Forgot your password?
New user?
New Window Opens on the Secret Life of Microbes: Scientists Develop First Microbial Profiles of Ecosystems
CAS announces the release of SciFinder Scholar for Mac OS X
Press Releases
Press Releases
| Check out our New Publishers' Select for Free Articles |
BBA - Molecular Cell Research (v.1783, #1)
Nuclear receptors and other nuclear transcription factors in mitochondria: Regulatory molecules in a new environment by Anna-Maria G. Psarra; Constantine E. Sekeris (pp. 1-11).
The mitochondrion is the major energy generating organelle of the cell and the site of other basic processes, including apoptosis. The mitochondrial functions are performed in concert with other cell compartments and are regulated by various extracellular and intracellular signals. Several nuclear receptors and other nuclear transcription factors, such as NF-κB, AP-1, CREB and p53, involved in growth, metabolic and developmental processes, have been detected in mitochondria. This finding raises the question as to the role of these regulatory molecules in their “new” environment. Experimental evidence supports the action of the mitochondrially localized transcription factors on mitochondrial transcription, energy yield and apoptosis, extending the known nuclear role of these molecules outside the nucleus. A principle of coordination of nuclear and mitochondrial gene transcription has been ascertained as regards the regulatory action of steroid and thyroid hormones on energy yield. Accordingly, the same nuclear receptors, localized in the two compartments–nuclei and mitochondria–regulate transcription of genes serving a common function by way of interaction with common binding sites in the two genomes. This principle is now expanding to encompass other nuclearly and mitochondrially localized transcription factors.
Keywords: Nuclear receptor; Transcription factor; Mitochondrial transcription; Mitochondrial DNA; Apoptosis; Hormone
Nuclear receptors and other nuclear transcription factors in mitochondria: Regulatory molecules in a new environment by Anna-Maria G. Psarra; Constantine E. Sekeris (pp. 1-11).
The mitochondrion is the major energy generating organelle of the cell and the site of other basic processes, including apoptosis. The mitochondrial functions are performed in concert with other cell compartments and are regulated by various extracellular and intracellular signals. Several nuclear receptors and other nuclear transcription factors, such as NF-κB, AP-1, CREB and p53, involved in growth, metabolic and developmental processes, have been detected in mitochondria. This finding raises the question as to the role of these regulatory molecules in their “new” environment. Experimental evidence supports the action of the mitochondrially localized transcription factors on mitochondrial transcription, energy yield and apoptosis, extending the known nuclear role of these molecules outside the nucleus. A principle of coordination of nuclear and mitochondrial gene transcription has been ascertained as regards the regulatory action of steroid and thyroid hormones on energy yield. Accordingly, the same nuclear receptors, localized in the two compartments–nuclei and mitochondria–regulate transcription of genes serving a common function by way of interaction with common binding sites in the two genomes. This principle is now expanding to encompass other nuclearly and mitochondrially localized transcription factors.
Keywords: Nuclear receptor; Transcription factor; Mitochondrial transcription; Mitochondrial DNA; Apoptosis; Hormone
Thrombin peptide TP508 prevents nitric oxide mediated apoptosis in chondrocytes in the endochondral developmental pathway by M. Zhong; L.J. Wike; J.T. Ryaby; D.H. Carney; B.D. Boyan; Z. Schwartz (pp. 12-22).
TP508 is a 23-amino acid peptide derived from human prothrombin that increases cartilage matrix production and reduces alkaline phosphatase activity without changing chondrocyte proliferation. This study tested the hypothesis that TP508 acts by blocking the onset of apoptosis associated with hypertrophy. Rat costochondral resting zone chondrocytes and human auricular chondrocytes were cultured in DMEM containing 50μM ascorbic acid and 10% FBS. Apoptosis was induced by treatment of confluent cultures with chelerythrine, tamoxifen, or inorganic phosphate (Pi) for 24h. One half of the cultures received TP508 (0, 0.7, or 7μg/ml). Apoptosis was assessed as a function of DNA fragmentation ([3H]-thymidine labeled DNA fragments), TUNEL staining, and cell viability using the MTT assay, as well as by assessing the Bcl-2/Bax mRNA and protein ratios and caspase-3 activity. The universal NO synthase inhibitorl-NMMA was used to assess the effect of NO production on chondrocyte apoptosis and specific NO synthase subspecies were identified using iNOS inhibitor 1400W and nNOS inhibitor vinyl-l-NIO, as well asl-NAME, which inhibits both iNOS and eNOS. Finally, we assessed if TP508 would block NO production induced by the apoptogens. Chelerythrine, tamoxifen and Pi-induced apoptosis and this was reversed by TP508. All apoptogens increased NO production and this was reduced by TP508. TP508 reduced NO levels to the same extent as 1400W but not to the same extent asl-NAME, suggesting that its effects are mediated primarily by iNOS. In addition, TP508 reduced the effect of chelerythrine to the same extent as 1400W andl-NAME, again indicating that it acts via inhibition of an iNOS pathway. TP508 also regulated Bcl-2/Bax mRNA in a time and dose-dependent manner. The Bcl-2/Bax mRNA ratio was 0.11 in the absence of TP508 at 1h and 4.95 at 7μg/ml TP508; by 3h the ratio was approximately 1 in both groups. The Bcl-2/Bax protein ratio also increased by 63% at 1h. TP508 did not affect caspase-3 activity. TP508 also caused a dose-dependent increase in protein kinase C (PKC) activity within 9min that was maximal at 270min. These results show that TP508 prevents apoptosis in growth plate chondrocytes via inhibition of iNOS-dependent NO and suggest a possible role for PKC in the mechanism.
Keywords: Chondrocytes; TP508; Thrombin peptide; Apoptosis; Growth plate; Endochondral ossification
Thrombin peptide TP508 prevents nitric oxide mediated apoptosis in chondrocytes in the endochondral developmental pathway by M. Zhong; L.J. Wike; J.T. Ryaby; D.H. Carney; B.D. Boyan; Z. Schwartz (pp. 12-22).
