scholarly journals Identification and Characterization of SAP25, a Novel Component of the mSin3 Corepressor Complex

2006 ◽  
Vol 26 (4) ◽  
pp. 1386-1397 ◽  
Author(s):  
Yuzuru Shiio ◽  
David W. Rose ◽  
Radin Aur ◽  
Sam Donohoe ◽  
Ruedi Aebersold ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Histone Deacetylases ◽  
Subcellular Location ◽  
Nuclear Bodies ◽  
Nuclear Accumulation ◽  
Affinity Tag ◽  
Pml Nuclear Bodies ◽  
Corepressor Complex

ABSTRACT The transcriptional corepressor mSin3 is associated with histone deacetylases (HDACs) and is utilized by many DNA-binding transcriptional repressors. We have cloned and characterized a novel mSin3A-binding protein, SAP25. SAP25 binds to the PAH1 domain of mSin3A, associates with the mSin3A-HDAC complex in vivo, and represses transcription when tethered to DNA. SAP25 is required for mSin3A-mediated, but not N-CoR-mediated, repression. SAP25 is a nucleocytoplasmic shuttling protein, actively exported from the nucleus by a CRM1-dependent mechanism. A fraction of SAP25 is located in promyelocytic leukemia protein (PML) nuclear bodies, and PML induces a striking nuclear accumulation of SAP25. An isotope-coded affinity tag quantitative proteomic analysis of the SAP25 complex revealed that SAP25 is associated with several components of the mSin3 complex, nuclear export machinery, and regulators of transcription and cell cycle. These results suggest that SAP25 is a novel core component of the mSin3 corepressor complex whose subcellular location is regulated by PML.

scholarly journals Adenovirus E1B 55-Kilodalton Protein Is a p53-SUMO1 E3 Ligase That Represses p53 and Stimulates Its Nuclear Export through Interactions with Promyelocytic Leukemia Nuclear Bodies

2010 ◽  
Vol 84 (23) ◽  
pp. 12210-12225 ◽  
Author(s):  
Mario A. Pennella ◽  
Yue Liu ◽  
Jennifer L. Woo ◽  
Chongwoo A. Kim ◽  
Arnold J. Berk
Keyword(s):  
Nuclear Export ◽  
P53 Mutation ◽  
Promyelocytic Leukemia ◽  
Nuclear Bodies ◽  
Live Cells ◽  
High Concentration ◽  
Sumoylation Site ◽  
Pml Nuclear Bodies ◽  
Induced Apoptosis

ABSTRACT Oncogenic transformation by adenovirus E1A and E1B-55K requires E1B-55K inhibition of p53 activity to prevent E1A-induced apoptosis. During viral infection, E1B-55K and E4orf6 substitute for the substrate-binding subunits of the host cell cullin 5 class of ubiquitin ligases, resulting in p53 polyubiquitinylation and proteasomal degradation. Here we show that E1B-55K alone also functions as an E3 SUMO1-p53 ligase. Fluorescence microscopy studies showed that E1B-55K alone, in the absence of other viral proteins, causes p53 to colocalize with E1B-55K in promyelocytic leukemia (PML) nuclear bodies, nuclear domains with a high concentration of sumoylated proteins. Photobleaching experiments with live cells revealed that E1B-55K tethering of p53 in PML nuclear bodies decreases the in vivo nuclear mobility of p53 nearly 2 orders of magnitude. E1B-55K-induced p53 sumoylation contributes to maximal inhibition of p53 function since mutation of the major p53 sumoylation site decreases E1B-55K-induced p53 sumoylation, tethering in PML nuclear bodies, and E1B-55K inhibition of p53 activity. Mutation of the E1B-55K sumoylation site greatly inhibits E1B-55K association with PML nuclear bodies and the p53 nuclear export to cytoplasmic aggresomes observed in E1A-E1B-transformed cells. Purified E1B-55K and p53 form high-molecular-weight complexes potentially through the formation of a network of E1B-55K dimers bound to the N termini of p53 tetramers. In support of this model, a p53 mutation that prevents tetramer formation greatly reduces E1B-55K-induced tethering in PML nuclear bodies and p53 nuclear export. These data indicate that E1B-55K's association with PML nuclear bodies inactivates p53 by first sequestering it in PML nuclear bodies and then greatly facilitating its nuclear export.

scholarly journals MicroRNA-10a is crucial for endothelial response to different flow patterns via interaction of retinoid acid receptors and histone deacetylases

