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1
Noriega, Guillermo O., Adela A. Juknat, and Alcira M. del C. Batlle. "Non-Essential Activation of Rat Liver Porphobilinogen-Deaminase by Folic Acid." Zeitschrift für Naturforschung C 47, no. 5-6 (June 1, 1992): 416–19. http://dx.doi.org/10.1515/znc-1992-0616.
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This report demonstrates the ability of folic acid to activate rat liver porphobilinogen-deaminase (PBG -D). Lineweaver-Burk analysis revealed an increase in Vmax (38%) without affecting the Km. In the concentration range assayed, secondary replots of 1/Δslope and 1/Δintersect versus 1/[folic acid] yielded straight lines, indicating the binding of a single molecule of activator to the enzyme PBG-D , with a KA = 1.66 mᴍ. Results presented here show that folic acid acts as a non-essential activator (α = 1; β = 1.6). The activating effect of folic acid has been observed employing the 35-70% ammonium sulphate precipitated fraction, desalted by dialysis or gel filtration, whereas no action was detected when other partially purified PBG -D preparations were utilized as the enzyme source, suggesting either the presence of sites saturated for the activator, or the existence of a different structural protein conformation, or both.
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Das-Panja, Kaberi, Vidya Jonnalagadda, and Sobhanaditya Jonnalagadda. "Orthophosphate is a non-essential activator of Vigna radiata flavokinase." IUBMB Life 47, no. 4 (April 1999): 547–54. http://dx.doi.org/10.1080/15216549900201583.
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Cassels, R., R. Fears, and R. A. Smith. "The interaction of streptokinase.plasminogen activator complex, tissue-type plasminogen activator, urokinase and their acylated derivatives with fibrin and cyanogen bromide digest of fibrinogen. Relationship to fibrinolytic potency in vitro." Biochemical Journal 247, no. 2 (October 15, 1987): 395–400. http://dx.doi.org/10.1042/bj2470395.
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The effects of purified soluble fibrin and of fibrinogen fragments (fibrin mimic) on the activation of Lys-plasminogen (i.e. plasminogen residues 77-790) to plasmin by streptokinase.plasminogen activator complex and by tissue-type plasminogen activator were studied. Dissociation constants of both activators were estimated to lie in the range 90-160 nM (fibrin) and 16-60 nM (CNBr-cleavage fragments of fibrinogen). The kinetic mechanism for both types of activator comprised non-essential enzyme activation via a Rapid Equilibrium Ordered Bireactant sequence. In order to relate the fibrin affinity of plasminogen activators to their fibrinolytic potency, the rate of lysis of supported human plasma clots formed in the presence of unmodified or active-centre-acylated precursors of plasminogen activators was studied as a function of the concentration of enzyme derivative. The concentrations of unmodified enzyme giving 50% lysis/h in this assay were 0.9, 2.0 and 11.0 nM for tissue-type plasminogen activator, streptokinase.plasmin(ogen) and urokinase respectively. However, the potencies of active-centre-acylated derivatives of these enzymes suggested that acylated-tissue plasminogen activator and streptokinase.plasminogen complexes of comparable hydrolytic stability were of comparable potency. Both types of acyl-enzyme were significantly more potent than acyl-urokinases.
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Speijer, Han, José W. P. Govers-Riemslag, Robert F. A. Zwaal, and Jan Rosing. "Platelet Procoagulant Properties Studied with Snake Venom Prothrombin Activators." Thrombosis and Haemostasis 57, no. 03 (1987): 349–55. http://dx.doi.org/10.1055/s-0038-1651132.
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SummaryPurified snake venom prothrombin activators were used to probe the procoagulant properties of platelet membranes. Human platelets were able to stimulate prothrombin activation by the venom activators from Oxyuranus scutellatus and Notechis scutatus, while the prothrombin activator from Echis carinatus was not affected by the presence of platelets. The prothrombinconverting activity of platelets was further studied with the venom activator from Oxyuranus scutellatus and with the factor Xa-Va complex as prothrombin activating enzymes. Stimulation of platelets with collagen, collagen plus thrombin or with the Ca-ionophore A23187 resulted in a considerable increase of platelet prothrombin converting activity probed with the factor Xa-Va complex as well as with the prothrombin activator from Oxyuranus scutellatus. The stimulatory effect of activated platelets on the rates of prothrombin activation by Oxyuranus scutellatus was similar to that determined for factor Xa-Va-catalyzed prothrombin activation. Compared to non-stimulated platelets, platelets stimulated with thrombin plus collagen exposed 20-times more procoagulant sites for as well the factor Xa-Va complex, as for the venom activator from Oxyuranus scutellatus. The actual number of procoagulant sites per platelet determined with the factor Xa-Va complex was in close agreement with the number of sites determined with the venom activator. Also the time course of appearance of procoagulant activity during platelet stimulation by collagen plus thrombin was comparable for both activator complexes. Phospholipase A2 treatment of stimulated platelets resulted in an almost complete loss of their ability to stimulate prothrombin activation by the enzyme from Oxyuranus scutellatus or by factor Xa-Va complex. The findings presented in this paper suggest: a) that the factor Xa-Va complex and the prothrombin activator from Oxyuranus scutellatus recognize the same procoagulant sites on both stimulated and unstimulated platelets and b) that negatively-charged phospholipids are essential components of these procoagulant sites.
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Gai, Lili, Yuting Zhu, Chun Zhang, and Xianfang Meng. "Targeting Canonical and Non-Canonical STAT Signaling Pathways in Renal Diseases." Cells 10, no. 7 (June 27, 2021): 1610. http://dx.doi.org/10.3390/cells10071610.
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Signal transducer and activator of transcription (STAT) plays an essential role in the inflammatory reaction and immune response of numerous renal diseases. STATs can transmit the signals of cytokines, chemokines, and growth factors from the cell membrane to the nucleus. In the canonical STAT signaling pathways, upon binding with their cognate receptors, cytokines lead to a caspase of Janus kinases (JAKs) and STATs tyrosine phosphorylation and activation. Besides receptor-associated tyrosine kinases JAKs, receptors with intrinsic tyrosine kinase activities, G-protein coupled receptors, and non-receptor tyrosine kinases can also activate STATs through tyrosine phosphorylation or, alternatively, other post-translational modifications. Activated STATs translocate into the nucleus and mediate the transcription of specific genes, thus mediating the progression of various renal diseases. Non-canonical STAT pathways consist of preassembled receptor complexes, preformed STAT dimers, unphosphorylated STATs (U-STATs), and non-canonical functions including mitochondria modulation, microtubule regulation and heterochromatin stabilization. Most studies targeting STAT signaling pathways have focused on canonical pathways, but research extending into non-canonical STAT pathways would provide novel strategies for treating renal diseases. In this review, we will introduce both canonical and non-canonical STAT pathways and their roles in a variety of renal diseases.
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Qiu, Yi, Min Guo, Suming Huang, and Roland Stein. "Insulin Gene Transcription Is Mediated by Interactions between the p300 Coactivator and PDX-1, BETA2, and E47." Molecular and Cellular Biology 22, no. 2 (January 15, 2002): 412–20. http://dx.doi.org/10.1128/mcb.22.2.412-420.2002.
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ABSTRACT Pancreatic β-cell-type-specific expression of the insulin gene requires both ubiquitous and cell-enriched activators, which are organized within the enhancer region into a network of protein-protein and protein-DNA interactions to promote transcriptional synergy. Protein-protein-mediated communication between DNA-bound activators and the RNA polymerase II transcriptional machinery is inhibited by the adenovirus E1A protein as a result of E1A’s binding to the p300 coactivator. E1A disrupts signaling between the non-DNA-binding p300 protein and the basic helix-loop-helix DNA-binding factors of insulin’s E-element activator (i.e., the islet-enriched BETA2 and generally distributed E47 proteins), as well as a distinct but unidentified enhancer factor. In the present report, we show that E1A binding to p300 prevents activation by insulin’s β-cell-enriched PDX-1 activator. p300 interacts directly with the N-terminal region of the PDX-1 homeodomain protein, which contains conserved amino acid sequences essential for activation. The unique combination of PDX-1, BETA2, E47, and p300 was shown to promote synergistic activation from a transfected insulin enhancer-driven reporter construct in non-β cells, a process inhibited by E1A. In addition, E1A inhibited the level of PDX-1 and BETA2 complex formation in β cells. These results indicate that E1A inhibits insulin gene transcription by preventing communication between the p300 coactivator and key DNA-bound activators, like PDX-1 and BETA2:E47.
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Geletu, Mulu, Zaid Taha, Patrick T. Gunning, and Leda Raptis. "PI3k and Stat3: Oncogenes that are Required for Gap Junctional, Intercellular Communication." Cancers 11, no. 2 (February 1, 2019): 167. http://dx.doi.org/10.3390/cancers11020167.
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Gap junctional, intercellular communication (GJIC) is interrupted in cells transformed by oncogenes such as activated Src. The Src effector, Ras, is required for this effect, so that Ras inhibition restores GJIC in Src-transformed cells. Interestingly, the inhibition of the Src effector phosphatidyl-inositol-3 kinase (PI3k) or Signal Transducer and Activator of Transcription-3 (Stat3) pathways does not restore GJIC. In the contrary, inhibition of PI3k or Stat3 in non-transformed rodent fibroblasts or epithelial cells or certain human lung carcinoma lines with extensive GJIC inhibits communication, while mutational activation of PI3k or Stat3 increases GJIC. Therefore, it appears that oncogenes such as activated Src have a dual role upon GJIC; acting as inhibitors of communication through the Ras pathway, and as activators through activation of PI3k or Stat3. In the presence of high Src activity the inhibitory functions prevail so that the net effect is gap junction closure. PI3k and Stat3 constitute potent survival signals, so that their inhibition in non-transformed cells triggers apoptosis which, in turn, has been independently demonstrated to suppress GJIC. The interruption of gap junctional communication would confine the apoptotic event to single cells and this might be essential for the maintenance of tissue integrity. We hypothesize that the GJIC activation by PI3k or Stat3 may be linked to their survival function.
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Wang, Xinjiang, Junru Wang, and Xuejun Jiang. "MdmX Protein Is Essential for Mdm2 Protein-mediated p53 Polyubiquitination." Journal of Biological Chemistry 286, no. 27 (May 13, 2011): 23725–34. http://dx.doi.org/10.1074/jbc.m110.213868.
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Genetic evidence has implicated both Mdm2 and MdmX as essential in negative regulation of p53. However, the exact role of MdmX in this Mdm2-dependent protein degradation is not well understood. Most, if not all, previous Mdm2 studies used GST-Mdm2 fusion proteins in the in vitro assays. Here, we show that the p53 polyubiquitination activity of GST-Mdm2 is conferred by the GST tag and non-GST-tagged Mdm2 only catalyzes monoubiquitination of p53 even at extremely high concentrations. We further demonstrate that MdmX is a potent activator of Mdm2, facilitating dose-dependent p53 polyubiquitination. This activation process requires the RING domains of both MdmX and Mdm2 proteins. The polyubiquitination activity of Mdm2/MdmX is Mdm2-dependent. Unlike Mdm2 or MdmX overexpression alone, co-overexpression of MdmX and Mdm2 consistently triggered p53 degradation in cells. Moreover, cellular polyubiquitination of p53 was only observable in the cytoplasm where both Mdm2 and MdmX are readily detectable. Importantly, RNAi knockdown of MdmX increased levels of endogenous p53 accompanied by reduced p53 polyubiquitination. In conclusion, our work has resolved a major confusion in the field derived from using GST-Mdm2 and demonstrated that MdmX is the cellular activator that converts Mdm2 from a monoubiquitination E3 ligase to a polyubiquitination E3 ligase toward p53. Together, our findings provide a biochemical basis for the requirement of both Mdm2 and MdmX in the dynamic regulation of p53 stability.
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Peluso, Heather, Julie A. Caffrey, and Stephen M. Milner. "4G/4G PAI-1 gene variant in a patient with non-healing ulcers." Journal of Epidemiological Research 2, no. 1 (November 11, 2015): 91. http://dx.doi.org/10.5430/jer.v2n1p91.
