High affinity promoter binding of STOP1 is essential for the early aluminum-inducible expression of novel Al resistance genes GDH1 and GDH2 in Arabidopsis

Abstract Malate-efflux from roots, which is regulated by the transcription factor STOP1 (SENSITIVE-TO-PROTON-RHIZOTOXICITY1), which mediates aluminum-induced expression of ALUMINUM-ACTIVATED-MALATE-TRANSPORTER1 (AtALMT1), is critical for aluminum-resistance in Arabidopsis thaliana. Several studies showed that root AtALMT1 expression is rapidly observed in response to aluminum (within 1-hour), this early induction is an important mechanism to immediately protect roots from aluminum-toxicity. Additionally, identifying the molecular mechanisms that underlie rapid aluminum-resistance responses should lead to a better understanding of plant aluminum-sensing and -signal transduction mechanisms. In this study, histochemical analyses using GFP-tagged STOP1 proteins showed that STOP1 proteins were accumulated in the nucleus soon after aluminum-treatment. The rapid aluminum-induced STOP1-nuclear localization and AtALMT1-induction were observed in the presence of the protein synthesis inhibitor, suggesting that post-translational regulation is involved in these events. STOP1 also regulated rapid aluminum-induced expression for other genes that carry a functional/high-affinity STOP1-binding site in their promoter, including STOP2, GLUTAMATE-DEHYDROGENASE1 and 2 (GDH1 and 2), but not for Al resistance genes which have no functional STOP1-binding site such as ALUMINUM-SENSITIVE3, suggesting that the binding of STOP1 in the promoter is essential for the early induction. Finally, we report that GDH1 and 2 which are the target of STOP1 are novel aluminum-resistance genes in Arabidopsis.

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Structure of the translating Neurospora ribosome arrested by cycloheximide

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2111862118 ◽

2021 ◽

Vol 118 (48) ◽

pp. e2111862118

Author(s):

Lunda Shen ◽

Zhaoming Su ◽

Kailu Yang ◽

Cheng Wu ◽

Thomas Becker ◽

...

Keyword(s):

Binding Site ◽

Messenger Rna ◽

Large Subunit ◽

Structural Data ◽

Binding Pocket ◽

Protein Synthesis Inhibitor ◽

Synthesis Inhibitor ◽

Translation Elongation ◽

Elongation Step ◽

A Site

Ribosomes translate RNA into proteins. The protein synthesis inhibitor cycloheximide (CHX) is widely used to inhibit eukaryotic ribosomes engaged in translation elongation. However, the lack of structural data for actively translating polyribosomes stalled by CHX leaves unanswered the question of which elongation step is inhibited. We elucidated CHX’s mechanism of action based on the cryo-electron microscopy structure of actively translating Neurospora crassa ribosomes bound with CHX at 2.7-Å resolution. The ribosome structure from this filamentous fungus contains clearly resolved ribosomal protein eL28, like higher eukaryotes but unlike budding yeast, which lacks eL28. Despite some differences in overall structures, the ribosomes from Neurospora, yeast, and humans all contain a highly conserved CHX binding site. We also sequenced classic Neurospora CHX-resistant alleles. These mutations, including one at a residue not previously observed to affect CHX resistance in eukaryotes, were in the large subunit proteins uL15 and eL42 that are part of the CHX-binding pocket. In addition to A-site transfer RNA (tRNA), P-site tRNA, messenger RNA, and CHX that are associated with the translating N. crassa ribosome, spermidine is present near the CHX binding site close to the E site on the large subunit. The tRNAs in the peptidyl transferase center are in the A/A site and the P/P site. The nascent peptide is attached to the A-site tRNA and not to the P-site tRNA. The structural and functional data obtained show that CHX arrests the ribosome in the classical PRE translocation state and does not interfere with A-site reactivity.