2017 ◽  
Vol 114 (8) ◽  
pp. 2072-2077 ◽  
Author(s):  
Ding-Yu Lee ◽  
Ting-Er Lin ◽  
Chih-I Lee ◽  
Jing Zhou ◽  
Yi-Hsuan Huang ◽  
...  
Keyword(s):  
Shear Stress ◽  
Histone Deacetylases ◽  
Inflammatory Responses ◽  
Nuclear Accumulation ◽  
Hemodynamic Forces ◽  
Endothelial Response ◽  
Vascular Physiology ◽  
Responsive Element ◽  
Retinoid Acid

Histone deacetylases (HDACs) and microRNAs (miRs) have emerged as two important epigenetic factors in the regulation of vascular physiology. This study aimed to elucidate the relationship between HDACs and miRs in the hemodynamic modulation of endothelial cell (EC) dysfunction. We found that miR-10a has the lowest expression among all examined shear-responsive miRs in ECs under oscillatory shear stress (OS), and a relatively high expression under pulsatile shear stress (PS). PS and OS alter EC miR-10a expression to regulate the expression of its direct target GATA6 and downstream vascular cell adhesion molecule (VCAM)-1. PS induces the expression, nuclear accumulation, and association of retinoid acid receptor-α (RARα) and retinoid X receptor-α (RXRα). RARα and RXRα serve as a “director” and an “enhancer,” respectively, to enhance RARα binding to RA-responsive element (RARE) and hence miR-10a expression, thus down-regulating GATA6/VCAM-1 signaling in ECs. In contrast, OS induces associations of “repressors” HDAC-3/5/7 with RARα to inhibit the RARα-directed miR-10a signaling. The flow-mediated miR-10a expression is regulated by Krüppel-like factor 2 through modulation in RARα–RARE binding, with the consequent regulation in GATA6/VCAM-1 in ECs. These results are confirmed in vivo by en face staining on the aortic arch vs. the straight thoracic aorta of rats. Our findings identify a mechanism by which HDACs and RXRα modulate the hormone receptor RARα to switch miR-10a expression and hence the proinflammatory vs. anti-inflammatory responses of vascular endothelium under different hemodynamic forces.

scholarly journals Nuclear and cytoplasmic shuttling of TRADD induces apoptosis via different mechanisms

2002 ◽  
Vol 157 (6) ◽  
pp. 975-984 ◽  
Author(s):  
Michael Morgan ◽  
Jacqueline Thorburn ◽  
Pier Paolo Pandolfi ◽  
Andrew Thorburn
Keyword(s):  
Nuclear Export ◽  
Tumor Necrosis Factor Receptor ◽  
Promyelocytic Leukemia ◽  
Dominant Negative ◽  
Nuclear Bodies ◽  
Death Domain ◽  
Apoptosis Pathway ◽  
Promyelocytic Leukemia Protein ◽  
Caspase Inhibitors ◽  
Pml Nuclear Bodies

The adapter protein tumor necrosis factor receptor (TNFR)1–associated death domain (TRADD) plays an essential role in recruiting signaling molecules to the TNFRI receptor complex at the cell membrane. Here we show that TRADD contains a nuclear export and import sequence that allow shuttling between the nucleus and the cytoplasm. In the absence of export, TRADD is found within nuclear structures that are associated with promyelocytic leukemia protein (PML) nuclear bodies. In these structures, the TRADD death domain (TRADD-DD) can activate an apoptosis pathway that is mechanistically distinct from its action at the membrane-bound TNFR1 complex. Apoptosis by nuclear TRADD-DD is promyelocytic leukemia protein dependent, involves p53, and is inhibited by Bcl-xL but not by caspase inhibitors or dominant negative FADD (FADD-DN). Conversely, apoptosis induced by TRADD in the cytoplasm is resistant to Bcl-xL, but sensitive to caspase inhibitors and FADD-DN. These data indicate that nucleocytoplasmic shuttling of TRADD leads to the activation of distinct apoptosis mechanisms that connect the death receptor apparatus to nuclear events.

scholarly journals PML is a ROS sensor activating p53 upon oxidative stress

2017 ◽  
Vol 214 (11) ◽  
pp. 3197-3206 ◽  
Author(s):  
Michiko Niwa-Kawakita ◽  
Omar Ferhi ◽  
Hassane Soilihi ◽  
Morgane Le Bras ◽  
Valérie Lallemand-Breitenbach ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cell Biology ◽  
Antioxidant Properties ◽  
Promyelocytic Leukemia ◽  
Nuclear Bodies ◽  
Glutathione Depletion ◽  
Pml Nuclear Bodies ◽  
P53 Activation ◽  
Induced Changes