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Plasminogen activator inhibitor is a serine protease inhibitor from the serpin gene family that modulates fibrin clot breakdown.PAI-1 irreversibly inhibits tissue plasminogen activator (t-PA) and urokinase plasminogen activator (u-PA) from activatingplasminogen. PAI-1 also inhibits integrin-vitronectin and vitronectin-vitronectin interactions that are essential for cell migration,adhesion, and angiogenesis. We describe a patient, who developed chronic non-healing ulcers after minimal trauma to severalareas of his body. Genetic testing revealed the 4G/4G homozygous genotype for the polymorphism in the promoter regionof the PAI-1 gene. Increased PAI-1 activity prevents the breakdown of the fibrin clot and cell migration to remodel damagedtissue. A combination of poor clot fibrinolysis and cell recruitment to the site of injury may explain our patient’s non-healingulcers following minor traumatic injury. Early treatment with excision and skin grafting may benefit patients presenting withnon-healing ulcers and the homozygous 4G/4G PAI-1 variant. To our knowledge, there have been no reports in the literatureassociating PAI-1 overexpression and chronic non-healing wounds.
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Liu, Yudan, Meghan Harding, Andrea Pittman, Jules Dore, Jörg Striessnig, Anjali Rajadhyaksha, and Xihua Chen. "Cav1.2 and Cav1.3 L-type calcium channels regulate dopaminergic firing activity in the mouse ventral tegmental area." Journal of Neurophysiology 112, no. 5 (September 1, 2014): 1119–30. http://dx.doi.org/10.1152/jn.00757.2013.
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Dopaminergic projections from the ventral tegmental area (VTA) constitute the mesolimbocortical system that underlies addiction and psychosis primarily as a result of increased dopaminergic transmission. Dopamine release is spike dependent. L-type calcium channels (LTCCs) play an important role in regulating firing activities, but the contribution of specific subtypes remains unclear. This article describes different functions of Cav1.2 and Cav1.3 subtypes in regulating firing properties with two transgenic mouse strains. For basal firing, Cav1.3-deficient (Cav1.3−/−) mice had a lower basal firing frequency. The dihydropyridine (DHP) channel blocker nifedipine reduced single-spike firing in mice expressing DHP-insensitive Cav1.2 channels (Cav1.2DHP−/− mice), confirming the significant contribution from the Cav1.3 subtype in basal firing. Moreover, the DHP channel activator ( S)-(−)-Bay K8644 and the non-DHP channel activator FPL 64176 converted firing patterns from single spiking to bursting in Cav1.2DHP−/− mice. Nifedipine inhibited burst firing induced by both activators, suggesting that Cav1.3 also serves an essential role in burst firing. However, FPL 64176 also induced bursting in Cav1.3−/− mice. These results indicate that the Cav1.3 subtype is crucial to regulation of basal single-spike firing, while activation of both Cav1.2 and Cav1.3 can support burst firing of VTA neurons.
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Bethea, Emily K., Billy J. Carver, Anthony E. Montedonico, and Todd B. Reynolds. "The inositol regulon controls viability in Candida glabrata." Microbiology 156, no. 2 (February 1, 2010): 452–62. http://dx.doi.org/10.1099/mic.0.030072-0.
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Inositol is essential in eukaryotes, and must be imported or synthesized. Inositol biosynthesis in Saccharomyces cerevisiae is controlled by three non-essential genes that make up the inositol regulon: ScINO2 and ScINO4, which together encode a heterodimeric transcriptional activator, and ScOPI1, which encodes a transcriptional repressor. ScOpi1p inhibits the ScIno2-ScIno4p activator in response to extracellular inositol levels. An important gene controlled by the inositol regulon is ScINO1, which encodes inositol-3-phosphate synthase, a key enzyme in inositol biosynthesis. In the pathogenic yeast Candida albicans, homologues of the S. cerevisiae inositol regulon genes are ‘transcriptionally rewired’. Instead of regulating the CaINO1 gene, CaINO2 and CaINO4 regulate ribosomal genes. Another Candida species that is a prevalent cause of infections is Candida glabrata; however, C. glabrata is phylogenetically more closely related to S. cerevisiae than C. albicans. Experiments were designed to determine if C. glabrata homologues of the inositol regulon genes function similarly to S. cerevisiae or are transcriptionally rewired. CgINO2, CgINO4 and CgOPI1 regulate CgINO1 in a manner similar to that observed in S. cerevisiae. However, unlike in S. cerevisiae, CgOPI1 is essential. Genetic data indicate that CgOPI1 is a repressor that affects viability by regulating activation of a target of the inositol regulon.
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Ma, Shuai, Cheng Cao, Shiyou Che, Yuejiao Wang, Dongxue Su, Shuai Liu, Wenchen Gong, et al. "PHF8-promoted TOPBP1 demethylation drives ATR activation and preserves genome stability." Science Advances 7, no. 19 (May 2021): eabf7684. http://dx.doi.org/10.1126/sciadv.abf7684.
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The checkpoint kinase ATR [ATM (ataxia-telangiectasia mutated) and rad3-related] is a master regulator of DNA damage response. Yet, how ATR activity is regulated remains to be investigated. We report here that histone demethylase PHF8 (plant homeodomain finger protein 8) plays a key role in ATR activation and replication stress response. Mechanistically, PHF8 interacts with and demethylates TOPBP1 (DNA topoisomerase 2-binding protein 1), an essential allosteric activator of ATR, under unperturbed conditions, but replication stress results in PHF8 phosphorylation and dissociation from TOPBP1. Consequently, hypomethylated TOPBP1 facilitates RAD9 (RADiation sensitive 9) binding and chromatin loading of the TOPBP1-RAD9 complex to fully activate ATR and thus safeguard the genome and protect cells against replication stress. Our study uncovers a demethylation and phosphorylation code that controls the assembly of TOPBP1-scaffolded protein complex, and provides molecular insight into non-histone methylation switch in ATR activation.
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HACKENG, Tilman M., Guido TANS, Stefan J. KOPPELMAN, Philip G. de GROOT, Jan ROSING, and Bonno N. BOUMA. "Protein C activation on endothelial cells by prothrombin activation products generated in situ: meizothrombin is a better protein C activator than α-thrombin." Biochemical Journal 319, no. 2 (October 15, 1996): 399–405. http://dx.doi.org/10.1042/bj3190399.
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The conversion of protein C into activated protein C (APC) by the thrombin-thrombomodulin complex on the surface of endothelial cells initiates an essential negative feedback reaction on blood coagulation. APC, together with its non-enzymic cofactor protein S, inactivates factors Va and VIIIa, the non-enzymic protein cofactors of the prothrombinase and intrinsic tenase complex, by proteolytic degradation. In this study we report that prothrombin activation products, generated by the prothrombinase complex on the surface of quiescent endothelial cells, are able to activate protein C. Subsequent inactivation of factor Va by the APC that was formed decreased the rate of prothrombin activation, thus demonstrating in vitro the negative feedback loop on coagulation factor activation. The anticoagulant feedback reaction of APC on the prothrombinase complex was stimulated 3–4-fold by the addition of protein S but not by thrombin-cleaved protein S or by protein S complexed with C4b-binding protein. Stimulation of endothelial cells with 50 pM tumour necrosis factor (TNF) or 500 pM interleukin 1 (IL-1) resulted in a 70% decrease in activation of protein C by exogenously added α-thrombin, which seemed to be due to down-regulation of thrombomodulin activity on the surface of endothelial cells. However, when prothrombin activation products generated in situ were allowed to activate protein C, stimulation of endothelial cells with TNF and IL-1 resulted in only a 25% decrease in activation of protein C. Stimulation with TNF or IL-1 did not affect the ability of endothelial cells to support prothrombinase activity. We investigated whether the differences in extent of protein C activation by exogenously added α-thrombin and by prothrombin activation products generated in situ were due to meizothrombin formed during prothrombin activation. Previous reports from our groups revealed that meizothrombin is generated as a transient intermediate during prothrombin activation on phospholipid vesicles and endothelial cells. Here we show that meizothrombin is at least a 6-fold better activator of protein C on the surface of endothelial cells than is α-thrombin. These results demonstrate that meizothrombin, formed during the initial phase of prothrombin activation, efficiently down-regulates both its own formation and that of thrombin.
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Bonif, Marianne, Marie-Alice Meuwis, Pierre Close, Valérie Benoit, Karen Heyninck, Jean-Paul Chapelle, Vincent Bours, et al. "TNFα- and IKKβ-mediated TANK/I-TRAF phosphorylation: implications for interaction with NEMO/IKKγ and NF-κB activation." Biochemical Journal 394, no. 3 (February 24, 2006): 593–603. http://dx.doi.org/10.1042/bj20051659.
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Pro-inflammatory cytokines trigger signalling cascades leading to NF-κB (nuclear factor-κB)-dependent gene expression through IKK [IκB (inhibitory κB) kinase]-dependent phosphorylation and subsequent degradation of the IκB proteins and via induced phosphorylation of p65. These signalling pathways rely on sequentially activated kinases which are assembled by essential and non-enzymatic scaffold proteins into functional complexes. Here, we show that the pro-inflammatory cytokine TNFα (tumour necrosis factor α) promotes TANK [TRAF (TNF receptor-associated factor) family member associated NF-κB activator] recruitment to the IKK complex via a newly characterized C-terminal zinc finger. Moreover, we show that TANK is phosphorylated by IKKβ upon TNFα stimulation and that this modification negatively regulates TANK binding to NEMO (NF-κB essential modulator). Interestingly, reduced TANK expression by RNA interference attenuates TNFα-mediated induction of a subset of NF-κB target genes through decreased p65 transactivation potential. Therefore the scaffold protein TANK is required for the cellular response to TNFα by connecting upstream signalling molecules to the IKKs and p65, and its subsequent IKKβ-mediated phosphorylation may be a mechanism to terminate the TANK-dependent wave of NF-κB activation.
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Kim, Mihwa, Liza Morales, Ik-Soon Jang, Yong-Yeon Cho, and Dae Kim. "Protein Tyrosine Phosphatases as Potential Regulators of STAT3 Signaling." International Journal of Molecular Sciences 19, no. 9 (September 11, 2018): 2708. http://dx.doi.org/10.3390/ijms19092708.
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The signal transducer and activator of transcription 3 (STAT3) protein is a major transcription factor involved in many cellular processes, such as cell growth and proliferation, differentiation, migration, and cell death or cell apoptosis. It is activated in response to a variety of extracellular stimuli including cytokines and growth factors. The aberrant activation of STAT3 contributes to several human diseases, particularly cancer. Consequently, STAT3-mediated signaling continues to be extensively studied in order to identify potential targets for the development of new and more effective clinical therapeutics. STAT3 activation can be regulated, either positively or negatively, by different posttranslational mechanisms including serine or tyrosine phosphorylation/dephosphorylation, acetylation, or demethylation. One of the major mechanisms that negatively regulates STAT3 activation is dephosphorylation of the tyrosine residue essential for its activation by protein tyrosine phosphatases (PTPs). There are seven PTPs that have been shown to dephosphorylate STAT3 and, thereby, regulate STAT3 signaling: PTP receptor-type D (PTPRD), PTP receptor-type T (PTPRT), PTP receptor-type K (PTPRK), Src homology region 2 (SH-2) domain-containing phosphatase 1(SHP1), SH-2 domain-containing phosphatase 2 (SHP2), MEG2/PTP non-receptor type 9 (PTPN9), and T-cell PTP (TC-PTP)/PTP non-receptor type 2 (PTPN2). These regulators have great potential as targets for the development of more effective therapies against human disease, including cancer.
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MATSUURA, Kazuya, Yoshiyuki TAMADA, Yoshihiro DEYASHIKI, Yoshiyuki MIYABE, Masayuki NAKANISHI, Isao OHYA, and Akira HARA. "Activation of human liver 3α-hydroxysteroid dehydrogenase by sulphobromophthalein." Biochemical Journal 313, no. 1 (January 1, 1996): 179–84. http://dx.doi.org/10.1042/bj3130179.
