Aurora Kinase B, a novel regulator of TERF1 binding and telomeric integrity.
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ABSTRACT:
SUBMITTER: Chan FL
PROVIDER: S-EPMC5716096 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:ObjectiveThe disruption of the endothelial cell barrier is a critical feature of inflammation and an important contributing factor to acute lung injury (ALI), an inflammatory condition that is a major cause of morbidity and mortality in critically ill patients. We evaluated the role of the extracellular serine protease, hyaluronic acid binding protein 2 (HABP2), in vascular barrier regulation.Methods and resultsBy using immunoblot and immunohistochemical analysis, we observed that lipopolysaccharide (LPS) induces HABP2 expression in murine lung endothelium in vivo and in human pulmonary microvascular endothelial cells (ECs) in vitro. High-molecular-weight hyaluronan (HMW-HA, approximately 1x10(6) Da) decreased HABP2 protein expression in human pulmonary microvascular ECs and decreased purified HABP2 enzymatic activity, whereas low-molecular-weight HA (LMW-HA, approximately 2500 Da) increased these activities. The effects of LMW-HA, but not HMW-HA, on HABP2 activity were inhibited with a peptide of the polyanion-binding domain of HABP2. Silencing (small interfering RNA) HABP2 expression augmented HMW-HA-induced EC barrier enhancement and inhibited LPS and LMW-HA-mediated EC barrier disruption, results that were reversed with overexpression of HABP2. Silencing protease-activated receptor 1 and 3, RhoA, or Rho kinase expression attenuated LPS-, LMW-HA-, and HABP2-mediated EC barrier disruption. By using murine models of acute lung injury, we observed that LPS- and ventilator-induced pulmonary vascular hyperpermeability was significantly reduced with vascular silencing (small interfering RNA) of HABP2.ConclusionsHABP2 negatively regulates vascular integrity via activation of protease-activated receptor/RhoA/Rho kinase signaling and represents a potentially useful therapeutic target for syndromes of increased vascular permeability.
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Project description:Previously, we demonstrated that Bcl-2-like 10 (Bcl2l10) is associated with meiotic spindle assembly and that the gene that is most strongly down-regulated by Bcl2l10 RNAi is targeting protein for Xklp2 (Tpx2). Tpx2 is a well-known cofactor that controls the activity and localization of Aurora kinase A (Aurka) during mitotic spindle assembly. Therefore, this study was conducted (1) to identify the associations among Bcl2l10, Tpx2, and Aurka and (2) to understand how Bcl2l10 regulates meiotic spindle assembly in mouse oocytes. Bcl2l10, Tpx2, and Aurka co-localized on the meiotic spindles, and Bcl2l10 was present in the same complex with Tpx2. Tpx2 and Aurka expression decreased whereas phospho-Aurka increased in Bcl2l10 RNAi-treated oocytes. Counterbalancing changes in the levels of these 2 activators, Tpx2 and phospho-Aurka, resulted in decreased Aurka catalytic activity after Bcl2l10 RNAi treatment. Bcl2l10 RNAi decreased the expression of microtubule organizing center (MTOC)-related proteins, disturbed MTOC formation and disrupted meiotic spindle assembly. Our data demonstrate that Bcl2l10 is a binding partner of Tpx2 and a new regulator of the complex controlling the organization of microtubules and MTOC biogenesis in meiotic spindle assembly. The discovery of Bcl2l10 as a new effector of Aurka suggests that Bcl2l10 may have diverse functions in mitotic cells.
| S-EPMC5176144 | biostudies-literature
Project description:Aurora kinases are mitotic enzymes involved in centrosome maturation and separation, spindle assembly and stability, and chromosome condensation, segregation, and cytokinesis and represent well known targets for cancer therapy because their deregulation has been linked to tumorigenesis. The availability of suitable markers is of crucial importance to investigate the functions of Auroras and monitor kinase inhibition in in vivo models and in clinical trials. Extending the knowledge on Aurora substrates could help to better understand their biology and could be a source for clinical biomarkers. Using biochemical, mass spectrometric, and cellular approaches, we identified MYBBP1A as a novel Aurora B substrate and serine 1303 as the major phosphorylation site. MYBBP1A is phosphorylated in nocodazole-arrested cells and is dephosphorylated upon Aurora B silencing or by treatment with Danusertib, a small molecule inhibitor of Aurora kinases. Furthermore, we show that MYBBP1A depletion by RNA interference causes mitotic progression delay and spindle assembly defects. MYBBP1A has until now been described as a nucleolar protein, mainly involved in transcriptional regulation. The results presented herein show MYBBP1A as a novel Aurora B kinase substrate and reveal a not yet recognized link of this nucleolar protein to mitosis.
