BBA - Molecular Cell Research (v.1863, #4)

The effects of autophagy on cell death are highly contextual and either beneficial or deleterious. One prime example for this dual function of autophagy is evidenced by the cell responses to the BH3 mimetic AT-101 that is known to induce either apoptotic or autophagy-dependent cell death in different settings. Based on previous reports, we hypothesized that the expression levels of pro-survival Bcl-2 family members may be key determinants for the respective death mode induced by AT-101. Here we investigated the role of autophagy in the response of MCF7 breast cancer cells to AT-101. AT-101 treatment induced a prominent conversion of LC3-I to LC3-II and apoptotic cell death characterized by the appearance of Annexin-positive/PI-negative early apoptotic cells and PARP cleavage. Inhibition of the autophagy pathway, either through application of 3-MA or by lentiviral knockdown of ATG5, strongly potentiated cell death, indicating a pro-survival function of autophagy. Overexpression of wild type Bcl-xL significantly diminished the net amount of AT-101-induced cell death, but failed to alter the death-enhancing effects of the ATG5 knockdown. This was also observed with the organelle-specific variants Bcl-xL-ActA and Bcl-2-ActA (mitochondrial) as well as Bcl-xL-cb5 and Bcl-2-cb5 (ER) which all reduced AT-101-induced cell death, but did not affect the death-enhancing effects of 3-MA. Collectively, our data indicate that in apoptosis-proficient MCF7 cells, AT-101 triggers Bcl-2- and Bcl-xL-dependent apoptosis and a cytoprotective autophagy response that is independent of the expression and subcellular localization of Bcl-xL and Bcl-2.
Keywords: Apoptosis; Autophagy; BH3 mimetic; Bcl-2 family; Caspases;

Skp2 inhibits osteogenesis by promoting ubiquitin–proteasome degradation of Runx2 by Gatha Thacker; Yogesh Kumar; Mohd. Parvez Khan; Nidhi Shukla; Isha Kapoor; Jitendra Kumar Kanaujiya; Savita Lochab; Shakil Ahmed; Sabyasachi Sanyal; Naibedya Chattopadhyay; Arun Kumar Trivedi (510-519).
Osteogenic transcription factor Runx2 is essential for osteoblast differentiation. The activity of Runx2 is tightly regulated at transcriptional as well as post-translational level. However, regulation of Runx2 stability by ubiquitin mediated proteasomal degradation by E3 ubiquitin ligases is little-known. Here, for the first time we demonstrate that Skp2, an SCF family E3 ubiquitin ligase negatively targets Runx2 by promoting its polyubiquitination and proteasome dependent degradation. Co-immunoprecipitation studies revealed that Skp2 physically interacts with Runx2 both in a heterologous as well as physiologically relevant system. Functional consequences of Runx2–Skp2 physical interaction were then assessed by promoter reporter assay. We show that Skp2-mediated downregulation of Runx2 led to reduced Runx2 transactivation and osteoblast differentiation. On the contrary, inhibition of Skp2 restored Runx2 levels and promoted osteoblast differentiation. We further show that Skp2 and Runx2 proteins are co-expressed and show inverse relation in vivo such as in lactating, ovariectomized and estrogen-treated ovariectomized animals. Together, these data demonstrate that Skp2 targets Runx2 for ubiquitin mediated degradation and hence negatively regulate osteogenesis. Therefore, the present study provides a plausible therapeutic target for osteoporosis or cleidocranial dysplasia caused by the heterozygous mutation of Runx2 gene.
Keywords: Runx2; Osteoblast; Skp2; Polyubiquitination; Osteogenesis;

miR-29c-3p promotes senescence of human mesenchymal stem cells by targeting CNOT6 through p53–p21 and p16–pRB pathways by Jin Shang; Yuan Yao; Xin Fan; Lei Shangguan; Jie Li; Huan Liu; Yue Zhou (520-532).
Mesenchymal stem cells (MSCs) are important seed cells for tissue engineering and are promising targets for cell-based therapies. However, the replicative senescence of MSCs during in vitro culture limits their research and clinical applications. The molecular mechanisms underlying the replicative senescence of MSCs are not fully understood. Evidence suggests that miRNAs play important roles in replicative senescence. A microarray analysis found that the miR-29c-3p level was significantly increased during the MSC senescence process. In our study, we investigated the roles of miR-29c-3p in senescence of MSCs. We cultured MSCs for long periods of time, up and down-regulated the miR-29c-3p expression in MSCs, and examined the senescent phenotype changes. The over-expression of miR-29c-3p led to enhanced senescence-associated-β-galactosidase (SA-β-gal) staining, senescence associated secretory phenotype (SASP), senescence associated heterochromatic foci (SAHF), reduced proliferation ability, retarded osteogenic differentiation and corresponding changes in senescence markers, whereas the miR-29c-3p down-regulation had the opposite results. Dual-luciferase reporter assays demonstrated that CNOT6 is the target gene of miR-29c-3p. Knockdown of CNOT6 confirmed its inhibitory effects on the senescence of MSCs. In addition, Western blot results showed that both the p53–p21 and the p16–pRB pathways were activated during the miR-29c-3p-induced senescence of MSCs. In conclusion, our results demonstrate that miR-29c-3p promotes the senescence of MSCs by targeting CNOT6 through p53–p21 and p16–pRB pathways and highlight the contribution of post-transcriptional regulation to stem cell senescence.
Keywords: Replicative senescence; Mesenchymal stem cell; MicroRNA; Tissue engineering;

