Phospholipase D (PLD) signaling plays a critical role in cell growth and proliferation, vesicular trafficking, secretion, and endocytosis. At the cellular level, PLD and its reaction product, phosphatidate, interact with a large number of protein partners that are directly related to the actin cytoskeleton and cell migration. Cancer invasion and metastasis rely heavily on cellular motility, and as such, they have put PLD at center stage in cancer research. This minireview series highlights some of the molecular mechanisms that provide evidence for the emerging tumorigenic potential of PLD, the role of the microenvironment, and putative connections with inflammation. PLD represents a potential target for the rational development of therapeutics against cancer and other diseases.
Phospholipase D (PLD) enzymes play a double vital role in cells: they maintain the integrity of cellular membranes and they participate in cell signaling including intracellular protein trafficking, cytoskeletal dynamics, cell migration, and cell proliferation. The particular involvement of PLD in cell migration is accomplished: (a) through the actions of its enzymatic product of reaction, phosphatidic acid, and its unique shape-binding role on membrane geometry; (b) through a particular guanine nucleotide exchange factor (GEF) activity (the first of its class assigned to a phospholipase) in the case of the mammalian isoform PLD2; and (c) through protein-protein interactions with a wide network of molecules: Wiskott–Aldrich syndrome protein (WASp), Grb2, ribosomal S6 kinase (S6K), and Rac2. Further, PLD interacts with a variety of kinases (PKC, FES, EGF receptor (EGFR), and JAK3) that are activated by it, or PLD becomes the target substrate. Out of these myriads of functions, PLD is becoming recognized as a major player in cell migration, cell invasion, and cancer metastasis. This is the story of the evolution of PLD from being involved in a large number of seemingly unrelated cellular functions to its most recent role in cancer signaling, a subfield that is expected to grow exponentially.
Phospholipase D enzymes have long been proposed to play multiple cell biological roles in cancer. With the generation of phospholipase D1 (PLD1)-deficient mice and the development of small molecule PLD-specific inhibitors, in vivo roles for PLD1 in cancer are now being defined, both in the tumor cells and in the tumor environment. We review here tools now used to explore in vivo roles for PLD1 in cancer and summarize recent findings regarding functions in angiogenesis and metastasis.
Phospholipase D (PLD) regulates downstream effectors by generating phosphatidic acid. Growing links of dysregulation of PLD to human disease have spurred interest in therapeutics that target its function. Aberrant PLD expression has been identified in multiple facets of complex pathological states, including cancer and inflammatory diseases. Thus, it is important to understand how the signaling network of PLD expression is regulated and contributes to progression of these diseases. Interestingly, small molecule PLD inhibitors can suppress PLD expression as well as enzymatic activity of PLD and have been shown to be effective in pathological mice models, suggesting the potential for use of PLD inhibitors as therapeutics against cancer and inflammation. Here, we summarize recent scientific developments regarding the regulation of PLD expression and its role in cancer and inflammatory processes.
Phosphatidic acid (PA) is a critical metabolite at the heart of membrane phospholipid biosynthesis. However, PA also serves as a critical lipid second messenger that regulates several proteins implicated in the control of cell cycle progression and cell growth. Three major metabolic pathways generate PA: phospholipase D (PLD), diacylglycerol kinase (DGK), and lysophosphatidic acid acyltransferase (LPAAT). The LPAAT pathway is integral to de novo membrane phospholipid biosynthesis, whereas the PLD and DGK pathways are activated in response to growth factors and stress. The PLD pathway is also responsive to nutrients. A key target for the lipid second messenger function of PA is mTOR, the mammalian/mechanistic target of rapamycin, which integrates both nutrient and growth factor signals to control cell growth and proliferation. Although PLD has been widely implicated in the generation of PA needed for mTOR activation, it is becoming clear that PA generated via the LPAAT and DGK pathways is also involved in the regulation of mTOR. In this minireview, we highlight the coordinated maintenance of intracellular PA levels that regulate mTOR signals stimulated by growth factors and nutrients, including amino acids, lipids, glucose, and Gln. Emerging evidence indicates compensatory increases in one source of PA when another source is compromised, highlighting the importance of being able to adapt to stressful conditions that interfere with PA production. The regulation of PA levels has important implications for cancer cells that depend on PA and mTOR activity for survival.
CREPT (cell cycle-related and expression elevated protein in tumor)/RPRD1B (regulation of nuclear pre-mRNA domain-containing protein 1B), highly expressed during tumorigenesis, was shown to enhance transcription of CCND1 and to promote cell proliferation by interacting with RNA polymerase II. However, which signaling pathway is involved in CREPT-mediated activation of gene transcription remains unclear. In this study, we reveal that CREPT participates in transcription of the Wnt/β-catenin signaling activated genes through the β-catenin and the TCF4 complex. Our results demonstrate that CREPT interacts with both β-catenin and TCF4, and enhances the association of β-catenin with TCF4, in response to Wnt stimulation. Furthermore, CREPT was shown to occupy at TCF4 binding sites (TBS) of the promoters of Wnt-targeted genes under Wnt stimulation. Interestingly, depletion of CREPT resulted in decreased occupancy of β-catenin on TBS, and over-expression of CREPT enhances the activity of the β-catenin·TCF4 complex to initiate transcription of Wnt target genes, which results in up-regulated cell proliferation and invasion. Our study suggests that CREPT acts as an activator to promote transcriptional activity of the β-catenin·TCF4 complex in response to Wnt signaling.
Emerging evidence has shown that microRNAs have key roles in regulating various normal physiological processes, whereas their deregulated expression is correlated with various diseases. The miR-146 family includes miR-146a and miR-146b, with a distinct expression spectrum in different hematopoietic cells. Recent work indicated that miR-146a has a close relationship with inflammation and autoimmune diseases. miR-146-deficient mice have developed some abnormal hematopoietic phenotypes, suggesting the potential functions of miR-146 in hematopoietic development. In this study, we found that miR-146b was consistently up-regulated in both K562 and CD34+ hematopoietic stem/progenitor cells (HSPCs) undergoing either erythroid or megakaryocytic differentiation. Remarkably, erythroid and megakaryocytic maturation of K562 cells was induced by excess miR-146b but inhibited by decreased miR-146b levels. More importantly, an mRNA encoding receptor tyrosine kinase, namely platelet-derived growth factor receptor α (PDGFRA), was identified and validated as a direct target of miR-146b in hematopoietic cells. Gain-of-function and loss-of-function assays showed that PDGFRA functioned as a negative regulator in erythroid and megakaryocytic differentiation. miR-146b could ultimately affect the expression of the GATA-1 gene, which is regulated by HEY1 (Hairy/enhancer-of-split related with YRPW motif protein 1), a transcriptional repressor, via inhibition of the PDGFRA/JNK/JUN/HEY1 pathway. Lentivirus-mediated gene transfer also demonstrated that the overexpression of miR-146b promoted erythropoiesis and megakaryocytopoiesis of HSPCs via its regulation on the PDGFRA gene and effects on GATA-1 expression. Moreover, we confirmed that the binding of GATA-1 to the miR-146b promoter and induction of miR-146b during hematopoietic maturation were dependent on GATA-1. Therefore, miR-146b, PDGFRA, and GATA-1 formed a regulatory circuit to promote erythroid and megakaryocytic differentiation.
Yin Yang 1 (YY1) is a transcription factor regulating proliferation and differentiation and is involved in cancer development. Oligomers of recombinant YY1 have been observed before, but their structure and DNA binding properties are not well understood. Here we find that YY1 assembles several homo-oligomeric species built from the association of a bell-shaped dimer, a process we characterized by electron microscopy. Moreover, we find that YY1 self-association also occurs in vivo using bimolecular fluorescence complementation. Unexpectedly, these oligomers recognize several DNA substrates without the consensus sequence for YY1 in vitro, and DNA binding is enhanced in the presence of RuvBL1-RuvBL2, two essential AAA+ ATPases. YY1 oligomers bind RuvBL1-RuvBL2 hetero-oligomeric complexes, but YY1 interacts preferentially with RuvBL1. Collectively, these findings suggest that YY1-RuvBL1-RuvBL2 complexes could contribute to functions beyond transcription, and we show that YY1 and the ATPase activity of RuvBL2 are required for RAD51 foci formation during homologous recombination.
The immune regulatory functions of B cells are not fully understood yet. The present study aims to characterize a subtype of B cells that expresses CX3CR1. In this study, peripheral blood samples were collected from patients with food allergies and healthy subjects. Peripheral B cells were analyzed by flow cytometry. T cell proliferation was assessed by carboxyfluorescein succinimidyl ester dilution assay. The results showed that the CX3CR1+ B cells were detected in the peripheral blood samples of healthy subjects and were significantly less in patients with food allergies. CX3CR1+ B cells expressed high levels of TGF-β and integrin αvβ6. CX3CR1+ B cells could efficiently suppress other effector CD4+ T cell activation. We conclude that human peripheral CX3CR1+ B cells have immune suppressor properties.
Fibrillar type I collagen is the major organic component in bone, providing a stable template for mineralization. During collagen biosynthesis, specific hydroxylysine residues become glycosylated in the form of galactosyl- and glucosylgalactosyl-hydroxylysine. Furthermore, key glycosylated hydroxylysine residues, α1/2-87, are involved in covalent intermolecular cross-linking. Although cross-linking is crucial for the stability and mineralization of collagen, the biological function of glycosylation in cross-linking is not well understood. In this study, we quantitatively characterized glycosylation of non-cross-linked and cross-linked peptides by biochemical and nanoscale liquid chromatography-high resolution tandem mass spectrometric analyses. The results showed that glycosylation of non-cross-linked hydroxylysine is different from that involved in cross-linking. Among the cross-linked species involving α1/2-87, divalent cross-links were glycosylated with both mono- and disaccharides, whereas the mature, trivalent cross-links were primarily monoglycosylated. Markedly diminished diglycosylation in trivalent cross-links at this locus was also confirmed in type II collagen. The data, together with our recent report (Sricholpech, M., Perdivara, I., Yokoyama, M., Nagaoka, H., Terajima, M., Tomer, K. B., and Yamauchi, M. (2012) Lysyl hydroxylase 3-mediated glucosylation in type I collagen: molecular loci and biological significance. J. Biol. Chem. 287, 22998–23009), indicate that the extent and pattern of glycosylation may regulate cross-link maturation in fibrillar collagen.
