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BBA - Molecular Cell Research (v.1793, #3)
Inhibition of extracellular signal-regulated kinase (ERK) signaling participates in repression of nuclear factor (NF)-κB activity by glucocorticoids by Lars-Göran Bladh; Krishan Johansson-Haque; Ingalill Rafter; Stefan Nilsson; Sam Okret ⁎ (pp. 439-446).
Glucocorticoid (GC) effects are mediated via the GC-receptor (GR), which either stimulates or represses gene expression. Repression of target genes often involves negative cross-talk between the GR and other transcription factors e.g. NF-κB, important for gene activation. Using HEK293 cells we here describe that repression of NF-κB requires functions of the GR that are dependent on the signaling pathways employed to activate NF-κB. While a GR mutant was able to repress NF-κB activity following activation by TNFα, it did not so following activation by the phorbol ester TPA. In these cells, TPA stimulation but not TNFα, activated extracellular signal-regulated kinase (ERK). We demonstrated that the ability of the dexamethasone activated GR mutant to repress TPA-induced NF-κB activity was restored in conjunction with ERK1/2 inhibition. Previous reports have shown GC-mediated inhibition of ERK1/2 phosphorylation to involve GC induction of MAPK phosphatase-1 (MKP-1). Here, we demonstrated that the GRR488Q mutant was incapable of inducing gene expression of endogenous MKP-1 following dexamethasone treatment, in contrast to the GRwt. However, TPA treatment alone resulted in much stronger MKP-1 expression in both GRwt and GRR488Q containing cells than that of dexamethasone suggesting that the inability of GRR488Q to inhibit TPA-induced NF-κB activity did not involve a lack of MKP-1 expression. In line with this, RNAi targeted towards MKP-1 did not abolish or inhibit the ability of the GRwt to repress NF-κB activity. Importantly, we observed no difference in activated ERK1/2 (phospho-ERK1/2) expression over time between GRwt and GRR488Q containing cells following co-treatment with TPA and dexamethasone. Based on these results we suggest that GRwt does not directly regulate ERK1/2 but rather alters ERK1/2-mediated effects allowing it to repress NF-κB activity, a capacity lacked by the GRR488Q mutant.
Keywords: Glucocorticoid; Cross-talk; NF-κB; MAPK signaling; ERK; MKP-1 DUSP1
Membrane localization of RasGRP1 is controlled by an EF-hand, and by the GEF domain by Ghazaleh Tazmini; Nadine Beaulieu; Ada Woo; Bari Zahedi; Rebecca E. Goulding; Robert J. Kay (pp. 447-461).
RasGRP1 is an exchange factor for membrane-localized Ras GTPases. Activation of RasGRP1 requires its translocation to membranes, which can be directly mediated by either its PT or C1 domains. RasGRP1 also has a pair of EF-hands which have been proposed to regulate RasGRP1 by sensing receptor-induced calcium fluxes. We determined that one of these EF-hands, EF1, is required for receptor-induced translocation of RasGRP1 to the plasma membrane in B cell lines. EF1 enables plasma membrane targeting of RasGRP1 by counteracting the SuPT domain, a negative regulator of the PT domain. Contrary to expectations, EF1-mediated translocation of RasGRP1 does not involve antigen receptor-induced intracellular calcium flux. Instead, alternative splicing affecting EF1 serves to modulate RasGRP1 localization. Excision of an exon encoding part of EF1 selectively disables PT domain-mediated plasma membrane targeting of RasGRP1, without affecting C1 domain-mediated localization to endomembranes. While EF1 specifically controls PT-mediated plasma membrane targeting, the Ras binding site in the catalytic GEF domain of RasGRP1 is required for both PT-mediated plasma membrane targeting and C1-mediated localization to endomembranes. Positive feedback between its GEF domain and membrane-binding domains could be important for full activation of RasGRP1, with occupation of the Ras binding sites in the GEF domain resulting in functional liberation of the PT and C1 domains, and membrane binding by these domains serving to maintain the Ras–GEF interaction.
