scholarly journals DEAD-box protein family member DDX28 is a negative regulator of HIF-2α and eIF4E2-directed hypoxic translation

2019 ◽  
Author(s):  
Sonia L. Evagelou ◽  
Olivia Bebenek ◽  
Erin J. Specker ◽  
James Uniacke
Keyword(s):  
Family Member ◽  
Mammalian Cells ◽  
Protein Family ◽  
Negative Regulator ◽  
Protein Levels ◽  
Oxygen Homeostasis ◽  
Dead Box ◽  
Nuclear Abundance ◽  
Potential Tumor Suppressor ◽  
Proliferative Advantage

ABSTRACTHypoxia occurs when there is a deficiency in oxygen delivery to tissues and is connected to physiological and pathophysiological processes such as embryonic development, wound healing, heart disease and cancer. The master regulators of oxygen homeostasis in mammalian cells are the heterodimeric hypoxia-inducible transcription factors HIF-1 and HIF-2. The oxygen-labile HIF-2α subunit has not only been implicated in transcription, but also as a regulator of eIF4E2-directed hypoxic translation. Here, we have identified the DEAD-box protein family member DDX28 as a novel interactor and negative regulator of HIF-2α that suppresses its ability to activate eIF4E2-directed translation. We demonstrate that stable silencing of DDX28 via shRNA in hypoxic human U87MG glioblastoma cells caused an increase, relative to control, to: HIF-2α protein levels, the ability of eIF4E2 to bind the m7GTP cap structure, and the translation of select eIF4E2 target mRNAs. DDX28 depletion elevated both nuclear and cytoplasmic HIF-2α, but HIF-2α transcriptional activity did not increase possibly due to its already high nuclear abundance in hypoxic control cells. Depletion of DDX28 conferred a proliferative advantage to hypoxic, but not normoxic cells, which is likely a consequence of the translational upregulation of a subset of hypoxia-response mRNAs. DDX28 protein levels are reduced in several cancers, including glioma, relative to normal tissue. Therefore, we uncover a regulatory mechanism for this potential tumor suppressor in the repression of HIF-2α- and eIF4E2-mediated translation activation of oncogenic mRNAs.

scholarly journals DEAD Box Protein Family Member DDX28 Is a Negative Regulator of Hypoxia-Inducible Factor 2α- and Eukaryotic Initiation Factor 4E2-Directed Hypoxic Translation

2020 ◽  
Vol 40 (6) ◽  
Author(s):  
Sonia L. Evagelou ◽  
Olivia Bebenek ◽  
Erin J. Specker ◽  
James Uniacke
Keyword(s):  
Family Member ◽  
Mammalian Cells ◽  
Hypoxia Inducible Factor ◽  
Protein Family ◽  
Negative Regulator ◽  
Initiation Factor ◽  
Eukaryotic Initiation Factor ◽  
Protein Levels ◽  
Oxygen Homeostasis ◽  
Dead Box

ABSTRACT Hypoxia is a deficiency in oxygen delivery to tissues and is connected to physiological and pathophysiological processes such as embryonic development and cancer. The master regulators of oxygen homeostasis in mammalian cells are the heterodimeric hypoxia-inducible transcription factors 1 and 2 (HIF-1 and HIF-2, respectively). The oxygen-labile HIF-2α subunit has been implicated not only in transcription but also as a regulator of eukaryotic initiation factor 4E2 (eIF4E2)-directed hypoxic translation. Here, we have identified the DEAD box protein family member DDX28 as an interactor and negative regulator of HIF-2α that suppresses HIF-2α’s ability to activate eIF4E2-directed translation. Stable silencing of DDX28 via short hairpin RNA (shRNA) in hypoxic human U87MG glioblastoma cells caused an increase of eIF4E2 binding to the m7GTP cap structure and the translation of eIF4E2 target mRNAs (including the HIF-2α mRNA itself). DDX28 depletion elevated nuclear and cytoplasmic HIF-2α protein, but HIF-2α transcriptional activity did not increase, possibly due to its already high nuclear abundance in hypoxic control cells. Depletion of DDX28 conferred a proliferative advantage to hypoxic, but not normoxic, cells. DDX28 protein levels are reduced in several cancers, including gliomas, relative to levels in normal tissue. Therefore, we uncover a regulatory mechanism for this potential tumor suppressor in the repression of HIF-2α- and eIF4E2-mediated translation activation of oncogenic mRNAs.