A long non-coding RNA, BC048612 and a microRNA, miR-203 coordinate the gene expression of neuronal growth regulator 1 (NEGR1) adhesion protein by Prameet Kaur; Jun Rong Tan; Dwi Setyowati Karolina; Sugunavathi Sepramaniam; Arunmozhiarasi Armugam; Peter T-H Wong; Kandiah Jeyaseelan (533-543).
The regulatory roles for non-coding RNAs, the long non-coding RNAs and microRNAs, are emerging as crucial determinants of central nervous system development and function. Neuronal growth regulator 1 (NEGR1) is a cell adhesion molecule that has been shown to play an important role in neurite outgrowth during neuronal development. Precise expression of the Negr1 gene is crucial for proper brain development and is dysregulated during brain injury. Hence, we attempted to elucidate the non-coding RNAs that control Negr1 gene expression.A long non-coding RNA, BC048612, transcribed from the bidirectional GC-rich Negr1 gene promoter was found to influence Negr1 mRNA expression. In vitro knockdown of the long non-coding RNA resulted in significant down-regulation of Negr1 mRNA expression, NEGR1 protein levels and neurite length whereas over-expression enhanced Negr1 mRNA expression, NEGR1 protein levels and increased neurite length. Meanwhile, another non-coding RNA, microRNA-203, was found to target the 3′ untranslated region of the Negr1 mRNA. Inhibition of microRNA-203 led to increased expression of Negr1 mRNA, elevated NEGR1 protein levels and increased neurite length. Conversely, microRNA-203 over-expression decreased the level of Negr1 mRNA, NEGR1 protein and neurite length. Neither microRNA-203 nor the long non-coding RNA, BC048612 could influence each other's expression. Hence, the long non-coding RNA, BC048612, and microRNA-203 were determined to be positive and negative regulators of Negr1 gene expression respectively. These processes have a direct effect on NEGR1 protein levels and neurite length, thus highlighting the importance of the regulatory non-coding RNAs in modulating Negr1 gene expression for precise neuronal development.
Keywords: Cell adhesion molecules; Negr1; Non-coding RNA; lncRNA; miRNA; Neuronal maturation;

The signaling module cAMP/Epac/Rap1/PLCε/IP3 mobilizes acrosomal calcium during sperm exocytosis by Ornella Lucchesi; María C. Ruete; Matías A. Bustos; María F. Quevedo; Claudia N. Tomes (544-561).
Exocytosis of the sperm's single secretory granule, or acrosome, is a regulated exocytosis triggered by components of the egg's investments. In addition to external calcium, sperm exocytosis (termed the acrosome reaction) requires cAMP synthesized endogenously and calcium mobilized from the acrosome through IP3-sensitive channels. The relevant cAMP target is Epac. In the first part of this paper, we present a novel tool (the TAT-cAMP sponge) to investigate cAMP-related signaling pathways in response to progesterone as acrosome reaction trigger. The TAT-cAMP sponge consists of the cAMP-binding sites of protein kinase A regulatory subunit RIβ fused to the protein transduction domain TAT of the human immunodeficiency virus-1. The sponge permeated into sperm, sequestered endogenous cAMP, and blocked exocytosis. Progesterone increased the population of sperm with Rap1-GTP, Rab3-GTP, and Rab27-GTP in the acrosomal region; pretreatment with the TAT-cAMP sponge prevented the activation of all three GTPases. In the second part of this manuscript, we show that phospholipase Cε (PLCε) is required for the acrosome reaction downstream of Rap1 and upstream of intra-acrosomal calcium mobilization. Last, we present direct evidence that cAMP, Epac, Rap1, and PLCε are necessary for calcium mobilization from sperm's secretory granule. In summary, we describe here a pathway that connects cAMP to calcium mobilization from the acrosome during sperm exocytosis. Never before had direct evidence for each step of the cascade been put together in the same study.
Keywords: Acrosome reaction; Calcium; TAT-cAMP sponge; Epac; Exocytosis; Protein transduction;

