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Journal of Biological Chemistry, The

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Journal of Biological Chemistry
The recent discovery of numerous human short open reading frame (sORF)-encoded polypeptides (SEPs) has raised important questions about the functional roles of these molecules in cells. Here, we show that a 69-amino acid SEP, MRI-2, physically interacts with the Ku heterodimer to stimulate DNA double-strand break ligation via nonhomologous end joining. The characterization of MRI-2 suggests that this SEP may participate in DNA repair and underscores the potential of SEPs to serve important biological functions in mammalian cells.

βγ-Crystallin-type double clamp (N/D)(N/D)XX(S/T)S motif is an established but sparsely investigated motif for Ca2+ binding. A βγ-crystallin domain is formed of two Greek key motifs, accommodating two Ca2+-binding sites. βγ-Crystallins make a separate class of Ca2+-binding proteins (CaBP), apparently a major group of CaBP in bacteria. Paralleling the diversity in βγ-crystallin domains, these motifs also show great diversity, both in structure and in function. Although the expression of some of them has been associated with stress, virulence, and adhesion, the functional implications of Ca2+ binding to βγ-crystallins in mediating biological processes are yet to be elucidated.

Correct positioning of neurotransmitter-gated receptors at postsynapses is essential for synaptic transmission. At Caenorhabditis elegans neuromuscular junctions, clustering of levamisole-sensitive acetylcholine receptors (L-AChRs) requires the muscle-secreted scaffolding protein LEV-9, a multidomain factor containing complement control protein (CCP) modules. Here we show that LEV-9 needs to be cleaved at its C terminus to exert its function. LEV-9 cleavage is not required for trafficking nor secretion but directly controls scaffolding activity. The cleavage site is evolutionarily conserved, and post-translational cleavage ensures the structural and functional decoupling between different isoforms encoded by the lev-9 gene. Data mining indicates that most human CCP-containing factors are likely cleaved C-terminally from CCP tandems, suggesting that not only domain architectures but also cleavage location can be conserved in distant architecturally related proteins.

The metabotropic glutamate receptor subtype 7 (mGlu7) is an important presynaptic regulator of neurotransmission in the mammalian CNS. mGlu7 function has been linked to autism, drug abuse, anxiety, and depression. Despite this, it has been difficult to develop specific blockers of native mGlu7 signaling in relevant brain areas such as amygdala and limbic cortex. Here, we present the mGlu7-selective antagonist 7-hydroxy-3-(4-iodophenoxy)-4H-chromen-4-one (XAP044), which inhibits lateral amygdala long term potentiation (LTP) in brain slices from wild type mice with a half-maximal blockade at 88 nm. There was no effect of XAP044 on LTP of mGlu7-deficient mice, indicating that this pharmacological effect is mGlu7-dependent. Unexpectedly and in contrast to all previous mGlu7-selective drugs, XAP044 does not act via the seven-transmembrane region but rather via a binding pocket localized in mGlu7's extracellular Venus flytrap domain, a region generally known for orthosteric agonist binding. This was shown by chimeric receptor studies in recombinant cell line assays. XAP044 demonstrates good brain exposure and wide spectrum anti-stress and antidepressant- and anxiolytic-like efficacy in rodent behavioral paradigms. XAP044 reduces freezing during acquisition of Pavlovian fear and reduces innate anxiety, which is consistent with the phenotypes of mGlu7-deficient mice, the results of mGlu7 siRNA knockdown studies, and the inhibition of amygdala LTP by XAP044. Thus, we present an mGlu7 antagonist with a novel molecular mode of pharmacological action, providing significant application potential in psychiatry. Modeling the selective interaction between XAP044 and mGlu7's Venus flytrap domain, whose three-dimensional structure is already known, will facilitate future drug development supported by computer-assisted drug design.

♦ See referenced article, J. Biol. Chem. 2014, 289, 10975–10987 A receptor in the central nervous system called metabotropic glutamate receptor subtype 7 (mGlu7) has been implicated in a number of neurological disorders such as depression, drug abuse, and autism. There are no suitable molecules that specifically block mGlu7 in critical brain areas involved in emotion. In this Paper of the Week, a team led by Peter Flor at the University of Regensburg in Germany described a molecule called XAP044 that selectively targets mGlu7 in the amygdala of mouse brains. The investigators demonstrated that XAP044 acts on an extracellular domain of the receptor, a mode of action not seen with other mGlu7 drugs. When XAP044 acts on this extracellular domain, it disrupts a G protein signaling pathway in which mGlu7 is involved. The investigators also showed that the molecule works in mice to reduce stress, depressive, and anxiety behaviors. The authors concluded, “We present an mGlu7 antagonist with a novel molecular mode of pharmacological action, providing significant application potential in psychiatry.” jbc;289/16/10988/FU1F1FU1 XAP044 selectively antagonizes [35S]GTPγS binding via mGlu7.

Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease in the world, and it is thought to be the hepatic manifestation of the metabolic syndrome. Excess dietary fructose causes both metabolic syndrome and NAFLD in rodents and humans, but the pathogenic mechanisms of fructose-induced metabolic syndrome and NAFLD are poorly understood. GLUT8 (Slc2A8) is a facilitative glucose and fructose transporter that is highly expressed in liver, heart, and other oxidative tissues. We previously demonstrated that female mice lacking GLUT8 exhibit impaired first-pass hepatic fructose metabolism, suggesting that fructose transport into the hepatocyte, the primary site of fructose metabolism, is in part mediated by GLUT8. Here, we tested the hypothesis that GLUT8 is required for hepatocyte fructose uptake and for the development of fructose-induced NAFLD. We demonstrate that GLUT8 is a cell surface-localized transporter and that GLUT8 overexpression or GLUT8 shRNA-mediated gene silencing significantly induces and blocks radiolabeled fructose uptake in cultured hepatocytes. We further show diminished fructose uptake and de novo lipogenesis in fructose-challenged GLUT8-deficient hepatocytes. Finally, livers from long term high-fructose diet-fed GLUT8-deficient mice exhibited attenuated fructose-induced hepatic triglyceride and cholesterol accumulation without changes in hepatocyte insulin-stimulated Akt phosphorylation. GLUT8 is thus essential for hepatocyte fructose transport and fructose-induced macrosteatosis. Fructose delivery across the hepatocyte membrane is thus a proximal, modifiable disease mechanism that may be exploited to prevent NAFLD.

Membrane asymmetry is essential for generating second messengers that act in the cytosol and for trafficking of membrane proteins and membrane lipids, but the role of asymmetry in regulating membrane protein function remains unclear. Here we show that the signaling lipid phosphoinositide 4,5-bisphosphate (PI(4,5)P2) has opposite effects on the function of TRPV1 ion channels depending on which leaflet of the cell membrane it resides in. We observed potentiation of capsaicin-activated TRPV1 currents by PI(4,5)P2 in the intracellular leaflet of the plasma membrane but inhibition of capsaicin-activated currents when PI(4,5)P2 was in both leaflets of the membrane, although much higher concentrations of PI(4,5)P2 in the extracellular leaflet were required for inhibition compared with the concentrations of PI(4,5)P2 in the intracellular leaflet that produced activation. Patch clamp fluorometry using a synthetic PI(4,5)P2 whose fluorescence reports its concentration in the membrane indicates that PI(4,5)P2 must incorporate into the extracellular leaflet for its inhibitory effects to be observed. The asymmetry-dependent effect of PI(4,5)P2 may resolve the long standing controversy about whether PI(4,5)P2 is an activator or inhibitor of TRPV1. Our results also underscore the importance of membrane asymmetry and the need to consider its influence when studying membrane proteins reconstituted into synthetic bilayers.

Amyloid precursor protein (APP) undergoes post-translational modification, including O- and N-glycosylation, ubiquitination, and phosphorylation as it traffics through the secretory pathway. We have previously reported that copper promotes a change in the cellular localization of APP. We now report that copper increases the phosphorylation of endogenous APP at threonine 668 (Thr-668) in SH-SY5Y neuronal cells. The level of APPT668-p (detected using a phospho-site-specific antibody) exhibited a copper-dependent increase. Using confocal microscopy imaging we demonstrate that the phospho-deficient mutant, Thr-668 to alanine (T668A), does not exhibit detectable copper-responsive APP trafficking. In contrast, mutating a serine to an alanine at residue 655 does not affect copper-responsive trafficking. We further investigated the importance of the Thr-668 residue in copper-responsive trafficking by treating SH-SY5Y cells with inhibitors for glycogen synthase kinase 3-β (GSK3β) and cyclin-dependent kinases (Cdk), the main kinases that phosphorylate APP at Thr-668 in neurons. Our results show that the GSK3β kinase inhibitors LiCl, SB 216763, and SB 415286 prevent copper-responsive APP trafficking. In contrast, the Cdk inhibitors Purvalanol A and B had no significant effect on copper-responsive trafficking in SH-SY5Y cells. In cultured primary hippocampal neurons, copper promoted APP re-localization to the axon, and this effect was inhibited by the addition of LiCl, indicating that a lithium-sensitive kinase(s) is involved in copper-responsive trafficking in hippocampal neurons. This is consistent with APP axonal transport to the synapse, where APP is involved in a number of functions. We conclude that copper promotes APP trafficking by promoting a GSK3β-dependent phosphorylation in SH-SY5Y cells.

