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.

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 Villin Severing Activity Enhances Actin-based Motility In Vivo

2007 ◽  
Vol 18 (3) ◽  
pp. 827-838 ◽  
Author(s):  
Céline Revenu ◽  
Matthieu Courtois ◽  
Alphée Michelot ◽  
Cécile Sykes ◽  
Daniel Louvard ◽  
...  
Keyword(s):  
Shigella Flexneri ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Loss Of Function ◽  
Mesenchymal Transition ◽  
Capping Protein ◽  
Function Strategy ◽  
In Cells

Villin, an actin-binding protein associated with the actin bundles that support microvilli, bundles, caps, nucleates, and severs actin in a calcium-dependant manner in vitro. We hypothesized that the severing activity of villin is responsible for its reported role in enhancing cell plasticity and motility. To test this hypothesis, we chose a loss of function strategy and introduced mutations in villin based on sequence comparison with CapG. By pyrene-actin assays, we demonstrate that this mutant has a strongly reduced severing activity, whereas nucleation and capping remain unaffected. The bundling activity and the morphogenic effects of villin in cells are also preserved in this mutant. We thus succeeded in dissociating the severing from the three other activities of villin. The contribution of villin severing to actin dynamics is analyzed in vivo through the actin-based movement of the intracellular bacteria Shigella flexneri in cells expressing villin and its severing variant. The severing mutations abolish the gain of velocity induced by villin. To further analyze this effect, we reconstituted an in vitro actin-based bead movement in which the usual capping protein is replaced by either the wild type or the severing mutant of villin. Confirming the in vivo results, villin-severing activity enhances the velocity of beads by more than two-fold and reduces the density of actin in the comets. We propose a model in which, by severing actin filaments and capping their barbed ends, villin increases the concentration of actin monomers available for polymerization, a mechanism that might be paralleled in vivo when an enterocyte undergoes an epithelio-mesenchymal transition.

scholarly journals The Ubiquitously Expressed Csk Adaptor Protein Cbp Is Dispensable for Embryogenesis and T-Cell Development and Function

2005 ◽  
Vol 25 (23) ◽  
pp. 10533-10542 ◽  
Author(s):  
Marc-Werner Dobenecker ◽  
Christian Schmedt ◽  
Masato Okada ◽  
Alexander Tarakhovsky
Keyword(s):  
T Cell ◽  
Adaptor Protein ◽  
Regulatory Function ◽  
Loss Of Function ◽  
Thymic Development ◽  
Cell Functions ◽  
Carboxy Terminal ◽  
And Function

ABSTRACT Regulation of Src family kinase (SFK) activity is indispensable for a functional immune system and embryogenesis. The activity of SFKs is inhibited by the presence of the carboxy-terminal Src kinase (Csk) at the cell membrane. Thus, recruitment of cytosolic Csk to the membrane-associated SFKs is crucial for its regulatory function. Previous studies utilizing in vitro and transgenic models suggested that the Csk-binding protein (Cbp), also known as phosphoprotein associated with glycosphingolipid microdomains (PAG), is the membrane adaptor for Csk. However, loss-of-function genetic evidence to support this notion was lacking. Herein, we demonstrate that the targeted disruption of the cbp gene in mice has no effect on embryogenesis, thymic development, or T-cell functions in vivo. Moreover, recruitment of Csk to the specialized membrane compartment of “lipid rafts” is not impaired by Cbp deficiency. Our results indicate that Cbp is dispensable for the recruitment of Csk to the membrane and that another Csk adaptor, yet to be discovered, compensates for the loss of Cbp.

In Vitro Biochemical Characterization of Cytokinesis Actin-Binding Proteins

2016 ◽  
pp. 151-179 ◽  
Author(s):  
Dennis Zimmermann ◽  
Alisha N. Morganthaler ◽  
David R. Kovar ◽  
Cristian Suarez
Keyword(s):  
Binding Proteins ◽  
Biochemical Characterization ◽  
Actin Binding ◽  
Actin Binding Proteins

scholarly journals Dysbindin deficiency Alters Cardiac BLOC-1 Complex and Myozap Levels in Mice

Cells ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 2390
Author(s):  
Ankush Borlepawar ◽  
Nesrin Schmiedel ◽  
Matthias Eden ◽  
Lynn Christen ◽  
Alexandra Rosskopf ◽  
...  
Keyword(s):  
Direct Interaction ◽  
Cardiomyocyte Hypertrophy ◽  
Loss Of Function ◽  
Interaction Partners ◽  
Lysosome Related Organelles ◽  
The Stability ◽  
Schizophrenia Susceptibility