Combination of galectin inhibitor GCS-100 and BH3 mimetics eliminates both p53 wild type and p53 null AML cells by Peter P. Ruvolo; Vivian R. Ruvolo; Christopher B. Benton; Ahmed AlRawi; Jared K. Burks; Wendy Schober; James Rolke; George Tidmarsh; Numsen Hail; R. Eric Davis; Michael Andreeff (562-571).
Galectin 3 (LGALS3) expression is prognostic for poor survival in acute myeloid leukemia (AML) patients. GCS-100 is a novel galectin inhibitor that may prove useful for AML therapy. In this study, we found that GCS-100 induced apoptosis in AML cells. The agent reduced MCL-1 expression suggesting that GCS-100 could be more effective when combined with a BH3 mimetic. Indeed, potent synergistic cytotoxicity was achieved when GCS-100 was combined with ABT-737 or ABT-199. Furthermore, the GCS-100/ABT-199 combination was effective against primary AML blast cells from patients with FLT3 ITD mutations, which is another prognostic factor for poor outcome in AML. This activity may involve wild-type p53 as shRNA knockdown of LGALS3 or galectin 1 (LGALS1) sensitized wild-type p53 OCI-AML3 cells to GCS-100/ABT-737-induced apoptosis to a much greater extent than p53 null THP-1 cells. Suppression of LGALS3 by shRNA inhibited MCL-1 expression in OCI-AML3 cells, but not THP-1 cells, suggesting the induced sensitivity to ABT-737 may involve a MCL-1 mediated mechanism. OCI-AML3 cells with LGALS1 shRNA were also sensitized to ABT-737. However, these cells exhibited increased MCL-1 expression, so MCL-1 reduction is apparently not required in this process. A role for p53 appears important as GCS-100 induces p53 expression and shRNA knockdown of p53 protected OCI-AML3 cells from the cytotoxic effects of the GCS-100/ABT-737 treatment combination. Our results suggest that galectins regulate a survival axis in AML cells, which may be targeted via combined inhibition with drugs such as GCS-100 and ABT-199.
Keywords: Galectin; GCS-100; MCL-1; p53; Signal transduction; Leukemia;

Cellular functions of programmed cell death 5 by Ge Li; Dalong Ma; Yingyu Chen (572-580).
Programmed cell death 5 (PDCD5) was originally identified as an apoptosis-accelerating protein that is widely expressed and has been well conserved during the process of evolution. PDCD5 has complex biological functions, including programmed cell death and immune regulation. It can accelerate apoptosis in different type of cells in response to different stimuli. During this process, PDCD5 rapidly translocates from the cytoplasm to the nucleus. PDCD5 regulates the activities of TIP60, HDAC3, MDM2 and TP53 transcription factors. These proteins form part of a signaling network that is disrupted in most, if not all, cancer cells. Recent evidence suggests that PDCD5 participates in immune regulation by promoting regulatory T cell function via the PDCD5–TIP60–FOXP3 pathway. The stability and expression of PDCD5 are finely regulated by other molecules, such as NF-κB p65, OTUD5, YAF2 and DNAJB1. PDCD5 is phosphorylated by CK2 at Ser119, which is required for nuclear translocation in response to genotoxic stress. In this review, we describe what is known about PDCD5 and its cellular functions.
Keywords: Programmed cell death 5; PDCD5; Apoptosis; Tumor; Immune regulation; Autoimmune diseases;

Melanoma antigen-D2: A nucleolar protein undergoing delocalization during cell cycle and after cellular stress by Céline Pirlot; Marc Thiry; Charlotte Trussart; Emmanuel Di Valentin; Jacques Piette; Yvette Habraken (581-595).
Melanoma antigen D2 (MAGE-D2) is recognized as a cancer diagnostic marker; however, it has poorly characterized functions. Here, we established its intracellular localization and shuttling during cell cycle progression and in response to cellular stress. In normal conditions, MAGE-D2 is present in the cytoplasm, nucleoplasm, and nucleoli. Within the latter, MAGE-D2 is mostly found in the granular and the dense fibrillar components, and it interacts with nucleolin. Transfection of MAGE-D2 deletion mutants demonstrated that Δ203–254 leads to confinement of MAGE-D2 to the cytoplasm, while Δ248–254 prevents its accumulation in nucleoli but still allows its presence in the nucleoplasm. Consequently, this short sequence belongs to a nucleolar localization signal. MAGE-D2 deletion does not alter the nucleolar organization or rRNA levels. However, its intracellular localization varies with the cell cycle in a different kinetic than nucleolin. After genotoxic and nucleolar stresses, MAGE-D2 is excluded from nucleoli and concentrates in the nucleoplasm. We demonstrated that its camptothecin-related delocalization results from two distinct events: a rapid nucleolar release and a slower phospho-ERK-dependent cytoplasm to nucleoplasm translocation, which results from an increased flux from the cytoplasm to nucleoplasm. In conclusion, MAGE-D2 is a dynamic protein whose shuttling properties could suggest a role in cell cycle regulation.
Keywords: Melanoma antigen protein; Nucleolus; Cell cycle; Cellular stress; Camptothecin; MAP kinases;

