Post-ovulatory ageing of mouse oocytes affects the distribution of specific spindle-associated proteins and Akt expression levels

2014 ◽  
Vol 26 (4) ◽  
pp. 562 ◽  
Author(s):  
Sandra Cecconi ◽  
Gianna Rossi ◽  
Hamid Deldar ◽  
Valerio Cellini ◽  
Felice Patacchiola ◽  
...  
Keyword(s):  
Histone H3 ◽  
Meiotic Spindle ◽  
Post Translational Modifications ◽  
Expression Levels ◽  
Acetylated Tubulin ◽  
Protein Levels ◽  
Histone 3 ◽  
Phosphorylated Akt ◽  
Associated Proteins

The aim of this study has been to determine the effects of in vivo post-ovulatory ageing (POA) on the distribution of spindle-associated proteins, histone H3/H4 post-translational modifications and on v-akt murine thymoma viral oncogene homolog 1 (Akt) expression levels. To this end, oocytes were retrieved 13, 29 and 33 h after human chorionic gonadotrophin (hCG) treatment. The presence and distribution at the meiotic spindle of acetylated tubulin, γ-tubulin, polo kinase-1 and Ser473/Thr308 phosphorylated Akt (pAkt) as well as histone H3 and H4 acetylation and phosphorylation levels were assayed via immunofluorescence. Akt expression levels were determined via reverse transcription–polymerase chain reaction and western blotting analyses. Spindles from oocytes recovered 13 h and 29 h after hCG treatment showed similar levels of acetylated tubulin but ageing induced: (1) translocation of γ-tubulin from spindle poles to microtubules, (2) absence of Thr308- and Ser473-pAkt in 76% and 30% of oocytes, respectively, and (3) a significant reduction in phosphorylation levels of serine 10 on histone 3. At 29 h, a significant decrease in Akt mRNA, but not in pAkt or Akt protein levels, was recorded. By contrast, protein content significantly decreased 33 h after hCG. We conclude that POA impairs oocyte viability and fertilisability by altering the expression levels and spindle distribution of proteins that are implicated in cell survival and chromosome segregation. Together, these events could play a role in oocyte apoptosis.

scholarly journals A role for the MLL fusion partner ENL in transcriptional elongation and chromatin modification

Blood ◽  
2007 ◽  
Vol 110 (13) ◽  
pp. 4445-4454 ◽  
Author(s):  
Dorothee Mueller ◽  
Christian Bach ◽  
Deniz Zeisig ◽  
Maria-Paz Garcia-Cuellar ◽  
Sara Monroe ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Chromatin Modification ◽  
Histone H3 ◽  
Elongation Factor ◽  
Polycomb Group ◽  
Fusion Partner ◽  
Transcriptional Elongation ◽  
Associated Proteins ◽  
Group Members

Chimeric proteins joining the histone methyltransferase MLL with various fusion partners trigger distinctive lymphoid and myeloid leukemias. Here, we immunopurified proteins associated with ENL, a protein commonly fused to MLL. Identification of these ENL-associated proteins (EAPs) by mass spectrometry revealed enzymes with a known role in transcriptional elongation (RNA polymerase II C-terminal domain kinase [RNAPolII CTD] positive transcription elongation factor b [pTEFb]), and in chromatin modification (histone-H3 methyltransferase DOT1L) as well as other frequent MLL partners (AF4, AF5q31, and LAF4), and polycomb group members (RING1, CBX8, and BCoR). The composition of EAP was further verified by coimmunoprecipitation, 2-hybrid analysis, pull-down, and colocalization experiments. Purified EAP showed a histone H3 lysine 79–specific methylase activity, displayed a robust RNAPolII CTD kinase function, and counteracted the effect of the pTEFb inhibitor 5,6-dichloro-benzimidazole-riboside. In vivo, an ENL knock-down diminished genome-wide as well as gene-specific H3K79 dimethylation, reduced global run-on elongation, and inhibited transient transcriptional reporter activity. According to structure-function data, DOT1L recruitment was important for transformation by the MLL-ENL fusion derivative. These results suggest a function of ENL in histone modification and transcriptional elongation.

scholarly journals Intestinal differentiation involves cleavage of histone H3 N-terminal tails by multiple proteases

2021 ◽  
Author(s):  
Karin Johanna Ferrari ◽  
Simona Amato ◽  
Roberta Noberini ◽  
Cecilia Toscani ◽  
Daniel Fernández-Pérez ◽  
...  
Keyword(s):  
Transcriptional Control ◽  
Cathepsin L ◽  
Histone H3 ◽  
Regulatory Function ◽  
Post Translational Modifications ◽  
Cellular Processes ◽  
Differentiated Cells ◽  
Proteolytic Activities