E1 enzymes activate ubiquitin or ubiquitin-like proteins (Ubl) via an adenylate intermediate and initiate the enzymatic cascade of Ubl conjugation to target proteins or lipids. Ubiquitin-fold modifier 1 (Ufm1) is activated by the E1 enzyme Uba5, and this pathway is proposed to play an important role in the endoplasmic reticulum (ER) stress response. However, the mechanisms of Ufm1 activation by Uba5 and subsequent transfer to the conjugating enzyme (E2), Ufc1, have not been studied in detail. In this work, we found that Uba5 activated Ufm1 via a two-step mechanism and formed a binary covalent complex of Uba5∼Ufm1 thioester. This feature contrasts with the three-step mechanism and ternary complex formation in ubiquitin-activating enzyme Uba1. Uba5 displayed random ordered binding with Ufm1 and ATP, and its ATP-pyrophosphate (PPi) exchange activity was inhibited by both AMP and PPi. Ufm1 activation and Uba5∼Ufm1 thioester formation were stimulated in the presence of Ufc1. Furthermore, binding of ATP to Uba5∼Ufm1 thioester was required for efficient transfer of Ufm1 from Uba5 to Ufc1 via transthiolation. Consistent with the two-step activation mechanism, the mechanism-based pan-E1 inhibitor, adenosine 5′-sulfamate (ADS), reacted with the Uba5∼Ufm1 thioester and formed a covalent, tight-binding Ufm1-ADS adduct in the active site of Uba5, which prevented further substrate binding or catalysis. ADS was also shown to inhibit the Uba5 conjugation pathway in the HCT116 cells through formation of the Ufm1-ADS adduct. This suggests that further development of more selective Uba5 inhibitors could be useful in interrogating the roles of the Uba5 pathway in cells.
Variation in the topology of mitochondrial DNA (mtDNA) in eukaryotes evokes the question if differently structured DNAs are replicated by a common mechanism. RNA-primed DNA synthesis has been established as a mechanism for replicating the circular animal/mammalian mtDNA. In yeasts, circular mtDNA molecules were assumed to be templates for rolling circle DNA-replication. We recently showed that in Candida albicans, which has circular mapping mtDNA, recombination driven replication is a major mechanism for replicating a complex branched mtDNA network. Careful analyses of C. albicans-mtDNA did not reveal detectable amounts of circular DNA molecules. In the present study we addressed the question of how the unit sized linear mtDNA of Candida parapsilosis terminating at both ends with arrays of tandem repeats (mitochondrial telomeres) is replicated. Originally, we expected to find replication intermediates diagnostic of canonical bi-directional replication initiation at the centrally located bi-directional promoter region. However, we found that the linear mtDNA of Candida parapsilosis also employs recombination for replication initiation. The most striking findings were that the mitochondrial telomeres appear to be hot spots for recombination driven replication, and that stable RNA:DNA hybrids, with a potential role in mtDNA replication, are also present in the mtDNA preparations.
VOLUME 287 (2012) PAGES 33198–33214
The curcumin nanoparticle binding assay described failed to indicate that 1 ml of chitosan (5 mg/ml stock at pH 5) was combined with PEG, curcumin, and phosphate-buffered saline in the first step and that hydrolyzed tetramethylorthosilicate (TMOS) was used, not trimethoxysilane. This protocol was based on the platform developed by Friedman et al. (Friedman, A. J., Han, G., Navati, M. S., Chacko, M., Gunther, L., Alfieri, A., and Friedman, J. M. (2008) Sustained release nitric oxide releasing nanoparticles: characterization of a novel delivery platform based on nitrite containing hydrogel/glass composites. Nitric Oxide 19, 12–20).
The curcumin nanoparticles described in Fig. 5B were developed and synthesized by Mahantesh H. Navati and Joel M. Friedman (Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, NY) and Adam J. Friedman (Division of Dermatology, Department of Medicine, Albert Einstein College of Medicine, Bronx, NY). Madiha Sewani also assisted in synthesizing the nanoparticles.
Growing clinical and experimental evidence suggests that sterile inflammation contributes to alcoholic liver disease (ALD). High mobility group box-1 (HMGB1) is highly induced during liver injury; however, a link between this alarmin and ALD has not been established. Thus, the aim of this work was to determine whether HMGB1 contributes to the pathogenesis of ALD. Liver biopsies from patients with ALD showed a robust increase in HMGB1 expression and translocation, which correlated with disease stage, compared with healthy explants. Similar findings were observed in chronic ethanol-fed wild-type (WT) mice. Using primary cell culture, we validated the ability of hepatocytes from ethanol-fed mice to secrete a large amount of HMGB1. Secretion was time- and dose-dependent and responsive to prooxidants and antioxidants. Selective ablation of Hmgb1 in hepatocytes protected mice from alcohol-induced liver injury due to increased carnitine palmitoyltransferase-1, phosphorylated 5′AMP-activated protein kinase-α, and phosphorylated peroxisome proliferator-activated receptor-α expression along with elevated LDL plus VLDL export. Native and post-translationally modified HMGB1 were detected in humans and mice with ALD. In liver and serum from control mice and in serum from healthy volunteers, the lysine residues within the peptides containing nuclear localization signals (NLSs) 1 and 2 were non-acetylated, and all cysteine residues were reduced. However, in livers from ethanol-fed mice, in addition to all thiol/non-acetylated isoforms of HMGB1, we observed acetylated NLS1 and NLS2, a unique phosphorylation site in serine 35, and an increase in oxidation of HMGB1 to the disulfide isoform. In serum from ethanol-fed mice and from patients with ALD, there was disulfide-bonded hyperacetylated HMGB1, disulfide-bonded non-acetylated HMGB1, and HMGB1 phosphorylated in serine 35. Hepatocytes appeared to be a major source of these HMGB1 isoforms. Thus, hepatocyte HMGB1 participates in the pathogenesis of ALD and undergoes post-translational modifications (PTMs) that could condition its toxic effects.
Ribosome formation in Saccharomyces cerevisiae requires a large number of transiently associated assembly factors that coordinate processing and folding of pre-rRNA and binding of ribosomal proteins. Krr1 and Faf1 are two interacting proteins present in early 90 S precursor particles of the small ribosomal subunit. Here, we determined a co-crystal structure of the core domain of Krr1 bound to a 19-residue fragment of Faf1 at 2.8 Å resolution. The structure reveals that Krr1 consists of two packed K homology (KH) domains, KH1 and KH2, and resembles archaeal Dim2-like proteins. We show that KH1 is a divergent KH domain that lacks the RNA-binding GXXG motif and is involved in binding another assembly factor, Kri1. KH2 contains a canonical RNA-binding surface and additionally associates with an α-helix of Faf1. Specific disruption of the Krr1-Faf1 interaction impaired early 18 S rRNA processing at sites A0, A1, and A2 and caused cell lethality, but it did not prevent incorporation of the two proteins into pre-ribosomes. The Krr1-Faf1 interaction likely maintains a critical conformation of 90 S pre-ribosomes required for pre-rRNA processing. Our results illustrate the versatility of KH domains in protein interaction and provide insight into the role of Krr1-Faf1 interaction in ribosome biogenesis.
Ion channels are an attractive class of drug targets, but progress in developing inhibitors for therapeutic use has been limited largely due to challenges in identifying subtype selective small molecules. Animal venoms provide an alternative source of ion channel modulators, and the venoms of several species, such as scorpions, spiders and snails, are known to be rich sources of ion channel modulating peptides. Importantly, these peptides often bind to hyper-variable extracellular loops, creating the potential for subtype selectivity rarely achieved with small molecules. We have engineered scorpion venom peptides and incorporated them in fusion proteins to generate highly potent and selective Kv1.3 inhibitors with long in vivo half-lives. Kv1.3 has been reported to play a role in human T cell activation, and therefore, these Kv1.3 inhibitor fusion proteins may have potential for the treatment of autoimmune diseases. Our results support an emerging approach to generating subtype selective therapeutic ion channel inhibitors.
Macrophage phagocytosis plays an important role in host defense. The molecular mechanism, especially factors regulating the phagocytosis, however, is not completely understood. In the present study, we found that response gene to complement 32 (RGC-32) is an important regulator of phagocytosis. Although RGC-32 is induced and abundantly expressed in macrophage during monocyte-macrophage differentiation, RGC-32 appears not to be important for this process because RGC-32-deficient bone marrow progenitor can normally differentiate to macrophage. However, both peritoneal macrophages and bone marrow-derived macrophages with RGC-32 deficiency exhibit significant defects in phagocytosis, whereas RGC-32-overexpressed macrophages show increased phagocytosis. Mechanistically, RGC-32 is recruited to macrophage membrane where it promotes F-actin assembly and the formation of phagocytic cups. RGC-32 knock-out impairs F-actin assembly. RGC-32 appears to interact with PKC to regulate PKC-induced phosphorylation of F-actin cross-linking protein myristoylated alanine-rich protein kinase C substrate. Taken together, our results demonstrate for the first time that RGC-32 is a novel membrane regulator for macrophage phagocytosis.
