Atp7b-dependent choroid plexus dysfunction causes transient copper deficit and metabolic changes in the developing mouse brain.

Copper (Cu) has a multifaceted role in brain development, function, and metabolism. Two homologous Cu transporters, Atp7a (Menkes disease protein) and Atp7b (Wilson disease protein), maintain Cu homeostasis in the tissue. Atp7a mediates Cu entry into the brain and activates Cu-dependent enzymes, whereas the role of Atp7b is less clear. We show that during postnatal development Atp7b is necessary for normal morphology and function of choroid plexus (ChPl). Inactivation of Atp7b causes reorganization of ChPl' cytoskeleton and cell-cell contacts, loss of Slc31a1 from the apical membrane, and a decrease in the length and number of microvilli and cilia. In ChPl lacking Atp7b, Atp7a is upregulated but remains intracellular, which limits Cu transport into the brain and results in significant Cu deficit, which is reversed only in older animals. Cu deficiency is associated with down-regulation of Atp7a in locus coeruleus and catecholamine imbalance, despite normal expression of dopamine-ß-hydroxylase. In addition, there are notable changes in the brain lipidome, which can be attributed to inhibition of diacylglyceride-to-phosphatidylethanolamine conversion. These results identify the new role for Atp7b in developing brain and identify metabolic changes that could be exacerbated by Cu chelation therapy.

NEDD4L intramolecular interactions regulate its auto and substrate NaV1.5 ubiquitination.

NEDD4L is a HECT-type E3 ligase that catalyzes the addition of ubiquitin to intracellular substrates such as the cardiac voltage-gated sodium channel, NaV1.5. The intramolecular interactions of NEDD4L regulate its enzymatic activity which is essential for proteostasis. For NaV1.5, this process is critical as alterations in Na+ current is involved in cardiac diseases including arrhythmias and heart failure. In this study, we perform extensive biochemical and functional analyses that implicate the C2 domain and the first WW-linker (1,2-linker) in the autoregulatory mechanism of NEDD4L. Through in vitro and electrophysiological experiments, the NEDD4L 1,2-linker was determined to be important in substrate ubiquitination of NaV1.5. We establish the preferred sites of ubiquitination of NEDD4L to be in the second WW-linker (2,3-linker). Interestingly, NEDD4L ubiquitinates the cytoplasmic linker between the first and second transmembrane domains of the channel (DI-DII) of NaV1.5. Moreover, we design a genetically encoded modulator of Nav1.5 that achieves Na+ current reduction using the NEDD4L HECT domain as cargo of a NaV1.5-binding nanobody. These investigations elucidate the mechanisms regulating the NEDD4 family and furnish a new molecular framework for understanding NaV1.5 ubiquitination.

Lack of the MHC class II chaperone H2-O causes susceptibility to autoimmune diseases.

DO (HLA-DO, in human; murine H2-O) is a highly conserved nonclassical major histocompatibility complex class II (MHC II) accessory molecule mainly expressed in the thymic medulla and B cells. Previous reports have suggested possible links between DO and autoimmunity, Hepatitis C (HCV) infection, and cancer, but the mechanism of how DO contributes to these diseases remains unclear. Here, using a combination of various in vivo approaches, including peptide elution, mixed lymphocyte reaction, T-cell receptor (TCR) deep sequencing, tetramer-guided naïve CD4 T-cell precursor enumeration, and whole-body imaging, we report that DO affects the repertoire of presented self-peptides by B cells and thymic epithelium. DO induces differential effects on epitope presentation and thymic selection, thereby altering CD4 T-cell precursor frequencies. Our findings were validated in two autoimmune disease models by demonstrating that lack of DO increases autoreactivity and susceptibility to autoimmune disease development.

Fluid shear stress enhances differentiation of jejunal human enteroids in Intestine-Chip.