Abstract The proteolytic cleavage of histone tails, also termed histone clipping, has been described as a mechanism for permanent removal of post-translational modifications (PTMs) from histone proteins. Such activity has been ascribed to ensure regulatory function in key cellular processes such as differentiation, senescence and transcriptional control, for which different histone-specific proteases have been described. However, all these studies were exclusively performed using cell lines cultured in vitro and no clear evidence that histone clipping is regulated in vivo has been reported. Here we show that histone H3 N-terminal tails undergo extensive cleavage in the differentiated cells of the villi in mouse intestinal epithelium. Combining biochemical methods, 3D organoid cultures and in vivo approaches, we demonstrate that intestinal H3 clipping is the result of multiple proteolytic activities. We identified Trypsins and Cathepsin L as specific H3 tail proteases active in small intestinal differentiated cells and showed that their proteolytic activity is differentially affected by the PTM pattern of histone H3 tails. Together, our findings provide in vivo evidence of H3 tail proteolysis in mammalian tissues, directly linking H3 clipping to cell differentiation.

scholarly journals Phosphoregulation of HORMA domain protein HIM-3 promotes asymmetric synaptonemal complex disassembly in meiotic prophase in C. elegans

2020 ◽  
Author(s):  
Aya Sato-Carlton ◽  
Chihiro Nakamura-Tabuchi ◽  
Xuan Li ◽  
Hendrik Boog ◽  
Madison K Lehmer ◽  
...  
Keyword(s):  
Synaptonemal Complex ◽  
Post Translational Modifications ◽  
C Elegans ◽  
Phosphorylated Proteins ◽  
C Terminus ◽  
The Hierarchical Structure ◽  
Associated Proteins ◽  
Domain Protein

AbstractIn the two cell divisions of meiosis, diploid genomes are reduced into complementary haploid sets through the discrete, two-step removal of chromosome cohesion, a task carried out in most eukaryotes by protecting cohesion at the centromere until the second division. In eukaryotes without defined centromeres, however, alternative strategies have been innovated. The best-understood of these is that used by the nematode Caenorhabditis elegans, where upon division of the chromosome into two segments or arms by the single off-center crossover, several chromosome-associated proteins or post-translational modifications become specifically partitioned to either the short or long arm, where they affect the timing of cohesion loss through as-yet unknown mechanisms. Here, we investigate the meiotic axis HORMA-domain protein HIM-3 and show that it becomes phosphorylated at its C-terminus, within the conserved “closure motif” region bound by the related HORMA-domain proteins HTP-1 and HTP-2. Binding of HTP-2 is abrogated by phosphorylation of the closure motif in in vitro assays, strongly suggesting that in vivo phosphorylation of HIM-3 likely modulates the hierarchical structure of the chromosome axis. Phosphorylation of HIM-3 only occurs on synapsed chromosomes, and similarly to previously-described phosphorylated proteins of the synaptonemal complex, becomes restricted to the short arm after designation of crossover recombination sites. Regulation of HIM-3 phosphorylation status is required for timely disassembly of synaptonemal complex central elements from the long arm, and is also required for proper timing of HTP-1 and HTP-2 dissociation from the short arm. Phosphorylation of HIM-3 thus plays a role in establishing the identity of short and long arms, thereby contributing to the robustness of the two-step chromosome segregation.

scholarly journals Genistein Inhibits Human Colorectal Cancer Growth and Suppresses MiR-95, Akt and SGK1

2015 ◽  
Vol 35 (5) ◽  
pp. 2069-2077 ◽  
Author(s):  
Jian Qin ◽  
Jia Xin Chen ◽  
Zhou Zhu ◽  
Jia An Teng
Keyword(s):  
Colorectal Cancer ◽  
Cell Proliferation ◽  
Molecular Mechanisms ◽  
Xenograft Model ◽  
Inhibitory Effect ◽  
Protein Levels ◽  
Phosphorylated Akt ◽  
Hct 116 ◽  
Hct 116 Cells