Keywords: Exchange factor; Ras GTPase; EF-hand; C1 domain; Antigen receptor; Membrane localization
The polo-like kinase 1 regulates CDC25B-dependent mitosis entry by Valerie Lobjois; Denis Jullien; Jean-Pierre Bouché; Bernard Ducommun (pp. 462-468).
Activation of cyclin-dependent kinase complexes (CDK) at key cell cycle transitions is dependent on their dephosphorylation by CDC25 dual-specificity phosphatases (CDC25A, B and C in human). The CDC25B phosphatase plays an essential role in controlling the activity of CDK1-cyclin B complexes at the entry into mitosis and together with polo-like kinase 1 (PLK1) in regulating the resumption of cell cycle progression after DNA damage-dependent checkpoint arrest in G2. In this study, we analysed the regulation of CDC25B-dependent mitosis entry by PLK1. We demonstrate that PLK1 activity is essential for the relocation of CDC25B from the cytoplasm to the nucleus. By gain and loss of function analyses, we show that PLK1 stimulates CDC25B-induced mitotic entry in both normal conditions and after DNA-damage induced G2/M arrest. Our results support a model in which the relocalisation of CDC25B to the nucleus at the G2-M transition by PLK1 regulates its mitotic inducing activity.
Keywords: CDC25B phosphatase; Polo-like kinase 1; Mitosis; G2/M checkpoint
Direct binding of cytosolic NDP kinases to membrane lipids is regulated by nucleotides by Kimberly A.P. Mitchell; Gabor Szabo ⁎; Angela de S. Otero (pp. 469-476).
In spite of their complete lack of any structural features that characterize membrane proteins, cytosolic nucleoside-diphosphate kinases (NDPKs) have been found repeatedly to associate with membranes. In some instances the recruitment of cytosolic NDPKs to membranes was attributed to interactions with peripheral or integral membrane proteins, but in many cases the mechanism underlying the association of NDPKs with membranes remained unknown. We show here that cytosolic NDPKs bind directly to membrane lipids in a dynamic process that is controlled by its substrates, nucleoside tri- and diphosphates, and can be fully reconstituted with chemically defined, protein-free phospholipids and recombinant NDPK, or with purified NDPK. Our results uncover a novel mechanism for the reversible targeting of soluble NDPKs to membranes, where they may act as a reservoir of high energy phosphate, supporting the operation of membrane-based processes that utilize nucleotides other than ATP, such as intracellular traffic and phospholipid biosynthesis.
Keywords: Nucleoside-diphosphate kinase; nm23; Cytosol; Membrane; Phospholipid; Nucleotide
Expression of gastrin precursors by CD133-positive colorectal cancer cells is crucial for tumour growth by Audrey Ferrand; Mauro S. Sandrin; Arthur Shulkes; Graham S. Baldwin (pp. 477-488).
Precursors of the hormone gastrin, progastrin and glycine-extended gastrin (G-gly), have been detected in colorectal polyps and tumours, and in the blood of patients with colorectal cancer (CRC), while their expression is lower in healthy subjects. The surface glycoproteins CD133 and CD44 have been identified as possible markers for CRC stem cells. Our aims were to investigate whether progastrin and G-gly are expressed by CD133-positive cells in human CRC tissues and in the human CRC cell line DLD-1, and to determine whether this expression is biologically relevant. The great majority of the cells expressing CD133 also expressed gastrin precursors in both DLD-1 cells, which retain a stem cell-like subpopulation, and human CRC specimens. The CD133high/CD44high/progastrinhigh cells gave rise to larger tumours in SCID mice compared to CD133low/CD44low/progastrinlow cells. The CD133high/CD44high/progastrinhigh cells displayed enhanced activation of the signalling molecules JAK2, STAT3, ERK1/2 and Akt, known to regulate the induction of proliferation and/or survival by gastrin precursors. Moreover, downregulation of the gastrin gene in DLD-1 cells reduced the expression of cancer stem cell markers and abolished tumour development in SCID mice. We conclude that gastrin precursors may provide a target for therapies directed against the cells responsible for tumour development and recurrence.
