telomerase in endothelial cells (ECs) may be an effective approach to the treatment of vascular disorders associated with telomere attrition and EC senescence. However, overexpression of human telomerase reverse transcriptase (hTERT) does not prevent net telomere loss in ECs grown in standard culture medium with exposure to atmospheric oxygen (21% O2). Since these culture conditions are hyperoxic relative to normal tissue in vivo, where oxygen tension is estimated to be 1%–6%, we examined the effects of reduced exposure to oxidative stress (OS) on telomere length maintenance in hTERT-transduced bone marrow endothelial (BMhTERT) cells. Propagation of BMhTERT cells in the free radical scavenger, tert-butylhydroxylamine (tBN), and/or in 5% O2 increased telomerase enzyme activity and facilitated telomere length maintenance. The enhancement of telomerase activity correlated with higher levels of the telomerase RNA component (hTR). We also investigated the role of the telomere binding protein, TRF1, in telomere length regulation under alternate OS conditions. Inhibition of TRF1 function had no effect on telomere length in BMhTERT cells grown under standard culture conditions. However, alleviation of OS by growth in tBN plus 5% O2, elevated hTR levels, enhanced telomerase enzyme activity, and enabled progressive telomere lengthening. The direct impact of hTR levels on telomerase-mediated telomere lengthening was demonstrated by overexpression of hTR. BMhTERT cells transduced with hTR exhibited very high telomerase enzyme activity and underwent dramatic telomere lengthening under standard culture conditions. Overall, these results demonstrate that hTR levels are reduced by mild hyperoxia and limit telomerase-mediated telomere lengthening in hTERT-transduced ECs.►Alleviation of oxidative stress (OS) enhanced telomerase activity and facilitated telomere length maintenance in hTERT-transduced bone marrow endothelial cells (BMECs). ►Alterations in telomerase enzyme activity associated with varied exposure to OS correlated with hTR levels in hTERT-transduced BMECs. ►Inhibition of the telomere binding protein, TRF1, enabled telomere lengthening in hTERT-transduced BMECs only when OS was alleviated. ►Overexpression of hTR elevated telomerase activity and promoted telomere lengthening in hTERT-transduced BMECs maintained under standard conditions.
Keywords: Telomerase; Telomeres; Endothelial cell; TRF1; Oxidative stress; TERT; Telomerase RNA;

Absence of ataxin-3 leads to cytoskeletal disorganization and increased cell death by Ana-João Rodrigues; Maria do Carmo Costa; Teresa-Luísa Silva; Daniela Ferreira; Fernanda Bajanca; Elsa Logarinho; Patrícia Maciel (1154-1163).
Ataxin-3 (ATXN3) is a widely expressed protein that binds to ubiquitylated proteins, has deubiquitylating activity in vitro and is thought to modulate substrate degradation through the ubiquitin–proteasome pathway. Expansion of a polyglutamine tract in ATXN3 causes Machado–Joseph disease, a late-onset neurodegenerative disorder characterized by ubiquitin-positive aggregate formation and specific neuronal death. Although ATXN3 has been involved in transcriptional repression and in the ubiquitin–proteasome pathway, its biological function is still unknown.In this work, we show that depletion of ATXN3 using small-interference RNA (siRNA) causes a prominent phenotype in both human and mouse cell lines. A mild increase in ubiquitylation occurs and cells exhibit ubiquitin-positive foci, which is consistent with ATXN3 putative function as a deubiquitylating enzyme. In addition, siATXN3-silenced cells exhibit marked morphological changes such as rounder shape and loss of adhesion protrusions. At a structural level, the microtubule, microfilament and intermediate filament networks are severely compromised and disorganized. This cytoskeletal phenotype is reversible and dependent on ATXN3 levels. Cell–extracellular matrix connection is also affected in ATXN3-depleted cells as talin expression is reduced in the focal adhesions and lower levels of α-1 integrin subunit are expressed at their surface. Although the cytoskeletal and adhesion problems do not originate any major change in the cell cycle of siATXN3-depleted cells, cell death is increased in siATXN3 cultures compared to controls.In summary, in this work we show that the absence of ATXN3 leads to an overt cytoskeletal/adhesion defect raising the possibility that this protein may play a role in the cytoskeleton.
Keywords: Polyglutamine; Ubiquitin–proteasome; Sinocerebellar ataxia type 3; Tubulin;

