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1
Tweardy, David John, Xu Xuejun, Naijie Jing, and Huang Shao. "Structural Determinants for Signal Transducer and Activator of Transcription (STAT) 3 Recruitment and Activation by the Granulocyte Colony-Stimulating Factor Receptor (G-CSFR) at Phosphotyrosine Ligands 704 and 744." Blood 104, no. 11 (November 16, 2004): 2169. http://dx.doi.org/10.1182/blood.v104.11.2169.2169.
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Abstract Four tyrosine (Y) residues within the cytoplasmic domain of the G-CSFR (Y704, Y228, Y744 and Y764 in the human receptor; Y703, Y227, Y743 and Y763 in the murine receptor) become phosphorylated by Jak kinases upon ligand binding leading to recruitment of Src homology (SH) 2 domain-containing proteins that link to programs for myeloid cell survival and differentiation (Stat3 recruitment to Y704 and Y744) and proliferation (SHP-2 and PI3K recruitment to Y04; Grb2, Shc and SHP-2 recruitment to Y764). While the preference of SH2 domain binding to specific phospho (p) Y peptide ligands was shown to map to the three residues immediately C-terminal to the pY (+1, +2, +3 residues), the structural basis for these preferences is poorly understood but could be exploited to specifically target deleterious G-CSFR-mediated signaling events such as aberrant Stat3 activation, which has been demonstrated in a subset of acute myelogenous leukemia (AML) patients whose cells contain Flt3 internal tandem duplications and who suffer relapse following initial chemotherapy. To establish the structural basis for Stat3 recruitment and activation by the G-CSFR at Y704 and Y744, we generated purified recombinant full-length Stat3 and phosphododecapeptides based on the sequence surrounding each Y within the G-CSFR. In peptide pull-down assays, recombinant Stat3 bound only to Y704 and Y744 phosphododecapeptide, which contain core pY motifs consisting of pYVLQ and pYLRC, respectively. In mirror resonance affinity assays employed to obtain quantitative binding information, Stat3 bound to each phosphododecapeptide with similar kinetics (e.g. KDs = 0.703 and 0.95 μM, respectively). We tested three models for Stat3 SH2-pY ligand binding proposed by us and others using wild type and mutant recombinant Stat3 proteins in peptide pull-down and mirror resonance affinity assays along with computer modeling of this interaction using the known structures of Statβ SH2 and EGFR pY ligand (EpY1068INQ). Our results revealed loss of binding of Stat3 to Y704 and Y744 phosphododecapeptides only in Stat3 mutated within the SH2 domain at K591 or R609, whose side chains interacted with the pY phosphate group, and in Stat3 mutated within the SH2 domain at E638, whose amide hydrogen bonded with oxygen within the +3 Q side chain (or with sulfur within the +3 C side chain) when the pY ligand assumes a β turn. G-CSF stimulation of cells co-expressing full-length G-CSFR and either wild type or mutant Stat3 constructs confirmed the requirements for the side chain of R609 and the amide hydrogen of E638 within the Stat3 SH2 domain for binding to the G-CSFR and subsequent phosphorylation of Stat3 on Y705. Thus, our findings identify for the first time the structural basis for recruitment and activation of Stat3 by the G-CSFR and reveal unique features of their interaction at Y704 and Y744 i.e. a β turn within the receptor pY motif and a key hydrogen bond formed between the polar side chain of the +3 residue and the amide hydrogen of E638 within the Stat3 SH2 domain. These features explain the preference of the Stat3 SH2 domain for pY peptide ligands with +3 Q or C as well as +3 T (pY705LKT within Stat3) and +3 S (pY743IRS within the murine G-CSFR) and can be exploited using a structure-assisted drug design strategy to develop new therapies for a subset of AML patients with poor prognosis whose cells demonstrate aberrant activation of Stat3.
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Stojanovic, Aleksandra, Panagiotis Flevaris, Xiaodong Xi, Athar Chisti, David Phillips, Stephen C. T. Lam, and Xiaoping Du. "Tyrosine Phosphorylation of the Integrin β3 Subunit Regulates β3 Cleavage by Calpain." Blood 108, no. 11 (November 16, 2006): 1524. http://dx.doi.org/10.1182/blood.v108.11.1524.1524.
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Abstract Outside-in signaling of β3 integrins induces and requires phosphorylation at tyrosine-747 (Y747) and tyrosine-759 (Y759) of the β3 subunit, but the mechanism for this requirement is unclear. On the other hand, a key consequence of integrin signaling, cell spreading, is inhibited by calpain cleavage of β3 cytoplasmic domain. Here we show that tyrosine phosphorylation in the synthetic β3 cytoplasmic domain peptide inhibits calpain cleavage. In platelets, tyrosine phosphates inhibitor, sodium vanadate, enhances thrombin-induced phosphorylation at Y747 and Y759, which is associated with the reduced integrin cleavage by calpain. The effects of sodium vanadate is unlikely to be caused by its effects on calpain activity but is likely to be caused by the susceptibility of integrin cytoplasmic domain, because sodium vanadate did not affect the calpain cleavage of another substrate, fodrin, in platelets. To further support the protective effect of tyrosine phosphorylation against calpain cleavage, we show that mouse β3 (DiYF) with both Y759 and Y747 mutated to phenylalanine is more susceptible to calpain cleavage than wild type during thrombin-induced platelet aggregation. Furthermore, phosphorylation at Y747 and Y759 of β3 in the focal adhesion sites and the leading edge of spreading platelets was differentially regulated. Selective dephosphorylation of Y759 is associated with calpain cleavage at Y759. Thus, one mechanism by which tyrosine phosphorylation promotes integrin signaling and cell spreading is its inhibition of calpain cleavage of the β3 cytoplasmic domain.
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Chakraborty, Arup, Kevin F. Dyer, Michael Cascio, Timothy A. Mietzner, and David J. Tweardy. "Identification of a Novel Stat3 Recruitment and Activation Motif Within the Granulocyte Colony-Stimulating Factor Receptor." Blood 93, no. 1 (January 1, 1999): 15–24. http://dx.doi.org/10.1182/blood.v93.1.15.
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Abstract Stat3 is essential for early embryonic development and for myeloid differentiation induced by the cytokines granulocyte colony-stimulating factor (G-CSF) and interleukin-6 (IL-6). Two isoforms of Stat3 have been identified, (p92) and β (p83), which have distinct transcriptional and biological functions. Activation of both Stat3 and Stat3β requires the distal cytoplasmic domain of the G-CSFR, which contains four Tyr at positions 704, 729, 744, and 764. The studies reported here were undertaken to determine which, if any, of these tyrosine residues participated in Stat3/β recruitment and activation. We showed that Stat3 and Stat3β were affinity purified using phosphopeptides containing Y704 and Y744 but not by nonphosphorylated peptide analogues or by phosphopeptides containing Y729 and Y764. Complementary results were obtained in studies examining the ability of these peptides to destabilize and inhibit DNA binding of activated Stat3. Both Y704 and Y744 contributed to optimal activation of Stat3/β in M1 murine myeloid leukemia cells containing wild-type and Y-to-F mutant G-CSFR constructs. Carboxy-terminal to Y704 at the +3 position is Gln; YXXQ represents a consensus Stat3 recruitment and activation motif. Y744 is followed at the +3 position by Cys (C); YXXC, represents a novel motif implicated in the recruitment and activation of Stat3. Modeling of the SH2 domain of Stat3 based on homologous SH2 domains of known structure revealed polar residues whose side chains contact the +3 position. This substitution may confer specificity for the Y704- and Y744-based ligands by allowing H-bond formation between the binding surface and the Gln or Cys found at the respective +3 position.
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Chakraborty, Arup, Kevin F. Dyer, Michael Cascio, Timothy A. Mietzner, and David J. Tweardy. "Identification of a Novel Stat3 Recruitment and Activation Motif Within the Granulocyte Colony-Stimulating Factor Receptor." Blood 93, no. 1 (January 1, 1999): 15–24. http://dx.doi.org/10.1182/blood.v93.1.15.401a46_15_24.
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Stat3 is essential for early embryonic development and for myeloid differentiation induced by the cytokines granulocyte colony-stimulating factor (G-CSF) and interleukin-6 (IL-6). Two isoforms of Stat3 have been identified, (p92) and β (p83), which have distinct transcriptional and biological functions. Activation of both Stat3 and Stat3β requires the distal cytoplasmic domain of the G-CSFR, which contains four Tyr at positions 704, 729, 744, and 764. The studies reported here were undertaken to determine which, if any, of these tyrosine residues participated in Stat3/β recruitment and activation. We showed that Stat3 and Stat3β were affinity purified using phosphopeptides containing Y704 and Y744 but not by nonphosphorylated peptide analogues or by phosphopeptides containing Y729 and Y764. Complementary results were obtained in studies examining the ability of these peptides to destabilize and inhibit DNA binding of activated Stat3. Both Y704 and Y744 contributed to optimal activation of Stat3/β in M1 murine myeloid leukemia cells containing wild-type and Y-to-F mutant G-CSFR constructs. Carboxy-terminal to Y704 at the +3 position is Gln; YXXQ represents a consensus Stat3 recruitment and activation motif. Y744 is followed at the +3 position by Cys (C); YXXC, represents a novel motif implicated in the recruitment and activation of Stat3. Modeling of the SH2 domain of Stat3 based on homologous SH2 domains of known structure revealed polar residues whose side chains contact the +3 position. This substitution may confer specificity for the Y704- and Y744-based ligands by allowing H-bond formation between the binding surface and the Gln or Cys found at the respective +3 position.