TP508 is a 23-amino acid peptide derived from human prothrombin that increases cartilage matrix production and reduces alkaline phosphatase activity without changing chondrocyte proliferation. This study tested the hypothesis that TP508 acts by blocking the onset of apoptosis associated with hypertrophy. Rat costochondral resting zone chondrocytes and human auricular chondrocytes were cultured in DMEM containing 50μM ascorbic acid and 10% FBS. Apoptosis was induced by treatment of confluent cultures with chelerythrine, tamoxifen, or inorganic phosphate (Pi) for 24h. One half of the cultures received TP508 (0, 0.7, or 7μg/ml). Apoptosis was assessed as a function of DNA fragmentation ([3H]-thymidine labeled DNA fragments), TUNEL staining, and cell viability using the MTT assay, as well as by assessing the Bcl-2/Bax mRNA and protein ratios and caspase-3 activity. The universal NO synthase inhibitorl-NMMA was used to assess the effect of NO production on chondrocyte apoptosis and specific NO synthase subspecies were identified using iNOS inhibitor 1400W and nNOS inhibitor vinyl-l-NIO, as well asl-NAME, which inhibits both iNOS and eNOS. Finally, we assessed if TP508 would block NO production induced by the apoptogens. Chelerythrine, tamoxifen and Pi-induced apoptosis and this was reversed by TP508. All apoptogens increased NO production and this was reduced by TP508. TP508 reduced NO levels to the same extent as 1400W but not to the same extent asl-NAME, suggesting that its effects are mediated primarily by iNOS. In addition, TP508 reduced the effect of chelerythrine to the same extent as 1400W andl-NAME, again indicating that it acts via inhibition of an iNOS pathway. TP508 also regulated Bcl-2/Bax mRNA in a time and dose-dependent manner. The Bcl-2/Bax mRNA ratio was 0.11 in the absence of TP508 at 1h and 4.95 at 7μg/ml TP508; by 3h the ratio was approximately 1 in both groups. The Bcl-2/Bax protein ratio also increased by 63% at 1h. TP508 did not affect caspase-3 activity. TP508 also caused a dose-dependent increase in protein kinase C (PKC) activity within 9min that was maximal at 270min. These results show that TP508 prevents apoptosis in growth plate chondrocytes via inhibition of iNOS-dependent NO and suggest a possible role for PKC in the mechanism.
Keywords: Chondrocytes; TP508; Thrombin peptide; Apoptosis; Growth plate; Endochondral ossification
Nox1-dependent superoxide production controls colon adenocarcinoma cell migration by Amine Sadok; Véronique Bourgarel-Rey; Florence Gattacceca; Claude Penel; Maxime Lehmann; Hervé Kovacic (pp. 23-33).
Reactive oxygen species are well-known mediators of various biological responses. Recently, new homologues of the catalytic subunit of NADPH oxidase have been discovered in non-phagocytic cells. These new homologues (Nox1–Nox5) produce low levels of superoxides compared to the phagocytic homologue Nox2/gp91phox. Using Nox1 siRNA, we show that Nox1-dependent superoxide production affects the migration of HT29-D4 colonic adenocarcinoma cells on collagen-I. Nox1 inhibition or down-regulation led to a decrease of superoxide production and α2β1 integrin membrane availability. An addition of arachidonic acid stimulated Nox1-dependent superoxide production and HT29-D4 cell migration. Pharmacological evidences using phospholipase A2, lipoxygenases and protein kinase C inhibitors show that upstream regulation of Nox1 relies on arachidonic acid metabolism. Inhibition of 12-lipoxygenase decreased basal and arachidonic acid induced Nox1-dependent superoxide production and cell migration. Migration and ROS production inhibited by a 12-lipoxygenase inhibitor were restored by the addition of 12(S)-HETE, a downstream product of 12-lipoxygenase. Protein kinase C δ inhibition by rottlerin (and also GO6983) prevented Nox1-dependent superoxide production and inhibited cell migration, while other protein kinase C inhibitors were ineffective. We conclude that Nox1 activation by arachidonic acid metabolism occurs through 12-lipoxygenase and protein kinase C δ, and controls cell migration by affecting integrin α2 subunit turn-over.
Keywords: Abbreviations; AA; arachidonic acid; Bim-I; Bisindolylmaleimide I; CDC; cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate; Col-I; Collagen-I; Cox; cyclooxygenase; DEC; diethylcarbamazine; DPI; diphenylene iodonium; DMEM; Dulbecco's modified Eagle's medium; ERK; Extracellular signal-regulated kinase; 12(S)-HETE; 12-S-hydroxyeicosatetraenoic acid; JNK; Jun-N-terminal kinase; Lox; lipoxygenase; MAPK; mitogen activated protein kinase, OA, Okadaic acid; PKC; protein kinase C; PLA2; phospholipase A2; PP2A; protein phosphatase 2A; ROS; reactive oxygen species; siRNA; small interfering RNANox1; Migration; Arachidonic acid; ROS; Colon; NADPH oxidase
Nox1-dependent superoxide production controls colon adenocarcinoma cell migration by Amine Sadok; Véronique Bourgarel-Rey; Florence Gattacceca; Claude Penel; Maxime Lehmann; Hervé Kovacic (pp. 23-33).
Reactive oxygen species are well-known mediators of various biological responses. Recently, new homologues of the catalytic subunit of NADPH oxidase have been discovered in non-phagocytic cells. These new homologues (Nox1–Nox5) produce low levels of superoxides compared to the phagocytic homologue Nox2/gp91phox. Using Nox1 siRNA, we show that Nox1-dependent superoxide production affects the migration of HT29-D4 colonic adenocarcinoma cells on collagen-I. Nox1 inhibition or down-regulation led to a decrease of superoxide production and α2β1 integrin membrane availability. An addition of arachidonic acid stimulated Nox1-dependent superoxide production and HT29-D4 cell migration. Pharmacological evidences using phospholipase A2, lipoxygenases and protein kinase C inhibitors show that upstream regulation of Nox1 relies on arachidonic acid metabolism. Inhibition of 12-lipoxygenase decreased basal and arachidonic acid induced Nox1-dependent superoxide production and cell migration. Migration and ROS production inhibited by a 12-lipoxygenase inhibitor were restored by the addition of 12(S)-HETE, a downstream product of 12-lipoxygenase. Protein kinase C δ inhibition by rottlerin (and also GO6983) prevented Nox1-dependent superoxide production and inhibited cell migration, while other protein kinase C inhibitors were ineffective. We conclude that Nox1 activation by arachidonic acid metabolism occurs through 12-lipoxygenase and protein kinase C δ, and controls cell migration by affecting integrin α2 subunit turn-over.
Keywords: Abbreviations; AA; arachidonic acid; Bim-I; Bisindolylmaleimide I; CDC; cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate; Col-I; Collagen-I; Cox; cyclooxygenase; DEC; diethylcarbamazine; DPI; diphenylene iodonium; DMEM; Dulbecco's modified Eagle's medium; ERK; Extracellular signal-regulated kinase; 12(S)-HETE; 12-S-hydroxyeicosatetraenoic acid; JNK; Jun-N-terminal kinase; Lox; lipoxygenase; MAPK; mitogen activated protein kinase, OA, Okadaic acid; PKC; protein kinase C; PLA2; phospholipase A2; PP2A; protein phosphatase 2A; ROS; reactive oxygen species; siRNA; small interfering RNANox1; Migration; Arachidonic acid; ROS; Colon; NADPH oxidase
Disruption of the latent transforming growth factor-β binding protein-1 gene causes alteration in facial structure and influences TGF-β bioavailability by Falko Drews; Sebastian Knöbel; Markus Moser; Kai G. Muhlack; Simone Mohren; Christian Stoll; Andreas Bosio; Axel M. Gressner; Ralf Weiskirchen (pp. 34-48).