The catalytic domain of XynCDBFV, a glycoside hydrolase family 11 (GH11) xylanase from ruminal fungus Neocallimastix patriciarum previously engineered to exhibit higher specific activity and broader pH adaptability, holds great potential in commercial applications. Here, the crystal structures of XynCDBFV and its complex with substrate were determined to 1.27–1.43 Å resolution. These structures revealed a typical GH11 β-jelly-roll fold and detailed interaction networks between the enzyme and ligands. Notably, an extended N-terminal region (NTR) consisting of 11 amino acids was identified in the XynCDBFV structure, which is found unique among GH11 xylanases. The NTR is attached to the catalytic core by hydrogen bonds and stacking forces along with a disulfide bond between Cys-4 and Cys-172. Interestingly, the NTR deletion mutant retained 61.5% and 19.5% enzymatic activity at 55 °C and 75 °C, respectively, compared with the wild-type enzyme, whereas the C4A/C172A mutant showed 86.8% and 23.3% activity. These results suggest that NTR plays a role in XynCDBFV thermostability, and the Cys-4/Cys-172 disulfide bond is critical to the NTR-mediated interactions. Furthermore, we also demonstrated that Pichia pastoris produces XynCDBFV with higher catalytic activity at higher temperature than Escherichia coli, in which incorrect NTR folding and inefficient disulfide bond formation might have occurred. In conclusion, these structural and functional analyses of the industrially favored XynCDBFV provide a molecular basis of NTR contribution to its thermostability.

ANO1, a calcium-activated chloride channel, is highly expressed and amplified in human cancers and is a critical survival factor in these cancers. The ANO1 inhibitor CaCCinh-A01 decreases proliferation of ANO1-amplified cell lines; however, the mechanism of action remains elusive. We explored the mechanism behind the inhibitory effect of CaCCinh-A01 on cell proliferation using a combined experimental and in silico approach. We show that inhibition of ANO1 function is not sufficient to diminish proliferation of ANO1-dependent cancer cells. We report that CaCCinh-A01 reduces ANO1 protein levels by facilitating endoplasmic reticulum-associated, proteasomal turnover of ANO1. Washout of CaCCinh-A01 rescued ANO1 protein levels and resumed cell proliferation. Proliferation of newly derived CaCCinh-A01-resistant cell pools was not affected by CaCCinh-A01 as compared with the parental cells. Consistently, CaCCinh-A01 failed to reduce ANO1 protein levels in these cells, whereas ANO1 currents were still inhibited by CaCCinh-A01, indicating that CaCCinh-A01 inhibits cell proliferation by reducing ANO1 protein levels. Furthermore, we employed in silico methods to elucidate novel biological functions of ANO1 inhibitors. Specifically, we derived a pharmacophore model to describe inhibitors capable of promoting ANO1 degradation and report new inhibitors of ANO1-dependent cell proliferation. In summary, our data demonstrate that inhibition of the channel activity of ANO1 is not sufficient to inhibit ANO1-dependent cell proliferation, indicating that the role of ANO1 in cancer only partially depends on its function as a channel. Our results provide an impetus for gaining a deeper understanding of ANO1 modulation in cells and introduce a new targeting approach for antitumor therapy in ANO1-amplified cancers.

Dps (DNA-binding protein from starved cells) are dodecameric assemblies belonging to the ferritin family that can bind DNA, carry out ferroxidation, and store iron in their shells. The ferritin-like trimeric pore harbors the channel for the entry and exit of iron. By representing the structure of Dps as a network we have identified a charge-driven interface formed by a histidine aspartate cluster at the pore interface unique to Mycobacterium smegmatis Dps protein, MsDps2. Site-directed mutagenesis was employed to generate mutants to disrupt the charged interactions. Kinetics of iron uptake/release of the wild type and mutants were compared. Crystal structures were solved at a resolution of 1.8–2.2 Å for the various mutants to compare structural alterations vis à vis the wild type protein. The substitutions at the pore interface resulted in alterations in the side chain conformations leading to an overall weakening of the interface network, especially in cases of substitutions that alter the charge at the pore interface. Contrary to earlier findings where conserved aspartate residues were found crucial for iron release, we propose here that in the case of MsDps2, it is the interplay of negative-positive potentials at the pore that enables proper functioning of the protein. In similar studies in ferritins, negative and positive patches near the iron exit pore were found to be important in iron uptake/release kinetics. The unique ionic cluster in MsDps2 makes it a suitable candidate to act as nano-delivery vehicle, as these gated pores can be manipulated to exhibit conformations allowing for slow or fast rates of iron release.

The small GTPase Rab27A is a crucial regulator of actin-based melanosome transport in melanocytes, and functionally defective Rab27A causes human Griscelli syndrome type 2, which is characterized by silvery hair. A GTPase-deficient, constitutively active Rab27A(Q78L) mutant has been shown to act as an inhibitor of melanosome transport and to induce perinuclear aggregation of melanosomes, but the molecular mechanism by which Rab27A(Q78L) inhibits melanosome transport remained to be determined. In this study, we attempted to identify the primary cause of the perinuclear melanosome aggregation induced by Rab27A(Q78L). The results showed that Rab27A(Q78L) is unable to localize on mature melanosomes and that its inhibitory activity on melanosome transport is completely dependent on its binding to the Rab27A effector Slac2-a/melanophilin. When we forcibly expressed Rab27A(Q78L) on mature melanosomes by using a novel melanosome-targeting tag that we developed in this study and named the MST tag, the MST-Rab27A(Q78L) fusion protein behaved in the same manner as wild-type Rab27A. It localized on mature melanosomes without inducing melanosome aggregation and restored normal peripheral melanosome distribution in Rab27A-deficient cells. These findings indicate that the GTPase activity of Rab27A is required for its melanosome localization but is not required for melanosome transport.

Recruitment of mesenchymal stem cells (MSC) following cardiac injury, such as myocardial infarction, plays a critical role in tissue repair and may contribute to myocardial recovery. However, the mechanisms that regulate migration of MSC to the site of tissue damage remain elusive. Here, we demonstrate in vitro that activated platelets substantially inhibit recruitment of MSC toward apoptotic cardiac myocytes and fibroblasts. The alarmin high mobility group box 1 (HMGB1) was released by platelets upon activation and mediated inhibition of the cell death-dependent migratory response through Toll-like receptor (TLR)-4 expressed on the MSC. Migration of MSC to apoptotic cardiac myocytes and fibroblasts was driven by hepatocyte growth factor (HGF), and platelet activation was followed by HMGB1/TLR-4-dependent down-regulation of HGF receptor MET on MSC, thereby impairing HGF-driven MSC recruitment. We identify a novel mechanism by which platelets, upon activation, interfere with MSC recruitment to apoptotic cardiac cells, a process that may be of particular relevance for myocardial repair and regeneration.

The bifunctional major autolysin AtlA of Staphylococcus aureus cleaves the bacterium's peptidoglycan network (PGN) at two distinct sites during cell division. Deletion of the enzyme results in large cell clusters with disordered division patterns, indicating that AtlA could be a promising target for the development of new antibiotics. One of the two functions of AtlA is performed by the N-acetylmuramyl-l-alanine amidase AmiA, which cleaves the bond between the carbohydrate and the peptide moieties of PGN. To establish the structural requirements of PGN recognition and the enzymatic mechanism of cleavage, we solved the crystal structure of the catalytic domain of AmiA (AmiA-cat) in complex with a peptidoglycan-derived ligand at 1.55 Å resolution. The peptide stem is clearly visible in the structure, forming extensive contacts with protein residues by docking into an elongated groove. Less well defined electron density and the analysis of surface features indicate likely positions of the carbohydrate backbone and the pentaglycine bridge. Substrate specificity analysis supports the importance of the pentaglycine bridge for fitting into the binding cleft of AmiA-cat. PGN of S. aureus with l-lysine tethered with d-alanine via a pentaglycine bridge is completely hydrolyzed, whereas PGN of Bacillus subtilis with meso-diaminopimelic acid directly tethered with d-alanine is not hydrolyzed. An active site mutant, H370A, of AmiA-cat was completely inactive, providing further support for the proposed catalytic mechanism of AmiA. The structure reported here is not only the first of any bacterial amidase in which both the PGN component and the water molecule that carries out the nucleophilic attack on the carbonyl carbon of the scissile bond are present; it is also the first peptidoglycan amidase complex structure of an important human pathogen.

Oxysterols, oxidized metabolites of cholesterol, are endogenous small molecules that regulate lipid metabolism, immune function, and developmental signaling. Although the cell biology of cholesterol has been intensively studied, fundamental questions about oxysterols, such as their subcellular distribution and trafficking pathways, remain unanswered. We have therefore developed a useful method to image intracellular 20(S)-hydroxycholesterol with both high sensitivity and spatial resolution using click chemistry and fluorescence microscopy. The metabolic labeling of cells with an alkynyl derivative of 20(S)-hydroxycholesterol has allowed us to directly visualize this oxysterol by attaching an azide fluorophore through cyclo-addition. Unexpectedly, we found that this oxysterol selectively accumulates in the Golgi membrane using a pathway that is sensitive to ATP levels, temperature, and lysosome function. Although previous models have proposed nonvesicular pathways for the rapid equilibration of oxysterols between membranes, direct imaging of oxysterols suggests that a vesicular pathway is responsible for differential accumulation of oxysterols in organelle membranes. More broadly, clickable alkynyl sterols may represent useful tools for sterol cell biology, both to investigate the functions of these important lipids and to decipher the pathways that determine their cellular itineraries.

