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Transient opening of fibronectin type III (FNIII) domains: the interaction of the third FNIII domain of FN with anastellin.

ABSTRACT: We previously reported that the fibronectin (FN) type III domains of FN may unfold to interact with anastellin and form FN aggregates. In the present study, we have focused on the interaction between anastellin and the third FN type III domain (III3), which is a key anastellin binding site on FN. Anastellin binding to III3 was monitored by 8-anilino-1-naphthalene sulfonate (ANS) fluorescence. ANS binding to anastellin dramatically increased its emission intensity, but this was reduced to half by the addition of III3, suggesting that ANS and III3 share a common hydrophobic binding site on anastellin. An engineered mutant of III3 that was stabilized by an intrachain disulfide bond did not interact with anastellin, as seen by its failure to interfere with ANS binding to anastellin. We also mutated hydrophobic core residues to destabilize III3 and found that these mutants were still capable of interacting with anastellin. Anastellin binding to III3 was also monitored using an intramolecular green fluorescent protein (GFP)-based fluorescence resonance energy transfer (FRET) construct, in which III3 was flanked by two GFP variants (III3-FRET). Anastellin bound to III3-FRET and caused an increase in the FRET signal. The dissociation constant was estimated to be approximately 210 nM. The binding kinetics of anastellin to III3-FRET fit a first-order reaction with a half-time of approximately 30 s; the kinetics with destabilized III3 mutants were even faster. Matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry suggested that the middle part of III3 became destabilized and protease sensitive upon anastellin binding. Thus, the stability of III3 seems to be a key factor in anastellin binding.

SUBMITTER: Ohashi T

PROVIDER: S-EPMC2680933 | biostudies-literature | 2009 May

REPOSITORIES: biostudies-literature

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Transient opening of fibronectin type III (FNIII) domains: the interaction of the third FNIII domain of FN with anastellin.

Ohashi Tomoo T Augustus Anne Marie AM Erickson Harold P HP

Biochemistry 20090501 19

We previously reported that the fibronectin (FN) type III domains of FN may unfold to interact with anastellin and form FN aggregates. In the present study, we have focused on the interaction between anastellin and the third FN type III domain (III3), which is a key anastellin binding site on FN. Anastellin binding to III3 was monitored by 8-anilino-1-naphthalene sulfonate (ANS) fluorescence. ANS binding to anastellin dramatically increased its emission intensity, but this was reduced to half by ...[more]

PMID: 19320499

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Project description:BACKGROUND:The histone methyltransferase SETDB1 (also known as ESET) represses genes and various types of transposable elements, such as endogenous retroviruses (ERVs) and integrated exogenous retroviruses, through a deposition of trimethylation on lysine 9 of histone H3 (H3K9me3) in mouse embryonic stem cells (mESCs). ATF7IP (also known as MCAF1 or AM), a binding partner of SETDB1, regulates the nuclear localization and enzymatic activities of SETDB1 and plays a crucial role in SETDB1-mediated transcriptional silencing. In this study, we further dissected the ATF7IP function with its truncated mutants in Atf7ip knockout (KO) mESCs. RESULTS:We demonstrated that the SETDB1-interaction region within ATF7IP is essential for ATF7IP-dependent SETDB1 nuclear localization and silencing of both ERVs and integrated retroviral transgenes, whereas its C-terminal fibronectin type-III (FNIII) domain is dispensable for both these functions; rather, it has a role in efficient silencing mediated by the SETDB1 complex. Proteomic analysis identified a number of FNIII domain-interacting proteins, some of which have a consensus binding motif. We showed that one of the FNIII domain-binding proteins, ZMYM2, was involved in the efficient silencing of a transgene by ATF7IP. RNA-seq analysis of Atf7ip KO and WT or the FNIII domain mutant of ATF7IP-rescued Atf7ip KO mESCs showed that the FNIII domain mutant re-silenced most de-repressed SETDB1/ATF7IP-targeted ERVs compared to the WT. However, the silencing activity of the FNIII domain mutant was weaker than that of the ATF7IP WT, and some of the de-repressed germ cell-related genes in Atf7ip KO mESCs were not silenced by the FNIII domain mutant. Such germ cell-related genes are targeted and silenced by the MAX/MGA complex, and MGA was also identified as another potential binding molecule of the ATF7IP FNIII domain in the proteomic analysis. This suggests that the FNIII domain of ATF7IP acts as a binding hub of ATF7IP-interacting molecules possessing a specific interacting motif we named FAM and contributes to one layer of the SETDB1/ATF7IP complex-mediated silencing mechanisms. CONCLUSIONS:Our findings contributed to further understanding the function of ATF7IP in the SETDB1 complex, revealed the role of the FNIII domain of ATF7IP in transcriptional silencing, and suggested a potential underlying molecular mechanism for it.