Latent transforming growth factor-β binding proteins are a family of extracellular matrix proteins comprising four isoforms (LTBP-1, -2, -3, -4) with different structures, tissue expression patterns and affinity for TGF-β. So far, respective knockout models have highlighted some essential functions for LTBP-2, LTBP-3 and LTBP-4, while the physiological significance of LTBP-1 is only superficially known. Here we report for the first time the generation and characterization of a mouse model lacking both the long and short LTBP-1 isoform. Surprisingly, respective mice are viable and fertile. However, detailed X-ray analysis of the skull revealed a modified facial profile. In addition, the gene disruption induces a reduced biological activity of TGF-β that became evident in an experimental model of hepatic fibrogenesis in which the LTBP-1 knockout animals were less prone to hepatic fibrogenesis. Furthermore, comparative cDNA microarray gene expression profiling of cultured hepatic stellate cells confirmed that respective nulls were less receptive to cellular activation and transdifferentiation into myofibroblasts. Therefore, we conclude that LTBP-1 has essential functions in the control of TGF-β activation.
Keywords: Abbreviations; α-SMA; α-smooth muscle actin; ECM; extracellular matrix; EGF; epidermal growth factor; ES; embryonic stem cells; FCS; fetal calf serum; GAPDH; glyceraldehyde-3-phosphate dehydrogenase; HBSS; Hank's buffered saline solution; HRP; horse radish peroxidase; HSC; hepatic stellate cell(s); Ko; knockout(s); LAP; latency associated protein; Ltbp-1/-2/-3/-4; genes encoding the LTBP-1/-2/-3/-4 proteins; LTBP; latent transforming growth factor-β binding protein; TGF-β; transforming growth factor-β; Wt; wild type(s)LTBP-1; Microarray; Stellate cells; Liver fibrogenesis
Disruption of the latent transforming growth factor-β binding protein-1 gene causes alteration in facial structure and influences TGF-β bioavailability by Falko Drews; Sebastian Knöbel; Markus Moser; Kai G. Muhlack; Simone Mohren; Christian Stoll; Andreas Bosio; Axel M. Gressner; Ralf Weiskirchen (pp. 34-48).
Latent transforming growth factor-β binding proteins are a family of extracellular matrix proteins comprising four isoforms (LTBP-1, -2, -3, -4) with different structures, tissue expression patterns and affinity for TGF-β. So far, respective knockout models have highlighted some essential functions for LTBP-2, LTBP-3 and LTBP-4, while the physiological significance of LTBP-1 is only superficially known. Here we report for the first time the generation and characterization of a mouse model lacking both the long and short LTBP-1 isoform. Surprisingly, respective mice are viable and fertile. However, detailed X-ray analysis of the skull revealed a modified facial profile. In addition, the gene disruption induces a reduced biological activity of TGF-β that became evident in an experimental model of hepatic fibrogenesis in which the LTBP-1 knockout animals were less prone to hepatic fibrogenesis. Furthermore, comparative cDNA microarray gene expression profiling of cultured hepatic stellate cells confirmed that respective nulls were less receptive to cellular activation and transdifferentiation into myofibroblasts. Therefore, we conclude that LTBP-1 has essential functions in the control of TGF-β activation.
Keywords: Abbreviations; α-SMA; α-smooth muscle actin; ECM; extracellular matrix; EGF; epidermal growth factor; ES; embryonic stem cells; FCS; fetal calf serum; GAPDH; glyceraldehyde-3-phosphate dehydrogenase; HBSS; Hank's buffered saline solution; HRP; horse radish peroxidase; HSC; hepatic stellate cell(s); Ko; knockout(s); LAP; latency associated protein; Ltbp-1/-2/-3/-4; genes encoding the LTBP-1/-2/-3/-4 proteins; LTBP; latent transforming growth factor-β binding protein; TGF-β; transforming growth factor-β; Wt; wild type(s)LTBP-1; Microarray; Stellate cells; Liver fibrogenesis
VRK3-mediated inactivation of ERK signaling in adult and embryonic rodent tissues by Tae-Hong Kang; Kyong-Tai Kim (pp. 49-58).
Vaccinia-related kinase 3 (VRK3), previously characterized as a direct activator of vaccinia H1-related (VHR) phosphatase, inactivates extracellular signal-regulated kinase (ERK) in the nucleus of neuronal cells. Here we show that VRK3 is expressed in various other rodent tissues and in embryos, and regulates VHR phosphatase activity in these tissues. We observed colocalization of VRK3 and VHR in the testis tissue and could detect protein complex containing VRK3, VHR and ERK in immunoprecipitation analysis. Notably, the addition of recombinant VRK3 protein to total protein lysates, obtained either from adult tissues or embryos, enhanced the phosphatase activity of VHR, but not the activity of MKP3. The results further indicate that the VHR–VRK3 complex is a phosphatase-active form. In addition, we found that VRK3 can regulate EGF-induced cellular growth signaling that is mediated by ERK activation. Our results suggest that in addition to neuronal cells, various other rodent adult tissues and embryos possess a common signaling mechanism which is involved in an indirect regulation of ERK activity by VRK3-mediated VHR activity.
Keywords: Vaccinia-related kinase 3; Vaccinia H1-related phosphatase; Extracellular signal-regulated kinase; Mitogen-activated protein kinase phosphatase
VRK3-mediated inactivation of ERK signaling in adult and embryonic rodent tissues by Tae-Hong Kang; Kyong-Tai Kim (pp. 49-58).
Vaccinia-related kinase 3 (VRK3), previously characterized as a direct activator of vaccinia H1-related (VHR) phosphatase, inactivates extracellular signal-regulated kinase (ERK) in the nucleus of neuronal cells. Here we show that VRK3 is expressed in various other rodent tissues and in embryos, and regulates VHR phosphatase activity in these tissues. We observed colocalization of VRK3 and VHR in the testis tissue and could detect protein complex containing VRK3, VHR and ERK in immunoprecipitation analysis. Notably, the addition of recombinant VRK3 protein to total protein lysates, obtained either from adult tissues or embryos, enhanced the phosphatase activity of VHR, but not the activity of MKP3. The results further indicate that the VHR–VRK3 complex is a phosphatase-active form. In addition, we found that VRK3 can regulate EGF-induced cellular growth signaling that is mediated by ERK activation. Our results suggest that in addition to neuronal cells, various other rodent adult tissues and embryos possess a common signaling mechanism which is involved in an indirect regulation of ERK activity by VRK3-mediated VHR activity.