There is increasing evidence that the study of normal human enteroids duplicates many known aspects of human intestinal physiology. However, this epithelial cell-only model lacks the many nonepithelial intestinal cells present in the gastrointestinal tract and exposure to the mechanical forces to which the intestine is exposed. We tested the hypothesis that physical shear forces produced by luminal and blood flow would provide an intestinal model more closely resembling normal human jejunum. Jejunal enteroid monolayers were studied in the Emulate, Inc. Intestine-Chip under conditions of constant luminal and basolateral flow that was designed to mimic normal intestinal fluid flow, with human umbilical vein endothelial cells (HUVECs) on the basolateral surface and with Wnt3A, R-spondin, and Noggin only on the luminal surface. The jejunal enteroids formed monolayers that remained confluent for 6-8 days, began differentiating at least as early as day 2 post plating, and demonstrated continuing differentiation over the entire time of the study, as shown by quantitative real-time polymerase chain reaction and Western blot analysis. Differentiation impacted villus genes and proteins differently with early expression of regenerating family member 1α (REG1A), early reduction to a low but constant level of expression of Na+-K+-2Cl- cotransporter 1 (NKCC1), and increasing expression of sucrase-isomaltase (SI) and downregulated in adenoma (DRA). These results were consistent with continual differentiation, as was shown to occur in mouse villus enterocytes. Compared with differentiated enteroid monolayers grown on Transwell inserts, enteroids exposed to flow were more differentiated but exhibited increased apoptosis and reduced carbohydrate metabolism, as shown by proteomic analysis. This study of human jejunal enteroids-on-chip suggests that luminal and basolateral flow produce a model of continual differentiation over time and NaCl absorption that mimics normal intestine and should provide new insights in intestinal physiology.NEW & NOTEWORTHY This study showed that polarized enteroid models in which there is no basolateral Wnt3a, are differentiated, regardless of the Wnt3a status of the apical media. The study supports the concept that in the human intestine villus differentiation is not an all or none phenomenon, demonstrating that at different days after lack of basolateral Wnt exposure, clusters of genes and proteins exist geographically along the villus with different domains having different functions.

Valsartan nano-filaments alter mitochondrial energetics and promote faster healing in diabetic rat wounds.

Chronic wounds are a common and debilitating condition associated with aging populations that impact more than 6.5 million patients in the United States. We have previously demonstrated the efficacy of daily topical 1% valsartan in treating wounds in diabetic mouse and pig models. Despite these promising results, there remains a need to develop an extended-release formulation that would reduce patient burden by decreasing the frequency of daily applications. Here, we used nanotechnology to self-assemble valsartan amphiphiles into a filamentous structure (val-filaments) that would serve as a scaffold in wound beds and allow for steady, localised and tunable release of valsartan amphiphiles over 24 days. Two topical treatments of this peptide-based hydrogel on full-thickness wounds in Zucker Diabetic Fatty rats resulted in faster rates of wound closure. By day 23, all val-filament treated wounds were completely closed, as compared to one wound closed in the placebo group. Mechanistically, we observed enrichment of proteins involved in cell adhesion and energetics pathways, downregulation of Tgf-ß signalling pathway mediators (pSmad2, pSmad3 and Smad4) and increased mitochondrial metabolic pathway intermediates. This study demonstrates the successful synthesis of a sustained-release valsartan filament hydrogel, its impact on mitochondrial energetics and efficacy in treating diabetic wounds.

Lysyl oxidase-like 2 depletion is protective in age-associated vascular stiffening.

Vascular stiffening and its sequelae are major causes of morbidity and mortality in the elderly. The increasingly accepted concept of "smooth muscle cell (SMC) stiffness syndrome" along with matrix deposition has emerged in vascular biology to account for the mechanical phenotype of arterial aging, but the molecular targets remain elusive. In this study, using an unbiased proteomic analysis, we identified lysyl oxidase-like 2 (LOXL2) as a critical SMC mediator for age-associated vascular stiffening. We tested the hypothesis that loss of LOXL2 function is protective in aging-associated vascular stiffening. We determined that exogenous and endogenous nitric oxide markedly decreased LOXL2 abundance and activity in the extracellular matrix of isolated SMCs and LOXL2 endothelial cells suppress LOXL2 abundance in the aorta. In a longitudinal study, LOXL2+/- mice were protected from age-associated increase in pulse-wave velocity, an index of vascular stiffening, as occurred in littermate wild-type mice. Using isolated aortic segments, we found that LOXL2 mediates vascular stiffening in aging by promoting SMC stiffness, augmented SMC contractility, and vascular matrix deposition. Together, these studies establish LOXL2 as a nodal point for a new therapeutic approach to treat age-associated vascular stiffening. NEW & NOTEWORTHY Increased central vascular stiffness augments risk of major adverse cardiovascular events. Despite significant advances in understanding the genetic and molecular underpinnings of vascular stiffening, targeted therapy has remained elusive. Here, we show that lysyl oxidase-like 2 (LOXL2) drives vascular stiffening during aging by promoting matrix remodeling and vascular smooth muscle cell stiffening. Reduced LOXL2 expression protects mice from age-associated vascular stiffening and delays the onset of isolated systolic hypertension, a major consequence of stiffening.

