scholarly journals Model-driven experimental approach reveals the complex regulatory distribution of p53 by the circadian factor Period 2

2016 ◽  
Vol 113 (47) ◽  
pp. 13516-13521 ◽  
Author(s):  
Tetsuya Gotoh ◽  
Jae Kyoung Kim ◽  
Jingjing Liu ◽  
Marian Vila-Caballer ◽  
Philip E. Stauffer ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Time Course ◽  
Experimental Approach ◽  
Ectopic Expression ◽  
Phase Relationship ◽  
Genotoxic Stress ◽  
Period 2 ◽  
Nuclear Shuttling ◽  
The Stability

The circadian clock and cell cycle networks are interlocked on the molecular level, with the core clock loop exerting a multilevel regulatory role over cell cycle components. This is particularly relevant to the circadian factor Period 2 (Per2), which modulates the stability of the tumor suppressor p53 in unstressed cells and transcriptional activity in response to genotoxic stress. Per2 binding prevents Mdm2-mediated ubiquitination of p53 and, therefore, its degradation, and oscillations in the peaks of Per2 and p53 were expected to correspond. However, our findings showed that Per2 and p53 rhythms were significantly out-of-phase relative to each other in cell lysates and in purified cytoplasmic fractions. These seemingly conflicting experimental data motivated the use of a combined theoretical and experimental approach focusing on the role played by Per2 in dictating the phase of p53 oscillations. Systematic modeling of all possible regulatory scenarios predicted that the observed phase relationship between Per2 and p53 could be simulated if (i) p53 was more stable in the nucleus than in the cytoplasm, (ii) Per2 associates to various ubiquitinated forms of p53, and (iii) Per2 mediated p53 nuclear import. These predictions were supported by a sevenfold increase in p53’s half-life in the nucleus and by in vitro binding of Per2 to the various ubiquitinated forms of p53. Last, p53’s nuclear shuttling was significantly favored by ectopic expression of Per2 and reduced because of Per2 down-regulation. Our combined theoretical/mathematical approach reveals how clock regulatory nodes can be inferred from oscillating time course data.

Studies on the mechanism of acute inhibition of thyroglobulin hydrolysis by iodine

1985 ◽  
Vol 108 (4) ◽  
pp. 511-517 ◽  
Author(s):  
Nandalal Bagchi ◽  
Birdie Shivers ◽  
Thomas R. Brown
Keyword(s):  
Time Course ◽  
Period 2 ◽  
Iodotyrosine Deiodinase ◽  
Rate Of Hydrolysis ◽  
Underlying Mechanisms ◽  
Excess Iodide ◽  
Sites Of Action ◽  
Hydrolysis Of

Abstract. Iodine in excess is known to acutely inhibit thyroidal secretion. In the present study we have characterized the time course of the iodine effect in vitro and investigated the underlying mechanisms. Labelled thyroid glands were cultured in vitro in medium containing mononitrotyrosine, an inhibitor of iodotyrosine deiodinase. The rate of hydrolysis of labelled thyroglobulin was measured as the proportion of labelled iodotyrosines and iodothyronines recovered at the end of culture and was used as an index of thyroidal secretion. Thyrotrophin (TSH) administered in vivo acutely stimulated the rate of thyroglobulin hydrolysis. Addition of Nal to the culture medium acutely inhibited both basal and TSH-stimulated thyroglobulin hydrolysis. The effect of iodide was demonstrable after 2 h, maximal after 6 h and was not reversible upon removal of iodide. Iodide abolished the dibutyryl cAMP induced stimulation of thyroglobulin hydrolysis. Iodide required organic binding of iodine for its effect but new protein or RNA synthesis was not necessary. The inhibitory effects of iodide and lysosomotrophic agents such as NH4C1 and chloroquin on thyroglobulin hydrolysis were additive suggesting different sites of action. Iodide added in vitro altered the distribution of label in prelabelled thyroglobulin in a way that suggested increased coupling in the thyroglobulin molecule. These data indicate that 1) the iodide effect occurs progressively over a 6 h period, 2) continued presence of iodide is not necessary once the inhibition is established, 3) iodide exerts its action primarily at a post cAMP, prelysosomal site and 4) the effect requires organic binding of iodine, but not new RNA or protein synthesis. Our data are consistent with the hypothesis that excess iodide acutely inhibits thyroglobulin hydrolysis by increasing the resistance of thyroglobulin to proteolytic degradation through increased iodination and coupling.

scholarly journals Growth Suppression by Acute PromyelocyticLeukemia-Associated Protein PLZF Is Mediated by Repression ofc-mycExpression