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Project description:A hallmark of advanced maternal age is a significant increase in meiotic chromosome segregation errors, resulting in early miscarriages and congenital disorders. These errors most frequently occur during meiosis I (MI). The spindle assembly checkpoint (SAC) prevents chromosome segregation errors by arresting the cell cycle until proper chromosome alignment is achieved. Unlike in mitosis, the SAC in oocytes is desensitized, allowing chromosome segregation in the presence of improperly aligned chromosomes. Whether SAC integrity further deteriorates with advancing maternal age, and if this decline contributes to increased segregation errors remains a fundamental question. In somatic cells, activation of the SAC depends upon Aurora kinase B (AURKB), which functions to monitor kinetochore-microtubule attachments and recruit SAC regulator proteins. In mice, oocyte-specific deletion of AURKB (Aurkb cKO) results in an increased production of aneuploid metaphase II-arrested eggs and premature age-related infertility. Here, we aimed to understand the cause of the short reproductive lifespan and hypothesized that SAC integrity was compromised. In comparing oocytes from young and sexually mature Aurkb cKO females, we found that SAC integrity becomes compromised rapidly with maternal age. We show that the increased desensitization of the SAC is driven by reduced expression of MAD2, ZW10 and Securin proteins, key contributors to the SAC response pathway. The reduced expression of these proteins is the result of altered protein homeostasis, likely caused by the accumulation of reactive oxygen species. Taken together, our results demonstrate a novel function for AURKB in preserving the female reproductive lifespan possibly by protecting oocytes from oxidative stress.
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Project description:We investigated the influence of telomere proximity and composition on the expression of an EGFP reporter gene in human cells. In transient transfection assays, telomeric DNA does not repress EGFP but rather slightly increases its expression. In contrast, in stable cell lines, the same reporter construct is repressed when inserted at a subtelomeric location. The telomeric repression is transiently alleviated by increasing the dosage of the TTAGGG repeat factor 1 (TRF1). Upon a prolongated treatment with trichostatin A, the derepression of the subtelomeric reporter gene correlates with the delocalization of HP1alpha and HP1beta. In contrast, treating the cells with 5 azacytidin, a demethylating agent, or with sirtinol, an inhibitor of the Sir2 family of deacetylase, has no apparent effect on telomeric repression. Overall, position effects at human chromosome ends are dependent on a specific higher-order organization of the telomeric chromatin. The possible involvement of HP1 isoforms is discussed.
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Project description:Aurora-A, a centrosomal serine-threonine kinase, orchestrates several key aspects of cell division. However, the regulatory pathways for the protein stability and kinase activity of Aurora-A are still not completely understood. In this study, PUM2, an RNA-binding protein, is identified as a novel substrate and interacting protein of Aurora-A. Overexpression of the PUM2 mutant which fails to interact with Aurora-A, and depletion of PUM2 result in a decrease in the amount of Aurora-A. PUM2 physically binds to the D-box of Aurora-A, which is recognized by APC/C(Cdh1). Overexpression of PUM2 prevents ubiquitination and enhances the protein stability of Aurora-A, suggesting that PUM2 protects Aurora-A from APC/C(Cdh1)-mediated degradation. Moreover, association of PUM2 with Aurora-A not only makes Aurora-A more stable but also enhances the kinase activity of Aurora-A. Our study suggests that PUM2 plays two different but important roles during cell cycle progression. In interphase, PUM2 localizes in cytoplasm and plays as translational repressor through its RNA binding domain. However, in mitosis, PUM2 physically associates with Aurora-A to ensure enough active Aurora-A at centrosomes for mitotic entry. This is the first time to reveal the moonlight role of PUM2 in mitosis.