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Yoon, Jeong Hyun, Min-Kyu Song, and Jang-Yeon Kwon. "Detection of Proton Carriers in Tyrosine-Rich Peptide Thin Film." ECS Meeting Abstracts MA2022-01, no. 18 (July 7, 2022): 1048. http://dx.doi.org/10.1149/ma2022-01181048mtgabs.
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Traditional Von Neumann structure, where memory and computational units are separated, is now facing significant difficulties in terms of power consumption and computing speed in big data era. Therefore, human brain-like computing architecture is now paid attention in research field owing to its high parallelism, fault tolerance and robustness1-2. In this biological system, synaptic weight which plays crucial role in memory and learning is modulated by ionic species, especially protons transmitted between synapses. Here, we focused on short length peptide Y7C (YYACAYY) thin film which induces proton coupled electron transfer (PCET) due to its Tyrosine-rich structure. The Y7C film has shown an interesting behavior in which electrical conductivity varies with humidity3-4. By this modulation, bimodal memristor using both of voltage and humidity5 input and synaptic transistor which shows dynamic reponses through humidity have been demonstrated, but direct relation between current and proton conduction is still ambiguous. Therefore, we measured transient current flow between Pd and Au electrode and detected proton conduction of Y7C film. In previous studies, voltage & humidity controlled Y7C bimodal memristor has been reported. However, since the current regulation through humidity showed insufficient response than that through voltage, a definite relationship between moisture and current needs to be defined. To achieve that, thin film of the peptide material dissolved in Trifluoroacetic acid (TFA) 99% solution was spin coated on SiO2/Si substrate. By measuring the transient current of the Y7C film, presence of temporary charge carriers was observed. Also, the electrical impedance spectroscopy (EIS) of peptide layer showed a semicircle and a nonvertical tail, which indicate ionic conduction through the film. Lastly, film characterization of Y7C thin layer unveiled that the corresponding ions are protons as originally targeted. This research has showed principles of peptide thin film’s current increment driven by both of humidity and voltage through electrochemical reaction. Fig.1. Transient charge flowing through Y7C thin film measured in various atmospheric conditions and electrodes Acknowledgment This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. 2020R1A2C2004864). References Pakkenberg B, Pelvig D, Marner L, Bundgaard M J, Gundersen H J G, Nyengaard J R and Regeur L 2003 Exp. Gerontol. 38 95 Abbott L F and Nelson S B 2000 Nat. Neurosci. 3 1178 Lee, Jaehun, et al. "Proton conduction in a tyrosine‐rich peptide/manganese oxide hybrid nanofilm." Advanced Functional Materials 27.35 (2017): 1702185. Sung, Taehoon, et al. "Effects of proton conduction on dielectric properties of peptides." RSC advances 8.59 (2018): 34047-34055. Song, Min-Kyu, et al. "Proton-enabled activation of peptide materials for biological bimodal memory." Nature Communications 11.1 (2020): 1-8. Figure 1
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Boettiger, David, Francois Huber, Laura Lynch, and Scott Blystone. "Activation of αvβ3-Vitronectin Binding Is a Multistage Process in which Increases in Bond Strength Are Dependent on Y747 and Y759 in the Cytoplasmic Domain of β3." Molecular Biology of the Cell 12, no. 5 (May 2001): 1227–37. http://dx.doi.org/10.1091/mbc.12.5.1227.
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Integrin receptors serve as mechanical links between the cell and its structural environment. Using αvβ3 integrin expressed in K562 cells as a model system, the process by which the mechanical connection between αvβ3 and vitronectin develops was analyzed by measuring the resistance of these bonds to mechanical separation. Three distinct stages of activation, as defined by increases in the αvβ3-vitronectinbinding strength, were defined by mutational, biochemical, and biomechanical analyses. Activation to the low binding strength stage 1 occurs through interaction with the vitronectin ligand and leads to the phosphorylation of Y747 in the β3 subunit. Stage 2 is characterized by a 4-fold increase in binding strength and is dependent on stage1 and the phosphorylation of Y747. Stage 3 is characterized by a further 2.5-fold increase in binding strength and is dependent on stage 2 events and the availability of Y759 for interaction with cellular proteins. The Y747F mutant blocked the transition from stage 1 to stage 2, and the Y759F blocked the transition from stage 2 to stage 3. The data suggest a model for tension-induced activation of αvβ3 integrin.
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Xi, Xiaodong, Richard J. Bodnar, Zhenyu Li, Stephen C. T. Lam, and Xiaoping Du. "Critical roles for the COOH-terminal NITY and RGT sequences of the integrin β3 cytoplasmic domain in inside-out and outside-in signaling." Journal of Cell Biology 162, no. 2 (July 8, 2003): 329–39. http://dx.doi.org/10.1083/jcb.200303120.
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Bidirectional signaling of integrin αIIbβ3 requires the β3 cytoplasmic domain. To determine the sequence in the β3 cytoplasmic domain that is critical to integrin signaling, cell lines were established that coexpress the platelet receptor for von Willebrand factor (vWF), glycoprotein Ib-IX, integrin αIIb, and mutants of β3 with truncations at sites COOH terminal to T741, Y747, F754, and Y759. Truncation at Y759 did not affect integrin activation, as indicated by vWF-induced fibrinogen binding, but affected cell spreading and stable adhesion. Thus, the COOH-terminal RGT sequence of β3 is important for outside-in signaling but not inside-out signaling. In contrast, truncation at F754, Y747, or T741 completely abolished integrin activation. A point mutation replacing Y759 with alanine also abolished integrin activation. Thus, the T755NITY759 sequence of β3, containing an NXXY motif, is critical to inside-out signaling, whereas the intact COOH terminus is important for outside-in signaling. In addition, we found that the calcium-dependent protease calpain preferentially cleaves at Y759 in a population of β3 during platelet aggregation and adhesion, suggesting that calpain may selectively regulate integrin outside-in signaling.
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Katayama, Takane, Hideyuki Suzuki, Takashi Koyanagi, and Hidehiko Kumagai. "Cloning and Random Mutagenesis of the Erwinia herbicola tyrR Gene for High-Level Expression of Tyrosine Phenol-Lyase." Applied and Environmental Microbiology 66, no. 11 (November 1, 2000): 4764–71. http://dx.doi.org/10.1128/aem.66.11.4764-4771.2000.
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ABSTRACT Tyrosine phenol-lyase (Tpl), which can synthesize 3,4-dihydroxyphenylalanine from pyruvate, ammonia, and catechol, is a tyrosine-inducible enzyme. Previous studies demonstrated that thetpl promoter of Erwinia herbicola is activated by the TyrR protein of Escherichia coli. In an attempt to create a high-Tpl-expressing strain, we cloned the tyrRgene of E. herbicola and then randomly mutagenized it. Mutant TyrR proteins with enhanced ability to activate tplwere screened for by use of the lac reporter system inE. coli. The most increased transcription oftpl was observed for the strain with the mutanttyrR allele involving amino acid substitutions of alanine, cysteine, and glycine for valine-67, tyrosine-72, and glutamate-201, respectively. A tyrR-deficient derivative of E. herbicola was constructed and transformed with a plasmid carrying the mutant tyrR allele (V67A Y72C E201G substitutions). The resultant strain expressed Tpl without the addition of tyrosine to the medium and produced as much of it as was produced by the wild-type strain grown under tyrosine-induced conditions. The regulatory properties of the mutant TyrRV67A, TyrRY72C, TyrRE201G, and TyrRV67A Y72C E201G proteins were examined in vivo. Interestingly, as opposed to the wild-type TyrR protein, the mutant TyrRV67A protein had a repressive effect on the tyrP promoter in the presence of phenylalanine as the coeffector.
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García-Peydró, Marina, Alberto Paradela, José R. Lamas, and José A. López de Castro. "Peptide Presentation to an Alloreactive CTL Clone Is Modulated Through Multiple Mechanisms Involving Polymorphic and Conserved Residues in HLA-B27." Journal of Immunology 163, no. 11 (December 1, 1999): 6060–64. http://dx.doi.org/10.4049/jimmunol.163.11.6060.
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Abstract This study addressed the mechanisms by which HLA class I polymorphism modulates allorecognition. CTL 27S69 is an alloreactive clone raised against HLA-B*2705, with a known peptide epitope. This CTL cross-reacts with B*2702, which differs from B*2705 in the D77N, T80I, and L81A changes, but not with B*2701, which has D74Y, D77N, and L81A changes. To explain this differential recognition, B*2705 mutants mimicking subtype changes were used. The A81 mutant was not recognized, despite binding the natural epitope in vivo, suggesting that, when bound to this mutant, this peptide adopts an inappropriate conformation. The N77 and I80 mutations restored recognition in the N77A81 or I80A81 mutants. These compensatory effects explain the cross-reaction with B*2702. The Y74 and the Y74N77 mutants were weakly recognized or not recognized by CTL 27S69. This correlated with the absence or marginal presence of the peptide epitope in the Y74N77-bound pool. As with B*2701, exogenous addition of the peptide epitope sensitized Y74 and Y74N77 targets for lysis, indicating that failure to cross-react with B*2701 or these mutants was due to poor binding of the peptide in vivo and not to inappropriate presentation. The abrogating effect of Y74 was critically dependent upon the K70 residue, conserved among subtypes, as demonstrated with mutants at this position. Thus, HLA polymorphism affects allorecognition by modulating peptide binding or the conformation of bound peptides. Compensatory mutations and indirect effects of a polymorphic residue on residues conserved play a critical role.