Promyelocytic leukemia (PML) nuclear bodies (NBs) recruit partner proteins, including p53 and its regulators, thereby controlling their abundance or function. Investigating arsenic sensitivity of acute promyelocytic leukemia, we proposed that PML oxidation promotes NB biogenesis. However, physiological links between PML and oxidative stress response in vivo remain unexplored. Here, we identify PML as a reactive oxygen species (ROS) sensor. Pml−/− cells accumulate ROS, whereas PML expression decreases ROS levels. Unexpectedly, Pml−/− embryos survive acute glutathione depletion. Moreover, Pml−/− animals are resistant to acetaminophen hepatotoxicity or fasting-induced steatosis. Molecularly, Pml−/− animals fail to properly activate oxidative stress–responsive p53 targets, whereas the NRF2 response is amplified and accelerated. Finally, in an oxidative stress–prone background, Pml−/− animals display a longevity phenotype, likely reflecting decreased basal p53 activation. Thus, similar to p53, PML exerts basal antioxidant properties but also drives oxidative stress–induced changes in cell survival/proliferation or metabolism in vivo. Through NB biogenesis, PML therefore couples ROS sensing to p53 responses, shedding a new light on the role of PML in senescence or stem cell biology.

scholarly journals MondoA, a Novel Basic Helix-Loop-Helix–Leucine Zipper Transcriptional Activator That Constitutes a Positive Branch of a Max-Like Network

2000 ◽  
Vol 20 (23) ◽  
pp. 8845-8854 ◽  
Author(s):  
Andrew N. Billin ◽  
Alanna L. Eilers ◽  
Kathryn L. Coulter ◽  
Jennifer S. Logan ◽  
Donald E. Ayer
Keyword(s):  
Transcriptional Activation ◽  
Nuclear Export ◽  
Mammalian Cells ◽  
Leucine Zipper ◽  
Functional Characterization ◽  
Nuclear Accumulation ◽  
Cytoplasmic Localization ◽  
Basic Helix Loop Helix ◽  
Helix Loop Helix

ABSTRACT Max is a common dimerization partner for a family of transcription factors (Myc, Mad [or Mxi]), and Mnt [or Rox] proteins) that regulate cell growth, proliferation, and apoptosis. We recently characterized a novel Max-like protein, Mlx, which interacts with Mad1 and Mad4. Here we describe the cloning and functional characterization of a new family of basic helix-loop-helix–leucine zipper heterodimeric partners for Mlx termed the Mondo family. MondoA forms homodimers weakly and does not interact with Max or members of the Myc or Mad families. MondoA and Mlx associate in vivo, and surprisingly, they are localized primarily to the cytoplasm of cultured mammalian cells. Treatment of cells with the nuclear export inhibitor leptomycin B results in the nuclear accumulation of MondoA and Mlx, demonstrating that they shuttle between the cytoplasmic and nuclear compartments rather than having exclusively cytoplasmic localization. MondoA preferentially forms heterodimers with Mlx, and this heterocomplex can bind to, and activate transcription from, CACGTG E-boxes when targeted to the nucleus via a heterologous nuclear localization signal. The amino termini of the Mondo proteins are highly conserved among family members and contain separable and autonomous cytoplasmic localization and transcription activation domains. Therefore, Mlx can mediate transcriptional repression in conjunction with the Mad family and can mediate transcriptional activation via the Mondo family. We propose that Mlx, like Max, functions as the center of a transcription factor network.

Ratjadone C-Mediated Nuclear Accumulation of HDAC4: Implications on Runx2-Induced Osteoblast Differentiation of C3H10T1/2 Mesenchymal Stem Cells

2014 ◽  
Vol 69 (11-12) ◽  
pp. 471-478 ◽  
Author(s):  
Ping Mu ◽  
Vishwa Deepak ◽  
Liheng Kang ◽  
Qing Jiang ◽  
Rong Liu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Nuclear Export ◽  
Histone Deacetylases ◽  
Osteoblast Differentiation ◽  
Cellular Differentiation ◽  
Screening Tool ◽  
Nuclear Accumulation ◽  
Myoblast Differentiation ◽  
Nuclear Retention ◽  
Lysine Residues