Keywords: CD133; Cancer stem cell; Tumour-initiating cell; Colon; Cancer; Gastrin
A novel mode of translocation for cytolethal distending toxin by Lina Guerra; Kathleen N. Nemec; Shane Massey; Suren A. Tatulian; Monica Thelestam; Teresa Frisan; Ken Teter (pp. 489-495).
Thermal instability in the toxin catalytic subunit may be a common property of toxins that exit the endoplasmic reticulum (ER) by exploiting the mechanism of ER-associated degradation (ERAD). The Haemophilus ducreyi cytolethal distending toxin (HdCDT) does not utilize ERAD to exit the ER, so we predicted the structural properties of its catalytic subunit (HdCdtB) would differ from other ER-translocating toxins. Here, we document the heat-stable properties of HdCdtB which distinguish it from other ER-translocating toxins. Cell-based assays further suggested that HdCdtB does not unfold before exiting the ER and that it may move directly from the ER lumen to the nucleoplasm. These observations suggest a novel mode of ER exit for HdCdtB.
Keywords: Abbreviations; CT; cholera toxin; CD; circular dichroism; CDT; cytolethal distending toxin; ER; endoplasmic reticulum; ERAD; endoplasmic reticulum-associated degradation; GST; glutathione S-transferase; HdCDT; Haemophilus ducreyi; cytolethal distending toxin; EC; 50; half-maximal effective concentration; PT; pertussis toxinCytolethal distending toxin; Circular dichroism; Endoplasmic reticulum; Fluorescence spectroscopy; 20S proteasome; Toxin translocation
Bcl-XL mediates epidermal growth factor dependent cell survival in HC11 mammary epithelial cells by Leonardo Romorini; Omar A. Coso; Adali Pecci ⁎ (pp. 496-505).
Apoptosis is the predominant process controlling cell deletion during post-lactational mammary gland remodeling. The members of the Bcl-2 protein family, whose expression levels are under the control of lactogenic hormones, internally control this mechanism. Epidermal growth factor (EGF) belongs to a family of proteins that act as survival factors for mammary epithelial cells upon binding to specific membrane tyrosine kinase receptors. Expression of EGF peaks during lactation and dramatically decreases in the involuting mammary gland. Though it was suggested that the protective effect of EGF is mediated through the phosphatidylinositol-3-kinase (PI3K) or MEK/ERK kinases activities, little is known about the downstream mechanisms involved on the anti-apoptotic effect of EGF on mammary epithelial cells; particularly the identity of target genes controlling apoptosis. Here, we focused on the effect of EGF on the survival of mammary epithelial cells. We particularly aimed at the characterization of the signaling pathways that were triggered by this growth factor, impinge upon expression of Bcl-2 family members and therefore have an impact on the regulation of cell survival. We demonstrate that EGF provokes the induction of the anti-apoptotic isoform Bcl-XL and the phosphorylation and down-regulation of the pro-apoptotic protein Bad. The activation of JNK and PI3K/AKT signaling pathways promotes the induction of Bcl-XL while AKT activation also leads to Bad phosphorylation and down-regulation. This protective effect of EGF correlates mainly with the up-regulation of Bcl-XL than with the down-regulation of Bad. In fact, HC11 cells unable to express bcl-X, die even in the presence of EGF. In this context, Bcl-XL emerges as a key anti-apoptotic molecule critical for mediating EGF cell survival.