2003 ◽  
Vol 23 (24) ◽  
pp. 9375-9388 ◽  
Author(s):  
Melanie J. McConnell ◽  
Nathalie Chevallier ◽  
Windy Berkofsky-Fessler ◽  
Jena M. Giltnane ◽  
Rupal B. Malani ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Growth ◽  
Cell Cycle Arrest ◽  
Target Genes ◽  
Ectopic Expression ◽  
Growth Suppression ◽  
Quiescent State ◽  
Cycle Arrest

ABSTRACT The transcriptional repressor PLZF was identified by its translocation with retinoic acid receptor alpha in t(11;17) acute promyelocytic leukemia (APL). Ectopic expression of PLZF leads to cell cycle arrest and growth suppression, while disruption of normal PLZF function is implicated in the development of APL. To clarify the function of PLZF in cell growth and survival, we used an inducible PLZF cell line in a microarray analysis to identify the target genes repressed by PLZF. One prominent gene identified was c-myc. The array analysis demonstrated that repression of c-myc by PLZF led to a reduction in c-myc-activated transcripts and an increase in c-myc-repressed transcripts. Regulation of c-myc by PLZF was shown to be both direct and reversible. An interaction between PLZF and the c-myc promoter could be detected both in vitro and in vivo. PLZF repressed the wild-type c-myc promoter in a reporter assay, dependent on the integrity of the binding site identified in vitro. PLZF binding in vivo was coincident with a decrease in RNA polymerase occupation of the c-myc promoter, indicating that repression occurred via a reduction in the initiation of transcription. Finally, expression of c-myc reversed the cell cycle arrest induced by PLZF. These data suggest that PLZF expression maintains a cell in a quiescent state by repressing c-myc expression and preventing cell cycle progression. Loss of this repression through the translocation that occurs in t(11;17) would have serious consequences for cell growth control.

scholarly journals Porcine Haemagglutinating Encephalomyelitis Virus Triggers Neural Autophagy Independent of ULK1

2021 ◽  
Author(s):  
Zi Li ◽  
Feng Gao ◽  
Yungang Lan ◽  
Jiyu Guan ◽  
Jing Zhang ◽  
...  
Keyword(s):  
Time Course ◽  
Ectopic Expression ◽  
Neuroblastoma Cells ◽  
Neurological Dysfunction ◽  
Global Public Health ◽  
Complex Interactions ◽  
Encephalomyelitis Virus ◽  
Bona Fide

Uncoordinated 51-like kinase 1 (ULK1) is a well-characterized initiator of canonical autophagy under basal or pathological conditions. Porcine haemagglutinating encephalomyelitis virus (PHEV), a neurotropic betacoronavirus (β-CoV), impairs ULK1 kinase but hijacks autophagy to facilitate viral proliferation. However, the machinery of PHEV-induced autophagy initiation upon ULK1 kinase deficiency remains unclear. Here, the time course of PHEV infection showed a significant accumulation of autophagosomes (APs) in nerve cells in vivo and in vitro. Utilizing the ULK1-knockout neuroblastoma cells, we have identified that ULK1 was not essential for productive AP formation induced by PHEV. In vitro phosphorylation studies discovered that mTORC1-regulated ULK1 activation stalls during PHEV infection, whereas the AP biogenesis was controlled by AMPK-driven BECN1 phosphorylation. A lack of BECN1 is sufficient to block LC3 lipidation and disrupt recruitment of the LC3-ATG14 complex. Moreover, BECN1 acts as a bona fide substrate for ULK1-independent neural autophagy, and ectopic expression of BECN1 somewhat enhances PHEV replication. These findings highlight a novel machinery of non-canonical autophagy independent of ULK1 that bypasses the conserved initiation circuit of AMPK-mTORC1-ULK1, providing new insights into the interplay between neurotropic β-CoV and the host. IMPORTANCE The ongoing COVID-19 pandemic alongside the outbreaks of SARS and MERS pose betacoronavirus (β-CoV) as a global public health challenge. Coronaviruses subvert, haijack, or utilize autophagy to promote proliferation, thus exploring the cross-talk between β-CoV and autophagy of great significance in confronting future β-CoV outbreaks. Porcine haemagglutinating encephalomyelitis virus (PHEV) is a highly neurotropic β-CoV and invades the central nervous system (CNS) in pigs, but understanding of the pathogenesis for PHEV-induced neurological dysfunction yet limited. Here, we discovered a novel regulatory principle of neural autophagy initiation during PHEV infection, where productive autophagosome (AP) biogenesis bypassing the multifaceted regulation of ULK1 kinase. The PHEV-triggered non-canonical autophagy underscores the complex interactions of virus-host, and will help in the development of therapeutic strategies targeting non-canonical autophagy to treat β-CoV disease.