Telomeres are dynamic DNA–protein complexes that protect the ends of linear chromosome. Telomere-binding proteins play crucial role in the maintenance of telomeres. HnRNP A3 has been shown recently to bind specifically to single-stranded telomeric DNA in vitro, although its in vivo telomere function remains unknown. In this study, the DNA-binding properties of hnRNP A3 in vitro as well as its putative role of telomere maintenance in vivo were investigated. The minimal sequence for hnRNP A3 binding to DNA was determined as an undecamer with the following consensus sequence 5′-[T/C]AG[G/T]NN[T/C]AG[G/T]N-3′. Confocal microscopy and chromatin-immunoprecipitation (ChIP) analyses showed that hnRNP A3 is associated with telomere in vivo. Knocking-down the expression of hnRNP A3 had no effect on telomere length maintenance and did not affect cell proliferation. In contrast, overexpression of hnRNP A3 resulted in the production of steady-state short telomeres in OECM1 cells. These results suggest that hnRNP A3 is associated with telomere in vivo and acts as a negative regulator of telomere length maintenance.
Keywords: Telomere; Telomerase; hnRNP A3; Telomere-binding proteins; RNA recognition motif;

Cell cycle control pathways act as conditioning factors for TK/GCV sensitivity in pancreatic cancer cells by Daniel Abate-Daga; Laura Garcia-Rodríguez; Lauro Sumoy; Cristina Fillat (1175-1185).
The suicide system TK/GCV is an enzyme/prodrug therapy that involves the transfer of the cDNA for the herpes simplex virus thymidine kinase gene (TK) into tumor cells which then sensitizes the cells to the non-toxic antiviral drug ganciclovir. Although extensively characterized, the suicide system TK/GCV conceals the details of its mechanism of action. In order to shed some light on this issue, we conducted experiments designed to identify key features of sensitive cells, as compared to cells that displayed reduced sensitivity to TK/GCV. Cell lines displaying different degrees of sensitivity underwent apoptotic cell death upon treatment with TK/GCV. S-phase delay, however, was almost exclusively restricted to sensitive cells and was impaired in a model of treatment-induced resistance. In this model genes with differential expression associated to induced resistance were identified. Noteworthy, two cell cycle-related genes (CCNE1 and GADD45) were functionally validated as conditioners of cellular sensitivity to TK/GCV. The relevance of cell cycle control was further demonstrated by experiments showing the association of Chk1 activation with greater TK/GCV cytotoxicity. Combination treatment with Chk1 inhibitor UCN-01 induced, in sensitive cells, an antagonistic effect on TK/GCV cytotoxicity highlighting the relevance of Chk1's activity on TK/GCV mechanism of action. These results reveal the relevance of cell cycle control pathways in the cytotoxicity induced by the TK/GCV system identifying candidate genes as conditioners of TK/GCV sensitivity. Moreover it points out, for the first time at Chk1 activation as a key factor to mediate TK/GCV cytotoxicity.►TK/GCV sensitive and resistant cancer cells display different properties in cell cycle control. ►Cell cycle control genes are deregulated in TK/GCV treatment-induced resistant cells. ►The cell cycle related genes CCNE1 and GADD45 act as conditioners of cellular sensitivity to TK/GCV. ►Chk1 activation is a key factor to mediate TK/GCV cytotoxicity.
Keywords: Thymidine kinase; Ganciclovir; Pancreatic cancer; Cell cycle; Chk1;

