scholarly journals Inhibition of Nuclear Import by the Proapoptotic Protein CC3

2004 ◽  
Vol 24 (16) ◽  
pp. 7091-7101 ◽  
Author(s):  
Frank W. King ◽  
Emma Shtivelman
Keyword(s):  
Nuclear Import ◽  
Nuclear Translocation ◽  
Cellular Protein ◽  
Dominant Negative ◽  
Transport Pathways ◽  
Proapoptotic Protein ◽  
Dominant Negative Form ◽  
Increased Sensitivity

ABSTRACT We report here that the normal cellular protein CC3/TIP30, when in excess, inhibits nuclear import in vitro and in vivo. CC3 binds directly to the karyopherins of the importin β family in a RanGTP-insensitive manner and associates with nucleoporins in vivo. CC3 inhibits the nuclear import of proteins possessing either the classical nuclear localization signal or the M9 signal recognized by transportin. CC3 also inhibits nuclear translocation of transportin itself. Cells modified to express higher levels of CC3 have a slower rate of nuclear import and, as described earlier, show an increased sensitivity to death signals. A mutant CC3 protein lacking proapoptotic activity has a lower affinity for transportin, is displaced from it by RanGTP, and fails to inhibit nuclear import in vitro and in vivo. Together, our results support a correlation between the ability of CC3 to form a RanGTP-resistant complex with importins, inhibit nuclear import, and induce apoptosis. Significantly, a dominant-negative form of importin β1 shown previously to inhibit multiple transport pathways induces rapid cell death, strongly indicating that inhibition of nuclear transport serves as a potent apoptotic signal.

scholarly journals Nuclear Import of IκBα Is Accomplished by a Ran-Independent Transport Pathway

2000 ◽  
Vol 20 (5) ◽  
pp. 1571-1582 ◽  
Author(s):  
Shrikesh Sachdev ◽  
Sriparna Bagchi ◽  
Donna D. Zhang ◽  
Angela C. Mings ◽  
Mark Hannink
Keyword(s):  
Nuclear Import ◽  
Nuclear Export ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Nuclear Pore ◽  
Transport Pathway ◽  
Repeat Domain ◽  
Nuclear Export Receptor

ABSTRACT The inhibitor of kappa B alpha (IκBα) protein is able to shuttle between the cytoplasm and the nucleus. We have utilized a combination of in vivo and in vitro approaches to provide mechanistic insight into nucleocytoplasmic shuttling by IκBα. IκBα contains multiple functional domains that contribute to shuttling of IκBα between the cytoplasm and the nucleus. Nuclear import of IκBα is mediated by the central ankyrin repeat domain. Similar to previously described nuclear import pathways, nuclear import of IκBα is temperature and ATP dependent and is blocked by a dominant-negative mutant of importin β. However, in contrast to classical nuclear import pathways, nuclear import of IκBα is independent of soluble cytosolic factors and is not blocked by the dominant-negative RanQ69L protein. Nuclear export of IκBα is mediated by an N-terminal nuclear export sequence. Nuclear export of IκBα requires the CRM1 nuclear export receptor and is blocked by the dominant-negative RanQ69L protein. Our results are consistent with a model in which nuclear import of IκBα is mediated through direct interactions with components of the nuclear pore complex, while nuclear export of IκBα is mediated via a CRM1-dependent pathway.

Regulation of Hox target genes by a DNA bound Homothorax/Hox/Extradenticle complex

Development ◽  
1999 ◽  
Vol 126 (22) ◽  
pp. 5137-5148 ◽  
Author(s):  
H.D. Ryoo ◽  
T. Marty ◽  
F. Casares ◽  
M. Affolter ◽  
R.S. Mann
Keyword(s):  
Nuclear Localization ◽  
Target Genes ◽  
Dominant Negative ◽  
Homeodomain Protein ◽  
Hox Proteins ◽  
Trimeric Complex ◽  
Binding Residue ◽  
Dominant Negative Form