Clock genes-dependent acetylation of complex I sets rhythmic activity of mitochondrial OxPhos by Olga Cela; Rosella Scrima; Valerio Pazienza; Giuseppe Merla; Giorgia Benegiamo; Bartolomeo Augello; Sabino Fugetto; Marta Menga; Rosa Rubino; Luise Fuhr; Angela Relógio; Claudia Piccoli; Gianluigi Mazzoccoli; Nazzareno Capitanio (596-606).
Physiology of living beings show circadian rhythms entrained by a central timekeeper present in the hypothalamic suprachiasmatic nuclei. Nevertheless, virtually all peripheral tissues hold autonomous molecular oscillators constituted essentially by circuits of gene expression that are organized in negative and positive feed-back loops. Accumulating evidence reveals that cell metabolism is rhythmically controlled by cell-intrinsic molecular clocks and the specific pathways involved are being elucidated. Here, we show that in vitro-synchronized cultured cells exhibit BMAL1-dependent oscillation in mitochondrial respiratory activity, which occurs irrespective of the cell type tested, the protocol of synchronization used and the carbon source in the medium. We demonstrate that the rhythmic respiratory activity is associated to oscillation in cellular NAD content and clock-genes-dependent expression of NAMPT and Sirtuins 1/3 and is traceable back to the reversible acetylation of a single subunit of the mitochondrial respiratory chain Complex I. Our findings provide evidence for a new interlocked transcriptional-enzymatic feedback loop controlling the molecular interplay between cellular bioenergetics and the molecular clockwork.Display Omitted
Keywords: Mitochondria; Clock-genes; Oxidative phosphorylation; Complex I; NAD; Sirtuins;

CXCR4 signaling is controlled by immobilization at the plasma membrane by Elena Beletkaia; Susanne F. Fenz; Wim Pomp; B. Ewa Snaar-Jagalska; Pancras W.C. Hogendoorn; Thomas Schmidt (607-616).
Understanding of the regulation mechanisms of CXCR4 signaling is essential for revealing its role in physiological and pathological processes. Though biochemical pathways following CXCR4 activation by its ligand CXCL12 are well established, knowledge about the receptor dynamics on the plasma membrane remains limited. Here we used Ewing sarcoma-derived cells to unravel the processes that are involved in regulating CXCR4 dynamics on the plasma membrane during receptor signaling. Single-molecule epi-fluorescence microscopy showed that CXCR4 was present in monomeric state on the plasma membrane independent of receptor stimulation. However, upon activation freely diffusing receptors were immobilized in a ligand concentration-dependent manner. CXCR4 immobilization was strongly correlated with the ability for G-protein signaling and was a precursor of subsequent endocytotic events. Our data suggest that, a balanced regulation of G-protein dependent and independent pathways is required for controlling CXCR4 receptor mobility, and potentially subsequent controlled signal transduction.
Keywords: CXCR4; Single molecule; Signaling; Endocytosis;

A stromal interaction molecule 1 variant up-regulates matrix metalloproteinase-2 expression by strengthening nucleoplasmic Ca2+ signaling by Fengrong Chen; Liping Zhu; Lei Cai; Jiwei Zhang; Xianqin Zeng; Jiansha Li; Yuan Su; Qinghua Hu (617-629).
Very recent studies hold promise to reveal the role of stromal interaction molecule 1 (STIM1) in non-store-operated Ca2+ entry. Here we showed that in contrast to cytoplasmic membrane redistribution as previously noted, human umbilical vein endothelial STIM1 with a T-to-C nucleotide transition resulting in an amino acid substitution of leucine by proline in the signal peptide sequence translocated to perinuclear membrane upon intracellular Ca2+ depletion, amplified nucleoplasmic Ca2+ signaling through ryanodine receptor-dependent pathway, and enhanced the subsequent cAMP responsive element binding protein activity, matrix metalloproteinase-2 (MMP-2) gene expression, and endothelial tube forming. The abundance of mutated STIM1 and the MMP-2 expression were higher in native human umbilical vein endothelial cells of patients with gestational hypertension than controls and were significantly correlated with blood pressure. These findings broaden our understanding about structure–function bias of STIM1 and offer unique insights into its application in nucleoplasmic Ca2+, MMP-2 expression, endothelial dysfunction, and pathophysiological mechanism(s) of gestational hypertension.
Keywords: STIM1; Matrix metalloproteinase-2; Nucleoplasmic Ca2+;  CAMP responsive element binding protein; Gene expression;

TCTP regulates spindle microtubule dynamics by stabilizing polar microtubules during mouse oocyte meiosis by Hyuk-Joon Jeon; Seung Yeop You; Yong Seok Park; Jong Wook Chang; Jae-Sung Kim; Jeong Su Oh (630-637).
Dynamic changes in spindle structure and function are essential for maintaining genomic integrity during the cell cycle. Spindle dynamics are highly dependent on several microtubule-associated proteins that coordinate the dynamic behavior of microtubules, including microtubule assembly, stability and organization. Here, we show that translationally controlled tumor protein (TCTP) is a novel microtubule-associated protein that regulates spindle dynamics during meiotic maturation. TCTP was expressed and widely distributed in the cytoplasm with strong enrichment at the spindle microtubules during meiosis. TCTP was found to be phosphorylated during meiotic maturation, and was exclusively localized to the spindle poles. Knockdown of TCTP impaired spindle organization without affecting chromosome alignment. These spindle defects were mostly due to the destabilization of the polar microtubules. However, the stability of kinetochore microtubules attached to chromosomes was not affected by TCTP knockdown. Overexpression of a nonphosphorylable mutant of TCTP disturbed meiotic maturation, stabilizing the spindle microtubules. In addition, Plk1 was decreased by TCTP knockdown. Taken together, our results demonstrate that TCTP is a microtubule-associating protein required to regulate spindle microtubule dynamics during meiotic maturation in mouse oocytes.Display Omitted
Keywords: Oocyte; Meiosis; TCTP; Microtubule; Spindle;