Project description:BackgroundElevated intraocular pressure (IOP) is a major risk factor for the development and progression of primary open angle glaucoma and is due to trabecular meshwork (TM) damage. Here, we investigate the role of an endogenous Toll-like receptor 4 (TLR4) ligand, FN-EDA, in the development of glaucoma utilizing a transgenic mouse strain (B6.EDA+/+) that constitutively expresses only FN containing the EDA isoform.MethodsEyes from C57BL6/J (wild-type), B6.EDA+/+ (constitutively active EDA), B6.EDA-/- (EDA null) mice were processed for electron microscopy and consecutive images of the entire length of the TM and Schlemm's canal (SC) from anterior to posterior were collected and montaged into a single image. ECM accumulation, basement membrane length, and size and number of giant vacuoles were quantified by ImageJ analysis. Tlr4 and Iba1 expression in the TM and ONH cells was conducted using RNAscope in situ hybridization and immunohistochemistry protocols. IOP was measured using a rebound tonometer, ON damage assessed by PPD stain, and RGC loss quantified in RBPMS labeled retina flat mounts.ResultsUltrastructure analyses show the TM of B6.EDA+/+ mice have significantly increased accumulation of ECM between TM beams with few empty spaces compared to C57BL/6 J mice (p < 0.05). SC basement membrane is thicker and more continuous in B6.EDA+/+ mice compared to C57BL/6 J. No significant structural differences are detected in the TM of EDA null mice. Tlr4 and Iba1 expression is increased in the TM of B6.EDA+/+ mice compared to C57BL/6 J eyes (p < 0.05). IOP is significantly higher in B6.EDA+/+ mice compared to C57BL/6 J eyes (p < 0.001), and significant ON damage (p < 0.001) and RGC loss (p < 0.05) detected at 1 year of age. Tlr4 mRNA is expressed in mouse ONH cells, and is present in ganglion cell axons, microglia, and astrocytes. There is a significant increase in the area occupied by Iba-1 positive microglia cells in the ONH of B6.EDA+/+ mice compared to C57BL/6 J control eyes (p < 0.01).ConclusionsB6.EDA+/+ mice have increased ECM accumulation in the TM, elevated IOP, enhanced proinflammatory changes in the ONH, loss of RGCs, and ONH damage. These data suggest B6.EDA+/+ mice recapitulate many aspects of glaucomatous damage.

Project description:Angiogenesis is regulated by integrin-dependent cell adhesion and the activation of specific cell surface receptors on vascular endothelial cells by angiogenic factors. Lysophosphatidic acid (LPA) and sphingosine-1 phosphate (S1P) are bioactive lysophospholipids that activate G protein-coupled receptors that stimulate phosphatidylinositol 3-kinase (PI3K), Ras, and Rho effector pathways involved in vascular cell survival, proliferation, adhesion, and migration. Previous studies have shown that anastellin, a fragment of the first type III module of fibronectin, functions as an antiangiogenic peptide suppressing tumor growth and metastasis. We have previously shown that anastellin blocks serum-dependent proliferation of microvessel endothelial cells (MVEC) by affecting extracellular signal-regulated kinase (ERK)-dependent G(1)-S transition. However, the mechanism by which anastellin regulates endothelial cell function remains unclear. In the present study, we mapped several lysophospholipid-mediated signaling pathways in MVEC and examined the effects of anastellin on LPA- and S1P-induced MVEC proliferation, migration, and cytoskeletal organization. Both LPA and S1P activated PI3K, Ras/ERK, and Rho/Rho kinase pathways, leading to migration, G(1)-S cell cycle progression, and stress fiber formation, respectively. Stimulation of proliferation by LPA/S1P occurred through a G(i)-dependent Ras/ERK pathway, which was independent of growth factor receptors and PI3K and Rho/Rho kinase signaling. Although LPA and S1P activated both PI3K/Akt and Ras/ERK signaling through G(i), anastellin inhibited only the Ras/ERK pathway. Stress fiber formation in response to LPA was dependent on Rho/Rho kinase but independent of G(i) and unaffected by anastellin. These results suggest that lysophospholipid mediators of G(i) activation leading to PI3K/Akt and Ras/ERK signaling bifurcate downstream of G(i) and that anastellin selectively inhibits the Ras/ERK arm of the pathway.

Project description:Objective- Alterations in extracellular matrix quantity and composition contribute to atherosclerosis, with remodeling of the subendothelial basement membrane to an FN (fibronectin)-rich matrix preceding lesion development. Endothelial cell interactions with FN prime inflammatory responses to a variety of atherogenic stimuli; however, the mechanisms regulating early atherogenic FN accumulation remain unknown. We previously demonstrated that oxLDL (oxidized low-density lipoprotein) promotes endothelial proinflammatory gene expression by activating the integrin α5β1, a classic mediator of FN fibrillogenesis. Approach and Results- We now show that oxLDL drives robust endothelial FN deposition and inhibiting α5β1 (blocking antibodies, α5 knockout cells) completely inhibits oxLDL-induced FN deposition. Consistent with this, inducible endothelial-specific α5 integrin deletion in ApoE knockout mice significantly reduces atherosclerotic plaque formation, associated with reduced early atherogenic inflammation. Unlike TGFβ (transforming growth factor β)-induced FN deposition, oxLDL does not induce FN expression (mRNA, protein) or the endothelial-to-mesenchymal transition phenotype. In addition, we show that cell-derived and plasma-derived FN differentially affect endothelial function, with only cell-derived FN capable of supporting oxLDL-induced VCAM-1 (vascular cell adhesion molecule 1) expression, despite plasma FN deposition by oxLDL. The inclusion of alternative exon EIIIA (EDA) of FN (EIIIA) and alternative exon EIIIB (EDB) of FN (EIIIB) domains in cell-derived FN mediates this effect, as EIIIA/EIIIB knockout endothelial cells show diminished oxLDL-induced inflammation. Furthermore, our data suggest that EIIIA/EIIIB-positive cellular FN is required for maximal α5β1 recruitment to focal adhesions and FN fibrillogenesis. Conclusions- Taken together, our data demonstrate that endothelial α5 integrins drive oxLDL-induced FN deposition and early atherogenic inflammation. Additionally, we show that α5β1-dependent endothelial FN deposition mediates oxLDL-dependent endothelial inflammation and FN fibrillogenesis.

Dataset Information

Transient opening of fibronectin type III (FNIII) domains: the interaction of the third FNIII domain of FN with anastellin.

Publications

Transient opening of fibronectin type III (FNIII) domains: the interaction of the third FNIII domain of FN with anastellin.

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