Journal articles on the topic 'Beta arrestin isoforms'
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Rakib, Ahmed, Taslima Akter Eva, Saad Ahmed Sami, Saikat Mitra, Iqbal Hossain Nafiz, Ayan Das, Abu Montakim Tareq, et al. "Beta-Arrestins in the Treatment of Heart Failure Related to Hypertension: A Comprehensive Review." Pharmaceutics 13, no. 6 (June 5, 2021): 838. http://dx.doi.org/10.3390/pharmaceutics13060838.
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Heart failure (HF) is a complicated clinical syndrome that is considered an increasingly frequent reason for hospitalization, characterized by a complex therapeutic regimen, reduced quality of life, and high morbidity. Long-standing hypertension ultimately paves the way for HF. Recently, there have been improvements in the treatment of hypertension and overall management not limited to only conventional medications, but several novel pathways and their pharmacological alteration are also conducive to the treatment of hypertension. Beta-arrestin (β-arrestin), a protein responsible for beta-adrenergic receptors’ (β-AR) functioning and trafficking, has recently been discovered as a potential regulator in hypertension. β-arrestin isoforms, namely β-arrestin1 and β-arrestin2, mainly regulate cardiac function. However, there have been some controversies regarding the function of the two β-arrestins in hypertension regarding HF. In the present review, we try to figure out the paradox between the roles of two isoforms of β-arrestin in the treatment of HF.
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Taghert, Paul H. "The incidence of candidate binding sites for β-arrestin in Drosophila neuropeptide GPCRs." PLOS ONE 17, no. 11 (November 1, 2022): e0275410. http://dx.doi.org/10.1371/journal.pone.0275410.
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To support studies of neuropeptide neuromodulation, I have studied beta-arrestin binding sites (BBS’s) by evaluating the incidence of BBS sequences among the C terminal tails (CTs) of each of the 49 Drosophila melanogaster neuropeptide GPCRs. BBS were identified by matches with a prediction derived from structural analysis of rhodopsin:arrestin and vasopressin receptor: arrestin complexes [1]. To increase the rigor of the identification, I determined the conservation of BBS sequences between two long-diverged species D. melanogaster and D. virilis. There is great diversity in the profile of BBS’s in this group of GPCRs. I present evidence for conserved BBS’s in a majority of the Drosophila neuropeptide GPCRs; notably some have no conserved BBS sequences. In addition, certain GPCRs display numerous conserved compound BBS’s, and many GPCRs display BBS-like sequences in their intracellular loop (ICL) domains as well. Finally, 20 of the neuropeptide GPCRs are expressed as protein isoforms that vary in their CT domains. BBS profiles are typically different across related isoforms suggesting a need to diversify and regulate the extent and nature of GPCR:arrestin interactions. This work provides the initial basis to initiate future in vivo, genetic analyses in Drosophila to evaluate the roles of arrestins in neuropeptide GPCR desensitization, trafficking and signaling.
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Buechler, Christa, Susanne Feder, Elisabeth Haberl, and Charalampos Aslanidis. "Chemerin Isoforms and Activity in Obesity." International Journal of Molecular Sciences 20, no. 5 (March 5, 2019): 1128. http://dx.doi.org/10.3390/ijms20051128.
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Overweight and adiposity are risk factors for several diseases, like type 2 diabetes and cancer. White adipose tissue is a major source for adipokines, comprising a diverse group of proteins exerting various functions. Chemerin is one of these proteins whose systemic levels are increased in obesity. Chemerin is involved in different physiological and pathophysiological processes and it regulates adipogenesis, insulin sensitivity, and immune response, suggesting a vital role in metabolic health. The majority of serum chemerin is biologically inert. Different proteases are involved in the C-terminal processing of chemerin and generate diverse isoforms that vary in their activity. Distribution of chemerin variants was analyzed in adipose tissues and plasma of lean and obese humans and mice. The Tango bioassay, which is suitable to monitor the activation of the beta-arrestin 2 pathway, was used to determine the ex-vivo activation of chemerin receptors by systemic chemerin. Further, the expression of the chemerin receptors was analyzed in adipose tissue, liver, and skeletal muscle. Present investigations assume that increased systemic chemerin in human obesity is not accompanied by higher biologic activity. More research is needed to fully understand the pathways that control chemerin processing and chemerin signaling.
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Parruti, G., F. Peracchia, M. Sallese, G. Ambrosini, M. Masini, D. Rotilio, and A. De Blasi. "Molecular analysis of human beta-arrestin-1: cloning, tissue distribution, and regulation of expression. Identification of two isoforms generated by alternative splicing." Journal of Biological Chemistry 268, no. 13 (May 1993): 9753–61. http://dx.doi.org/10.1016/s0021-9258(18)98412-7.
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Idriss, Maya, Mohammad Hassan Hodroj, Rajaa Fakhoury, and Sandra Rizk. "Beta-Tocotrienol Exhibits More Cytotoxic Effects than Gamma-Tocotrienol on Breast Cancer Cells by Promoting Apoptosis via a P53-Independent PI3-Kinase Dependent Pathway." Biomolecules 10, no. 4 (April 9, 2020): 577. http://dx.doi.org/10.3390/biom10040577.
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Studies on tocotrienols have progressively revealed the benefits of these vitamin E isoforms on human health. Beta-tocotrienol (beta-T3) is known to be less available in nature compared to other vitamin E members, which may explain the restricted number of studies on beta-T3. In the present study, we aim to investigate the anti-proliferative effects and the pro-apoptotic mechanisms of beta-T3 on two human breast adenocarcinoma cell lines MDA-MB-231 and MCF7. To assess cell viability, both cell lines were incubated for 24 and 48 h, with different concentrations of beta-T3 and gamma-T3, the latter being a widely studied vitamin E isoform with potent anti-cancerous properties. Cell cycle progression and apoptosis induction upon treatment with various concentrations of the beta-T3 isoform were assessed. The effect of beta-T3 on the expression level of several apoptosis-related proteins p53, cytochrome C, cleaved-PARP-1, Bax, Bcl-2, and caspase-3, in addition to key cell survival proteins p-PI3K and p-GSK-3 α/β was determined using western blot analysis. Beta-tocotrienol exhibited a significantly more potent anti-proliferative effect than gamma-tocotrienol on both cell lines regardless of their hormonal receptor status. Beta-T3 induced a mild G1 arrest on both cell lines, and triggered a mitochondrial stress-mediated apoptotic response in MDA-MB-231 cells. Mechanistically, beta-T3′s anti-neoplastic activity involved the downregulation of phosphorylated PI3K and GSK-3 cell survival proteins. These findings suggest that vitamin E beta-T3 should be considered as a promising anti-cancer agent, more effective than gamma-T3 for treating human breast cancer and deserves to be further studied to investigate its effects in vitro and on other cancer types.
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Carneiro de Morais, Carla P., Juliano Z. Polidoro, Donna L. Ralph, Thaissa D. Pessoa, Maria Oliveira-Souza, Valério G. Barauna, Nancy A. Rebouças, Gerhard Malnic, Alicia A. McDonough, and Adriana C. C. Girardi. "Proximal tubule NHE3 activity is inhibited by beta-arrestin-biased angiotensin II type 1 receptor signaling." American Journal of Physiology-Cell Physiology 309, no. 8 (October 15, 2015): C541—C550. http://dx.doi.org/10.1152/ajpcell.00072.2015.
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Physiological concentrations of angiotensin II (ANG II) upregulate the activity of Na+/H+ exchanger isoform 3 (NHE3) in the renal proximal tubule through activation of the ANG II type I (AT1) receptor/G protein-coupled signaling. This effect is key for maintenance of extracellular fluid volume homeostasis and blood pressure. Recent findings have shown that selective activation of the beta-arrestin-biased AT1 receptor signaling pathway induces diuresis and natriuresis independent of G protein-mediated signaling. This study tested the hypothesis that activation of this AT1 receptor/beta-arrestin signaling inhibits NHE3 activity in proximal tubule. To this end, we determined the effects of the compound TRV120023, which binds to the AT1R, blocks G-protein coupling, and stimulates beta-arrestin signaling on NHE3 function in vivo and in vitro. NHE3 activity was measured in both native proximal tubules, by stationary microperfusion, and in opossum proximal tubule (OKP) cells, by Na+-dependent intracellular pH recovery. We found that 10−7 M TRV120023 remarkably inhibited proximal tubule NHE3 activity both in vivo and in vitro. Additionally, stimulation of NHE3 by ANG II was completely suppressed by TRV120023 both in vivo as well as in vitro. Inhibition of NHE3 activity by TRV120023 was associated with a decrease in NHE3 surface expression in OKP cells and with a redistribution from the body to the base of the microvilli in the rat proximal tubule. These findings indicate that biased signaling of the beta-arrestin pathway through the AT1 receptor inhibits NHE3 activity in the proximal tubule at least in part due to changes in NHE3 subcellular localization.