Background: Genistein, a major isoflavonoid isolated from dietary soybean, has been shown to suppress the growth of several cancers through modulation of various pathways. However, the molecular mechanisms by which genistein elicit its effects on colorectal cancer (CRC) cells have not been fully elucidated. In this study, we aimed to investigate the anti-tumor activities of genistein on CRC and its potential mechanism. Methods: Effects of genistein on the cell proliferation were tested in HCT-116 cells by MTT assay, and apoptosis was measured by Flow cytometry. Real-time PCR was also used to evaluate the regulation of genistein on miR-95, Akt and SGK1 expression. The protein levels of total Akt (T-Akt), and phosphorylated Akt (P-Akt) were assessed by western blot. A nude mice xenograft model was employed to determine whether genistein could have an anti-tumor effect on CRC in vivo. Results: We found that treatment of HCT-116 cells with genistein caused an inhibition of cell proliferation and induction of apoptosis. Meanwhile, genistein down-regulated the mRNA expression of Akt, SGK1 and miR-95, and inhibited the phosphorylation of Akt in HCT-116 cells. The experiment in vivo also showed that genistein significantly suppressed the growth of mouse xenograft tumor. Conclusion: This study demonstrates that genistein has an inhibitory effect on CRC involved in reducing miR-95, Akt and SGK1, offering novel insights into the mechanisms of genistein therapeutic actions.

scholarly journals Membrane-Bound CD40L Promotes Senescence and Initiates Senescence-Associated Secretory Phenotype via NF-κB Activation in Lung Adenocarcinoma

2018 ◽  
Vol 48 (4) ◽  
pp. 1793-1803 ◽  
Author(s):  
Wei Xu ◽  
Yue Li ◽  
Wei-Wei Yuan ◽  
Yuan Yin ◽  
Wei-Wei Song ◽  
...  
Keyword(s):  
Lung Adenocarcinoma ◽  
Cellular Senescence ◽  
Mutant Form ◽  
Dependent Manner ◽  
Expression Levels ◽  
Migration Assay ◽  
Protein Levels ◽  
Membrane Bound ◽  
Secretory Phenotype

Background/Aims: Cellular senescence acts as a barrier against tumorigenesis. The CD40L transgene, expressed in some tumor cells, not only becomes visible to antigen-presenting cells but also actively catalyzes its own termination. Here, we evaluated the effect of a membrane-bound mutant form of human CD40L (CD40L-M) on senescence and the senescence-associated secretory phenotype (SASP) in non-small cell lung cancer (NSCLC). Methods: CD40 expression levels in the NSCLC cell lines A549/TR, A549/DDP and H460 were examined by flow cytometry. Senescent cells and tissues were identified via SA-β-gal activity. Cell proliferation was visualized by EdU labeling. qRT-PCR, Western blotting, and immunofluorescence staining were conducted to assess mRNA and protein expression levels of CD40L, γ-H2A.X, p65, p-p65, IκBα, p53, p21 and p16. Cytokines secreted from transfected cells were tested by ELISA and cell migration assay. Capsid tyrosine-modified rAAV5-CD40L-M was packaged and carried out in vivo. Results: Overexpression of CD40L-M promoted senescence, inhibited proliferation, increased DNA damage-associated γ-H2A.X, and initiated the SASP in CD40-positive NSCLC cells. NF-κB signaling was activated by CD40L-M overexpression in these cells. Knockdown of NF-κB partially overcame senescence and failed to induce SASP. Furthermore, increased p53 and p21 protein levels induced by CD40L-M were also reduced following NF-κB suppression. Conclusions: These data showed that the membrane-bound CD40L mutant may promote cellular senescence and initiate the SASP of NSCLC cells in an NF-κB-dependent manner. Therefore, CD40L-M-induced senescence may be a potential approach to protect against lung adenocarcinoma.

scholarly journals Phosphoregulation of HORMA domain protein HIM-3 promotes asymmetric synaptonemal complex disassembly in meiotic prophase in Caenorhabditis elegans

PLoS Genetics ◽  
2020 ◽  
Vol 16 (11) ◽  
pp. e1008968
Author(s):  
Aya Sato-Carlton ◽  
Chihiro Nakamura-Tabuchi ◽  
Xuan Li ◽  
Hendrik Boog ◽  
Madison K. Lehmer ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Synaptonemal Complex ◽  
Post Translational Modifications ◽  
Phosphorylated Proteins ◽  
C Terminus ◽  
The Hierarchical Structure ◽  
Associated Proteins ◽  
Domain Protein