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de Koning, John P., Amrita A. Soede-Bobok, Anita M. Schelen, Louise Smith, Daphne van Leeuwen, Valeria Santini, Boudewijn M. T. Burgering, Johannes L. Bos, Bob Löwenberg, and Ivo P. Touw. "Proliferation Signaling and Activation of Shc, p21Ras, and Myc Via Tyrosine 764 of Human Granulocyte Colony-Stimulating Factor Receptor." Blood 91, no. 6 (March 15, 1998): 1924–33. http://dx.doi.org/10.1182/blood.v91.6.1924.
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Abstract The membrane-distal region of the cytoplasmic domain of human granulocyte colony-stimulating factor receptor (G-CSF-R) contains four conserved tyrosine residues: Y704, Y729, Y744, and Y764. Three of these (Y729, Y744, and Y764) are located in the C-terminal part of G-CSF-R, previously shown to be essential for induction of neutrophilic differentiation. To determine the role of the tyrosines in G-CSF–mediated responses, we constructed tyrosine-to-phenylalanine (Y-to-F) substitution mutants and expressed these in a differentiation competent subclone of 32D cells that lacks endogenous G-CSF-R. We show that all tyrosines can be substituted essentially without affecting the differentiation signaling properties of G-CSF-R. However, substitution of one specific tyrosine, ie, Y764, markedly influenced proliferation signaling as well as the timing of differentiation. 32D cells expressing wild-type (WT) G-CSF-R (or mutants Y704F, Y729F, or Y744F) proliferated in G-CSF–containing cultures until day 8 and then developed into mature neutrophils. In contrast, 32D/Y764F cells arrested in the G1 phase of the cell cycle within 24 hours and showed complete neutrophilic differentiation after 3 days of culture. This resulted in an average 30-fold reduction of neutrophil production as compared with the 32D/WT controls. Importantly, G-CSF–mediated activation of Shc, p21Ras and the induction of c-myc were severely reduced by substitution of Y764. These findings indicate that Y764 of G-CSF-R is crucial for maintaining the proliferation/differentiation balance during G-CSF–driven neutrophil development and suggest a role for multiple signaling mechanisms in maintaining this balance.
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de Koning, John P., Amrita A. Soede-Bobok, Anita M. Schelen, Louise Smith, Daphne van Leeuwen, Valeria Santini, Boudewijn M. T. Burgering, Johannes L. Bos, Bob Löwenberg, and Ivo P. Touw. "Proliferation Signaling and Activation of Shc, p21Ras, and Myc Via Tyrosine 764 of Human Granulocyte Colony-Stimulating Factor Receptor." Blood 91, no. 6 (March 15, 1998): 1924–33. http://dx.doi.org/10.1182/blood.v91.6.1924.1924_1924_1933.
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The membrane-distal region of the cytoplasmic domain of human granulocyte colony-stimulating factor receptor (G-CSF-R) contains four conserved tyrosine residues: Y704, Y729, Y744, and Y764. Three of these (Y729, Y744, and Y764) are located in the C-terminal part of G-CSF-R, previously shown to be essential for induction of neutrophilic differentiation. To determine the role of the tyrosines in G-CSF–mediated responses, we constructed tyrosine-to-phenylalanine (Y-to-F) substitution mutants and expressed these in a differentiation competent subclone of 32D cells that lacks endogenous G-CSF-R. We show that all tyrosines can be substituted essentially without affecting the differentiation signaling properties of G-CSF-R. However, substitution of one specific tyrosine, ie, Y764, markedly influenced proliferation signaling as well as the timing of differentiation. 32D cells expressing wild-type (WT) G-CSF-R (or mutants Y704F, Y729F, or Y744F) proliferated in G-CSF–containing cultures until day 8 and then developed into mature neutrophils. In contrast, 32D/Y764F cells arrested in the G1 phase of the cell cycle within 24 hours and showed complete neutrophilic differentiation after 3 days of culture. This resulted in an average 30-fold reduction of neutrophil production as compared with the 32D/WT controls. Importantly, G-CSF–mediated activation of Shc, p21Ras and the induction of c-myc were severely reduced by substitution of Y764. These findings indicate that Y764 of G-CSF-R is crucial for maintaining the proliferation/differentiation balance during G-CSF–driven neutrophil development and suggest a role for multiple signaling mechanisms in maintaining this balance.
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Jones, Susan, John S. Reader, Maria Healy, D. Alastair Smith, and Sheena E. Radford. "Unfolding of the β-sheet Protein Y74W Apo-pseudoazurin: Evidence of an Equilibrium Intermediate Species." Biochemical Society Transactions 28, no. 3 (June 1, 2000): A69. http://dx.doi.org/10.1042/bst028a069b.
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Jones, Susan, John S. Reader, Maria Healy, Andrew P. Capaldi, Alison E. Ashcroft, Arnout P. Kalverda, D. Alastair Smith, and Sheena E. Radford. "Partially Unfolded Species Populated during Equilibrium Denaturation of the β-Sheet Protein Y74W Apo-Pseudoazurin†." Biochemistry 39, no. 19 (May 2000): 5672–82. http://dx.doi.org/10.1021/bi9923959.
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Gruslova, Aleksandra, Iurii Semenov, and Bin Wang. "An extracellular domain of the accessory β1 subunit is required for modulating BK channel voltage sensor and gate." Journal of General Physiology 139, no. 1 (December 12, 2011): 57–67. http://dx.doi.org/10.1085/jgp.201110698.
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A family of tissue-specific auxiliary β subunits modulates large conductance voltage- and calcium-activated potassium (BK) channel gating properties to suit their diverse functions. Paradoxically, β subunits both promote BK channel activation through a stabilization of voltage sensor activation and reduce BK channel openings through an increased energetic barrier of the closed-to-open transition. The molecular determinants underlying β subunit function, including the dual gating effects, remain unknown. In this study, we report the first identification of a β1 functional domain consisting of Y74, S104, Y105, and I106 residues located in the extracellular loop of β1. These amino acids reside within two regions of highest conservation among related β1, β2, and β4 subunits. Analysis in the context of the Horrigan-Aldrich gating model revealed that this domain functions to both promote voltage sensor activation and also reduce intrinsic gating. Free energy calculations suggest that the dual effects of the β1 Y74 and S104–I106 domains can be largely accounted for by a relative destabilization of channels in open states that have few voltage sensors activated. These results suggest a unique and novel mechanism for β subunit modulation of voltage-gated potassium channels wherein interactions between extracellular β subunit residues with the external portions of the gate and voltage sensor regulate channel opening.
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Malkia, Annika, María Pertusa, Gregorio Fernández-Ballester, Antonio Ferrer-Montiel, and Félix Viana. "Differential Role of the Menthol-Binding Residue Y745 in the Antagonism of Thermally Gated TRPM8 Channels." Molecular Pain 5 (January 2009): 1744–8069. http://dx.doi.org/10.1186/1744-8069-5-62.
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Phillips, Robert S., Nancy Johnson, and Ajith V. Kamath. "Formation in Vitro of Hybrid Dimers of H463F and Y74F MutantEscherichia coliTryptophan Indole-lyase Rescues Activity withl-Tryptophan†." Biochemistry 41, no. 12 (March 2002): 4012–19. http://dx.doi.org/10.1021/bi015838t.
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Chen, Jian-Xin, Mao-Yuan Yao, Jian-Jun Guo, Tian-Ci Yi, and Dao-Chao Jin. "Lepidocunaxoides bisetosus, a new species of Cunaxidae (Acariformes: Prostigmata) from China." Acarologia 63, no. 1 (February 6, 2023): 231–40. http://dx.doi.org/10.24349/7rzt-y74u.
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A new species of genus Lepidocunaxoides, namely, L. bisetosus Chen and Jin sp. nov. is described and illustrated based on female and male from China. Additionally, an identification key to known species of Lepidocunaxoides is updated.
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Bertagnolo, Valeria, Silvia Grassilli, Simona D’Aguanno, Federica Brugnoli, Alberto Bavelloni, Irene Faenza, Ervin Nika, Andrea Urbani, Lucio Cocco, and Silvano Capitani. "Mass Spectrometry-Based Identification of Y745 of Vav1 as a Tyrosine Residue Crucial in Maturation of Acute Promyelocytic Leukemia-Derived Cells." Journal of Proteome Research 9, no. 2 (February 5, 2010): 752–60. http://dx.doi.org/10.1021/pr900581y.
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Liu, Yihui, Jiang Xu, Wanyun Tao, Changbiao Fu, Jiangbing Liu, Rong Yu, and Xinjiang Zhang. "Exome Sequencing Identifies a Mutation (Y740C) in Spastic Paraplegia 7 Gene Associated with Adult-Onset Primary Lateral Sclerosis in a Chinese Family." European Neurology 81, no. 1-2 (2019): 87–93. http://dx.doi.org/10.1159/000500672.