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Malapert, M., H. Guizouarn, B. Fievet, R. Jahns, F. Garcia-Romeu, R. Motais, and F. Borgese. "Regulation of Na+/H+ antiporter in trout red blood cells." Journal of Experimental Biology 200, no. 2 (January 1, 1997): 353–60. http://dx.doi.org/10.1242/jeb.200.2.353.
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The trout red blood cell Na+/H+ antiporter (beta NHE) plays two interesting properties: it is the only NHE own to be activated by cyclic AMP, and the activation process is followed by a desensitisation of the transport system itself. Cloning and expression of beta NHE have provided inificant information about Na+/H+ activation, in particular that activation by cyclic AMP is directly dependent upon the presence of two protein kinase A consensus sites in the cytoplasmic tail of the antiporter. Expression of beta NHE in fibroblasts demonstrates that the protein kinase A (PKA) and protein kinase C (PKC) activation pathways are independent and do not converge a common kinase. Moreover, the hydrophilic C-terminal fragment is essential to the mediation of the various hormonal responses. NHE1 (the human ubiquitous isoform) is not activated by cyclic AMP, but a "NHE1 transmembrane domain/beta NHE cytoplasmic domain' chimera is fully activated by cyclic AMP. In red cells, activation of beta NHE is the result of phosphorylation by PKA of at least two independent sites. Desensitisation, inhibited by the phosphatase inhibitor okadaic acid, may consist of the dephosphorylation of one of these two sites. Furthermore, Calyculin A (CIA), another specific protein phosphatase inhibitor, induces in unstimulated cells a Na+/H+ exchange activity whose exchange properties are very different from those of the adrenergically stimulated antiporter. It is suggested that CIA may be able to revive "sequestered' antiporters. We propose that the molecular events underlying beta NHE desensitisation could be similar to those involved in rhodopsin desensitisation. Antibodies were generated against trout red cell arrestin in order to analyse the binding of arrestin to the activated exchanger. Recombinant trout arrestin was produced in a protease-deficient strain of Escherichia coli and its functionality tested in a reconstituted rhodopsin assay.
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Mischak, H., W. Kolch, J. Goodnight, W. F. Davidson, U. Rapp, S. Rose-John, and J. F. Mushinski. "Expression of protein kinase C genes in hemopoietic cells is cell-type- and B cell-differentiation stage specific." Journal of Immunology 147, no. 11 (December 1, 1991): 3981–87. http://dx.doi.org/10.4049/jimmunol.147.11.3981.
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Abstract We have studied the expression of mRNA encoding all known protein kinase C (PKC) isozymes (alpha, beta, gamma, delta, epsilon, zeta, and eta) in murine tumor cell lines that exemplify hemopoietic cells arrested at different stages of development as well as in normal hemopoietic cells. We demonstrate that some of the isozymes, PKC-alpha, -beta, and -eta, are differentially expressed in different lineages. PKC-alpha and -beta generally are not detectable in myeloid cell lines, where PKC-delta is the predominant isoform. Both PKC-alpha and -beta are abundant in most T and B lymphocytic lines, but steady state levels of PKC-beta mRNA are lowest in plasma cell tumors, which exemplify the terminally differentiated B lymphocyte. In contrast, the levels of PKC-alpha mRNA remain high in plasma cell tumors, and a novel, 2.5-kb PKC-alpha mRNA gains prominence. PKC-eta mRNA is the major PKC isoform expressed in T lymphocytes, but it also is highly abundant in some myeloid lines. PKC-delta is expressed at high levels in all the lines we studied, whereas PKC-epsilon and -zeta are found in most cells but only at rather low levels. Analysis of myeloid clones derived from bipotential B lineage progenitor cell lines suggests that the B cell phenotype is associated with the expression of PKC-alpha. The close correlation of protein levels with mRNA levels indicates that PKC expression in hemopoietic cells is mainly regulated at the level of mRNA. The lineage- and differentiation stage-specific patterns of PKC-isozyme expression presented here suggest the involvement of specific PKC isozymes in differentiation as well as lineage determination of hemopoietic cells.
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Aletta, J. M., and L. A. Greene. "Sequential phosphorylation of chartin microtubule-associated proteins is regulated by the presence of microtubules." Journal of Cell Biology 105, no. 1 (July 1, 1987): 277–90. http://dx.doi.org/10.1083/jcb.105.1.277.
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Chartins are a unique class of three families of microtubule-associated proteins, each consisting of several isoforms possessing varying degrees of phosphorylation. The most highly phosphorylated chartin isoforms are highly enriched in neuronal cell fractions containing microtubules and there is evidence that their phosphorylation may play a role in promoting neurite outgrowth. The present work describes the relationship between the phosphorylation state of chartins and the presence of intact microtubules in long-term cultures of NGF-treated, neurite-bearing PC12 cells. Cultures were depleted of microtubules by exposure to high concentrations of depolymerizing agents for 2-24 h. Radiolabeling of cellular proteins with [32P]orthophosphate or [35S]methionine revealed that both the ongoing and steady-state phosphorylation of chartins is markedly altered under these conditions. Two-dimensional isoelectric focusing by SDS-PAGE of whole cell extracts demonstrated that the more acidic, highly phosphorylated isoforms are diminished with a concomitant increase in the more basic, less phosphorylated isoforms. These phosphorylation changes were relatively specific for the chartins and were not observed for phosphorylated MAP 1.2, phospho-beta-tubulin, or most other phosphoproteins. Thus, the phosphorylation state of chartins, but not of other phosphoproteins, is regulated by the presence of native microtubules. Despite depolymerization of microtubules, neurites remained extended for at least 24 h. Neurite elongation, however, was arrested. Microtubules, therefore, may be required for extension, but not for short-term maintenance of well-established neurites. Taxol, which promotes tubule assembly and stability, does not, conversely, drive phosphorylation of the chartins. Instead, taxol appeared to decrease the turnover of phosphate in microtubule-associated, acidic chartin isoforms. These data suggest several models as to how chartin phosphorylation is regulated in neurite-bearing cells and indicate that phosphorylation of cytoplasmic and microtubule-associated chartins occurs via different mechanisms.
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Li, Pengfei, James A. Cook, Gary S. Gilkeson, Louis M. Luttrell, Liping Wang, Keith T. Borg, Perry V. Halushka, and Hongkuan Fan. "Increased expression of beta-arrestin 1 and 2 in murine models of rheumatoid arthritis: Isoform specific regulation of inflammation." Molecular Immunology 49, no. 1-2 (October 2011): 64–74. http://dx.doi.org/10.1016/j.molimm.2011.07.021.
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Huang, Shengjian, Loretta J. Nastoupil, Hui Guo, Taylor Bell, Makhdum Ahmed, Carrie J. Li, Jack Wang, et al. "Pre-Clinical Evaluation of the PI3K-p110β/δ Inhibitor KA2237 in Mantle Cell Lymphoma." Blood 128, no. 22 (December 2, 2016): 1837. http://dx.doi.org/10.1182/blood.v128.22.1837.1837.
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Abstract Background: Mantle cell lymphoma (MCL) accounts for 6% of all non-Hodgkin lymphoma and is a therapeutic challenge. Phosphoinositide-3 kinase (PI3K) has been shown to be an alternative survival pathway in relapsed/refractory MCL. KA2237 (designed by Karus Therapeutics Ltd, Oxfordshire, United Kingdom) is a dual inhibitor of the class I beta and delta isoforms of the 110 kDa catalytic subunit of PI3K. By selectively targeting PI3K-beta and -delta isoforms and preventing their activation, KA2237 may decrease proliferation and induce cell death in susceptible tumor cells. Methods: We assessed the effects of KA2237 on the in vitro cell proliferation of both ibrutinib-sensitive (Mino, Jeko-1, and Rec-1) and primary ibrutinib-resistant (Z-138 and Maver-1) cell lines, and acquired ibrutinib-resistant MCL cell line, Jeko-R. We also tested the viability of patient-derived xenograft (PDX) tumor cells to KA2237. We compared the efficacy of KA2237 with two other commercial PI3K inhibitors, duvelisib (IPI-145, Selleck) and idelalisib (Cal-101, Selleck). Also, we paired these three inhibitors (KA2237, duvelisib and idelalisib) each with ibrutinib to evaluate the potential synergistic effects of these combinations. Lastly, we also tested in vivo efficacy of KA2237 and its combination with ibrutinib in PDX tumor cells. Results: KA2237 inhibited cell proliferation in both ibrutinib-sensitive and ibrutinib-resistant cell lines in a dose-dependent and time-dependent manner. For Mino and Jeko-1, the IC50 was 4.8 uM and 2.9 uM and for Z-138 and Maver-1 cell lines, the IC50 was 0.6 uM and 0.1 uM, respectively. KA2237 also decreased cell viability of ibrutinib-sensitive and ibrutinib-resistant MCL PDX tumor cells. However, KA2237 did not decrease the cell viability of normal human peripheral blood mono-nuclear cells. KA2237 arrested phase G0/G1 in Rec-1 and Jeko-R cell lines. We detected the expression of PI3K isoforms in MCL, finding higher expression of PI3K β and δ in MCL-resistant cell lines as compared with sensitive cell lines. We found that KA2237 induced MCL cell apoptosis in a time-dependent and dose-dependent manner. In comparison with duvelisib and idelalisib, KA2237 achieved greater inhibition of cell viability, cell apoptosis and cell cycle arrest. Furthermore, we found synergistic effects of KA2237 and ibrutinib combination in several MCL cell lines and in PDX models. In an ibrutinib-resistant PDX model, KA2237 treated mice reduced tumor burden significantly compared with vehicle control, and higher tumor growth inhibition was achieved as compared with ibrutinib. Conclusion: The novel PI3K inhibitor, KA2237 may be a potential candidate for MCL therapy, especially in the ibrutinib-resistant cases. Disclosures Wang: Acerta Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Juno Therapeutics: Research Funding; Pharmacyclics: Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Onyx: Research Funding; BeiGene: Research Funding; Asana BioSciences: Research Funding; Kite Pharma: Research Funding; Celgene: Research Funding.