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Project description:To investigate the underlying mechanism of pulmonary hypertension, the model of monocrotaline (MCT)-treated pulmonary arterial hypertension (PAH) rats were constructed to detect the differentially expressed profile of genes in lung tissue of PAH rat.
2024-01-11 | GSE252564 | GEO
Project description:Mammalian telomeric DNA contains duplex TTAGGG repeats and single-stranded overhangs. POT1 (protection of telomeres 1) is a telomere-specific single-stranded DNA-binding protein, highly conserved in eukaryotes. The biological function of human POT1 is not well understood. In the present study, we demonstrate that POT1 plays a key role in telomeric end protection. The reduction of POT1 by RNA interference led to the loss of telomeric single-stranded overhangs and induced apoptosis, chromosomal instability, and senescence in cells. POT1 and TRF2 interacted with each other to form a complex with telomeric DNA. A dominant negative TRF2, TRF2(DeltaBDeltaM), bound to POT1 and prevented it from binding to telomeres. POT1 overexpression protected against TRF2(DeltaBDeltaM)-induced loss of telomeric single-stranded overhangs, chromosomal instability, and senescence. These results demonstrate that POT1 and TRF2 share in part in the same pathway for telomere capping and suggest that POT1 binds to the telomeric single-stranded DNA in the D-loop and cooperates with TRF2 in t-loop maintenance.
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Project description:The metalloprotease meprin β (Mep1b) is capable of cleaving cell-adhesion molecules in different tissues (e.g. skin, kidney and intestine) and is dysregulated in several diseases associated with barrier breakdown (Alzheimer´s disease, kidney disruption, inflammatory bowel disease). In this study, we demonstrate that Mep1b is a novel regulator of tight junction (TJ) composition and blood-brain barrier (BBB) integrity in brain endothelium. In Mep1b-transfected mouse brain endothelial cells (bEnd.3), we observed a reduction of the TJ protein claudin-5, decreased transendothelial electrical resistance (TEER) and an elevated permeability to paracellular diffusion marker [14C]-inulin. Analysis of global Mep1b knock-out (Mep1b-/-) mice showed increased TJ protein expression (claudin-5, occludin, ZO-1) in cerebral microvessels and increased TEER in cultivated primary mouse brain endothelial compared to wild-type (wt) mice. Furthermore, we investigated the IgG levels in cerebrospinal fluid (CSF) and the brain water content as additional permeability markers and detected lower IgG levels and reduced brain water content in Mep1b-/- mice compared to wt mice. Showing opposing features in overexpression and knock-out, we conclude that Mep1b plays a role in regulating brain endothelial TJ-proteins and therefore affecting BBB tightness in vitro and in vivo.
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Project description:Human RecQL4 helicase plays critical roles in the maintenance of genomic stability. Mutations in RecQL4 helicase results in three clinically related autosomal recessive disorders: Rothmund-Thomson syndrome (RTS), RAPADILINO, and Baller-Gerold syndrome. In addition to several premature aging features, RTS patients are characterized by aneuploidy involving either loss or gain of a single chromosome. Chromosome mosaicism and isochromosomes involving chromosomes 2, 7, and 8 have been reported in RecQL4-deficient RTS patients, but the precise role of RecQL4 in chromosome segregation/stability remains to be elucidated. Here, we demonstrate that RecQL4 physically and functionally interacts with Aurora B kinase (AURKB) and stabilizes its expression by inhibiting its ubiquitination process. Our study indicates that the N-terminus of RecQL4 interacts with the catalytic domain of AURKB. Strikingly, RecQL4 suppression reduces the expression of AURKB leading to mitotic irregularities and apoptotic cell death. RecQL4 suppression increases the proportion of cells at the G2/M phase followed by an extensive cell death, presumably owing to the accumulation of mitotic irregularities. Both these defects (accumulation of cells at G2/M phase and an improper mitotic exit to sub-G1) are complemented by the ectopic expression of AURKB. Finally, evidence is provided for the requirement of both human telomerase reverse transcriptase and RecQL4 for stable immortalization and longevity of RTS fibroblasts. Collectively, our study suggests that the RecQL4-AURKB axis is essential for cellular proliferation, cell cycle progression, and mitotic stability in human cells.
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