scholarly journals LARP7 is a BRCA1 ubiquitinase substrate and regulates genome stability and tumorigenesis

2019 ◽  
Author(s):  
Fang Zhang ◽  
Pengyi Yan ◽  
Huijing Yu ◽  
Huangying Le ◽  
Zixuan li ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Rna Binding ◽  
Tumor Therapy ◽  
Genotoxic Stress ◽  
Therapy Resistance ◽  
List Type ◽  
M Cell

SummaryAttenuated DNA repair leads to genomic instability and tumorigenesis. BRCA1/BARD1 are the best known tumor suppressors that promote homology recombination (HR) and arrest cell cycle at G2/M checkpoint. As E3 ubiquitin ligases, their ubiquitinase activity has been known to involve in the HR and tumor suppression, but the mechanism remains ambiguous. Here, we demonstrated upon genotoxic stress, BRCA1 together with BARD1 catalyzed the K48 ployubiquitination on LARP7, a 7SK RNA binding protein known to control RNAPII pausing, and thereby degraded it through 26S ubiquitin-proteasome pathway. Depleting LARP7 suppressed the expression of CDK1 complex, arrested cell at G2/M DNA damage checkpoint and reduced BRCA2 phosphorylation which thereby facilitated RAD51 recruitment to damaged DNA to enhance HR. Importantly, LARP7 depletion observed in breast patients lead to the chemoradiotherapy resistance both in vitro and in vivo. Together, this study unveils a mechanism by which BRCA1/BARD1 utilizes their E3 ligase activity to control HR and cell cycle, and highlights LARP7 as a potential target for cancer prevention and therapy.HighlightsDNA damage response downregulates LARP7 through BRCA1/BARD1BRCA1/BARD1 catalyzes the K48 polyubiquitination on LARP7LARP7 promotes G2/M cell cycle transition and tumorigenesis via CDK1 complexLARP7 disputes homology-directed repair that leads to tumor therapy resistance

ELAV protein HuA (HuR) can redistribute between nucleus and cytoplasm and is upregulated during serum stimulation and T cell activation

1998 ◽  
Vol 111 (21) ◽  
pp. 3145-3156 ◽  
Author(s):  
U. Atasoy ◽  
J. Watson ◽  
D. Patel ◽  
J.D. Keene
Keyword(s):  
Cell Cycle ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Ectopic Expression ◽  
Cyclin B1 ◽  
3T3 Cells ◽  
Gm Csf ◽  
Serum Stimulation ◽  
The Stability

ELAV proteins are implicated in regulating the stability and translation of cytokine and growth regulatory mRNAs such as GM-CSF, IL-2, c-myc, c-fos and GLUT1 by binding to their AU-rich 3′UTRs. The tissue-specific ELAV protein HuB (aka. Hel-N1) is predominantly cytoplasmic and has been shown to stabilize GLUT1 and c-myc mRNAs and to increase their translation following ectopic expression in 3T3-L1 cells. We report that the most widely expressed mouse ELAV protein, mHuA, is predominately nuclear in cultured NIH-3T3 cells, but is localized in the cytoplasm during early G1 of the cell cycle. Therefore, much like the primarily cytoplasmic HuB, HuA becomes temporally localized in the cytoplasm where it can potentially regulate the stability or translation of bound mRNAs. Moreover, we report that stimulation of mouse spleen cells using either mitogenic or sub-mitogenic levels of anti-CD3/CD28 resulted in a dramatic increase in the level of HuA. Upregulation of HuA corresponds to previously documented increases in cytokine expression which are due to increased mRNA stability following T cell activation. Consistent with these findings, HuA was down regulated in quiescent cells and upregulated in 3T3 cells following serum stimulation. The increase of murine HuA during the cell cycle closely resembles that of cyclin B1 which peaks in G2/M. Together with our earlier studies, these data indicate that mammalian ELAV proteins function during cell growth and differentiation due in part to their effects on posttranscriptional stability and translation of multiple growth regulatory mRNAs. This supports the hypothesis that ELAV proteins can function as transacting factors which affect a default pathway of mRNA degradation involved in the expression of growth regulatory proteins.