Gtpase Immunity-Associated Protein Family Member 4 Contributes to the Enhanced Expansion of HSPCs in RUNX1 Deficient Mice

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4344-4344
Author(s):  
Amanda Scholl ◽  
Kentson Lam ◽  
Alex Muselman ◽  
Tingdong Tang ◽  
Shinobu Matsuura ◽  
...  
Keyword(s):  
Family Member ◽  
Conflicts Of Interest ◽  
Protein Family ◽  
Dominant Negative ◽  
Negative Regulator ◽  
Wild Type ◽  
Double Knockout ◽  
Hematopoietic Stem ◽  
Myeloid Progenitor

Abstract RUNX1 is the transcription factor described as the master regulator of hematopoiesis. Due to its central role during blood development, numerous RUNX1 mutations have been reported in hematologic abnormalities. Mice null for Runx1 die during embryogenesis, lacking definitive HSCs. Conditional Runx1Δ/Δ mice are viable, but exhibit a variety of blood abnormalities. The most salient defect in these Runx1Δ/Δ mice is expansion of the hematopoietic stem and progenitor cell (HSPC) population, measured as an increase in number of lineage negative, Sca1 positive, cKit positive (LSK) cells. A shortened form of RUNX1 (RUNX1SF) lacking the C-terminal and part of the N-terminal domain (41-214) acts as a dominant negative regulator of RUNX1 and hence also models RUNX1 loss-of-function. A differential gene expression analysis of HSPCs derived from Runx1Δ/Δ compared to wild type mice uncovered GTPase immunity-associated protein family member 4 (GIMAP4) as one of the genes most highly upregulated. Previous studies have focused almost exclusively on the role of GIMAP4 as a pro-apoptotic protein during T-cell development. This study illuminates a novel non-apoptotic role of GIMAP4 in a formerly unstudied HSPC context. Runx1Δ/Δ mice were crossed with Gimap4-/- mice to generate a double knockout (dKO) mouse line. These dKO mice exhibited attenuated HSPC proliferation in comparison to Runx1Δ/Δ mice, suggesting that GIMAP4 functions in this HSPC expansion phenotype. BMT experiments using lethally irradiated C57 mice and RUNX1SF transduced wild type versus Gimap4-/-bone marrow confirmed this result. GIMAP4 also worked independently and coordinately with RUNX1 to influence individual progenitor populations. Common lymphoid progenitors (CLP) were affected only by GIMAP4. Gimap4-/- mice exhibited an expansion of the CLP population, consistent with its pro-apoptotic role in lymphoid populations. Conversely, both RUNX1 and GIMAP4 coordinately exerted an effect on myeloid progenitor populations. Runx1Δ/Δ mice harbored expanded granulocyte-macrophage progenitor (GMP) and common myeloid progenitor (CMP) populations. This expansion was not observed when GIMAP4 was also ablated. This suggests a pro-proliferative role of GIMAP4 specifically in myeloid populations. These opposing roles of GIMAP4 in lymphoid versus myeloid cells suggest a more contextual, cell-specific role of this GTPase protein. Ultimately, this study provides insight into how RUNX1 and GIMAP4 may coordinate to maintain HSPC homeostasis. Disclosures No relevant conflicts of interest to declare.

scholarly journals A long hypoxia-inducible factor 3 isoform 2 is a transcription activator that regulates erythropoietin

2019 ◽  
Vol 77 (18) ◽  
pp. 3627-3642 ◽  
Author(s):  
Jussi-Pekka Tolonen ◽  
Minna Heikkilä ◽  
Marjo Malinen ◽  
Hang-Mao Lee ◽  
Jorma J. Palvimo ◽  
...  
Keyword(s):  
Target Genes ◽  
Hypoxia Inducible Factor ◽  
Negative Regulator ◽  
Luciferase Reporter ◽  
Transcription Activator ◽  
Promoter Regions ◽  
Α Subunit ◽  
Hypoxia Response ◽  
Protein Levels ◽  
Oxygen Homeostasis

AbstractHypoxia-inducible factor (HIF), an αβ dimer, is the master regulator of oxygen homeostasis with hundreds of hypoxia-inducible target genes. Three HIF isoforms differing in the oxygen-sensitive α subunit exist in vertebrates. While HIF-1 and HIF-2 are known transcription activators, HIF-3 has been considered a negative regulator of the hypoxia response pathway. However, the human HIF3A mRNA is subject to complex alternative splicing. It was recently shown that the long HIF-3α variants can form αβ dimers that possess transactivation capacity. Here, we show that overexpression of the long HIF-3α2 variant induces the expression of a subset of genes, including the erythropoietin (EPO) gene, while simultaneous downregulation of all HIF-3α variants by siRNA targeting a shared HIF3A region leads to downregulation of EPO and additional genes. EPO mRNA and protein levels correlated with HIF3A silencing and HIF-3α2 overexpression. Chromatin immunoprecipitation analyses showed that HIF-3α2 binding associated with canonical hypoxia response elements in the promoter regions of EPO. Luciferase reporter assays showed that the identified HIF-3α2 chromatin-binding regions were sufficient to promote transcription by all three HIF-α isoforms. Based on these data, HIF-3α2 is a transcription activator that directly regulates EPO expression.