To regulate their target genes, the Hox proteins of Drosophila often bind to DNA as heterodimers with the homeodomain protein Extradenticle (EXD). For EXD to bind DNA, it must be in the nucleus, and its nuclear localization requires a third homeodomain protein, Homothorax (HTH). Here we show that a conserved N-terminal domain of HTH directly binds to EXD in vitro, and is sufficient to induce the nuclear localization of EXD in vivo. However, mutating a key DNA binding residue in the HTH homeodomain abolishes many of its in vivo functions. HTH binds to DNA as part of a HTH/Hox/EXD trimeric complex, and we show that this complex is essential for the activation of a natural Hox target enhancer. Using a dominant negative form of HTH we provide evidence that similar complexes are important for several Hox- and exd-mediated functions in vivo. These data suggest that Hox proteins often function as part of a multiprotein complex, composed of HTH, Hox, and EXD proteins, bound to DNA.

scholarly journals Heparan Sulfate-dependent RAGE oligomerization is indispensable for pathophysiological functions of RAGE

2021 ◽  
Author(s):  
Miaomiao Li ◽  
Chih Yean Ong ◽  
Christophe J Langouet-Astrie ◽  
Lisi TAN ◽  
Ashwni Verma ◽  
...  
Keyword(s):  
Heparan Sulfate ◽  
Genetic Model ◽  
Point Mutations ◽  
Active Role ◽  
Dominant Negative ◽  
Unexpected Finding ◽  
Rna Seq ◽  
Dominant Negative Form

RAGE, a druggable inflammatory receptor, is known to function as an oligomer but the exact oligomerization mechanism remains poorly understood. Previously we have shown that heparan sulfate (HS) plays an active role in RAGE oligomerization. To understand the physiological significance of HS-induced RAGE oligomerization in vivo, we generated RAGE knock-in mice (RageAHA/AHA) by introducing point mutations to specifically disrupt HS-RAGE interaction. The RAGE mutant demonstrated normal ligand-binding but impaired capacity of HS-binding and oligomerization. Remarkably, RageAHA/AHA mice phenocopied Rage-/- mice in two different pathophysiological processes, namely bone remodeling and neutrophil-mediated liver injury, which demonstrates that HS-induced RAGE oligomerization is essential for RAGE signaling. Our findings suggest that it should be possible to block RAGE signaling by inhibiting HS-RAGE interaction. To test this, we generated a monoclonal antibody that targets the HS-binding site of RAGE. This antibody blocks RAGE signaling in vitro and in vivo, recapitulating the phenotype of RageAHA/AHA mice. By inhibiting HS-RAGE interaction genetically and pharmacologically, our work validated an alternative strategy to antagonize RAGE. Finally, we have performed RNA-seq analysis of neutrophils and lungs and found that while Rage-/- mice had a broad alteration of transcriptome in both tissues compared to wild-type mice, the changes of transcriptome in RageAHA/AHA mice were much more restricted. This unexpected finding suggests that by preserving the expression of RAGE protein (in a dominant-negative form), RageAHA/AHA mouse might represent a cleaner genetic model to study physiological roles of RAGE in vivo compared to Rage-/- mice.

scholarly journals CSIG-15. INHIBITION OF THE bHLH TRANSCRIPTIONAL NETWORKS BY A MUTATED E47 PROTEIN LEADS TO A STRONG ANTI-GLIOMA ACTIVITY IN VITRO AND IN VIVO

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi47-vi47
Author(s):  
Marilin Koch ◽  
Stefan Czemmel ◽  
Felix Lennartz ◽  
Sarah Beyeler ◽  
Justyna Przystal ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Nuclear Translocation ◽  
Clonogenic Survival ◽  
Dominant Negative ◽  
Molecular Profile ◽  
Transcriptional Networks ◽  
Molecular Alterations ◽  
Helix Loop Helix