Proteolysis by Granzyme B Enhances Presentation of Autoantigenic Peptidylarginine Deiminase 4 Epitopes in Rheumatoid Arthritis.

Proteolysis of autoantigens can alter normal MHC class II antigen processing and has been implicated in the induction of autoimmune diseases. Many autoantigens are substrates for the protease granzyme B (GrB), but the mechanistic significance of this association is unknown. Peptidylarginine deiminase 4 (PAD4) is a frequent target of autoantibodies in patients with rheumatoid arthritis (RA) and a substrate for GrB. RA is strongly associated with specific MHC class II alleles, and elevated levels of GrB and PAD4 are found in the joints of RA patients, suggesting that GrB may alter the presentation of PAD4 by RA-associated class II alleles. In this study, complementary proteomic and immunologic approaches were utilized to define the effects of GrB cleavage on the structure, processing, and immunogenicity of PAD4. Hydrogen-deuterium exchange and a cell-free MHC class II antigen processing system revealed that proteolysis of PAD4 by GrB induced discrete structural changes in PAD4 that promoted enhanced presentation of several immunogenic peptides capable of stimulating PAD4-specific CD4+ T cells from patients with RA. This work demonstrates the existence of PAD4-specific T cells in patients with RA and supports a mechanistic role for GrB in enhancing the presentation of autoantigenic CD4+ T cell epitopes.

Combined Antibody/Lectin Enrichment Identifies Extensive Changes in the O-GlcNAc Sub-proteome upon Oxidative Stress.

O-Linked N-acetyl-ß-d-glucosamine (O-GlcNAc) is a dynamic post-translational modification that modifies and regulates over 3000 nuclear, cytoplasmic, and mitochondrial proteins. Upon exposure to stress and injury, cells and tissues increase the O-GlcNAc modification, or O-GlcNAcylation, of numerous proteins promoting the cellular stress response and thus survival. The aim of this study was to identify proteins that are differentially O-GlcNAcylated upon acute oxidative stress (H2O2) to provide insight into the mechanisms by which O-GlcNAc promotes survival. We achieved this goal by employing Stable Isotope Labeling of Amino Acids in Cell Culture (SILAC) and a novel "G5-lectibody" immunoprecipitation strategy that combines four O-GlcNAc-specific antibodies (CTD110.6, RL2, HGAC39, and HGAC85) and the lectin WGA. Using the G5-lectibody column in combination with basic reversed phase chromatography and C18 RPLC-MS/MS, 990 proteins were identified and quantified. Hundreds of proteins that were identified demonstrated increased (>250) or decreased (>110) association with the G5-lectibody column upon oxidative stress, of which we validated the O-GlcNAcylation status of 24 proteins. Analysis of proteins with altered glycosylation suggests that stress-induced changes in O-GlcNAcylation cluster into pathways known to regulate the cell's response to injury and include protein folding, transcriptional regulation, epigenetics, and proteins involved in RNA biogenesis. Together, these data suggest that stress-induced O-GlcNAcylation regulates numerous and diverse cellular pathways to promote cell and tissue survival.

OxLDL triggers retrograde translocation of arginase2 in aortic endothelial cells via ROCK and mitochondrial processing peptidase.