Phospholipase D1b and D2a generate structurally identical phosphatidic acid species in mammalian cells

2001 ◽  
Vol 360 (3) ◽  
pp. 707-715 ◽  
Author(s):  
Trevor R. PETTITT ◽  
Mark McDERMOTT ◽  
Khalid M. SAQIB ◽  
Neil SHIMWELL ◽  
Michael J. O. WAKELAM
Keyword(s):  
Liquid Chromatography ◽  
Phosphatidic Acid ◽  
Phospholipase D ◽  
Mammalian Cells ◽  
Inducible Promoter ◽  
In Vitro Assays ◽  
Protein Levels ◽  
Intracellular Messenger

Mammalian cells contain different phospholipase D enzymes (PLDs) whose distinct physiological roles are poorly understood and whose products have not been characterized. The development of porcine aortic endothelial (PAE) cell lines able to overexpress PLD-1b or −2a under the control of an inducible promoter has enabled us to characterize both the substrate specificity and the phosphatidic acid (PtdOH) product of these enzymes under controlled conditions. Liquid chromatography–MS analysis showed that PLD1b- and PLD2a-transfected PAE cells, as well as COS7 and Rat1 cells, generate similar PtdOH and, in the presence of butan-1-ol, phosphatidylbutanol (PtdBut) profiles, enriched in mono- and di-unsaturated species, in particular 16:0/18:1. Although PtdBut mass increased, the species profile did not change in cells stimulated with ATP or PMA. Overexpression of PLD made little difference to basal or stimulated PtdBut formation, indicating that activity is tightly regulated in vivo and that factors other than just PLD protein levels limit hydrolytic function. In vitro assays using PLD-enriched lysates showed that the enzyme could utilize both phosphatidylcholine and, much less efficiently, phosphatidylethanolamine, with slight selectivity towards mono- and di-unsaturated species. Phosphatidylinositol was not a substrate. Thus PLD1b and PLD2a hydrolyse a structurally similar substrate pool to generate an identical PtdOH product enriched in mono- and di-unsaturated species that we propose to function as the intracellular messenger forms of this lipid.

scholarly journals Dicer is involved in protection against influenza A virus infection

2007 ◽  
Vol 88 (10) ◽  
pp. 2627-2635 ◽  
Author(s):  
Alexey A. Matskevich ◽  
Karin Moelling
Keyword(s):  
Virus Infection ◽  
Influenza A Virus ◽  
Mammalian Cells ◽  
Influenza A ◽  
Virus Production ◽  
Vero Cells ◽  
Antiviral Response ◽  
Protein Levels ◽  
Infected Cells ◽  
Influenza A Virus Infection

In mammals the interferon (IFN) system is a central innate antiviral defence mechanism, while the involvement of RNA interference (RNAi) in antiviral response against RNA viruses is uncertain. Here, we tested whether RNAi is involved in the antiviral response in mammalian cells. To investigate the role of RNAi in influenza A virus-infected cells in the absence of IFN, we used Vero cells that lack IFN-α and IFN-β genes. Our results demonstrate that knockdown of a key RNAi component, Dicer, led to a modest increase of virus production and accelerated apoptosis of influenza A virus-infected cells. These effects were much weaker in the presence of IFN. The results also show that in both Vero cells and the IFN-producing alveolar epithelial A549 cell line influenza A virus targets Dicer at mRNA and protein levels. Thus, RNAi is involved in antiviral response, and Dicer is important for protection against influenza A virus infection.

scholarly journals Coordinated Regulation of Insulin Signaling by the Protein Tyrosine Phosphatases PTP1B and TCPTP

2005 ◽  
Vol 25 (2) ◽  
pp. 819-829 ◽  
Author(s):  
Sandra Galic ◽  
Christine Hauser ◽  
Barbara B. Kahn ◽  
Fawaz G. Haj ◽  
Benjamin G. Neel ◽  
...  
Keyword(s):  
Insulin Signaling ◽  
Tyrosine Phosphatase ◽  
Protein Tyrosine Phosphatases ◽  
Negative Regulator ◽  
Mitogen Activated Protein Kinase ◽  
Protein Tyrosine ◽  
Akt Activation ◽  
Protein Levels ◽  
Mitogen Activated Protein