The filovirus Ebola (EBOV) causes the most severe hemorrhagic fever known. The EBOV RNA-dependent polymerase complex includes a filovirus-specific VP30, which is critical for the transcriptional but not replication activity of EBOV polymerase; to support transcription, VP30 must be in a dephosphorylated form. Here we show that EBOV VP30 is phosphorylated not only at the N-terminal serine clusters identified previously but also at the threonine residues at positions 143 and 146. We also show that host cell protein phosphatase 1 (PP1) controls VP30 dephosphorylation because expression of a PP1-binding peptide cdNIPP1 increased VP30 phosphorylation. Moreover, targeting PP1 mRNA by shRNA resulted in the overexpression of SIPP1, a cytoplasm-shuttling regulatory subunit of PP1, and increased EBOV transcription, suggesting that cytoplasmic accumulation of PP1 induces EBOV transcription. Furthermore, we developed a small molecule compound, 1E7-03, that targeted a non-catalytic site of PP1 and increased VP30 dephosphorylation. The compound inhibited the transcription but increased replication of the viral genome and completely suppressed replication of EBOV in cultured cells. Finally, mutations of Thr143 and Thr146 of VP30 significantly inhibited EBOV transcription and strongly induced VP30 phosphorylation in the N-terminal Ser residues 29–46, suggesting a novel mechanism of regulation of VP30 phosphorylation. Our findings suggest that targeting PP1 with small molecules is a feasible approach to achieve dysregulation of the EBOV polymerase activity. This novel approach may be used for the development of antivirals against EBOV and other filovirus species.
Rad17 is a subunit of the Rad9-Hus1-Rad1 clamp loader complex, which is required for Chk1 activation after DNA damage. Rad17 has been shown to be regulated by the ubiquitin-proteasome system. We have identified a deubiquitylase, USP20 that is required for Rad17 protein stability in the steady-state and post DNA damage. We demonstrate that USP20 and Rad17 interact, and that this interaction is enhanced by UV exposure. We show that USP20 regulation of Rad17 is at the protein level in a proteasome-dependent manner. USP20 depletion results in poor activation of Chk1 protein by phosphorylation, consistent with Rad17 role in ATR-mediated phosphorylation of Chk1. Similar to other DNA repair proteins, USP20 is phosphorylated post DNA damage, and its depletion sensitizes cancer cells to damaging agents that form blocks ahead of the replication forks. Similar to Chk1 and Rad17, which enhance recombinational repair of collapsed replication forks, we demonstrate that USP20 depletion impairs DNA double strand break repair by homologous recombination. Together, our data establish a new function of USP20 in genome maintenance and DNA repair.
Kv7.1 to Kv7.5 α-subunits belong to the family of voltage-gated potassium channels (Kv). Assembled with the β-subunit KCNE1, Kv7.1 conducts the slowly activating potassium current IKs, which is one of the major currents underlying repolarization of the cardiac action potential. A known regulator of Kv7 channels is the lipid phosphatidylinositol 4,5-bisphosphate (PIP2). PIP2 increases the macroscopic current amplitude by stabilizing the open conformation of 7.1/KCNE1 channels. However, knowledge about the exact nature of the interaction is incomplete. The aim of this study was the identification of the amino acids responsible for the interaction between Kv7.1 and PIP2. We generated 13 charge neutralizing point mutations at the intracellular membrane border and characterized them electrophysiologically in complex with KCNE1 under the influence of diC8-PIP2. Electrophysiological analysis of corresponding long QT syndrome mutants suggested impaired PIP2 regulation as the cause for channel dysfunction. To clarify the underlying structural mechanism of PIP2 binding, molecular dynamics simulations of Kv7.1/KCNE1 complexes containing two PIP2 molecules in each subunit at specific sites were performed. Here, we identified a subset of nine residues participating in the interaction of PIP2 and Kv7.1/KCNE1. These residues may form at least two binding pockets per subunit, leading to the stabilization of channel conformations upon PIP2 binding.
Phagocyte superoxide production by a multicomponent NADPH oxidase is important in host defense against microbial invasion. However inappropriate NADPH oxidase activation causes inflammation. Endothelial cells express NADPH oxidase and endothelial oxidative stress due to prolonged NADPH oxidase activation predisposes many diseases. Discovering the mechanism of NADPH oxidase activation is essential for developing novel treatment of these diseases. The p47phox is a key regulatory subunit of NADPH oxidase; however, due to the lack of full protein structural information, the mechanistic insight of p47phox phosphorylation in NADPH oxidase activation remains incomplete. Based on crystal structures of three functional domains, we generated a computational structural model of the full p47phox protein. Using a combination of in silico phosphorylation, molecular dynamics simulation and protein/protein docking, we discovered that the C-terminal tail of p47phox is critical for stabilizing its autoinhibited structure. Ser-379 phosphorylation disrupts H-bonds that link the C-terminal tail to the autoinhibitory region (AIR) and the tandem Src homology 3 (SH3) domains, allowing the AIR to undergo phosphorylation to expose the SH3 pocket for p22phox binding. These findings were confirmed by site-directed mutagenesis and gene transfection of p47phox−/− coronary microvascular cells. Compared with wild-type p47phox cDNA transfected cells, the single mutation of S379A completely blocked p47phox membrane translocation, binding to p22phox and endothelial O2⨪ production in response to acute stimulation of PKC. p47phox C-terminal tail plays a key role in stabilizing intramolecular interactions at rest. Ser-379 phosphorylation is a molecular switch which initiates p47phox conformational changes and NADPH oxidase-dependent superoxide production by cells.
Chromosome ends contain nucleoprotein structures known as telomeres. Damage to chromosome ends during interphase elicits a DNA damage response (DDR) resulting in cell cycle arrest. However, little is known regarding the signaling from damaged chromosome ends (designated here as “TIPs”) during mitosis. In the present study, we investigated the consequences of DNA damage induced at a single TIP in mitosis. We used laser microirradiation to damage mitotic TIPs or chromosome arms (non-TIPs) in PtK2 kidney epithelial cells. We found that damage to a single TIP, but not a non-TIP, delays anaphase onset. This TIP-specific checkpoint response is accompanied by differential recruitment of DDR proteins. Although phosphorylation of H2AX and the recruitment of several repair factors, such as Ku70-Ku80, occur in a comparable manner at both TIP and non-TIP damage sites, DDR factors such as ataxia telangiectasia mutated (ATM), MDC1, WRN, and FANCD2 are specifically recruited to TIPs but not to non-TIPs. In addition, Nbs1, BRCA1, and ubiquitin accumulate at damaged TIPs more rapidly than at damaged non-TIPs. ATR and 53BP1 are not detected at either TIPs or non-TIPs in mitosis. The observed delay in anaphase onset is dependent on the activity of DDR kinases ATM and Chk1, and the spindle assembly checkpoint kinase Mps1. Cells damaged at a single TIP or non-TIP eventually exit mitosis with unrepaired lesions. Damaged TIPs are segregated into micronuclei at a significantly higher frequency than damaged non-TIPs. Together, these findings reveal a mitosis-specific DDR uniquely associated with chromosome ends.
Hypoxia-inducible factor 1 (HIF1) is a heterodimeric transcription factor containing an inducibly expressed HIF1α subunit and a constitutively expressed HIF1β subunit. Under hypoxic conditions, the HIF1α subunit accumulates because of a decrease in the rate of proteolytic degradation, and the resulting HIF1α–HIF1β heterodimers undergo post-translational modifications that promote transactivation. Previous reports suggest that amplified signaling through PI3K enhances HIF1-dependent gene expression; however, its role is controversial, and the mechanism is unclear. Using genetically engineered PTEN-deficient cell lines, we demonstrate that PTEN specifically inhibited the accumulation of HIF1α in response to hypoxia. Furthermore, we report that in glioblastoma cell lines, inhibition of PI3K pathway, using pan as well as isoform-specific PI3K inhibitors SF1126, PF4691502, BEZ-235, GDC0941, and TGX221 blocked the induction of HIF1α protein and its targets vascular endothelial growth factor, HK1, and GLUT1 mRNA in response to hypoxia. Herein, we describe the first evidence that HIF1α can be degraded under hypoxic conditions via the 26 S proteasome and that MDM2 is the E3 ligase that induces the hypoxic degradation of HIF1α. Moreover, the action of MDM2 on HIF1α under hypoxia occurs in the cytoplasm and is controlled by the PTEN-PI3K-AKT signaling axis. These data strongly suggest a new role for PTEN in the regulation of HIF1α and importantly that PI3K-AKT activation is required for the hypoxic stabilization of HIF1α and that hypoxia alone is not sufficient to render HIF1α resistant to proteasomal cleavage and degradation. Moreover, these findings suggest new therapeutic considerations for PI3K and/or AKT inhibitors for cancer therapeutics.
DHX9 is an ATP-dependent DEXH box helicase with a multitude of cellular functions. Its ability to unwind both DNA and RNA, as well as aberrant, noncanonical polynucleotide structures, has implicated it in transcriptional and translational regulation, DNA replication and repair, and maintenance of genome stability. We report that loss of DHX9 in primary human fibroblasts results in premature senescence, a state of irreversible growth arrest. This is accompanied by morphological defects, elevation of senescence-associated β-galactosidase levels, and changes in gene expression closely resembling those encountered during replicative (telomere-dependent) senescence. Activation of the p53 signaling pathway was found to be essential to this process. ChIP analysis and investigation of nascent DNA levels revealed that DHX9 is associated with origins of replication and that its suppression leads to a reduction of DNA replication. Our results demonstrate an essential role of DHX9 in DNA replication and normal cell cycle progression.