The ARF family of regulatory GTPases, within the RAS superfamily, is composed of ∼30 members in mammals, including up to six ARF and at least 18 ARF-like (ARL) proteins. They exhibit significant structural and biochemical conservation and regulate a variety of essential cellular processes, including membrane traffic, cell division, and energy metabolism; each with links to human diseases. We previously identified members of the ELMOD family as GTPase-activating proteins (GAPs) for ARL2 that displayed crossover activity for ARFs as well. To further characterize the GAP activities of the three human ELMODs as GAPs we developed new preparations of each after overexpression in human embryonic kidney (HEK293T) cells. This allowed much higher specific activities and enhanced stability and solubility of the purified proteins. The specificities of ELMOD1–3 as GAPs for six different members of the ARF family were determined and found to display wide variations, which we believe will reveal differences in cellular functions of family members. The non-opioid sigma-1 receptor (S1R) was identified as a novel effector of GAP activity of ELMOD1–3 proteins as its direct binding to either ELMOD1 or ELMOD2 resulted in loss of GAP activity. These findings are critical to understand the roles of ELMOD proteins in cell signaling in general and in the inner ear specifically, and open the door to exploration of the regulation of their GAP activities via agonists or antagonists of the S1R.

Amyloid β (Aβ) fibrillar deposits in the brain are a hallmark of Alzheimer disease (AD). Curcumin, a common ingredient of Asian spices, is known to disrupt Aβ fibril formation and to reduce AD pathology in mouse models. Understanding the structural changes induced by curcumin can potentially lead to AD pharmaceutical agents with inherent bio-compatibility. Here, we use solid-state NMR spectroscopy to investigate the structural modifications of amyloid β(1–42) (Aβ42) aggregates induced by curcumin. We find that curcumin induces major structural changes in the Asp-23–Lys-28 salt bridge region and near the C terminus. Electron microscopy shows that the Aβ42 fibrils are disrupted by curcumin. Surprisingly, some of these alterations are similar to those reported for Zn2+ ions, another agent known to disrupt the fibrils and alter Aβ42 toxicity. Our results suggest the existence of a structurally related family of quasi-fibrillar conformers of Aβ42, which is stabilized both by curcumin and by Zn2+.

The transfer of N-acetylglucosamine (GlcNAc) to Ser or Thr in cytoplasmic and nuclear proteins is a well known post-translational modification that is catalyzed by the O-GlcNAc transferase OGT. A more recently identified O-GlcNAc transferase, EOGT, functions in the secretory pathway and transfers O-GlcNAc to proteins with epidermal growth factor-like (EGF) repeats. A number of antibodies that detect O-GlcNAc in cytosolic and nuclear extracts have been described previously. Here we compare seven of these antibodies (CTD110.6, 10D8, RL2, HGAC85, 18B10.C7(#3), 9D1.E4(#10), and 1F5.D6 (#14) for detection of the O-GlcNAc modification on extracellular domains of membrane or secreted glycoproteins that may also carry various N- and O-glycans. We found that CTD110.6 binds not only to O-GlcNAc on proteins but also to terminal β-GlcNAc on the complex N-glycans of Lec8 Chinese hamster ovary (CHO) cells that lack UDP-Gal transporter activity and express GlcNAc-terminating, complex N-glycans. We show that CTD110.6, #3, and #10 antibodies can be used to detect cell surface glycoproteins bearing O-GlcNAc. Cell surface glycoproteins recognized by CTD110.6 antibody included NOTCH1 that possesses many EGF repeats with a consensus site for EOGT. Knockdown of CHO Eogt reduced binding of CTD110.6 to Lec1 CHO cells, and expression of a human EOGT cDNA increased the O-GlcNAc signal on Lec1 cells and the extracellular domain of NOTCH1. Thus, with careful controls, antibodies CTD110.6 (IgM), #3 (IgG), and #10 (IgG) can be used to detect membrane and secreted proteins modified by O-GlcNAc on EGF repeats.

Gdown1, the substoichiometric 13th subunit of RNA polymerase II (pol II), has an important role in pausing during the initial stage of transcript elongation. However, Gdown1 quantitatively displaces the essential initiation factor TFIIF from free pol II and elongating pol II. Thus, it is not clear how or even if pol II can initiate in the presence of Gdown1. Using an in vitro transcription system with purified factors and pol II lacking Gdown1, we found that although Gdown1 is strongly inhibitory to transcription when prebound to pol II, a fraction of complexes do remain active. Surprisingly, when Gdown1 is added to complete preinitiation complexes (PICs), it does not inhibit initiation or functionally associate with the PICs. Gdown1 does associate with pol II during the early stage of transcript elongation but this association is competitive with TFIIF. By phosphorylating TFIIF, PICs can be assembled that do not retain TFIIF. Gdown1 also fails to functionally associate with these TFIIF-less PICs, but once polymerase enters transcript elongation, complexes lacking TFIIF quantitatively bind Gdown1. Our results provide a partial resolution of the paradox of the competition between Gdown1 and TFIIF for association with pol II. Although Gdown1 completely displaces TFIIF from free pol II and elongation complexes, Gdown1 does not functionally associate with the PIC. Gdown1 can enter the transcription complex immediately after initiation. Modification of TFIIF provides one pathway through which efficient Gdown1 loading can occur early in elongation, allowing downstream pausing to be regulated.

The greatest challenge for the seeding of cancer in metastatic sites is integration into the ectopic microenvironment despite the lack of an orthotopic supportive environment and presence of pro-death signals concomitant with a localized “foreign-body” inflammatory response. In this metastatic location, many carcinoma cells display a reversion of the epithelial-to-mesenchymal transition that marks dissemination in the primary tumor mass. This mesenchymal to epithelial reverting transition (MErT) is thought to help seeding and colonization by protecting against cell death. We have previously shown that hepatocyte coculture induces the re-expression of E-cadherin via abrogation of autocrine EGFR signaling pathway in prostate cancer (PCa) cells and that this confers a survival advantage. Herein, we show that hepatocytes educate PCa to undergo MErT by modulating the activity of p38 and ERK1/2. Hepatocytes inhibited p38 and ERK1/2 activity in prostate cancer cells, which allowed E-cadherin re-expression. Introduction of constitutively active MEK6 and MEK1 to DU145 cells cocultured with hepatocytes abrogated E-cadherin re-expression. At least a partial phenotypic reversion can be achieved by suppression of p38 and ERK1/2 activation in DU145 cells even in the absence of hepatocytes. Interestingly, these mitogen-activated protein kinase activities were also triggered by re-expressed E-cadherin leading to p38 and ERK1/2 activity in PCa cells; these signals provide protection to PCa cells upon challenge with chemotherapy and cell death-inducing cytokines. We propose that distinct p38/ERK pathways are related to E-cadherin levels and function downstream of E-cadherin allowing, respectively, for hepatocyte-mediated MErT and tumor cell survival in the face of death signals.

Calcium phosphate precipitates (CPPs) form complexes with DNA, which enter cells via endocytosis. Under this condition CPPs induce autophagy via the canonic autophagy machinery. Here we showed that CPP-induced autophagy was also dependent on endocytosis as the process was significantly inhibited by methyl-β-cyclodextrin and dynasore, which suppress clathrin-dependent endocytosis. Consistently, CPP treatment triggered the formation of filipin-positive intracellular vesicles whose membranes are rich in cholesterol. Unexpectedly, these vesicles were also positive for galectin 3, suggesting that they were damaged and the membrane glycans became accessible to galectins to bind. Endosome damage was caused by endocytosis of CPPs and was reversed by calcium chelators or by endocytosis inhibitors. Notably, CPP-induced LC3-positive autophagosomes were colocalized with galectin 3, ubiquitin, and p62/SQSTM1. Inhibition of galectin 3 reduced p62 puncta and autophagosome formation. Knockdown of p62 additionally inhibited the colocalization of autophagosomes with galectins. Furthermore, most of the galectin 3-positive vesicles were colocalized with Rab7 or LAMP1. Agents that affect endosome/lysosome maturation and function, such as bafilomycin A1, also significantly affected CPP-induced tubulovesicular autophagosome formation. These findings thus indicate that endocytosed CPPs caused endosome damage and recruitment of galectins, particularly at the later endosome stage, which led to the interaction of the autophagosomal membranes with the damaged endosome in the presence of p62.

Excitatory amino acid transporters remove synaptically released glutamate and maintain its concentrations below neurotoxic levels. EAATs also mediate a thermodynamically uncoupled substrate-gated anion conductance that may modulate cell excitability. A structure of an archeal homologue, which reflects an early intermediate on the proposed substrate translocation path, has been suggested to be similar to an anion conducting conformation. To probe this idea by functional studies, we have introduced two cysteine residues in the neuronal glutamate transporter EAAC1 at positions predicted to be close enough to form a disulfide bond only in outward-facing and early intermediate conformations of the homologue. Upon treatment of Xenopus laevis oocytes expressing the W441C/K269C double mutant with dithiothreitol, radioactive transport was stimulated >2-fold but potently inhibited by low micromolar concentrations of the oxidizing reagent copper(II)(1,10-phenanthroline)3. The substrate-induced currents by the untreated double mutant, reversed at approximately −20 mV, close to the reversal potential of chloride, but treatment with dithiothreitol resulted in transport currents with the same voltage dependence as the wild type. It appears therefore that in the oocyte expression system the introduced cysteine residues in many of the mutant transporters are already cross-linked and are only capable of mediating the substrate-gated anion conductance. Reduction of the disulfide bond now allows these transporters to execute the full transport cycle. Our functional data support the idea that the anion conducting conformation of the neuronal glutamate transporter is associated with an early step of the transport cycle.