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Human liver contains at least two isoenzymes (DD2 and DD4) of 3α-hydroxysteroid/dihydrodiol dehydrogenase. The NADP(H)-linked oxidoreductase activities of DD4 were activated more than 4-fold by sulphobromophthalein at concentrations above 20 μM and under physiological pH conditions. Sulphobromophthalein did not stimulate the activities of DD2 and human liver aldehyde reductase, which are functionally and/or structurally related to DD4. No stimulatory effect on the activity of DD4 was observed with other organic anions such as Indocyanine Green, haematin and Rose Bengal. The binding of sulphobromophthalein to DD4 was instantaneous and reversible, and was detected by fluorescence and ultrafiltration assays. The activation by sulphobromophthalein decreased the activation energy in the dehydrogenation reaction for the enzyme, and increased both kcat. and Km values for the coenzymes and substrates. Kinetic analyses with respect to concentrations of NADP+ and (S)-(+)-indan-1-ol indicated that sulphobromophthalein was a non-essential activator of mixed type showing a dissociation constant of 2.6 μM. Thus, the human 3α-hydroxysteroid dehydrogenase isoenzyme has a binding site specific to sulphobromophthalein, and the hepatic metabolism mediated by this isoenzyme may be influenced when this drug is administered.
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WACHTEL, Marco, Karl FREI, Elisabeth EHLER, Christian BAUER, Max GASSMANN, and Sergio M. GLOOR. "Extracellular signal-regulated protein kinase activation during reoxygenation is required to restore ischaemia-induced endothelial barrier failure." Biochemical Journal 367, no. 3 (November 1, 2002): 873–79. http://dx.doi.org/10.1042/bj20020746.
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During an ischaemic insult, oedema formation occurs as a consequence of increased vascular permeability. To study mechanisms leading to vascular barrier failure, endothelial cells were exposed to ischaemia (1% O2 in serum- and glucose-free medium) for 5h. In in vitro conditions, ischaemia increased paracellular permeability, disassembled actin stress fibres, displaced focal adhesion kinase (FAK) from focal adhesions and enhanced cytoskeletal association of occludin. Reoxygenation restored paracellular barrier function, actin organization and FAK distribution. The mitogen-activated protein kinase (MAPK)/extracellular signal-regulated protein kinase (ERK) was rapidly activated after 30min, strongly inhibited after 5h of continuous ischaemia and reactivated 3 times more than control during reoxygenation. Inhibition of ERK activation during reoxygenation with U0126, an inhibitor of the ERK activator, MAPK/ERK kinase 1/2, prevented both barrier restoration and stress-fibre formation, but did not prevent recruitment of FAK to focal contacts. Under normoxic conditions, ERK inhibition led to barrier failure and disassembly of stress fibres only in the absence of serum. These results demonstrate that ERK activity is essential to rebuild a disrupted endothelial barrier after ischaemia and to maintain barrier function in cells exposed to non-ischaemic stress.
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Guertin, Michael J., Xuesen Zhang, Scott A. Coonrod, and Gordon L. Hager. "Transient Estrogen Receptor Binding and p300 Redistribution Support a Squelching Mechanism for Estradiol-Repressed Genes." Molecular Endocrinology 28, no. 9 (September 1, 2014): 1522–33. http://dx.doi.org/10.1210/me.2014-1130.
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Proper gene regulation is essential for proper organismal development and appropriate responses to external stimuli. Specialized factors, termed master regulators, are often responsible for orchestrating the molecular events that result from signaling cascades. Master regulators coordinate the activation and repression of specific gene classes. Estrogen receptor α (ER) precipitates the signaling cascade that results from endogenous or exogenous estrogen hormones. ER is a classic transcriptional activator and the mechanisms by which ER coordinates gene activation are well characterized. However, it remains unclear how ER coordinates the immediate repression of genes. We integrated genomic transcription, chromosome looping, transcription factor binding, and chromatin structure data to analyze the molecular cascade that results from estradiol (E2)-induced signaling in human MCF-7 breast cancer cells and addressed the context-specific nature of gene regulation. We defined a class of genes that are immediately repressed upon estrogen stimulation, and we compared and contrasted the molecular characteristics of these repressed genes vs activated and unregulated genes. The most striking and unique feature of the repressed gene class is transient binding of ER at early time points after estrogen stimulation. We also found that p300, a coactivator and acetyltransferase, quantitatively redistributes from non-ER enhancers to ER enhancers after E2 treatment. These data support an extension of the classic physiological squelching model, whereby ER hijacks coactivators from repressed genes and redistributes the coactivators to ER enhancers that activate transcription.
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Richter, HE, T. Albrektsen, and N. Billestrup. "The role of signal transducer and activator of transcription 5 in the inhibitory effects of GH on adipocyte differentiation." Journal of Molecular Endocrinology 30, no. 2 (April 1, 2003): 139–50. http://dx.doi.org/10.1677/jme.0.0300139.
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GH inhibits primary rat preadipocyte differentiation and expression of late genes required for terminal differentiation. Here we show that GH-mediated inhibition of fatty acid-binding protein aP2 gene expression correlates with the activation of the Janus kinase-2/signal transducer and activator of transcription (STAT)-5 signalling pathway. Within minutes of treatment, GH induced the tyrosine phosphorylation, nuclear localization and DNA binding of STAT5. Importantly, there was no evidence that STAT5 acted via an interaction with peroxisome proliferator-activated receptor gamma. To further understand the mechanism of STAT5 action, we reconstituted the inhibition of aP2 in a non-adipogenic cell line. Using this system, we showed that the ability of GH to inhibit a 520 bp aP2 reporter was largely dependent upon the presence of either STAT5A or STAT5B. Mutant analysis confirmed that the tyrosine phosphorylation of STAT5 was essential for this signalling. However, STAT5's C-terminal transactivation domain was fully dispensable for this inhibition. Taken together, these data confirm a key regulatory role of STAT5 in adipose tIssue and point to STAT5 as the repressing modulator of GH-mediated inhibition in primary preadipocytes.
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Alexandre, C., M. Lecourtois, and J. Vincent. "Wingless and Hedgehog pattern Drosophila denticle belts by regulating the production of short-range signals." Development 126, no. 24 (December 15, 1999): 5689–98. http://dx.doi.org/10.1242/dev.126.24.5689.
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The secreted proteins Wingless and Hedgehog are essential to the elaboration of the denticle pattern in the epidermis of Drosophila embryos. We show that signaling by Wingless and Hedgehog regulates the expression of veinlet (rhomboid) and Serrate, two genes expressed in prospective denticle belts. Thus, Serrate and veinlet (rhom) partake in the last layer of the segmentation cascade. Ultimately, Wingless, Hedgehog, Veinlet (an indirect activator of the Egfr) and Serrate (an activator of Notch) are expressed in non-overlapping narrow stripes. The interface between any two stripes allows a reliable prediction of individual denticle types and polarity suggesting that contact-dependent signaling modulates individual cell fates. Attributes of a morphogen can be ascribed to Hedgehog in this system. However, no single morphogen organises the whole denticle pattern.
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Nishimura, Wataru, Therese Salameh, Takuma Kondo, and Arun Sharma. "Regulation of insulin gene expression by overlapping DNA-binding elements." Biochemical Journal 392, no. 1 (November 8, 2005): 181–89. http://dx.doi.org/10.1042/bj20050970.
Full textAbstract:
The transcription factor MafA/RIPE3b1 is an important regulator of insulin gene expression. MafA binds to the insulin enhancer element RIPE3b (C1-A2), now designated as insulin MARE (Maf response element). The insulin MARE element shares an overlapping DNA-binding region with another insulin enhancer element A2. A2.2, a β-cell-specific activator, like the MARE-binding factor MafA, binds to the overlapping A2 element. Our previous results demonstrated that two nucleotides in the overlapping region are required for the binding of both factors. Surprisingly, instead of interfering with each other's binding activity, the MafA and the A2-binding factors co-operatively activated insulin gene expression. To understand the molecular mechanisms responsible for this functional co-operation, we have determined the nucleotides essential for the binding of the A2.2 factor. Using this information, we have constructed non-overlapping DNA-binding elements and their derivatives, and subsequently analysed the effect of these modifications on insulin gene expression. Our results demonstrate that the overlapping binding site is essential for maximal insulin gene expression. Furthermore, the overlapping organization is critical for MafA-mediated transcriptional activation, but has a minor effect on the activity of A2-binding factors. Interestingly, the binding affinities of both MafA and A2.2 to the overlapping or non-overlapping binding sites were not significantly different, implying that the overlapping binding organization may increase the activation potential of MafA by physical/functional interactions with A2-binding factors. Thus our results demonstrate a novel mechanism for the regulation of MafA activity, and in turn β-cell function, by altering expression and/or binding of the A2.2 factor. Our results further suggest that the major downstream targets of MafA will in addition to the MARE element have a binding site for the A2.2 factor.
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Reimer, Raylene A. "Meat hydrolysate and essential amino acid-induced glucagon-like peptide-1 secretion, in the human NCI-H716 enteroendocrine cell line, is regulated by extracellular signal-regulated kinase1/2 and p38 mitogen-activated protein kinases." Journal of Endocrinology 191, no. 1 (October 2006): 159–70. http://dx.doi.org/10.1677/joe.1.06557.
Full textAbstract:
Glucagon-like peptide-1 (GLP-1) is a potent insulin secretagogue released from L-cells in the intestine. Meat hydrolysate (MH) is a powerful activator of GLP-1 secretion in the human enteroendocrine NCI-H716 cell line, but the mechanisms involved in nutrient-stimulated GLP-1 secretion are poorly understood. The objective of this study was to characterize the intracellular signalling pathways regulating MH- and amino acid-induced GLP-1 secretion. Individually, the pharmacological inhibitors, SB203580 (inhibitor of p38 mitogen-activated protein kinase (MAPK)), wortmannin (inhibitor of phosphatidyl inositol 3-kinase) and U0126 (inhibitor of mitogen activated or extracellular signal-regulated protein kinase (MEK1/2) upstream of extracellular signal-regulated kinase (ERK)1/2) all inhibited MH-induced GLP-1 secretion. Further examination of the MAPK pathway showed that MH increased the phosphorylation of ERK1/2, but not p38 or c-Jun N-terminal kinase over 2–15 min. Incubation with SB203580 resulted in a decrease in phosphorylated p38 MAPK and a concomitant increase in the phosphorylation of ERK1/2. Phosphorylation of ERK1/2 was augmented by co-incubation of MH with SB203580. Inhibitors of protein kinase A and protein kinase C did not inhibit MH-induced GLP-1 secretion. In contrast to non-essential amino acids, essential amino acids (EAAs) increased GLP-1 secretion and similar to MH, activated ERK1/2. However, they also activated p38-suggesting type of protein may affect GLP-1 secretion. In conclusion, there appears to be a crosstalk between p38 and ERK1/2 MAPK in the human enteroendocrine cell with the activation of ERK1/2 common to both MH and EAA. Understanding the cellular pathways involved in nutrient-stimulated GLP-1 secretion has important implications for the design of new treatments aimed at increasing endogenous GLP-1 release in type-2 diabetes and obesity.
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Kim, Min Young, Bowen Yan, Suming Huang, and Yi Qiu. "Regulating the Regulators: The Role of Histone Deacetylase 1 (HDAC1) in Erythropoiesis." International Journal of Molecular Sciences 21, no. 22 (November 11, 2020): 8460. http://dx.doi.org/10.3390/ijms21228460.