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Differential Glucocorticoid-Dependent Regulation and Function of the ERRFI1 Gene in Triple-Negative Breast Cancer

Endocrinology ◽

10.1210/endocr/bqaa082 ◽

2020 ◽

Vol 161 (7) ◽

Author(s):

Chromewell Agustin R Mojica ◽

Weand S Ybañez ◽

Kevin Christian V Olarte ◽

Alyssa Beatrice C Poblete ◽

Pia D Bagamasbad

Keyword(s):

Breast Cancer ◽

Molecular Mechanisms ◽

Triple Negative ◽

Growth Factor Receptor ◽

In Silico Analysis ◽

Normal Breast ◽

Protein Synthesis Inhibitor ◽

Synthesis Inhibitor ◽

Mechanistic Basis ◽

And Function

Abstract Glucocorticoids (GCs; eg, hydrocortisone [CORT]) are routinely used as chemotherapeutic, anti-emetic, and palliative agents in breast cancer (BCa) therapy. The effects of GC signaling on BCa progression, however, remain a contentious topic as GC treatment seems to be beneficial for receptor-positive subtypes but elicits unfavorable responses in triple-negative BCa (TNBC). The mechanistic basis for these conflicting effects of GC in BCa is poorly understood. In this study, we sought to decipher the molecular mechanisms that govern the GC-dependent induction of the tumor suppressor ERRFI1 gene, an inhibitor of epidermal growth factor receptor (EGFR) signaling, and characterize the role of the GC-ERRFI1 regulatory axis in TNBC. Treatment of TNBC cell lines with a protein synthesis inhibitor or GC receptor (GR) antagonist followed by gene expression analysis suggests that ERRFI1 is a direct GR target. Using in silico analysis coupled with enhancer-reporter assays, we identified a putative ERRFI1 enhancer that supports CORT-dependent transactivation. In orthogonal assays for cell proliferation, survival, migration, and apoptosis, CORT mostly facilitated an oncogenic phenotype regardless of malignancy status. Lentiviral knockdown and overexpression of ERRFI1 showed that the CORT-enhanced oncogenic phenotype is restricted by ERRFI1 in the normal breast epithelial model MCF10A and to a lesser degree in the metastatic TNBC line MDA-MB-468. Conversely, ERRFI1 conferred pro-tumorigenic effects in the highly metastatic TNBC model MDA-MB-231. Taken together, our findings suggest that the progressive loss of the GC-dependent regulation and anti-tumorigenic function of ERRFI1 influences BCa progression and may contribute to the unfavorable effects of GC therapy in TNBC.

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ACTH-induced c-myc proto-oncogene expression precedes antimitogenic effect during differentiation of fetal rat adrenocortical cells

Journal of Endocrinology ◽

10.1677/joe.0.1450379 ◽

1995 ◽

Vol 145 (2) ◽

pp. 379-385 ◽

Cited By ~ 4

Author(s):

P Heikkilä ◽

J Arola ◽

A Salmi ◽

A I Kahri

Keyword(s):

Mrna Expression ◽

Inhibitory Effect ◽

Zona Glomerulosa ◽

Protein Synthesis Inhibitor ◽

Mrna Levels ◽

Synthesis Inhibitor ◽

Adrenocortical Cells ◽

Induced Expression ◽

Inhibition Of Proliferation ◽

Fetal Rat

Abstract The regulation of proto-oncogenes has been connected with proliferation and differentiation in various cell types. In the present study, the ACTH-induced expression of c-myc mRNA and proliferation of fetal rat adrenocortical cells have been studied. Low levels of c-myc mRNA were detected in undifferentiated zona glomerulosa-like cells. Stimulation with ACTH for 2 to 6 h transiently increased the c-myc mRNA levels. Both basal and ACTH-induced expression levels were increased by the protein synthesis inhibitor cycloheximide. Treatment with a protein kinase C (PKC) activator 12–0-tetradecanoyl phorbol-13-acetate mimicked the effect of ACTH, whereas c-myc mRNA levels decreased by inhibiting the PKC with H-7. ACTH inhibited proliferation of fetal rat adrenocortical cells during the first 24 h of stimulation. The inhibitory effect began from 6 h, reached its maximum at 12 h and slowly vanished at 24 h. Our data demonstrated that ACTH transiently increased c-myc mRNA expression. Adrenocortical c-myc expression was mediated via PKC. In contrast to previous reports, where c-myc expression precedes proliferation of various cells, ACTH-induced c-myc mRNA expression of cultured fetal rat adrenocortical cells was followed by inhibition of proliferation. Journal of Endocrinology (1995) 145, 379–385

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Oxidative and Electrophilic Stresses Activate Nrf2 through Inhibition of Ubiquitination Activity of Keap1

Molecular and Cellular Biology ◽

10.1128/mcb.26.1.221-229.2006 ◽

2006 ◽

Vol 26 (1) ◽

pp. 221-229 ◽

Cited By ~ 544

Author(s):

Akira Kobayashi ◽

Moon-Il Kang ◽

Yoriko Watai ◽

Kit I. Tong ◽

Takahiro Shibata ◽

...