Protein-protein interactions are driving forces in cellular processes. As a prime example, transmembrane integrins link extracellular matrix and intracellular proteins, resulting in bidirectional signaling that regulates cell migration, proliferation, differentiation, and survival. Here we provide the first evidence that interaction between the integrin β1 cytoplasmic tail and kindlin-2, a member of a family of adapters implicated in human disease pathogenesis, is mainly governed by the β1 C-terminal carboxylate moiety and is required for laterality organ development in zebrafish. Affinity measurements indicate that this unusual protein-protein interaction mode is coordinated by a putative carboxylate-binding motif in the kindlin-2 FERM subdomain F3. Contrary to the C terminus of proteins that engage PDZ domains, the C-terminal three residues of β1, per se, do not contribute to kindlin-2 binding or to laterality organ development. Thus, by employing zebrafish as an in situ physiological tool to correlate protein structure and function, we have discovered an unexpected association chemistry between an integrin and a key adapter involved in integrin signaling.

Epithelial-mesenchymal transition (EMT) is required for the specification of tissues during embryonic development and is recapitulated during the metastatic progression of tumors. The miR-200 family plays a critical role in enforcing the epithelial state with their expression lost in cells undergoing EMT. EMT can be mediated by activation of the ZEB1 and ZEB2 (ZEB) transcription factors, which repress miR-200 expression via a self-reinforcing double negative feedback loop to promote the mesenchymal state. However, it remains unclear what factors drive and maintain epithelial-specific expression of miR-200 in the absence of EMT-inducing factors. Here, we show that the transcription factor Specificity Protein 1 (Sp1) binds to the miR-200b∼200a∼429 proximal promoter and activates miR-200 expression in epithelial cells. In mesenchymal cells, Sp1 expression is maintained, but its ability to activate the miR-200 promoter is perturbed by ZEB-mediated repression. Reduction of Sp1 expression caused changes in EMT-associated markers in epithelial cells. Furthermore, we observed co-expression of Sp1 and miR-200 during mouse embryonic development wherein miR-200 expression was only lost in regions with high ZEB expression. Together, these findings indicate that miR-200 family members require Sp1 to drive basal expression and to maintain an epithelial state.

Phospholipase Cβ (PLCβ) enzymes are activated by G protein-coupled receptors through receptor-catalyzed guanine nucleotide exchange on Gαβγ heterotrimers containing Gq family G proteins. Here we report evidence for a direct interaction between M3 muscarinic receptor (M3R) and PLCβ3. Both expressed and endogenous M3R interacted with PLCβ in coimmunoprecipitation experiments. Stimulation of M3R with carbachol significantly increased this association. Expression of M3R in CHO cells promoted plasma membrane localization of YFP-PLCβ3. Deletion of the PLCβ3 C terminus or deletion of the PLCβ3 PDZ ligand inhibited coimmunoprecipitation with M3R and M3R-dependent PLCβ3 plasma membrane localization. Purified PLCβ3 bound directly to glutathione S-transferase (GST)-fused M3R intracellular loops 2 and 3 (M3Ri2 and M3Ri3) as well as M3R C terminus (M3R/H8-CT). PLCβ3 binding to M3Ri3 was inhibited when the PDZ ligand was removed. In assays using reconstituted purified components in vitro, M3Ri2, M3Ri3, and M3R/H8-CT potentiated Gαq-dependent but not Gβγ-dependent PLCβ3 activation. Disruption of key residues in M3Ri3N and of the PDZ ligand in PLCβ3 inhibited M3Ri3-mediated potentiation. We propose that the M3 muscarinic receptor maximizes the efficiency of PLCβ3 signaling beyond its canonical role as a guanine nucleotide exchange factor for Gα.

Cancer cell proliferation is a metabolically demanding process, requiring high glycolysis, which is known as “Warburg effect,” to support anabolic growth. Steroid receptor coactivator-3 (SRC-3), a steroid receptor coactivator, is overexpressed and/or amplified in multiple cancer types, including non-steroid targeted cancers, such as urinary bladder cancer (UBC). However, whether SRC-3 regulates the metabolic reprogramming for cancer cell growth is unknown. Here, we reported that overexpression of SRC-3 accelerated UBC cell growth, accompanied by the increased expression of genes involved in glycolysis. Knockdown of SRC-3 reduced the UBC cell glycolytic rate under hypoxia, decreased tumor growth in nude mice, with reduction of proliferating cell nuclear antigen and lactate dehydrogenase expression levels. We further revealed that SRC-3 could interact with hypoxia inducible factor 1α (HIF1α), which is a key transcription factor required for glycolysis, and coactivate its transcriptional activity. SRC-3 was recruited to the promoters of HIF1α-target genes, such as glut1 and pgk1. The positive correlation of expression levels between SRC-3 and Glut1 proteins was demonstrated in human UBC patient samples. Inhibition of glycolysis through targeting HK2 or LDHA decelerated SRC-3 overexpression-induced cell growth. In summary, overexpression of SRC-3 promoted glycolysis in bladder cancer cells through HIF1α to facilitate tumorigenesis, which may be an intriguing drug target for bladder cancer therapy.

Wnt signaling plays a pivotal role in cell proliferation, tissue homeostasis, and tumorigenesis. Dishevelled (Dvl) is a central node of Wnt signaling. Insulin receptor substrates (IRSs), as a critical component of insulin signaling, are involved in cell proliferation, metabolism, and cancer development. In this study, we report that IRS1/2 promotes Wnt/β-catenin signaling by stabilizing Dvl2. We found that IRS1/2 interacts with Dvl2. Overexpression of IRS1/2 increased the protein level of Dvl2 and promoted canonical Wnt signaling, as evidenced by the increased T cell-specific factor 4 transcriptional activity and the up-regulation of expression of CYCLIN D1 and c-MYC, two Wnt target genes critical for cell growth, whereas depletion of IRS1/2 reduced the level of Dvl2 and attenuated Wnt/β-catenin signaling. Biochemical analyses revealed that IRS1/2 decreased Lys-63-linked ubiquitination of Dvl2 and stabilized Dvl2 protein via suppressing its autophagy-mediated degradation. We further revealed that IRS1/2 blocks autophagy-induced formation of the Dvl2-p62/SQSTM1 complex, resulting in disabled association of Dvl2 to autophagosomes. We demonstrated that IRS1/2 promoted the induction of epithelial-mesenchymal transition (EMT) and cell proliferation in response to Wnt stimulation, whereas depletion of Dvl2 impaired the IRS1/2-mediated EMT and cell growth. Our findings revealed that IRS1/2 promotes EMT and cell proliferation through stabilizing Dvl2.

In this work we present and compare the results of extensive molecular dynamics simulations of model systems comprising an Aβ1–40 peptide in water in interaction with short peptides (β-sheet breakers) mimicking the 17–21 region of the Aβ1–40 sequence. Various systems differing in the customized β-sheet breaker structure have been studied. Specifically we have considered three kinds of β-sheet breakers, namely Ac-LPFFD-NH2 and two variants thereof, one obtained by substituting the acetyl group with the sulfonic amino acid taurine (Tau-LPFFD-NH2) and a second novel one in which the aspartic acid is substituted by an asparagine (Ac-LPFFN-NH2). Thioflavin T fluorescence, circular dichroism, and mass spectrometry experiments have been performed indicating that β-sheet breakers are able to inhibit in vitro fibril formation and prevent the β sheet folding of portions of the Aβ1–40 peptide. We show that molecular dynamics simulations and far UV circular dichroism provide consistent evidence that the new Ac-LPFFN-NH2 β-sheet breaker is more effective than the other two in stabilizing the native α-helix structure of Aβ1–40. In agreement with these results thioflavin T fluorescence experiments confirm the higher efficiency in inhibiting Aβ1–40 aggregation. Furthermore, mass spectrometry data and molecular dynamics simulations consistently identified the 17–21 Aβ1–40 portion as the location of the interaction region between peptide and the Ac-LPFFN-NH2 β-sheet breaker.

Expression of glycosyltransferase genes is essential for glycosylation. However, the detailed mechanisms of how glycosyltransferase gene expression is regulated in a specific tissue or during disease progression are poorly understood. In particular, epigenetic studies of glycosyltransferase genes are limited, although epigenetic mechanisms, such as histone and DNA modifications, are central to establish tissue-specific gene expression. We previously found that epigenetic histone activation is essential for brain-specific expression of N-acetylglucosaminyltransferase-IX (GnT-IX, also designated GnT-Vb), but the mechanism of brain-specific chromatin activation around GnT-IX gene (Mgat5b) has not been clarified. To reveal the mechanisms regulating the chromatin surrounding GnT-IX, we have investigated the epigenetic factors that are specifically involved with the mouse GnT-IX locus by comparing their involvement with other glycosyltransferase loci. We first found that a histone deacetylase (HDAC) inhibitor enhanced the expression of GnT-IX but not of other glycosyltransferases tested. By overexpression and knockdown of a series of HDACs, we found that HDAC11 silenced GnT-IX. We also identified the O-GlcNAc transferase (OGT) and ten-eleven translocation-3 (TET3) complex as a specific chromatin activator of GnT-IX gene. Moreover, chromatin immunoprecipitation (ChIP) analysis in combination with OGT or TET3 knockdown showed that this OGT-TET3 complex facilitates the binding of a potent transactivator, NeuroD1, to the GnT-IX promoter, suggesting that epigenetic chromatin activation by the OGT-TET3 complex is a prerequisite for the efficient binding of NeuroD1. These results reveal a new epigenetic mechanism of brain-specific GnT-IX expression regulated by defined chromatin modifiers, providing new insights into the tissue-specific expression of glycosyltransferases.