Dysbindin, a schizophrenia susceptibility marker and an essential constituent of BLOC-1 (biogenesis of lysosome-related organelles complex-1), has recently been associated with cardiomyocyte hypertrophy through the activation of Myozap-RhoA-mediated SRF signaling. We employed sandy mice (Dtnbp1_KO), which completely lack Dysbindin protein because of a spontaneous deletion of introns 5–7 of the Dtnbp1 gene, for pathophysiological characterization of the heart. Unlike in vitro, the loss-of-function of Dysbindin did not attenuate cardiac hypertrophy, either in response to transverse aortic constriction stress or upon phenylephrine treatment. Interestingly, however, the levels of hypertrophy-inducing interaction partner Myozap as well as the BLOC-1 partners of Dysbindin like Muted and Pallidin were dramatically reduced in Dtnbp1_KO mouse hearts. Taken together, our data suggest that Dysbindin’s role in cardiomyocyte hypertrophy is redundant in vivo, yet essential to maintain the stability of its direct interaction partners like Myozap, Pallidin and Muted.

Biochemical characterization of bona fide polycystin-1 in vitro and in vivo

2001 ◽  
Vol 38 (6) ◽  
pp. 1421-1429 ◽  
Author(s):  
Alessandra Boletta ◽  
Feng Qian ◽  
Luiz F. Onuchic ◽  
Alessandra Bragonzi ◽  
Marina Cortese ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Bona Fide

scholarly journals Characterization of a Spontaneous 9.5-Kilobase-Deletion Mutant of Murine Gammaherpesvirus 68 Reveals Tissue-Specific Genetic Requirements for Latency

2002 ◽  
Vol 76 (13) ◽  
pp. 6532-6544 ◽  
Author(s):  
Eric T. Clambey ◽  
Herbert W. Virgin ◽  
Samuel H. Speck
Keyword(s):  
Deletion Mutant ◽  
Latent Infection ◽  
Loss Of Function ◽  
Murine Gammaherpesvirus ◽  
Peritoneal Cells ◽  
Gammaherpesvirus 68 ◽  
Murine Gammaherpesvirus 68

ABSTRACT Murine gammaherpesvirus 68 (γHV68 [also known as MHV-68]) establishes a latent infection in mice, providing a small-animal model with which to identify host and viral factors that regulate gammaherpesvirus latency. While γHV68 establishes a latent infection in multiple tissues, including splenocytes and peritoneal cells, the requirements for latent infection within these tissues are poorly defined. Here we report the characterization of a spontaneous 9.5-kb-deletion mutant of γHV68 that lacks the M1, M2, M3, and M4 genes and eight viral tRNA-like genes. Previously, this locus has been shown to contain the latency-associated M2, M3, and viral tRNA-like genes. Through characterization of this mutant, we found that the M1, M2, M3, M4 genes and the viral tRNA-like genes are dispensable for (i) in vitro replication and (ii) the establishment and maintenance of latency in vivo and reactivation from latency following intraperitoneal infection. In contrast, following intranasal infection with this mutant, there was a defect in splenic latency at both early and late times, a phenotype not observed in peritoneal cells. These results indicate (i) that there are different genetic requirements for the establishment of latency in different latent reservoirs and (ii) that the genetic requirements for latency depend on the route of infection. While some of these phenotypes have been observed with specific mutations in the M1 and M2 genes, other phenotypes have never been observed with the available γHV68 mutants. These studies highlight the importance of loss-of-function mutations in defining the genetic requirements for the establishment and maintenance of herpesvirus latency.

scholarly journals Isolation and characterization of a regulated form of actin depolymerizing factor

1993 ◽  
Vol 122 (3) ◽  
pp. 623-633 ◽  
Author(s):  
TE Morgan ◽  
RO Lockerbie ◽  
LS Minamide ◽  
MD Browning ◽  
JR Bamburg
Keyword(s):  
Muscle Development ◽  
Peptide Mapping ◽  
Actin Binding ◽  
Actin Assembly ◽  
Actin Depolymerizing Factor ◽  
Isolation And Characterization ◽  
Relative Molecular Weight

Actin depolymerizing factor (ADF) is an 18.5-kD protein with pH-dependent reciprocal F-actin binding and severing/depolymerizing activities. We previously showed developing muscle down-regulates ADF (J. R. Bamburg and D. Bray. 1987. J. Cell Biol. 105: 2817-2825). To further study this process, we examined ADF expression in chick myocytes cultured in vitro. Surprisingly, ADF immunoreactivity increases during the first 7-10 d in culture. This increase is due to the presence of a new ADF species with higher relative molecular weight which reacts identically to brain ADF with antisera raised against either brain ADF or recombinant ADF. We have purified both ADF isoforms from myocytes and have shown by peptide mapping and partial sequence analysis that the new isoform is structurally related to ADF. Immunoprecipitation of both isoforms from extracts of cells prelabeled with [32P]orthophosphate showed that the new isoform is radiolabeled, predominantly on a serine residue, and hence is called pADF. pADF can be converted into a form which comigrates with ADF on 1-D and 2-D gels by treatment with alkaline phosphatase. pADF has been quantified in a number of cells and tissues where it is present from approximately 18% to 150% of the amount of unphosphorylated ADF. pADF, unlike ADF, does not bind to G-actin, or affect the rate or extent of actin assembly. Four ubiquitous protein kinases failed to phosphorylate ADF in vitro suggesting that ADF phosphorylation in vivo is catalyzed by a more specific kinase. We conclude that the ability to regulate ADF activity is important to muscle development since myocytes have both pre- and posttranslational mechanisms for regulating ADF activity. The latter mechanism is apparently a general one for cell regulation of ADF activity.