Abstract OBJECTIVE The transcription factor E47 heterodimerizes with helix-loop-helix (HLH) and basic helix-loop-helix transcription (bHLH) factors like ID-1 and Olig2 that are overexpressed in glioblastoma. A dominant-negative variant of the E47 (dnE47) lacking the nuclear translocation signal, leads to cytoplasmatic sequestration of HLH and bHLH transcription factors. Here, we investigated combinations of dnE47-mediated inhibition of the bHLH transcriptional network with temozolomide and irradiation and explored the underlying molecular mechanisms. METHODS Long-term and stem cell glioma lines were transduced with a Doxycycline-inducible dnE47 lentivirus. Functional characterizations included immunocytochemistry, immunoblots, cytotoxicity and clonogenicity assays in vitro and latency until the onset of symptoms in vivo. CAGE and RNASeq were conducted for analyzing the dnE47-induced molecular profile. RESULTS The induction of dnE47 led to cytoplasmatic sequestration of HLH/bHLH transcription, reduced proliferation, increased cytotoxicity and reduced clonogenic survival in vitro and a prolonged latency until the onset of neurological symptoms in vivo. CAGE and RNASeq data revealed alterations in several cancer-relevant pathways. CONCLUSIONS A dnE47-mediated inhibition of the bHLH transcription network induced actionable molecular alterations in glioma cells that could be exploited for the design of novel therapies.

High glucose promotes nascent nephron apoptosis via NF-κB and p53 pathways

2011 ◽  
Vol 300 (1) ◽  
pp. F147-F156 ◽  
Author(s):  
Yun-Wen Chen ◽  
Isabelle Chenier ◽  
Shiao-Ying Chang ◽  
Stella Tran ◽  
Julie R. Ingelfinger ◽  
...  
Keyword(s):  
High Glucose ◽  
Nuclear Translocation ◽  
Dominant Negative ◽  
In Vivo Studies ◽  
Ros Generation ◽  
Small Interfering Rnas ◽  
Kidney Size ◽  
P53 Signaling

A hyperglycemic environment in utero reduces kidney size and nephron number due to nascent nephron apoptosis. However, the underlying mechanisms are incompletely understood. The present study investigated whether the nascent nephron apoptosis promoted by high glucose is mediated via the transcription factor NF-κB and p53 signaling pathways. Neonatal mouse kidneys from the offspring of nondiabetic, diabetic, and insulin-treated diabetic dams were used for in vivo studies, and MK4 cells, an embryonic metanephric mesenchymal (MM) cell line, were used for in vitro studies. Neonatal kidneys of the offspring of diabetic mothers exhibited an increased number of apoptotic cells and reactive oxygen species (ROS) generation, enhanced NF-κB activation, and nuclear translocation of its subunits (p50 and p65 subunits) as well as phosphorylation (Ser 15) of p53 compared with kidneys of offspring of nondiabetic mothers. Insulin treatment of diabetic dams normalized these parameters in the offspring. In vitro, high-glucose (25 mM) induced ROS generation and significantly increased MK4 cell apoptosis and caspase-3 activity via activation of NF-κB pathway, with p53 phosphorylation and nuclear translocation compared with normal glucose (5 mM). These changes in a high-glucose milieu were prevented by transient transfection of small interfering RNAs for dominant negative IκBα or IKK or p53. Our data demonstrate that high glucose-induced nascent nephron apoptosis is mediated, at least in part, via ROS generation and the activation of NF-κB and p53 pathways.

scholarly journals The mechanism of inhibition of Ran-dependent nuclear transport by cellular ATP depletion

2002 ◽  
Vol 157 (6) ◽  
pp. 963-974 ◽  
Author(s):  
Eric D. Schwoebel ◽  
Thai H. Ho ◽  
Mary Shannon Moore
Keyword(s):  
Nuclear Import ◽  
Nuclear Transport ◽  
Intact Cell ◽  
Transport Processes ◽  
Atp Depletion ◽  
Transport Pathways ◽  
Atp Production ◽  
Import And Export

Rran-dependent nuclear transport requires a nuclear pool of RanGTP both for the assembly of export complexes and the disassembly of import complexes. Accordingly, in order for these processes to proceed, Ran-dependent nuclear import and export assays in vitro require the addition of GTP to produce RanGTP. Notably, no ATP requirement can be detected for these transport processes in vitro. But in vivo, when cells are depleted of ATP by the addition of sodium azide and 2-deoxyglucose to block ATP production by oxidative phosphorylation and glycolysis, respectively, Ran-dependent nuclear import and export are rapidly inhibited. This raised the question of whether there is an ATP requirement for these nuclear transport pathways in an intact cell that has remained undetected in vitro. Here we report that the free (but not total) GTP concentration rapidly drops to an undetectable level upon ATP depletion as does the availability of RanGTP. Our conclusion is that the inhibition of Ran-dependent nuclear transport observed upon ATP depletion in vivo results from a shortage of RanGTP rather than the inhibition of some ATP-dependent process.