In the two cell divisions of meiosis, diploid genomes are reduced into complementary haploid sets through the discrete, two-step removal of chromosome cohesion, a task carried out in most eukaryotes by protecting cohesion at the centromere until the second division. In eukaryotes without defined centromeres, however, alternative strategies have been innovated. The best-understood of these is found in the nematode Caenorhabditis elegans: after the single off-center crossover divides the chromosome into two segments, or arms, several chromosome-associated proteins or post-translational modifications become specifically partitioned to either the shorter or longer arm, where they promote the correct timing of cohesion loss through as-yet unknown mechanisms. Here, we investigate the meiotic axis HORMA-domain protein HIM-3 and show that it becomes phosphorylated at its C-terminus, within the conserved “closure motif” region bound by the related HORMA-domain proteins HTP-1 and HTP-2. Binding of HTP-2 is abrogated by phosphorylation of the closure motif in in vitro assays, strongly suggesting that in vivo phosphorylation of HIM-3 likely modulates the hierarchical structure of the chromosome axis. Phosphorylation of HIM-3 only occurs on synapsed chromosomes, and similarly to other previously-described phosphorylated proteins of the synaptonemal complex, becomes restricted to the short arm after designation of crossover sites. Regulation of HIM-3 phosphorylation status is required for timely disassembly of synaptonemal complex central elements from the long arm, and is also required for proper timing of HTP-1 and HTP-2 dissociation from the short arm. Phosphorylation of HIM-3 thus plays a role in establishing the identity of short and long arms, thereby contributing to the robustness of the two-step chromosome segregation.

scholarly journals PRL1 promotes glioblastoma invasion and tumorigenesis via activating USP36-mediated Snail2 deubiquitination

2021 ◽  
Author(s):  
Wenjin Qiu ◽  
Xiaomin Cai ◽  
Kaya Xu ◽  
Shibin Song ◽  
Zumu Xiao ◽  
...  
Keyword(s):  
Cell Lines ◽  
Glioma Cell ◽  
Ectopic Expression ◽  
Tumor Grade ◽  
Migration And Invasion ◽  
Expression Levels ◽  
Protein Levels ◽  
Glioma Cell Lines

Abstract Background Regenerating liver phosphatase 1 (PRL1) is an established oncogene in various cancers, although its biological functions and the underlying mechanisms in glioblastoma multiforme (GBM) remain unclear. Methods PRL1 expression levels were analyzed in glioma tissues and cell lines. Multiple glioma cell lines were transfected with PRL1-overexpressing and shRNA constructs. In vitro proliferation, migration and invasion assays were conducted. Western blot and ubiquitylation assays were performed for molecular and mechanistic analyses. PRL1 expression levels were correlated with downstream ubiquitin pathway and clinical parameters using archival GBM samples. Results PRL1 was significantly upregulated in glioma tissues and cell lines, and positively correlated with the tumor grade. Ectopic expression of PRL1 in glioma cell lines significantly enhanced their tumorgenicity and invasion both in vitro and in vivo by promoting EMT. Conversely, knocking down PRL1 blocked EMT in the GBM cells, and inhibited their invasion, migration and tumorigenic growth. PRL1 also stabilized Snail2 through deubiquitination by activating USP36. Snail2 was identified as a crucial mediator of the oncogenic effects of PRL1 in GBM. Finally, PRL1 protein levels were positively correlated with that of Snail2 and predicted poor outcome of GBMs. Conclusions PRL1 promotes GBM progression by activating USP36-mediated Snail2 deubiquitination. This novel PRL1/USP36/Snail2 axis may be a promising therapeutic target for glioblastoma.

scholarly journals Phosphorylation of Histone H3 Thr-45 Is Linked to Apoptosis

2009 ◽  
Vol 284 (24) ◽  
pp. 16575-16583 ◽  
Author(s):  
Paul J. Hurd ◽  
Andrew J. Bannister ◽  
Karen Halls ◽  
Mark A. Dawson ◽  
Michiel Vermeulen ◽  
...  
Keyword(s):  
Histone H3 ◽  
Histone Tails ◽  
Post Translational Modifications ◽  
Nucleosome Structure ◽  
Cytokine Inhibition ◽  
Dna Nicking ◽  
A Site ◽  
Histone Core

Numerous post-translational modifications have been identified in histones. Most of these occur within the histone tails, but a few have been identified within the histone core sequences. Histone core post-translational modifications have the potential to directly modulate nucleosome structure and consequently DNA accessibility. Here, we identify threonine 45 of histone H3 (H3T45) as a site of phosphorylation in vivo. We find that phosphorylation of H3T45 (H3T45ph) increases dramatically in apoptotic cells, around the time of DNA nicking. To further explore this connection, we analyzed human neutrophil cells because they are short-lived cells that undergo apoptosis in vivo. Freshly isolated neutrophils contain very little H3T45ph, whereas cells cultured for 20 h possess significant amounts; the kinetics of H3T45ph induction closely parallel those of caspase-3 activation. Cytokine inhibition of neutrophil apoptosis leads to reduced levels of H3T45ph. We identify protein kinase C-δ as the kinase responsible for H3T45ph in vitro and in vivo. Given the nucleosomal position of H3T45, we postulate that H3T45ph induces structural change within the nucleosome to facilitate DNA nicking and/or fragmentation.