scholarly journals RNA triphosphatase and guanylyl transferase activities are associated with the RNA polymerase protein L of rinderpest virus

2009 ◽  
Vol 90 (7) ◽  
pp. 1748-1756 ◽  
Author(s):  
M. Gopinath ◽  
M. S. Shaila
Keyword(s):  
Biochemical Characterization ◽  
Covalent Bond ◽  
Rinderpest Virus ◽  
Viral Mrnas ◽  
L Protein ◽  
Rna Triphosphatase ◽  
Rnp Complex

Rinderpest virus (RPV) large (L) protein is an integral part of the ribonucleoprotein (RNP) complex of the virus that is responsible for transcription and replication of the genome. Previously, we have shown that recombinant L protein coexpressed along with P protein (as the L–P complex) catalyses the synthesis of all viral mRNAs in vitro and the abundance of mRNAs follows a gradient of polarity, similar to the occurrence in vivo. In the present work, we demonstrate that the viral mRNAs synthesized in vitro by the recombinant L or purified RNP are capped and methylated at the N7 guanine position. RNP from the purified virions, as well as recombinant L protein, shows RNA triphosphatase (RTPase) and guanylyl transferase (GT) activities. L protein present in the RNP complex catalyses the removal of γ-phosphate from triphosphate-ended 25 nt RNA generated in vitro representing the viral N-terminal mRNA 5′ sequence. The L protein forms a covalent enzyme–guanylate intermediate with the GMP moiety of GTP, whose formation is inhibited by the addition of pyrophosphate; thus, it exhibits characteristics of cellular GTs. The covalent bond between the enzyme and nucleotide is acid labile and alkali stable, indicating the presence of phosphoamide linkage. The C-terminal region (aa 1717–2183) of RPV L protein alone exhibits the first step of GT activity needed to form a covalent complex with GMP, though it lacks the ability to transfer GMP to substrate RNA. Here, we describe the biochemical characterization of the newly found RTPase/GT activity of L protein.

scholarly journals Genetic and Biochemical Characterization of the Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) Synthase in Haloferax mediterranei

2008 ◽  
Vol 190 (12) ◽  
pp. 4173-4180 ◽  
Author(s):  
Qiuhe Lu ◽  
Jing Han ◽  
Ligang Zhou ◽  
Jian Zhou ◽  
Hua Xiang
Keyword(s):  
Biochemical Characterization ◽  
Complete Loss ◽  
Pha Synthase ◽  
Significant Activity ◽  
Class Iii ◽  
Haloferax Mediterranei ◽  
Thermal Asymmetric Interlaced Pcr

ABSTRACT The haloarchaeon Haloferax mediterranei has shown promise for the economical production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), a desirable bioplastic. However, little is known at present about the genes involved in PHBV synthesis in the domain Archaea. In this study, we cloned the gene cluster (phaEC Hme) encoding a polyhydroxyalkanoate (PHA) synthase in H. mediterranei CGMCC 1.2087 via thermal asymmetric interlaced PCR. Western blotting revealed that the phaE Hme and phaC Hme genes were constitutively expressed, and both the PhaEHme and PhaCHme proteins were strongly bound to the PHBV granules. Interestingly, CGMCC 1.2087 could synthesize PHBV in either nutrient-limited medium (supplemented with 1% starch) or nutrient-rich medium, up to 24 or 18% (wt/wt) in shaking flasks. Knockout of the phaEC Hme genes in CGMCC 1.2087 led to a complete loss of PHBV synthesis, and only complementation with the phaEC Hme genes together (but not either one alone) could restore to this mutant the capability for PHBV accumulation. The known haloarchaeal PhaC subunits are much longer at their C termini than their bacterial counterparts, and the C-terminal extension of PhaCHme was proven to be indispensable for its function in vivo. Moreover, the mixture of purified PhaEHme/PhaCHme (1:1) showed significant activity of PHA synthase in vitro. Taken together, our results indicated that a novel member of the class III PHA synthases, composed of PhaCHme and PhaEHme, accounted for the PHBV synthesis in H. mediterranei.

Sign in / Sign up

Export Citation Format

Share Document