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He, Yi, Salvador Alejo, Prabhakar Pitta Venkata, Jessica D. Johnson, Ilanna Loeffel, Uday P. Pratap, Yi Zou, et al. "Therapeutic Targeting of Ovarian Cancer Stem Cells Using Estrogen Receptor Beta Agonist." International Journal of Molecular Sciences 23, no. 13 (June 28, 2022): 7159. http://dx.doi.org/10.3390/ijms23137159.
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Ovarian cancer (OCa) is the deadliest gynecologic cancer. Emerging studies suggest ovarian cancer stem cells (OCSCs) contribute to chemotherapy resistance and tumor relapse. Recent studies demonstrated estrogen receptor beta (ERβ) exerts tumor suppressor functions in OCa. However, the status of ERβ expression in OCSCs and the therapeutic utility of the ERβ agonist LY500307 for targeting OCSCs remain unknown. OCSCs were enriched from ES2, OV90, SKOV3, OVSAHO, and A2780 cells using ALDEFLUOR kit. RT-qPCR results showed ERβ, particularly ERβ isoform 1, is highly expressed in OCSCs and that ERβ agonist LY500307 significantly reduced the viability of OCSCs. Treatment of OCSCs with LY500307 significantly reduced sphere formation, self-renewal, and invasion, while also promoting apoptosis and G2/M cell cycle arrest. Mechanistic studies using RNA-seq analysis demonstrated that LY500307 treatment resulted in modulation of pathways related to cell cycle and apoptosis. Western blot and RT-qPCR assays demonstrated the upregulation of apoptosis and cell cycle arrest genes such as FDXR, p21/CDKN1A, cleaved PARP, and caspase 3, and the downregulation of stemness markers SOX2, Oct4, and Nanog. Importantly, treatment of LY500307 significantly attenuated the tumor-initiating capacity of OCSCs in orthotopic OCa murine xenograft models. Our results demonstrate that ERβ agonist LY500307 is highly efficacious in reducing the stemness and promoting apoptosis of OCSCs and shows significant promise as a novel therapeutic agent in treating OCa.
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Million, K., J. Larcher, J. Laoukili, D. Bourguignon, F. Marano, and F. Tournier. "Polyglutamylation and polyglycylation of alpha- and beta-tubulins during in vitro ciliated cell differentiation of human respiratory epithelial cells." Journal of Cell Science 112, no. 23 (December 1, 1999): 4357–66. http://dx.doi.org/10.1242/jcs.112.23.4357.
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Tubulins are the major proteins within centriolar and axonemal structures. In all cell types studied so far, numerous alpha- and beta-tubulin isoforms are generated both by expression of a multigenic family and various post-translational modifications. We have developed a primary culture of human nasal epithelial cells where the ciliated cell differentiation process has been observed and quantified. We have used this system to study several properties concerning polyglutamylation and polyglycylation of tubulin. GT335, a monoclonal antibody directed against glutamylated tubulins, stained the centriole/basal bodies and the axonemes of ciliated cells, and the centrioles of non-ciliated cells. By contrast, axonemal but not centriolar tubulins were polyglycylated. Several polyglutamylated and polyglycylated tubulin isotypes were detected by two-dimensional electrophoresis, using GT335 and a specific monoclonal antibody (TAP952) directed against short polyglycyl chains. Immunoelectron microscopy experiments revealed that polyglycylation only affected axonemal tubulin. Using the same technical approach, polyglutamylation was shown to be an early event in the centriole assembly process, as gold particles were detected in fibrogranular material corresponding to the first cytoplasmic structures involved in centriologenesis. In a functional assay, GT335 and TAP952 had a dose-dependent inhibitory effect on ciliary beat frequency. TAP952 had only a weak effect while GT335 treatment led to a total arrest of beating. These results strongly suggest that in human ciliated epithelial cells, tubulin polyglycylation has only a structural role in cilia axonemes, while polyglutamylation may have a function both in centriole assembly and in cilia activity.
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Ohoka, Y., T. Kuwata, A. Asada, Y. Zhao, M. Mukai, and M. Iwata. "Regulation of thymocyte lineage commitment by the level of classical protein kinase C activity." Journal of Immunology 158, no. 12 (June 15, 1997): 5707–16. http://dx.doi.org/10.4049/jimmunol.158.12.5707.
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Abstract Thymocyte-positive selection involves signaling through TCR and accessory molecules, and the signaling intensity appears to be critical for this event. The specific inhibitor of classical Ca2+-dependent protein kinase C (cPKC), Gö 6976, inhibited positive selection in fetal thymus organ culture, indicating that cPKC activation is essential for positive selection. The major protein kinase C isoforms in CD4+ CD8+ thymocytes are cPKC-alpha, cPKC-beta, and the novel Ca2+-independent protein kinase C, nPKC-epsilon. To analyze the effect of cPKC activation level on positive selection, we used thymocytes from TCR transgenic mice with nonselecting and RAG-2 -/- backgrounds as they were developmentally arrested at the CD4+ CD8+ stage without positive selection signals. These thymocytes survived and acquired CD4/CD8 lineage commitment in suspension culture upon transient stimulation with limited concentrations of the selective activator of cPKC-alpha and -beta, thymeleatoxin, and the calcium ionophore, ionomycin. However, neither 12-deoxyphorbol 13-phenylacetate 20-acetate, which selectively activates cPKC-beta, nor ingenol 3,20-dibenzoate, which selectively activates nPKC-epsilon, exerted such an effect. The thymeleatoxin/ionomycin concentrations corresponded to those that inhibit glucocorticoid-induced apoptosis in thymocytes and were lower than those that induce proliferation of mature T cells. The CD4 lineage commitment required a higher level of cPKC activity than the CD8 lineage commitment. CD8alpha or CD4 mRNA expression was down-regulated. Functional helper and killer T cells were induced from the CD4 and CD8 lineage-committed cells, respectively, by additional stimulation. These results suggest that thymocyte lineage commitment in positive selection is regulated by the level of cPKC-alpha activity or by the levels of cPKC-alpha and -beta activities.
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Azab, Feda, Shireen Vali, Nicholas Potter, Joseph Abraham, Barbara Muz, Pilar De La Puente, Taher Abbasi, Ravi Vij, and Abdel Kareem Azab. "PI3K-Alpha Plays A Major Role In Multiple Myeloma and Its Inhibition With BYL917 Decreases Proliferation, Synergizes With Other Therapies and Overcomes Stroma-Induced Resistance." Blood 122, no. 21 (November 15, 2013): 3215. http://dx.doi.org/10.1182/blood.v122.21.3215.3215.