Mutations in the NPHS2 gene are a major cause of steroid-resistant nephrotic syndrome, a severe human kidney disorder. The NPHS2 gene product podocin is a key component of the slit diaphragm cell junction at the kidney filtration barrier and part of a multiprotein-lipid supercomplex. A similar complex with the podocin ortholog MEC-2 is required for touch sensation in Caenorhabditis elegans. Although podocin and MEC-2 are membrane-associated proteins with a predicted hairpin-like structure and amino and carboxyl termini facing the cytoplasm, this membrane topology has not been convincingly confirmed. One particular mutation that causes kidney disease in humans (podocinP118L) has also been identified in C. elegans in genetic screens for touch insensitivity (MEC-2P134S). Here we show that both mutant proteins, in contrast to the wild-type variants, are N-glycosylated because of the fact that the mutant C termini project extracellularly. PodocinP118L and MEC-2P134S did not fractionate in detergent-resistant membrane domains. Moreover, mutant podocin failed to activate the ion channel TRPC6, which is part of the multiprotein-lipid supercomplex, indicative of the fact that cholesterol recruitment to the ion channels, an intrinsic function of both proteins, requires C termini facing the cytoplasmic leaflet of the plasma membrane. Taken together, this study demonstrates that the carboxyl terminus of podocin/MEC-2 has to be placed at the inner leaflet of the plasma membrane to mediate cholesterol binding and contribute to ion channel activity, a prerequisite for mechanosensation and the integrity of the kidney filtration barrier.

The 26S proteasome is the end point of the ubiquitin- and ATP-dependent degradation pathway. The 26S proteasome complex (26S PC) integrity and function has been shown to be highly dependent on ATP and its homolog nucleotides. We report here that the redox molecule NADH binds the 26S PC and is sufficient in maintaining 26S PC integrity even in the absence of ATP. Five of the 19S proteasome complex subunits contain a putative NADH binding motif (GxGxxG) including the AAA-ATPase subunit, Psmc1 (Rpt2). We demonstrate that recombinant Psmc1 binds NADH via the GxGxxG motif. Introducing the ΔGxGxxG Psmc1 mutant into cells results in reduced NADH-stabilized 26S proteasomes and decreased viability following redox stress induced by the mitochondrial inhibitor rotenone. The newly identified NADH binding of 26S proteasomes advances our understanding of the molecular mechanisms of protein degradation and highlights a new link between protein homeostasis and the cellular metabolic/redox state.

Apolipoprotein E (apoE) is the major cholesterol transport protein in the brain. Among the three human APOE alleles (APOE2, APOE3, and APOE4), APOE4 is the strongest genetic risk factor for late-onset Alzheimer disease (AD). The accumulation of amyloid-β (Aβ) is a central event in AD pathogenesis. Increasing evidence demonstrates that apoE isoforms differentially regulate AD-related pathways through both Aβ-dependent and -independent mechanisms; therefore, modulating apoE secretion, lipidation, and function might be an attractive approach for AD therapy. We performed a drug screen for compounds that modulate apoE production in immortalized astrocytes derived from apoE3-targeted replacement mice. Here, we report that retinoic acid (RA) isomers, including all-trans-RA, 9-cis-RA, and 13-cis-RA, significantly increase apoE secretion to ∼4-fold of control through retinoid X receptor (RXR) and RA receptor. These effects on modulating apoE are comparable with the effects recently reported for the RXR agonist bexarotene. Furthermore, all of these compounds increased the expression of the cholesterol transporter ABCA1 and ABCG1 levels and decreased cellular uptake of Aβ in an apoE-dependent manner. Both bexarotene and 9-cis-RA promote the lipidation status of apoE, in which 9-cis-RA promotes a stronger effect and exhibits less cytotoxicity compared with bexarotene. Importantly, we showed that oral administration of bexarotene and 9-cis-RA significantly increases apoE, ABCA1, and ABCG1 levels in mouse brains. Taken together, our results demonstrate that RXR/RA receptor agonists, including several RA isomers, are effective modulators of apoE secretion and lipidation and may be explored as potential drugs for AD therapy.

Isoform 3 of the Na+-Ca2+ exchanger (NCX3) is crucial for maintaining intracellular calcium ([Ca2+]i) homeostasis in excitable tissues. In this sense NCX3 plays a key role in neuronal excitotoxicity and Ca2+ extrusion during skeletal muscle relaxation. Alternative splicing generates two variants (NCX3-AC and NCX3-B). Here, we demonstrated that NCX3 variants display a tissue-specific distribution in mice, with NCX3-B as mostly expressed in brain and NCX-AC as predominant in skeletal muscle. Using Fura-2-based Ca2+ imaging, we measured the capacity and regulation of the two variants during Ca2+ extrusion and uptake in different conditions. Functional studies revealed that, although both variants are activated by intracellular sodium ([Na+]i), NCX3-AC has a higher [Na+]i sensitivity, as Ca2+ influx is observed in the presence of extracellular Na+. This effect could be partially mimicked for NCX3-B by mutating several glutamate residues in its cytoplasmic loop. In addition, NCX3-AC displayed a higher capacity of both Ca2+ extrusion and uptake compared with NCX3-B, together with an increased sensitivity to intracellular Ca2+. Strikingly, substitution of Glu580 in NCX3-B with its NCX3-AC equivalent Lys580 recapitulated the functional properties of NCX3-AC regarding Ca2+ sensitivity, Lys580 presumably acting through a structure stabilization of the Ca2+ binding site. The higher Ca2+ uptake capacity of NCX3-AC compared with NCX3-B is in line with the necessity to restore Ca2+ levels in the sarcoplasmic reticulum during prolonged exercise. The latter result, consistent with the high expression in the slow-twitch muscle, suggests that this variant may contribute to the Ca2+ handling beyond that of extruding Ca2+.

Rhomboid proteases occur in all domains of life; however, their physiological role is not completely understood, and nothing is known of the biology of these enzymes in Archaea. One of the two rhomboid homologs of Haloferax volcanii (RhoII) is fused to a zinc finger domain. Chromosomal deletion of rhoII was successful, indicating that this gene is not essential for this organism; however, the mutant strain (MIG1) showed reduced motility and increased sensitivity to novobiocin. Membrane preparations of MIG1 were enriched in two glycoproteins, identified as the S-layer glycoprotein and an ABC transporter component. The H. volcanii S-layer glycoprotein has been extensively used as a model to study haloarchaeal protein N-glycosylation. HPLC analysis of oligosaccharides released from the S-layer glycoprotein after PNGase treatment revealed that MIG1 was enriched in species with lower retention times than those derived from the parent strain. Mass spectrometry analysis showed that the wild type glycoprotein released a novel oligosaccharide species corresponding to GlcNAc-GlcNAc(Hex)2-(SQ-Hex)6 in contrast to the mutant protein, which contained the shorter form GlcNAc2(Hex)2-SQ-Hex-SQ. A glycoproteomics approach of the wild type glycopeptide fraction revealed Asn-732 peptide fragments linked to the sulfoquinovose-containing oligosaccharide. This work describes a novel N-linked oligosaccharide containing a repeating SQ-Hex unit bound to Asn-732 of the H. volcanii S-layer glycoprotein, a position that had not been reported as glycosylated. Furthermore, this study provides the first insight on the biological role of rhomboid proteases in Archaea, suggesting a link between protein glycosylation and this protease family.

The molecular chaperone ClpC/Hsp93 is essential for chloroplast function in vascular plants. ClpC has long been held to act both independently and as the regulatory partner for the ATP-dependent Clp protease, and yet this and many other important characteristics remain unclear. In this study, we reveal that of the two near-identical ClpC paralogs (ClpC1 and ClpC2) in Arabidopsis chloroplasts, along with the closely related ClpD, it is ClpC1 that is the most abundant throughout leaf maturation. An unexpectedly large proportion of both chloroplast ClpC proteins (30% of total ClpC content) associates to envelope membranes in addition to their stromal localization. The Clp proteolytic core is also bound to envelope membranes, the amount of which is sufficient to bind to all the similarly localized ClpC. The role of such an envelope membrane Clp protease remains unclear although it appears uninvolved in preprotein processing or Tic subunit protein turnover. Within the stroma, the amount of oligomeric ClpC protein is less than that of the Clp proteolytic core, suggesting most if not all stromal ClpC functions as part of the Clp protease; a proposal supported by the near abolition of Clp degradation activity in the clpC1 knock-out mutant. Overall, ClpC appears to function primarily within the Clp protease, as the principle stromal protease responsible for maintaining homeostasis, and also on the envelope membrane where it possibly confers a novel protein quality control mechanism for chloroplast preprotein import.

Receptor-mediated activation of the Gα subunit of heterotrimeric G proteins requires allosteric communication between the receptor binding site and the guanine nucleotide binding site, which are separated by >30 Å. Structural changes in the allosteric network connecting these sites are predicted to be transient in the wild-type Gα subunit, making studies of these connections challenging. In the current work, site-directed mutants that alter the energy barriers between the activation states are used as tools to better understand the transient features of allosteric signaling in the Gα subunit. The observed differences in relative receptor affinity for intact Gαi1 subunits versus C-terminal Gαi1 peptides harboring the K345L mutation are consistent with this mutation modulating the allosteric network in the protein subunit. Measurement of nucleotide exchange rates, affinity for metarhodopsin II, and thermostability suggest that the K345L Gαi1 variant has reduced stability in both the GDP-bound and nucleotide-free states as compared with wild type but similar stability in the GTPγS-bound state. High resolution x-ray crystal structures reveal conformational changes accompanying the destabilization of the GDP-bound state. Of these, the conformation for Switch I was stabilized by an ionic interaction with the phosphate binding loop. Further site-directed mutagenesis suggests that this interaction between Switch I and the phosphate binding loop is important for receptor-mediated nucleotide exchange in the wild-type Gαi1 subunit.