The acetylenic tricyclic bis(cyano enone), TBE-31, targets microtubule dynamics and cell polarity in migrating cells by Eddie Chan; Akira Saito; Tadashi Honda; Gianni M. Di Guglielmo (638-649).
Cell migration is dependent on the microtubule network for structural support as well as for the proper delivery and positioning of polarity proteins at the leading edge of migrating cells. Identification of drugs that target cytoskeletal-dependent cell migration and protein transport in polarized migrating cells is important in understanding the cell biology of normal and tumor cells and can lead to new therapeutic targets in disease processes. Here, we show that the tricyclic compound TBE-31 directly binds to tubulin and interferes with microtubule dynamics, as assessed by end binding 1 (EB1) live cell imaging. Interestingly, this interference is independent of in vitro tubulin polymerization. Using immunofluorescence microscopy, we also observed that TBE-31 interferes with the polarity of migratory cells. The polarity proteins Rac1, IQGAP and Tiam1 were localized at the leading edge of DMSO-treated migrating cell, but were observed to be in multiple protrusions around the cell periphery of TBE-31-treated cells. Finally, we observed that TBE-31 inhibits the migration of Rat2 fibroblasts with an IC50 of 0.75 μM. Taken together, our results suggest that the inhibition of cell migration by TBE-31 may result from the improper maintenance of cell polarity of migrating cells.
Keywords: Cytoskeleton; Rac1; Live cell imaging; Immunofluorescence microscopy;

Posttranslational modifications including histone methylation regulate gene transcription through directly affecting the structure of chromatin. Trimethylation of histone H3K27 (H3K27me3) contributes to gene silencing and the histone demethylase Jumonji domain-containing 3 (Jmjd3) specifically removes the methylation of H3K27me3, followed by the activation of gene expression. In the present study, we explored the roles of Jmjd3 in regulating osteoblast apoptosis. Knockdown of Jmjd3 promoted osteoblast apoptosis induced by serum deprivation with decreased mitochondrial membrane potential and increased levels of caspase-3 activation, PARP cleavage, and DNA fragmentation. B cell lymphoma-2 (Bcl-2), an anti-apoptotic protein, was down-regulated by knockdown of Jmjd3 through retaining H3K27me3 on its promoter region. Knockdown of Jmjd3 increased the pro-apoptotic activity of Bim through inhibiting ERK-dependent phosphorylation of Bim. Protein kinase D1 (PKD1), which stimulates ERK phosphorylation, decreased in the Jmjd3-knockdown cells and introduction of PKD1 relieved osteoblast apoptosis in the Jmjd3-knockdown cells through increasing ERK-regulated Bim phosphorylation. These results suggest that Jmjd3 regulates osteoblast apoptosis through targeting Bcl-2 expression and Bim phosphorylation.Display Omitted
Keywords: Jmjd3; Osteoblast apoptosis; Bcl-2; Bim; Protein kinase D1; Histone demethylation;

The lipopolysaccharide-binding protein (LBP) is critically involved in innate immune responses to Gram-negative infections. We show here that human peripheral blood-derived monocytes, but not lymphocytes, stain positive for endogenous LBP on the cell surface. Studies on human macrophages demonstrate LBP binding at normal serum concentrations of 1–10 μg/ml. Binding was increased in a concentration-dependent manner by lipopolysaccharide (LPS). Fluorescence quenching experiments and confocal microscopy revealed constitutive and LPS-induced internalization of LBP by macrophages. Experiments with macrophages and HEK293 cell lines showed that binding and uptake of LBP do not depend on the LPS receptors CD14 and TLR4/MD-2. Fractionation of Triton X-100 solubilized cytoplasmic membranes revealed that LBP was primarily localized in non-raft domains under resting conditions. Cellular LPS stimulation elevated LBP levels and induced enrichment in fractions marking the transition between non-raft and raft domains. LBP was found to colocalize with LPS at the cytoplasmic membrane and in intracellular compartments of macrophages. In macrophages stimulated with LPS and ATP for inflammasome activation, LBP was observed in close vicinity to activated caspases. Furthermore, LBP conferred IL-1β production by LPS in the absence of ATP. These data establish that LBP serves not only as an extracellular LPS shuttle but in addition facilitates intracellular transport of LPS. This observation adds a new function to this central immune regulator of LPS biology and raises the possibility for a role of LBP in the delivery of LPS to TLR4-independent intracellular receptors.
Keywords: Lipopolysaccharide; Lipopolysaccharide-binding protein; Macrophage; TLR4;