scholarly journals Selection of a Dominant Negative Retinoblastoma Protein (RB) Inhibiting Satellite Myoblast Differentiation Implies an Indirect Interaction between MyoD and RB

2000 ◽  
Vol 20 (14) ◽  
pp. 5129-5139 ◽  
Author(s):  
Feng-Qian Li ◽  
Archie Coonrod ◽  
Marshall Horwitz
Keyword(s):  
Myogenic Differentiation ◽  
Retinoblastoma Protein ◽  
Dominant Negative ◽  
Myoblast Differentiation ◽  
Loss Of Function ◽  
Lysosomal Protease ◽  
Dominant Negative Form ◽  
Genes Encoding

ABSTRACT Satellite myoblasts serve as stem cells in postnatal skeletal muscle, but the genes responsible for choosing between growth versus differentiation are largely undefined. We have used a novel genetic approach to identify genes encoding proteins whose dominant negative inhibition is capable of interrupting the in vitro differentiation of C2C12 murine satellite myoblasts. The screen is based on fusion of a library of cDNA fragments with the lysosomal protease cathepsin B (CB), such that the fusion protein intracellularly diverts interacting factors to the lysosome. Among other gene fragments selected in this screen, including those of known and novel sequence, is the retinoblastoma protein (RB) pocket domain. This unique dominant negative form of RB allows us to genetically determine if MyoD and RB associate in vivo. The dominant negative CB-RB fusion produces a cellular phenotype indistinguishable from recessive loss of function RB mutations. The fact that the dominant negative RB inhibits myogenic differentiation in the presence of nonlimiting concentrations of either RB or MyoD suggests that these two proteins do not directly interact. We further show that the dominant negative RB inhibits E2F1 but cannot inhibit a forced E2F1-RB dimer. Therefore, E2F1 is a potential mediator of the dominant negative inhibition of MyoD by CB-RB during satellite cell differentiation. We propose this approach to be generally suited to the investigation of gene function, even when little is known about the pathway being studied.

scholarly journals Ribosomal Protein L12 Uses a Distinct Nuclear Import Pathway Mediated by Importin 11

2002 ◽  
Vol 22 (4) ◽  
pp. 1266-1275 ◽  
Author(s):  
Scott M. Plafker ◽  
Ian G. Macara
Keyword(s):  
Ribosomal Protein ◽  
Nuclear Import ◽  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Protein Import ◽  
Nuclear Translocation ◽  
Nuclear Accumulation ◽  
Transient Transfection Assay

ABSTRACT Ribosome biogenesis requires the nuclear translocation of ribosomal proteins from their site of synthesis in the cytoplasm to the nucleus. Analyses of the import mechanisms have revealed that most ribosomal proteins can be delivered to the nucleus by multiple transport receptors (karyopherins or importins). We now provide evidence that ribosomal protein L12 (rpL12) is distinguished from the bulk of ribosomal proteins because it accesses the importin 11 pathway as a major route into the nucleus. rpL12 specifically and directly interacted with importin 11 in vitro and in vivo. Both rpL12 binding to and import by importin 11 were inhibited by another importin 11 substrate, UbcM2, indicating that these two cargoes may bind overlapping sites on the transport receptor. In contrast, the import of rpL23a, a ribosomal protein that uses the general ribosomal protein import system, was not competed by UbcM2, and in an in vitro binding assay, importin 11 did not bind to the nuclear localization signal of rpL23a. Furthermore, in a transient transfection assay, the nuclear accumulation of rpL12 was increased by coexpressed importin 11, but not by other importins. These data are consistent with importin 11 being a mediator of rpL12 nuclear import. Taken together, these results indicate that rpL12 uses a distinct nuclear import pathway that may contribute to a mechanism for regulating ribosome synthesis and/or maturation.