Environmental control of the cell cycle in Drosophila: nutrition activates mitotic and endoreplicative cells by distinct mechanisms

Development ◽  
1998 ◽  
Vol 125 (11) ◽  
pp. 2149-2158 ◽  
Author(s):  
J.S. Britton ◽  
B.A. Edgar
Keyword(s):  
Cell Cycle ◽  
Fat Body ◽  
Environmental Control ◽  
Ectopic Expression ◽  
Cell Types ◽  
Quiescent State ◽  
Independent Manner ◽  
Cell Cycle Activation ◽  
E2f Transcription Factor

In newly hatched Drosophila larvae, quiescent cells reenter the cell cycle in response to dietary amino acids. To understand this process, we varied larval nutrition and monitored effects on cell cycle initiation and maintenance in the mitotic neuroblasts and imaginal disc cells, as well as the endoreplicating cells in other larval tissues. After cell cycle activation, mitotic and endoreplicating cells respond differently to the withdrawal of nutrition: mitotic cells continue to proliferate in a nutrition-independent manner, while most endoreplicating cells reenter a quiescent state. We also show that ectopic expression of Drosophila Cyclin E or the E2F transcription factor can drive quiescent endoreplicating cells, but not quiescent imaginal neuroblasts, into S-phase. Conversely, we demonstrate that quiescent imaginal neuroblasts, but not quiescent endoreplicating cells, can be induced to enter the cell cycle when co-cultured with larval fat body in vitro. These results demonstrate a fundamental difference in the control of cell cycle activation and maintenance in these two cell types, and imply the existence of a novel mitogen generated by the larval fat body in response to nutrition.

Phosphorylation-deficient Stat1 inhibits retinoic acid–induced differentiation and cell cycle arrest in U-937 monoblasts

Blood ◽  
2000 ◽  
Vol 96 (8) ◽  
pp. 2870-2878
Author(s):  
Anna Dimberg ◽  
Kenneth Nilsson ◽  
Fredrik Öberg
Keyword(s):  
Cell Cycle ◽  
Retinoic Acid ◽  
Cell Cycle Arrest ◽  
Nuclear Translocation ◽  
Ectopic Expression ◽  
Leukemic Cell ◽  
Potent Inducer ◽  
Cycle Arrest

All-trans retinoic acid (ATRA) is a potent inducer of terminal differentiation of immature leukemic cell lines in vitro and of acute promyelocytic leukemia (APL) cells in vivo. Recent reports have shown that ATRA induces the expression of several interferon-regulated genes, including signal transducer and activator of transcription (Stat)1. To investigate the role of Stat1 activation in ATRA signaling, sublines were established for the human monoblastic cell line U-937 constitutively expressing wild-type or phosphorylation-defective Stat1, mutated in the conserved tyrosine 701 required for dimerization and nuclear translocation. Results showed that ATRA induction leads to activation of Stat1 by the phosphorylation of tyrosine 701 and subsequent nuclear translocation. Consistent with a functional importance of this activation, ectopic expression of Stat1Y701F suppressed ATRA-induced morphologic differentiation and expression of the monocytic surface markers CD11c and the granulocyte colony-stimulating factor receptor. Moreover, ATRA-induced growth arrest in the G0/G1phase of the cell cycle was inhibited by phosphorylation-deficient Stat1. Taken together, these results indicate that Stat1 is a key mediator of ATRA-induced cell cycle arrest and differentiation of U-937 cells.

scholarly journals Plk is a functional homolog of Saccharomyces cerevisiae Cdc5, and elevated Plk activity induces multiple septation structures.

1997 ◽  
Vol 17 (6) ◽  
pp. 3408-3417 ◽  
Author(s):  
K S Lee ◽  
R L Erikson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Cell Division ◽  
Specific Activity ◽  
Ectopic Expression ◽  
Sf9 Cells ◽  
Wild Type ◽  
C Terminus ◽  
M Phase