Direct non transcriptional role of NF-Y in DNA replication by Paolo Benatti; Silvia Belluti; Benoit Miotto; Julia Neusiedler; Diletta Dolfini; Marjorie Drac; Valentina Basile; Etienne Schwob; Roberto Mantovani; J. Julian Blow; Carol Imbriano (673-685).
NF-Y is a heterotrimeric transcription factor, which plays a pioneer role in the transcriptional control of promoters containing the CCAAT-box, among which genes involved in cell cycle regulation, apoptosis and DNA damage response. The knock-down of the sequence-specific subunit NF-YA triggers defects in S-phase progression, which lead to apoptotic cell death.Here, we report that NF-Y has a critical function in DNA replication progression, independent from its transcriptional activity. NF-YA colocalizes with early DNA replication factories, its depletion affects the loading of replisome proteins to DNA, among which Cdc45, and delays the passage from early to middle-late S phase. Molecular combing experiments are consistent with a role for NF-Y in the control of fork progression. Finally, we unambiguously demonstrate a direct non-transcriptional role of NF-Y in the overall efficiency of DNA replication, specifically in the DNA elongation process, using a Xenopus cell-free system.Our findings broaden the activity of NF-Y on a DNA metabolism other than transcription, supporting the existence of specific TFs required for proper and efficient DNA replication.
Keywords: NF-Y; CCAAT-binding factor; Transcription factors; DNA replication; Xenopus cell-free system;

How does α-actinin-3 deficiency alter muscle function? Mechanistic insights into ACTN3, the ‘gene for speed’ by Fiona X.Z. Lee; Peter J. Houweling; Kathryn N. North; Kate G.R. Quinlan (686-693).
An estimated 1.5 billion people worldwide are deficient in the skeletal muscle protein α-actinin-3 due to homozygosity for the common ACTN3 R577X polymorphism. α-Actinin-3 deficiency influences muscle performance in elite athletes and the general population. The sarcomeric α-actinins were originally characterised as scaffold proteins at the muscle Z-line. Through studying the Actn3 knockout mouse and α-actinin-3 deficient humans, significant progress has been made in understanding how ACTN3 genotype alters muscle function, leading to an appreciation of the diverse roles that α-actinins play in muscle. The α-actinins interact with a number of partner proteins, which broadly fall into three biological pathways—structural, metabolic and signalling. Differences in functioning of these pathways have been identified in α-actinin-3 deficient muscle that together contributes to altered muscle performance in mice and humans. Here we discuss new insights that have been made in understanding the molecular mechanisms that underlie the consequences of α-actinin-3 deficiency.
Keywords: Athletic performance; Common human polymorphism; Molecular mechanism; Protein interactions; α-Actinin-3 deficiency; Muscle function;

Activation of autophagy by globular adiponectin is required for muscle differentiation by Tania Gamberi; Alessandra Modesti; Francesca Magherini; Donna M. D'Souza; Thomas Hawke; Tania Fiaschi (694-702).
Regulated autophagy is a critical component for a healthy skeletal muscle mass, such that dysregulation of the autophagic processes correlates with severe myopathies. Thus, defining the biological molecules involved in the autophagic processes within skeletal muscle is of great importance. Here we demonstrate that globular adiponectin (gAd) activates autophagy in skeletal muscle myoblasts via an AMPK-dependent mechanism. Activation of autophagy through gAd promotes myoblast survival and apoptosis inhibition during serum starvation and the gAd-activated autophagy orchestrates the myogenic properties of the hormone. Consistent with this conclusion, inhibition of gAd-activated autophagy by both a pharmacological (chloroquine) or siRNA approach greatly inhibited muscle differentiation, as demonstrated by reductions in myosin heavy chain expression and myotube formation. Further support for the role of adiponectin in autophagy comes from the skeletal muscles of adiponectin KO mice which display decreased LC3 II expression and a myopathic phenotype (heterogeneous fiber sizes, numerous central nuclei). Overall, these findings demonstrate that gAd activates autophagy in myoblasts and that gAd-activated autophagy drives the myogenic properties of this hormone.
Keywords: Globular adiponectin; Myogenesis; Autophagy;

Perrin and Förster unified: Dual-laser triple-polarization FRET (3polFRET) for interactions at the Förster-distance and beyond by Tamás Ungvári; Péter Gogolák; Miklós Bagdány; László Damjanovich; László Bene (703-716).
Dual laser flow cytometric energy transfer (FCET) – elaborated by Trón et al. in 1984 – is an efficient and rapid way of measuring FRET on large cell populations. FRET efficiency and the donor and acceptor concentrations are determined from one donor and two acceptor signals. In this communication this method is extended towards the domain of receptor dynamics by the detection of polarized components of the three intensities. By enabling a complete description of the proximity and dynamics of FRET-systems, the new measuring scheme allows a more refined description of both the structure and dynamics of cell surface receptor clusters at the nano-scale and beyond. Associated donor fraction, limiting anisotropy and rotational correlation time of the donor, acceptor anisotropy and cell-by-cell estimation of the orientation factor for FRET (κ2) are available in the steady state on a single FRET sample in a very rapid and statistically efficient way offered by flow cytometry. For a more sensitive detection of conformational changes the “polarized FRET indices” – quantities composed from FRET efficiency and anisotropies – are proposed. The method is illustrated by measurements on a FRET system with changing FRET-fraction and on a two donor-one acceptor-system, when the existence of receptor trimers are proven by the detection of “hetero-FRET induced homo-FRET relief”, i.e. the diminishing of homo-FRET between the two donors in the presence of a donor quencher. The method also offers higher sensitivity for assessing conformational changes at the nano-scale, due to its capability for the simultaneous detection of changes of proximity and relative orientations of the FRET donor and acceptor. Although the method has been introduced in the context of FRET, it is more general: It can be used for monitoring triple-anisotropy correlations also in those cases when FRET actually does not occur, e.g. for interactions occuring beyond the Förster-distance R0. Interpretation of κ2 has been extended.
Keywords: Triple-anisotropy correlations; Donor anisotropy; Acceptor anisotropy; Orientation factor for FRET; Homo-FRET relief; FRET-fraction;

