Epidermal Growth Factor Receptor Substrate Research Articles

EPS15-AS1 Inhibits AKR1B1 Expression to Enhance Ferroptosis in Hepatocellular Carcinoma Cells.

Epidermal growth factor receptor substrate 15 (EPS15) is part of the EGFR pathway and has been implicated in various tumorigenesis. Increasing evidence suggests that long noncoding RNA (lncRNA) plays an essential role in liver hepatocellular carcinoma (LIHC) by regulating the expression of proteins and genes. Through analysis of the cancer genome atlas (TCGA) database, we found that EPS15 is highly expressed in LIHC tissue, and lncRNA EPS15-antisense1 (EPS15-AS1) decreased in LIHC cell lines. However, the function of EPS15-AS1 in LIHC is still unknown. When EPS15-AS1 was overexpressed in HepG2 cell lines, the expression of EPS15 was reduced and cell activity and invasiveness were inhibited. In addition, we observed an increase in Fe2+ ion and lipid peroxidation after overexpression of EPS15-AS1, and further analysis showed that the susceptibility to ferroptosis increased. Aldo-keto reductase family 1 member B 1 (AKR1B1) belongs to the aldo/keto reductase superfamily and is involved in maintaining the cellular redox balance. Survival analysis revealed that patients with a higher level of AKR1B1 have a lower survival rate in the TCGA database. We also found that EPS15 enhanced the AKR1B1 expression in LIHC, and AKR1B1 had the ability to promote cell invasiveness. Moreover, overexpression of AKR1B1 alleviated the promoting effect of EPS15-AS1 on ferroptosis. Therefore, EPS15-AS1 can induce ferroptosis in hepatocellular carcinoma cells by inhibiting the expression of EPS15 and AKR1B1 and disrupting the redox balance. EPS15 and AKR1B1 may serve as biomarkers for diagnosis and lncRNA EPS15-AS1 potential drug for LIHC.

Proteomic map of the differentially expressed proteins in the skin of Ctenopharyngodon idella against Aeromonas hydrophila infection

The skin mucus of fish is an important part of the innate immune system, which is poorly understood at the proteomic level. The study established a complete map of the proteins in the skin mucus of Ctenopharangdon idella (C. idella) and discussed the Differentially Expressed Proteins (DEPs) after Aeromonas hydrophila (A. hydrophila) infection. Using Label Free Liquid Chromatography-Mass Spectrometry (LC–MS/MS) analysis, a total of 126 proteins were identified as differentially expressed, 89 proteins of which were upregulated, and 37 proteins were downregulated. Functional annotations of DEPs showed that the upregulated proteins in the skin mucus of the treated group were mostly associated with complement system and cytoskeleton proteins, whereas downregulated proteins were associated with metabolism. The key upregulated immune proteins were transferrin variant C, lysozyme g, annexin A11, 26S proteasome non-ATPase regulatory subunit 8, hypothetical protein ROHU_000884, 60S ribosomal L7a, calpain-2 catalytic subunit-like protein, calpain-9-like protein, complement component C9, complement C3, cathepsin S, cathepsin Z, 14 kDa apolipo, heat shock protein and intelectin, whereas, leukocyte elastase inhibitor, annexin A11, C-factor-like protein, biotinidase isoform X1 and epidermal growth factor receptor substrate 15-like were the downregulated proteins. Moreover, we for the first-time report proteins such as coactosin, lamin-B2 and kelch 12, which were never reported in fish. Our study directly pointing out the possible immunological biomarkers in the skin mucus of C. idella after A. hydrophila treatment. Each of the protein we report in this study could be used as base to establish their mechanism of action during bacterial infection that may contribute to the strategies against bacterial prevention and control in fishes.

EHBP1L1, an apicobasal polarity regulator, is critical for nuclear polarization during enucleation of erythroblasts.

