Small-molecule agonists of SHIP1 inhibit the phosphoinositide 3-kinase pathway in hematopoietic cells

Blood ◽  
2007 ◽  
Vol 110 (6) ◽  
pp. 1942-1949 ◽  
Author(s):  
Christopher J. Ong ◽  
Andrew Ming-Lum ◽  
Matt Nodwell ◽  
Ali Ghanipour ◽  
Lu Yang ◽  
...  
Keyword(s):  
Small Molecule ◽  
Hematopoietic Cells ◽  
Pi3k Pathway ◽  
Cellular Activation ◽  
Phosphoinositide 3 Kinase ◽  
Identification And Characterization ◽  
Kinase Pathway

Abstract Because phosphoinositide 3-kinase (PI3K) plays a central role in cellular activation, proliferation, and survival, pharmacologic inhibitors targeting components of the PI3K pathway are actively being developed as therapeutics for the treatment of inflammatory disorders and cancer. These targeted drugs inhibit the activity of either PI3K itself or downstream protein kinases. However, a previously unexplored, alternate strategy is to activate the negative regulatory phosphatases in this pathway. The SH2-containing inositol-5′-phosphatase SHIP1 is a normal physiologic counter-regulator of PI3K in immune/hematopoietic cells that hydrolyzes the PI3K product phosphatidylinositiol-3,4,5-trisphosphate (PIP3). We now describe the identification and characterization of potent and specific small-molecule activators of SHIP1. These compounds represent the first small-molecule activators of a phosphatase, and are able to activate recombinant SHIP1 enzyme in vitro and stimulate SHIP1 activity in intact macrophage and mast cells. Mechanism of activation studies with these compounds suggest that they bind a previously undescribed, allosteric activation domain within SHIP1. Furthermore, in vivo administration of these compounds was protective in mouse models of endotoxemia and acute cutaneous anaphylaxis, suggesting that SHIP1 agonists could be used therapeutically to inhibit the PI3K pathway.

scholarly journals The Human FSGS-Causing ANLN R431C Mutation Induces Dysregulated PI3K/AKT/mTOR/Rac1 Signaling in Podocytes

2018 ◽  
Vol 29 (8) ◽  
pp. 2110-2122 ◽  
Author(s):  
Gentzon Hall ◽  
Brandon M. Lane ◽  
Kamal Khan ◽  
Igor Pediaditakis ◽  
Jianqiu Xiao ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Adaptor Protein ◽  
Pi3k Pathway ◽  
Actin Binding ◽  
Loss Of Function ◽  
Signaling Axis ◽  
Phosphoinositide 3 Kinase

BackgroundWe previously reported that mutations in the anillin (ANLN) gene cause familial forms of FSGS. ANLN is an F-actin binding protein that modulates podocyte cell motility and interacts with the phosphoinositide 3-kinase (PI3K) pathway through the slit diaphragm adaptor protein CD2-associated protein (CD2AP). However, it is unclear how the ANLN mutations cause the FSGS phenotype. We hypothesized that the R431C mutation exerts its pathogenic effects by uncoupling ANLN from CD2AP.MethodsWe conducted in vivo complementation assays in zebrafish to determine the effect of the previously identified missense ANLN variants, ANLNR431C and ANLNG618C during development. We also performed in vitro functional assays using human podocyte cell lines stably expressing wild-type ANLN (ANLNWT) or ANLNR431C.ResultsExperiments in anln-deficient zebrafish embryos showed a loss-of-function effect for each ANLN variant. In human podocyte lines, expression of ANLNR431C increased cell migration, proliferation, and apoptosis. Biochemical characterization of ANLNR431C-expressing podocytes revealed hyperactivation of the PI3K/AKT/mTOR/p70S6K/Rac1 signaling axis and activation of mTOR-driven endoplasmic reticulum stress in ANLNR431C-expressing podocytes. Inhibition of mTOR, GSK-3β, Rac1, or calcineurin ameliorated the effects of ANLNR431C. Additionally, inhibition of the calcineurin/NFAT pathway reduced the expression of endogenous ANLN and mTOR.ConclusionsThe ANLNR431C mutation causes multiple derangements in podocyte function through hyperactivation of PI3K/AKT/mTOR/p70S6K/Rac1 signaling. Our findings suggest that the benefits of calcineurin inhibition in FSGS may be due, in part, to the suppression of ANLN and mTOR. Moreover, these studies illustrate that rational therapeutic targets for familial FSGS can be identified through biochemical characterization of dysregulated podocyte phenotypes.

scholarly journals Oligonucleotide uptake in human hematopoietic cells is increased in leukemia and is related to cellular activation