Keywords: Vaccinia-related kinase 3; Vaccinia H1-related phosphatase; Extracellular signal-regulated kinase; Mitogen-activated protein kinase phosphatase
Agonist-mediated docking of androgen receptor onto the mitotic chromatin platform discriminates intrinsic mode of action of prostate cancer drugs by Sanjay Kumar; Nagendra K. Chaturvedi; Subodh Kumar; Rakesh K. Tyagi (pp. 59-73).
This study documents the analysis of a hitherto unreported dynamic behavior of androgen receptor (AR), a member of the nuclear receptor superfamily. Employing GFP-tagged AR, we observed agonist-mediated docking of AR onto the mitotic chromatin during all the stages of mitosis. When bound to therapeutic drugs with intrinsically absolute or partial agonistic properties, AR concomitantly associated with the mitotic chromatin. Conversely, pure antagonists known to bind and subsequently translocate unliganded AR from cytoplasm to nuclear compartment did not provoke such association. The agonist-mediated docking of AR could not be competed with other transcription factors that constitutively preoccupied the chromosomal docking sites. Amongst the previously reported proteins, AR is first example of a transcription factor whose response on mitotic chromatin platform can be modulated in a ligand-specific manner. However, data from live cell imaging revealed that co-activators of agonist-activated receptor that are recruited into “nuclear foci” of interphase chromatin are dislodged from the mitotic chromatin during cell division. This implies that in absence of critical co-activators, AR transverses mitotic phase in transcriptionally silenced state. Finally, our results indicate that ligand-mediated dynamic relationship of nuclear receptors with mitotic chromatin can be effectively exploited to study, analyze and authenticate therapeutic ligands.
Keywords: Steroid receptor; Mitotic chromatin; Antiandrogen; Prostate cancer
Agonist-mediated docking of androgen receptor onto the mitotic chromatin platform discriminates intrinsic mode of action of prostate cancer drugs by Sanjay Kumar; Nagendra K. Chaturvedi; Subodh Kumar; Rakesh K. Tyagi (pp. 59-73).
This study documents the analysis of a hitherto unreported dynamic behavior of androgen receptor (AR), a member of the nuclear receptor superfamily. Employing GFP-tagged AR, we observed agonist-mediated docking of AR onto the mitotic chromatin during all the stages of mitosis. When bound to therapeutic drugs with intrinsically absolute or partial agonistic properties, AR concomitantly associated with the mitotic chromatin. Conversely, pure antagonists known to bind and subsequently translocate unliganded AR from cytoplasm to nuclear compartment did not provoke such association. The agonist-mediated docking of AR could not be competed with other transcription factors that constitutively preoccupied the chromosomal docking sites. Amongst the previously reported proteins, AR is first example of a transcription factor whose response on mitotic chromatin platform can be modulated in a ligand-specific manner. However, data from live cell imaging revealed that co-activators of agonist-activated receptor that are recruited into “nuclear foci” of interphase chromatin are dislodged from the mitotic chromatin during cell division. This implies that in absence of critical co-activators, AR transverses mitotic phase in transcriptionally silenced state. Finally, our results indicate that ligand-mediated dynamic relationship of nuclear receptors with mitotic chromatin can be effectively exploited to study, analyze and authenticate therapeutic ligands.
Keywords: Steroid receptor; Mitotic chromatin; Antiandrogen; Prostate cancer
DNA-PK contributes to the phosphorylation of AIRE: Importance in transcriptional activity by Ingrid Liiv; Ana Rebane; Tõnis Org; Mario Saare; Julia Maslovskaja; Kai Kisand; Erkki Juronen; Leena Valmu; Matthew James Bottomley; Nisse Kalkkinen; Pärt Peterson (pp. 74-83).
The autoimmune regulator (AIRE) protein is a key mediator of the central tolerance for tissue specific antigens and is involved in transcriptional control of many antigens in thymic medullary epithelial cells (mTEC). Mutations in the AIRE gene cause a rare disease named autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED). Here we report using GST pull-down assay, mass-spectrometry and co-immunoprecipitation that a heterotrimeric complex of DNA-Dependent Protein Kinase (DNA-PK), consisting of Ku70, Ku80 and DNA-PK catalytic subunit (DNA-PKcs), is a novel interaction partner for AIRE. In vitro phosphorylation assays show that the residues Thr68 and Ser156 are DNA-PK phosphorylation sites in AIRE. In addition, we demonstrate that DNA-PKcs is expressed in AIRE positive mTEC cell population and that introduction of mutations into the AIRE phosphorylation sites decrease the capacity of AIRE to activate transcription from reporter promoters. In conclusion, our results suggest that phosphorylation of the AIRE protein at Thr68 and Ser156 by DNA-PK influences AIRE transactivation ability and might have impact on other aspects of the functional regulation of the AIRE protein.
Keywords: APECED; HSR domain; PHD finger; Phosphorylation
DNA-PK contributes to the phosphorylation of AIRE: Importance in transcriptional activity by Ingrid Liiv; Ana Rebane; Tõnis Org; Mario Saare; Julia Maslovskaja; Kai Kisand; Erkki Juronen; Leena Valmu; Matthew James Bottomley; Nisse Kalkkinen; Pärt Peterson (pp. 74-83).
The autoimmune regulator (AIRE) protein is a key mediator of the central tolerance for tissue specific antigens and is involved in transcriptional control of many antigens in thymic medullary epithelial cells (mTEC). Mutations in the AIRE gene cause a rare disease named autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED). Here we report using GST pull-down assay, mass-spectrometry and co-immunoprecipitation that a heterotrimeric complex of DNA-Dependent Protein Kinase (DNA-PK), consisting of Ku70, Ku80 and DNA-PK catalytic subunit (DNA-PKcs), is a novel interaction partner for AIRE. In vitro phosphorylation assays show that the residues Thr68 and Ser156 are DNA-PK phosphorylation sites in AIRE. In addition, we demonstrate that DNA-PKcs is expressed in AIRE positive mTEC cell population and that introduction of mutations into the AIRE phosphorylation sites decrease the capacity of AIRE to activate transcription from reporter promoters. In conclusion, our results suggest that phosphorylation of the AIRE protein at Thr68 and Ser156 by DNA-PK influences AIRE transactivation ability and might have impact on other aspects of the functional regulation of the AIRE protein.
Keywords: APECED; HSR domain; PHD finger; Phosphorylation
Phosphatidylinositol 4,5-bisphosphate enhances store-operated calcium entry through hTRPC6 channel in human platelets by Isaac Jardín; Pedro C. Redondo; Ginés M. Salido; Juan A. Rosado (pp. 84-97).