Histone deacetylase 7 (HDAC7) regulates myocyte migration and differentiation by Chengzhuo Gao; Yu Liu; Minh Lam; Hung-Ying Kao (1186-1197).
Class IIa HDACs including HDAC7 play a role in gene expression, cell differentiation, and animal development through their association with transcription factors such as myogenic enhancer factors 2 (MEF2s). In this study, we show that endogenous HDAC7 localizes to both the nucleus and the cytoplasm of C2C12 myoblasts but is exclusively retained in the cytoplasm of myotubes after completion of differentiation process. To elucidate the role of differential distribution of HDAC7 during myogenesis, we examined the effects of stably expressed HDAC7 mutants on myogenesis. Expression of nuclear-retained HDAC7 mutants significantly inhibits myogenesis in C2C12 cells and reduces the expression of muscle-specific myosin heavy chain (MHC) and myogenin. The inhibition in myocyte differentiation can be partially relieved by introduction of a mutation disrupting HDAC7:MEF2 interaction. Since phosphorylation of HDAC7 plays an important role in its nucleocytoplasmic shuttling, we further investigated the expression and distribution of phosphorylated HDAC7. To our surprise, the phosphorylation levels of HDAC7 at S344 and S479 were slightly decreased upon differentiation, whereas the phosphorylation of S178 was unchanged. Interestingly, a significant fraction of pS344- and/or pS479-HDAC7 localized to plasma membrane of myotubes. In addition, Ser178-phosphorylated (pS178) HDAC7 displays a predominately actin filament-like structure before muscle differentiation. Consistent with this notion, HDAC7 partially colocalized with actin filaments; in particular, pS178-HDAC7 largely colocalized with actin filaments as indicated by phalloidin counter staining in myocytes. Furthermore, C2C12 cells expressing nuclear-retained HDAC7 display defects in migration. Our results provide novel insight into the mechanisms that regulate myocyte differentiation and migration by controlling the subcellular distribution of HDAC7 in differentiating myoblasts.
Keywords: Nucleocytoplasmic shuttling; Histone deacetylase 7 (HDAC7); Phosphorylation; Myocyte differentiation; Cell migration; Myogenin;

Galectin-3: A novel substrate for c-Abl kinase by Vitaly Balan; Pratima Nangia-Makker; Young Suk Jung; Yi Wang; Avraham Raz (1198-1205).
Galectin-3, a ß-galactoside-binding lectin, is found in cellular and extracellular location of the cell and has pleiotropic biological functions such as cell growth, cell adhesion and cell-cell interaction. It may exhibit anti- or pro-apoptotic activity depending on its localization and post-translational modifications. Two important post-translational modifications of galectin-3 have been reported: its cleavage and phosphorylation. Cleavage of galectin-3 was reported to be involved with angiogenic potential and apoptotic resistance. Phosphorylation of galectin-3 regulates its sugar-binding ability. In this report we have identified novel tyrosine phosphorylation sites in galectin-3 as well as the kinase responsible for its phosphorylation. Our results demonstrate that tyrosines at positions 79, 107 and 118 can be phosphorylated in vitro and in vivo by c-Abl kinase. Tyrosine 107 is the main target of c-Abl. Expression of galectin-3 Y107F mutant in galectin-3 null SK-Br-3 cells leads to morphological changes and increased motility compared to wild type galectin-3. Further investigation is needed to better understand the functional significance of the novel tyrosine phosphorylated sites of galectin-3.►Galectin-3 Y79, 107 and 118 phosphorylated in vitro and in vivo by c-Abl kinase ►Y107 is the main target of c-Abl ►Expression of galectin-3 Y107F mutant leads to morphological changes and increased motility
Keywords: Phosphorylation; Kinase; Galectin-3;