scholarly journals Naringin protects cardiomyocytes against hyperglycemia-induced injuries in vitro and in vivo

2016 ◽  
Vol 230 (2) ◽  
pp. 197-214 ◽  
Author(s):  
Qiong You ◽  
Zijun Wu ◽  
Bin Wu ◽  
Chang Liu ◽  
Ruina Huang ◽  
...  
Keyword(s):  
Caspase 3 ◽  
Mapk Pathway ◽  
Diabetic Rats ◽  
Pyrrolidine Dithiocarbamate ◽  
Sod Activity ◽  
Expression Levels ◽  
Protein Levels ◽  
And Oxidative Stress

We previously reported that naringin (NRG) protects cardiomyocytes against high glucose (HG)-induced injuries by inhibiting the MAPK pathway. The aim of this study was to test the hypothesis that NRG prevents cardiomyocytes from hyperglycemia-induced insult through the inhibition of the nuclear factor kappa B (NF-κB) pathway and the upregulation of ATP-sensitive K+ (KATP) channels. Our results showed that exposure of cardiomyocytes to HG for 24h markedly induced injuries, as evidenced by a decrease in cell viability and oxidative stress, and increases in apoptotic cells as well as the dissipation of mitochondrial membrane potential (MMP). These injuries were markedly attenuated by the pretreatment of cells with either NRG or pyrrolidine dithiocarbamate (PDTC) before exposure to HG. Furthermore, in streptozotocin (STZ)-induced diabetic rats and in HG-induced cardiomyocytes, the expression levels of caspase-3, bax and phosphorylated (p)-NF-κB p65 were increased. The increased protein levels were ameliorated by pretreatment with both NRG and PDTC. However, the expression levels of bcl-2 and KATP and superoxide dismutase (SOD) activity were decreased by hyperglycemia; the expression level of Nox4 and the ADP/ATP ratio were increased by hyperglycemia. These hyperglycemia-induced indexes were inhibited by the pretreatment of cardiomyocytes with NRG or PDTC. In addition, in STZ-induced diabetic rats, we also observed that NRG or PDTC contributed to protecting mitochondrial injury and myocardium damage. This study demonstrated that NRG protects cardiomyocytes against hyperglycemia-induced injury by upregulating KATP channels in vitro and inhibiting the NF-κB pathway in vivo and in vitro.

scholarly journals Ca2+/Calmodulin-NOS/NO-TNFs Pathway Hallmarks the Inflammation Response of Oyster During Aerial Exposure

2021 ◽  
Vol 7 ◽  
Author(s):  
Hao Chen ◽  
Lusheng Xin ◽  
Lin Wang ◽  
Huan Zhang ◽  
Rui Liu ◽  
...  
Keyword(s):  
Aerial Exposure ◽  
Inflammation Response ◽  
Expression Levels ◽  
Protein Levels ◽  
Downstream Effector ◽  
Intracellular Ca ◽  
Oxide Synthase ◽  
Mollusk Species

Aerial exposure (emersion) due to the periodical ebb and flow of tides is a major stressor for intertidal organisms and a key environmental factor in shaping their local communities. Oysters are among the most emersion-tolerant mollusk species and can survive for several days under aerial exposure. Noticeably, overwhelming inflammation responses could occur during the emersion stress. However, mechanisms beneath the activation and modulation of emersion-induced inflammation response have remained largely unknown. Ca2+ is an important intracellular second messenger that plays indispensable roles in inflammation response by cooperation with calmodulin (CaM) genes. Here, we showed that intracellular Ca2+ accumulates rapidly in oyster hemocytes during emersion stress along with the changes in the protein levels of three CaM genes, which function as intracellular sensors of Ca2+. As downstream effector of Ca2+/CaM complex, nitric oxide synthase (NOS) activity in hemocytes was enhanced during the emersion stress, facilitating a greater production of nitrite oxide (NO). Augmentation of NO concentration was associated with the increased mRNA expression levels of two oyster cytokines (CgTNFs) during aerial exposure. The robust accumulation of cytokines and severe injury of tissues in oysters have been regarded as potential cause and marker of their death in prolonged emersion stress. Here, both the expression levels of CgTNFs and the tissue injuries of oysters were attenuated when Ca2+/CaM complex or NOS activity were repressed in vivo during the emersion stress. These findings indicate that Ca2+/CaM-NOS/NO-CgTNFs pathway is critically involved in the emersion-induced inflammation response in oysters and plays a role in the resistance against long-term aerial exposure.

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