RATIONALE: Increased arginase activity contributes to endothelial dysfunction by competition for l-arginine substrate and reciprocal regulation of nitric oxide synthase (NOS). The rapid increase in arginase activity in human aortic endothelial cells exposed to oxidized low-density lipoprotein (OxLDL) is consistent with post-translational modification or subcellular trafficking. OBJECTIVE: To test the hypotheses that OxLDL triggers reverse translocation of mitochondrial arginase 2 (Arg2) to cytosol and Arg2 activation, and that this process is dependent on mitochondrial processing peptidase, lectin-like OxLDL receptor-1 receptor, and rho kinase. METHODS AND RESULTS: OxLDL-triggered translocation of Arg2 from mitochondria to cytosol in human aortic endothelial cells and in murine aortic intima with a concomitant rise in arginase activity. All of these changes were abolished by inhibition of mitochondrial processing peptidase or by its siRNA-mediated knockdown. Rho kinase inhibition and the absence of the lectin-like OxLDL receptor-1 in knockout mice also ablated translocation. Aminoterminal sequencing of Arg2 revealed 2 candidate mitochondrial targeting sequences, and deletion of either of these confined Arg2 to the cytoplasm. Inhibitors of mitochondrial processing peptidase or lectin-like OxLDL receptor-1 knockout attenuated OxLDL-mediated decrements in endothelial-specific NO production and increases in superoxide generation. Finally, Arg2(-/-) mice bred on an ApoE(-/-) background showed reduced plaque load, reduced reactive oxygen species production, enhanced NO, and improved endothelial function when compared with ApoE(-/-) controls. CONCLUSIONS: These data demonstrate dual distribution of Arg2, a protein with an unambiguous mitochondrial targeting sequence, in mammalian cells, and its reverse translocation to cytoplasm by alterations in the extracellular milieu. This novel molecular mechanism drives OxLDL-mediated arginase activation, endothelial NOS uncoupling, endothelial dysfunction, and atherogenesis.

Occurrence of a multimeric high-molecular-weight glyceraldehyde-3-phosphate dehydrogenase in human serum.

Cellular glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a phylogenetically conserved, ubiquitous enzyme that plays an indispensable role in energy metabolism. Although a wealth of information is available on cellular GAPDH, there is a clear paucity of data on its extracellular counterpart (i.e., the secreted or extracellular GAPDH). Here, we show that the extracellular GAPDH in human serum is a multimeric, high-molecular-weight, yet glycolytically active enzyme. The high-molecular-weight multimers of serum GAPDH were identified by immunodetection on one- and two-dimensional gel electrophoresis using multiple antibodies specific for various epitopes of GAPDH. Partial purification of serum GAPDH by DEAE Affigel affinity/ion exchange chromatography further established the multimeric composition of serum GAPDH. In vitro data demonstrated that human cell lines secrete a multimeric, high-molecular-weight enzyme similar to that of serum GAPDH. Furthermore, LC-MS/MS analysis of extracellular GAPDH from human cell lines confirmed the presence of unique peptides of GAPDH in the high-molecular-weight subunits. Furthermore, data from pulse-chase experiments established the presence of high-molecular-weight subunits in the secreted, extracellular GAPDH. Taken together, our findings demonstrate the presence of a high-molecular-weight, enzymatically active secretory GAPDH in human serum that may have a hitherto unknown function in humans.

The plasma proteome identifies expected and novel proteins correlated with micronutrient status in undernourished Nepalese children.