Full textAbstract:
Histone deacetylases (HDACs) play important roles in transcriptional regulation in eukaryotic cells. Class I deacetylase HDAC1/2 often associates with repressor complexes, such as Sin3 (Switch Independent 3), NuRD (Nucleosome remodeling and deacetylase) and CoREST (Corepressor of RE1 silencing transcription factor) complexes. It has been shown that HDAC1 interacts with and modulates all essential transcription factors for erythropoiesis. During erythropoiesis, histone deacetylase activity is dramatically reduced. Consistently, inhibition of HDAC activity promotes erythroid differentiation. The reduction of HDAC activity not only results in the activation of transcription activators such as GATA-1 (GATA-binding factor 1), TAL1 (TAL BHLH Transcription Factor 1) and KLF1 (Krüpple-like factor 1), but also represses transcription repressors such as PU.1 (Putative oncogene Spi-1). The reduction of histone deacetylase activity is mainly through HDAC1 acetylation that attenuates HDAC1 activity and trans-repress HDAC2 activity through dimerization with HDAC1. Therefore, the acetylation of HDAC1 can convert the corepressor complex to an activator complex for gene activation. HDAC1 also can deacetylate non-histone proteins that play a role on erythropoiesis, therefore adds another layer of gene regulation through HDAC1. Clinically, it has been shown HDACi can reactivate fetal globin in adult erythroid cells. This review will cover the up to date research on the role of HDAC1 in modulating key transcription factors for erythropoiesis and its clinical relevance.
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Lee, Kwangwon, Jay C. Dunlap, and Jennifer J. Loros. "Roles for WHITE COLLAR-1 in Circadian and General Photoperception inNeurospora crassa." Genetics 163, no. 1 (January 1, 2003): 103–14. http://dx.doi.org/10.1093/genetics/163.1.103.
Full textAbstract:
AbstractThe transcription factors WHITE COLLAR-1 (WC-1) and WHITE COLLAR-2 (WC-2) interact to form a heterodimeric complex (WCC) that is essential for most of the light-mediated processes in Neurospora crassa. WCC also plays a distinct non-light-related role as the transcriptional activator in the FREQUENCY (FRQ)/WCC feedback loop that is central to the N. crassa circadian system. Although an activator role was expected for WC-1, unanticipated phenotypes resulting from some wc-1 alleles prompted a closer examination of an allelic series for WC-1 that has uncovered roles for this central regulator in constant darkness and in response to light. We analyzed the phenotypes of five different wc-1 mutants for expression of FRQ and WC-1 in constant darkness and following light induction. While confirming the absolute requirement of WC-1 for light responses, the data suggest multiple levels of control for light-regulated genes.
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Polonio-Alcalá, Emma, Sònia Palomeras, Daniel Torres-Oteros, Joana Relat, Marta Planas, Lidia Feliu, Joaquim Ciurana, Santiago Ruiz-Martínez, and Teresa Puig. "Fatty Acid Synthase Inhibitor G28 Shows Anticancer Activity in EGFR Tyrosine Kinase Inhibitor Resistant Lung Adenocarcinoma Models." Cancers 12, no. 5 (May 19, 2020): 1283. http://dx.doi.org/10.3390/cancers12051283.
Full textAbstract:
Epidermal growth factor receptor (EGFR) tyrosine kinases inhibitors (TKIs) are effective therapies for non-small cell lung cancer (NSCLC) patients whose tumors harbor an EGFR activating mutation. However, this treatment is not curative due to primary and secondary resistance such as T790M mutation in exon 20. Recently, activation of transducer and activator of transcription 3 (STAT3) in NSCLC appeared as an alternative resistance mechanism allowing cancer cells to elude the EGFR signaling. Overexpression of fatty acid synthase (FASN), a multifunctional enzyme essential for endogenous lipogenesis, has been related to resistance and the regulation of the EGFR/Jak2/STAT signaling pathways. Using EGFR mutated (EGFRm) NSCLC sensitive and EGFR TKIs’ resistant models (Gefitinib Resistant, GR) we studied the role of the natural polyphenolic anti-FASN compound (−)-epigallocatechin-3-gallate (EGCG), and its derivative G28 to overcome EGFR TKIs’ resistance. We show that G28’s cytotoxicity is independent of TKIs’ resistance mechanisms displaying synergistic effects in combination with gefitinib and osimertinib in the resistant T790M negative (T790M−) model and showing a reduction of activated EGFR and STAT3 in T790M positive (T790M+) models. Our results provide the bases for further investigation of G28 in combination with TKIs to overcome the EGFR TKI resistance in NSCLC.
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Fontes, R., J. M. Ribeiro, and A. Sillero. "Inhibition and activation of enzymes. The effect of a modifier on the reaction rate and on kinetic parameters." Acta Biochimica Polonica 47, no. 1 (March 31, 2000): 233–57. http://dx.doi.org/10.18388/abp.2000_4082.
Full textAbstract:
A combined analysis of enzyme inhibition and activation is presented, based on a rapid equilibrium model assumption in which one molecule of enzyme binds one molecule of substrate (S) and/or one molecule of a modifier X. The modifier acts as activator (essential or non-essential), as inhibitor (total or partial), or has no effect on the reaction rate (v), depending on the values of the equilibrium constants, the rate constants of the limiting velocity steps, and the concentration of substrate ([S]). Different possibilities have been analyzed from an equation written to emphasize that v = f([X]) is, in general and at a fixed [S], a hyperbolic function. Formulas for Su (the value of [S], different from zero, at which v is unaffected by the modifier) and v(su) (v at that particular [S]) were deduced. In Lineweaver-Burk plots, the straight lines related to different [X] generally cross in a point (P) with coordinates (Su, v(su)). In certain cases, point P is located in the first quadrant which implies that X acts as activator, as inhibitor, or has no effect, depending on [S]. Furthermore, we discuss: (1) the apparent Vmax and Km displayed by the enzyme in different situations; (2) the degree of effect (inhibition or activation) observed at different concentrations of substrate and modifier; (3) the concept of Ke, a parameter that depends on the concentration of substrate and helps to evaluate the effect of the modifier: it equals the value of [X] at which the increase or decrease in the reaction rate is half of that achieved at saturating [X]. Equations were deduced for the general case and for particular situations, and used to obtain computer-drawn graphs that are presented and discussed. Formulas for apparent Vmax, Km and Ke have been written in a way making it evident that these parameters can be expressed as pondered means.
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Sundaram, Kumaran, Srinivasan Shanmugarajan, D. Sudhaker Rao, and Sakamuri V. Reddy. "Mutant p62P392L Stimulation of Osteoclast Differentiation in Paget's Disease of Bone." Endocrinology 152, no. 11 (August 30, 2011): 4180–89. http://dx.doi.org/10.1210/en.2011-1225.
Full textAbstract:
Paget's disease of the bone (PDB) is an autosomal dominant trait with genetic heterogeneity, characterized by abnormal osteoclastogenesis. Sequestosome 1 (p62) is a scaffold protein that plays an important role in receptor activator of nuclear factor κB (RANK) signaling essential for osteoclast (OCL) differentiation. p62P392L mutation in the ubiquitin-associated (UBA) domain is widely associated with PDB; however, the mechanisms by which p62P392L stimulate OCL differentiation in PDB are not completely understood. Deubiquitinating enzyme cylindromatosis (CYLD) has been shown to negatively regulate RANK ligand-RANK signaling essential for OCL differentiation. Here, we report that CYLD binds with the p62 wild-type (p62WT), non-UBA mutant (p62A381V) but not with the UBA mutant (p62P392L) in OCL progenitor cells. Also, p62P392L induces expression of c-Fos (2.8-fold) and nuclear factor of activated T cells c1 (6.0-fold) transcription factors critical for OCL differentiation. Furthermore, p62P392L expression results in accumulation of polyubiquitinated TNF receptor-associated factor (TRAF)6 and elevated levels of phospho-IκB during OCL differentiation. Retroviral transduction of p62P392L/CYLD short hairpin RNA significantly increased TRAP positive multinucleated OCL formation/bone resorption activity in mouse bone marrow cultures. Thus, the p62P392L mutation abolished CYLD interaction and enhanced OCL development/bone resorption activity in PDB.
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Bowman, Teresa V., Rosannah C. Cameron, Kathryn S. Potts, Mia McKinstry, Varun Gupta, and Xiaoying Bai. "Sf3b1 Regulation of Jak/Stat Signaling Is Essential for Hematopoietic Stem Cell Formation." Blood 132, Supplement 1 (November 29, 2018): 1268. http://dx.doi.org/10.1182/blood-2018-99-118158.
Full textAbstract:
Abstract Hematopoietic stem cells (HSCs) maintain the hematopoietic system throughout the lifetime of an organism. During embryonic development, HSCs emerge through an endothelial-to-hematopoietic transition (EHT) from specialized hemogenic endothelial (HE) cells in the dorsal aorta. HSC fate specification depends on gene expression, which is the culmination of coordinated transcription, RNA splicing, and translation. Although transcriptional regulation of HSC fate choice is well studied, the regulatory role of RNA splicing in this process is poorly understood. Using zebrafish loss-of-function mutants for the spliceosomal component splicing factor 3b, subunit 1 (sf3b1), we identified that impaired splicing hindered HSC production. Surprisingly, we found that this constitutive splicing factor selectively regulates the fate of hemogenic endothelium while leaving the identity of closely-related non-hemogenic endothelium unperturbed. To identify Sf3b1-regulated transcripts important in EHT, we performed RNA-sequencing on purified kdrl:gfp+ endothelial cells from sf3b1 mutant and wild-type siblings at 24 hpf. Approximately 900 genes were mis-spliced, 144 of which were differentially expressed. Ingenuity Pathway Analysis identified Janus Kinase (Jak)/Signaling Transducer and Activator of Transcription (Stat) signaling, in particular Stat3, as one of the top perturbed pathways in the mis-spliced gene set. Stat3 is a transcription factor activated in response to several cytokine and inflammatory signals. To determine if altered splicing of stat3 was critical for HSC formation, we injected antisense splice-blocking morpholinos (MO) targeting the Sf3b1-sensitive stat3 exon19 into wild-type and sf3b1 heterozygous embryos, which normally generate equivalent levels of HSCs. We observed an impairment of HSC production in stat3 morpholino-injected sf3b1 heterozygotes, but not wild-type siblings, indicating a synthetic lethal interaction between sf3b1 and stat3. We also found that overexpression of a constitutively active form of Stat3 significantly suppressed the HSC defects in sf3b1 homozygous mutants. Together, these data indicate that Sf3b1-mediated splicing regulation of the Jak/Stat pathway is critical for HSC emergence. Disclosures No relevant conflicts of interest to declare.
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Osuegbu, Osborne Ikechuckwu, Foluke Olukemi Adeniji, Golden Chukwuemeka Owhonda, Rogers Bariture Kanee, and Eric Osamudiamwen Aigbogun. "Exploring the Essential Stroke Care Structures in Tertiary Healthcare Facilities in Rivers State, Nigeria." INQUIRY: The Journal of Health Care Organization, Provision, and Financing 59 (January 2022): 004695802110679. http://dx.doi.org/10.1177/00469580211067939.
Full textAbstract:
This study evaluated the essential stroke care structure available in the two Tertiary Health Facilities in Rives State, Nigeria. This was a descriptive survey involving the Stroke Care Survey and Assessment Tool (checklist/questionnaire) developed by the World Stroke Organisation to obtain information about the available essential stroke care structure (facilities, equipment, personnel and management protocol) at the two tertiary health facilities (RSUTH & UPTH). The study gathered relevant information, which was summarised into tables and graphs using Microsoft Excel 2016. From the results, although facilities had A and E departments, dedicated stroke units (fixed or mobile) were unavailable, and there was no locally developed protocol to support rapid triage of stroke patients. The facilities and equipment were either unavailable or insufficient. Only one health facility (RSUTH) provided 24 hrs/7 days laboratory services. The workforces were a mix between regular clinical staff and some specialists. Tissue plasminogen activator (tPA) use was non-existent, though specialists were trained on its administration. There was no locally developed or adopted stroke-specific clinical guidelines. In conclusion, the structural services available for stroke care within the studied tertiary health facilities were poor, unavailable or grossly insufficient. The state facility (RSUTH) suffered the most in terms of unavailable national support and staff development.