Keywords: Mammary epithelial cell; Apoptosis; EGF; MAPKs; Bcl-X; L; Bad
Dispensable role of protein 4.1B/DAL-1 in rodent adrenal medulla regarding generation of pheochromocytoma and plasmalemmal localization of TSLC1 by Nobuhiko Ohno; Nobuo Terada; Masayuki Komada; Sei Saitoh; Frank Costantini; Virgilio Pace; Paul-Georg Germann; Klaus Weber; Hisashi Yamakawa; Osamu Ohara; Shinichi Ohno (pp. 506-515).
Protein 4.1B is a membrane skeletal protein expressed in various organs, and is associated with tumor suppressor in lung cancer-1 (TSLC1) in vitro. Although involvement of 4.1B in the intercellular junctions and tumor-suppression was suggested, some controversial results posed questions to the general tumor-suppressive function of 4.1B and its relation to TSLC1 in vivo. In this study, the expression of 4.1B and its interaction with TSLC1 were examined in rodent adrenal gland, and the involvement of 4.1B in tumorigenesis and the effect of 4.1B deficiency on TSLC1 distribution were also investigated using rodent pheochromocytoma and 4.1B-knockout mice. Although plasmalemmal immunolocalization of 4.1B was shown in chromaffin cells of rodent adrenal medulla, expression of 4.1B was maintained in developed pheochromocytoma, and morphological abnormality or pheochromocytoma generation could not be found in 4.1B-deficient mice. Furthermore, molecular interaction and colocalization of 4.1B and TSLC1 were observed in mouse adrenal gland, but the immunolocalization of TSLC1 along chromaffin cell membranes was not affected in the 4.1B-deficient mice. These results suggest that the function of 4.1B as tumor suppressor might significantly differ among organs and species, and that plasmalemmal retention of TSLC1 would be maintained by molecules other than 4.1B interacting in rodent chromaffin cells.
Keywords: Adrenal medulla; Chromaffin cell; Pheochromocytoma; Protein 4.1B; TSLC1
Identification, characterization and essentiality of the unusual peroxin 13 from Trypanosoma brucei by Emilie Verplaetse; Daniel J. Rigden; Paul A.M. Michels ⁎ (pp. 516-527).
Peroxin 13 (PEX13) is one of the components of a peroxisomal membrane complex involved in import of proteins into the matrix of the organelles and has previously been characterized in a variety of organisms. Trypanosomatids ( Trypanosoma, Leishmania), protozoan parasites having peroxisome-like organelles designated glycosomes, possess an unusual PEX13 which shares very low sequence identity with others and lacks some typical PEX13 characteristics. It was identified in the databases through its multiple YGx motifs present in a glycine-rich N-terminal region of low sequence complexity. Like other PEX13s, it contains predicted transmembrane segments and a SH3 domain in its C-terminal half. The localization of T. brucei PEX13 in the glycosomal membrane was confirmed by expression of a fusion construct with Green Fluorescent Protein, and western blot analysis of purified organelles and membranes. The C-terminal half of the protein was shown to interact with the third of three pentapeptide repeats of the previously characterized PEX5, the receptor of glycosomal proteins with a type 1 peroxisome-targeting signal, and with PEX14, another component of the same peroxisomal protein import complex in the membrane. PEX13 is essential for the parasite; depletion by RNA interference results in mislocalization of glycosomal proteins and death of the parasites.
Keywords: Trypanosome; Glycosome; Peroxisome; Biogenesis; Peroxin; PEX13
Lysophospholipids stimulate prostate cancer cell migration via TRPV2 channel activation by Michaël Monet; Dimitra Gkika; V'yacheslav Lehen'kyi; Albin Pourtier; Fabien Vanden Abeele; Gabriel Bidaux; Véronique Juvin; François Rassendren; Sandrine Humez; Natalia Prevarsakaya ⁎ (pp. 528-539).