Blood ◽  
1996 ◽  
Vol 88 (5) ◽  
pp. 1788-1795 ◽  
Author(s):  
Q Zhao ◽  
X Song ◽  
T Waldschmidt ◽  
E Fisher ◽  
AM Krieg
Keyword(s):  
T Cells ◽  
Cell Activation ◽  
Hematopoietic Cells ◽  
Leukemic Cells ◽  
Cellular Activation ◽  
Therapeutic Drugs ◽  
Normal Human ◽  
Novel Strategy

Abstract The use of antisense oligonucleotides as tools for modulating gene expression represents a novel strategy for designing drugs to treat a variety of diseases. Several factors, including cellular uptake and internalization of the oligonucleotides, are important parameters in determining the effectiveness of antisense agents such as therapeutic drugs. We have studied oligonucleotides uptake in normal and leukemic human hematopoietic cells, such as peripheral blood, bone marrow (BM), and HL-60 cell line; and have found that, in normal human blood and BM, myeloid cells and B cells preferably took up more oligonucleotides than T cells. There was no marked difference in oligonucleotide uptake between CD4+ helper T cells and CD8+ cytolytic T cells. Leukemic cells had greater oligonucleotide uptake than their normal counterparts. Furthermore, oligonucleotide uptake was closely related to cell activation status and can be modulated by growth factors or inhibitors. These studies provide a basis for using oligonucleotides as therapeutic drugs both in vitro and in vivo.

scholarly journals Identification and characterization of T reg–like cells in zebrafish

2017 ◽  
Vol 214 (12) ◽  
pp. 3519-3530 ◽  
Author(s):  
Melissa Kasheta ◽  
Corrie A. Painter ◽  
Finola E. Moore ◽  
Riadh Lobbardi ◽  
Alysia Bryll ◽  
...  
Keyword(s):  
T Lymphocytes ◽  
Live Cell Imaging ◽  
Genetic Ablation ◽  
Functional Studies ◽  
Normal Functions ◽  
Inflammatory Phenotype ◽  
Identification And Characterization

Regulatory T (T reg) cells are a specialized sublineage of T lymphocytes that suppress autoreactive T cells. Functional studies of T reg cells in vitro have defined multiple suppression mechanisms, and studies of T reg–deficient humans and mice have made clear the important role that these cells play in preventing autoimmunity. However, many questions remain about how T reg cells act in vivo. Specifically, it is not clear which suppression mechanisms are most important, where T reg cells act, and how they get there. To begin to address these issues, we sought to identify T reg cells in zebrafish, a model system that provides unparalleled advantages in live-cell imaging and high-throughput genetic analyses. Using a FOXP3 orthologue as a marker, we identified CD4-enriched, mature T lymphocytes with properties of T reg cells. Zebrafish mutant for foxp3a displayed excess T lymphocytes, splenomegaly, and a profound inflammatory phenotype that was suppressed by genetic ablation of lymphocytes. This study identifies T reg–like cells in zebrafish, providing both a model to study the normal functions of these cells in vivo and mutants to explore the consequences of their loss.

scholarly journals Identification and Characterization of Two Bordetella avium Gene Products Required for Hemagglutination

2010 ◽  
Vol 78 (6) ◽  
pp. 2370-2376 ◽  
Author(s):  
Louise M. Temple ◽  
David M. Miyamoto ◽  
Manju Mehta ◽  
Christian M. Capitini ◽  
Stephen Von Stetina ◽  
...  
Keyword(s):  
Whooping Cough ◽  
Bioinformatic Analysis ◽  
Tracheal Ring ◽  
Tracheal Cell ◽  
Bordetella Avium ◽  
Identification And Characterization

ABSTRACT Bordetella avium causes bordetellosis in birds, a disease similar to whooping cough caused by Bordetella pertussis in children. B. avium agglutinates guinea pig erythrocytes via an unknown mechanism. Loss of hemagglutination ability results in attenuation. We report the use of transposon mutagenesis to identify two genes required for hemagglutination. The genes (hagA and hagB) were adjacent and divergently oriented and had no orthologs in the genomes of other Bordetella species. Construction of in-frame, unmarked mutations in each gene allowed examination of the role of each in conferring erythrocyte agglutination, explanted tracheal cell adherence, and turkey poult tracheal colonization. In all of the in vitro and in vivo assays, the requirement for the trans-acting products of hagA and hagB (HagA and HagB) was readily shown. Western blotting, using antibodies to purified HagA and HagB, revealed proteins of the predicted sizes of HagA and HagB in an outer membrane-enriched fraction. Antiserum to HagB, but not HagA, blocked B. avium erythrocyte agglutination and explanted turkey tracheal ring binding. Bioinformatic analysis indicated the similarity of HagA and HagB to several two-component secretory apparatuses in which one product facilitates the exposition of the other. HagB has the potential to serve as a useful immunogen to protect turkeys against colonization and subsequent disease.