We have examined the distribution of ribosomes and nucleoids in live Escherichia coli cells under conditions of growth, division, and in quiescence. In exponentially growing cells translating ribosomes are interspersed among and around the nucleoid lobes, appearing as alternative bands under a fluorescence microscope. In contrast, inactive ribosomes either in stationary phase or after treatment with translation inhibitors such as chloramphenicol, tetracycline, and streptomycin gather predominantly at the cell poles and boundaries with concomitant compaction of the nucleoid. However, under all conditions, spatial segregation of the ribosomes and the nucleoids is well maintained. In dividing cells, ribosomes accumulate on both sides of the FtsZ ring at the mid cell. However, the distribution of the ribosomes among the new daughter cells is often unequal. Both the shape of the nucleoid and the pattern of ribosome distribution are also modified when the cells are exposed to rifampicin (transcription inhibitor), nalidixic acid (gyrase inhibitor), or A22 (MreB-cytoskeleton disruptor). Thus we conclude that the intracellular organization of the ribosomes and the nucleoids in bacteria are dynamic and critically dependent on cellular growth processes (replication, transcription, and translation) as well as on the integrity of the MreB cytoskeleton.

Riboswitches are a class of cis-acting regulatory RNAs normally characterized from the 5′-UTR of bacterial transcripts that bind a specific ligand to regulate expression of associated genes by forming alternative conformations. Here, we present a riboswitch that contributes to transcriptional regulation through sensing Mn2+ in Salmonella typhimurium. We characterized a 5′-UTR (UTR1) from the mntH locus encoding a Mn2+ transporter, which forms a Rho-independent terminator to implement transcription termination with a high Mn2+ selectivity both in vivo and in vitro. Nucleotide substitutions that cause disruption of the terminator interfere with the regulatory function of UTR1. RNA probing analyses outlined a specific UTR1 conformation that favors the terminator structure in Mn2+-replete condition. Switch sequence GCUAUG can alternatively base pair duplicated hexanucleotide CAUAGC to form either a pseudoknot or terminator stem. Mn2+, but not Mg2+, and Ca2+, can enhance cleavage at specific nucleotides in UTR1. We conclude that UTR1 is a riboswitch that senses cytoplasmic Mn2+ and therefore participates in Mn2+-responsive mntH regulation in Salmonella. This riboswitch domain is also conserved in several Gram-negative enteric bacteria, indicating that this Mn2+-responsive mechanism could have broader implications in bacterial gene expression. Additionally, a high level of cytoplasmic Mn2+ can down-regulate transcription of the Salmonella Mg2+ transporter mgtA locus in a Mg2+ riboswitch-dependent manner. On the other hand, these two types of cation riboswitches do not share similarity at the primary or secondary structural levels. Taken together, characterization of Mn2+-responsive riboswitches should expand the scope of RNA regulatory elements in response to inorganic ions.

The spindle assembly checkpoint (SAC) ensures the faithful segregation of the genome during mitosis by ensuring that sister chromosomes form bipolar attachments with microtubules of the mitotic spindle. p31Comet is an antagonist of the SAC effector Mad2 and promotes silencing of the SAC and mitotic progression. However, p31Comet interacts with Mad2 throughout the cell cycle. We show that p31Comet binds Mad2 solely in an inhibitory manner. We demonstrate that attenuating the affinity of p31Comet for Mad2 by phosphorylation promotes SAC activity in mitosis. Specifically, phosphorylation of Ser-102 weakens p31Comet-Mad2 binding and enhances p31Comet-mediated bypass of the SAC. Our results provide the first evidence for regulation of p31Comet and demonstrate a previously unknown event controlling SAC activity.

The replication fork temporarily stalls when encountering an obstacle on the DNA, and replication resumes after the barrier is removed. Simultaneously, activation of the replication checkpoint delays the progression of S phase and inhibits late origin firing. Camptothecin (CPT), a topoisomerase I (Top1) inhibitor, acts as a DNA replication barrier by inducing the covalent retention of Top1 on DNA. The Timeless-Tipin complex, a component of the replication fork machinery, plays a role in replication checkpoint activation and stabilization of the replication fork. However, the role of the Timeless-Tipin complex in overcoming the CPT-induced replication block remains elusive. Here, we generated viable TIPIN gene knock-out (KO) DT40 cells showing delayed S phase progression and increased cell death. TIPIN KO cells were hypersensitive to CPT. However, homologous recombination and replication checkpoint were activated normally, whereas DNA synthesis activity was markedly decreased in CPT-treated TIPIN KO cells. Proteasome-dependent degradation of chromatin-bound Top1 was induced in TIPIN KO cells upon CPT treatment, and pretreatment with aphidicolin, a DNA polymerase inhibitor, suppressed both CPT sensitivity and Top1 degradation. Taken together, our data indicate that replication forks formed without Tipin may collide at a high rate with Top1 retained on DNA by CPT treatment, leading to CPT hypersensitivity and Top1 degradation in TIPIN KO cells.

Experiments were carried out to probe the details of the hydration-initiated hydrolysis catalyzed by the Clostridium perfringens unsaturated glucuronyl hydrolase of glycoside hydrolase family 88 in the CAZy classification system. Direct 1H NMR monitoring of the enzymatic reaction detected no accumulated reaction intermediates in solution, suggesting that rearrangement of the initial hydration product occurs on-enzyme. An attempt at mechanism-based trapping of on-enzyme intermediates using a 1,1-difluoro-substrate was unsuccessful because the probe was too deactivated to be turned over by the enzyme. Kinetic isotope effects arising from deuterium-for-hydrogen substitution at carbons 1 and 4 provide evidence for separate first-irreversible and overall rate-determining steps in the hydration reaction, with two potential mechanisms proposed to explain these results. Based on the positioning of catalytic residues in the enzyme active site, the lack of efficient turnover of a 2-deoxy-2-fluoro-substrate, and several unsuccessful attempts at confirmation of a simpler mechanism involving a covalent glycosyl-enzyme intermediate, the most plausible mechanism is one involving an intermediate bearing an epoxide on carbons 1 and 2.

Glycine receptors (GlyRs) mediate inhibitory neurotransmission in spinal cord and brainstem. They are clustered at inhibitory postsynapses via a tight interaction of their β subunits (GlyRβ) with the scaffolding protein gephyrin. In an attempt to isolate additional proteins interacting with GlyRβ, we performed pulldown experiments with rat brain extracts using a glutathione S-transferase fusion protein encompassing amino acids 378–455 of the large intracellular loop of GlyRβ as bait. This identified syndapin I (SdpI) as a novel interaction partner of GlyRβ that coimmunoprecipitates with native GlyRs from brainstem extracts. Both SdpI and SdpII bound efficiently to the intracellular loop of GlyRβ in vitro and colocalized with GlyRβ upon coexpression in COS-7 cells. The SdpI-binding site was mapped to a proline-rich sequence of 22 amino acids within the intracellular loop of GlyRβ. Deletion and point mutation analysis disclosed that SdpI binding to GlyRβ is Src homology 3 domain-dependent. In cultured rat spinal cord neurons, SdpI immunoreactivity was found to partially colocalize with marker proteins of inhibitory and excitatory synapses. When SdpI was acutely knocked down in cultured spinal cord neurons by viral miRNA expression, postsynaptic GlyR clusters were significantly reduced in both size and number. Similar changes in GlyR cluster properties were found in spinal cultures from SdpI-deficient mice. Our results are consistent with a role of SdpI in the trafficking and/or cytoskeletal anchoring of synaptic GlyRs.

Isolated rat bone marrow stromal cells cultured in osteogenic medium in which the normal 5.6 mm glucose is changed to hyperglycemic 25.6 mm glucose greatly increase lipid formation between 21–31 days of culture that is associated with decreased biomineralization, up-regulate expression of cyclin D3 and two adipogenic markers (CCAAT/enhancer binding protein α and peroxisome proliferator-activated receptor γ) within 5 days of culture, increase neutral and polar lipid synthesis within 5 days of culture, and form a monocyte-adhesive hyaluronan matrix through an endoplasmic reticulum stress-induced autophagic mechanism. Evidence is also provided that, by 4 weeks after diabetes onset in the streptozotocin-induced diabetic rat model, there is a large loss of trabecular bone mineral density without apparent proportional changes in underlying collagen matrices, a large accumulation of a hyaluronan matrix within the trabecular bone marrow, and adipocytes and macrophages embedded in this hyaluronan matrix. These results support the hypothesis that hyperglycemia in bone marrow diverts dividing osteoblastic precursor cells (bone marrow stromal cells) to a metabolically stressed adipogenic pathway that induces synthesis of a hyaluronan matrix that recruits inflammatory cells and establishes a chronic inflammatory process that demineralizes trabecular cancellous bone.