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Background: Primary lateral sclerosis (PLS) is considered a rare variant of motor neuron disease (MND) characterized by selective upper motor neuron dysfunction leading to limb weakness, spasticity, and even bulbar symptoms. Previous studies have demonstrated that mutations in ALSIN, spastic paraplegia 7 (SPG7), TBK1, ALS2, ERLIN2, and FIG4 are responsible for PLS. Most of them occurred in childhood to young-adult onset patients. The aim of this study was to identify the genetic lesion of patients with adult-onset PLS. Methods: We applied whole-exome sequencing (WES) and MND and ataxia-related genes filtering strategies to discover the genetic factors in a Chinese adult-onset PLS family. Sanger sequencing was used in the cosegregation analysis in the affected family members. Results: A mutation (c.2219A>G/p.Y740C) in exon 17 of SPG7 was identified in an adult-onset PLS patient and cosegregated with the affected members in this family. Meanwhile, the mutation was predicted to be deleterious by 3 bioinformatics programs (Polymorphism phenotyping-2, sorting intolerant from tolerant and MutationTaster). This variant may cause the structure changes of paraplegin protein. Conclusions: We employed WES to detect a missense mutation of SPG7 gene in a PLS family. This finding expands the spectrum of known SPG7 mutations, and it may contribute to novel approaches to genetic diagnosis and counseling of families with PLS.
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Xiao, Juanjuan, Qiuhong Duan, Zhe Wang, Wei Yan, Huimin Sun, Peipei Xue, Xiaoming Fan, et al. "Phosphorylation of TOPK at Y74, Y272 by Src increases the stability of TOPK and promotes tumorigenesis of colon." Oncotarget 7, no. 17 (March 21, 2016): 24483–94. http://dx.doi.org/10.18632/oncotarget.8231.
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Deschuyter, Marlène, Florian Pennarubia, Emilie Pinault, Sébastien Legardinier, and Abderrahman Maftah. "Functional Characterization of POFUT1 Variants Associated with Colorectal Cancer." Cancers 12, no. 6 (May 31, 2020): 1430. http://dx.doi.org/10.3390/cancers12061430.
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Background: Protein O-fucosyltransferase 1 (POFUT1) overexpression, which is observed in many cancers such as colorectal cancer (CRC), leads to a NOTCH signaling dysregulation associated with the tumoral process. In rare CRC cases, with no POFUT1 overexpression, seven missense mutations were found in human POFUT1. Methods: Recombinant secreted forms of human WT POFUT1 and its seven mutated counterparts were produced and purified. Their O-fucosyltransferase activities were assayed in vitro using a chemo-enzymatic approach with azido-labeled GDP-fucose as a donor substrate and NOTCH1 EGF-LD26, produced in E. coli periplasm, as a relevant acceptor substrate. Targeted mass spectrometry (MS) was carried out to quantify the O-fucosyltransferase ability of all POFUT1 proteins. Findings: MS analyses showed a significantly higher O-fucosyltransferase activity of six POFUT1 variants (R43H, Y73C, T115A, I343V, D348N, and R364W) compared to WT POFUT1. Interpretation: This study provides insights on the possible involvement of these seven missense mutations in colorectal tumors. The hyperactive forms could lead to an increased O-fucosylation of POFUT1 protein targets such as NOTCH receptors in CRC patients, thereby leading to a NOTCH signaling dysregulation. It is the first demonstration of gain-of-function mutations for this crucial glycosyltransferase, modulating NOTCH activity, as well as that of other potential glycoproteins.
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Su, Xiaoyu, Jianqing Mi, Jinsong Yan, Panagiotis Flevaris, Yuanjing Lu, Hongchen Liu, Zheng Ruan, et al. "RGT, a synthetic peptide corresponding to the integrin β3 cytoplasmic C-terminal sequence, selectively inhibits outside-in signaling in human platelets by disrupting the interaction of integrin αIIbβ3 with Src kinase." Blood 112, no. 3 (August 1, 2008): 592–602. http://dx.doi.org/10.1182/blood-2007-09-110437.
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Abstract Mutational analysis has established that the cytoplasmic tail of the integrin β3 subunit binds c-Src (termed as Src in this study) and is critical for bidirectional integrin signaling. Here we show in washed human platelets that a cell-permeable, myristoylated RGT peptide (myr-RGT) corresponding to the integrin β3 C-terminal sequence dose-dependently inhibited stable platelet adhesion and spreading on immobilized fibrinogen, and fibrin clot retraction as well. Myr-RGT also inhibited the aggregation-dependent platelet secretion and secretion-dependent second wave of platelet aggregation induced by adenosine diphosphate, ristocetin, or thrombin. Thus, myr-RGT inhibited integrin outside-in signaling. In contrast, myr-RGT had no inhibitory effect on adenosine diphosphate-induced soluble fibrinogen binding to platelets that is dependent on integrin inside-out signaling. Furthermore, the RGT peptide induced dissociation of Src from integrin β3 and dose-dependently inhibited the purified recombinant β3 cytoplasmic domain binding to Src-SH3. In addition, phosphorylation of the β3 cytoplasmic tyrosines, Y747 and Y759, was inhibited by myr-RGT. These data indicate an important role for β3-Src interaction in outside-in signaling. Thus, in intact human platelets, disruption of the association of Src with β3 and selective blockade of integrin αIIbβ3 outside-in signaling by myr-RGT suggest a potential new antithrombotic strategy.
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Karas, M., A. P. Koval, Y. Zick, and D. LeRoith. "The Insulin-Like Growth Factor I Receptor-Induced Interaction of Insulin Receptor Substrate-4 and Crk-II." Endocrinology 142, no. 5 (May 1, 2001): 1835–40. http://dx.doi.org/10.1210/endo.142.5.8135.
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Abstract Stimulation of the insulin or insulin-like growth factor (IGF)-I receptor results in activation of several signaling pathways. Proteins of the insulin receptor substrate (IRS) family play important roles in mediating these signaling cascades. To date, four members of the IRS family of docking proteins have been characterized. Recently, we have reported that stimulation of the IGF-I receptor in 293 HEK cells regulates interaction of the newly discovered IRS-4 molecule with the Crk family of proteins. In the present study, we characterize the molecular basis of these interactions. C- and N termini truncation analysis of IRS-4 demonstrated that the region between amino acids 678 and 800 of the IRS-4 molecule is involved in this interaction. This region contains a cluster of four tyrosines (Y700, Y717, Y743, and Y779). We hypothesize that one or more of these tyrosines are involved in the interaction between the SH2 domain of the Crk-II molecule when IRS-4 is phosphorylated upon IGF-I receptor activation. Additional mutational analyses confirmed this hypothesis. Interestingly, none of these four tyrosines was individually critical for the interaction between Crk-II and IRS-4, but when all four tyrosines were simultaneously mutated to phenylalanine, the IGF-I induced interaction between these molecules was abolished. Taken together, these results suggest a novel mechanism of Crk-II binding to tyrosine phosphorylated proteins.
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Borodin, E. A., A. P. Chupalov, P. D. Timkin, E. A. Timofeev, and N. Yu Leusova. "Selection of potential ligands for TRPM8 using deep neural networks and intermolecular docking." Bulletin Physiology and Pathology of Respiration, no. 80 (July 16, 2021): 26–33. http://dx.doi.org/10.36604/1998-5029-2021-80-26-33.
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Introduction. TRPM8 has been implicated in the development of bronchial hypersensitivity to cold and is considered a potential target for computer-generated drugs.Aim. Development of a strategy for the selection of ligands for TRPM8 by in silico methods.Materials and methods. Using machine learning tools based on deep neural networks and further verification by intermolecular docking, a strategy has been proposed for predicting potential ligands for TRPM8, which consists in using a neural network to screen out potential drug candidates and thereby reduce the list of candidate ligands for verification using AutoDock program, which allows assessing the affinity of a protein for a ligand by the minimum binding energy and identifying possible conformations of a ligand upon binding to certain centers (amino acid residues) of a protein. The latter were used: Y745 (tyrosine 745 is a critical center for TRPM8), R1008 (phenylalanine 1008) and L1009 (alanine 1009).Results. Of the 10 potential ligands predicted by the neural network, eight showed a high minimum binding energy and a greater number of conformations compared to the classic TRPM8 ligand, menthol, when verified by the AutoDock program. The two predicted ligands did not show the ability to interact with TRPM8, which may be due to insufficient allocated memory of the computing device for successful docking or other technical problems.Conclusion. The proposed strategy is universal; it will accelerate the search for ligands for various proteins and will facilitate the accelerated search for potential drugs by in silico methods.
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Ruusala, A., C. Sundberg, A. K. Arvidsson, E. Rupp-Thuresson, C. H. Heldin, and L. Claesson-Welsh. "Platelet-derived growth factor (PDGF)-induced actin rearrangement is deregulated in cells expressing a mutant Y778F PDGF beta-receptor." Journal of Cell Science 111, no. 1 (January 1, 1998): 111–20. http://dx.doi.org/10.1242/jcs.111.1.111.