Cell polarity, the asymmetric distribution of proteins and organelles, is permanently or transiently established in various cell types and plays an important role in many physiological events. EH domain-binding protein 1 like 1 (EHBP1L1) is an adaptor protein that is localized on recycling endosomes and regulates apical-directed transport in polarized epithelial cells. However, the role of EHBP1L1 in non-epithelial cells remains unknown. In this study, Ehbp1l1-/- mice showed impaired erythroblast enucleation. Further analyses showed that nuclear polarization prior to enucleation was impaired in Ehbp1l1-/- erythroblasts. It was also revealed that EHBP1L1 interactors Rab10, Bin1, and dynamin were involved in erythroblast enucleation. In addition, Ehbp1l1-/- erythrocytes exhibited stomatocytic morphology and dehydration. These defects in erythroid cells culminated in early postnatal anemic lethality in Ehbp1l1-/- mice. Moreover, we found the mislocalization of nuclei and mitochondria in the skeletal muscle cells of Ehbp1l1-/- mice, as observed in patients with centronuclear myopathy with genetic mutations in Bin1 or dynamin 2. Taken together, our findings indicate that the Rab8/10-EHBP1L1-Bin1-dynamin axis plays an important role in multiple cell polarity systems in epithelial and non-epithelial cells.

Transcriptome analysis showed the metabolic pathway of differentially expressed genes (DEGs) in resistant and susceptible soybean (Glycine max) to sclerotinia stem rot (SSR) and candidate gene mining

Context Sclerotinia stem rot (SSR) is one of the diseases that seriously affect soybean yield, leading to heavy losses all over the world. A well-known SSR resistant variety is ‘Maple Arrow’. Aims In this study, transcriptome sequencing analysis of resistant variety ‘Maple Arrow’ and susceptible variety ‘Hefeng25’ was conducted to understand the resistance mechanism of resistant and susceptible soybean varieties to SSR and to look for candidate genes. Methods RNA sequencing of Maple Arrow and Hefeng25 generated 75.09 GB and 64.97 GB clean readings, respectively. In total, 417 differentially expressed genes (DEGs) were found among the different comparable groups. Gene ontology enrichment, Kyoto Encyclopedia of Genes and Genomes analysis and haplotype analysis were performed for genes with different expression levels in Maple Arrow and Hefeng25. Key results It was found that DEGs from Maple Arrow and Hefeng25 were involved in the regulation of ‘oxidation–reduction process’, ‘regulation of transcription’, ‘amino acid metabolism’, ‘methylation’ and ‘membrane’, ‘integral component of membrane’ and ‘epidermal growth-factor receptor substrate 15’. In total, 31 haplotypes of 12 genes were screened out with significant or extremely significant differences among soybeans with different levels of SSR resistance. Conclusions These genes may be involved in the relevant pathways of soybean sclerotiniose. Implications To provide excellent gene resources for further disease-resistance breeding.

Endocytic Adaptors in Cardiovascular Disease.

Endocytosis is the process of actively transporting materials into a cell by membrane engulfment. Traditionally, endocytosis was divided into three forms: phagocytosis (cell eating), pinocytosis (cell drinking), and the more selective receptor-mediated endocytosis (clathrin-mediated endocytosis); however, other important endocytic pathways (e.g., caveolin-dependent endocytosis) contribute to the uptake of extracellular substances. In each, the plasma membrane changes shape to allow the ingestion and internalization of materials, resulting in the formation of an intracellular vesicle. While receptor-mediated endocytosis remains the best understood pathway, mammalian cells utilize each form of endocytosis to respond to their environment. Receptor-mediated endocytosis permits the internalization of cell surface receptors and their ligands through a complex membrane invagination process that is facilitated by clathrin and adaptor proteins. Internalized vesicles containing these receptor-ligand cargoes fuse with early endosomes, which can then be recycled back to the plasma membrane, delivered to other cellular compartments, or destined for degradation by fusing with lysosomes. These intracellular fates are largely determined by the interaction of specific cargoes with adaptor proteins, such as the epsins, disabled-homolog 2 (Dab2), the stonin proteins, epidermal growth factor receptor substrate 15, and adaptor protein 2 (AP-2). In this review, we focus on the role of epsins and Dab2 in controlling these sorting processes in the context of cardiovascular disease. In particular, we will focus on the function of epsins and Dab2 in inflammation, cholesterol metabolism, and their fundamental contribution to atherogenicity.