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Yuan, Meng, Iain W. McNae, Yiyuan Chen, Elizabeth A. Blackburn, Martin A. Wear, Paul A. M. Michels, Linda A. Fothergill-Gilmore, Ted Hupp, and Malcolm D. Walkinshaw. "An allostatic mechanism for M2 pyruvate kinase as an amino-acid sensor." Biochemical Journal 475, no. 10 (May 31, 2018): 1821–37. http://dx.doi.org/10.1042/bcj20180171.
Full textAbstract:
We have tested the effect of all 20 proteinogenic amino acids on the activity of the M2 isoenzyme of pyruvate kinase (M2PYK) and show that, within physiologically relevant concentrations, phenylalanine, alanine, tryptophan, methionine, valine, and proline act as inhibitors, while histidine and serine act as activators. Size exclusion chromatography has been used to show that all amino acids, whether activators or inhibitors, stabilise the tetrameric form of M2PYK. In the absence of amino-acid ligands an apparent tetramer–monomer dissociation Kd is estimated to be ∼0.9 µM with a slow dissociation rate (t1/2 ∼ 15 min). X-ray structures of M2PYK complexes with alanine, phenylalanine, and tryptophan show the M2PYK locked in an inactive T-state conformation, while activators lock the M2PYK tetramer in the active R-state conformation. Amino-acid binding in the allosteric pocket triggers rigid body rotations (11°) stabilising either T or R states. The opposing inhibitory and activating effects of the non-essential amino acids serine and alanine suggest that M2PYK could act as a rapid-response nutrient sensor to rebalance cellular metabolism. This competition at a single allosteric site between activators and inhibitors provides a novel regulatory mechanism by which M2PYK activity is finely tuned by the relative (but not absolute) concentrations of activator and inhibitor amino acids. Such ‘allostatic’ regulation may be important in metabolic reprogramming and influencing cell fate.
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van der Velden, Gisela J., Monique A. Vink, Ben Berkhout, and Atze T. Das. "Tat has a dual role in simian immunodeficiency virus transcription." Journal of General Virology 93, no. 10 (October 1, 2012): 2279–89. http://dx.doi.org/10.1099/vir.0.044511-0.
Full textAbstract:
Tat has a pivotal role in human and simian immunodeficiency virus (HIV and SIV) replication because it stimulates transcription by binding to the trans-activator response (TAR) element. In addition, several other Tat functions have been proposed. Most studies have focused on HIV-1 Tat and much less is known about SIV Tat. An SIVmac239 variant was constructed previously in which the Tat–TAR transcription mechanism is functionally replaced by the doxycycline-inducible Tet-On gene expression mechanism (SIV-rtTA). In this study, SIV-rtTA variants were used to analyse the functions of SIV Tat. It was shown that Tat-minus SIV-rtTA variants replicated efficiently in PM1 T-cells, ruling out an additional essential Tat function. Nevertheless, replication was suboptimal in other cells, and evolutionary pressure to repair Tat expression was documented. It was demonstrated that SIV-rtTA required Tat for optimal gene expression, despite the absence of the Tat–TAR axis. This Tat effect was lost upon replacement of the long terminal repeat promoter region by a non-related promoter. These results indicate that Tat can activate SIV transcription via TAR RNA and U3 DNA elements but has no other essential function in replication in cultured cells. The experiments were limited to cell lines and PBMCs, and did not exclude an accessory Tat function under specific conditions or in vivo.
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Chernov-Rogan, Tania, Tianbo Li, Gang Lu, Henry Verschoof, Kuldip Khakh, Steven W. Jones, Maureen H. Beresini, et al. "Mechanism-specific assay design facilitates the discovery of Nav1.7-selective inhibitors." Proceedings of the National Academy of Sciences 115, no. 4 (January 8, 2018): E792—E801. http://dx.doi.org/10.1073/pnas.1713701115.
Full textAbstract:
Many ion channels, including Nav1.7, Cav1.3, and Kv1.3, are linked to human pathologies and are important therapeutic targets. To develop efficacious and safe drugs, subtype-selective modulation is essential, but has been extremely difficult to achieve. We postulate that this challenge is caused by the poor assay design, and investigate the Nav1.7 membrane potential assay, one of the most extensively employed screening assays in modern drug discovery. The assay uses veratridine to activate channels, and compounds are identified based on the inhibition of veratridine-evoked activities. We show that this assay is biased toward nonselective pore blockers and fails to detect the most potent, selective voltage-sensing domain 4 (VSD4) blockers, including PF-05089771 (PF-771) and GX-936. By eliminating a key binding site for pore blockers and replacing veratridine with a VSD-4 binding activator, we directed the assay toward non–pore-blocking mechanisms and discovered Nav1.7-selective chemical scaffolds. Hence, we address a major hurdle in Nav1.7 drug discovery, and this mechanistic approach to assay design is applicable to Cav3.1, Kv1.3, and many other ion channels to facilitate drug discovery.
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KRETZSCHMAR, Antje K., Michaela C. DINGER, Christian HENZE, Katja BROCKE-HEIDRICH, and Friedemann HORN. "Analysis of Stat3 (signal transducer and activator of transcription 3) dimerization by fluorescence resonance energy transfer in living cells." Biochemical Journal 377, no. 2 (January 15, 2004): 289–97. http://dx.doi.org/10.1042/bj20030708.
Full textAbstract:
Signal transducer and activator of transcription 3 (Stat3) dimerization is commonly thought to be triggered by its tyrosine phosphorylation in response to interleukin-6 (IL-6) or other cytokines. Accumulating evidence from in vitro studies, however, suggests that cytoplasmic Stat3 may be associated with high-molecular-mass protein complexes and/or dimerize prior to its activation. To directly study Stat3 dimerization and subcellular localization upon cytokine stimulation, we used live-cell fluorescence spectroscopy and imaging microscopy combined with fluorescence resonance energy transfer (FRET). Stat3 fusion proteins with spectral variants of green fluorescent protein (GFP), cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) were constructed and expressed in human hepatoma cells (HepG2) and human embryonic kidney cells (HEK-293). Like wild-type Stat3, the fusion proteins redistributed from a preferentially cytoplasmic to nuclear localization upon IL-6 stimulation and supported IL-6-dependent target gene expression. FRET studies in cells co-expressing Stat3–CFP and Stat3–YFP demonstrated that Stat3 dimers exist in the absence of tyrosine phosphorylation. IL-6 induced a 2-fold increase of this basal FRET signal, indicating that tyrosine phosphorylation either increases the dimer/monomer ratio of Stat3 or induces a conformational change of the dimer yielding a higher FRET efficiency. Studies using a mutated Stat3 with a non-functional src-homology 2 (SH2) domain showed that the SH2 domain is essential for dimer formation of phosphorylated as well as non-phosphorylated Stat3. Furthermore, our data show that visualization of normalized FRET signals allow insights into the spatiotemporal dynamics of Stat3 signal transduction.
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Joseph, S., and J. MacDermot. "Thrombin promotes actin polymerization in U937 human monocyte-macrophage cells. Analysis of the signalling mechanisms mediating actin polymerization." Biochemical Journal 286, no. 3 (September 15, 1992): 945–50. http://dx.doi.org/10.1042/bj2860945.
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The U937 human monocyte-macrophage cell line was used to examine the effect of thrombin, an ill-defined chemoattractant, on the polymerization of actin, a process essential for cell motility. In differentiated macrophage-like U937 cells, thrombin (0.5-50 units/ml) caused a rapid dose-dependent increase in the formation of filamentous (F-) actin, detected by the staining of F-actin with the fluorescent toxin, 7-nitrobenz-2-oxa-1,3-diazole-phallacidin. In contrast with other chemoattractants such as N-formylmethionyl-leucylphenylalanine or C5a, actin polymerization in response to thrombin occurred via a pertussis-toxin-insensitive G1-(inhibitory G-protein) independent signalling pathway. Further, this response was not affected by the Ca2+ chelator EGTA or by the specific protein kinase C (PKC) inhibitor RO-31-8220. The response to thrombin was not mimicked by the Ca2+ ionophore ionomycin or by the direct PKC activator phorbol 12-myristate 13-acetate. The thrombin response was, however, inhibited by the non-specific protein kinase inhibitor staurosporine. The present results suggest that in U937 cells thrombin stimulates the formation of F-actin via a signalling pathway independent of (i) the activation of PKC, (ii) the mobilization of intracellular Ca2+ and (iii) the activation of Ca(2+)-dependent protein kinases, but dependent on the activation of an undefined staurosporine-sensitive protein kinase.
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Simões, Filipa B., Margarida C. Quaresma, Luka A. Clarke, Iris AL Silva, Ines Pankonien, Violeta Railean, Arthur Kmit, and Margarida D. Amaral. "TMEM16A chloride channel does not drive mucus production." Life Science Alliance 2, no. 6 (November 15, 2019): e201900462. http://dx.doi.org/10.26508/lsa.201900462.
Full textAbstract:
Airway mucus obstruction is the main cause of morbidity in cystic fibrosis, a disease caused by mutations in the CFTR Cl− channel. Activation of non-CFTR Cl− channels such as TMEM16A can likely compensate for defective CFTR. However, TMEM16A was recently described as a key driver in mucus production/secretion. Here, we have examined whether indeed there is a causal relationship between TMEM16A and MUC5AC production, the main component of respiratory mucus. Our data show that TMEM16A and MUC5AC are inversely correlated during differentiation of human airway cells. Furthermore, we show for the first time that the IL-4–induced TMEM16A up-regulation is proliferation-dependent, which is supported by the correlation found between TMEM16A and Ki-67 proliferation marker during wound healing. Consistently, the notch signaling activator DLL4 increases MUC5AC levels without inducing changes neither in TMEM16A nor in Ki-67 expression. Moreover, TMEM16A inhibition decreased airway surface liquid height. Altogether, our findings demonstrate that up-regulation of TMEM16A and MUC5AC is only circumstantial under cell proliferation, but with no causal relationship between them. Thus, although essential for airway hydration, TMEM16A is not required for MUC5AC production.
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Xu, Jie, Yue Zhang, Philip A. Berry, Jing Jiang, Peter E. Lobie, John F. Langenheim, Wen Y. Chen, and Stuart J. Frank. "Growth Hormone Signaling in Human T47D Breast Cancer Cells: Potential Role for a Growth Hormone Receptor-Prolactin Receptor Complex." Molecular Endocrinology 25, no. 4 (April 1, 2011): 597–610. http://dx.doi.org/10.1210/me.2010-0255.
Full textAbstract:
GH receptor (GHR) and prolactin (PRL) receptor (PRLR) are structurally similar cytokine receptor superfamily members that are highly conserved among species. GH has growth-promoting and metabolic effects in various tissues in vertebrates, including humans. PRL is essential for regulation of lactation in mammals. Recent studies indicate that breast tissue bears GHR and PRLR and that both GH and PRL may impact development or behavior of breast cancer cells. An important facet of human GH (hGH) and human PRL (hPRL) biology is that although hPRL interacts only with hPRLR, hGH binds well to both hGHR and hPRLR. Presently, we investigated potential signaling effects of both hormones in the estrogen receptor- and progesterone receptor-positive human T47D breast cancer cell line. We found that this cell type expresses ample GHR and PRLR and responds well to both hGH and hPRL, as evidenced by activation of the Janus kinase 2/signal transducer and activator of transcription 5 pathway. Immunoprecipitation studies revealed specific GHR-PRLR association in these cells that was acutely enhanced by GH treatment. Although GH caused formation of disulfide-linked and chemically cross-linked GHR dimers in T47D cells, GH preferentially induced tyrosine phosphorylation of PRLR rather than GHR. Notably, both a GHR-specific ligand antagonist (B2036) and a GHR-specific antagonist monoclonal antibody (anti-GHRext-mAb) failed to inhibit GH-induced signal transducer and activator of transcription 5 activation. In contrast, although the non-GHR-specific GH antagonist (G120R) and the PRL antagonist (G129R) individually only partially inhibited GH-induced activation, combined treatment with these two antagonists conferred greater inhibition than either alone. These data indicate that endogenous GHR and PRLR associate (possibly as a GHR-PRLR heterodimer) in human breast cancer cells and that GH signaling in these cells is largely mediated by the PRLR in the context of both PRLR-PRLR homodimers and GHR-PRLR heterodimers, broadening our understanding of how these related hormones and their related receptors may function in physiology and pathophysiology.