scholarly journals Superoxide scavenging and Akt inhibition in myocardium ameliorate pressure overload-induced NF-κB activation and cardiac hypertrophy

2010 ◽  
Vol 41 (2) ◽  
pp. 127-136 ◽  
Author(s):  
Shawn D. Hingtgen ◽  
Zhenbo Li ◽  
William Kutschke ◽  
Xin Tian ◽  
Ram V. Sharma ◽  
...  
Keyword(s):  
Cardiac Hypertrophy ◽  
Viral Vectors ◽  
Pressure Overload ◽  
Dominant Negative ◽  
Cardiomyocyte Hypertrophy ◽  
Luciferase Expression ◽  
Akt Activation ◽  
Dominant Negative Form

Recent studies from our laboratory and others have shown that increases in cytoplasmic superoxide (O2·−) levels and Akt activation play a key role in agonist-stimulated NF-κB activation and cardiomyocyte hypertrophy in vitro. In this study, we tested the hypothesis that adenovirus (Ad)-mediated intramyocardial gene transfer of cytoplasmic superoxide dismutase (AdCu/ZnSOD) or a dominant-negative form of Akt (AdDNAkt) in mice would attenuate pressure overload-induced increases in activation of the redox-sensitive transcription factor NF-κB and cardiac hypertrophy. Adult C57BL/6 mice were subjected to thoracic aortic banding (TAB) or sham surgery, and intramyocardial injections of viral vectors (AdCu/ZnSOD, AdDNAkt, or control) were performed. There was robust transgene expression in the heart, which peaked 6–7 days after injection and then declined to undetectable levels by 12–14 days. In mice injected with AdBgL II, TAB caused a significant increase in O2·− generation and cardiac mass at 1 wk, and these responses were markedly attenuated by AdCu/ZnSOD. In addition, TAB induced time-dependent activation of NF-κB in the myocardium as measured longitudinally by in vivo bioluminescent imaging of NF-κB-dependent luciferase expression. This was also abolished by intracardiac AdCu/ZnSOD or AdDNAkt, but not the control vector. The inhibition of Akt and O2·−-mediated NF-κB activation in TAB hearts was associated with an attenuation of cardiac hypertrophy. Since a direct cause-and-effect relationship between NF-κB activation and cardiomyocyte hypertrophy has been established previously, our data support the hypothesis that increased O2·− generation and Akt activation are key signaling intermediates in pressure overload-induced activation of NF-κB and cardiac hypertrophy.

scholarly journals FOXA1 plays a role in regulating secretoglobin 1a1 expression in the absence of CCAAT/enhancer binding protein activities in lung in vivo

2011 ◽  
Vol 300 (3) ◽  
pp. L441-L452 ◽  
Author(s):  
Taketomo Kido ◽  
Takeshi Tomita ◽  
Minoru Okamoto ◽  
Yan Cai ◽  
Yoshimi Matsumoto ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Regulation Of Gene Expression ◽  
Dominant Negative ◽  
Clara Cell ◽  
Small Molecular Weight ◽  
Dominant Negative Form

Secretoglobin (SCGB) 1A1, also called Clara cell secretor protein (CCSP) or Clara cell-specific 10-kDa protein (CC10), is a small molecular weight secreted protein mainly expressed in lung, with anti-inflammatory/immunomodulatory properties. Previous in vitro studies demonstrated that CCAAT/enhancer-binding proteins (C/EBPs) are the major transcription factors for the regulation of Scbg1a1 gene expression, whereas FOXA1 had a minimum effect on the transcription. To determine the in vivo role of C/EBPs in the regulation of SCGB1A1 expression, experiments were performed in which A-C/EBP, a dominant-negative form of C/EBP that interferes with DNA binding activities of all C/EBPs, was specifically expressed in lung. Surprisingly, despite the in vitro findings, expression of SCGB1A1 mRNA was not decreased in vivo in the absence of C/EBPs. This may be due to a compensatory role assumed by FOXA1 in the regulation of Scgb1a1 gene expression in lung in the absence of active C/EBPs. This disconnect between in vitro and in vivo results underscores the importance of studies using animal models to determine the role of specific transcription factors in the regulation of gene expression in intact multicellular complex organs such as lung.

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