Keyword(s):

Molecular Mechanisms ◽

De Novo ◽

Degradation Mechanism ◽

Nuclear Accumulation ◽

Protein Synthesis Inhibitor ◽

Synthesis Inhibitor ◽

Nrf2 Activation ◽

Nrf2 Protein

ABSTRACT The Keap1-Nrf2 system is the major regulatory pathway of cytoprotective gene expression against oxidative and/or electrophilic stresses. Keap1 acts as a stress sensor protein in this system. While Keap1 constitutively suppresses Nrf2 activity under unstressed conditions, oxidants or electrophiles provoke the repression of Keap1 activity, inducing the Nrf2 activation. However, the precise molecular mechanisms behind the liberation of Nrf2 from Keap1 repression in the presence of stress remain to be elucidated. We hypothesized that oxidative and electrophilic stresses induce the nuclear accumulation of Nrf2 by affecting the Keap1-mediated rapid turnover of Nrf2, since such accumulation was diminished by the protein synthesis inhibitor cycloheximide. While both the Cys273 and Cys288 residues of Keap1 are required for suppressing Nrf2 nuclear accumulation, treatment of cells with electrophiles or mutation of these cysteine residues to alanine did not affect the association of Keap1 with Nrf2 either in vivo or in vitro. Rather, these treatments impaired the Keap1-mediated proteasomal degradation of Nrf2. These results support the contention that Nrf2 protein synthesized de novo after exposure to stress accumulates in the nucleus by bypassing the Keap1 gate and that the sensory mechanism of oxidative and electrophilic stresses is closely linked to the degradation mechanism of Nrf2.

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Molecular mechanisms of initiation of fibrinolysis by fibrin

Thrombosis and Haemostasis ◽

10.1055/s-0037-1613368 ◽

2003 ◽

Vol 89 (03) ◽

pp. 409-419 ◽

Cited By ~ 126

Author(s):

Willem Nieuwenhuizen ◽

Leonid Medved

Keyword(s):

Binding Site ◽

Conformational Changes ◽

Binding Sites ◽

Molecular Mechanisms ◽

Intramolecular Interaction ◽

Affinity Binding ◽

Fibrin Deposition ◽

High Affinity ◽

Physiological Processes ◽

D Region

SummaryFibrinogen is rather inert in the circulation, however, after conversion into fibrin it participates in various physiological processes including fibrinolysis. Initiation of fibrinolysis occurs through a number of orchestrated interactions between fibrin, plasminogen and its activator tPA which result in generation of plasmin. Numerous studies localized a set of specific low affinity tPA- and plasminogen-binding sites in each D region of fibrin(ogen). The tPA-binding site includes residues γ312-324 and the plasminogen-binding site includes residues Aα148-160; they bind tPA and plasminogen with a Kd of about 1 μM. Another set of high affinity tPA- and plasminogen-binding sites (Kds = 16-33 nM) was identified in the compact portion of each fibrin(ogen) αC-domain within residues Aα392-610. All these sites are cryptic in fibrinogen and become exposed in fibrin. Recent studies with recombinant and proteolytic fibrin(ogen) fragments clarified the molecular mechanisms by which these sites become exposed. Namely, upon fibrin assembly, the interaction between the D and E regions causes conformational changes in the former that expose the low affinity binding sites. The exposure of the high affinity binding sites in the αC-domains is connected most probably with their switch from an intramolecular interaction in fibrinogen to an intermolecular one in fibrin. These mechanisms serve to minimize degradation of circulating fibrinogen and confine fibrinolysis to places of fibrin deposition.