Characterizing Proteins That Mediate CALM-AF10 Leukemogenesis

Background: Leukemia is the most common type of childhood cancer. Although the prognosis for many pediatric leukemias has improved, leukemias associated with the t(10;11) CALM-AF10 translocation remain difficult to treat. CALM-AF10 leukemias account for ~5-10% of childhood T-cell acute lymphoid leukemia (T-ALL) as well as a subset of acute myeloid leukemia (AML). CALM-AF10 leukemias exhibit increased expression of proleukemic HOXA genes, but relatively little is known about the cellular mechanisms that drive CALM-AF10 leukemogenesis. Our laboratory has demonstrated that the CALM protein contains a nuclear export signal (NES) that is critical for CALM-AF10-dependent leukemogenesis. The NES interacts with the CRM1/XPO1 nuclear export receptor, which shuttles proteins from the nucleus to the cytoplasm through the nuclear pore complex. We have shown that transcriptional activation of HOXA genes by CALM-AF10 is critically dependent on its interaction with CRM1. Importantly, CRM1 does not contain a recognized DNA binding domain, and it is not currently understood how the CALM-AF10/CRM1 complex interacts with regulatory regions of HOXAgenes. In order to identify proteins that mediate the interaction between the CALM-AF10/CRM1 complex and DNA, we took advantage of a proximity-based labeling approach using BioID2, a second-generation biotin ligase. When fused to a protein of interest and in the presence of biotin, BioID2 biotinylates proteins in close proximity to the ligase. These biotinylated proteins can then be identified by mass spectrometry (MS). Methods: We prepared an expression plasmid in which BioID2 was cloned in-frame with CALM-AF10. We then transiently transfected Human Embryonic Kidney 293 (HEK293) cells with the BioID2-CALM-AF10 plasmid, grew them in the presence or absence of biotin, and performed streptavidin-pulldown followed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) to identify candidate interacting proteins. Proteins were considered candidates if they had a peptide spectrum match (PSM) score > 10 and at least a two-fold greater PSM score versus negative control. We validated direct interactions of candidate proteins with CALM-AF10 by performing co-immunoprecipitation experiments. Results: We first confirmed that the addition of BioID2 to CALM-AF10 does not affect the transcriptional activation of HOXA genes or CALM-AF10 mediated immortalization of hematopoietic stem cells. We carried out three independent transfections/LC-MS/MS experiments, which identified 71, 95 and 61 proteins, respectively. Of the proteins identified, 11 candidates were common to all three experiments.Of particular interest, we identified Disruptor Of Telomeric silencing 1-Like (DOT1L), a protein known to interact with AF10, and Nuclear pore complex protein 214 (NUP214), a protein that has been identified in leukemogenic translocations. The nine additional candidate proteins included: EPS15, DVL2, DVL3, and DDX3X -all known to play a role in leukemogenesis. We performed initial validation of direct interactions via co-immunoprecipitation and found that Epidermal Growth Factor Receptor Substrate 15(EPS15) co-precipitates with CALM-AF10. Conclusion: We used biotin ligase-dependent proximity-based labeling to identify candidate proteins that potentially interact with the CALM-AF10 fusion protein. Our identification of DOT1L validates the approach, since DOT1L is known to interact with CALM-AF10. We have started to investigate other candidate proteins, focusing on known translocation partners in various leukemias. Our screen identified EPS15, a protein involved in receptor-mediated endocytosis of epidermal growth factor and a known translocation partner for MLL/KMT2A. KMT2A-EPS15 translocations (t(1;11)(p32;q23)) have been identified in both AML and ALL, and KMT2A-EPS15 is among the eight most common KMT2A rearrangements. We have shown that EPS15 co-immunoprecipitates with CALM-AF10, suggesting that EPS15 may also play a role in CALM-AF10 leukemogenesis. Further evaluation of this interaction is underway, and may lead to identification of novel pathways involved in CALM-AF10 leukemogenesis. Disclosures No relevant conflicts of interest to declare.