A functional voltage-gated K+ (Kv) channel comprises four pore-forming α-subunits, and only members of the same Kv channel subfamily may co-assemble to form heterotetramers. The ether-à-go-go family of Kv channels (KCNH) encompasses three distinct subfamilies: Eag (Kv10), Erg (Kv11), and Elk (Kv12). Members of different ether-à-go-go subfamilies, such as Eag and Erg, fail to form heterotetramers. Although a short stretch of amino acid sequences in the distal C-terminal section has been implicated in subfamily-specific subunit assembly, it remains unclear whether this region serves as the sole and/or principal subfamily recognition domain for Eag and Erg. Here we aim to ascertain the structural basis underlying the subfamily specificity of ether-à-go-go channels by generating various chimeric constructs between rat Eag1 and human Erg subunits. Biochemical and electrophysiological characterizations of the subunit interaction properties of a series of different chimeric and truncation constructs over the C terminus suggested that the putative C-terminal recognition domain is dispensable for subfamily-specific assembly. Further chimeric analyses over the N terminus revealed that the N-terminal region may also harbor a subfamily recognition domain. Importantly, exchanging either the N-terminal or the C-terminal domain alone led to a virtual loss of the intersubfamily assembly boundary. By contrast, simultaneously swapping both recognition domains resulted in a reversal of subfamily specificity. Our observations are consistent with the notion that both the N-terminal and the C-terminal recognition domains are required to sustain the subfamily-specific assembly of rat Eag1 and human Erg.
Desmoplastic malignancies such as cholangiocarcinoma (CCA) are characterized by a dense stroma containing an abundance of myofibroblasts termed cancer-associated fibroblasts (CAF). The CAF phenotype represents an “activated state” in which cells are primed for cell death triggered by BH3 mimetics. Accordingly, this primed state may be therapeutically exploited. To elucidate the mechanisms underlying this poorly understood apoptotic priming, we examined the role of platelet-derived growth factor (PDGF) in CAF priming for cell death given its prominent role in CAF activation. PDGF isomers PDGF-B and PDGF-D are abundantly expressed in CCA cells derived from human specimens. Either isomer sensitizes myofibroblasts to cell death triggered by BH3 mimetics. Similar apoptotic sensitization was observed with co-culture of myofibroblasts and CCA cells. Profiling of Bcl-2 proteins expressed by PDGF-primed myofibroblasts demonstrated an increase in cellular levels of Puma. PDGF-mediated increases in cellular Puma levels induced proapoptotic changes in Bak, which resulted in its binding to Bcl-2. Short hairpin RNA-mediated down-regulation of Puma conferred resistance to PDGF-mediated apoptotic priming. Conversely, the BH3 mimetic navitoclax disrupted Bcl-2/Bak heterodimers, allowing Bak to execute the cell death program. Treatment with a Bcl-2-specific BH3 mimetic, ABT-199, reduced tumor formation and tumor burden in a murine model of cholangiocarcinoma. Collectively, these findings indicate that apoptotic priming of CAF by PDGF occurs via Puma-mediated Bak activation, which can be converted to active full-blown apoptosis by navitoclax or ABT-199 for therapeutic benefit.
Rapid tumor growth can establish metabolically stressed microenvironments that activate 5′-AMP-activated protein kinase (AMPK), a ubiquitous regulator of ATP homeostasis. Previously, we investigated the importance of AMPK for the growth of experimental tumors prepared from HRAS-transformed mouse embryo fibroblasts and for primary brain tumor development in a rat model of neurocarcinogenesis. Here, we used triple-negative human breast cancer cells in which AMPK activity had been knocked down to investigate the contribution of AMPK to experimental tumor growth and core glucose metabolism. We found that AMPK supports the growth of fast-growing orthotopic tumors prepared from MDA-MB-231 and DU4475 breast cancer cells but had no effect on the proliferation or survival of these cells in culture. We used in vitro and in vivo metabolic profiling with [13C]glucose tracers to investigate the contribution of AMPK to core glucose metabolism in MDA-MB-231 cells, which have a Warburg metabolic phenotype; these experiments indicated that AMPK supports tumor glucose metabolism in part through positive regulation of glycolysis and the nonoxidative pentose phosphate cycle. We also found that AMPK activity in the MDA-MB-231 tumors could systemically perturb glucose homeostasis in sensitive normal tissues (liver and pancreas). Overall, our findings suggest that the contribution of AMPK to the growth of aggressive experimental tumors has a critical microenvironmental component that involves specific regulation of core glucose metabolism.
Regulated family II pyrophosphatases (CBS-PPases) contain a nucleotide-binding insert comprising a pair of cystathionine β-synthase (CBS) domains, termed a Bateman module. By binding with high affinity to the CBS domains, AMP and ADP usually inhibit the enzyme, whereas ATP activates it. Here, we demonstrate that AMP, ADP, and ATP bind in a positively cooperative manner to CBS-PPases from four bacteria: Desulfitobacterium hafniense, Clostridium novyi, Clostridium perfringens, and Eggerthella lenta. Enzyme interaction with substrate as characterized by the Michaelis constant (Km) also exhibited positive catalytic cooperativity that decreased in magnitude upon nucleotide binding. The degree of both types of cooperativity increased with increasing concentration of the cofactor Mg2+ except for the C. novyi PPase where Mg2+ produced the opposite effect on kinetic cooperativity. Further exceptions from these general rules were ADP binding to C. novyi PPase and AMP binding to E. lenta PPase, neither of which had any effect on activity. A genetically engineered deletion variant of D. hafniense PPase lacking the regulatory insert was fully active but differed from the wild-type enzyme in that it was insensitive to nucleotides and bound substrate non-cooperatively and with a smaller Km value. These results indicate that the regulatory insert acts as an internal inhibitor and confers dual positive cooperativity to CBS domain-containing PPases, making them highly sensitive regulators of the PPi level in response to the changes in cell energy status that control adenine nucleotide distribution. These regulatory features may be common among other CBS domain-containing proteins.
Ki67 is a protein widely used as cell-proliferation marker, with its cellular functions being hardly unveiled. In this paper, we present the direct interaction between Ki67 and PP1γ, a protein phosphatase showing characteristic accumulation on anaphase chromosomes via the canonical PP1-binding motif within Ki67. In cells depleted of Ki67, PP1γ is targeted to anaphase chromosomes less efficiently. Additionally, overexpression of Ki67, but not a mutant form without the ability to bind PP1γ, induced ectopic localization of PP1γ οn metaphase chromosomes. These observations demonstrate that Ki67 is one factor that defines the cellular behavior of PP1γ in anaphase. To explore the specific roles of the subset of PP1γ recruited on chromosome via its interaction with Ki67 (PP1γ-Ki67), endogenous Ki67 was replaced with a Ki67 mutant deficient in its ability to interact with PP1γ. Although no obvious defects in the progression of mitosis were observed, the timing of dephosphorylation of the mutant Ki67 in anaphase was delayed, indicating that Ki67 itself is one of the substrates of PP1γ-Ki67.
Hyaluronan (HA) may have proinflammatory roles in the context of CNS autoimmunity. It accumulates in demyelinated multiple sclerosis (MS) lesions, promotes antigen presentation, and enhances T-cell activation and proliferation. HA facilitates lymphocyte binding to vessels and CNS infiltration at the CNS vascular endothelium. Furthermore, HA signals through Toll-like receptors 2 and 4 to stimulate inflammatory gene expression. We assessed the role of HA in experimental autoimmune encephalomyelitis (EAE), an animal model of MS by administration of 4-methylumbelliferone (4MU), a well established inhibitor of HA synthesis. 4MU decreased hyaluronan synthesis in vitro and in vivo. It was protective in active EAE of C57Bl/6 mice, decreased spinal inflammatory infiltrates and spinal infiltration of Th1 cells, and increased differentiation of regulatory T-cells. In adoptive transfer EAE, feeding of 4MU to donor mice significantly decreased the encephalitogenicity of lymph node cells. The transfer of proteolipid protein (PLP)-stimulated lymph node cells to 4MU-fed mice resulted in a delayed EAE onset and delayed spinal T-cell infiltration. Expression of CXCL12, an anti-inflammatory chemokine, is reduced in MS patients in CSF cells and in spinal cord tissue during EAE. Hyaluronan suppressed production of CXCL12, whereas 4MU increased spinal CXCL12 in naive animals and during neuroinflammation. Neutralization of CXCR4, the most prominent receptor of CXCL12, by administration of AMD3100 diminished the protective impact of 4MU in adoptive transfer EAE. In conclusion, hyaluronan exacerbates CNS autoimmunity, enhances encephalitogenic T-cell responses, and suppresses the protective chemokine CXCL12 in CNS tissue. Inhibition of hyaluronan synthesis with 4MU protects against an animal model of MS and may represent an important therapeutic option in MS and other neuroinflammatory diseases.
Following activation, the cytoplasmic pattern recognition receptor nucleotide-binding oligomerization domain-containing protein 1 (NOD1) interacts with its adaptor protein receptor-interacting protein 2 (RIP2) to propagate immune signaling and initiate a proinflammatory immune response. This interaction is mediated by the caspase recruitment domain (CARD) of both proteins. Polymorphisms in immune proteins can affect receptor function and predispose individuals to specific autoinflammatory disorders. In this report, we show that mutations in helix 2 of the CARD of NOD1 disrupted receptor function but did not interfere with RIP2 interaction. In particular, N43S, a rare polymorphism, resulted in receptor dysfunction despite retaining normal cellular localization, protein folding, and an ability to interact with RIP2. Mutation of Asn-43 resulted in an increased tendency to form dimers, which we propose is the source of this dysfunction. We also demonstrate that mutation of Lys-443 and Tyr-474 in RIP2 disrupted the interaction with NOD1. Mapping the key residues involved in the interaction between NOD1 and RIP2 to the known structures of CARD complexes revealed the likely involvement of both type I and type III interfaces in the NOD1·RIP2 complex. Overall we demonstrate that the NOD1-RIP2 signaling axis is more complex than previously assumed, that simple engagement of RIP2 is insufficient to mediate signaling, and that the interaction between NOD1 and RIP2 constitutes multiple CARD-CARD interfaces.