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Abstract Introduction Multiple myeloma (MM) is the second most prevalent hematologic malignancy and is still incurable. The PI3K pathway is activated and correlated with drug resistance in MM. Pan-inhibition of the PI3K pathway resulted in serious side effects, therefore, we focused on specific inhibition of the PI3K-alpha isoform. We investigated the role of PI3K- alpha in the progression and drug resistance of MM, by inhibiting the PI3K- alpha with a novel specific inhibitor BYL719. This is the first study to describe the preclinical effect of BYL719 on MM in particular and in hematologic malignancies in general. Methods and Results We analyzed the gene expression of PI3K isoforms in MM patients based on published datasets from the Gene Expression Omnibus by Zhan et al and found that PI3K-alpha and beta isoforms were highly expressed in MM. However, the fold-change of expression of the alpha isoform in MM patients was higher than the beta isoform, compared to healthy subjects. BYL719 inhibited the survival of MM cells isolated from three MM patients by MTT, at an IC50 around 1uM, but had no effect on normal PBMCs. Furthermore, BYL719 inhibited survival of all MM cell lines in a different manner. In silico-predicted activity of PI3K-alpha in the cell lines was exponentially correlated with the killing induced by the PI3K inhibitor BYL719 in vitro. BYL719 significantly decreased the activation of the PI3K signaling related proteins (pAKT, pS6R, and pGSK) by western blotting. Moreover, BYL719 inhibited cell cycle of MM cells detected by PI staining showed induction of G1-phase cell cycle arrest. BYL719 inhibited MM proliferation of MM1s cells in a dose dependent manner, through decreasing the levels of pCyclin-E1 and pRb, and increasing of P27 levels. The results were in agreement with the in silico studies that predicted the inhibition of other cell-cycle proteins including CDK4-Cyclin D complex, Myc-Max complex and CDK1-Cyclin B complex by BYL719 in a dose dependent manner. Moreover, BYL719 (0-2.5uM) increased the fraction of apoptotic MM cells in a dose depended manner, as detected by Annexin/PI staining. BYL719 induced apoptosis signaling by inducing the cleavage of Caspase-3, Caspase-9 and PARP by western blotting, in a dose dependent manner. We tested the effect of the combination of BYL719 with other drugs (Bortezomib and Carfilzomib) on survival of MM cells by MTT. We found that the combination of the two drugs decreased the surviving fraction of MM cells more than each of the drugs alone. Mechanistically, Bortezomib increased pAKT and pS6R as a resistance mechanism, and BYL719 abolished the Bortezomib-induced increase of pAKT and pS6R. Moreover, BYL719 enhanced the activation of pJNK induced by Carfilzomib and the combination of each drug increased cleavage of PARP, caspase-3 and caspase-9 more than each of the drugs alone. We tested the effect of BYL719 on the interaction of MM cells with BM stromal cells (BMSCs), and it was found that BYL719 decreased the adhesion of MM cells to BMSCs in a dose-dependent manner. Mechanistically, BYL719 decreased the activation of adhesion signaling such as pFAK, pSRC and pCofilin in a dose dependent manner. The in silico studies predicted the inhibition of the small GTPases Rho, Rac and Cdc42. To test the effect of BYL719 on drug resistance induced by the BM stroma, MM cells were co-Cultured with BMSCs, treated with BYL719 in combination with Bortezomib or Carfilzomib. It was found that co-culture with BMSCs increased the surviving fraction of MM cells after treatment with Bortezomib and Carfilzomib, as a drug resistance mechanism. The combination of the two drugs with BYL719 overcame the resistance induced by the stroma and reduced the surviving fraction to the values observed for treatment without presence of stroma. Conclusion This is the first study to describe preclinical effect of BYL719 on MM in particular and hematologic malignancies in general. The PI3K-alpha isoform plays a major role in the progression and drug resistance in MM cells, and it's inhibition with BYL719 reduces proliferation, inhibits cell cycle and induces apoptosis in MM cells. Moreover, it showed that BYL719 synergizes with Bortezomib and Carfilzomib, and overcomes drug resistance induced by BM stroma. Our findings provide a preclinical basis of future clinical trial of BYL719 in MM as a single agent or in combination with other drugs. Disclosures: Vali: Cellworks Group Inc., San Jose, CA, USA: Employment. Abbasi:Cellworks Group Inc: Employment.
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Aagaard, L., M. Schmid, P. Warburton, and T. Jenuwein. "Mitotic phosphorylation of SUV39H1, a novel component of active centromeres, coincides with transient accumulation at mammalian centromeres." Journal of Cell Science 113, no. 5 (March 1, 2000): 817–29. http://dx.doi.org/10.1242/jcs.113.5.817.
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Centromeres of eukaryotes are frequently associated with constitutive heterochromatin and their activity appears to be coregulated by epigenetic modification of higher order chromatin. Recently, we isolated murine (Suv39h1) and human (SUV39H1) homologues of the dominant Drosophila suppressor of position effect variegation Su(var)3-9, which is also related to the S. pombe silencing factor Clr4. We have shown that mammalian Su(var)3-9 homologues encode novel centromeric proteins on metaphase-arrested chromosomes. Here, we describe a detailed analysis of the chromatin distribution of human SUV39H1 during the cell cycle. Although there is significant heterochromatic overlap between SUV39H1 and M31 (HP1(beta)) during interphase, mitotic SUV39H1 displays a more restricted spatial and temporal association pattern with metaphase chromosomes than M31 (HP1(beta)), or the related HP1(α) gene product. SUV39H1 specifically accumulates at the centromere during prometaphase but dissociates from centromeric positions at the meta- to anaphase transition. In addition, SUV39H1 selectively associates with the active centromere of a dicentric chromosome and also with a neocentromere. Interestingly, SUV39H1 is shown to be a phosphoprotein with modifications at serine and, to a lesser degree, also at threonine residues. Whereas SUV39H1 steady-state protein levels appear constant during the cell cycle, two additional phosphorylated isoforms are detected in mitotic extracts. This intriguing localisation and modification pattern would be consistent with a regulatory role(s) for SUV39H1 in participating in higher order chromatin organisation at mammalian centromeres.
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Clayton, L. K., A. Bauer, Y. J. Jin, L. D'Adamio, S. Koyasu, and E. L. Reinherz. "Characterization of thymus-derived lymphocytes expressing Ti alpha-beta CD3 gamma delta epsilon zeta-zeta, Ti alpha-beta CD3 gamma delta epsilon eta-eta or Ti alpha-beta CD3 gamma delta epsilon zeta-zeta/zeta-eta antigen receptor isoforms: analysis by gene transfection." Journal of Experimental Medicine 172, no. 4 (October 1, 1990): 1243–53. http://dx.doi.org/10.1084/jem.172.4.1243.
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To characterize the function of the CD3 eta subunit of the T cell receptor (TCR), we have used cDNAs encoding CD3 zeta, CD3 eta, or both to reconstitute a variant of a cytochrome c-specific, I-Ek-restricted murine T cell hybridoma, termed MA5.8, which lacks CD3 zeta and CD3 eta proteins. We provide direct evidence that assembly and surface expression of TCRs can be mediated by either of these subunits separately or together. However, the level of TCR expression on zeta transfectants is up to one order of magnitude greater than that on eta transfectants, implying that CD3 eta is weakly associated with the pentameric Ti alpha-beta CD3 gamma delta epsilon complex and/or inefficient at salvaging the incomplete TCR from lysosomal degradation. As a component of the TCR, the CD3 eta subunit preferentially forms a heterodimer with CD3 zeta, but is also able to form a CD3 eta-eta homodimer. Crosslinking of Ti alpha-beta CD3 gamma delta epsilon zeta-zeta, Ti alpha-beta CD3 gamma delta epsilon eta-eta, or Ti alpha-beta CD3 gamma delta epsilon zeta-zeta/zeta-eta TCR isotypes with anti-CD3 epsilon monoclonal antibody or a cytochrome c peptide epitope on I-Ek antigen-presenting cells mediates signal transduction resulting in reversible cell-cycle arrest of transfected clones. Given the potential for diversity of signals generated by these functional TCR isotypes and the expression of the CD3 eta gene product in the thymus, CD3 eta is likely to play a role in selection and/or activation of thymocytes during development.
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Hurtado, Antoni, Tomàs Pinós, Anna Barbosa-Desongles, Sandra López-Avilés, Jordi Barquinero, Jordi Petriz, Albert Santamaria-Martínez, et al. "Estrogen Receptor Beta Displays Cell Cycle-Dependent Expression and Regulates the G1 Phase through a Non-Genomic Mechanism in Prostate Carcinoma Cells." Analytical Cellular Pathology 30, no. 4 (January 1, 2008): 349–65. http://dx.doi.org/10.1155/2008/129726.
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Background: It is well known that estrogens regulate cell cycle progression, but the specific contributions and mechanisms of action of the estrogen receptor beta (ERβ) remain elusive.Methods: We have analyzed the levels of ERβ1 and ERβ2 throughout the cell cycle, as well as the mechanisms of action and the consequences of the over-expression of ERβ1 in the human prostate cancer LNCaP cell line.Results: Both ERβ1 mRNA and protein expression increased from the G1 to the S phase and decreased before entering the G2/M phase, whereas ERβ2 levels decreased during the S phase and increased in the G2/M phase. ERβ1 protein was detected in both the nuclear and non-nuclear fractions, and ERβ2 was found exclusively in the nucleus. Regarding the mechanisms of action, endogenous ERβ was able to activate transcription via ERE during the S phase in a ligand-dependent manner, whereas no changes in AP1 and NFκB transactivation were observed after exposure to estradiol or the specific inhibitor ICI 182,780. Over-expression of either wild type ERβ1 or ERβ1 mutated in the DNA-binding domain caused an arrest in early G1. This arrest was accompanied by the interaction of over-expressed ERβ1 with c-Jun N-terminal protein kinase 1 (JNK1) and a decrease in c-Jun phosphorylation and cyclin D1 expression. The administration of ICI impeded the JNK1–ERβ1 interaction, increased c-Jun phosphorylation and cyclin D1 expression and allowed the cells to progress to late G1, where they became arrested.Conclusions: Our results demonstrate that, in LNCaP prostate cancer cells, both ERβ isoforms are differentially expressed during the cell cycle and that ERβ regulates the G1 phase by a non-genomic mechanism.