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Platelet-derived growth factor-stimulated actin rearrangement and edge ruffle formation have previously been shown to be dependent on activation of phosphatidylinositol 3′-kinase, the activity of which also is important for directed migration of cells. This lipid kinase binds to phosphorylated tyrosine residues Y740 and Y751 in the kinase insert of the human platelet-derived growth factor ss-receptor. We examined the role of two other tyrosine residues in the kinase insert of this receptor, Y775 and Y778, for ligand-induced actin rearrangement. Both were shown to be phosphorylation sites; Y775 was only marginally phosphorylated in cells expressing the wild-type ss-receptor, whereas Y778 was phosphorylated at higher stoichiometry. Mutant receptors Y775F, Y778F and Y775/778F were active kinases and mediated proliferative responses when expressed in porcine aortic endothelial cells. Fluorescence staining of actin in platelet-derived growth factor-stimulated PAE cells revealed that Y778 is involved in regulation of the actin cytoskeleton since the cells contained, apart from edge ruffles and circular ruffles, a novel type of giant ruffle on the dorsal side of the cell, which consisted of irregular multilayered actin structures. Mutation at Y778 had no effect on activation of phosphatidylinositol 3′-kinase, nor on the GTPase activating protein of Ras and phospholipase C(gamma), and the extent of directed migration towards platelet-derived growth factor of these cells was not changed. We conclude that actin rearrangement is regulated in part by Y778 in the platelet-derived growth factor ss-receptor, potentially through binding of a novel signaling molecule to this site.
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Holland, Audrey L. "Living Successfully with Aphasia: Three Variations on the Theme." Topics in Stroke Rehabilitation 13, no. 1 (January 2006): 44–51. http://dx.doi.org/10.1310/13d7-r31r-8a0d-y74g.
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Akbarzadeh, Shiva, Alister C. Ward, Dora O. M. McPhee, Warren S. Alexander, Graham J. Lieschke, and Judith E. Layton. "Tyrosine residues of the granulocyte colony-stimulating factor receptor transmit proliferation and differentiation signals in murine bone marrow cells." Blood 99, no. 3 (February 1, 2002): 879–87. http://dx.doi.org/10.1182/blood.v99.3.879.
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Abstract Granulocyte colony-stimulating factor (G-CSF) is the major regulator of granulopoiesis and acts through binding to its specific receptor (G-CSF-R) on neutrophilic granulocytes. Previous studies of signaling from the 4 G-CSF-R cytoplasmic tyrosine residues used model cell lines that may have idiosyncratic, nonphysiological responses. This study aimed to identify specific signals transmitted by the receptor tyrosine residues in primary myeloid cells. To bypass the presence of endogenous G-CSF-R, a chimeric receptor containing the extracellular domain of the epidermal growth factor receptor in place of the entire extracellular domain of the G-CSF-R was used. A series of chimeric receptors containing tyrosine mutations to phenylalanine, either individually or collectively, was constructed and expressed in primary bone marrow cells from G-CSF–deficient mice. Proliferation and differentiation responses of receptor-expressing bone marrow cells stimulated by epidermal growth factor were measured. An increased 50% effective concentration to stimulus of the receptor Ynullmutant indicated that specific signals from tyrosine residues were required for cell proliferation, particularly at low concentrations of stimulus. Impaired responses by mutant receptors implicated G-CSF-R Y764 in cell proliferation and Y729 in granulocyte differentiation signaling. In addition, different sensitivities to ligand stimulation between mutant receptors indicated that G-CSF-R Y744 and possibly Y729 have an inhibitory role in cell proliferation. STAT activation was not affected by tyrosine mutations, whereas ERK activation appeared to depend, at least in part, on Y764. These observations have suggested novel roles for the G-CSF-R tyrosine residues in primary cells that were not observed previously in studies in cell lines.
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Huang, Jiansong, Xiaofeng Shi, Wenda Xi, Ping Liu, and Xiaodong Xi. "CRGT Peptide In Cytoplasm Regulates Thrombus Formation Via Dissociating c-Src-β3 Interaction." Blood 122, no. 21 (November 15, 2013): 3501. http://dx.doi.org/10.1182/blood.v122.21.3501.3501.
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Abstract The RGT sequences of the integrin β3 tail directly and constitutively bind the inactive c-Src, regulating integrin αIIbβ3 signaling and platelet function. Previous work has shown that disrupting the interaction of c-Src with β3 via myristoylated RGT peptide or deletion of the RGT sequences in β3 selectively inhibits integrin αIIbβ3 outside-in signaling in platelets. However, the precise molecular mechanisms by which the Src-β3 association regulates integrin αIIbβ3 signaling need to be clarified. We found that active c-Src phosphoylated the Y747 and Y759 residues of β3 directly at the in vitro protein/protein level or in CHO cell models bearing Tac-β3 chimeras, which were devoid of the intact β3 signal transduction. Furthermore, data from mass spectrometry, [γ-32P] ATP incorporation assays and CHO cell/Tac-β3 chimeras demonstrated that the direct phosphorylation of Y747 and Y759 by active c-Src did not depend on the binding of c-Src to the RGT sequences of the β3 tail. To further investigate the biological functions of Src-β3 association in signal transduction we employed a cell-permeable and reduction-sensitive peptide (myr-AC∼CRGT), which disrupted the Src-β3 association in platelets independent of membrane-anchorage, and found that when platelets were stimulated by thrombin the c-Src activation and the phosphorylation of the tyrosine residues of the β3 tail were substantially inhibited by the presence of the peptide. These results suggest that one of the crucial biological functions of Src-β3 association is to serve as a “bridge” linking integrin signaling with the c-Src full activation and phosphorylation of the tyrosines of the β3 tail. To answer whether the RGT peptide binding to Src is able to alter the enzymatic activity of c-Src, we examined the Src-Csk association, the phosphorylation status of Y416 and Y527 of c-Src and the c-Src kinase catalytic activity. Results showed that myr-AC∼CRGT did not dissociate Csk from c-Src in resting platelets and the phosphorylation level of Y416 and Y527 of c-Src remained unaltered. Consistent data were also obtained from in vitro analysis of the c-Src kinase catalytic activity in the presence of CRGT peptide. These results suggest that myr-AC∼CRGT peptide per se does not fully activate c-Src. Myr-AC∼CRGT was also found to inhibit integrin αIIbβ3 outside-in signaling in human platelets. To examine the effect of the myr-AC∼CRGT on platelet adhesion and aggregation under flow conditions, we measured the platelet thrombus formation under different shear rates. Myr-AC∼CRGT did not affect the platelet adhesion at a wall shear rate of 125 s-1. The inability of myr-AC∼CRGT to affect platelet adhesion and aggregation remained at 500 s-1 shear rates. At 1,500 s-1, or 5,000 s-1 rates, myr-AC∼CRGT partially inhibited platelet adhesion and aggregation. These observations indicate that the Src-regulated outside-in signaling plays a pivotal role in the stable thrombus formation and the thrombus growth under flow conditions. The present study reveals novel insights into the molecular mechanisms by which c-Src regulates integrin αIIbβ3 signaling, particularly the phorsphorylation of the β3 cytoplasmic tyrosines, and provides first evidence in human platelets that the RGT peptide or derivatives regulate thrombus formation through dissociating the Src-β3 interaction. The data of this work allow us to anticipate that intracellular delivery of the RGT peptide or its analogues may have potential in the development of a new antithrombotic strategy where only the Src-β3 interaction is specifically interrupted so as to provide an effective inhibition on thrombosis together with a decent hemostasis. Disclosures: No relevant conflicts of interest to declare.
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Sarmiento Saavedra, Juan Carlos, María Angélica Troya Loor, Luis Alfonso Núñez Freire, and Eugenio Rafael Mora Zambrano. "La evaluación del docente de matemática y su incidencia en los procesos de enseñanza aprendizaje." Ciencia Digital 2, no. 1 (April 13, 2018): 223–23. http://dx.doi.org/10.33262/cienciadigital.v2i1.16.
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La educación es un derecho que tienen todas personas a nivel de todas las naciones del mundo y por ende a nivel latinoamericano, la cual busca el desarrollo y mejoramiento del nivel de vida, de esta manera dejar de ser países subdesarrollados y dependientes. Por tal motivo la educación impartida por docentes en conjunto con autoridades y administrativos de los planteles educativos debe ser de calidad y nos debería conducir a una constante evaluación de los procesos de enseñanza aprendizaje, enfatizando en el proceso docente y administrativo, el Colegio Nacional Mixto Santo Domingo de los Colorados, es un establecimiento de educación media que funciona en la ciudad del mismo nombre. la formación académica se está innovando constantemente, la investigación aplicada es de Campo, con un método descriptivo, tomando en cuenta el contacto directo con el objeto, se aplicó un muestra y entrevista a 22 docentes, 22 estudiantes y74 padres de familia, con esta evaluación que en su esencia busca el análisis permanente e integral del estudiante no para que de manera consciente asuma sus aciertos y errores; se ha convertido en la medición de la repetición de lo enseñado por el profesor transformándose en una evaluación netamente cuantitativa que busca la promoción del estudiante a nivel científico, producto de la visión que los maestros tienen al respecto.