The neuropeptides orexin-A/hypocretin-1 (Ox-A) and orexin-B/hypocretin-2 play an important role in the control of energy metabolism via either of two G-protein-coupled receptors, orexin receptor 1 (Ox1R) and 2. Despite its significant physiological functions, signaling via orexin receptors is still poorly characterized. The aim of this study was to improve our understanding of early signaling events triggered by the binding of Ox-A to Ox1R. Using phosphospecific antibodies, we observed that early kinase activation by Ox-A in a HEK293 cell line stably expressing Ox1R (HEKOx1R) included ERK1/2, PKCδ, and PKD1. Elevation of intracellular Ca2+ is a well-characterized response to Ox1R activation. Comparison of Ox-A-induced calcium elevation and PKD1 activation demonstrated that both responses are detectable soon after stimulation and increase in a dose-dependent manner, but inhibition of protein kinase C, when low Ox-A concentrations are used, affects them differently. PKD family of protein kinases has 3 members: PKD1, 2, and 3, which are all expressed in HEKOx1R cells. In response to stimulation of the cells with 1 nM Ox-A, both PKD1 and PKD3 are activated and increased in the plasma membrane, pointing at a possible role for these kinases in that cell compartment. Overexpression of either kinase-dead PKD1 or kinase-dead PKD3 disrupts Ox-A-induced calcium oscillations demonstrating the functional role of these kinases in modulating physiological responses to Ox-A.►Orexin-A/hypocretin-1 binding to orexin receptor 1 triggers a rapid intracellular calcium rise and activation of the protein kinases PKD1 and 3. This calcium response consists of oscillations which frequency and pattern are modulated by PKD1 and PKD3.
Keywords: Orexin (hypocretin) receptor 1; Orexin-A (hypocretin-1); PKC; PKD1 (PKCμ); PKD3 (PKCν);

A protein phosphatase feedback mechanism regulates the basal phosphorylation of Chk2 kinase in the absence of DNA damage by Luigi Carlessi; Giacomo Buscemi; Enrico Fontanella; Domenico Delia (1213-1223).
The checkpoint kinase Chk2 is an effector component of the ATM-dependent DNA damage response (DDR) pathway. The activation of Chk2 by genotoxic stress involves its phosphorylation on T68 by ATM and additional auto/transphosphorylations. Here we demonstrate that in unperturbed cells, chemical inhibition of Chk2 by VRX0466617 (VRX) enhances the phosphorylation of Chk2-T68 throughout the cell cycle phases. This event, dependent on the presence of ATM and catalytically functional Chk2, is not consequential to DNA damage, as neither γ-H2AX nuclear foci nor increased ATM activation is detected in VRX-treated cells, suggesting the involvement of other regulatory proteins. As serine/threonine protein phosphatases (PPs) regulate the phosphorylation and deactivation of proteins of the DDR pathway, we analyzed their role in phospho-T68-Chk2 regulation. We found that intracellular inhibition of PP1 and PP2A-like activities by okadaic acid markedly raised the accumulation of Chk2-pT68 without DNA damage induction, and this phenomenon was also seen when PP1-C, PP2A-C, and Wip1/PPM1D were simultaneously knockdown by siRNA. Altogether, these data indicate a novel mechanism in undamaged cells where PPs function to maintain the balance between ATM and its direct substrate Chk2 through a regulatory circuit.
Keywords: Cell cycle checkpoints; DNA damage responses; Chk2; Kinase inhibitors; Protein phosphatases;

Subdiffraction fluorescence imaging of biomolecular structure and distributions with quantum dots by Meike Heidbreder; Ulrike Endesfelder; Sebastian van de Linde; Simon Hennig; Darius Widera; Barbara Kaltschmidt; Christian Kaltschmidt; Mike Heilemann (1224-1229).
We introduce semiconductor quantum dot-based fluorescence imaging with ∼ 2-fold increased optical resolution in three dimensions as a method that allows both studying cellular structures and spatial organization of biomolecules in membranes and subcellular organelles. Target biomolecules are labelled with quantum dots via immunocytochemistry. The resolution enhancement is achieved by three-photon absorption of quantum dots and subsequent fluorescence emission from a higher-order excitonic state. Different from conventional multiphoton microscopy, this approach can be realized on any confocal microscope without the need for pulsed excitation light. We demonstrate quantum dot triexciton imaging (QDTI) of the microtubule network of U373 cells, 3D imaging of TNF receptor 2 on the plasma membrane of HeLa cells, and multicolor 3D imaging of mitochondrial cytochrome c oxidase and actin in COS-7 cells.
Keywords: Fluorescence microscopy; Superresolution; Quantum dot triexciton imaging; TNF receptor; Mitochondrial inner membrane;