EHD1 Modulates Cx43 Gap Junction Remodeling Associated With Cardiac Diseases.

Efficient communication between heart cells is vital to ensure the anisotropic propagation of electrical impulses, a function mainly accomplished by gap junctions (GJ) composed of Cx43 (connexin 43). Although the molecular mechanisms remain unclear, altered distribution and function of gap junctions have been associated with acute myocardial infarction and heart failure. A recent proteomic study from our laboratory identified EHD1 (Eps15 [endocytic adaptor epidermal growth factor receptor substrate 15] homology domain-containing protein 1) as a novel interactor of Cx43 in the heart. In the present work, we demonstrate that knockdown of EHD1 impaired the internalization of Cx43, preserving gap junction-intercellular coupling in cardiomyocytes. Interaction of Cx43 with EHD1 was mediated by Eps15 and promoted by phosphorylation and ubiquitination of Cx43. Overexpression of wild-type EHD1 accelerated internalization of Cx43 and exacerbated ischemia-induced lateralization of Cx43 in isolated adult cardiomyocytes. In addition, we show that EHDs associate with Cx43 in human and murine failing hearts. Overall, we identified EHDs as novel regulators of endocytic trafficking of Cx43, participating in the pathological remodeling of gap junctions, paving the way to innovative therapeutic strategies aiming at preserving intercellular communication in the heart.

Dietary Zinc Supplementation from Organic and Inorganic Sources Induce Differential Intestinal Gene Expression in Swine

BackgroundZinc (Zn) is an essential mineral supplemented to animal diets from organic (bound to organic compounds) and inorganic sources (inorganic salts). Diets supplemented with organic Zn result in better animal performance than inorganic sources especially under challenges like heat stress. The mechanisms by which sources of Zn differ in their physiological effects are unknown. A study in piglets showed that organic Zn sources increase mRNA expression of Zn transporters compared to inorganic Zn. A cell line study demonstrated that amino acid‐bound Zn can be absorbed by amino acid transporters, increasing Zn bioavailability in a model of acrodermatitis enteropathica. Here, we used in vitro and in vivo models to test the hypothesis that intestinal luminal exposure to organic and inorganic Zn sources differentially affects nutrient absorption and transport in the intestine.MethodsForty‐five pigs (body weight = 77.5 ± 2.5 kg) were allocated to pens (9 pigs/pen) and randomly assigned to five different diets: a corn‐soybean meal basal diet without added Zn (control diet), or control diet supplemented with 50 ppm (low) and 100 ppm (high) of ZnCl2 and amino acid‐bound Zn (LQ, Zinpro Corp.) as inorganic and organic sources respectively. After 32 days, ileum samples were collected and RNA was extracted for RNAseq analysis. To define if responses to Zn are due to luminal or basal signaling, mouse enteroids were microinjected with 50 ppm, 100 ppm, 150 ppm and 1000 ppm of either ZnCl2 or LQ‐Zn to model luminal exposure or Zn sources were added to the media to identify changes induced by circulating Zn. Enteroid gene expression of relevant genes was measured by qPCR.ResultsCompared to control diet, ZIP4 expression was increased by high and low LQ‐Zn and high ZnCl2, while low ZnCl2 induced ZIP10 expression. High ZnCl2 diet induced expression of SLC2A6 (GLUT6) compared with control. Comparison of inorganic to organic Zn sources showed 292 differentially expressed genes (DEG's) between low ZnCl2 (n=8) and low LQ‐Zn (n=9) and 370 DEG's between high ZnCl2 (n=8) and high LQ‐Zn (n=8). Of note, phospholipase A2 expression was repressed by high ZnCl2 compared to high LQ‐Zn. Aminopeptidase‐N, metallothionein‐1A (MT1), vitamin D 25‐ hydroxylase, interleukin 33 and epidermal growth factor receptor substrate 15‐like 1 genes showed higher expression induced by ZnCl2 compared to LQ‐Zn. Enteroids luminally exposed to ZnCl2 showed increased expression of metallothionein, Sglt1 and amino acid transporter Pept1. Both ZnCl2 and LQ‐Zn increased the expression of zinc transporter Zip7 in the enteroids. Zn in the enteroid media increased the expression of Glut2, ZnT1 and Mt1, and decreased the expression of Sglt1, Pept1 and Zip 4 compared with no additional Zn. However, no differences were found between ZnCl2 and LQ‐Zn treatments.ConclusionsOur results suggest that Zn supplementation modulates the intestinal expression of nutrients and Zn transporters. Findings in our enteroid experiment suggest that the changes are dependent on the source of Zn only when Zn is supplemented luminally. Overall, our findings provide information on the potential mechanisms by which Zn sources can affect animal performance and health.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Chronic tinnitus and unipolar brush cell alterations in the cerebellum and dorsal cochlear nucleus