5-Aminolevulinate (ALA), an essential metabolite in all heme-synthesizing organisms, results from the pyridoxal 5′-phosphate (PLP)-dependent enzymatic condensation of glycine with succinyl-CoA in non-plant eukaryotes and α-proteobacteria. The predicted chemical mechanism of this ALA synthase (ALAS)-catalyzed reaction includes a short-lived glycine quinonoid intermediate and an unstable 2-amino-3-ketoadipate intermediate. Using liquid chromatography coupled with tandem mass spectrometry to analyze the products from the reaction of murine erythroid ALAS (mALAS2) with O-methylglycine and succinyl-CoA, we directly identified the chemical nature of the inherently unstable 2-amino-3-ketoadipate intermediate, which predicates the glycine quinonoid species as its precursor. With stopped-flow absorption spectroscopy, we detected and confirmed the formation of the quinonoid intermediate upon reacting glycine with ALAS. Significantly, in the absence of the succinyl-CoA substrate, the external aldimine predominates over the glycine quinonoid intermediate. When instead of glycine, l-serine was reacted with ALAS, a lag phase was observed in the progress curve for the l-serine external aldimine formation, indicating a hysteretic behavior in ALAS. Hysteresis was not detected in the T148A-catalyzed l-serine external aldimine formation. These results with T148A, a mALAS2 variant, which, in contrast to wild-type mALAS2, is active with l-serine, suggest that active site Thr-148 modulates ALAS strict amino acid substrate specificity. The rate of ALA release is also controlled by a hysteretic kinetic mechanism (observed as a lag in the ALA external aldimine formation progress curve), consistent with conformational changes governing the dissociation of ALA from ALAS.
Host defense peptides play an important host-protective role by their microcidal action, immunomodulatory functions, and tissue repair activities. Proteolysis is a common strategy of pathogens used to neutralize host defense peptides. Here, we show that actin, the most abundant structural protein in eukaryotes, binds the LL-37 host defense peptide, protects it from degradation by the proteases of Pseudomonas aeruginosa and Porphyromonas gingivalis, and enables its antimicrobial activity despite the presence of the proteases. Co-localization of LL-37 with extracellular actin was observed in necrotized regions of samples from oral lesions. Competition assays, cross-linking experiments, limited proteolysis, and mass spectrometry revealed that LL-37 binds by specific hydrophobic interactions to the His-40–Lys-50 segment of actin, located in the DNase I binding loop. The integrity of the binding site of both LL-37 and actin is a prerequisite to the binding. Our results demonstrate that actin, presumably released by dead cells and abundant in infected sites, might be utilized by the immune system to enhance spatio-temporal immunity in an attempt to arrest infection and control inflammation.
Current views on the control of IL-23 production focus on the regulation of il23a, the gene encoding IL-23 p19, by NF-κB in combination with other transcription factors. C/EBP homologous protein (CHOP), X2-Box-binding protein 1 (XBP1), activator protein 1 (AP1), SMAD, CCAAT/enhancer-binding protein (C/EBPβ), and cAMP-response element-binding protein (CREB) have been involved in response to LPS, but no data are available regarding the mechanism triggered by the fungal mimic and β-glucan-containing stimulus zymosan, which produces IL-23 and to a low extent the related cytokine IL-12 p70. Zymosan induced the mobilization of CHOP from the nuclear fractions to phagocytic vesicles. Hypha-forming Candida also induced the nuclear disappearance of CHOP. Assay of transcription factor binding to the il23a promoter showed an increase of Thr(P)-71–Thr(P)-69-activating transcription factor 2 (ATF2) binding in response to zymosan. PKC and PKA/mitogen- and stress-activated kinase inhibitors down-regulated Thr(P)-71–ATF2 binding to the il23a promoter and il23a mRNA expression. Consistent with the current concept of complementary phosphorylations on N-terminal Thr-71 and Thr-69 of ATF2 by ERK and p38 MAPK, MEK, and p38 MAPK inhibitors blunted Thr(P)-69–ATF2 binding. Knockdown of atf2 mRNA with siRNA correlated with inhibition of il23a mRNA, but it did not affect the expression of il12/23b and il10 mRNA. These data indicate the following: (i) zymosan decreases nuclear proapoptotic CHOP, most likely by promoting its accumulation in phagocytic vesicles; (ii) zymosan-induced il23a mRNA expression is best explained through coordinated κB- and ATF2-dependent transcription; and (iii) il23a expression relies on complementary phosphorylation of ATF2 on Thr-69 and Thr-71 dependent on PKC and MAPK activities.
Human trophoblast invasion of decidualized endometrium is essential for placentation and is tightly regulated and involves trophoblast-decidual cell interaction. High temperature requirement A4 (HtrA4) is a secreted serine protease highly expressed in the invasive extravillous trophoblasts that invade decidua. In contrast, both HtrA1 and HtrA3 have been shown to inhibit trophoblast invasion. Here we provide evidence that decidua-secreted HtrA1 and HtrA3 antagonize HtrA4-mediated trophoblast invasion. We demonstrated that HtrA1 and HtrA3 interact with and degrade HtrA4 and thereby inhibit trophoblast-like JAR cell invasion. Specifically, HtrA1 and HtrA3 expression is up-regulated under decidualization conditions in endometrial stromal and epithelial cells, T-HESCs and Ishikawa cells, respectively. Conditioned media from these two cell lines after decidualization treatment suppress HtrA4-expressing JAR cell invasion in an HtrA1- or HtrA3-dependent manner. Co-culture of the HtrA4-expressing JAR cells with decidualization stimuli-treated T-HESC or Ishikawa monolayer also impairs JAR cell invasion, which can be reversed by HtrA1 or HtrA3 knockdown, supporting that HtrA1 and HtrA3 are crucial for trophoblast-decidual cell interaction in the control of trophoblast invasion. Our study reveals a novel regulatory mechanism of trophoblast invasion through physical and functional interaction between HtrA family members.
Defensins are components of the innate immune system that promote the directional migration and activation of dendritic cells, thereby modulating the adaptive immune response. Because matrix glycosaminoglycan (GAG) is known to be important for these functions, we characterized the structural features of human β-defensin 6 (hBD6) and GAG interaction using a combination of structural and in silico analyses. Our results showed that GAG model compounds, a pentasaccharide (fondaparinux, FX) and an octasaccharide heparin derivative (dp8) bind to the α-helix and in the loops between the β2 and β3 strands, inducing the formation of a ternary complex with a 2:1 hBD6:FX stoichiometry. Competition experiments indicated an overlap of GAG and chemokine receptor CCR2 binding sites. An NMR-derived model of the ternary complex revealed that FX interacts with hBD6 along the dimerization interface, primarily contacting the α-helices and β2-β3 loops from each monomer. We further demonstrated that high-pressure NMR spectroscopy could capture an intermediate stage of hBD6-FX interaction, exhibiting features of a cooperative binding mechanism. Collectively, these data suggest a “sandwich-like” model in which two hBD6 molecules bind a single FX chain and provide novel structural insights into how defensin orchestrates leukocyte recruitment through GAG binding and G protein-coupled receptor activation. Despite the similarity to chemokines and hBD2, our data indicate different properties for the hBD6-GAG complex. This work adds significant information to the currently limited data available for the molecular structures and dynamics of defensin carbohydrate binding.
Programmed cell death protein 4 (PDCD4) is a tumor suppressor and has also been shown to suppress production of the immunomodulatory cytokine IL-10. The precise role of PDCD4 in IL-10 induction in macrophages is still not fully understood. Incubation of macrophages with inhibitors of PI3K and mTOR blocked LPS-stimulated PDCD4 degradation and expression of c-Maf and IL-10 production. PDCD4 and the transcription factor Twist2 were shown to form a complex in untreated cells. LPS disrupted the complex allowing Twist2 to bind to the c-Maf promoter. PI3K and mTOR inhibitors prevented this disruption by stabilizing PDCD4 and thereby decreased Twist2 binding to the c-Maf promoter and induction of c-Maf mRNA. These results indicate a regulatory role for PDCD4 and Twist2 in LPS-induced IL-10 production in macrophages. LPS promotes PDCD4 degradation via a pathway involving PI3K and mTOR, releasing Twist2, which induces IL-10 via c-Maf.
The starch debranching enzymes isoamylase 1 and 2 (ISA1 and ISA2) are known to exist in a large complex and are involved in the biosynthesis and crystallization of starch. It is suggested that the function of the complex is to remove misplaced branches of growing amylopectin molecules, which would otherwise prevent the association and crystallization of adjacent linear chains. Here, we investigate the function of ISA1 and ISA2 from starch producing alga Chlamydomonas. Through complementation studies, we confirm that the STA8 locus encodes for ISA2 and sta8 mutants lack the ISA1·ISA2 heteromeric complex. However, mutants retain a functional dimeric ISA1 that is able to partly sustain starch synthesis in vivo. To better characterize ISA1, we have overexpressed and purified ISA1 from Chlamydomonas reinhardtii (CrISA1) and solved the crystal structure to 2.3 Å and in complex with maltoheptaose to 2.4 Å. Analysis of the homodimeric CrISA1 structure reveals a unique elongated structure with monomers connected end-to-end. The crystal complex reveals details about the mechanism of branch binding that explains the low activity of CrISA1 toward tightly spaced branches and reveals the presence of additional secondary surface carbohydrate binding sites.