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Temporal pattern of rat small intestinal gene expression with refeeding

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1994.266.1.g83 ◽

1994 ◽

Vol 266 (1) ◽

pp. G83-G89 ◽

Cited By ~ 8

Author(s):

R. A. Hodin ◽

J. R. Graham ◽

S. Meng ◽

M. P. Upton

Keyword(s):

Gene Expression ◽

Molecular Mechanisms ◽

Early Response ◽

Protein Synthesis Inhibitor ◽

Chow Diet ◽

Intestinal Alkaline Phosphatase ◽

Synthesis Inhibitor ◽

Adult Rats ◽

Fed State ◽

Small Intestinal

Studies were carried out to elucidate the molecular mechanisms underlying small intestinal epithelial growth. Adult rats were fasted for 4 days and then refed a chow diet for up to 48 h. Histological examination confirmed the sequential occurrence of mucosal atrophy and hyperplasia. Northern blot analyses of RNA derived from small intestinal mucosal scrapings revealed a striking pattern of alterations in the expression of two different categories of genes. There were very early increases in the expression of c-fos and c-jun, reflecting the mitogenic response to refeeding that occurs within the crypt compartment. Studies using the protein synthesis inhibitor cycloheximide suggest that c-fos and c-jun are part of the “immediate-early” response of the small intestine. At later time points after the refeeding stimulus, differential changes occurred in the expression of the brush-border enzymes, lactase, and intestinal alkaline phosphatase (IAP). Refeeding caused a decrease in lactase gene expression and an increase in the expression of the 3.0-kb IAP mRNA species, reflecting a return of the villus phenotype to the normal fed state. Thus we have demonstrated a complex and temporally related pattern of gene expression within the small intestinal epithelium upon refeeding. The results provide insight into the relationship between the processes of intestinal growth and differentiation.

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Modulation of Arachidonic Acid Metabolism in Human Endothelial Cells by Glucocorticoids

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661566 ◽

1986 ◽

Vol 55 (03) ◽

pp. 369-374 ◽

Cited By ~ 15

Author(s):

Raffaele De Caterina ◽

Babette B Weksler

Keyword(s):

Endothelial Cells ◽

Vascular Endothelial Cells ◽

Umbilical Vein ◽

Arachidonic Acid Metabolism ◽

Protein Synthesis Inhibitor ◽

Synthesis Inhibitor ◽

Prolonged Incubation ◽

Dependent Inhibition ◽

Human Thrombin ◽

Umbilical Vein Endothelial Cells

SummaryTo learn whether glucocorticoids inhibit prostaglandin (PG) production in vascular endothelial cells, we investigated the effects of glucocorticoids on PG synthesis by cultured human umbilical vein endothelial cells (EC). Pretreatment of EC with dexamethasone (DX, 10-9 to 5 x 10-5 M) caused a dose-dependent inhibition of PGI2 production when PG synthesis from endogenous arachidonate was stimulated by human thrombin (0.25-2 U/ml) or ionophore A 23187 (1-5 μM). The inhibition was detectable at 10-7 M DX and maximal at 10-5 M (4.0 ± 0.7 vs. control: 7.7 ± 1.9 ng/ml, mean ± S.D., P <0.01). The production of PGE2 and the release of radiolabelled arachidonate (AA) from prelabelled cells were similarly inhibited. Prolonged incubation of EC with glucocorticoids was required to inhibit PG production or arachidonate release: ranging from 8% inhibition at 5 h to 44% at 38 h. In contrast, prostaglandin formation from exogenous AA was not altered by DX treatment. When thrombin or ionophore-stimulated EC were restimulated with exogenous AA (25 μM), DX-treated cells released more PGI2 than control cells (5.7 ± 0.5 vs. 4.1 ± 0.6 ng/ml, P <0.01). Both the decrease in PGI2 production after thrombin/ionophore and the increase after re-stimulation with AA were blunted in the presence of the protein synthesis inhibitor cycloheximide (0.1-0.2 μg/ml). Thus, incubation of EC with glucocorticoids inhibits PG production at the step of phospholipase activation. The time requirement for these steroid effects and their blunting by cycloheximide are consistent with the induction of regulatory proteins, possibly lipocortins, in endothelial cells.

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