Animal model research has shown that the central features of tinnitus, the perception of sound without an acoustic correlate, include elevated spontaneous and stimulus-driven activity, enhanced burst-mode firing, decreased variance of inter-spike intervals, and distortion of tonotopic frequency representation. Less well documented are cell-specific correlates of tinnitus. Unipolar brush cell (UBC) alterations in animals with psychophysical evidence of tinnitus has recently been reported. UBCs are glutamatergic interneurons that appear to function as local-circuit signal amplifiers. UBCs are abundant in the dorsal cochlear nucleus (DCN) and very abundant in the flocculus (FL) and paraflocculus (PFL) of the cerebellum. In the present research, two indicators of UBC structure and function were examined: Doublecortin (DCX) and epidermal growth factor receptor substrate 8 (Eps8). DCX is a protein that binds to microtubules where it can modify their assembly and growth. Eps8 is a cell-surface tyrosine kinase receptor mediating the response to epidermal growth factor; it appears to have a role in actin polymerization as well as cytoskeletal protein interactions. Both functions could contribute to synaptic remodeling. In the present research UBC Eps8 and DCX immunoreactivity (IR) were determined in 4 groups of rats distinguished by their exposure to high-level sound and psychophysical performance: Unexposed, exposed to high-level sound with behavioral evidence of tinnitus, and two exposed groups without behavioral evidence of tinnitus. Compared to unexposed controls, exposed animals with tinnitus had Eps8 IR elevated in their PFL; other structures were not affected, nor was DCX IR affected. This was interpreted as UBC upregulation in animals with tinnitus. Exposure that failed to produce tinnitus did not increase either Eps8 or DCX IR. Rather Eps8 IR was decreased in the FL and DCN of one subgroup (Least-Tinnitus), while DCX IR decreased in the FL of the other subgroup (No-Tinnitus). Neuron degeneration was also documented in the cochlear nucleus and PFL of exposed animals, both with and without tinnitus. Degeneration was not found in unexposed animals. Implications for tinnitus neuropathy are discussed in the context of synaptic remodeling and cerebellar sensory modulation.

Conditional deletion of Eps8 reduces hippocampal synaptic plasticity and impairs cognitive function

Epidermal growth factor receptor substrate 8 (Eps8) is a multifunctional protein involved in actin cytoskeleton regulation and is abundantly expressed in many brain regions. However, the functional significance of Eps8 in the brain has only just begun to be elucidated. Here, we demonstrate that genetic deletion of Eps8 (Eps8−/−) from excitatory neurons leads to impaired performance in a novel object recognition test. Consistently, Eps8−/− mice displayed a deficit in the maintenance of long-term potentiation in the CA1 region of hippocampal slices, which was rescued by bath application of N-methyl-d-aspartate receptor (NMDAR) antagonist 2-amino-5-phosphonopentanoate. While Eps8−/− mice showed normal basal synaptic transmission, a significant increase in the amplitude and a significantly slower decay kinetic of NMDAR-mediated excitatory postsynaptic currents (EPSCs) were observed in hippocampal CA1 neurons. Furthermore, a significant increase in the expression of ifenprodil-sensitive NMDAR-mediated EPSCs was observed in neurons from Eps8−/− mice compared with those from wild-type mice. Eps8 deletion led to decreased mature mushroom-shaped dendritic spine density but increased complexity of basal dendritic trees of hippocampal CA1 pyramidal neurons. These results implicate NMDAR hyperfunction in the cognitive deficits observed in Eps8−/− mice and demonstrate a novel role for Eps8 in regulating hippocampal long-term synaptic plasticity and cognitive function.This article is part of the Special Issue entitled ‘Ionotropic glutamate receptors’.