The physiological role, the mechanisms of activation, as well as the endogenous regulators for the non-selective cationic channel TRPV2 are not known so far. In the present work we report that endogenous lysophospholipids such as lysophosphatidylcholine (LPC) and lysophosphatidylinositol (LPI) induce a calcium influx via TRPV2 channel. This activation is dependent on the length of the side-chain and the nature of the lysophospholipid head-group. TRPV2-mediated calcium uptake stimulated by LPC and LPI occurred via Gq/Go-protein and phosphatidylinositol-3,4 kinase (PI3,4K) signalling. We have shown that the mechanism of TRPV2 activation induced by LPC and LPI is due to the TRPV2 channel translocation to the plasma membrane. The activation of TRPV2 channel by LPC and LPI leads to an increase in the cell migration of the prostate cancer cell line PC3. We have demonstrated that TRPV2 is directly involved in both steady-state and lysophospholipid-stimulated cancer cell migration. Thus, for the first time, we have identified one of the natural regulators of TRPV2 channel, one of the mechanisms of TRPV2 activation and regulation, as well as its pathophysiological role in cancer.
Keywords: TRPV2; Lysophospholipid; Calcium; Translocation; Prostate cancer; Cell migration
Vacuolar functions determine the mode of cell death by Alexandra Schauer; Heide Knauer; Christoph Ruckenstuhl; Heike Fussi; Michael Durchschlag; Ulrike Potocnik; Kai-Uwe Fröhlich (pp. 540-545).
The yeast vacuole plays a crucial role in cell homeostasis including pH regulation and degradation of proteins and organelles. Class C VPS genes code for proteins essential for vacuolar and endosomal vesicle fusion, their deletion results in the absence of a detectable vacuole. We found that single gene deletions of class C VPS genes result in a drastically enhanced sensitivity to treatment with acetic acid whereas sensitivity towards H2O2 remains largely unaffected. Interestingly acetic acid treatment known as an established inducer of yeast apoptosis leads to necrosis in class C VPS deletion strains. Their intracellular pH drops from 6.7 to 5.5 after acetic acid treatment, while in wild type the pH drops to just 6.3. When the intracellular pH in wild type is lowered below pH 5.5 using a higher concentration of acetic acid, the survival rate is similarly low as in the class C VPS mutants, however, the death phenotype is predominantly apoptotic. Hence, the vacuole not only prevents acetic acid induced cell death by buffering the cytosolic pH, but it also has a proapoptotic function.
Keywords: Vacuole; Necrosis; Apoptosis; Acetic acid; pH regulation
Role of BNIP3 in TNF-induced cell death — TNF upregulates BNIP3 expression by Saeid Ghavami; Mehdi Eshraghi; Kamran Kadkhoda; Mark M. Mutawe; Subbareddy Maddika; Graham H. Bay; Sebastian Wesselborg; Andrew J. Halayko; Thomas Klonisch; Marek Los ⁎ (pp. 546-560).
Tumor necrosis factor alpha (TNF) is a cytokine that induces caspase-dependent (apoptotic) and caspase-independent (necrosis-like) cell death in different cells. We used the murine fibrosarcoma cell line model L929 and a stable L929 transfectant over-expressing a mutated dominant-negative form of BNIP3 lacking the C-terminal transmembrane (TM) domain (L929-ΔTM-BNIP3) to test if TNF-induced cell death involved pro-apoptotic Bcl2 protein BNIP3. Treatment of cells with TNF in the absence of actinomycin D caused a rapid fall in the mitochondrial membrane potential (Δ Ψm) and a prompt increase in reactive oxygen species (ROS) production, which was significantly less pronounced in L929-ΔTM-BNIP3. TNF did not cause the mitochondrial release of apoptosis inducing factor (AIF) and Endonuclease G (Endo-G) but provoked the release of cytochrome c, Smac/Diablo, and Omi/HtrA2 at similar levels in both L929 and in L929-ΔTM-BNIP3 cells. We observed TNF-associated increase in the expression of BNIP3 in L929 that was mediated by nitric oxide and significantly inhibited by nitric oxide synthase inhibitor N5-(methylamidino)-l-ornithine acetate. In L929, lysosomal swelling and activation were markedly increased as compared to L929-ΔTM-BNIP3 and could be inhibited by treatment with inhibitors to vacuolar H+-ATPase and cathepsins −B/−L. Together, these data indicate that TNF-induced cell death involves BNIP3, ROS production, and activation of the lysosomal death pathway.