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Valgeirsdottir, S., L. Claesson-Welsh, E. Bongcam-Rudloff, U. Hellman, B. Westermark, and C. H. Heldin. "PDGF induces reorganization of vimentin filaments." Journal of Cell Science 111, no. 14 (July 30, 1998): 1973–80. http://dx.doi.org/10.1242/jcs.111.14.1973.
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In this study we demonstrate that stimulation with platelet-derived growth factor (PDGF) leads to a marked reorganization of the vimentin filaments in porcine aortic endothelial (PAE) cells ectopically expressing the PDGF beta-receptor. Within 20 minutes after stimulation, the well-spread fine fibrillar vimentin was reorganized as the filaments aggregated into a dense coil around the nucleus. The solubility of vimentin upon Nonidet-P40-extraction of cells decreased considerably after PDGF stimulation, indicating that PDGF caused a redistribution of vimentin to a less soluble compartment. In addition, an increased tyrosine phosphorylation of vimentin was observed. The redistribution of vimentin was not a direct consequence of its tyrosine phosphorylation, since treatment of cells with an inhibitor for the cytoplasmic tyrosine kinase Src, attenuated phosphorylation but not redistribution of vimentin. These changes in the distribution of vimentin occurred in conjunction with reorganization of actin filaments. In PAE cells expressing a Y740/751F mutant receptor that is unable to bind and activate phosphatidylinositol 3′-kinase (PI3-kinase), the distribution of vimentin was virtually unaffected by PDGF stimulation. Thus, PI3-kinase is important for vimentin reorganization, in addition to its previously demonstrated role in actin reorganization. The small GTPase Rac has previously been shown to be involved downstream of PI3-kinase in the reorganization of actin filaments. In PAE cells overexpressing dominant negative Rac1 (N17Rac1), no change in the fine fibrillar vimentin network was seen after PDGF-BB stimulation, whereas in PAE cells overexpressing constitutively active Rac1 (V12Rac1), there was a dramatic change in vimentin filament organization independent of PDGF stimulation. These data indicate that PDGF causes a reorganization of microfilaments as well as intermediate filaments in its target cells and suggest an important role for Rac downstream of PI3-kinase in the PDGF stimulated reorganization of both actin and vimentin filaments.
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Ward, Alister C., Mirjam H. A. Hermans, Louise Smith, Yvette M. van Aesch, Anita M. Schelen, Claudia Antonissen, and Ivo P. Touw. "Tyrosine-Dependent and -Independent Mechanisms of STAT3 Activation by the Human Granulocyte Colony-Stimulating Factor (G-CSF) Receptor Are Differentially Utilized Depending on G-CSF Concentration." Blood 93, no. 1 (January 1, 1999): 113–24. http://dx.doi.org/10.1182/blood.v93.1.113.401k33_113_124.
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The granulocyte colony-stimulating factor receptor (G-CSF-R) activates multiple STAT proteins. Although the membrane-proximal cytoplasmic region of the G-CSF-R is necessary and sufficient for activation of STAT1 and STAT5, activation of STAT3 requires the membrane distal region that contains four tyrosines. Although one of these (Y704) has previously been shown to be involved in STAT3 activation from a truncated G-CSF-R derived from a patient with severe chronic neutropenia (SCN), this tyrosine is not required for STAT3 activation by the full-length G-CSF-R. To investigate possible alternative mechanisms of STAT3 activation, we generated a series of Ba/F3 cell transfectants expressing the wild-type G-CSF-R or mutant receptors that either completely lack tyrosines or retain just one of the four cytoplasmic tyrosines of the G-CSF-R. We show that, at saturating G-CSF concentrations, STAT3 activation from the full-length G-CSF-R is efficiently mediated by the C-terminal domain in a manner independent of receptor tyrosines. In contrast, at low G-CSF concentrations, Y704 and Y744 of the G-CSF-R play a major role in STAT3 activation. Both tyrosine-dependent and -independent mechanisms of STAT3 activation are sensitive to the Jak2 inhibitor AG-490, follow similar kinetics, and lead to transactivation of a STAT3 reporter construct, indicating functional equivalence. STAT3 activation is also impaired, particularly at nonsaturating G-CSF concentrations, in bone marrow cells from mice expressing a truncated G-CSF-R (gcsfr-▵715). These findings suggest that G-CSF–induced STAT3 activation during basal granulopoiesis (low G-CSF) and “emergency” granulopoiesis (high G-CSF) are differentially controlled. In addition, the data establish the importance of the G-CSF-R C-terminus in STAT3 activation in primary cells, which has implications for understanding why truncated G-CSF-R derived from SCN patients are defective in maturation signaling.
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Lee, Jintae, Toshinari Maeda, Seok Hoon Hong, and Thomas K. Wood. "Reconfiguring the Quorum-Sensing Regulator SdiA of Escherichia coli To Control Biofilm Formation via Indole and N-Acylhomoserine Lactones." Applied and Environmental Microbiology 75, no. 6 (January 23, 2009): 1703–16. http://dx.doi.org/10.1128/aem.02081-08.
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ABSTRACT SdiA is a homolog of quorum-sensing regulators that detects N-acylhomoserine lactone (AHL) signals from other bacteria. Escherichia coli uses SdiA to reduce its biofilm formation in the presence of both AHLs and its own signal indole. Here we reconfigured SdiA (240 amino acids) to control biofilm formation using protein engineering. Four SdiA variants were obtained with altered biofilm formation, including truncation variants SdiA1E11 (F7L, F59L, Y70C, M94K, and K153X) and SdiA14C3 (W9R, P49T, N87T, frameshift at N96, and L123X), which reduced biofilm formation by 5- to 20-fold compared to wild-type SdiA in the presence of endogenous indole. Whole-transcriptome profiling revealed that wild-type SdiA reduced biofilm formation by repressing genes related to indole synthesis and curli synthesis compared to when no SdiA was expressed, while variant SdiA1E11 induced genes related to indole synthesis in comparison to wild-type SdiA. These results suggested altered indole metabolism, and corroborating the DNA microarray results in regard to indole synthesis, variant SdiA1E11 produced ninefold more indole, which led to reduced swimming motility and cell density. Also, wild-type SdiA decreased curli production and tnaA transcription, while SdiA1E11 increased tnaA transcription (tnaA encodes tryptophanase, which forms indole) compared to wild-type SdiA. Hence, wild-type SdiA decreased biofilm formation by reducing curli production and motility, and SdiA1E11 reduced biofilm formation via indole. Furthermore, an AHL-sensitive variant (SdiA2D10, having four mutations at E31G, Y42F, R116H, and L165Q) increased biofilm formation sevenfold in the presence of N-octanoyl-dl-homoserine lactone and N-(3-oxododecatanoyl)-l-homoserine lactone. Therefore, SdiA can be evolved to increase or decrease biofilm formation, and biofilm formation may be controlled by altering sensors rather than signals.
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Ward, Alister C., Mirjam H. A. Hermans, Louise Smith, Yvette M. van Aesch, Anita M. Schelen, Claudia Antonissen, and Ivo P. Touw. "Tyrosine-Dependent and -Independent Mechanisms of STAT3 Activation by the Human Granulocyte Colony-Stimulating Factor (G-CSF) Receptor Are Differentially Utilized Depending on G-CSF Concentration." Blood 93, no. 1 (January 1, 1999): 113–24. http://dx.doi.org/10.1182/blood.v93.1.113.
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Abstract The granulocyte colony-stimulating factor receptor (G-CSF-R) activates multiple STAT proteins. Although the membrane-proximal cytoplasmic region of the G-CSF-R is necessary and sufficient for activation of STAT1 and STAT5, activation of STAT3 requires the membrane distal region that contains four tyrosines. Although one of these (Y704) has previously been shown to be involved in STAT3 activation from a truncated G-CSF-R derived from a patient with severe chronic neutropenia (SCN), this tyrosine is not required for STAT3 activation by the full-length G-CSF-R. To investigate possible alternative mechanisms of STAT3 activation, we generated a series of Ba/F3 cell transfectants expressing the wild-type G-CSF-R or mutant receptors that either completely lack tyrosines or retain just one of the four cytoplasmic tyrosines of the G-CSF-R. We show that, at saturating G-CSF concentrations, STAT3 activation from the full-length G-CSF-R is efficiently mediated by the C-terminal domain in a manner independent of receptor tyrosines. In contrast, at low G-CSF concentrations, Y704 and Y744 of the G-CSF-R play a major role in STAT3 activation. Both tyrosine-dependent and -independent mechanisms of STAT3 activation are sensitive to the Jak2 inhibitor AG-490, follow similar kinetics, and lead to transactivation of a STAT3 reporter construct, indicating functional equivalence. STAT3 activation is also impaired, particularly at nonsaturating G-CSF concentrations, in bone marrow cells from mice expressing a truncated G-CSF-R (gcsfr-▵715). These findings suggest that G-CSF–induced STAT3 activation during basal granulopoiesis (low G-CSF) and “emergency” granulopoiesis (high G-CSF) are differentially controlled. In addition, the data establish the importance of the G-CSF-R C-terminus in STAT3 activation in primary cells, which has implications for understanding why truncated G-CSF-R derived from SCN patients are defective in maturation signaling.