Plk is a mammalian serine/threonine protein kinase whose activity peaks at the onset of M phase. It is closely related to other mammalian kinases, Snk, Fnk, and Prk, as well as to Xenopus laevis Plx1, Drosophila melanogaster polo, Schizosaccharomyces pombe Plo1, and Saccharomyces cerevisiae Cdc5. The M phase of the cell cycle is a highly coordinated process which insures the equipartition of genetic and cellular materials during cell division. To enable understanding of the function of Plk during M phase progression, various Plk mutants were generated and expressed in Sf9 cells and budding yeast. In vitro kinase assays with Plk immunoprecipitates prepared from Sf9 cells indicate that Glu206 and Thr210 play equally important roles for Plk activity and that replacement of Thr210 with a negatively charged residue elevates Plk specific activity. Ectopic expression of wild-type Plk (Plk WT) complements the cell division defect associated with the cdc5-1 mutation in S. cerevisiae. The degree of complementation correlates closely with the Plk activity measured in vitro, as it is enhanced by a mutationally activated Plk, T210D, but is not observed with the inactive forms K82M, D194N, and D194R. In a CDC5 wild-type background, expression of Plk WT or T210D, but not of inactive forms, induced a sharp accumulation of cells in G1. Consistent with elevated Plk activity, this phenomenon was enhanced by the C-terminally deleted forms WT deltaC and T210D deltaC. Expression of T210D also induced a class of cells with unusually elongated buds which developed multiple septal structures. This was not observed with the C-terminally deleted form T210D deltaC, however. It appears that the C terminus of Plk is not required for the observed cell cycle influence but may be important for polarized cell growth and septal structure formation.

Hemolytic phospholipaseRv0183of Mycobacterium tuberculosis induces inflammatory response and apoptosis in alveolar macrophage RAW264.7 cells

2010 ◽  
Vol 56 (11) ◽  
pp. 916-924 ◽  
Author(s):  
Guangxian Xu ◽  
Hao Jia ◽  
Yong Li ◽  
Xiaoming Liu ◽  
Min Li ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Inflammatory Response ◽  
Alveolar Macrophage ◽  
Cell Apoptosis ◽  
Time Course ◽  
Ectopic Expression ◽  
Host Cells ◽  
Raw264.7 Cells ◽  
Raw264.7 Cell

The metabolic pathway of phospholipids is one of the most important physiologic pathways in Mycobacterium tuberculosis , a typical intracellular bacterium. The hemolytic phospholipase lip gene (Rv0183) is one of 24 phospholipase genes that have been demonstrated to play critical roles in the metabolism of phospholipids in M. tuberculosis. Quantitative RT–PCR and flow cytometry were used to elucidate the immunological and pathogenic implications of the Rv0183 gene on the inflammatory response following persistent expression of Rv0183 in mouse alveolar macrophage RAW264.7 cells. Our results demonstrate that a time-course-dependent ectopic expression of Rv0183 significantly elevated the expression of IL-6, NF-κB, TLR-2, TLR-6, TNFα, and MyD88 in these alveolar macrophage cells. Furthermore, the persistent expression of Rv0183 induced RAW264.7 cell apoptosis in vitro. These findings demonstrate that the expression of Rv0183 induces an inflammatory response and cell apoptosis in the host cells, suggesting that Rv0183 may play an important role in the virulence and pathogenesis of M. tuberculosis infection.

scholarly journals Requirement for p27KIP1 in Retinoblastoma Protein-Mediated Senescence

2001 ◽  
Vol 21 (11) ◽  
pp. 3616-3631 ◽  
Author(s):  
Kamilah Alexander ◽  
Philip W. Hinds
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Kinase Activity ◽  
Kinase Inhibitor ◽  
Cyclin E ◽  
Ectopic Expression ◽  
Retinoblastoma Protein ◽  
Morphological Transformation ◽  
Cycle Arrest

ABSTRACT In vivo and in vitro evidence indicate that cells do not divide indefinitely but instead stop growing and undergo a process termed cellular proliferative senescence. Very little is known about how senescence occurs, but there are several indications that the retinoblastoma protein (pRb) is involved, the most striking being that reintroduction of RB into RB −/−tumor cell lines induces senescence. In investigating the mechanism by which pRb induces senescence, we have found that pRb causes a posttranscriptional accumulation of the cyclin-dependent kinase inhibitor p27KIP1 that is accompanied by an increase in p27KIP1 specifically bound to cyclin E and a concomitant decrease in cyclin E-associated kinase activity. In contrast, pRb-related proteins p107 and p130, which also decrease cyclin E-kinase activity, do not cause an accumulation of p27KIP1 and induce senescence poorly. In addition, the use of pRb proteins mutated in the pocket domain demonstrates that pRb upregulation of p27KIP1 and senescence induction do not require the interaction of pRb with E2F. Furthermore, ectopic expression of p21CIP1 or p27KIP1 induces senescence but not the morphology change associated with pRb-mediated senescence, uncoupling senescence from the morphological transformation. Finally, the ability of pRb to maintain cell cycle arrest and induce senescence is reversibly abrogated by ablation of p27KIP1 expression. These findings suggest that prolonged cell cycle arrest through the persistent and specific inhibition of cdk2 activity by p27KIP1 is critical for pRb-induced senescence.

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