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NAKANO, Keiko, Akio MATSUSHITA, Shigekazu SASAKI, Hiroko MISAWA, Kozo NISHIYAMA, Yumiko KASHIWABARA, and Hirotoshi NAKAMURA. "Thyroid-hormone-dependent negative regulation of thyrotropin beta gene by thyroid hormone receptors: study with a new experimental system using CV1 cells." Biochemical Journal 378, no. 2 (March 1, 2004): 549–57. http://dx.doi.org/10.1042/bj20031592.
Full textAbstract:
The molecular mechanism involved in the liganded thyroid hormone receptor suppression of the TSHβ (thyroid-stimulating hormone β, or thyrotropin β) gene transcription is undetermined. One of the main reasons is the limitation of useful cell lines for the experiments. We have developed an assay system using non-pituitary CV1 cells and studied the negative regulation of the TSHβ gene. In CV1 cells, the TSHβ–CAT (chloramphenicol acetyltransferase) reporter was stimulated by Pit1 and GATA2 and suppressed by T3 (3,3´,5-tri-iodothyronine)-bound thyroid hormone receptor. The suppression was dependent on the amounts of T3 and the receptor. Unliganded receptor did not stimulate TSHβ activity, suggesting that the receptor itself is not an activator. Analyses using various receptor mutants revealed that the intact DNA-binding domain is crucial to the TSHβ gene suppression. Co-activators and co-repressors are not necessarily essential, but are required for the full suppression of the TSHβ gene. Among the three receptor isoforms, β2 exhibited the strongest inhibition and its protein level was the most predominant in a thyrotroph cell line, TαT1, in Western blotting. The dominant-negative effects of various receptor mutants measured on the TSHβ–CAT reporter were not simple mirror images of those in the positive regulation under physiological T3 concentration.
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Ni, Shuang, Hami Heal, Jie Xu, Robert Paulson, and Pamela H. Correll. "The Activation of Stat3 by the Stk Receptor Tyrosine Kinase Requires Src Kinases and Gab2, and Is Essential for Friend Virus Induced, Epo-Independent Growth of Primary Erythroid Cells." Blood 104, no. 11 (November 16, 2004): 820. http://dx.doi.org/10.1182/blood.v104.11.820.820.
Full textAbstract:
Abstract Friend virus-induced erythroleukemia is a model for the study of the multi-stage nature of carcinogenesis. The early stage of the disease is characterized by a polyclonal expansion of infected erythroid precursor cells, and the later stage is marked by the emergence of fully transformed cells in the spleen, blood, bone marrow and liver, leading to the development of erythroleukemia. Previous findings have demonstrated that a naturally occurring, N-terminally truncated form of the stk receptor tyrosine kinase (Sf-stk) provides signals necessary for the polycolonal expansion of infected cells. Signal transducer and activator of transcription (Stat) proteins play an important role in normal and malignant hematopoiesis. Moreover it has been shown that growth factors can induce Stat3 in a Src family kinase (SFK)-dependent manner. Here we show that dominant negative Stat3 and c-Src significantly inhibit the BFU-e and CFU-e colony number induced by friend virus. In addition, the non-specific SFK inhibitor PP1 also abrogated Epo-independent colony formation. Moreover we found that the inhibition of SFKs by PP1 suppresses Stat3 tyrosine phosphorylation activated by Sf-stk. To elucidate the signaling mechanism by which Sf-stk activates Stat3, we mutated the docking site tyrosines to phenylalanine. Our results indicated that the mutation of Y436F eliminates Sf-stk-induced Stat3 tyrosine phosphorylation. Studies from our lab have further demonstrated that tyrosine 436 of Sf-stk is essential for the cytokine-independent growth of primary erythroblasts induced by Friend virus, by providing a docking site for Grb2, which recruits Gab2 to induce erythroleukemia. To further determine the relationship between Gab2 and Stat3, we generated fusion proteins, in which Sf-stk lacking the docking site tyrosines is fused to Gab2 or Gab1. The Sf-stk/Gab2, but not Sf-stk/Gab1, fusion protein supported Epo-independent colony formation and induced Stat3 tyrosine phosphorylation. In conclusion, these results suggest that SFKs, Gab2 and Stat3 are all important in Friend virus induced erythroleukemia mediated by Sf-stk, and that Gab2 and SFKs lie upstream of Stat3 in this signaling pathway.
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Ranuncolo, Stella M., Vu Ngo, Georg Lenz, Wenming Xiao, George Wright, and Louis M. Staudt. "OCT2 (Octamer Binding Protein 2): An Essential Role in Germinal Center B Cell Differentiation and the “Achilles Heel” of Derived Lymphomas." Blood 114, no. 22 (November 20, 2009): 362. http://dx.doi.org/10.1182/blood.v114.22.362.362.
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Abstract Abstract 362 To identify genes requiered for the proliferation and survival of Diffuse Large B Cell Lymphomas (DLBCL) we conducted an “Achilles Heel” RNA interference screen in cell lines model of ABC (Activated B cell-like) and GCB (Germinal Center B-cell like) DLBCL subtypes. One of the most toxic small hairpins RNAs (shRNAs) in this screen targeted Oct2, encoding a POU domain transcriptional activator. Unlike Oct1, which is constitutively expressed in many cell types, Oct2 is primarily lymphoid restricted. It was identified by virtue of its ability to bind the highly conserved DNA octamer motif (ATGCAAAT) within immunoglobulin (Ig) genes promoters. The B cell specific co-activator OCA-B interacts with the POU domain of the octamer binding proteins enhancing their transactivation potential. Although Oct2 and OCA-B are not essential for Ig transcription, they are required for germinal center (GC) B cell differentiation. To understand the massive apoptotic cell death of DLBCL cells following shRNA Oct2 induction we investigated the genetic pathways controlled by Oct2. We profiled gene expression changes in DLBCL cell lines after knocking down Oct2 and merged this data set with data from genome-wide assessment of Oct2 and OCA-B binding sites, coupling chromatin immunoprecipitation (ChiP) with high-throughput sequencing technologies (ChIP-Seq). ChIP-Seq uncovered an extensive network of Oct2 target genes in DLBCL cells. More than 60% of the Oct2 target genes also showed OCA-B biding. This Oct2/OCA-B overlaping set of targets was enriched for genes selectively expressed in pan-B cells and GC B cells. We found that Oct2/OCA-B lie upstream many of the main transcription factors known to play an essential role in inducing and mantaining the GC stage of B cell development such as BCL6, MTA3, PU.1, IRF8, SpiB and OCA-B, among others. Oct2/OCA-B target these genes both in DLBCL cells and in normal human primary centroblasts. Strikingly, among Oct-2 downstream effectors, BCL6 cDNA was enough to rescue both ABC and GCB DLBCL cells from the Oct2 shRNA lethal effect. The Oct2/OCA-B binding of BCL6 promoter was confirmed in vivo by single locus ChIP in different GCB-DLBCL cell lines as well as in primary centroblasts isolated from human tonsils. Gel shifts experiments showed Oct2 binding to more than one non canonical octamer motif within the BCL6 promoter. Furthermore, computational analysis of the BCL6 promoter region bound by Oct2, showed PU.1 binding sites. Knocked down of PU.1 decreased Oct2 enrichment and viceversa suggesting cooperative Oct2/PU.1 biding to BCL6 pomoter. ChIP-Seq findings opened an entire and exciting new chapter in the Oct2 biology field. Pou transcription factors were suppossed to regulate the activity of octamer containing promoters. Interestingly, Oct2 binds to and control the expression of many GC specific genes that do not harbor a “canonical octamer” motif. Eventhough Oct2 is expressed throughout the different stages of B cell maturation, both mRNA and protein levels are enhanced in centroblasts as compared to pre-GC B cells. We found Oct2 capable of inducing its own expression as well as Oct-1 and OCA-B. This autoregulatory circuit might partially account for the Oct2 predominant role in GC specific genes expression control. By array cGH, high level amplification of Oct2 and OCA-B was found in less than 10% of non Hodgkin lymphoma patients samples. Nonetheless, these lymphoma cells become addicted to the Oct2 controlled network that sustain cell survival and proliferation, wich turns Oct2 into an attractive therapeutic target for non Hodking lymphoma patients. This critical Oct2 DLBCL cells dependency, is an example of “non oncogene addiction” that we recently defined based on our RNA interference screening. Oct-2 controls a network of regulatory relationships that sustain both normal GC B cells and malignant counterparts. In summary, we showed that Oct2 and OCA-B lie upstream of BCL6, one of the critical regulators of germinal center B cell differentiation. This suggests that Oct2-directed therapy should kill the same DLBCLs as BCL6-directed therapy. Furthermore, all GC and Post-GC B cells that were tested requiere Oct-2 for survival, indicating that Oct2-directed therapy might have a broder activity spectrum than the BCL6-directed therapy. The Oct2/OCA-B binding interface would be amenable to attack with potential manageable toxicity, since this interaction is exclusively required in GC B cells. Disclosures: No relevant conflicts of interest to declare.
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Remoli, Anna L., Giulia Marsili, Edvige Perrotti, Eleonora Gallerani, Ramona Ilari, Filomena Nappi, Aurelio Cafaro, Barbara Ensoli, Riccardo Gavioli, and Angela Battistini. "Intracellular HIV-1 Tat protein represses constitutive LMP2 transcription increasing proteasome activity by interfering with the binding of IRF-1 to STAT1." Biochemical Journal 396, no. 2 (May 15, 2006): 371–80. http://dx.doi.org/10.1042/bj20051570.
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The Tat protein is the transcriptional activator of HIV-1 gene expression, which is not only essential for viral replication, but also important in the complex HIV-induced pathogenesis of AIDS, as both an intracellular and an extracellular released protein. Accordingly, Tat is able to profoundly affect cellular gene expression, regulating several cellular functions, also in non-infected cells. We showed recently that Tat induces modification of immunoproteasomes in that it up-regulates LMP7 (low-molecular-mass polypeptide 7) and MECL1 (multicatalytic endopeptidase complex-like 1) subunits and down-modulates the LMP2 subunit, resulting in a change in the generation and presentation of epitopes in the context of MHC class I. In particular, Tat increases presentation of subdominant and cryptic epitopes. In the present study, we investigated the molecular mechanism responsible for the Tat-induced LMP2 down-regulation and show that intracellular Tat represses transcription of the LMP2 gene by competing with STAT1 (signal transducer and activator of transcription 1) for binding to IRF-1 (interferon-regulatory factor-1) on the overlapping ICS-2 (interferon consensus sequence-2)–GAS (γ-interferon-activated sequence) present in the LMP2 promoter. This element is constitutively occupied in vivo by the unphosphorylated STAT1–IRF-1 complex, which is responsible for the basal transcription of the gene. Sequestration of IRF-1 by intracellular Tat impairs the formation of the complex resulting in lower LMP2 gene transcription and LMP2 protein expression, which is associated with increased proteolytic activity. On the other hand, extracellular Tat induces the expression of LMP2. These effects of Tat provide another effective mechanism by which HIV-1 affects antigen presentation in the context of the MHC class I complex and may have important implications in the use of Tat for vaccination strategies.
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Rousselle, Anthony, Gabin Sihn, Martijn Rotteveel, and Michael Bader. "(Pro)renin receptor and V-ATPase: from Drosophila to humans." Clinical Science 126, no. 8 (December 17, 2013): 529–36. http://dx.doi.org/10.1042/cs20130307.