Cell surface syndecan-1 contributes to binding and function of macrophage migration inhibitory factor (MIF) on epithelial tumor cells by Tobias Pasqualon; Hongqi Lue; Sabine Groening; Jessica Pruessmeyer; Holger Jahr; Bernd Denecke; Jürgen Bernhagen; Andreas Ludwig (717-726).
Surface expressed proteoglycans mediate the binding of cytokines and chemokines to the cell surface and promote migration of various tumor cell types including epithelial tumor cells. We here demonstrate that binding of the chemokine-like inflammatory cytokine macrophage migration inhibitory factor (MIF) to epithelial lung and breast tumor cell lines A549 and MDA-MB231 is sensitive to enzymatic digestion of heparan sulphate chains and competitive inhibition with heparin. Moreover, MIF interaction with heparin was confirmed by chromatography and a structural comparison indicated a possible heparin binding site. These results suggested that proteoglycans carrying heparan sulphate chains are involved in MIF binding. Using shRNA-mediated gene silencing, we identified syndecan-1 as the predominant proteoglycan required for the interaction with MIF. MIF binding was decreased by induction of proteolytic shedding of syndecan-1, which could be prevented by inhibition of the metalloproteinases involved in this process. Finally, MIF induced the chemotactic migration of A549 cells, wound closure and invasion into matrigel without affecting cell proliferation. These MIF-induced responses were abrogated by heparin or by silencing of syndecan-1. Thus, our study indicates that syndecan-1 on epithelial tumor cells promotes MIF binding and MIF-mediated cell migration. This may represent a relevant mechanism through which MIF enhances tumor cell motility and metastasis.
Keywords: Proteoglycan; Syndecan; Chemokine; Heparin; Tumor cell migration; Macrophage migration inhibitory factor; Metalloproteinase;

Redox cycling metals: Pedaling their roles in metabolism and their use in the development of novel therapeutics by Danuta S. Kalinowski; Christian Stefani; Shinya Toyokuni; Tomas Ganz; Gregory J. Anderson; Nathan V. Subramaniam; Debbie Trinder; John K. Olynyk; Anita Chua; Patric J. Jansson; Sumit Sahni; Darius J.R. Lane; Angelica M. Merlot; Zaklina Kovacevic; Michael L.H. Huang; C. Soon Lee; Des R. Richardson (727-748).
Essential metals, such as iron and copper, play a critical role in a plethora of cellular processes including cell growth and proliferation. However, concomitantly, excess of these metal ions in the body can have deleterious effects due to their ability to generate cytotoxic reactive oxygen species (ROS). Thus, the human body has evolved a very well-orchestrated metabolic system that keeps tight control on the levels of these metal ions. Considering their very high proliferation rate, cancer cells require a high abundance of these metals compared to their normal counterparts. Interestingly, new anti-cancer agents that take advantage of the sensitivity of cancer cells to metal sequestration and their susceptibility to ROS have been developed. These ligands can avidly bind metal ions to form redox active metal complexes, which lead to generation of cytotoxic ROS. Furthermore, these agents also act as potent metastasis suppressors due to their ability to up-regulate the metastasis suppressor gene, N-myc downstream regulated gene 1. This review discusses the importance of iron and copper in the metabolism and progression of cancer, how they can be exploited to target tumors and the clinical translation of novel anti-cancer chemotherapeutics.
Keywords: Iron; Copper; Cancer; Thiosemicarbazones; Dp44mT; DpC; Bis(thiosemicarbazones);