Keywords: Abbreviations; TNF; tumor necrosis factor alpha; AIF; apoptosis inducing factor; Endo-G; Endonuclease G; tBid; truncated bid; BNIP3; Bcl2/E1B 19kD interacting protein; TM; trans-membrane; PTP; permeability transition pore; HIF-1α; hypoxia inducing factor-alpha-1; NO; nitric oxide; ROS; reactive oxygen species; MTT; 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; PARP-1; poly(ADP-ribose)polymerase-1; DAF-2DA; di-aminofluorescein-2 diacetate; GAPDH; glyceraldehyde-3-phosphate dehydrogenase; HSP60; heat shock protein 60; HDAC1; histone deacytalase 1; HtrA2; high-temperature requirement A2; Smac; second mitochondrial activator of caspases; DIABLO; direct inhibitor of apoptosis binding protein of low PI; LNMMA; N; 5; -(methylamidino)-; l; -ornithine acetate salt, JC-1, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide; DHR-123; dihydrorhodamine-123; Δ; Ψ; m; mitochondrial membrane potential changeCaspase; Cathepsin; Flow cytometry; Lysosome; ROS
Human initiator caspases trigger apoptotic and autophagic phenotypes in Saccharomyces cerevisiae by Patricia Lisa-Santamaría; Aaron M. Neiman; Álvaro Cuesta-Marbán; Faustino Mollinedo; José L. Revuelta ⁎; Alberto Jiménez (pp. 561-571).
Caspases are a family of proteases that participate in the progression and execution of the apoptotic program. However, regulation of the caspase activation and their substrates has not yet been fully elucidated. Here we explore the effect of the ectopic expression of the human initiator caspases-8 and -10 in Saccharomyces cerevisiae. Our results showed that the expression of human CASP10 and CASP8 triggers certain apoptotic markers such as a massive production of reactive oxygen species (ROS), chromatin condensation and phosphatidylserine externalization, finally leading to cell death. In response to hydroxyurea (HU), yeast cells expressing caspase-10 did not reduce the replication of DNA and escaped to the intra-S checkpoint of the cell cycle. In addition, caspase-10 expression induced yeast vacuolization and a vacuole-associated phenotype resembling autophagy. Other intracellular alterations such as disorganization of the actin cytoskeleton, cell wall damage, and aberrations within the endoplasmic reticulum lumen were also associated with caspase-10 expression. Furthermore, caspase-induced cell death was completely dependent on the proteolytic activation of the enzyme but, in contrast, was not dependent on either of the endogenous yeast apoptotic proteins Aif1 and Mca1 or the mitochondria.
Keywords: Caspase; Yeast; Oxidative stress; Cell death; Apoptosis; Autophagy
Role of intracellular calcium and S-glutathionylation in cell death induced by a mixture of isothiazolinones in HL60 cells by Simona Frosali; Alessandra Leonini; Anna Ettorre; Giuseppe Di Maio; Sandra Nuti; Simona Tavarini; Paolo Di Simplicio; Anna Di Stefano (pp. 572-583).