Using high-resolution MS-based proteomics in combination with multiple protease digestion, we profiled, with on average 90% sequence coverage, all 13 viral proteins present in an human adenovirus (HAdV) vector. This in-depth profile provided multiple peptide-based evidence on intrinsic protease activity affecting several HAdV proteins. Next, the generated peptide library was used to develop a targeted proteomics method using selected reaction monitoring (SRM) aimed at quantitative profiling of the stoichiometry of all 13 proteins present in the HAdV. We also used this method to probe the release of specific virus proteins initiated by thermal stimulation, mimicking the early stage of HAdV disassembly during entry into host cells. We confirmed the copy numbers of the most well characterized viral capsid components and established the copy numbers for proteins whose stoichiometry has so far not been accurately defined. We also found that heating HAdV induces the complete release of the penton base and fiber proteins as well as a substantial release of protein VIII and VI. For these latter proteins, maturational proteolysis by the adenoviral protease leads to the differential release of fragments with certain peptides being fully released and others largely retained in the AdV particles. This information is likely to be beneficial for the ongoing interpretation of high resolution cryoEM and x-ray electron density maps.

Palmitoylation at cysteine residues is the only known reversible form of lipidation and has been implicated in protein membrane association as well as function. Many palmitoylated proteins have regulatory roles in dynamic cellular processes, including membrane fusion. Recently, we identified Env7 as a conserved and palmitoylated protein kinase involved in negative regulation of membrane fusion at the lysosomal vacuole. Env7 contains a palmitoylation consensus sequence, and substitution of its three consecutive cysteines (Cys13–Cys15) results in a non-palmitoylated and cytoplasmic Env7. In this study, we further dissect and define the role(s) of individual cysteines of the consensus sequence in various properties of Env7 in vivo. Our results indicate that more than one of the cysteines serve as palmitoylation substrates, and any pairwise combination is essential and sufficient for near wild type levels of Env7 palmitoylation, membrane localization, and phosphorylation. Furthermore, individually, each cysteine can serve as a minimum requirement for distinct aspects of Env7 behavior and function in cells. Cys13 is sufficient for membrane association, Cys15 is essential for the fusion regulatory function of membrane-bound Env7, and Cys14 and Cys15 are redundantly essential for protection of membrane-bound Env7 from proteasomal degradation. A role for Cys14 and Cys15 in correct sorting at the membrane is also discussed. Thus, palmitoylation at the N-terminal cysteines of Env7 directs not only its membrane association but also its stability, phosphorylation, and cellular function.

PKCδ suppresses keratinocyte proliferation via a mechanism that involves increased expression of p21Cip1. However, the signaling mechanism that mediates this regulation is not well understood. Our present studies suggest that PKCδ activates p38δ leading to increased p21Cip1 promoter activity and p21Cip1 mRNA/protein expression. We further show that exogenously expressed p38δ increases p21Cip1 mRNA and protein and that p38δ knockdown or expression of dominant-negative p38 attenuates this increase. Moreover, p53 is an intermediary in this regulation, as p38δ expression increases p53 mRNA, protein, and promoter activity, and p53 knockdown attenuates the activation. We demonstrate a direct interaction of p38δ with PKCδ and MEK3 and show that exogenous agents that suppress keratinocyte proliferation activate this pathway. We confirm the importance of this regulation using a stratified epidermal equivalent model, which mimics in vivo-like keratinocyte differentiation. In this model, PKCδ or p38δ knockdown results in reduced p53 and p21Cip1 levels and enhanced cell proliferation. We propose that PKCδ activates a MEKK1/MEK3/p38δ MAPK cascade to increase p53 levels and p53 drives p21Cip1 gene expression.

In this study, the effects of cytokines on the activation of the DNA double strand break repair factors histone H2AX (H2AX) and ataxia telangiectasia mutated (ATM) were examined in pancreatic β cells. We show that cytokines stimulate H2AX phosphorylation (γH2AX formation) in rat islets and insulinoma cells in a nitric oxide- and ATM-dependent manner. In contrast to the well documented role of ATM in DNA repair, ATM does not appear to participate in the repair of nitric oxide-induced DNA damage. Instead, nitric oxide-induced γH2AX formation correlates temporally with the onset of irreversible DNA damage and the induction of apoptosis. Furthermore, inhibition of ATM attenuates cytokine-induced caspase activation. These findings show that the formation of DNA double strand breaks correlates with ATM activation, irreversible DNA damage, and ATM-dependent induction of apoptosis in cytokine-treated β cells.

The Burkholderia species utilize acetyl-CoA and oxaloacetate, substrates for citrate synthase in the TCA cycle, to produce oxalic acid in response to bacterial cell to cell communication, called quorum sensing. Quorum sensing-mediated oxalogenesis via a sequential reaction by ObcA and ObcB counteracts the population-collapsing alkaline pH of the stationary growth phase. Thus, the oxalic acid produced plays an essential role as an excreted public good for survival of the group. Here, we report structural and functional analyses of ObcA, revealing mechanistic features distinct from those of citrate synthase. ObcA exhibits a unique fold, in which a (β/α)8-barrel fold is located in the C-domain with the N-domain inserted into a loop following α1 in the barrel fold. Structural analyses of the complexes with oxaloacetate and with a bisubstrate adduct indicate that each of the oxaloacetate and acetyl-CoA substrates is bound to an independent site near the metal coordination shell in the barrel fold. In catalysis, oxaloacetate serves as a nucleophile by forming an enolate intermediate mediated by Tyr322 as a general base, which then attacks the thioester carbonyl carbon of acetyl-CoA to yield a tetrahedral adduct between the two substrates. Therefore, ObcA catalyzes its reaction by combining the enolase and acetyltransferase superfamilies, but the presence of the metal coordination shell and the absence of general acid(s) produces an unusual tetrahedral CoA adduct as a stable product. These results provide the structural basis for understanding the first step in oxalogenesis and constitute an example of the functional diversity of an enzyme for survival and adaptation in the environment.

Second-phase insulin secretion sustains insulin release in the face of hyperglycemia associated with insulin resistance, requiring the continued mobilization of insulin secretory granules to the plasma membrane. Cdc42, the small Rho family GTPase recognized as the proximal glucose-specific trigger to elicit second-phase insulin secretion, signals downstream to activate the p21-activated kinase (PAK1), which then signals to Raf-1/MEK/ERK to induce filamentous actin (F-actin) remodeling, to ultimately mobilize insulin granules to the plasma membrane. However, the steps required to initiate Cdc42 activation in a glucose-specific manner in β cells have remained elusive. Toward this, we identified the involvement of the Src family kinases (SFKs), based upon the ability of SFK inhibitors to block glucose-stimulated Cdc42 and PAK1 activation events as well as the amplifying pathway of glucose-stimulated insulin release, in MIN6 β cells. Indeed, subsequent studies performed in human islets revealed that SFK phosphorylation was induced only by glucose and within 1 min of stimulation before the activation of Cdc42 at 3 min. Furthermore, pervanadate treatment validated the phosphorylation event to be tyrosine-specific. Although RT-PCR showed β cells to express five different SFK proteins, only two of these, YES and Fyn kinases, were found localized to the plasma membrane, and of these two, only YES kinase underwent glucose-stimulated tyrosine phosphorylation. Immunodetection and RNAi analyses further established YES kinase as a proximal glucose-specific signal in the Cdc42-signaling cascade. Identification of YES kinase provides new insight into the mechanisms underlying the sustainment of insulin secretion via granule mobilization/replenishment and F-actin remodeling.

Newly synthesized thyroglobulin (Tg), the thyroid prohormone, forms detectable high molecular weight mixed disulfide adducts: until now, only Tg “adduct B” was identified as primarily engaging the endoplasmic reticulum oxidoreductases ERp57 and protein disulfide isomerase. Here, we demonstrate that the faster migrating Tg adduct C primarily engages the CaBP1/P5 oxidoreductase, whereas the slower migrating Tg adduct A primarily engages ERp72. Upon siRNA-mediated knockdown of CaBP1/P5 or ERp72, adducts C or A, respectively, are decreased. Within the three Tg adduct bands that do not exhibit a precursor-product relationship, Tg exhibits distinct oxidation patterns. We present evidence suggesting that disulfide maturation occurs within Tg monomers engaged in each of the adduct bands. Moreover, the same Tg substrate molecules can form simultaneous mixed disulfides with both CaBP1/P5 and protein disulfide isomerase, although these are generally viewed as components of distinct oxidoreductase-chaperone protein complexes. Such substrate-oxidoreductase combinations offer Tg the potential for simultaneous oxidative maturation along different parallel tracks leading to the native state.

Recent studies have suggested that phosphatidic acid (PA), a cone-shaped phospholipid that can generate negative curvature of lipid membranes, participates in mitochondrial fusion. However, precise mechanisms underling the production and consumption of PA on the mitochondrial surface are not fully understood. Phosphatidic acid-preferring phospholipase A1 (PA-PLA1)/DDHD1 is the first identified intracellular phospholipase A1 and preferentially hydrolyzes PA in vitro. Its cellular and physiological functions have not been elucidated. In this study, we show that PA-PLA1 regulates mitochondrial dynamics. PA-PLA1, when ectopically expressed in HeLa cells, induced mitochondrial fragmentation, whereas its depletion caused mitochondrial elongation. The effects of PA-PLA1 on mitochondrial morphology appear to counteract those of MitoPLD, a mitochondrion-localized phospholipase D that produces PA from cardiolipin. Consistent with high levels of expression of PA-PLA1 in testis, PA-PLA1 knock-out mice have a defect in sperm formation. In PA-PLA1-deficient sperm, the mitochondrial structure is disorganized, and an abnormal gap structure exists between the middle and principal pieces. A flagellum is bent at that position, leading to a loss of motility. Our results suggest a possible mechanism of PA regulation of the mitochondrial membrane and demonstrate an in vivo function of PA-PLA1 in the organization of mitochondria during spermiogenesis.