scholarly journals A Ral GAP complex links PI 3-kinase/Akt signaling to RalA activation in insulin action

2011 ◽  
Vol 22 (1) ◽  
pp. 141-152 ◽  
Author(s):  
Xiao-Wei Chen ◽  
Dara Leto ◽  
Tingting Xiong ◽  
Genggeng Yu ◽  
Alan Cheng ◽  
...  
Keyword(s):  
Glucose Transporter ◽  
Critical Role ◽  
Inhibitory Effect ◽  
Small Gtpase ◽  
Regulatory Subunit ◽  
Hydrolysis Of ◽  
Identification And Characterization

Insulin stimulates glucose transport in muscle  and adipose tissue by translocation of glucose transporter 4 (GLUT4) to the plasma membrane. We previously reported that activation of the small GTPase RalA downstream of PI 3-kinase plays a critical role in this process by mobilizing the exocyst complex for GLUT4 vesicle targeting in adipocytes. Here we report the identification and characterization of a Ral GAP complex (RGC) that mediates the activation of RalA downstream of the PI 3-kinase/Akt pathway. The complex is composed of an RGC1 regulatory subunit and an RGC2 catalytic subunit (previously identified as AS250) that directly stimulates the guanosine triphosphate hydrolysis of RalA. Knockdown of RGC proteins leads to increased RalA activity and glucose uptake in adipocytes. Insulin inhibits the GAP complex through Akt2-catalyzed phosphorylation of RGC2 in vitro and in vivo, while activated Akt relieves the inhibitory effect of RGC proteins on RalA activity. The RGC complex thus connects PI 3-kinase/Akt activity to the transport machineries responsible for GLUT4 translocation.

scholarly journals Computational Insight into the Effect of Natural Compounds on the Destabilization of Preformed Amyloid-β(1-40) Fibrils

2018 ◽  
Author(s):  
Francesco Tavanti ◽  
Alfonso Pedone ◽  
Maria Cristina Menziani
Keyword(s):  
Therapeutic Strategy ◽  
Therapeutic Potential ◽  
Structure Stability ◽  
Insoluble Form ◽  
The Brain ◽  
Identification And Characterization ◽  
Insight Into

One of the principal hallmarks of Alzheimer’s disease (AD) is related to the aggregation of amyloid-β fibrils in an insoluble form in the brain, also known as amyloidosis. Therefore, a prominent therapeutic strategy against AD consists either in blocking the amyloid aggregation and/or destroying the already formed aggregates. Natural products have shown significant therapeutic potential as amyloid inhibitors from in vitro studies as well as in vivo animal tests. In this study, the interaction of five natural biophenols (curcumin, dopamine, (-)-Epigallocatechin-3-gallate, Quercetin, and Rosmarinic acid) with the amyloid-β(1-40) fibrils has been studied through computational simulations. The results allowed the identification and characterization of the different binding modalities of each compounds and their consequences on fibril dynamics and aggregation. It emerges that the lateral aggregation of the fibrils is strongly influenced by the intercalation of the ligands, which modulate the double-layered structure stability.

scholarly journals Targeting the actin nucleation promoting factor WASp provides a therapeutic approach for hematopoietic malignancies

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Guy Biber ◽  
Aviad Ben-Shmuel ◽  
Elad Noy ◽  
Noah Joseph ◽  
Abhishek Puthenveetil ◽  
...  
Keyword(s):  
Small Molecule ◽  
Actin Polymerization ◽  
Hematopoietic Cells ◽  
Actin Nucleation ◽  
Hematopoietic Malignancies ◽  
Nucleation Promoting Factor ◽  
Syndrome Protein ◽  
Cytoskeleton Rearrangement

AbstractCancer cells depend on actin cytoskeleton rearrangement to carry out hallmark malignant functions including activation, proliferation, migration and invasiveness. Wiskott–Aldrich Syndrome protein (WASp) is an actin nucleation-promoting factor and is a key regulator of actin polymerization in hematopoietic cells. The involvement of WASp in malignancies is incompletely understood. Since WASp is exclusively expressed in hematopoietic cells, we performed in silico screening to identify small molecule compounds (SMCs) that bind WASp and promote its degradation. We describe here one such identified molecule; this WASp-targeting SMC inhibits key WASp-dependent actin processes in several types of hematopoietic malignancies in vitro and in vivo without affecting naïve healthy cells. This small molecule demonstrates limited toxicity and immunogenic effects, and thus, might serve as an effective strategy to treat specific hematopoietic malignancies in a safe and precisely targeted manner.

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