Abstract Histone deacetylases (HDACs) are a group of enzymes that deacetylate ε-N-acetyl lysine residues of histone and non-histone proteins and play an important role in gene regulation. HDAC4, a class-IIa HDAC, has been reported to shuttle between nucleus and cytoplasm in response to various cellular stimuli. The nucleo-cytoplasmic shuttling of HDAC4 is critical, and an anomalous nuclear localization might affect the cellular differentiation program. While the subcellular localization of HDAC4 has been reported to be vital for myoblast differentiation and chondrocyte hypertrophy, nuclear accumulation of HDAC4 during Runx2-induced osteoblast differentiation of stem cells has not been characterized. Ratjadone C is a natural compound that inhibits the nuclear export of proteins. Here, we show that Runx2 is a more potent transcription factor than Osterix in inducing osteoblast differentiation. Under the influence of ratjadone C, HDAC4 is retained in the nucleus and co-localizes with Runx2. However, forced nuclear accumulation of HDAC4 by ratjadone C or overexpression of the nuclear resident form of HDAC4 does not inhibit osteoblast differentiation, suggesting that the Runx2- induced osteogenic program of C3H10T1/2 cells is not affected by HDAC4. Even though phosphorylation of HDAC4 affects its compartmentalization and the stemness of progenitor cells, we found that total HDAC4 and phosphorylated HDAC4 remain cytoplasmic under both osteogenic and nonosteogenic conditions. Collectively, this work demonstrates that, regardless of the nucleo-cytoplasmic presence of HDAC4, the Runx2-induced osteoblast differentiation program of C3H10T1/2 cells remains unaffected. Additionally, the ratjadone C-mediated nuclear retention assay can potentially be used as a screening tool to identify novel regulatory mechanisms of HDAC4 and its functional partners in various pathophysiological conditions.

scholarly journals Resistance to Rabies Virus Infection Conferred by the PMLIV Isoform

2010 ◽  
Vol 84 (20) ◽  
pp. 10719-10726 ◽  
Author(s):  
Danielle Blondel ◽  
Sabrina Kheddache ◽  
Xavier Lahaye ◽  
Laurent Dianoux ◽  
Mounira K. Chelbi-Alix
Keyword(s):  
Rabies Virus ◽  
Single Gene ◽  
Antiviral Effect ◽  
Nuclear Bodies ◽  
Sumo Conjugation ◽  
Pml Nuclear Bodies ◽  
Protein Partners ◽  
Rabies Virus Infection ◽  
Vesicular Stomatitis

ABSTRACT Various reports implicate PML and PML nuclear bodies (NBs) in an intrinsic antiviral response targeting diverse cytoplasmic replicating RNA viruses. PML conjugation to the small ubiquitin-like modifier (SUMO) is required for its localization within NBs. PML displays antiviral effects in vivo, as PML deficiency renders mice more susceptible to infection with the rhabdovirus vesicular stomatitis virus (VSV). Cells derived from these mice are also more sensitive to infection with rabies virus, another member of the rhabdovirus family. Alternative splicing from a single gene results in the synthesis of several PML isoforms, and these are classified into seven groups, designated PMLI to -VII. We report here that expression of PMLIV or PMLIVa, which is missing exon 5, inhibited viral mRNA and protein synthesis, leading to a reduction in viral replication. However, the expression of other nuclear isoforms (PMLI to -VI) and cytoplasmic PMLVIIb failed to impair viral production. This antiviral effect required PMLIV SUMOylation, as it was not observed with PMLIV 3KR, in which the lysines involved in SUMO conjugation were mutated. Thus, PMLIV and PMLIVa may exert this isoform-specific function through interaction with specific NB protein partners via their common C-terminal region.

scholarly journals Capture of micrococcin biosynthetic intermediates reveals C-terminal processing as an obligatory step for in vivo maturation

2016 ◽  
Vol 113 (44) ◽  
pp. 12450-12455 ◽  
Author(s):  
Kathryn D. Bewley ◽  
Philip R. Bennallack ◽  
Mark A. Burlingame ◽  
Richard A. Robison ◽  
Joel S. Griffitts ◽  
...  
Keyword(s):  
Phase Ii ◽  
Oxidative Decarboxylation ◽  
Affinity Tag ◽  
Bioactive Natural Products ◽  
Precursor Peptide ◽  
Processing Step ◽  
Substrate Peptide ◽  
Processing Steps

Thiopeptides, including micrococcins, are a growing family of bioactive natural products that are ribosomally synthesized and heavily modified. Here we use a refactored, modular in vivo system containing the micrococcin P1 (MP1) biosynthetic genes (TclIJKLMNPS) from Macrococcus caseolyticus str 115 in a genetically tractable Bacillus subtilis strain to parse the processing steps of this pathway. By fusing the micrococcin precursor peptide to an affinity tag and coupling it with catalytically defective enzymes, biosynthetic intermediates were easily captured for analysis. We found that two major phases of molecular maturation are separated by a key C-terminal processing step. Phase-I conversion of six Cys residues to thiazoles (TclIJN) is followed by C-terminal oxidative decarboxylation (TclP). This TclP-mediated oxidative decarboxylation is a required step for the peptide to progress to phase II. In phase II, Ser/Thr dehydration (TclKL) and peptide macrocycle formation (TclM) occurs. A C-terminal reductase, TclS, can optionally act on the substrate peptide, yielding MP1, and is shown to act late in the pathway. This comprehensive characterization of the MP1 pathway prepares the way for future engineering efforts.