The serotonin transporter (SERT) plays a critical role in regulating serotonin signaling by mediating reuptake of serotonin from the extracellular space. The molecular and cellular mechanisms controlling SERT levels in the membrane remain poorly understood. To study trafficking of the surface resident SERT, two functional epitope-tagged variants were generated. Fusion of a FLAG-tagged one-transmembrane segment protein Tac to the SERT N terminus generated a transporter with an extracellular epitope suited for trafficking studies (TacSERT). Likewise, a construct with an extracellular antibody epitope was generated by introducing an HA (hemagglutinin) tag in the extracellular loop 2 of SERT (HA-SERT). By using TacSERT and HA-SERT in antibody-based internalization assays, we show that SERT undergoes constitutive internalization in a dynamin-dependent manner. Confocal images of constitutively internalized SERT demonstrated that SERT primarily co-localized with the late endosomal/lysosomal marker Rab7, whereas little co-localization was observed with the Rab11, a marker of the “long loop” recycling pathway. This sorting pattern was distinct from that of a prototypical recycling membrane protein, the β2-adrenergic receptor. Furthermore, internalized SERT co-localized with the lysosomal marker LysoTracker and not with transferrin. The sorting pattern was further confirmed by visualizing internalization of SERT using the fluorescent cocaine analog JHC1-64 and by reversible and pulse-chase biotinylation assays showing evidence for lysosomal degradation of the internalized transporter. Finally, we found that SERT internalized in response to stimulation with 12-myristate 13-acetate co-localized primarily with Rab7- and LysoTracker-positive compartments. We conclude that SERT is constitutively internalized and that the internalized transporter is sorted mainly to degradation.
Chitin synthases (CHS) produce chitin, an essential component of the fungal cell wall. The molecular mechanism of processive chitin synthesis is not understood, limiting the discovery of new inhibitors of this enzyme class. We identified the bacterial glycosyltransferase NodC as an appropriate model system to study the general structure and reaction mechanism of CHS. A high throughput screening-compatible novel assay demonstrates that a known inhibitor of fungal CHS also inhibit NodC. A structural model of NodC, on the basis of the recently published BcsA cellulose synthase structure, enabled probing of the catalytic mechanism by mutagenesis, demonstrating the essential roles of the DD and QXXRW catalytic motifs. The NodC membrane topology was mapped, validating the structural model. Together, these approaches give insight into the CHS structure and mechanism and provide a platform for the discovery of inhibitors for this antifungal target.
The epithelial sodium channel (ENaC) is activated upon endoproteolytic cleavage of specific segments in the extracellular domains of the α- and γ-subunits. Cleavage is accomplished by intracellular proteases prior to membrane insertion and by surface-expressed or extracellular soluble proteases once ENaC resides at the cell surface. These cleavage events are partially regulated by intracellular signaling through an unknown allosteric mechanism. Here, using a combination of computational and experimental techniques, we show that the intracellular N terminus of γ-ENaC undergoes secondary structural transitions upon interaction with phosphoinositides. From ab initio folding simulations of the N termini in the presence and absence of phosphatidylinositol 4,5-bisphosphate (PIP2), we found that PIP2 increases α-helical propensity in the N terminus of γ-ENaC. Electrophysiology and mutation experiments revealed that a highly conserved cluster of lysines in the γ-ENaC N terminus regulates accessibility of extracellular cleavage sites in γ-ENaC. We also show that conditions that decrease PIP2 or enhance ubiquitination sharply limit access of the γ-ENaC extracellular domain to proteases. Further, the efficiency of allosteric control of ENaC proteolysis is dependent on Tyr370 in γ-ENaC. Our findings provide an allosteric mechanism for ENaC activation regulated by the N termini and sheds light on a potential general mechanism of channel and receptor activation.
The E3 ubiquitin ligase CRL4Cdt2 targets proteins for destruction in S phase and after DNA damage by coupling ubiquitylation to DNA-bound proliferating cell nuclear antigen (PCNA). Coupling to PCNA involves a PCNA-interacting peptide (PIP) degron motif in the substrate that recruits CRL4Cdt2 while binding to PCNA. In vertebrates, CRL4Cdt2 promotes degradation of proteins whose presence in S phase is deleterious, including Cdt1, Set8, and p21. Here, we show that CRL4Cdt2 targets thymine DNA glycosylase (TDG), a base excision repair enzyme that is involved in DNA demethylation. TDG contains a conserved and nearly perfect match to the PIP degron consensus. TDG is ubiquitylated and destroyed in a PCNA-, Cdt2-, and PIP degron-dependent manner during DNA repair in Xenopus egg extract. The protein can also be destroyed during DNA replication in this system. During Xenopus development, TDG first accumulates during gastrulation, and its expression is down-regulated by CRL4Cdt2. Our results expand the group of vertebrate CRL4Cdt2 substrates to include a bona fide DNA repair enzyme.
Thymine DNA glycosylase (TDG) is an essential enzyme playing multiple roles in base excision repair, transcription regulation, and DNA demethylation. TDG mediates the cytotoxicity of the anti-cancer chemotherapeutic drug 5-fluorouracil (5-FU) by prolonging S phase, generating DNA strand breaks, and inducing DNA damage signaling. During S phase of the cell cycle, TDG is degraded via the proteasomal pathway. Here we show that CRL4Cdt2 E3 ubiquitin ligase promotes ubiquitination and proteasomal degradation of TDG in S phase in a reaction that is dependent on the interaction of TDG with proliferating cell nuclear antigen (PCNA). siRNA-mediated depletion of PCNA or components of CRL4Cdt2, specifically cullin4A/B or substrate adaptor Cdt2, stabilizes TDG in human cells. Mutations in the PCNA-interacting peptide (PIP) motif of TDG that disrupt the interaction of TDG with PCNA or change critical basic residues essential for the action of the PIP degron prevent the ubiquitination and degradation of TDG. Thus physical interaction of TDG with PCNA through the PIP degron is required for targeting TDG to the CRL4Cdt2 E3 ubiquitin ligase complex. Compared with forced expression of wild type TDG, CRL4Cdt2- resistant TDG (ΔPIP) slows cell proliferation and slightly increases the toxicity of 5-FU. Thus, CRL4Cdt2-dependent degradation of TDG occurs in S phase because of the requirement for TDG to interact with chromatin-loaded PCNA, and this degradation is important for preventing toxicity from excess TDG.
Inhaled β-agonists are effective at reversing bronchoconstriction in asthma, but the mechanism by which they exert this effect is unclear and controversial. PKA is the historically accepted effector, although this assumption is made on the basis of associative and not direct evidence. Recent studies have asserted that exchange protein activated by cAMP (Epac), not PKA, mediates the relaxation of airway smooth muscle (ASM) observed with β-agonist treatment. This study aims to clarify the role of PKA in the prorelaxant effects of β-agonists on ASM. Inhibition of PKA activity via expression of the PKI and RevAB peptides results in increased β-agonist-mediated cAMP release, abolishes the inhibitory effect of isoproterenol on histamine-induced intracellular calcium flux, and significantly attenuates histamine-stimulated MLC-20 phosphorylation. Analyses of ASM cell and tissue contraction demonstrate that PKA inhibition eliminates most, if not all, β-agonist-mediated relaxation of contracted smooth muscle. Conversely, Epac knockdown had no effect on the regulation of contraction or procontractile signaling by isoproterenol. These findings suggest that PKA, not Epac, is the predominant and physiologically relevant effector through which β-agonists exert their relaxant effects.
The control of IL-10 production in Toll-like receptor (TLR) signals remains to be elucidated. Here, we report that β-arrestin 2 positively regulates TLR-triggered IL-10 production in a p38 mitogen-activated protein kinase (MAPK)-dependent mechanism. In vitro studies with cells including peritoneal macrophages and HEK293/TLR4 cells have demonstrated that β-arrestin 2 forms complexes with p38 and facilitates p38 activation after lipopolysaccharide (LPS) stimulation. Deficiency of β-arrestin 2 and inhibition of p38 MAPK activity both ameliorate TLR4-stimulated IL-10 response. Additionally, in vivo experiments show that mice lacking β-arrestin 2 produce less amount of IL-10, and are more susceptible to LPS-induced septic shock which is further enhanced by blocking IL-10 signal. These results reveal a novel mechanism by which β-arrestin 2 negatively regulates TLR4-mediated inflammatory reactions.
Protein folding within the endoplasmic reticulum is assisted by molecular chaperones and folding catalysts that include members of the protein-disulfide isomerase and peptidyl-prolyl isomerase families. In this report, we examined the contributions of the cyclophilin subset of peptidyl-prolyl isomerases to protein folding and identified cyclophilin C as an endoplasmic reticulum (ER) cyclophilin in addition to cyclophilin B. Using albumin and transferrin as models of cis-proline-containing proteins in human hepatoma cells, we found that combined knockdown of cyclophilins B and C delayed transferrin secretion but surprisingly resulted in more efficient oxidative folding and secretion of albumin. Examination of the oxidation status of ER protein-disulfide isomerase family members revealed a shift to a more oxidized state. This was accompanied by a >5-fold elevation in the ratio of oxidized to total glutathione. This “hyperoxidation” phenotype could be duplicated by incubating cells with the cyclophilin inhibitor cyclosporine A, a treatment that triggered efficient ER depletion of cyclophilins B and C by inducing their secretion to the medium. To identify the pathway responsible for ER hyperoxidation, we individually depleted several enzymes that are known or suspected to deliver oxidizing equivalents to the ER: Ero1αβ, VKOR, PRDX4, or QSOX1. Remarkably, none of these enzymes contributed to the elevated oxidized to total glutathione ratio induced by cyclosporine A treatment. These findings establish cyclophilin C as an ER cyclophilin, demonstrate the novel involvement of cyclophilins B and C in ER redox homeostasis, and suggest the existence of an additional ER oxidative pathway that is modulated by ER cyclophilins.