Vaso-occlusive crises are the main acute complication in sickle cell disease. They are initiated by abnormal adhesion of circulating blood cells to vascular endothelium of the microcirculation. Several interactions involving an intricate network of adhesion molecules have been described between sickle red blood cells and the endothelial vascular wall. We have shown previously that young sickle reticulocytes adhere to resting endothelial cells through the interaction of α4β1 integrin with endothelial Lutheran/basal cell adhesion molecule (Lu/BCAM). In the present work, we investigated the functional impact of endothelial exposure to hydroxycarbamide (HC) on this interaction using transformed human bone marrow endothelial cells and primary human pulmonary microvascular endothelial cells. Adhesion of sickle reticulocytes to HC-treated endothelial cells was decreased despite the HC-derived increase of Lu/BCAM expression. This was associated with decreased phosphorylation of Lu/BCAM and up-regulation of the cAMP-specific phosphodiesterase 4A expression. Our study reveals a novel mechanism for HC in endothelial cells where it could modulate the function of membrane proteins through the regulation of phosphodiesterase expression and cAMP-dependent signaling pathways.

Mechanisms for cancer-related inflammation remain to be fully elucidated. Non-apoptotic functions of Fas signaling have been proposed to play an important role in promoting tumor progression. It has yet to be determined if targeting Fas signaling can control tumor progression through suppression of cancer-related inflammation. In the current study we found that breast cancer cells with constitutive Fas expression were resistant to apoptosis induction by agonistic anti-Fas antibody (Jo2) ligation or Fas ligand cross-linking. Higher expression of Fas in human breast cancer tissue has been significantly correlated with poorer prognosis in breast cancer patients. To determine whether blockade of Fas signaling in breast cancer could suppress tumor progression, we prepared an orthotopic xenograft mouse model with mammary cancer cells 4T1 and found that blockade of Fas signaling in 4T1 cancer cells markedly reduced tumor growth, inhibited tumor metastasis in vivo, and prolonged survival of tumor-bearing mice. Mechanistically, blockade of Fas signaling in cancer cells significantly decreased systemic or local recruitment of myeloid derived suppressor cells (MDSCs) in vivo. Furthermore, blockade of Fas signaling markedly reduced IL-6, prostaglandin E2 production from breast cancer cells by impairing p-p38, and activity of the NFκB pathway. In addition, administration of a COX-2 inhibitor and anti-IL-6 antibody significantly reduced MDSC accumulation in vivo. Therefore, blockade of Fas signaling can suppress breast cancer progression by inhibiting proinflammatory cytokine production and MDSC accumulation, indicating that Fas signaling-initiated cancer-related inflammation in breast cancer cells may be a potential target for treatment of breast cancer.

M-type potassium channels, encoded by the KCNQ family genes (KCNQ2–5), require calmodulin as an essential co-factor. Calmodulin bound to the KCNQ2 subunit regulates channel trafficking and stabilizes channel activity. We demonstrate that phosphorylation of calmodulin by protein kinase CK2 (casein kinase 2) rapidly and reversibly modulated KCNQ2 current. CK2-mediated phosphorylation of calmodulin strengthened its binding to KCNQ2 channel, caused resistance to phosphatidylinositol 4,5-bisphosphate depletion, and increased KCNQ2 current amplitude. Accordingly, application of CK2-selective inhibitors suppressed KCNQ2 current. This suppression was prevented by co-expression of CK2 phosphomimetic calmodulin mutants or pretreatment with a protein phosphatase inhibitor, calyculin A. We also demonstrated that functional CK2 and protein phosphatase 1 (PP1) were selectively tethered to the KCNQ2 subunit. We identified a functional KVXF consensus site for PP1 binding in the N-terminal tail of KCNQ2 subunit: mutation of this site augmented current density. CK2 inhibitor treatment suppressed M-current in rat superior cervical ganglion neurons, an effect negated by overexpression of phosphomimetic calmodulin or pretreatment with calyculin A Furthermore, CK2 inhibition diminished the medium after hyperpolarization by suppressing the M-current. These findings suggest that CK2-mediated phosphorylation of calmodulin regulates the M-current, which is tonically regulated by CK2 and PP1 anchored to the KCNQ2 channel complex.

Osteoclastogenesis is controlled by osteocytes; osteocytic osteoclastogenesis regulatory molecules are largely unknown. We searched for such factors using newly developed culture methods. Our culture system mimics the three-dimensional cellular structure of bone, consisting of collagen gel-embedded osteocytic MLO-Y4 cells, stromal ST2 cells on the gel as bone lining cells, and bone marrow cells. The gel-embedded MLO-Y4 cells inhibited the osteoclastogenesis induced by 1,25(OH)2D3 without modulating receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) production by ST2 cells, despite MLO-Y4 cells supported osteoclastogenesis in the absence of ST2 cells. In the bone marrow cell culture, the conditioned medium from MLO-Y4 cells decreased the capability of osteoclastic differentiation from the cells induced by macrophage colony-stimulating factor. This decreased capability was concomitant with an increase in protein kinase R mRNA expression and an inhibition of c-Fos translation. These changes were partially normalized by the simultaneous addition of an anti-interferon (IFN)-β neutralizing antibody to MLO-Y4 cell conditioned medium. To study primary osteocytes, we prepared non-osteocytic cell-free osteocyte-enriched bone fragments (OEBFs). When osteoclast precursors were induced by macrophage colony-stimulating factor in the presence of OEBFs, the generated cells exhibited a diminished capacity for osteoclastogenesis. OEBFs prepared from OPG-knock-out mice exhibited a similar effect, indicating OPG-independent inhibition. The addition of anti-IFN-β neutralizing antibody during the co-culture with OEBFs partially recovered the osteoclastogenic potential of the generated cells. The MLO-Y4 cells and OEBFs expressed IFN-β mRNA. Although osteocytic RANKL is known to be important for osteoclastogenesis, our data suggest that osteocytes also produce IFN-β as an inhibitor of osteoclastogenesis.

TREX1 is a 3′-deoxyribonuclease that degrades single- and double-stranded DNA (ssDNA and dsDNA) to prevent inappropriate nucleic acid-mediated immune activation. More than 40 different disease-causing TREX1 mutations have been identified exhibiting dominant and recessive genetic phenotypes in a spectrum of autoimmune disorders. Mutations in TREX1 at positions Asp-18 and Asp-200 to His and Asn exhibit dominant autoimmune phenotypes associated with the clinical disorders familial chilblain lupus and Aicardi-Goutières syndrome. Our previous biochemical studies showed that the TREX1 dominant autoimmune disease phenotype depends upon an intact DNA-binding process coupled with dysfunctional active site chemistry. Studies here show that the TREX1 Arg-62 residues extend across the dimer interface into the active site of the opposing protomer to coordinate substrate DNA and to affect catalysis in the opposing protomer. The TREX1R62A/R62A homodimer exhibits ∼50-fold reduced ssDNA and dsDNA degradation activities relative to TREX1WT. The TREX1 D18H, D18N, D200H, and D200N dominant mutant enzymes were prepared as compound heterodimers with the TREX1 R62A substitution in the opposing protomer. The TREX1D18H/R62A, TREX1D18N/R62A, TREX1D200H/R62A, and TREX1D200N/R62A compound heterodimers exhibit higher levels of ss- and dsDNA degradation activities than the homodimers demonstrating the requirement for TREX1 Arg-62 residues to provide necessary structural elements for full catalytic activity in the opposing TREX1 protomer. This concept is further supported by the loss of dominant negative effects in the TREX1 D18H, D18N, D200H, and D200N compound heterodimers. These data provide compelling evidence for the required TREX1 dimeric structure for full catalytic function.

VOLUME 288 (2013) PAGES 18811–18824 PAGE 18817: Lanes 3–6 of the original Western blot image in Fig. 3C contained the same bands as lanes 3–6 of the Western blot image in Fig. 3D, and both panels failed to indicate that separate lanes from the original blots had been spliced together. The correct Western blot image for Fig. 3C is now provided, and lines have been added to indicate the area where the blot images were joined. These changes do not affect the interpretation or conclusions of this work. jbc;289/16/11566/FU1F1FU1

VOLUME 288 (2013) PAGES 319–331 PAGE 319: The following grant information should have been included. “The clinical experiments were supported by a grant from the National Natural Science Foundation of China (81172592).”

VOLUME 285 (2010) PAGES 36865–36875 This article has been withdrawn by the authors.

VOLUME 288 (2013) PAGES 31902–31913 PAGE 31906: The revised Fig. 3E correctly shows one of the N-acetylglucosamines (Glc-NACs) as being bound to Asn-511, instead of Asn-110. The residue is correctly identified as Asn-511 in Fig. 1 and in the text of the manuscript. This correction does not change the interpretation of the results or the conclusions of this work. jbc;289/16/11569/FU1F1FU1

VOLUME 287 (2012) PAGES 44498–44507 PAGES 44504 and 44505: Panels D and E in Fig. 4 and panel E in Fig. 5 were inadvertently omitted from the final version of the article. The missing panels have now been provided. This correction does not change the interpretation of the results or the conclusions. jbc;289/16/11570/F1F1F1 FIGURE 4 jbc;289/16/11570/F2F2F2 FIGURE 5