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Shi, Jian, Baoen Shan, Ningning Luo, Lihua Li, Xing Zhang, Rongfeng Liu, Chao Song, Yujie Shan, and Yi Lu. "Novel mutations of SYK gene in solid tumors." Journal of Clinical Oncology 38, no. 15_suppl (May 20, 2020): e13519-e13519. http://dx.doi.org/10.1200/jco.2020.38.15_suppl.e13519.
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e13519 Background: Spleen tyrosine kinase ( SYK) is a non-receptor tyrosine kinase (NRTK) in cytoplasmic and plays a crucial role in multiple signaling pathways. The deletion of SYK gene expression will lead to the formation and metastasis of multiple solid tumors. Previous studies focus on the methylation of the SYK gene promoter and the protein isomers, including SYK (L) and SYK (S) which are differed by 23 amino acids in functional domains. Nevertheless, the knowledge of mutations in SYK remains poorly understood. Herein, next generation sequencing were performed to explore SYK mutations for further clinical research. Methods: We retrospectively analyzed the somatic mutations from a comprehensive genomic profiling in tumor tissue or blood ctDNA of 4,093 patients with pan-cancer, of which 2,213 patients using 528 genes panel and 1,880 patients using 539 genes panel, both of the panels contained all exon regions of SYK gene. SYK mutations analyzed by TCGA database was used as validation to our results. Results: In 4093 patients with pan-carcinoma, 36 patients were found mutations in SYK gene and the mutation frequency was 0.880%, of which 4 intron mutations and 32 cases of exon mutations were found in patients, respectively. A number of 9 mutations might affect protein function, which were stop-gained mutations and frameshift mutations and their proportion was 0.220%. The mutation frequency of SYK was consistent with TCGA. Among 9 mutations, 3 mutations were also found in TCGA, which were R574*, M34fs, E376*, respectively. And the rest 6 mutations were newly found, which were V395fs, N615fs, Y74*, G185*, Y74fs, Q145*, respectively. Colorectal cancer was the most common cancer type harboring SYK mutations, and TP53 was the most frequently co-mutated gene of SYK which was counted 5 both in our detected results and in TCGA database. Conclusions: Firstly report of 6 novel stop-gained and frame-shift mutations in SYK gene expanded the understanding of SYK in solid tumors. As a retrospective study in pan-cancer with a relatively small population and the mechanism is unclear, the conclusions of this study needed to be verified with a larger sample.
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Su, Xiaoyu, Jianqing Mi, Yuanjing Lu, Hongchen Liu, Saijuan Chen, and Xiaodong Xi. "RGT, a Sythetic Peptide, Selectively Inhibits Integrin αIIbβ3 Outside-In Signaling in Human Platelets through Disassociating SRC Kinase from β3 Subunit." Blood 110, no. 11 (November 16, 2007): 3633. http://dx.doi.org/10.1182/blood.v110.11.3633.3633.
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Abstract Integrin αIIbβ3 plays pivotal roles in platelet activation by interacting with ligands and by mediating bidirectional signal transduction as well. The molecular mechanisms underlying the signal transduction pathways by which integrin αIIbβ3 regulates platelet activation have not yet been completely understood. Previous truncational analyses with CHO cell model have established that the C-terminal RGT sequence of integrin β3 subunit is critical to outside-in signaling but not inside-out signaling. To examine whether this mechanism functions in intact human platelets, a synthetic cell-permeable peptide corresponding to C-terminal RGT sequence of integrin β3 was employed in this work in an attempt to compete with native integrin β3 subunit for downstream signaling molecules. Myristoylated RGT peptide (myr-RGT) dose-dependently inhibited adhesion and spreading of normal human platelets on immobilized fibrinogen and prevented fibrin clot retraction. Also, myr-RGT selectively inhibited the second wave of platelet aggregation induced by ADP, ristocetin or thrombin with unaffected first wave of aggregation, while it had no inhibitory effect on ADP-induced soluble fibrinogen binding. These results suggest that the integrin outside-in signaling pathway is impaired by the treatment of RGT peptide and that the inside-out pathway remained undisrupted. Thus we showed for the first time in intact platelets that the outside-in signaling could be selectively regulated by synthetic peptide with sequence corresponding to the last three amino acids of integrin β3 cytoplasmic tail. To further define the molecular basis of the roles of RGT peptide in signal transduction we examined the effect of RGT peptide on tyrosine phosphorylation of integrin β3 cytoplasmic domain. We found that when platelets were stimulated by thrombin the phosphorylation of both cytoplasmic tyrosine residues (Y747 and Y759) of integrin β3 was substantially inhibited by the presence of myr-RGT peptide. This inhibition could be attributed to the findings in immunoprecipitation experiments whereby RGT peptide attenuated the interaction of integrin β3 with Src kinase. On the contrary, the RGT peptide was unable to interfere talin binding to β3 subunit. These results support the conclusion that, in intact human platelets, the RGT peptide selectively blocked outside-in signaling by disrupting the constitutive interaction of Src kinase with integrin β3 and hence causing a decreased phosphorylation level of tyrosine residues at integrin β3 cytoplasmic domain. Thus, in platelets, the interaction of the RGT sequence in integrin β3 cytoplasmic tail with Src kinase is necessary and sufficient to transduce outside-in signals while the disassociation of Src from integrin β3 has no regulatory effect on talin binding to β3 cytoplasmic domain. The application of RGT peptide and derivatives in the practice of platelet studies enables us to regulate outside-in signaling without affecting inside-out pathway and to speculate the better understanding of the molecular basis of mechanisms for integrin signal transdution. These results also underline the potential use of RGT peptide as a new antithrombotic strategy.
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Mahabaleshwar, Ganapati H., David R. Philips, and Tatiana V. Byzova. "Beta 3 Integrin Phosphorylation and VEGF Signaling." Blood 106, no. 11 (November 16, 2005): 532. http://dx.doi.org/10.1182/blood.v106.11.532.532.
Full textAbstract:
Abstract The process of angiogenesis is critical for tumor survival as well as for ischemic remodeling of vasculature after thrombotic occlusion. This process is a result of finely tuned co-operation between angiogenic growth factors, primarily from the VEGF family, and their receptors and cell receptors for extracellular matrix, the integrins. The role of alpha v beta 3 integrin in neovasculature development appears to be controversial asthe blockade of this receptor with antibodies inhibited angiogenesis while the genetic ablation of the beta3 subunit resulted in increased angiogenesis due to the over-expression of VEGFR2 on endothelial cells (EC). In order to understand the role of beta 3 integrin in VEGF-induced responses, we utilized knock-in mice with a mutation within the beta 3 subunit (Y747/759 to F, DiYF) that results in beta 3 that is unable to undergo tyrosine phosphorylation, an event critical for integrin outside-in signaling. Using EC isolated from WT and DiYF mice, we have found that defective tyrosine phosphorylation resulted in impaired EC adhesion and spreading on vitronectin but not on fibronectin, laminin and collagen. Migration in response to VEGF was reduced in DIYF EC by 3 fold compared to WT when vitronectin and fibrinogen but not fibronectin and collagen were used as substrates. In a wound healing assay on vitronectin, DiYF but not WT EC failed to close the wound. Video microscopy of migrating EC revealed that WT EC form forward protrusions and then retract the back of the cell, which results in successful directed migration. DiYF EC also formed protrusions, but could not form stable adhesion and retract the tail of the cell. As a result, DiYF EC were moving forward and then back again, failing to migrate in the direction of the wound. Importantly, DiYF but not WT EC were not able to form complete capillaries on Matrigel in response to VEGF and the average length of EC tubes was 3.5 fold lower compared to WT. Interestingly, the DiYF mutation resulted in impaired integrin activation (inside-out signaling) in response to VEGF as well as PMA based on soluble fibrinogen and WOW-1 binding. We have found that VEGF treatment of WT EC resulted in beta 3 integrin phosphorylation, which occurred 5 minutes after stimulation and reached the maximum at 30 min. In DiYF EC, this response was absent. Most importantly, immediately after VEGF stimulation, VEGFR2 formed a complex with beta 3 integrin. This complex was not observed in DiYF EC. Moreover, phosphorylation of VEGFR2 (but not the total protein level) was significantly reduced in DiYF EC compared to WT. As a result, major intracellular signaling events triggered by VEGF stimulation, such as phoshorylation of Akt, ERK1/2 and p38 MAPK, were severely impaired in DiYF EC. The finding that beta 3 phosphorylation regulates an activation of VEGFR2 and subsequent signaling events in EC provides novel insights on EC biology and mechanisms of VEGF-induced angiogenesis.
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Nygren, Patrik, Lisa M. Span, David T. Moore, Paul C. Billings, and Joel S. Bennett. "Effect Of Lipids On The Sequence-Specific Interactions Of Kindlin-3 and Talin-1 With The Cytosolic Tail Of The Integrin β3 Subunit." Blood 122, no. 21 (November 15, 2013): 3502. http://dx.doi.org/10.1182/blood.v122.21.3502.3502.