Phosphatidylinositol 4,5-bisphosphate (PIP2) is a versatile regulator of TRP channels. We report that inclusion of a PIP2 analogue, PIP2 1,2-dioctanoyl, does not induce non-capacitative Ca2+ entry per se but enhanced Ca2+ entry stimulated either by thrombin or by selective depletion of the Ca2+ stores in platelets, the dense tubular system, using 10 nM TG, and the acidic stores, using 20 μM 2,5-di-( tert-butyl)-1,4-hydroquinone (TBHQ). Reduction of PIP2 levels by blocking PIP2 resynthesis with Li+ or introducing a monoclonal anti-PIP2 antibody, or sequestering PIP2 using poly-lysine, attenuated Ca2+ entry induced by thrombin, TG and TBHQ, and reduced thrombin-evoked, but not TG- or TBHQ-induced, Ca2+ release from the stores. Incubation with the anti-hTRPC1 antibody did not alter the stimulation of Ca2+ entry by PIP2, whilst introduction of anti-hTRPC6 antibody directed towards the C-terminus of hTRPC6 reduced Ca2+ and Mn2+ entry induced by thrombin, TG or TBHQ, and abolished the stimulation of Ca2+ entry by PIP2. The anti-hTRPC6 antibody, but not the anti-hTRPC1 antibody or PIP2, reduced non-capacitative Ca2+ entry by the DAG analogue 1-oleoyl-2-acetyl- sn-glycerol. In summary, hTRPC6 plays a role both in store-operated and in non-capacitative Ca2+ entry. PIP2 enhances store-operated Ca2+ entry in human platelets, most probably by stimulation of hTRPC6 channels.
Keywords: Abbreviations; BSA; bovine serum albumin; [Ca; 2+; ]; c; cytosolic free calcium concentration; DTS; dense tubular system; HBS; HEPES-buffered saline; hTRPC1; human canonical transient receptor potential 1; hTRPC6; human canonical transient receptor potential 6; IP; 3; inositol 1,4,5-trisphosphate; IP; 3; R; IP; 3; receptor; PIP; 2; Phosphatidylinositol 4,5-bisphosphate; PMCA; plasma membrane Ca; 2+; ATPase; TBHQ; 2,5-di-(; tert; -butyl)-1,4-hydroquinone; TG; thapsigarginCa; 2+; influx; PIP; 2; Platelet; Thrombin; TG; TBHQ; hTRPC6; hTRPC1
Phosphatidylinositol 4,5-bisphosphate enhances store-operated calcium entry through hTRPC6 channel in human platelets by Isaac Jardín; Pedro C. Redondo; Ginés M. Salido; Juan A. Rosado (pp. 84-97).
Phosphatidylinositol 4,5-bisphosphate (PIP2) is a versatile regulator of TRP channels. We report that inclusion of a PIP2 analogue, PIP2 1,2-dioctanoyl, does not induce non-capacitative Ca2+ entry per se but enhanced Ca2+ entry stimulated either by thrombin or by selective depletion of the Ca2+ stores in platelets, the dense tubular system, using 10 nM TG, and the acidic stores, using 20 μM 2,5-di-( tert-butyl)-1,4-hydroquinone (TBHQ). Reduction of PIP2 levels by blocking PIP2 resynthesis with Li+ or introducing a monoclonal anti-PIP2 antibody, or sequestering PIP2 using poly-lysine, attenuated Ca2+ entry induced by thrombin, TG and TBHQ, and reduced thrombin-evoked, but not TG- or TBHQ-induced, Ca2+ release from the stores. Incubation with the anti-hTRPC1 antibody did not alter the stimulation of Ca2+ entry by PIP2, whilst introduction of anti-hTRPC6 antibody directed towards the C-terminus of hTRPC6 reduced Ca2+ and Mn2+ entry induced by thrombin, TG or TBHQ, and abolished the stimulation of Ca2+ entry by PIP2. The anti-hTRPC6 antibody, but not the anti-hTRPC1 antibody or PIP2, reduced non-capacitative Ca2+ entry by the DAG analogue 1-oleoyl-2-acetyl- sn-glycerol. In summary, hTRPC6 plays a role both in store-operated and in non-capacitative Ca2+ entry. PIP2 enhances store-operated Ca2+ entry in human platelets, most probably by stimulation of hTRPC6 channels.
Keywords: Abbreviations; BSA; bovine serum albumin; [Ca; 2+; ]; c; cytosolic free calcium concentration; DTS; dense tubular system; HBS; HEPES-buffered saline; hTRPC1; human canonical transient receptor potential 1; hTRPC6; human canonical transient receptor potential 6; IP; 3; inositol 1,4,5-trisphosphate; IP; 3; R; IP; 3; receptor; PIP; 2; Phosphatidylinositol 4,5-bisphosphate; PMCA; plasma membrane Ca; 2+; ATPase; TBHQ; 2,5-di-(; tert; -butyl)-1,4-hydroquinone; TG; thapsigarginCa; 2+; influx; PIP; 2; Platelet; Thrombin; TG; TBHQ; hTRPC6; hTRPC1
Embryonic stem cell differentiation studied by FT-IR spectroscopy by Diletta Ami; Tui Neri; Antonino Natalello; Paolo Mereghetti; Silvia Maria Doglia; Mario Zanoni; Maurizio Zuccotti; Silvia Garagna; Carlo Alberto Redi (pp. 98-106).
We propose, here, an FT-IR method to monitor the spontaneous differentiation of murine embryonic stem (ES) cells in their early development. Principal component analysis and subsequent linear discriminant analysis enabled us to segregate stem cell spectra into separate clusters – corresponding to different differentiation times – and to identify the most significant spectral changes during differentiation. Between days 4 to 7 of differentiation, these spectral changes in the protein amide I band (1700–1600 cm−1) and in the nucleic acid absorption region (1050–850 cm−1) indicated that mRNA translation was taking place and that specific proteins were produced, reflecting the appearance of a new phenotype. The DNA/RNA hybrid bands (954 cm−1 and 899 cm−1) were also observed, suggesting that the transcriptional switch of the genome started at this stage of differentiation. As confirmed by cytochemical assays, the FT-IR approach presented here allows to detect at molecular level the biological events of ES cell differentiation as they take place and to monitor in a rapid way the temporal evolution of the ES cell culture.
Keywords: Cardiomyocyte; Cytodifferentiation; Embryonic stem cell; Fourier transform infrared spectroscopy; Linear discriminant analysis; Principal component analysis
Embryonic stem cell differentiation studied by FT-IR spectroscopy by Diletta Ami; Tui Neri; Antonino Natalello; Paolo Mereghetti; Silvia Maria Doglia; Mario Zanoni; Maurizio Zuccotti; Silvia Garagna; Carlo Alberto Redi (pp. 98-106).