The cardiac troponin I (cTnI) R21C (cTnI-R21C) mutation has been linked to hypertrophic cardiomyopathy and renders cTnI incapable of phosphorylation by PKA in vivo. Echocardiographic imaging of homozygous knock-in mice expressing the cTnI-R21C mutation shows that they develop hypertrophy after 12 months of age and have abnormal diastolic function that is characterized by longer filling times and impaired relaxation. Electrocardiographic analyses show that older R21C mice have elevated heart rates and reduced cardiovagal tone. Cardiac myocytes isolated from older R21C mice demonstrate that in the presence of isoproterenol, significant delays in Ca2+ decay and sarcomere relaxation occur that are not present at 6 months of age. Although isoproterenol and stepwise increases in stimulation frequency accelerate Ca2+-transient and sarcomere shortening kinetics in R21C myocytes from older mice, they are unable to attain the corresponding WT values. When R21C myocytes from older mice are treated with isoproterenol, evidence of excitation-contraction uncoupling is indicated by an elevation in diastolic calcium that is frequency-dissociated and not coupled to shorter diastolic sarcomere lengths. Myocytes from older mice have smaller Ca2+ transient amplitudes (2.3-fold) that are associated with reductions (2.9-fold) in sarcoplasmic reticulum Ca2+ content. This abnormal Ca2+ handling within the cell may be attributed to a reduction (2.4-fold) in calsequestrin expression in conjunction with an up-regulation (1.5-fold) of Na+-Ca2+ exchanger. Incubation of permeabilized cardiac fibers from R21C mice with PKA confirmed that the mutation prevents facilitation of mechanical relaxation. Altogether, these results indicate that the inability to enhance myofilament relaxation through cTnI phosphorylation predisposes the heart to abnormal diastolic function, reduced accessibility of cardiac reserves, dysautonomia, and hypertrophy.
Nck family proteins function as adaptors to couple tyrosine phosphorylation signals to actin cytoskeleton reorganization. Several lines of evidence indicate that Nck family proteins involve in regulating the activity of Rho family GTPases. In the present study, we characterized a novel interaction between Nck-1 with engulfment and cell motility 1 (ELMO1). GST pull-down and co-immunoprecipitation assay demonstrated that the Nck-1-ELMO1 interaction is mediated by the SH2 domain of Nck-1 and the phosphotyrosine residues at position 18, 216, 395, and 511 of ELMO1. A R308K mutant of Nck-1 (in which the SH2 domain was inactive), or a 4YF mutant of ELMO1 lacking these four phosphotyrosine residues, diminished Nck-1-ELMO1 interaction. Conversely, tyrosine phosphatase inhibitor treatment and overexpression of Src family kinase Hck significantly enhanced Nck-1-ELMO1 interaction. Moreover, wild type Nck-1, but not R308K mutant, significantly augmented the interaction between ELMO1 and constitutively active RhoG (RhoGV12A), thus promoted Rac1 activation and cell motility. Taken together, the present study characterized a novel Nck-1-ELMO1 interaction and defined a new role for Nck-1 in regulating Rac1 activity.
Within innate immune signaling pathways, interleukin-1 receptor-associated kinases (IRAKs) fulfill key roles downstream of multiple Toll-like receptors and the interleukin-1 receptor. Although human IRAK4 deficiency was shown to lead to severe immunodeficiency in response to pyogenic bacterial infection during childhood, little is known about the role of human IRAK2. We here identified a non-synonymous IRAK2 variant, rs35060588 (coding R214G), as hypofunctional in terms of NF-κB signaling and Toll-like receptor-mediated cytokine induction. This was due to reduced ubiquitination of TRAF6, a key step in signal transduction. IRAK2 rs35060588 occurs in 3–9% of individuals in different ethnic groups, and our studies suggested a genetic association of rs35060588 with colorectal cancer survival. This for the first time implicates human IRAK2 in a human disease and highlights the R214G IRAK2 variant as a potential novel and broadly applicable biomarker for disease or as a therapeutic intervention point.
Intestinal NPC1L1 transporter is essential for cholesterol absorption and the maintenance of cholesterol homeostasis in the body. NPC1L1 is differentially expressed along the gastrointestinal tract with very low levels in the colon as compared with the small intestine. This study was undertaken to examine whether DNA methylation was responsible for segment-specific expression of NPC1L1. Treatment of mice with 5-azacytidine (i.p.) resulted in a significant dose-dependent increase in NPC1L1 mRNA expression in the colon. The lack of expression of NPC1L1 in the normal colon was associated with high levels of methylation in the area flanking the 3-kb fragment upstream of the initiation site of the mouse NPC1L1 gene in mouse colon as analyzed by EpiTYPER® MassARRAY®. The high level of methylation in the colon was observed in specific CpG dinucleotides and was significantly decreased in response to 5-azacytidine. Similar to mouse NPC1L1, 5-azacytidine treatment also increased the level of human NPC1L1 mRNA expression in the intestinal HuTu-80 cell line in a dose- and time-dependent manner. Silencing the expression of DNA methyltransferase DNMT1, -2, -3A, and -3B alone by siRNA did not affect NPC1L1 expression in HuTu-80 cells. However, the simultaneous attenuation of DNMT1 and -3B expression caused a significant increase in NPC1L1 mRNA expression as compared with control. Also, in vitro methylation of the human NPC1L1 promoter significantly decreased NPC1L1 promoter activity in human intestinal Caco2 cells. In conclusion, our data demonstrated for the first time that DNA methylation in the promoter region of the NPC1L1 gene appears to be a major mechanism underlying differential expression of NPC1L1 along the length of the gastrointestinal tract.
The regulation of integrin-mediated adhesion is of vital importance to adaptive and innate immunity. Integrins are versatile proteins and mediate T cell migration and trafficking by binding to extracellular matrix or other cells as well as initiating intracellular signaling cascades promoting survival or activation. The MAPK pathway is known to be downstream from integrins and to regulate survival, differentiation, and motility. However, secondary roles for canonical MAPK pathway members are being discovered. We show that chemical inhibition of RAF by sorafenib or shRNA-mediated knockdown of B-Raf reduces T cell resistance to shear stress to α4β1 integrin ligands vascular cell adhesion molecule 1 (VCAM-1) and fibronectin, whereas inhibition of MEK/ERK by U0126 had no effect. Microscopy showed that RAF inhibition leads to significant inhibition of T cell spreading on VCAM-1. The association of α4β1 integrin with the actin cytoskeleton was shown to be dependent on B-Raf activity or expression, whereas α4β1 integrin affinity for soluble VCAM-1 was not. These effects were shown to be specific for α4β1 integrin and not other integrins, such as α5β1 or LFA-1, or a variety of membrane proteins. We demonstrate a novel role for B-Raf in the selective regulation of α4β1 integrin-mediated adhesion.
Transcription elongation has been recognized as a rate-limiting step for the expression of signal-inducible genes. Through recruitment of positive transcription elongation factor P-TEFb, the bromodomain-containing protein BRD4 plays critical roles in regulating the transcription elongation of a vast array of inducible genes that are important for multiple cellular processes. The diverse biological roles of BRD4 have been proposed to rely on its functional transition between chromatin targeting and transcription regulation. The signaling pathways and the molecular mechanism for regulating this transition process, however, are largely unknown. Here, we report a novel role of phosphorylated Ser10 of histone H3 (H3S10ph) in governing the functional transition of BRD4. We identified that the acetylated lysines 5 and 8 of nucleosomal histone H4 (H4K5ac/K8ac) is the BRD4 binding site, and the protein phosphatase PP1α and class I histone deacetylase (HDAC1/2/3) signaling pathways are essential for the stress-induced BRD4 release from chromatin. In the unstressed state, phosphorylated H3S10 prevents the deacetylation of nucleosomal H4K5ac/K8ac by HDAC1/2/3, thereby locking up the majority of BRD4 onto chromatin. Upon stress, PP1α-mediated dephosphorylation of H3S10ph allows the deacetylation of nucleosomal H4K5ac/K8ac by HDAC1/2/3, thereby leading to the release of chromatin-bound BRD4 for subsequent recruitment of P-TEFb to enhance the expression of inducible genes. Therefore, our study revealed a novel mechanism that the histone cross-talk between H3S10ph and H4K5ac/K8ac connects PP1α and HDACs to govern the functional transition of BRD4. Combined with previous studies on the regulation of P-TEFb activation, the intricate signaling network for the tight control of transcription elongation is established.
Fatty acid-binding protein 4 (FABP4) is an adipogenic protein and is implicated in atherosclerosis, insulin resistance, and cancer. In endothelial cells, FABP4 is induced by VEGFA, and inhibition of FABP4 blocks most of the VEGFA effects. We investigated the DLL4-NOTCH-dependent regulation of FABP4 in human umbilical vein endothelial cells by gene/protein expression and interaction analyses following inhibitor treatment and RNA interference. We found that FABP4 is directly induced by NOTCH. Stimulation of NOTCH signaling with human recombinant DLL4 led to FABP4 induction, independently of VEGFA. FABP4 induction by VEGFA was reduced by blockade of DLL4 binding to NOTCH or inhibition of NOTCH signal transduction. Chromatin immunoprecipitation of the NOTCH intracellular domain showed increased binding to two specific regions in the FABP4 promoter. The induction of FABP4 gene expression was dependent on the transcription factor FOXO1, which was essential for basal expression of FABP4, and FABP4 up-regulation following stimulation of the VEGFA and/or the NOTCH pathway. Thus, we show that the DLL4-NOTCH pathway mediates endothelial FABP4 expression. This indicates that induction of the angiogenesis-restricting DLL4-NOTCH can have pro-angiogenic effects via this pathway. It also provides a link between DLL4-NOTCH and FOXO1-mediated regulation of endothelial gene transcription, and it shows that DLL4-NOTCH is a nodal point in the integration of pro-angiogenic and metabolic signaling in endothelial cells. This may be crucial for angiogenesis in the tumor environment.