Previously we reported that brief exposure of HL60 cells to a mixture of 5-chloro-2-methyl-4-isothiazolin-3-one (CMI) and 2-methyl-4-isothiazolin-3-one (MI) shifts the cells into a state of oxidative stress that induces apoptosis and necrosis. In this study, flow cytometric analysis showed that CMI/MI induces early perturbation of calcium homeostasis, increasing cytosolic and mitochondrial calcium and depleting the intracellular endoplasmic reticulum (ER) stores. The calcium chelator BAPTA-AM reduced necrosis and secondary necrosis, the loss of Δ Ψm and S-glutathionylation induced by necrotic doses of CMI/MI, but did not protect against CMI/MI-induced apoptosis, mitochondrial calcium uptake and mitochondrial hyperpolarization. This indicates that increased cytoplasmic calcium does not have a causal role in the induction of apoptosis, while cross-talk between the ER and mitochondria could be responsible for the induction of apoptosis. GSH-OEt pretreatment, which enhances cellular GSH content, reduced S-glutathionylation and cytosolic and mitochondrial calcium levels, thus protecting against both apoptosis and necrosis shifting to apoptosis. Therefore, the degree of GSH depletion, paralleled by the levels of protein S-glutathionylation, may have a causal role in increasing calcium levels. The mitochondrial calcium increase could be responsible for apoptosis, while necrosis is associated with cytoplasmic calcium overload. These findings suggest that S-glutathionylation of specific proteins acts as a molecular linker between calcium and redox signalling.
Keywords: Isothiazolinones; Apoptosis; Necrosis; Mitochondrial transmembrane potential; Calcium; S-glutathionylation
PLTP is present in the nucleus, and its nuclear export is CRM1-dependent by Simona Vuletic; Weijiang Dong; Gertrud Wolfbauer; Joseph R. Day; John J. Albers ⁎ (pp. 584-591).
Phospholipid transfer protein (PLTP), one of the key lipid transfer proteins in plasma and cerebrospinal fluid, is nearly ubiquitously expressed in cells and tissues. Functions of secreted PLTP have been extensively studied. However, very little is known about potential intracellular PLTP functions. In the current study, we provide evidence for PLTP localization in the nucleus of cells that constitutively express PLTP (human neuroblastoma cells, SK-N-SH; and human cortical neurons, HCN2) and in cells transfected with human PLTP (Chinese hamster ovary and baby hamster kidney cells). Furthermore, we have shown that incubation of these cells with leptomycin B (LMB), a specific inhibitor of nuclear export mediated by chromosome region maintenance 1 (CRM1), leads to intranuclear accumulation of PLTP, suggesting that PLTP nuclear export is CRM1-dependent. We also provide evidence for entry of secreted PLTP into the cell and its translocation to the nucleus, and show that intranuclear PLTP is active in phospholipid transfer. These findings suggest that PLTP is involved in novel intracellular functions.
Keywords: Phospholipid transfer protein; PLTP; Nuclear localization; Neuronal cell; Leptomycin B
Self-activation of Caspase-6 in vitro and in vivo: Caspase-6 activation does not induce cell death in HEK293T cells by Guy Klaiman; Nathalie Champagne; Andréa C. LeBlanc ⁎ (pp. 592-601).
Caspase-6 (Casp6) is a short pro-domain caspase that is activated early in Alzheimer disease, yet, little is known on the mechanism of activation of this caspase. In this study, critical proteolytic processing events required for Casp6 activation in vitro and in vivo were evaluated by site directed mutagenesis of the D23 pro-domain, and D179 and D193 linker processing sites. We found that (1) Casp6 was self-processed and activated in vitro and in vivo, (2) uncleavable Casp6 possessed low activity in vitro but not in vivo, (3) the pro-domain of Casp6 entirely prevented self-processing and activation in vivo but not in vitro, (4) removal of the pro-domain promoted Casp6 activation, (5) cleavage at either D179 or D193 was sufficient to generate activity in vitro and in vivo, and (6) Casp6 activity did not induce cell death in HEK293T cells. We conclude that the Casp6 is activated through proteolytic cleavage, as are the effector Caspase-3 and -7. However, unlike other effector caspases, Casp6 can be entirely self-activated and its activation does not necessarily induce cell death.
Keywords: Caspase-6; Caspase self-activation; Effector caspase; HEK293T; Caspase activation; Caspase activity; Alzheimer disease