We propose, here, an FT-IR method to monitor the spontaneous differentiation of murine embryonic stem (ES) cells in their early development. Principal component analysis and subsequent linear discriminant analysis enabled us to segregate stem cell spectra into separate clusters – corresponding to different differentiation times – and to identify the most significant spectral changes during differentiation. Between days 4 to 7 of differentiation, these spectral changes in the protein amide I band (1700–1600 cm−1) and in the nucleic acid absorption region (1050–850 cm−1) indicated that mRNA translation was taking place and that specific proteins were produced, reflecting the appearance of a new phenotype. The DNA/RNA hybrid bands (954 cm−1 and 899 cm−1) were also observed, suggesting that the transcriptional switch of the genome started at this stage of differentiation. As confirmed by cytochemical assays, the FT-IR approach presented here allows to detect at molecular level the biological events of ES cell differentiation as they take place and to monitor in a rapid way the temporal evolution of the ES cell culture.
Keywords: Cardiomyocyte; Cytodifferentiation; Embryonic stem cell; Fourier transform infrared spectroscopy; Linear discriminant analysis; Principal component analysis
Maintenance and stabilization of mtDNA can be facilitated by the DNA-binding activity of Ilv5p by Malgorzata Macierzanka; Magdalena Plotka; Diana Pryputniewicz-Drobinska; Agnieszka Lewandowska; Robert Lightowlers; Jaroslaw Marszalek (pp. 107-117).
Mitochondrial DNA (mtDNA) is inherited as a protein–DNA complex (the nucleoid). Proteins associated with the nucleoid are not only components directly involved in maintenance and propagation of mtDNA but can also be bi-functional enzymes whose metabolic activities are not directly related to mtDNA stability. In the yeast Saccharomyces cerevisiae, one such enzyme, Ilv5p is required for branch chain amino acid biosynthesis but also associates with the nucleoid. Deletions of ILV5 lead not only to metabolic defects but also to destabilization of mtDNA. Further, minor overproduction of Ilv5p stabilizes mtDNA in strains lacking Abf2p, a major mtDNA binding and packaging protein. Here we show that Ilv5p binds double-stranded DNA in vitro and is unaffected by the presence of saturating concentrations of Abf2p. In cells lacking Abf2p the amount of Ilv5p associated with the nucleoid increases significantly and is proportional to the mitochondrial concentration of Ilv5p. Altogether, we conclude that direct binding of Ilv5p can aid in the maintenance and stabilization of mtDNA.
Keywords: Ilv5p; mtDNA; Nucleoid; Mitochondria; Yeast
Maintenance and stabilization of mtDNA can be facilitated by the DNA-binding activity of Ilv5p by Malgorzata Macierzanka; Magdalena Plotka; Diana Pryputniewicz-Drobinska; Agnieszka Lewandowska; Robert Lightowlers; Jaroslaw Marszalek (pp. 107-117).
Mitochondrial DNA (mtDNA) is inherited as a protein–DNA complex (the nucleoid). Proteins associated with the nucleoid are not only components directly involved in maintenance and propagation of mtDNA but can also be bi-functional enzymes whose metabolic activities are not directly related to mtDNA stability. In the yeast Saccharomyces cerevisiae, one such enzyme, Ilv5p is required for branch chain amino acid biosynthesis but also associates with the nucleoid. Deletions of ILV5 lead not only to metabolic defects but also to destabilization of mtDNA. Further, minor overproduction of Ilv5p stabilizes mtDNA in strains lacking Abf2p, a major mtDNA binding and packaging protein. Here we show that Ilv5p binds double-stranded DNA in vitro and is unaffected by the presence of saturating concentrations of Abf2p. In cells lacking Abf2p the amount of Ilv5p associated with the nucleoid increases significantly and is proportional to the mitochondrial concentration of Ilv5p. Altogether, we conclude that direct binding of Ilv5p can aid in the maintenance and stabilization of mtDNA.
Keywords: Ilv5p; mtDNA; Nucleoid; Mitochondria; Yeast
Differential splicing patterns of L-Type calcium channel Cav1.2 subunit in hearts of Spontaneously Hypertensive Rats and Wistar Kyoto rats by Zhen Zhi Tang; Ping Liao; Guang Li; Feng Li Jiang; Dejie Yu; Xin Hong; Tan Fong Yong; Gregory Tan; Songqing Lu; Jing Wang; Tuck Wah Soong (pp. 118-130).
Cav1.2 L-type calcium channels are essential in heart and smooth muscle contraction. Rat Cav1.2 gene contains 11 alternatively spliced exons (1a, 1, 8a, 8, 9⁎, 21, 22, 31, 32, 32-6nt and 33) which can be assorted to generate a large number of functionally distinct splice variants. Until now, it is unknown whether the utilization of these alternatively spliced exons is altered in the hypertrophied hearts of hypertensive rats. By comparing the assortments of these 11 exons in full-length Cav1.2 transcripts derived from Spontaneously Hypertensive Rats (SHRs) and Wistar Kyoto Rats (WKYs) hearts, we found that the inclusion of Cav1.2 alternative exons was significantly different between the two rats both at individual loci and in combinatorial arrangements. Functional characterizations of three Cav1.2 channel splice variants that were identified to be significantly altered in SHR hypertrophied cardiomyocytes demonstrated distinct whole-cell electrophysiological properties when expressed in HEK 293 cells. Interestingly, aberrant splice variants which included or excluded both mutually exclusive exons 21/22 or exons 31/32 were found to be increased in hypertensive rats. Two aberrant splice variants that included both exons 21 and 22 were found to be unable to conduct currents even though they expressed proteins with the predicted molecular mass. Characterization of one of the aberrant splice variants showed that it exerted a dominant negative effect on the functional Cav1.2 channels when co-expressed in HEK293 cells. The altered combinatorial splicing profiles of Cav1.2 transcripts identified in SHR hearts provide a different and new perspective in understanding the possible role of molecular remodeling of Cav1.2 channels in cardiac hypertrophy as a consequence of hypertension.
Keywords: Alternative splicing; Ca; v; 1.2; Voltage gated calcium channel; Spontaneously Hypertensive Rat; Hypertension; Hypertrophied heart
Differential splicing patterns of L-Type calcium channel Cav1.2 subunit in hearts of Spontaneously Hypertensive Rats and Wistar Kyoto rats by Zhen Zhi Tang; Ping Liao; Guang Li; Feng Li Jiang; Dejie Yu; Xin Hong; Tan Fong Yong; Gregory Tan; Songqing Lu; Jing Wang; Tuck Wah Soong (pp. 118-130).