In Escherichia coli, the biogenesis of both cytochrome bd-type quinol oxidases and periplasmic cytochromes requires the ATP-binding cassette-type cysteine/GSH transporter, CydDC. Recombinant CydDC was purified as a heterodimer and found to be an active ATPase both in soluble form with detergent and when reconstituted into a lipid environment. Two-dimensional crystals of CydDC were analyzed by electron cryomicroscopy, and the protein was shown to be made up of two non-identical domains corresponding to the putative CydD and CydC subunits, with dimensions characteristic of other ATP-binding cassette transporters. CydDC binds heme b. Detergent-solubilized CydDC appears to adopt at least two structural states, each associated with a characteristic level of bound heme. The purified protein in detergent showed a weak basal ATPase activity (approximately 100 nmol Pi/min/mg) that was stimulated ∼3-fold by various thiol compounds, suggesting that CydDC could act as a thiol transporter. The presence of heme (either intrinsic or added in the form of hemin) led to a further enhancement of thiol-stimulated ATPase activity, although a large excess of heme inhibited activity. Similar responses of the ATPase activity were observed with CydDC reconstituted into E. coli lipids. These results suggest that heme may have a regulatory role in CydDC-mediated transmembrane thiol transport.
Lysosomes contain abundant ATP, which is released through lysosomal exocytosis following exposure to various stimuli. However, the molecular mechanisms underlying lysosomal ATP accumulation remain unknown. The vesicular nucleotide transporter, also known as solute carrier family 17 member 9 (SLC17A9), has been shown to function in ATP transport across secretory vesicles/granules membrane in adrenal chromaffin cells, T cells, and pancreatic cells. Here, using mammalian cell lines, we report that SLC17A9 is highly enriched in lysosomes and functions as an ATP transporter in those organelles. SLC17A9 deficiency reduced lysosome ATP accumulation and compromised lysosome function, resulting in cell death. Our data suggest that SLC17A9 activity mediates lysosomal ATP accumulation and plays an important role in lysosomal physiology and cell viability.
Oxidative stress has been implicated in cardiac arrhythmia, although a causal relationship remains undefined. We have recently demonstrated a marked up-regulation of NADPH oxidase isoform 4 (NOX4) in patients with atrial fibrillation, which is accompanied by overproduction of reactive oxygen species (ROS). In this study, we investigated the impact on the cardiac phenotype of NOX4 overexpression in zebrafish. One-cell stage embryos were injected with NOX4 RNA prior to video recording of a GFP-labeled (myl7:GFP zebrafish line) beating heart in real time at 24–31 h post-fertilization. Intriguingly, NOX4 embryos developed cardiac arrhythmia that is characterized by irregular heartbeats. When quantitatively analyzed by an established LQ-1 program, the NOX4 embryos displayed much more variable beat-to-beat intervals (mean S.D. of beat-to-beat intervals was 0.027 s/beat in control embryos versus 0.038 s/beat in NOX4 embryos). Both the phenotype and the increased ROS in NOX4 embryos were attenuated by NOX4 morpholino co-injection, treatments of the embryos with polyethylene glycol-conjugated superoxide dismutase, or NOX4 inhibitors fulvene-5, 6-dimethylamino-fulvene, and proton sponge blue. Injection of NOX4-P437H mutant RNA had no effect on the cardiac phenotype or ROS production. In addition, phosphorylation of calcium/calmodulin-dependent protein kinase II was increased in NOX4 embryos but diminished by polyethylene glycol-conjugated superoxide dismutase, whereas its inhibitor KN93 or AIP abolished the arrhythmic phenotype. Taken together, our data for the first time uncover a novel pathway that underlies the development of cardiac arrhythmia, namely NOX4 activation, subsequent NOX4-specific NADPH-driven ROS production, and redox-sensitive CaMKII activation. These findings may ultimately lead to novel therapeutics targeting cardiac arrhythmia.
The aggregation of Tau into paired helical filaments is involved in the pathogenesis of several neurodegenerative diseases, including Alzheimer disease. The aggregation reaction is characterized by conformational conversion of the repeat domain, which partially adopts a cross-β-structure in the resulting amyloid-like fibrils. Here, we report the selection and characterization of an engineered binding protein, β-wrapin TP4, targeting the Tau repeat domain. TP4 was obtained by phage display using the four-repeat Tau construct K18ΔK280 as a target. TP4 binds K18ΔK280 as well as the longest isoform of human Tau, hTau40, with nanomolar affinity. NMR spectroscopy identified two alternative TP4-binding sites in the four-repeat domain, with each including two hexapeptide motifs with high β-sheet propensity. Both binding sites contain the aggregation-determining PHF6 hexapeptide within repeat 3. In addition, one binding site includes the PHF6* hexapeptide within repeat 2, whereas the other includes the corresponding hexapeptide Tau(337–342) within repeat 4, denoted PHF6**. Comparison of TP4-binding with Tau aggregation reveals that the same regions of Tau are involved in both processes. TP4 inhibits Tau aggregation at substoichiometric concentration, demonstrating that it interferes with aggregation nucleation. This study provides residue-level insight into the interaction of Tau with an aggregation inhibitor and highlights the structural flexibility of Tau.
Many essential cellular proteins fold only with the assistance of chaperonin machines like the GroEL-GroES system of Escherichia coli. However, the mechanistic details of assisted protein folding by GroEL-GroES remain the subject of ongoing debate. We previously demonstrated that GroEL-GroES enhances the productive folding of a kinetically trapped substrate protein through unfolding, where both binding energy and the energy of ATP hydrolysis are used to disrupt the inhibitory misfolded states. Here, we show that the intrinsically disordered yet highly conserved C-terminal sequence of the GroEL subunits directly contributes to substrate protein unfolding. Interactions between the C terminus and the non-native substrate protein alter the binding position of the substrate protein on the GroEL apical surface. The C-terminal tails also impact the conformational state of the substrate protein during capture and encapsulation on the GroEL ring. Importantly, removal of the C termini results in slower overall folding, reducing the fraction of the substrate protein that commits quickly to a productive folding pathway and slowing several kinetically distinct folding transitions that occur inside the GroEL-GroES cavity. The conserved C-terminal tails of GroEL are thus important for protein folding from the beginning to the end of the chaperonin reaction cycle.
Virus-associated RNA I (VA RNAI) is a short (∼160-nucleotide) non-coding RNA transcript employed by adenoviruses to subvert the innate immune system protein double-stranded RNA-activated protein kinase (PKR). The central domain of VA RNAI is proposed to contain a complex tertiary structure that contributes to its optimal inhibitory activity against PKR. Here we use a combination of VA RNAI mutagenesis, structural analyses, as well as PKR activity and binding assays to dissect this tertiary structure and assess its functional role. Our results support the existence of a pH- and Mg2+-dependent tertiary structure involving pseudoknot formation within the central domain. Unexpectedly, this structure appears to play no direct role in PKR inhibition. Deletion of central domain sequences within a minimal but fully active construct lacking the tertiary structure reveals a crucial role in PKR binding and inhibition for nucleotides in the 5′ half of the central domain. Deletion of the central domain 3′ half also significantly impacts activity but appears to arise indirectly by reducing its capacity to assist in optimally presenting the 5′ half sequence. Collectively, our results identify regions of VA RNAI critical for PKR inhibition and reveal that the requirements for an effective RNA inhibitor of PKR are simpler than appreciated previously.
Metformin, a well known antidiabetic agent that improves peripheral insulin sensitivity, also elicits anti-inflammatory actions, but its mechanism is unclear. Here, we investigated the mechanism responsible for the anti-inflammatory effect of metformin action in lipopolysaccharide (LPS)-stimulated murine macrophages. Metformin inhibited LPS-induced production of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in a concentration-dependent manner and in parallel induction of activating transcription factor-3 (ATF-3), a transcription factor and member of the cAMP-responsive element-binding protein family. ATF-3 knockdown abolished the inhibitory effects of metformin on LPS-induced proinflammatory cytokine production accompanied with reversal of metformin-induced suppression of mitogen-activated protein kinase (MAPK) phosphorylation. Conversely, AMP-activated protein kinase (AMPK) phosphorylation and NF-κB suppression by metformin were unaffected by ATF-3 knockdown. ChIP-PCR analysis revealed that LPS-induced NF-κB enrichments on the promoters of IL-6 and TNF-α were replaced by ATF-3 upon metformin treatment. AMPK knockdown blunted all the effects of metformin (ATF-3 induction, proinflammatory cytokine inhibition, and MAPK inactivation), suggesting that AMPK activation by metformin is required for and precedes ATF-3 induction. Oral administration of metformin to either mice with LPS-induced endotoxemia or ob/ob mice lowered the plasma and tissue levels of TNF-α and IL-6 and increased ATF-3 expression in spleen and lungs. These results suggest that metformin exhibits anti-inflammatory action in macrophages at least in part via pathways involving AMPK activation and ATF-3 induction.
The aspartate in the prototypical integrin-binding motif Arg-Gly-Asp binds the integrin βA domain of the β-subunit through a divalent cation at the metal ion-dependent adhesion site (MIDAS). An auxiliary metal ion at a ligand-associated metal ion-binding site (LIMBS) stabilizes the metal ion at MIDAS. LIMBS contacts distinct residues in the α-subunits of the two β3 integrins αIIbβ3 and αVβ3, but a potential role of this interaction on stability of the metal ion at LIMBS in β3 integrins has not been explored. Equilibrium molecular dynamics simulations of fully hydrated β3 integrin ectodomains revealed strikingly different conformations of LIMBS in unliganded αIIbβ3 versus αVβ3, the result of stronger interactions of LIMBS with αV, which reduce stability of the LIMBS metal ion in αVβ3. Replacing the αIIb-LIMBS interface residue Phe191 in αIIb (equivalent to Trp179 in αV) with Trp strengthened this interface and destabilized the metal ion at LIMBS in αIIbβ3; a Trp179 to Phe mutation in αV produced the opposite but weaker effect. Consistently, an F191/W substitution in cellular αIIbβ3 and a W179/F substitution in αVβ3 reduced and increased, respectively, the apparent affinity of Mn2+ to the integrin. These findings offer an explanation for the variable occupancy of the metal ion at LIMBS in αVβ3 structures in the absence of ligand and provide new insights into the mechanisms of integrin regulation.