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Fuchs, Ota, Gabriela Peslova, Dana Provaznikova, and Iuri Marinov. "Increased Expression of Genes for Protooncoproteins SnoN and SnoA in Human Myeloblastic Leukemia ML2 Cells and Resistance of ML2 Cells to Transforming Growth Factor-beta-Induced Growth Arrest." Blood 106, no. 11 (November 16, 2005): 4347. http://dx.doi.org/10.1182/blood.v106.11.4347.4347.
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Abstract Transforming growth factor-beta 1 (TGF-β1) is a multifunctional cytokine involved in a variety of biological processes including development, cell growth, differentiation, apoptosis, cell adhesion, migration, extracellular matrix deposition, and the immune response. The loss of a growth inhibitory response to TGF-β1 is a common feature of many cancers. Human myeloblastic ML2 cells originally obtained from Dr. Minowada (Palumbo A. et al., Blood64, 1059–1063, 1984) were pre-incubated with or without TGF-β1 (5 or 10 ng/ml) or with TGF-β1 antibody for 24 h or 72h and then incubated for further 4 h in the presence of [6-3H] thymidine. TGF-β1 did not decreased the proliferation of ML2 cells measured by incorporation of [6-3H] thymidine into DNA in comparison with control without TGF-β1 or with ML2 cells icubated in the presence of inactivating TGF-β1antibody. The resistance of ML2 cells to TGF-β1-induced growth arrest is not caused by mutation in TGF-β1 receptors (TβRII and ALK5) or Smad4 as we verified by direct sequencing of exons of these genes. After 24 h incubation TGF-β1 increased the levels of mRNA for some target proteins of TGF-β1 -plasminogen activator inhibitor-1, Smad7, SnoN and SnoA (ski novel related gene products), inhibitors of cyclin-dependent kinases (p15/INK4b, p21/WAF1/CIP1) and decreased the levels of mRNA for c-myc, transferrin receptor 1 and inhibitor of differentiation/DNA binding Id1. TGF-β1 did not affect the levels of mRNA for CDC25 phosphatase and RhoA GTPase. The levels of these mRNA were determined by real-time PCR or semiquantitative PCR using specific oligonucleotide primers. The increased expression of SnoN and SnoA genes and the inability of TGF-β1 to cause SnoN degradation may be the cause of ML2 cells resistance to TGF-β1 -induced growth arrest. Antiproliferative genes coding for p15/INK4b, p21/WAF1/CIP1 are not under control of SnoN and SnoA. SnoN (684 aminoacids) and SnoA (415 aminoacids) are the alternatively spliced isoforms. Both these isoforms contain the N-terminal region that is similar to the ski (sloan kettering virus gene product) protooncoprotein. These oncoproteins are incorporated into the histone deacetylase-1 complex through binding to the nuclear corepressor and Smad (Smad2, Smad3 and Smad4) proteins and repress the activity of Smad proteins. The addition of histone deacetylase inhibitors (0.5μM MS-275 or 1mM sodium butyrate) in combination with TGF-β1 (5 ng/ml or 10 ng/ml) in pre-incubation of ML2 cells for 24 h before the incorporation of [6-3H] thymidine into DNA measurement decreased proliferation of ML2 cells in comparison with ML2 cells without additions (control) or ML2 cells with TGF-β1 or histone deacetylase inhibitors. These results support the role of Sno protooncoproteins in resistance of ML2 cells to TGF-β1-induced growth arrest. This study was financially supported by the Internal Grant Agency of the Ministry of Health, Czech Republic (NC/7605-3).
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Robinson, L. C., M. M. Menold, S. Garrett, and M. R. Culbertson. "Casein kinase I-like protein kinases encoded by YCK1 and YCK2 are required for yeast morphogenesis." Molecular and Cellular Biology 13, no. 5 (May 1993): 2870–81. http://dx.doi.org/10.1128/mcb.13.5.2870-2881.1993.
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Casein kinase I is an acidotropic protein kinase class that is widely distributed among eukaryotic cell types. In the yeast Saccharomyces cerevisiae, the casein kinase I isoform encoded by the gene pair YCK1 and YCK2 is a 60- to 62-kDa membrane-associated form. The Yck proteins perform functions essential for growth and division; either alone supports growth, but loss of function of both is lethal. We report here that casein kinase I-like activity is associated with a soluble Yck2-beta-galactosidase fusion protein in vitro and that thermolabile protein kinase activity is exhibited by a protein encoded by fusion of a temperature-sensitive yck2 allele with lacZ. Cells carrying the yck2-2ts allele arrest at restrictive temperature with multiple, elongated buds containing multiple nuclei. This phenotype suggests that the essential functions of the Yck proteins include roles in bud morphogenesis, possibly in control of cell growth polarity, and in cytokinesis or cell separation. Further, a genetic relationship between the yck2ts allele and deletion of CDC55 indicates that the function of Yck phosphorylation may be related to that of protein phosphatase 2A activity.
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Robinson, L. C., M. M. Menold, S. Garrett, and M. R. Culbertson. "Casein kinase I-like protein kinases encoded by YCK1 and YCK2 are required for yeast morphogenesis." Molecular and Cellular Biology 13, no. 5 (May 1993): 2870–81. http://dx.doi.org/10.1128/mcb.13.5.2870.
Full textAbstract:
Casein kinase I is an acidotropic protein kinase class that is widely distributed among eukaryotic cell types. In the yeast Saccharomyces cerevisiae, the casein kinase I isoform encoded by the gene pair YCK1 and YCK2 is a 60- to 62-kDa membrane-associated form. The Yck proteins perform functions essential for growth and division; either alone supports growth, but loss of function of both is lethal. We report here that casein kinase I-like activity is associated with a soluble Yck2-beta-galactosidase fusion protein in vitro and that thermolabile protein kinase activity is exhibited by a protein encoded by fusion of a temperature-sensitive yck2 allele with lacZ. Cells carrying the yck2-2ts allele arrest at restrictive temperature with multiple, elongated buds containing multiple nuclei. This phenotype suggests that the essential functions of the Yck proteins include roles in bud morphogenesis, possibly in control of cell growth polarity, and in cytokinesis or cell separation. Further, a genetic relationship between the yck2ts allele and deletion of CDC55 indicates that the function of Yck phosphorylation may be related to that of protein phosphatase 2A activity.
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He, Yi, Salvador Alejo, Prabhakar P. Venkata, Jessica D. Johnson, Ilanna Loeffel, Julie Ann Martel, Uday P. Pratap, et al. "Abstract 891: Therapeutic targeting of ovarian cancer stem cells using estrogen receptor beta agonist." Cancer Research 82, no. 12_Supplement (June 15, 2022): 891. http://dx.doi.org/10.1158/1538-7445.am2022-891.
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Abstract Background: Ovarian cancer (OCa) is the deadliest gynecologic cancer. Emerging studies suggest that ovarian cancer stem cells (OCSCs) contribute to tumor relapse and chemotherapy resistance. Recent studies demonstrate that OCa cells express estrogen receptor beta (ERβ), which functions as a tumor suppressor. However, the status of ERβ expression in OCSCs and the therapeutic utility of ERβ agonist LY500307 for targeting OCSCs remain unknown. In this study, we tested the hypothesis that OCSCs express ERβ and that treatment with ERβ agonist reduces stemness and promotes apoptosis of OCSCs. Methods: We isolated subpopulations of OCSCs from SKOV3, A2780, and patient-derived ascites cells using ALDEFLUOR kit and examined the expression of ERβ in OCSCs using RT-qPCR. The effect of ERβ agonist LY500307 on OCSCs was examined utilizing cell viability, cell cycle, sphere formation, self-renewal, and apoptosis assays. Mechanistic studies were conducted using RNA-seq, RT-qPCR, and Western blot analysis. The efficacy of LY500307 on the tumor-initiating capacity of OCSCs was determined via in vivo limiting dilution assay studies using orthotopic intrabursal xenograft murine models. Results: RT-qPCR assay showed that ERβ, particularly ERβ isoform 1, is highly expressed in OCSCs compared to non-OCSCs. ERβ agonist LY500307 significantly reduced the viability of OCSCs compared to non-OCSCs. Treatment of OCSCs with LY500307 significantly reduced the sphere formation and self-renewal of OCSCs. Further, LY500307 treatment resulted in G2/M cell cycle arrest and promoted the apoptosis of OCSCs. Mechanistic studies using RNA-seq analysis demonstrated that ERβ agonist treatment resulted in the modulation of pathways related to cell cycle and apoptosis. Western blots and RT-qPCR assays confirmed the upregulation of genes involved in apoptosis and cell cycle arrest, such as FDXR and CDKN1A. Importantly, treatment of LY500307 significantly attenuated the tumor-initiating capacity of OCSCs in orthotopically implanted OCa murine models. Conclusions: Our results demonstrate that ERβ agonist LY500307 is highly efficacious in reducing the stemness and promoting apoptosis of OCSCs and may serve as a novel therapeutic agent in treating OCa Citation Format: Yi He, Salvador Alejo, Prabhakar P. Venkata, Jessica D. Johnson, Ilanna Loeffel, Julie Ann Martel, Uday P. Pratap, Suryavathi Viswanadhapalli, Rajeshwar R. Tekmal, Ratna K. Vadlamudi, Edward Kost, Gangadhara R. Sareddy. Therapeutic targeting of ovarian cancer stem cells using estrogen receptor beta agonist [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 891.