Micronutrient deficiencies are common in undernourished societies yet remain inadequately assessed due to the complexity and costs of existing assays. A plasma proteomics-based approach holds promise in quantifying multiple nutrient:protein associations that reflect biological function and nutritional status. To validate this concept, in plasma samples of a cohort of 500 6- to 8-y-old Nepalese children, we estimated cross-sectional correlations between vitamins A (retinol), D (25-hydroxyvitamin D), and E (α-tocopherol), copper, and selenium, measured by conventional assays, and relative abundance of their major plasma-bound proteins, measured by quantitative proteomics using 8-plex iTRAQ mass tags. The prevalence of low-to-deficient status was 8.8% (<0.70 µmol/L) for retinol, 19.2% (<50 nmol/L) for 25-hydroxyvitamin D, 17.6% (<9.3 µmol/L) for α-tocopherol, 0% (<10 µmol/L) for copper, and 13.6% (<0.6 µmol/L) for selenium. We identified 4705 proteins, 982 in >50 children. Employing a linear mixed effects model, we observed the following correlations: retinol:retinol-binding protein 4 (r = 0.88), 25-hydroxyvitamin D:vitamin D-binding protein (r = 0.58), α-tocopherol:apolipoprotein C-III (r = 0.64), copper:ceruloplasmin (r = 0.65), and selenium:selenoprotein P isoform 1 (r = 0.79) (all P < 0.0001), passing a false discovery rate threshold of 1% (based on P value-derived q values). Individual proteins explained 34-77% (R(2)) of variation in their respective nutrient concentration. Adding second proteins to models raised R(2) to 48-79%, demonstrating a potential to explain additional variation in nutrient concentration by this strategy. Plasma proteomics can identify and quantify protein biomarkers of micronutrient status in undernourished children. The maternal micronutrient supplementation trial, from which data were derived as a follow-up activity, was registered at clinicaltrials.gov as NCT00115271.

Electrophilic tuning of the chemoprotective natural product sulforaphane.

Sulforaphane [1-isothiocyanato-4-(methylsulfinyl)butane], a naturally occurring isothiocyanate derived from cruciferous vegetables, is a highly potent inducer of phase 2 cytoprotective enzymes and can protect against electrophiles including carcinogens, oxidative stress, and inflammation. The mechanism of action of sulforaphane is believed to involve modifications of critical cysteine residues of Keap1, which lead to stabilization of Nrf2 to activate the antioxidant response element of phase 2 enzymes. However, the dithiocarbamate functional group formed by a reversible reaction between isothiocyanate of sulforaphane and sulfhydryl nucleophiles of Keap1 is kinetically labile, and such modification in intact cells has not yet been demonstrated. Here we designed sulforaphane analogs with replacement of the reactive isothiocyanate by the more gentle electrophilic sulfoxythiocarbamate group that also selectively targets cysteine residues in proteins but forms stable thiocarbamate adducts. Twenty-four sulfoxythiocarbamate analogs were synthesized that retain the structural features important for high potency in sulforaphane analogs: the sulfoxide or keto group and its appropriate distance to electrophilic functional group. Evaluation in various cell lines including hepatoma cells, retinal pigment epithelial cells, and keratinocytes as well as in mouse skin shows that these analogs maintain high potency and efficacy for phase 2 enzyme induction as well as the inhibitory effect on lipopolysaccharide-induced nitric oxide formation like sulforaphane. We further show in living cells that a sulfoxythiocarbamate analog can label Keap1 on several key cysteine residues as well as other cellular proteins offering new insights into the mechanism of chemoprotection.

The C-Terminal of NaV1.7 Is Ubiquitinated by NEDD4L.

NaV1.7, the neuronal voltage-gated sodium channel isoform, plays an important role in the human body's ability to feel pain. Mutations within NaV1.7 have been linked to pain-related syndromes, such as insensitivity to pain. To date, the regulation and internalization mechanisms of the NaV1.7 channel are not well known at a biochemical level. In this study, we perform biochemical and biophysical analyses that establish that the HECT-type E3 ligase, NEDD4L, ubiquitinates the cytoplasmic C-terminal (CT) region of NaV1.7. Through in vitro ubiquitination and mass spectrometry experiments, we identify, for the first time, the lysine residues of NaV1.7 within the CT region that get ubiquitinated. Furthermore, binding studies with an NEDD4L E3 ligase modulator (ubiquitin variant) highlight the dynamic partnership between NEDD4L and NaV1.7. These investigations provide a framework for understanding how NEDD4L-dependent regulation of the channel can influence the NaV1.7 function.

A cell-free antigen processing system informs HIV-1 epitope selection and vaccine design.