Vestiges of Ent3p/Ent5p function in the giardial epsin homolog by Constanza Feliziani; Javier Valdez Taubas; Sofía Moyano; Gonzalo Quassollo; Joanna E. Poprawski; Beverly Wendland; Maria C. Touz (749-759).
An accurate way to characterize the functional potential of a protein is to analyze recognized protein domains encoded by the genes in a given group. The epsin N-terminal homology (ENTH) domain is an evolutionarily conserved protein module found primarily in proteins that participate in clathrin-mediated trafficking. In this work, we investigate the function of the single ENTH-containing protein from the protist Giardia lamblia by testing its function in Saccharomyces cerevisiae. This protein, named GlENTHp (for G. lamblia ENTH protein), is involved in Giardia in endocytosis and in protein trafficking from the ER to the vacuoles, fulfilling the function of the ENTH proteins epsin and epsinR, respectively. There are two orthologs of epsin, Ent1p and Ent2p, and two orthologs of epsinR, Ent3p and Ent5p in S. cerevisiae. Although the expression of GlENTHp neither complemented growth in the ent1Δent2Δ mutant nor restored the GFP-Cps1 vacuolar trafficking defect in ent3Δent5Δ, it interfered with the normal function of Ent3/5 in the wild-type strain. The phenotype observed is linked to a defect in Cps1 localization and α-factor mating pheromone maturation. The finding that GlENTHp acts as dominant negative epsinR in yeast cells reinforces the phylogenetic data showing that GlENTHp belongs to the epsinR subfamily present in eukaryotes prior to their evolution into different taxa.
Keywords: ENTH motif; Vacuole; Endocytosis; Giardia lamblia; Yeast; Vesicle transport;

VRK1 phosphorylates and protects NBS1 from ubiquitination and proteasomal degradation in response to DNA damage by Diana M. Monsalve; Ignacio Campillo-Marcos; Marcella Salzano; Marta Sanz-García; Lara Cantarero; Pedro A. Lazo (760-769).
NBS1 is an early component in DNA-Damage Response (DDR) that participates in the initiation of the responses aiming to repair double-strand breaks caused by different mechanisms. Early steps in DDR have to react to local alterations in chromatin that are induced by DNA damage. NBS1 participates in the early detection of DNA damage and functions as a platform for the recruitment and assembly of components that are sequentially required for the repair process. In this work we have studied whether the VRK1 chromatin kinase can affect the activation of NBS1 in response to DNA damage induced by ionizing radiation. VRK1 is forming a basal preassembled complex with NBS1 in non-damaged cells. Knockdown of VRK1 resulted in the loss of NBS1 foci induced by ionizing radiation, an effect that was also detected in cell-cycle arrested cells and in ATM (−/−) cells. The phosphorylation of NBS1 in Ser343 by VRK1 is induced by either doxorubicin or IR in ATM (−/−) cells. Phosphorylated NBS1 is also complexed with VRK1. NBS1 phosphorylation by VRK1 cooperates with ATM. This phosphorylation of NBS1 by VRK1 contributes to the stability of NBS1 in ATM (−/−) cells, and the consequence of its loss can be prevented by treatment with the MG132 proteasome inhibitor of RNF8. We conclude that VRK1 regulation of NBS1 contributes to the stability of the repair complex and permits the sequential steps in DDR.Display Omitted
Keywords: DNA damage foci; Phosphorylation; Signaling; DNA double strand breaks; Nibrin; NBN;

Roads to melanoma: Key pathways and emerging players in melanoma progression and oncogenic signaling by Jasmina Paluncic; Zaklina Kovacevic; Patric J. Jansson; Danuta Kalinowski; Angelika M. Merlot; Michael L.-H. Huang; Hiu Chuen Lok; Sumit Sahni; Darius J.R. Lane; Des R. Richardson (770-784).
Melanoma has markedly increased worldwide during the past several decades in the Caucasian population and is responsible for 80% of skin cancer deaths. Considering that metastatic melanoma is almost completely resistant to most current therapies and is linked with a poor patient prognosis, it is crucial to further investigate potential molecular targets. Major cell-autonomous drivers in the pathogenesis of this disease include the classical MAPK (i.e., RAS–RAF–MEK–ERK), WNT, and PI3K signaling pathways. These pathways play a major role in defining the progression of melanoma, and some have been the subject of recent pharmacological strategies to treat this belligerent disease. This review describes the latest advances in the understanding of melanoma progression and the major molecular pathways involved. In addition, we discuss the roles of emerging molecular players that are involved in melanoma pathogenesis, including the functional role of the melanoma tumor antigen, p97/MFI2 (melanotransferrin).
Keywords: Melanoma; MAPK; WNT; PI3K; BRAF; EMT;

Corrigendum to “Dominant negative Ras attenuates pathological ventricular remodeling in pressure overload cardiac hypertrophy” [Biochim. Biophys. Acta 1853/11 (2015) 2870–2884] by Manuel Ramos-Kuri; Kleopatra Rapti; Hind Mehel; Shihong Zhang; Perundurai S. Dhandapany; Lifan Liang; Alejandro García-Carrancá; Regis Bobe; Rodolphe Fischmeister; Serge Adnot; Djamel Lebeche; Roger J. Hajjar; Larissa Lipskaia; Elie R. Chemaly (785).

Corrigendum to “IRS2 and PTEN are key molecules in controlling insulin sensitivity in podocytes” [Biochim. Biophys. Acta 1853 (12) (2015) 3224–3234] by Beatriz Santamaria; Eva Marquez; Abigail Lay; RoseaMarie M. Carew; Águeda González-Rodríguez; Gavin I. Welsh; Lan Ni; Lorna J. Hale; Alberto Ortiz; Moin A. Saleem; Derek P. Brazil; Richard J. Coward; Ángela M. Valverde (786).