Cav1.2 L-type calcium channels are essential in heart and smooth muscle contraction. Rat Cav1.2 gene contains 11 alternatively spliced exons (1a, 1, 8a, 8, 9⁎, 21, 22, 31, 32, 32-6nt and 33) which can be assorted to generate a large number of functionally distinct splice variants. Until now, it is unknown whether the utilization of these alternatively spliced exons is altered in the hypertrophied hearts of hypertensive rats. By comparing the assortments of these 11 exons in full-length Cav1.2 transcripts derived from Spontaneously Hypertensive Rats (SHRs) and Wistar Kyoto Rats (WKYs) hearts, we found that the inclusion of Cav1.2 alternative exons was significantly different between the two rats both at individual loci and in combinatorial arrangements. Functional characterizations of three Cav1.2 channel splice variants that were identified to be significantly altered in SHR hypertrophied cardiomyocytes demonstrated distinct whole-cell electrophysiological properties when expressed in HEK 293 cells. Interestingly, aberrant splice variants which included or excluded both mutually exclusive exons 21/22 or exons 31/32 were found to be increased in hypertensive rats. Two aberrant splice variants that included both exons 21 and 22 were found to be unable to conduct currents even though they expressed proteins with the predicted molecular mass. Characterization of one of the aberrant splice variants showed that it exerted a dominant negative effect on the functional Cav1.2 channels when co-expressed in HEK293 cells. The altered combinatorial splicing profiles of Cav1.2 transcripts identified in SHR hearts provide a different and new perspective in understanding the possible role of molecular remodeling of Cav1.2 channels in cardiac hypertrophy as a consequence of hypertension.
Keywords: Alternative splicing; Ca; v; 1.2; Voltage gated calcium channel; Spontaneously Hypertensive Rat; Hypertension; Hypertrophied heart
DNMT1 interacts with the developmental transcriptional repressor HESX1 by Ezat Sajedi; Carles Gaston-Massuet; Cynthia L. Andoniadou; Massimo Signore; Paul J. Hurd; Mehul Dattani; Juan Pedro Martinez-Barbera (pp. 131-143).
Hesx1 is a highly conserved homeobox gene present in vertebrates, but absent from invertebrates. Gene targeting experiments in mice have shown that this transcriptional repressor is required for normal forebrain and pituitary development. In humans, mutations in HESX1 impairing either its repressing activity or DNA binding properties lead to a comparable phenotype to that observed in Hesx1 deficient mice. In an attempt to gain insights into the molecular function of HESX1, we have performed a yeast two-hybrid screen and identified DNA methyltransferase 1 (DNMT1) as a HESX1 binding protein. We show that Dnmt1 is co-expressed with Hesx1 within the anterior forebrain and in the developing Rathke's pouch. Mapping of the interacting regions indicates that the entire HESX1 protein is required to establish binding to a portion of the N-terminus of DNMT1 and its catalytic domain in the C-terminus. The HESX1–DNMT1 complexes can be immunoprecipitated in cells and co-localise in the nucleus. These results establish a link between HESX1 and DNMT1 and suggest a novel mechanism for the repressing properties of HESX1.
Keywords: Abbreviations; aa; amino acid; DNMT1; DNA methyltransferase 1; SAFB1; scaffold attachment factor beta 1; RNF2; ring finger protein 2; Lonp2; lon peptidase 2, peroxisomal; ZFP592; zinc finger protein 592; BTBD2; BTB (POZ) domain containing 2; SRFBP1; serum response factor binding protein 1; ZMIZ1; zinc finger MIZ-type containing 1; SOD; septo-optic dysplasia; TLE1; transducin-like enhancer of split 1; N-CoR; nuclear co-repressor; eh1; engrailed homology domain 1; GST; glutathione-; S; -transferase; Gal4DBD; Gal4 DNA binding domain; IVT; in vitro translated; HRP; horse radish peroxidase; PFA; paraformaldehyde; PcP; polycomb group; EZH2; enhancer of zeste homologue; ES cells; embryonic stem cellsDNA methylation; Repression; Homeobox; Forebrain; Pituitary; Mouse
DNMT1 interacts with the developmental transcriptional repressor HESX1 by Ezat Sajedi; Carles Gaston-Massuet; Cynthia L. Andoniadou; Massimo Signore; Paul J. Hurd; Mehul Dattani; Juan Pedro Martinez-Barbera (pp. 131-143).
Hesx1 is a highly conserved homeobox gene present in vertebrates, but absent from invertebrates. Gene targeting experiments in mice have shown that this transcriptional repressor is required for normal forebrain and pituitary development. In humans, mutations in HESX1 impairing either its repressing activity or DNA binding properties lead to a comparable phenotype to that observed in Hesx1 deficient mice. In an attempt to gain insights into the molecular function of HESX1, we have performed a yeast two-hybrid screen and identified DNA methyltransferase 1 (DNMT1) as a HESX1 binding protein. We show that Dnmt1 is co-expressed with Hesx1 within the anterior forebrain and in the developing Rathke's pouch. Mapping of the interacting regions indicates that the entire HESX1 protein is required to establish binding to a portion of the N-terminus of DNMT1 and its catalytic domain in the C-terminus. The HESX1–DNMT1 complexes can be immunoprecipitated in cells and co-localise in the nucleus. These results establish a link between HESX1 and DNMT1 and suggest a novel mechanism for the repressing properties of HESX1.
Keywords: Abbreviations; aa; amino acid; DNMT1; DNA methyltransferase 1; SAFB1; scaffold attachment factor beta 1; RNF2; ring finger protein 2; Lonp2; lon peptidase 2, peroxisomal; ZFP592; zinc finger protein 592; BTBD2; BTB (POZ) domain containing 2; SRFBP1; serum response factor binding protein 1; ZMIZ1; zinc finger MIZ-type containing 1; SOD; septo-optic dysplasia; TLE1; transducin-like enhancer of split 1; N-CoR; nuclear co-repressor; eh1; engrailed homology domain 1; GST; glutathione-; S; -transferase; Gal4DBD; Gal4 DNA binding domain; IVT; in vitro translated; HRP; horse radish peroxidase; PFA; paraformaldehyde; PcP; polycomb group; EZH2; enhancer of zeste homologue; ES cells; embryonic stem cellsDNA methylation; Repression; Homeobox; Forebrain; Pituitary; Mouse
Increase in Fru-2,6-P2 levels results in altered cell division in Schizosaccharomyces pombe by Silvia Fernández de Mattos; Vicenç Alemany; Rosa Aligué; Albert Tauler (pp. 144-152).