Full textAbstract:
Abstract Activating the platelet integrin αIIbβ3 is an essential step for primary hemostasis. Physiologic αIIbβ3 activation occurs when platelet agonist-generated inside-out signals induce binding of the FERM domains of the cytosolic proteins talin-1 and kindlin-3 to the cytosolic tail (CT) of the β3 subunit of αIIbβ3. While talin-1 binding is thought to activate αIIbβ3 by physically causing separation of the αIIb and β3 cytosolic and transmembrane domains, αIIbβ3 activation in platelets does not occur in the absence of kindlin-3 binding to the β3 CT. Nonetheless, it is unclear whether it is necessary for talin-1 and kindlin-3 to be concurrently bound to the β3 CT in order to activate αIIbβ3, and if that is the case, whether there is a preferred order of binding and whether binding is cooperative. It is noteworthy in this regard that the sequences of the core binding motifs on the β3 CT for the talin-1 and kindlin-3 FERM domains, N744PLY747 and N756ITY759, respectively, are quite similar. To begin to address these questions, we have expressed and purified recombinant forms of the integrin-binding talin-1 head domain (THD) and full-length kindlin-3 and measured their interaction with a peptide corresponding to the β3 CT by surface plasmon resonance (SPR). For these experiments, the β3 CT was anchored to the dextran matrix of a CM5 SPR sensor chip and the equilibrium kinetics of THD and kindlin-3 binding was measured. Analysis of the THD binding data was compatible with two classes of binding sites, a high affinity site with a Kd of 155 nM and a low affinity site with a Kd of 3.5 µM. Similar analysis of kindlin-3 binding was also consistent with two classes of binding sites, a high affinity site with a Kd of 5 nM and a lower affinity site with a Kd of 2.2 µM. Next, we tested the effect of mutating the core binding motifs for the THD and kindlin-3 on the β3 CT. We found that replacing Y759 in the core kindlin-3 binding motif with Ala eliminated high affinity kindlin-3 binding, whereas replacing Y747 in the core THD binding motif with Ala eliminated low affinity kindlin-3 binding. Conversely, the Y747A replacement eliminated high affinity THD binding, while the Y759A replacement eliminated low affinity THD binding. Thus, these experiments demonstrate that the talin-1 and kindlin-3 FERM domains each recognize the general NXXY motif, but their high affinity interactions with this motif are highly sequence-specific. Previously, we found that appending the β3 CT to acidic phospholipids increased its affinity for the THD by three orders of magnitude, likely through interactions involving an extended positively-charged surface on the THD F2 and F3 subdomains. Further, kindlins contain a pleckstrin homology domain with a conserved lipid-binding loop that has been found to be essential for αIIbβ3 activation. Accordingly, we investigated the effect on THD and kindlin-3 binding of tethering the β3 CT to DOPC-coated L1 SPR chips. Unexpectedly, we found that when the β3 CT was tethered to lipid, the Kd for THD binding increased to 430 nM, comparable to the Kd we previously found using isothermal titration calorimetry for THD binding to the β3 CT appended to liposomes. We also found that kindlin-3 binding to the β3 CT tethered to lipids was unexpectedly weaker than binding in the absence of lipid, but it remained approximately 3-fold stronger than THD binding under the same conditions. Previous NMR and hydrogen-deuterium exchange studies of the β3 CT appended to liposomes have revealed that the regions of the β3 CT containing the THD and kindlin-3 binding sites consist of two dynamic amphiphilic helices that are stabilized by interacting with lipid bilayers. Thus, the results presented here suggest that the folding of the β3 CT and the interaction of the folded structure with lipids are important determinants of the strength of the interaction of the THD and kindlin-3 with the β3 CT and consequently are important factors in the regulation of αIIbβ3 activation. Disclosures: No relevant conflicts of interest to declare.
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Gao, Xiu Kui, Zu Kang Sheng, Ye Hong Lu, Yu Ting Sun, Xi Sheng Rao, Lin Jing Shi, Xiao Xia Cong, et al. "VAPB-mediated ER-targeting stabilizes IRS-1 signalosomes to regulate insulin/IGF signaling." Cell Discovery 9, no. 1 (August 1, 2023). http://dx.doi.org/10.1038/s41421-023-00576-6.
Full textAbstract:
AbstractThe scaffold protein IRS-1 is an essential node in insulin/IGF signaling. It has long been recognized that the stability of IRS-1 is dependent on its endomembrane targeting. However, how IRS-1 targets the intracellular membrane, and what type of intracellular membrane is actually targeted, remains poorly understood. Here, we found that the phase separation-mediated IRS-1 puncta attached to endoplasmic reticulum (ER). VAPB, an ER-anchored protein that mediates tethers between ER and membranes of other organelles, was identified as a direct interacting partner of IRS-1. VAPB mainly binds active IRS-1 because IGF-1 enhanced the VAPB-IRS-1 association and replacing of the nine tyrosine residues of YXXM motifs disrupted the VAPB-IRS-1 association. We further delineated that the Y745 and Y746 residues in the FFAT-like motif of IRS-1 mediated the association with VAPB. Notably, VAPB targeted IRS-1 to the ER and subsequently maintained its stability. Consistently, ablation of VAPB in mice led to downregulation of IRS-1, suppression of insulin signaling, and glucose intolerance. The amyotrophic lateral sclerosis (ALS)-derived VAPB P56S mutant also impaired IRS-1 stability by interfering with the ER-tethering of IRS-1. Our findings thus revealed a previously unappreciated condensate-membrane contact (CMC), by which VAPB stabilizes the membraneless IRS-1 signalosome through targeting it to ER membrane.
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Xiao, Juanjuan, Lu Zhang, Huijun Yi, Ling Zou, Jianmei Mo, Feng Xue, Jinhua Zheng, et al. "Inhibiting ALK-TOPK signaling pathway promotes cell apoptosis of ALK-positive NSCLC." Cell Death & Disease 13, no. 9 (September 27, 2022). http://dx.doi.org/10.1038/s41419-022-05260-3.
Full textAbstract:
AbstractT-LAK cell-oriented protein kinase (TOPK) is a potential therapeutic target in tumors. However, its role in anaplastic lymphoma kinase (ALK)-positive non-small cell lung cancer (NSCLC) has not been reported. Here, we found that TOPK was highly expressed in ALK-positive NSCLC. Additionally, ALK was identified as another upstream kinase of TOPK by in vitro kinase assay screening. Then, it was proven that ALK phosphorylated TOPK at Y74 in vitro and ex vivo, and the pathways downstream of ALK-TOPK were explored by phosphoproteomic analysis. Subsequently, we demonstrated that inhibiting TOPK enhanced tumor sensitivity to alectinib (an ALK inhibitor). The combination of alectinib and HI-032 (a TOPK inhibitor) suppressed the growth and promoted the apoptosis of ALK-positive NSCLC cells ex vivo and in vivo. Our findings reveal a novel ALK-TOPK signaling pathway in ALK-positive NSCLC. The combination of alectinib and HI-032 might be a promising therapeutic strategy for improving the sensitivity of ALK-positive NSCLC to targeted therapy.
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"SARS-CoV-2 ORF8 gene CAA=TAA and AAA=TAA Termination Codon Mutations found mostly in B.1.1.7 Variants was Independent of Popular L84S Point Mutations." International Journal of Clinical and Medical Education Research 1, no. 6 (December 28, 2022). http://dx.doi.org/10.33140/ijcmer.01.06.01.
Full textAbstract:
Five VOCs of SARS-CoV-2 mainly caused million deaths worldwide and named as B.1.1.7 (U.K.), B.1.351 (South Africa), P.1 (Brazil), B.1.617.2 (India), and B.1.1.529 (Africa). In HIV mediated pathogenesis, small trans activator proteins (TAT, NEF, REV) modulate transcription of cellular genes. Similarly, preliminary reports indicated that corona virus ORF8 protein acts as histone mimics disrupting chromatic structure with many epigenetic changes and immune modulator functions. ORF8 protein had also some similarities to immunoglobulin domains and inhibited HMC-1 and IFN-beta functions. During evolution a 382-nucleotide deletion (∆382) in the ORF8 region of the corona virus genome leads to weak virus load and weak pathogenicity (accession no.MT374101). We BLAST searched deletion boundary and was selected few ORF8 protein truncated mutants. The C>T base change at 27972nt and another A>T base change at 28095nt created two termination codons (CAA=TAA and AAA=TAA) to produce 26AA and 67AA long ORF8 truncated proteins. Similar Blast-N search with oligonucleotides selected at the mutation boundaries gave many ORF8 mutants with distinct S24L, V32L, P38S, R52I, A65V, Y73C, L84S, K92E and V100L mutations with or without TAA termination mutations. Major mutations found in B.1.1.7 lineage which had spike 69HV and 145Y mutations and ORF1ab polyprotein 3675KSF deletion. However, one ORF8 mutant (accession no. OW221449) belongs to Omicron BA.2 variant with 24LPP spike deletion and others to Omicron BA.5 variants (accession nos. OP733645 and OP671680) with 24LPP and 69HV deletion in the spike protein. One termination codon mutant (accession no. OP711842) has also 63nt ORF7a/b deletions. Mutation did not change the hairpin structure in the ORF8 gene and ORF8 protein formed dimeric stable globular 3-D structure to interact with many host proteins. Clearly, generation of such abundant B.1.1.7 lineage ORF8 protein truncated mutants may be one of the causes for the extinction of Alpha variant of corona virus in 2021. Roles of ORF8 mutants as host proteins modulator were explained in light of other deletions and mutations in corona virus genome.