Distinct CD4+ T cell epitopes have been associated with spontaneous control of HIV-1 replication, but analysis of antigen-dependent factors that influence epitope selection is lacking. To examine these factors, we used a cell-free antigen processing system that incorporates soluble HLA-DR (DR1), HLA-DM (DM), cathepsins, and full-length protein antigens for epitope identification by LC-MS/MS. HIV-1 Gag, Pol, Env, Vif, Tat, Rev, and Nef were examined using this system. We identified 35 novel epitopes, including glycopeptides. Epitopes from smaller HIV-1 proteins mapped to regions of low protein stability and higher solvent accessibility. HIV-1 antigens associated with limited CD4+ T cell responses were processed efficiently, while some protective epitopes were inefficiently processed. 55% of epitopes obtained from cell-free processing induced memory CD4+ T cell responses in HIV-1+ donors, including eight of 19 novel epitopes tested. Thus, an in vitro processing system utilizing the components of Class II processing reveals factors influencing epitope selection of HIV-1 and represents an approach to understanding epitope selection from non-HIV-1 antigens.

Systemic deletion of Atp7b modifies the hepatocytes' response to copper overload in the mouse models of Wilson disease.

Wilson disease (WD) is caused by inactivation of the copper transporter Atp7b and copper overload in tissues. Mice with Atp7b deleted either globally (systemic inactivation) or only in hepatocyte recapitulate various aspects of human disease. However, their phenotypes vary, and neither the common response to copper overload nor factors contributing to variability are well defined. Using metabolic, histologic, and proteome analyses in three Atp7b-deficient mouse strains, we show that global inactivation of Atp7b enhances and specifically modifies the hepatocyte response to Cu overload. The loss of Atp7b only in hepatocytes dysregulates lipid and nucleic acid metabolisms and increases the abundance of respiratory chain components and redox balancing enzymes. In global knockouts, independently of their background, the metabolism of lipid, nucleic acid, and amino acids is inhibited, respiratory chain components are down-regulated, inflammatory response and regulation of chromosomal replication are enhanced. Decrease in glucokinase and lathosterol oxidase and elevation of mucin-13 and S100A10 are observed in all Atp7b mutant strains and reflect the extent of liver injury. The magnitude of proteomic changes in Atp7b-/- animals inversely correlates with the metallothioneins levels rather than liver Cu content. These findings facilitate identification of WD-specific metabolic and proteomic changes for diagnostic and treatment.

Oxidized CaMKII and O-GlcNAcylation cause increased atrial fibrillation in diabetic mice by distinct mechanisms.

Diabetes mellitus (DM) and atrial fibrillation (AF) are major unsolved public health problems, and diabetes is an independent risk factor for AF. However, the mechanism(s) underlying this clinical association is unknown. ROS and protein O-GlcNAcylation (OGN) are increased in diabetic hearts, and calmodulin kinase II (CaMKII) is a proarrhythmic signal that may be activated by ROS (oxidized CaMKII, ox-CaMKII) and OGN (OGN-CaMKII). We induced type 1 (T1D) and type 2 DM (T2D) in a portfolio of genetic mouse models capable of dissecting the role of ROS and OGN at CaMKII and global OGN in diabetic AF. Here, we showed that T1D and T2D significantly increased AF, and this increase required CaMKII and OGN. T1D and T2D both required ox-CaMKII to increase AF; however, we did not detect OGN-CaMKII or a role for OGN-CaMKII in diabetic AF. Collectively, our data affirm CaMKII as a critical proarrhythmic signal in diabetic AF and suggest ROS primarily promotes AF by ox-CaMKII, while OGN promotes AF by a CaMKII-independent mechanism(s). These results provide insights into the mechanisms for increased AF in DM and suggest potential benefits for future CaMKII and OGN targeted therapies.

In vitro enzymatic characterization of near full length EGFR in activated and inhibited states.

The epidermal growth factor receptor (EGFR) is a single-pass transmembrane protein with an extracellular ligand-binding region and a cytoplasmic tyrosine kinase. Ligand binding activates the tyrosine kinase, which in turn initiates signaling cascades that influence cell proliferation and differentiation. EGFR activity is essential for normal development of many multicellular organisms, and inappropriate activation of EGFR is associated with multiple human cancers. Several drugs targeting EGFR activity are approved cancer therapies, and new EGFR-targeted therapies are being actively pursued. Much of what is known about EGFR structure and function is derived from studies of soluble receptor fragments. We report here an approach to producing an active, membrane-spanning form of EGFR of suitable purity, homogeneity, and quantity for structural and functional studies. We show that EGFR is capable of direct autophosphorylation of tyrosine 845, which is located on its kinase activation loop, and that the kinase activity of EGFR is approximately 500-fold higher in the presence of EGF vs the inhibitory anti-EGFR antibody cetuximab. The potencies of the small molecule EGFR kinase inhibitors erlotinib and lapatinib for various forms of EGFR were measured, and the therapeutic and mechanistic implications of these results considered.