ABSTRACT The protein tyrosine phosphatase PTP1B is a negative regulator of insulin signaling and a therapeutic target for type 2 diabetes. Our previous studies have shown that the closely related tyrosine phosphatase TCPTP might also contribute to the regulation of insulin receptor (IR) signaling in vivo (S. Galic, M. Klingler-Hoffmann, M. T. Fodero-Tavoletti, M. A. Puryer, T. C. Meng, N. K. Tonks, and T. Tiganis, Mol. Cell. Biol. 23:2096-2108, 2003). Here we show that PTP1B and TCPTP function in a coordinated and temporally distinct manner to achieve an overall regulation of IR phosphorylation and signaling. Whereas insulin-induced phosphatidylinositol 3-kinase/Akt signaling was prolonged in both TCPTP−/− and PTP1B−/− immortalized mouse embryo fibroblasts (MEFs), mitogen-activated protein kinase ERK1/2 signaling was elevated only in PTP1B-null MEFs. By using phosphorylation-specific antibodies, we demonstrate that both IR β-subunit Y1162/Y1163 and Y972 phosphorylation are elevated in PTP1B−/− MEFs, whereas Y972 phosphorylation was elevated and Y1162/Y1163 phosphorylation was sustained in TCPTP−/− MEFs, indicating that PTP1B and TCPTP differentially contribute to the regulation of IR phosphorylation and signaling. Consistent with this, suppression of TCPTP protein levels by RNA interference in PTP1B−/− MEFs resulted in no change in ERK1/2 signaling but caused prolonged Akt activation and Y1162/Y1163 phosphorylation. These results demonstrate that PTP1B and TCPTP are not redundant in insulin signaling and that they act to control both common as well as distinct insulin signaling pathways in the same cell.

scholarly journals Cloning and expression of a novel cysteine-rich secreted protein family member expressed in thyroid and pancreatic mesoderm within the chicken embryo

2001 ◽  
Vol 102 (1-2) ◽  
pp. 223-226 ◽  
Author(s):  
Devyn M. Smith ◽  
Lisa A. Collins-Racie ◽  
Valeria A. Marigo ◽  
Drucilla J. Roberts ◽  
Nicole M. Davis ◽  
...  
Keyword(s):  
Family Member ◽  
Chicken Embryo ◽  
Protein Family ◽  
Cloning And Expression ◽  
Secreted Protein

scholarly journals FBXW7 mutations reduce binding of NOTCH1, leading to cleaved NOTCH1 accumulation and target gene activation in CLL

Blood ◽  
2019 ◽  
Vol 133 (8) ◽  
pp. 830-839 ◽  
Author(s):  
Viola Close ◽  
William Close ◽  
Sabrina Julia Kugler ◽  
Michaela Reichenzeller ◽  
Deyan Yordanov Yosifov ◽  
...  
Keyword(s):  
Target Gene ◽  
Hypoxia Inducible Factor ◽  
Gene Activation ◽  
Lymphocytic Leukemia ◽  
Negative Regulator ◽  
Missense Mutations ◽  
Protein Levels ◽  
In Silico Modeling ◽  
Functional Consequences ◽  
Wd40 Domain

Abstract NOTCH1 is mutated in 10% of chronic lymphocytic leukemia (CLL) patients and is associated with poor outcome. However, NOTCH1 activation is identified in approximately one-half of CLL cases even in the absence of NOTCH1 mutations. Hence, there appear to be additional factors responsible for the impairment of NOTCH1 degradation. E3-ubiquitin ligase F-box and WD40 repeat domain containing-7 (FBXW7), a negative regulator of NOTCH1, is mutated in 2% to 6% of CLL patients. The functional consequences of these mutations in CLL are unknown. We found heterozygous FBXW7 mutations in 36 of 905 (4%) untreated CLL patients. The majority were missense mutations (78%) that mostly affected the WD40 substrate binding domain; 10% of mutations occurred in the first exon of the α-isoform. To identify target proteins of FBXW7 in CLL, we truncated the WD40 domain in CLL cell line HG-3 via clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 (Cas9). Homozygous truncation of FBXW7 resulted in an increase of activated NOTCH1 intracellular domain (NICD) and c-MYC protein levels as well as elevated hypoxia-inducible factor 1-α activity. In silico modeling predicted that novel mutations G423V and W425C in the FBXW7-WD40 domain change the binding of protein substrates. This differential binding was confirmed via coimmunoprecipitation of overexpressed FBXW7 and NOTCH1. In primary CLL cells harboring FBXW7 mutations, activated NICD levels were increased and remained stable upon translation inhibition. FBXW7 mutations coincided with an increase in NOTCH1 target gene expression and explain a proportion of patients characterized by dysregulated NOTCH1 signaling.

scholarly journals Dual-controlled optogenetic system for the rapid down-regulation of protein levels in mammalian cells

2018 ◽  
Vol 8 (1) ◽  
Author(s):  
Julia Baaske ◽  
Patrick Gonschorek ◽  
Raphael Engesser ◽  
Alazne Dominguez-Monedero ◽  
Katrin Raute ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Down Regulation ◽  
Protein Levels
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