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Radeke, H. H., J. E. Gessner, P. Uciechowski, H. J. Mägert, R. E. Schmidt, and K. Resch. "Intrinsic human glomerular mesangial cells can express receptors for IgG complexes (hFc gamma RIII-A) and the associated Fc epsilon RI gamma-chain." Journal of Immunology 153, no. 3 (August 1, 1994): 1281–92. http://dx.doi.org/10.4049/jimmunol.153.3.1281.
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Abstract Immune complexes localize to the glomerular mesangium in many forms of human glomerulonephritis. In this investigation, we determined whether primary cultured human glomerular mesangial cells (HMC) express mRNA and functional protein of FcR for IgG. Performing reverse transcription-PCR with subsequent control Southern blots, we showed that in contrast to human monocytes or granulocytes, mRNA for Fc gamma RI and Fc gamma RII is not detectable in either growth-arrested (48 h serum free) or unstimulated proliferating (medium with 10% FCS) HMC. However, IFN-gamma in combination with LPSE.coli (LPS), but not LPS alone elicited a significant transcription of hFc gamma RIII mRNA in resting HMC, whereas cytokines, such as IL-1 beta or TGF-beta, failed to induce any of the three Fc gamma Rs in resting HMC. Proliferating HMC showed a basal transcription of Fc gamma RIII mRNA, which was enhanced by IFN-gamma in combination with LPS. Slot-blot analysis indicated that only the Fc gamma RIII-A gene encoding the transmembrane isoform of Fc gamma RIII was expressed by HMC. For the first time, transcripts for the gamma-chain of Fc epsilon RI were found in HMC. Fc gamma RIII protein expression was detected after LPS/IFN-gamma stimulation both by specific staining of paraformaldehyde-fixed HMC and immunoprecipitation of Fc gamma RIII protein from HMC membranes. Fc gamma RIII-A receptors are functionally active, as HMC IL-6 mRNA synthesis was stimulated by heat-aggregated IgG or F(ab')2 fragments of CLBGran1 mAb only after induction of Fc gamma RIII-A. These in vitro data suggest that HMC that are basically negative for Fc gamma RIII can be stimulated to express low affinity Fc gamma Rs, and thus may participate actively in human glomerulonephritis involving immune complexes.
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Yoon, Hyemin, Hoon Jang, Eun-Young Kim, Sohyeon Moon, Sangho Lee, Minha Cho, Hye Jung Cho, et al. "Knockdown of PRKAR2B Results in the Failure of Oocyte Maturation." Cellular Physiology and Biochemistry 45, no. 5 (2018): 2009–20. http://dx.doi.org/10.1159/000487978.
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Background/Aims: Cyclic adenosine monophosphate (cAMP)-dependent type 2 regulatory subunit beta (Prkar2b) is a regulatory isoform of cAMP-dependent protein kinase (PKA), which is the primary target for cAMP actions. In oocytes, PKA and the pentose phosphate pathway (PPP) have important roles during the germinal vesicle (GV) stage arrest of development. Although the roles of the PKA signal pathway have been studied in the development of oocyte, there has been no report on the function of PRKAR2B, a key regulator of PKA. Methods: Using reverse transcription polymerase chain reaction (RT-PCR), quantitative real-time PCR (qRT-PCR), immunohistochemistry, and immunofluorescence, we determined the relative expression of Prkar2b in various tissues, including ovarian follicles, during oocyte maturation. Prkar2b-interfering RNA (RNAi) microinjection was conducted to confirm the effect of Prkar2b knockdown, and immunofluorescence, qRT-PCR, and time-lapse video microscopy were used to analyze Prkar2b-deficient oocytes. Results: Prkar2b is strongly expressed in the ovarian tissues, particularly in the growing follicle. During oocyte maturation, the highest expression of Prkar2b was during metaphase I (MI), with a significant decrease at metaphase II (MII). RNAi-mediated Prkar2b suppression resulted in MI-stage arrest during oocyte development, and these oocytes exhibited abnormal spindle formation and chromosome aggregation. Expression of other members of the PKA family (except for Prkaca) were decreased, and the majority of the PPP factors were also reduced in Prkar2b-deficient oocytes. Conclusion: These results suggest that Prkar2b is closely involved in the maturation of oocytes by controlling spindle formation and PPP-mediated metabolism.
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Wieczorek, D. F., M. Periasamy, G. S. Butler-Browne, R. G. Whalen, and B. Nadal-Ginard. "Co-expression of multiple myosin heavy chain genes, in addition to a tissue-specific one, in extraocular musculature." Journal of Cell Biology 101, no. 2 (August 1, 1985): 618–29. http://dx.doi.org/10.1083/jcb.101.2.618.
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We have investigated the developmental transitions of myosin heavy chain (MHC) gene expression in the rat extraocular musculature (EOM) at the mRNA level using S1-nuclease mapping techniques and at the protein level by polypeptide mapping and immunochemistry. We have isolated a genomic clone, designated lambda 10B3, corresponding to an MHC gene which is expressed in the EOM fibers (recti and oblique muscles) of the adult rat but not in hind limb muscles. Using cDNA and genomic probes for MHC genes expressed in skeletal (embryonic, neonatal, fast oxidative, fast glycolytic, and slow/cardiac beta-MHC), cardiac (alpha-MHC), and EOM (lambda 10B3) muscles, we demonstrate the concomitant expression at the mRNA level of at least six different MHC genes in adult EOM. Protein and immunochemical analyses confirm the presence of at least four different MHC types in EOM. Immunocytochemistry demonstrates that different myosin isozymes tend to segregate into individual myofibers, although some fibers seem to contain more than one MHC type. The results also show that the EOM fibers exhibit multiple patterns of MHC gene regulation. One set of fibers undergoes a sequence of isoform transitions similar to the one described for limb skeletal muscles, whereas other EOM myofiber populations arrest the MHC transition at the embryonic, neonatal/adult, or adult EOM-specific stage. Thus, the MHC gene family is not under the control of a strict developmental clock, but the individual genes can modify their expression by tissue-specific and/or environmental factors.
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Wei, Sheng, Kathy Rocha, Ann Williams, Xianhong Chen, P. K. Burnette, Julie Y. Djeu, Qing Liu, et al. "Gene Dosage of the Cell Cycle Regulatory Phosphatases Cdc25C and PP2A Determines Sensitivity to Lenalidomide in del(5q) MDS." Blood 110, no. 11 (November 16, 2007): 118. http://dx.doi.org/10.1182/blood.v110.11.118.118.