Synthetic Circular RNA Functions as a miR-21 Sponge to Suppress Gastric Carcinoma Cell Proliferation.

MicroRNA (miR) sponges containing miR binding sequences constitute a potentially powerful molecular therapeutic strategy. Recently, naturally occurring circular RNAs (circRNAs) were shown to function as efficient miR sponges in cancer cells. We hypothesized that synthetic circRNA sponges could achieve therapeutic loss-of-function targeted against specific miRs. Linear RNA molecules containing miR-21 binding sites were transcribed in vitro; after dephosphorylation and phosphorylation, circularization was achieved using 5'-3' end-ligation by T4 RNA ligase 1. circRNA stability was assessed using RNase R and fetal bovine serum. Competitive inhibition of miR-21 activity by a synthetic circRNA sponge was assessed using luciferase reporter, cell proliferation, and cell apoptosis assays in three gastric cancer cell lines. circRNA effects on downstream proteins were also delineated by Tandem Mass Tag (TMT) labeling (data available via ProteomeXchange identifier PRIDE: PXD008584), followed by western blotting. We conclude that artificial circRNA sponges resistant to nuclease digestion can be synthesized using simple enzymatic ligation steps. These sponges inhibit cancer cell proliferation and suppress the activity of miR-21 on downstream protein targets, including the cancer protein DAXX. In summary, synthetic circRNA sponges represent a simple, effective, convenient strategy for achieving targeted loss of miR function in vitro, with potential future therapeutic application in human patients.

Distorted Immunodominance by Linker Sequences or other Epitopes from a Second Protein Antigen During Antigen-Processing.

The immune system focuses on and responds to very few representative immunodominant epitopes from pathogenic insults. However, due to the complexity of the antigen processing, understanding the parameters that lead to immunodominance has proved difficult. In an attempt to uncover the determinants of immunodominance among several dominant epitopes, we utilized a cell free antigen processing system and allowed the system to identify the hierarchies among potential determinants. We then tested the results in vivo; in mice and in human. We report here, that immunodominance of known sequences in a given protein can change if two or more proteins are being processed and presented simultaneously. Surprisingly, we find that new spacer/tag sequences commonly added to proteins for purification purposes can distort the capture of the physiological immunodominant epitopes. We warn against adding tags and spacers to candidate vaccines, or recommend cleaving it off before using for vaccination.

Endotoxemia-mediated activation of acetyltransferase P300 impairs insulin signaling in obesity.

Diabetes and obesity are characterized by insulin resistance and chronic low-grade inflammation. An elevated plasma concentration of lipopolysaccharide (LPS) caused by increased intestinal permeability during diet-induced obesity promotes insulin resistance in mice. Here, we show that LPS induces endoplasmic reticulum (ER) stress and protein levels of P300, an acetyltransferase involved in glucose production. In high-fat diet fed and genetically obese ob/ob mice, P300 translocates from the nucleus into the cytoplasm of hepatocytes. We also demonstrate that LPS activates the transcription factor XBP1 via the ER stress sensor IRE1, resulting in the induction of P300 which, in turn, acetylates IRS1/2, inhibits its association with the insulin receptor, and disrupts insulin signaling. Pharmacological inhibition of P300 acetyltransferase activity by a specific inhibitor improves insulin sensitivity and decreases hyperglycemia in obese mice. We suggest that P300 acetyltransferase activity may be a promising therapeutic target for the treatment of obese patients.Elevated plasma LPS levels have been associated with insulin resistance. Here Cao et al. show that LPS induces ER stress and P300 activity via the XBP1/IRE1 pathway. P300 acetylates IRS1/2 and inhibits its binding with the insulin receptor. The consequent impairment of insulin signaling can be rescued by pharmacological inhibition of P300.