Selective Inhibitors of Nuclear Export (SINE) Compounds Suppress Both HIV Replication and AIDS Related Lymphoma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2751-2751
Author(s):  
Dirk Daelemans ◽  
Eline Boons ◽  
Els Vanstreels ◽  
Maarten Jacquemyn ◽  
Tatiane C Nogueira ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Antiretroviral Drugs ◽  
Nuclear Accumulation ◽  
Core Antigen ◽  
Hiv Replication ◽  
Employment Equity ◽  
Strong Basis ◽  
Equity Ownership ◽  
Risk Of Cancer

Abstract Infection with human immunodeficiency virus (HIV) compromises the immune system leaving infected individuals vulnerable to other pathologies including cancer. HIV-infected patients have an increased risk of cancer development as compared to the general population. Primary effusion lymphoma (PEL), a high-grade non-Hodgkin lymphoma with very poor prognosis, typically affects individuals infected with HIV in advanced stages of acquired immune deficiency syndrome (AIDS). While the use of anti-HIV drugs is associated with better prognosis, treating cancer in HIV-infected patients is often challenging due to potential drug interactions between anti-cancer agents and antiretroviral drugs. We demonstrate that inhibition of the exportin-1 (CRM1/XPO1) mediated nuclear protein transport by SINE compounds results in effective suppression of HIV replication, which was quantified by measuring virus-associated core antigen (p24) by ELISA. In addition by using MTT, assays, Annexin staining, FACS and immunoblot analysis we demonstrated that XPO1 inhibition induced cell cycle arrest and apoptosis in PEL cell lines (BC-1, BCBL-1, and JSC-1) at similar doses. SINE blocked the nuclear export of the late viral partially and unspliced RNA species encoding for the viral structural proteins. In BC-1 PEL cells inhibition of XPO1 resulted in the nuclear accumulation of p53 tumor suppressor protein as well as inhibition of NF-κB activity. In vivo, oral administration twice a week for 4 weeks, 20 mg/kg of the clinical SINE compound selinexor (currently in phase I/II clinical trials) led to a significant suppression of the BC-1 PEL growth in vivo. These findings provide a strong basis for inhibiting XPO1 as a novel strategy for the combined treatment of HIV and PEL or other AIDS-related cancers. Disclosures Tamir: Karyopharm: Employment. Kauffman:Karyopharm: Employment, Equity Ownership. Shacham:Karyopharm: Employment, Equity Ownership. Landesman:Karyopharm: Employment.

scholarly journals PP2A Regulates HDAC4 Nuclear Import

2008 ◽  
Vol 19 (2) ◽  
pp. 655-667 ◽  
Author(s):  
Gabriela Paroni ◽  
Nadia Cernotta ◽  
Claudio Dello Russo ◽  
Paola Gallinari ◽  
Michele Pallaoro ◽  
...  
Keyword(s):  
Nuclear Import ◽  
Nuclear Export ◽  
Histone Deacetylases ◽  
Phosphorylation Site ◽  
Class Ii ◽  
Binding Studies ◽  
Nuclear Entry ◽  
Putative Phosphorylation Site

Different signal-regulated serine/threonine kinases phosphorylate class II histone deacetylases (HDACs) to promote nuclear export, cytosolic accumulation, and activation of gene transcription. However, little is known about mechanisms operating in the opposite direction, which, possibly through phosphatases, should promote class II HDACs nuclear entry and subsequent gene repression. Here we show that HDAC4 forms a complex with the PP2A holoenzyme Cα, Aα, B/PR55α. In vitro and in vivo binding studies demonstrate that the N-terminus of HDAC4 interacts with the catalytic subunit of PP2A. HDAC4 is dephosphorylated by PP2A and experiments using okadaic acid or RNA interference have revealed that PP2A controls HDAC4 nuclear import. Moreover, we identified serine 298 as a putative phosphorylation site important for HDAC4 nuclear import. The HDAC4 mutant mimicking phosphorylation of serine 298 is defective in nuclear import. Mutation of serine 298 to alanine partially rescues the defect in HDAC4 nuclear import observed in cells with down-regulated PP2A. These observations suggest that PP2A, via the dephosphorylation of multiple serines including the 14-3-3 binding sites and serine 298, controls HDAC4 nuclear import.

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