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Abstract MDS with chromosome 5q deletion [del(5q)] is uniquely sensitive to clonal suppression by lenalidomide (LEN), yielding high rates of cytogenetic and erythroid response. Although haploinsufficiency for a candidate tumor suppressor gene within the common deleted segment (CDS) has been implicated in the pathogenesis of del(5q) MDS, no specific gene products account for the karyotype-dependent LEN sensitivity. Using primary AML cells, we showed that LEN is selectively cytotoxic to del(5q) cells, inducing cell cycle arrest at the G2 > G1 junctures and apoptosis in a concentration-dependent fashion. Now we show that the CDS-encoded Cdc25C and PP2A phosphatases (PPTs) serve as molecular targets for LEN. Cdc25C and PP2A are dual specificity PPTs that regulate cell cycle. The genes encoding the catalytic subunit alpha (PP2ACa), the dominant catalytic isoform, and the regulatory subunit B (beta isoform PR 52) of the PP2A holoenzyme reside within the del(5q) CDS. Real-time RT-PCR showed that Cdc25C and PP2ACa gene expression is significantly reduced in primary del(5q) AML and MDS BM-MNC compared to non-del5q and normal controls. A Cdc25C FISH probe confirmed presence of only a single allele in del(5q) specimens [n=5] vs. normal karyotype [n=5] with no overlap with the EGR1 locus. We found that LEN inhibited rHu-Cdc25C activity with IC90 inhibitory concentrations ≤10nM. Similarly, PP2A PPT activity was inhibited by LEN in enzyme immunoprecipitates from Ramos cells at sub-micromolar concentrations. In U937 cells, we confirmed intracellular Cdc25C and PP2A inhibition, showing concentration-dependent retention of the inhibitory p-Tyr15 -Cdc2 substrates for Cdc25C with corresponding phosphorylation of the PP2A substrate Ser216 Cdc25C, findings reproduced by treatment with the PP2A selective antagonist, fostreicin. Cdc25C immunoprecipitation showed that phosphorylation of Ser216 Cdc25C increased binding to the 14-3-3 protein with consequent cytoplasmic sequestration confirmed by fluorescence microscopy. Phosphorylation of histone H2A.X Ser139 was unchanged after LEN treatment, indicating that phosphorylation of the inhibitory Cdc25C serine does not arise from upstream DNA damage activated kinases. Importantly, siRNA suppression of Cdc25C gene expression in U937 cells promoted LEN-specific apoptosis in 45% of cells, which increased to 68% after fostreicin inhibition of PP2A. These data indicate that haplodeficiency for the Cdc25C and PP2ACa genes promotes selective sensitivity of del(5q) cells to LEN-induced apoptosis. Haplodeficiency of critical drug-able targets may represent a novel strategy for tumor selective therapy.
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Buglio, Daniela, Manuela Lemoine, Jaymie Estrella, Sattva S. Neelapu, Richard Eric Davis, Laurence Doyle, Richard F. Little, Donald Berry, and Anas Younes. "The Allosteric AKT Inhibitor MK-2206 Demonstrates Potent Antiproliferative Activity in Lymphoma Cells and Synergizes with the HDAC Inhibitor Vorinostat,." Blood 118, no. 21 (November 18, 2011): 3729. http://dx.doi.org/10.1182/blood.v118.21.3729.3729.
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Abstract Abstract 3729 The serine/threonine kinase Akt plays a critical signaling role downstream of phosphatidylinositol-3-kinase (PI3K) and is important in promoting cell survival and inhibiting apoptosis. Indeed, Akt activation and overexpression is often associated with resistance to chemotherapy or radiotherapy. Previous studies demonstrated the potential therapeutic value of targeting the PI3K pathway in lymphoma, as both the selective PI3Kδ inhibitor CAL-101, and everolimus and temsirolimus, which target PI3K and mTOR, produce clinical responses in a variety of lymphomas. We evaluated the effect of the novel allosteric Akt inhibitor, MK-2206, in a panel of lymphoma cell lines and primary lymphoma cells. We found that Akt, and activated pAkt, are highly expressed in lymphoma cells. After 72 hours of incubation, the Akt inhibitor MK-2206 demonstrated antiproliferative activity in a variety of lymphoma cell lines, with an IC50 ranging between 0.1 and 5μM. There was no correlation between pre-treatment levels of pAKT, PI3K isoforms, or PTEN protein expression and sensitivity to MK-2206. Within the diffuse large B cell lymphoma cell lines, those of GCB cell of origin were more sensitive to MK-2206, compared with the ABC-derived cell lines. Resistant cell lines tended to had weak or absent expression of p-GSK3 and p-4EBPI. Mechanistically, MK-2206 treatment decreased the level of p-Akt (Ser473), and p-Akt (Thr308), irrespective of drug sensitivity. Furthermore, MK-2206 decreased the phosphorylation level of Akt downstream targets, including p-GSK3 beta and p-PRAS40, upregulated p27. and dephosphorylated p70S6K. Moreover, MK-2206 treatment decreased HIF-1 alpha and VEGF expression. Depending on the cell of origin, the antiproliferative effect resulted from cycle arrest at the G0/G1 phase, autophagy, orapoptosis. MK-2206 showed synergistic effect in combination with the HDAC inhibitor, Vorinostat. Using pathway-specific protein arrays focusing on apoptosis, kinases, and transcription factors, the combination of MK-2206 and Vorinostat effectively altered p53 and p27 levels, which were associated with increased PARP cleavage and induction of apoptosis. Our data demonstrate that AKT is a promising target for the treatment of lymphoma, and provide a rationale for an ongoing trial, evaluating MK-2206 for the treatment of patients with relapsed lymphoma. Disclosures: No relevant conflicts of interest to declare.
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Wade, James Lloyd, Corey J. Langer, Mary Redman, Charu Aggarwal, Jeffrey D. Bradley, Jeffrey Crawford, Jieling Miao, et al. "A phase II study of GDC-0032 (taselisib) for previously treated PI3K positive patients with stage IV squamous cell lung cancer (SqNSCLC): LUNG-MAP sub-study SWOG S1400B." Journal of Clinical Oncology 35, no. 15_suppl (May 20, 2017): 9054. http://dx.doi.org/10.1200/jco.2017.35.15_suppl.9054.
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9054 Background: Lung-MAP (S1400) is a National Clinical Trials Network “umbrella” trial for previously-treated SqNSCLC. Sub-study S1400B included patients (pts) with tumors harboring PI3K mutations.Taselisib (GDC-0032), a potent, small molecule inhibitor of Class 1 PI3K with beta isoform sparing selectivity, has been shown to be a potent inhibitor in preclinical models of PIK3CA-mutant tumors. Methods: Eligibility stipulated progressive SqNSCLC after primary platinum-based therapy and presence of a PIK3CA mutation as determined by Foundation Medicine (FMI+) NGS. . The primary analysis population was a subgroup of the total PIK3CA mutation group (GNE+) with alterations limited to substitutions: E542K, E545A, E545G, E545K, E545Q, H1047L, H1047R, H1047Y. Primary endpoint was response rate (RR) in GNE+ pts. The initial protocol randomized PIK3 mt (+) pts to taselisib 4 mg po daily or docetaxel, but was amended to single arm phase II trial of taselisib with interim analysis based on first 20 eligible GNE+ pts evaluable for response, stipulating closure for futility if < 2 responses were observed. Results: 26 eligible pts, 7% of those registered to S1400, received taselisib; of these , 21 (81%) were GNE+. Of the 20 eligible, response-evaluable GNE+ pts, one pt with PIK3CA E545K gene alteration responded (5% RR, 95% Confidence Interval [CI] 1%, 24%). 13 pts had stable disease. Median PFS was 2.5 mos (95% CI, 1.7-4.0 mos) and 2.7 mos (95% CI, 1.8-3.4 mos) among GNE+ and FMI+ pts, respectively. 26 FMI+ pts were evaluable for toxicity: two grade 5 events (cardiac arrest, respiratory failure), neither clearly attributable to treatment, were recorded, along with one instance each of grade 4 AEs (dyspnea, thrombocytopenia, pneumonitis). Grade 3 AEs included 5 pts each with hyperglycemia or diarrhea, and 3 with lymphopenia. Overall survival data is premature. Conclusions: Study S1400B failed to meet its primary endpoint and was closed December 2016 at interim analysis for futility. Toxicities were manageable. The trial is unique in cataloguing the diversity of mutations in the PI3K pathways in SqNSCLC Clinical trial information: NCT02785913.
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Mao, Xinliang, Tabitha W. Wood, Xiaoming Wang, Johnathan St-Germain, Michael F. Moran, Alessandro Datti, Jeff Wrana, Robert A. Batey, and Aaron D. Schimmer. "A Novel Chromene-Based Pan-PI3 Kinase Inhibitor Displays Preclinical Activity in Leukemia and Myeloma." Blood 112, no. 11 (November 16, 2008): 1605. http://dx.doi.org/10.1182/blood.v112.11.1605.1605.