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A decade ago, the (P)RR [(pro)renin receptor] was discovered and depicted as a potential activator of the tissue renin–angiotensin system. For this reason, the role of the (P)RR in cardiovascular diseases and diabetes has been particularly studied. However, the discovery of embryonic lethality after (P)RR gene deletion in mouse and zebrafish paved the way for additional roles of (P)RR in cell homoeostasis. Indeed, the (P)RR has been shown to associate with vacuolar H+-ATPase, hence its other name ATP6ap2. Developmental studies in Xenopus and Drosophila have revealed an essential role of this association to promote the canonical and non-canonical Wnt signalling pathways, whereas studies with tissue-specific gene deletion have pointed out a role in autophagy. The present review aims to summarize recent findings on the cellular functions of (P)RR emerging from various mutated and transgenic animal models.
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Samarakoon, Rohan, and Paul J. Higgins. "Integration of non-SMAD and SMAD signaling in TGF-β1-induced plasminogen activator inhibitor type-1 gene expression in vascular smooth muscle cells." Thrombosis and Haemostasis 100, no. 12 (2008): 976–83. http://dx.doi.org/10.1160/th08-05-0273.
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SummaryOverexpression of plasminogen activator inhibitor-1 (SER-PINE1, PAI-1), the major physiological inhibitor of pericellular plasmin generation, is a significant causative factor in the progression of vascular disorders (e.g. arteriosclerosis, thrombosis, perivascular fibrosis) as well as a biomarker and a predictor of cardiovascular-disease associated mortality. PAI-1 is a temporal/ spatial regulator of pericellular proteolysis and ECM accumulation impacting, thereby, vascular remodeling, smooth muscle cell migration, proliferation and apoptosis. Within the specific context of TGF-β1-initiated vascular fibrosis and neointima formation, PAI-1 is a member of the most prominently expressed subset of TGF-β1-induced transcripts. Recent findings implicate EGFR/pp60c-src →MEK/ERK1/2 and Rho/ROCK→SMAD2/3 signaling in TGF-β1-stimulated PAI-1 expression in vascular smooth muscle cells. The EGFR is a direct upstream regulator of MEK/ERK1/2 while Rho/ROCK modulate both the duration of SMAD2/3 phosphorylation and nuclear accumulation. E-box motifs (CACGTG) in the PE1/PE2 promoter regions of the human PAI-1 gene, moreover, are platforms for a MAP kinase-directed USF subtype switch (USF-1→USF-2) in response to growth factor addition suggesting that the EGFR→MEK/ERK axis impacts PAI-1 expression, at least partly, through USF-dependent transcriptional controls. This paper reviews recent data suggesting the essential cooperativity among the EGFR→MAP kinase cascade, the Rho/ROCK pathway and SMADs in TGF-β1-initiated PAI-1 expression. The continued clarification of mechanistic controls on PAI-1 transcription may lead to new targeted therapies and clinically-relevant options for the treatment of vascular diseases in which PAI-1 dysregulation is a major underlying pathogenic feature.
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Mellor, G. W., S. K. Sreedharan, D. Kowlessur, E. W. Thomas, and K. Brocklehurst. "Catalytic-site characteristics of the porcine calpain II 80 kDa/18 kDa heterodimer revealed by selective reaction of its essential thiol group with two-hydronic-state time-dependent inhibitors: evidence for a catalytic site Cys/His interactive system and an ionizing modulatory group." Biochemical Journal 290, no. 1 (February 15, 1993): 75–83. http://dx.doi.org/10.1042/bj2900075.
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1. Four calpain II heterodimers (80 kDa/30 kDa, 80 kDa/29 kDa, 80 kDa/26 kDa and 80 kDa/18 kDa) were isolated from fresh porcine kidney by (NH4)2SO4 precipitation, chromatography on DEAE-Sepharose CL-6B and subsequently on Reactive Red 120/agarose followed by f.p.l.c. on a Q-Sepharose Hi-Load 16/10 column. 2. The major component (80 kDa/30 kDa) was used to provide the catalytically active calpain II 80 kDa/18 kDa heterodimer by treatment with CaCl2; titration with trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E64) in the presence of monothioglycerol showed the preparation to have 1.0 +/- 0.05 catalytic sites per molecule of heterodimer. 3. The 80 kDa/30 kDa heterodimer was separated from monothioglycerol and other low-molecular-mass material by gel filtration on Sephadex G-25 without loss of catalytic activity towards sulphanilic acid/azocasein in the presence of added Ca2+. On storage overnight at a concentration of 3 microM in KCl at 4 degrees C in the absence of Ca2+ the activator-free preparation still produced fully active 80 kDa/18 kDa heterodimer on addition of Ca2+. 4. Activator-free 80 kDa/30 kDa heterodimer (in the absence of Ca2+) reacts relatively slowly with ethyl 2-pyridyl disulphide at pH 5.9; over 5000 s five thiol groups per molecule react, all at similar rates. In the presence of 8 mM CaCl2 under otherwise identical conditions (and also in the pH range 3.8-10.4) an initial faster phase of reaction corresponding to approx. one thiol group per molecule of heterodimer is generated, but it is not cleanly separated from the subsequent slower reactions on the stopped-flow trace. This fast phase of reaction does not occur when E64-inactivated calpain II is substituted for active 80 kDa/18 kDa heterodimer. 5. Greatly improved resolution of the fast phase of reaction involving the catalytic-site thiol group was achieved by using 2,2′-dipyridyl disulphide (2-Py-S-S-2-Py) instead of ethyl 2-pyridyl disulphide. 6. The pH-dependence of the second-order rate constant (k) for the reaction of the catalytically active activator-free 80 kDa/18 kDa calpain II heterodimer with 2-Py-S-S-2-Py was studied by stopped-flow spectral analysis in the pH range approx. 3-8 without interference from reactions of other thiol groups. 7. The form of the pH-k profile establishes for the first time the existence of an interactive catalytic site system [probably containing a (Cys)-S-/(His)-Im+H ion pair] analogous to those present in monomeric non-Ca(2+)-activated cysteine proteinases.(ABSTRACT TRUNCATED AT 400 WORDS)
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Hellmuth, Johannes C., Chi-Shuen Chu, Rajat Singh, Lucy A. Skrabanek, Ashley Doane, Olivier Elemento, Robert G. Roeder, and Ari Melnick. "An OCT2 / OCA-B / MEF2B Ternary Complex Controls the Activity and Architecture of an Essential Locus Control Region for Normal and Malignant Germinal Center B-Cells." Blood 134, Supplement_1 (November 13, 2019): 24. http://dx.doi.org/10.1182/blood-2019-130692.
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The transcriptional repressor BCL6 is considered the master regulator of the germinal center (GC) reaction and is a key proto-oncogene in GC-derived lymphoma pathogenesis. At GC initiation, chromatin architecture is dramatically remodeled around a BCL6-associated locus control region (LCR) with presumed enhancer function. The BCL6 LCR is located 150kb upstream of the BCL6 gene and is fundamentally required for GC formation. Herein, we functionally dissect the BCL6 LCR to uncover the crucial genetic elements and transcription factor binding events that drive BCL6 LCR function. We generated a custom gRNA library densely tiling the BCL6 LCR and surrounding regulatory elements. In total, the library covered a genomic region of 316.8kb with 25,698 gRNAs. We used a GC derived diffuse large B-cell lymphoma line, OCI-LY7, stably expressing dCas9-KRAB (CRISPRi) to screen for depletion of gRNAs. Surprisingly, BCL6 LCR function relied on only 4 of 21 constituent enhancers. These 4 essential enhancers were critically required for cell growth and BCL6 expression. In contrast, all other constituent enhancers were completely dispensable for LCR function. These results indicate that the BCL6 LCR is governed by a strong internal hierarchy and pinpoint the crucial genetic elements that drive LCR function. To understand what distinguishes essential enhancers from non-essential enhancers, we interrogated ChIP-seq profiles of key GC transcription factors (TF) and transcriptional co-activators. While most TFs and co-activators bound constituent enhancers indiscriminately, MEF2B specifically bound only to essential enhancers (p=0.01). Interestingly, essential enhancers did not contain MEF2B binding motifs. Furthermore, MEF2B did not bind to essential enhancer DNA in electrophoretic mobility shift (EMSA) and immobilized template assays. De novo motif analysis of MEF2B ChIP-seq data prominently featured the canonical octamer transcription factor motif (p=10-79) indicating a role for OCT2 in the recruitment of MEF2B to essential enhancers. Indeed, MEF2B binding to essential enhancer DNA in EMSA and immobilized template assays was dependent upon the presence of OCT2 and its co-activator OCA-B. We assayed occupancy of OCT2, OCA-B and MEF2B at essential enhancers in OCI-LY7 cells by qChIP and found that each factor required the other two for full binding activity. Furthermore, all three factors were required for BCL6 expression (p<0.001 for each factor). These results indicate that OCT2, OCA-B and MEF2B cooperatively bind to essential enhancer elements and act as an intimately linked ternary complex to drive BCL6 expression through the BCL6 LCR. To elucidate how the OCT2 / OCA-B / MEF2B complex promotes target gene transcription, we performed IP of OCA-B followed by mass spectrometry and found highly significant interactions with the majority of Mediator proteins (p<10-8). We furthermore showed that OCA-B directly and specifically interacts with MED1, through which it recruits the remainder of the Mediator complex and that OCA-B and OCT2 are required to recruit Mediator to essential enhancer elements. The Mediator complex is thought to serve as a drawbridge across enhancers and promoters to facilitate enhancer-promoter looping. Using 3C assays, we found that OCA-B is required for chromatin contacts between the BCL6 promoter and the LCR highlighting its importance in recruiting Mediator. Similarly, essential enhancer elements were crucially required for intact chromatin conformation at the BCL6 locus as determined by 3C. In summary, BCL6 LCR function completely relies on very few but highly essential enhancer elements. OCT2, OCA-B and MEF2B cooperatively bind these essential enhancers forming an intimately linked trimeric complex. By recruiting Mediator, OCA-B provides a direct link to the basal transcriptional machinery. Finally, essential enhancers as well as the OCT2 / OCA-B / MEF2B complex are required for BCL6 expression and intact chromatin conformation at the BCL6 locus - key determinants of the GC B cell state. Disclosures Melnick: Epizyme: Consultancy; Constellation: Consultancy; Janssen: Research Funding.
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Tello-Flores, Vianet Argelia, Fredy Omar Beltrán-Anaya, Marco Antonio Ramírez-Vargas, Brenda Ely Esteban-Casales, Napoleón Navarro-Tito, Luz del Carmen Alarcón-Romero, Carlos Aldair Luciano-Villa, Mónica Ramírez, Óscar del Moral-Hernández, and Eugenia Flores-Alfaro. "Role of Long Non-Coding RNAs and the Molecular Mechanisms Involved in Insulin Resistance." International Journal of Molecular Sciences 22, no. 14 (July 6, 2021): 7256. http://dx.doi.org/10.3390/ijms22147256.
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Long non-coding RNAs (lncRNAs) are single-stranded RNA biomolecules with a length of >200 nt, and they are currently considered to be master regulators of many pathological processes. Recent publications have shown that lncRNAs play important roles in the pathogenesis and progression of insulin resistance (IR) and glucose homeostasis by regulating inflammatory and lipogenic processes. lncRNAs regulate gene expression by binding to other non-coding RNAs, mRNAs, proteins, and DNA. In recent years, several mechanisms have been reported to explain the key roles of lncRNAs in the development of IR, including metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), imprinted maternal-ly expressed transcript (H19), maternally expressed gene 3 (MEG3), myocardial infarction-associated transcript (MIAT), and steroid receptor RNA activator (SRA), HOX transcript antisense RNA (HOTAIR), and downregulated Expression-Related Hexose/Glucose Transport Enhancer (DREH). LncRNAs participate in the regulation of lipid and carbohydrate metabolism, the inflammatory process, and oxidative stress through different pathways, such as cyclic adenosine monophosphate/protein kinase A (cAMP/PKA), phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT), polypyrimidine tract-binding protein 1/element-binding transcription factor 1c (PTBP1/SREBP-1c), AKT/nitric oxide synthase (eNOS), AKT/forkhead box O1 (FoxO1), and tumor necrosis factor-alpha (TNF-α)/c-Jun-N-terminal kinases (JNK). On the other hand, the mechanisms linked to the molecular, cellular, and biochemical actions of lncRNAs vary according to the tissue, biological species, and the severity of IR. Therefore, it is essential to elucidate the role of lncRNAs in the insulin signaling pathway and glucose and lipid metabolism. This review analyzes the function and molecular mechanisms of lncRNAs involved in the development of IR.