Mitogenic response to growth factors is concomitant with the modulation they exert on the levels of Fructose 2,6-bisphosphate (Fru-2,6-P2), an essential activator of the glycolytic flux. In mammalian cells, decreased Fru-2,6-P2 concentration causes cell cycle delay, whereas high levels of Fru-2,6-P2 sensitize cells to apoptosis. In order to analyze the cell cycle consequences due to changes in Fru-2,6-P2 levels, the bisphosphatase-dead mutant (H258A) of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase enzyme was over-expressed in Schizosaccharomyces pombe cells and the variation in cell phenotype was studied. The results obtained demonstrate that the increase in Fru-2,6-P2 levels results in a defective division of S. pombe, as revealed by an altered multisepted phenotype. The H258A-expressing cells showed impairment of cytokinesis, but normal nuclear division. In order to identify cellular mediators responsible for this effect, we transformed different S. pombe strains and observed that the cytokinetic defect was absent in cells defective for Wee1 kinase function. Therefore, in S. pombe, Wee1 integrates the metabolic signal emerging from changes in Fru-2,6-P2 content, thus coupling metabolism with cell proliferation. As the key regulators of the cell cycle checkpoints are conserved throughout evolution, these results may help to understand the experimental evidences obtained by manipulation of Fru-2,6-P2 levels in mammalian cells.
Keywords: Abbreviations; Fru-2,6-P; 2; Fructose 2,6-bisphosphate; PF2KFB; 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase; MAPK; mitogen-activated protein kinase; PI3K; phosphatidylinositol 3-kinase; ROS; reactive oxygen species; TIGAR; TP53-induced glycolysis and apoptosis regulator; HA; hemagglutinin; DAPI; 4′,6-Diamidino-2-phenylindoleGlycolysis; Fru-2,6-P; 2; Proliferation; Cell cycle control; Fission yeast
Increase in Fru-2,6-P2 levels results in altered cell division in Schizosaccharomyces pombe by Silvia Fernández de Mattos; Vicenç Alemany; Rosa Aligué; Albert Tauler (pp. 144-152).
Mitogenic response to growth factors is concomitant with the modulation they exert on the levels of Fructose 2,6-bisphosphate (Fru-2,6-P2), an essential activator of the glycolytic flux. In mammalian cells, decreased Fru-2,6-P2 concentration causes cell cycle delay, whereas high levels of Fru-2,6-P2 sensitize cells to apoptosis. In order to analyze the cell cycle consequences due to changes in Fru-2,6-P2 levels, the bisphosphatase-dead mutant (H258A) of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase enzyme was over-expressed in Schizosaccharomyces pombe cells and the variation in cell phenotype was studied. The results obtained demonstrate that the increase in Fru-2,6-P2 levels results in a defective division of S. pombe, as revealed by an altered multisepted phenotype. The H258A-expressing cells showed impairment of cytokinesis, but normal nuclear division. In order to identify cellular mediators responsible for this effect, we transformed different S. pombe strains and observed that the cytokinetic defect was absent in cells defective for Wee1 kinase function. Therefore, in S. pombe, Wee1 integrates the metabolic signal emerging from changes in Fru-2,6-P2 content, thus coupling metabolism with cell proliferation. As the key regulators of the cell cycle checkpoints are conserved throughout evolution, these results may help to understand the experimental evidences obtained by manipulation of Fru-2,6-P2 levels in mammalian cells.
Keywords: Abbreviations; Fru-2,6-P; 2; Fructose 2,6-bisphosphate; PF2KFB; 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase; MAPK; mitogen-activated protein kinase; PI3K; phosphatidylinositol 3-kinase; ROS; reactive oxygen species; TIGAR; TP53-induced glycolysis and apoptosis regulator; HA; hemagglutinin; DAPI; 4′,6-Diamidino-2-phenylindoleGlycolysis; Fru-2,6-P; 2; Proliferation; Cell cycle control; Fission yeast
Phosphatidic acid metabolism regulates the intracellular trafficking and retrotranslocation of CFTR by Yasuaki Hashimoto; Tsukasa Okiyoneda; Kazutsune Harada; Keiko Ueno; Takuya Sugahara; Atsushi Yamashita; Tsuyoshi Shuto; Mary Ann Suico; Hirofumi Kai (pp. 153-162).
The cystic fibrosis transmembrane conductance regulator (CFTR) is transported to the plasma membrane from endoplasmic reticulum (ER) through the Golgi. Crucial to these trafficking events is the role of not only the proteinous factors but also the membrane lipids. However, the involvement of lipids, such as phospholipids, on the regulation of CFTR trafficking has been largely unexplored. Here, we show that the inhibition of phospholipase D (PLD)-mediated phosphatidic acid (PA) formation by 1-butanol inhibited the maturation and export of CFTR from the ER. Exogenously added PA reversed these effects. Moreover, knock down of PLD1 by small interfering RNA decreased the expression of mature CFTR. Interestingly, sustaining the level of PA, by the addition of excess PA in the presence of PA phosphatase inhibitor, attenuated the transport of CFTR from the Golgi to plasma membrane and the retrograde transport of ΔF508 CFTR to the cytoplasm, a necessary step for the ER-associated degradation of ΔF508 CFTR. These results indicated that the metabolism of PA modulated the intracellular dynamics and trafficking of CFTR.
Keywords: Phosphatidic acid; Phospholipase D; CFTR; ΔF508 CFTR; Trafficking; Retrotranslocation
Phosphatidic acid metabolism regulates the intracellular trafficking and retrotranslocation of CFTR by Yasuaki Hashimoto; Tsukasa Okiyoneda; Kazutsune Harada; Keiko Ueno; Takuya Sugahara; Atsushi Yamashita; Tsuyoshi Shuto; Mary Ann Suico; Hirofumi Kai (pp. 153-162).
The cystic fibrosis transmembrane conductance regulator (CFTR) is transported to the plasma membrane from endoplasmic reticulum (ER) through the Golgi. Crucial to these trafficking events is the role of not only the proteinous factors but also the membrane lipids. However, the involvement of lipids, such as phospholipids, on the regulation of CFTR trafficking has been largely unexplored. Here, we show that the inhibition of phospholipase D (PLD)-mediated phosphatidic acid (PA) formation by 1-butanol inhibited the maturation and export of CFTR from the ER. Exogenously added PA reversed these effects. Moreover, knock down of PLD1 by small interfering RNA decreased the expression of mature CFTR. Interestingly, sustaining the level of PA, by the addition of excess PA in the presence of PA phosphatase inhibitor, attenuated the transport of CFTR from the Golgi to plasma membrane and the retrograde transport of ΔF508 CFTR to the cytoplasm, a necessary step for the ER-associated degradation of ΔF508 CFTR. These results indicated that the metabolism of PA modulated the intracellular dynamics and trafficking of CFTR.
Keywords: Phosphatidic acid; Phospholipase D; CFTR; ΔF508 CFTR; Trafficking; Retrotranslocation