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Abstract D-cyclins are universally dysregulated in multiple myeloma and frequently over-expressed in acute leukemia. Therefore, to better understand the regulation of the D-cyclins and identify leads for novel therapeutic agents for the treatment of hematologic malignancies, we conducted a high throughput screen of 50,000 novel chemical compounds to identify inhibitors of D-cyclin transactivation. From this screen, we identified a chromene-based compound 8-ethoxy-2-(4-fluorophenyl)-3-nitro-2H-chromene (pichromene). In secondary assays, pichromene reduced expression of cyclins D1, D2, and D3 in myeloma and leukemia cell lines at low micromolar concentrations. Furthermore, pichromene arrested the cells in the G0/G1 phase of the cell cycle. In myeloma and leukemia cell lines, pichromene decreased levels of phospho-AKT, but did not alter levels of total AKT. PI3 kinases regulate AKT phosphorylation that, in turn, regulate D-cyclin expression and cell cycle progression. Therefore, we evaluated the effects of pichromene on the enzymatic activity of PI3 kinases. In cell-free enzymatic assays, pichromene inhibited the enzymatic activity of all four isoforms of the PI3 kinase, PI3Kalpha, beta, delta and gamma, with similar efficacy. In contrast it did not markedly inhibit the enzymatic activities of unrelated kinases AKT 1, 2 or 3, PDK 1 or 2, or GSK3β or 3α at concentrations up to 300 μM in a similar cell-free assay. However, in intact cells, due to its inhibition of PI3 kinases, pichomene inhibited AKT activity as noted above. As inhibitors of PI3 kinases are pro-apoptotic and may have anti-cancer activity, we evaluated the effects of pichromene on the viability of leukemia and myeloma cells. Leukemia and myeloma cell lines were treated with increasing concentrations of pichromene and cell viability was measured after 72 hours by an MTS assay. Pichromene induced cell death in 9/10 leukemia and 9/10 myeloma cell lines with an ED50 < 10 μM. In contrast, it was less cytotoxic to primary normal hematopoietic cells obtained from volunteer donors of stem cells for allotransplant. Apoptosis was confirmed by Annexin V staining. Cell death was associated with caspase activation as demonstrated by the cleavage of caspase-3 and PARP through immunoblotting. Interestingly, U266 was the one myeloma cell line that was resistant to pichromene, and lacked detectable basal levels of phospho-AKT by immunoblotting. Given the effects of pichromene on malignant cells, we evaluated the efficacy of this compound in a leukemia xenograft mouse model. K562 cells were implanted subcutaneously into sublethally irradiated NOD/SCID mice. Mice were then treated with pichromene (50 mg/kg/day) or buffer control by oral gavage. Pichromene decreased tumor weight and volume by more than 35% as early as 8 days after treatment. No evidence of weight loss or gross organ toxicity was observed even when mice were treated with up to 500mg/kg/day of pichromene by oral gavage or intraperitoneally. Thus, in summary, we have identified a novel pan-inhibitor of PI3 kinases that displays preclinical efficacy in myeloma and leukemia.
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Blaine, Arryn T., Yiming Miao, Jinling Yuan, Sophia Palant, Rebecca J. Liu, Zhong-Yin Zhang, and Richard M. van Rijn. "Exploration of beta-arrestin isoform signaling pathways in delta opioid receptor agonist-induced convulsions." Frontiers in Pharmacology 13 (August 11, 2022). http://dx.doi.org/10.3389/fphar.2022.914651.
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The δ-opioid receptor (δOR) has been considered as a therapeutic target in multiple neurological and neuropsychiatric disorders particularly as δOR agonists are deemed safer alternatives relative to the more abuse-liable µ-opioid receptor drugs. Clinical development of δOR agonists, however, has been challenging in part due to the seizure-inducing effects of certain δOR agonists. Especially agonists that resemble the δOR-selective agonist SNC80 have well-established convulsive activity. Close inspection suggests that many of those seizurogenic δOR agonists efficaciously recruit β-arrestin, yet surprisingly, SNC80 displays enhanced seizure activity in β-arrestin 1 knockout mice. This finding led us to hypothesize that perhaps β-arrestin 1 is protective against, whereas β-arrestin 2 is detrimental for δOR-agonist-induced seizures. To investigate our hypothesis, we characterized three different δOR agonists (SNC80, ADL5859, ARM390) in cellular assays and in vivo in wild-type and β-arrestin 1 and β-arrestin 2 knockout mice for seizure activity. We also investigated downstream kinases associated with β-arrestin-dependent signal transduction. We discovered that δOR agonist-induced seizure activity strongly and positively correlates with β-arrestin 2 efficacy for the agonist, but that indirect inhibition of ERK activation using the MEK inhibitor SL327 did not inhibit seizure potency and duration. Inhibition of the PI3K/AKT/mTOR signaling with honokiol but not PQR530, attenuated SNC80 seizure duration in β-arrestin 1 knockout, but honokiol did not reduce SNC80-induced seizures in wild-type mice. Ultimately, our results indicate that β-arrestin 2 is correlated with δOR agonist-induced seizure intensity, but that global β-arrestin 1 knockout mice are a poor model system to investigate their mechanism of action.
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Chuderland, Dana, Zeev Dvashi, Ruth Kaplan-Kraicer, Daniella Ben-Meir, Ruth Shalgi, and Sara Lavi. "De novo synthesis of protein phosphatase 1A, magnesium dependent, alpha isoform (PPM1A) during oocyte maturation." Cellular and Molecular Biology Letters 17, no. 3 (January 1, 2012). http://dx.doi.org/10.2478/s11658-012-0022-7.
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AbstractOocyte maturation in mammals is a multiple-stage process that generates fertilizable oocytes. Ovarian oocytes are arrested at prophase of the first meiotic division characterized by the presence of a germinal vesicle. Towards ovulation, the oocytes resume meiosis and proceed to the second metaphase in a process known as maturation; they undergo nuclear and cytoplasmic changes that are accompanied by translation and degradation of mRNA. Protein phosphatase 1A, magnesium dependent, alpha isoform (PPM1A), which belongs to the metal-dependent serine/threonine protein phosphatase family, is highly conserved during evolution. PPM1A plays a significant role in many cellular functions such as cell cycle progression, apoptosis and cellular differentiation. It works through diverse signaling pathways, including p38 MAP kinase JNK and transforming growth factor beta (TGF-β). Herein we report that PPM1A is expressed in mouse oocytes and that its mRNA level rises during oocyte maturation. Using quantitative real-time polymerase chain reaction (qPCR) and western blot analysis, we found that PPM1A mRNA is synthesized at the beginning of the maturation process and remains elevated in the mature oocytes, promoting the accumulation of PPM1A protein. Since PPM1A function is mainly affected by its level, we propose that it might have an important role in oocyte maturation.
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Dubois, Julia, Aurélien Traversier, Thomas Julien, Blandine Padey, Bruno Lina, Jean-Christophe Bourdon, Virginie Marcel, Guy Boivin, Manuel Rosa-Calatrava, and Olivier Terrier. "The Nonstructural NS1 Protein of Influenza Viruses ModulatesTP53Splicing through Host Factor CPSF4." Journal of Virology 93, no. 7 (January 16, 2019). http://dx.doi.org/10.1128/jvi.02168-18.
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ABSTRACTInfluenza A viruses (IAV) are known to modulate and “hijack” several cellular host mechanisms, including gene splicing and RNA maturation machineries. These modulations alter host cellular responses and enable an optimal expression of viral products throughout infection. The interplay between the host protein p53 and IAV, in particular through the viral nonstructural protein NS1, has been shown to be supportive for IAV replication. However, it remains unknown whether alternatively spliced isoforms of p53, known to modulate p53 transcriptional activity, are affected by IAV infection and contribute to IAV replication. Using aTP53minigene, which mimics intron 9 alternative splicing, we have shown here that the NS1 protein of IAV changes the expression pattern of p53 isoforms. Our results demonstrate that CPSF4 (cellular protein cleavage and polyadenylation specificity factor 4) independently and the interaction between NS1 and CPSF4 modulate the alternative splicing ofTP53transcripts, which may result in the differential activation of p53-responsive genes. Finally, we report that CPSF4 and most likely beta and gamma spliced p53 isoforms affect both viral replication and IAV-associated type I interferon secretion. All together, our data show that cellular p53 and CPSF4 factors, both interacting with viral NS1, have a crucial role during IAV replication that allows IAV to interact with and alter the expression of alternatively spliced p53 isoforms in order to regulate the cellular innate response, especially via type I interferon secretion, and perform efficient viral replication.IMPORTANCEInfluenza A viruses (IAV) constitute a major public health issue, causing illness and death in high-risk populations during seasonal epidemics or pandemics. IAV are known to modulate cellular pathways to promote their replication and avoid immune restriction via the targeting of several cellular proteins. One of these proteins, p53, is a master regulator involved in a large panel of biological processes, including cell cycle arrest, apoptosis, or senescence. This “cellular gatekeeper” is also involved in the control of viral infections, and viruses have developed a wide diversity of mechanisms to modulate/hijack p53 functions to achieve an optimal replication in their hosts. Our group and others have previously shown that p53 activity is finely modulated by different multilevel mechanisms during IAV infection. Here, we characterized IAV nonstructural protein NS1 and the cellular factor CPSF4 as major partners involved in the IAV-induced modulation of theTP53alternative splicing that was associated with a strong modulation of p53 activity and notably the p53-mediated antiviral response.
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Boldizsar, Noelia M., Marta Sanchez‐Soto, Kayla Schardien, Julia Drube, Raphael Haider, R. B. Free, Carsten Hoffmann, and David Sibley. "G protein‐coupled receptor kinases regulate &[beta]‐arrestin interactions with the D2 dopamine receptor in an isoform‐specific manner and in the absence of direct receptor phosphorylation." FASEB Journal 36, S1 (May 2022). http://dx.doi.org/10.1096/fasebj.2022.36.s1.r3465.
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