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Taylor, Brett, Shayesteh Ferdosi, Rita Bybee, Nanyun Tang, Sen Peng, Krystine Garcia-Mansfield, Victoria David-Dirgo, et al. "EXTH-43. DYNAMIC MULTI-OMICS ANALYSIS OF MLN4924-TREATED GLIOBLASTOMA CELLS REVEALS ABSENCE OF PTEN AS A CANDIDATE DRIVER OF NEDDYLATION INHIBITION RESISTANCE." Neuro-Oncology 21, Supplement_6 (November 2019): vi91. http://dx.doi.org/10.1093/neuonc/noz175.375.
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Abstract Glioblastoma multiforme (GBM) is the most lethal primary brain tumor and calls for novel therapeutic development. The neddylation pathway may be a therapeutic target, as global over activation of neddylation has been found in GBM patients and has correlated with shorter patient survival. Analogous to ubiquitination in reaction scheme and enzyme classes used, neddylation is a post-translational modification that is essential to many protein regulation and biological processes. Although non-cullin NEDD8 substrates have been investigated in recent years, the most well-characterized substrates of neddylation are the cullin subunits of Cullin-RING ligases (CRLs), which act as a scaffold for substrates to be ubiquitinated and degraded. The neddylation inhibitor MLN4924 targets NEDD8 Activating Enzyme (NAE), an upstream activator of neddylation, and induces cell cycle arrest, apoptosis and senescence in cancer cells. In this work, we investigated the context of vulnerability to Pevonedistat (MLN4924) in GBM. Efficacy of MLN4924 in glioma cell models was evaluated by measuring cell viability (CellTiterGlo®), colony formation efficiency, and cell cycle progression (flow cytometry with propidium iodide staining). GB1 (IC50= 0.28 μM), LN18 (IC50 = 0.19 μM), and GBM43 (IC50= 0.45 μM) were established as sensitive and M059K (IC50 = 5.5 μM), SNU1105 (IC50 = 20.9 μM), and GBM39 (IC50= 10.3 μM) as non-sensitive cell lines based on IC50 values. To discover genomic and/or proteomic markers of differential response, we collected RNA and protein from sensitive and non-sensitive cell lines after 0, 2, 8 and 24 hour treatment with MLN4924 for RNA sequencing and mass spectroscopy analyses. Multi-OMICS data point towards the absence of functional PTEN as a driver of MLN4924 resistance. Uncovering the mechanism underlying GBM’s vulnerability to MLN4924 will expand the knowledge of neddylation’s role in cancer and may arm physicians with an understanding of whether a GBM patient would respond to MLN4924 treatment or not.
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Rúa, J., D. de Arriaga, F. Busto, and J. Soler. "Isocitrate lyase from Phycomyces blakesleeanus. The role of Mg2+ ions, kinetics and evidence for two classes of modifiable thiol groups." Biochemical Journal 272, no. 2 (December 1, 1990): 359–67. http://dx.doi.org/10.1042/bj2720359.
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Isocitrate lyase was purified from Phycomyces blakesleeanus N.R.R.L. 1555(-). The native enzyme has an Mr of 240,000. The enzyme appeared to be a tetramer with apparently identical subunits of Mr 62,000. The enzyme requires Mg2+ for activity, and the data suggest that the Mg2(+)-isocitrate complex is the true substrate and that Mg2+ ions act as a non-essential activator. The kinetic mechanism of the enzyme was investigated by using product and dead-end inhibitors of the cleavage and condensation reactions. The data indicated an ordered Uni Bi mechanism and the kinetic constants of the model were calculated. The spectrophotometric titration of thiol groups in Phycomyces isocitrate lyase with 5.5′-dithiobis-(2-nitrobenzoic acid) gave two free thiol groups per subunit of enzyme in the native state and three in the denatured state. The isocitrate lyase was completely inactivated by iodoacetate, with non-linear kinetics. The inactivation data suggest that the enzyme has two classes of modifiable thiol groups. The results are also in accord with the formation of a non-covalent enzyme-inhibitor complex before irreversible modification of the enzyme. Both the equilibrium constants for formation of the complex and the first-order rate constants for the irreversible modification step were determined. The partial protective effect of isocitrate and Mg2+ against iodoacetate inactivation was investigated in a preliminary form.
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Ye, Wenxiu, Eigo Ando, Mohammad Saidur Rhaman, Md Tahjib-Ul-Arif, Eiji Okuma, Yoshimasa Nakamura, Toshinori Kinoshita, and Yoshiyuki Murata. "Inhibition of light-induced stomatal opening by allyl isothiocyanate does not require guard cell cytosolic Ca2+ signaling." Journal of Experimental Botany 71, no. 10 (February 27, 2020): 2922–32. http://dx.doi.org/10.1093/jxb/eraa073.
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Abstract The glucosinolate–myrosinase system is a well-known defense system that has been shown to induce stomatal closure in Brassicales. Isothiocyanates are highly reactive hydrolysates of glucosinolates, and an isothiocyanate, allyl isothiocyanate (AITC), induces stomatal closure accompanied by elevation of free cytosolic Ca2+ concentration ([Ca2+]cyt) in Arabidopsis. It remains unknown whether AITC inhibits light-induced stomatal opening. This study investigated the role of Ca2+ in AITC-induced stomatal closure and inhibition of light-induced stomatal opening. AITC induced stomatal closure and inhibited light-induced stomatal opening in a dose-dependent manner. A Ca2+ channel inhibitor, La3+, a Ca2+chelator, EGTA, and an inhibitor of Ca2+ release from internal stores, nicotinamide, inhibited AITC-induced [Ca2+]cyt elevation and stomatal closure, but did not affect inhibition of light-induced stomatal opening. AITC activated non-selective Ca2+-permeable cation channels and inhibited inward-rectifying K+ (K+in) channels in a Ca2+-independent manner. AITC also inhibited stomatal opening induced by fusicoccin, a plasma membrane H+-ATPase activator, but had no significant effect on fusicoccin-induced phosphorylation of the penultimate threonine of H+-ATPase. Taken together, these results suggest that AITC induces Ca2+ influx and Ca2+ release to elevate [Ca2+]cyt, which is essential for AITC-induced stomatal closure but not for inhibition of K+in channels and light-induced stomatal opening.
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Chen, Si, Jin Tian, Zhijie Li, Hongtao Kang, Jikai Zhang, Jiapei Huang, Hang Yin, Xiaoliang Hu, and Liandong Qu. "Feline Infectious Peritonitis Virus Nsp5 Inhibits Type I Interferon Production by Cleaving NEMO at Multiple Sites." Viruses 12, no. 1 (December 30, 2019): 43. http://dx.doi.org/10.3390/v12010043.
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Feline infectious peritonitis (FIP), caused by virulent feline coronavirus, is the leading infectious cause of death in cats. The type I interferon (type I IFN)-mediated immune responses provide host protection from infectious diseases. Several coronaviruses have been reported to evolve diverse strategies to evade host IFN response. However, whether feline infectious peritonitis virus (FIPV) antagonizes the type I IFN signaling remains unclear. In this study, we demonstrated that FIPV strain DF2 infection not only failed to induce interferon-β (IFN-β) and interferon-stimulated gene (ISG) production, but also inhibited Sendai virus (SEV) or polyinosinic-polycytidylic acid (poly(I:C))-induced IFN-β production. Subsequently, we found that one of the non-structural proteins encoded by the FIPV genome, nsp5, interrupted type I IFN signaling in a protease-dependent manner by cleaving the nuclear factor κB (NF-κB) essential modulator (NEMO) at three sites—glutamine132 (Q132), Q205, and Q231. Further investigation revealed that the cleavage products of NEMO lost the ability to activate the IFN-β promoter. Mechanistically, the nsp5-mediated NEMO cleavage disrupted the recruitment of the TRAF family member-associated NF-κB activator (TANK) to NEMO, which reduced the phosphorylation of interferon regulatory factor 3 (IRF3), leading to the inhibition of type I IFN production. Our research provides new insights into the mechanism for FIPV to counteract host innate immune response.
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Li, Jiannan, Ilyssa Gordon, Dina Dejanovic, Sinan Lin, Jie Wang, Satya Kurada, Ren Mao, et al. "CADHERIN-11, AN ESSENTIAL REGULATOR OF CELL-CELL ADHESION UPREGULATED IN IBD PATIENTS, PLAYS ESSENTIAL ROLES IN PROMOTING FIBROBLAST ACTIVATION AND DEVELOPMENT OF INTESTINAL INFLAMMATION AND FIBROSIS." Inflammatory Bowel Diseases 27, Supplement_1 (January 1, 2021): S34—S35. http://dx.doi.org/10.1093/ibd/izaa347.084.
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Abstract Background Intestinal fibrosis is a severe complication of inflammatory bowel diseases (IBD) leading to intestinal strictures and need for surgery. No effective anti-fibrotic therapy is available. Cadherin-11 (Cad-11) is an adherens junction protein, which is upregulated in rheumatoid arthritis (RA), idiopathic pulmonary fibrosis (IPF) and skin fibrosis. Inhibition of cadherin-11 has shown beneficial effects in RA and IPF animal models. A phase II clinical trial of cadherin-11 inhibition in RA has shown a good safety profile. Our aim was to evaluate the expression levels and function of Cad-11 in IBD patients using intestinal tissues, primary human intestinal cells, and the murine dextran sulfate sodium (DSS)-induced chronic colitis model. Methods IBD (Crohn’s disease (CD) n=20; Ulcerative colitis (UC) (n=10) and control (n=10) full thickness resected intestinal tissues were procured from adults in accordance with IRB approval. Protein and mRNA were extracted for western blot (WB) and quantitative polymerase chain reaction (qPCR). Distribution of Cad-11 was evaluated by immunofluorescence (IF) and RNA hybridization in frozen and formalin-fixed paraffin-embedded (FFPE) tissue sections, respectively. Primary human intestinal myofibroblasts (HIMF) were used in functional experiments. Recombinant human Fc and Cad-11 extracellular domain (hCAD-11-Fc) was used as activator and siRNA as inhibitor of Cad-11 in HIMF. Murine chronic colitis was induced in wildtype BALB/c mice and cadherin-11 knockout mice by DSS. Anti-Cad-11 monoclonal antibody (H1M1) was used for the treatment of BALB/c mouse colitis. Results Increased gene and protein expression levels of Cad-11 were found in intestinal full thickness IBD tissue compared to controls (45-fold, p<0.01). Cad-11 colocalized with alpha smooth muscle actin (α-SMA) (Figure 1), indicating that Cad-11 is selectively expressed in intestinal myofibroblasts and smooth muscle cells. Among all the primary human intestinal cells, Cad-11 was expressed exclusively in HIMF and HIMC cells. Level of Cad-11 was increased in IBD HIMFs compared to non-IBD controls, and increased upon stimulation with TNF-α, IL-1β, b-FGF and TGF-β (all p<0.01). Knocking down Cad-11 with siRNA decreased FN expression, while hCAD-11-Fc increased the expression FN in a dose- and time-dependent manner as well as the proliferation of HIMF. Upon treatment with H1M1 antibody, DSS-treated mice showed lower clinical scores and weight loss compared to control mice (p<0.001. Figure 2), as well as less FN expression (p<0.001). Cadherin-11 knockout mice also showed lower clinical scores and weight loss compared to wild type mice (p<0.001). Conclusions Cad-11 expression is increased in CD stricture tissues and its blockade reduces profibrotic effects in HIMF in vitro. Inhibition of Cad-11 in vivo reduces clinical severity and fibrosis of experimental colitis.