Specifying the autoantibody-inducing CD4 T cell causing systemic lupus erythematosus out of PD-1+CD45RBlo122lo CD4 subpopulation using mass spectrometry (BA11P.135)

Abstract Objective: We have shown that repeated immunization of mice with antigen reproducibly led to development of experimental systemic lupus erythematosus (SLE), where a novel T cell type which we term an autoantibody-inducing CD4 T (aiCD4 T) cell was generated via TCR revision at periphery. The aiCD4 T cell induces varieties of autoantibodies and also helps full maturation of CTL to induce lupus tissue injuries. We have previously identified that aiCD4 T cell belongs to PD1+CD45RBlo122lo CD4 T subpopulation. Here we tried to further specify the aiCD4 T cell by membrane surface markers. Methods: BALB/c mice were repeatedly 12x immunized with OVA. To isolate aiCD4 T cell, we extracted cytoplasmic and membrane fractions of PD1+CD45RBlo122lo CD4 T subpopulation. They were electrophoresed and protein fractions unique to the PD1+CD45RBlo122lo CD4 T subpopulation were identified thru mass spectrometry. Results: Candidate membrane markers as suggested by mass spectrometry were DNA topoisomerase 2-beta (181 kDa), DOCK 8 (238 kDa), VWA5A (87 kDa), STAT5b (89 kDa), epidermal growth factor receptor substrate 15-like 1 (99 kDa) and FLAP (18 kDa) for aiCD4 T cell, among which we found that DOCK 8 was significantly increased in the membrane fraction of PD1+CD45RBlo122lo CD4 T subpopulation. Conclusion: We identified DOCK 8 as a possible candidate membrane marker characterizing the aiCD4 T cell as found within PD-1+CD45RBlo122lo CD4 T subpopulation.

Protein sorting to intracellular compartments.

The organization of eukaryotic cells depends on correct targeting of proteins and lipids to the organelles at which they function, but how organelle identity is maintained in the face of a constant flux of material is a major problem for the cell. New mechanisms addressing this question were revealed at the Minisymposium on “Membrane Traffic: Dynamic and Regulation.” Starting with the endoplasmic reticulum (ER), Silvere Pagant (Miller lab) showed that some plasma membrane (PM) proteins require multiple signals to drive capture into COPII vesicles. One signal comes from the cargo protein itself, and the second comes signal from a cargo receptor; both signals simultaneously engage the cargo adaptor for the COPII coat, Sec24. Prasanna Satpute-Krishnan (Lippincott-Schwartz and Hegde labs), recipient of the 2014 Merton Bernfield Award, presented her work on a novel quality-control mechanism for glycosylphosphatidylinositol (GPI)-anchored proteins called RESET (for rapid ER stress-induced export). ER stress causes receptor-mediated exit of GPI-anchored proteins from the ER through the secretory pathway to the PM, from which they are endocytosed and degraded in lysosomes. Only misfolded GPI-anchored proteins bind tightly to the cargo receptor, Tmp21, providing a new paradigm in ER stress clearance. Moving from the ER to the Golgi, Mie Wong (Munro lab) presented an elegant system to test the function of a family of diverse Golgi-localized coiled-coil proteins. When different golgins were ectopically attached to mitochondria, specific classes of vesicles were redirected to mitochondria, clearly demonstrating a vesicle capture role for these proteins. A possible mechanism of lipid transfer from the ER to the PM was revealed by a structural study presented by Chris Schauder (Reinisch and de Camilli groups). The synaptotagmin-like mitochondrial-lipid binding protein (SMP) domain of the ER–PM tether E-Syt2 forms a long hydrophobic channel that can harbor multiple phospholipid molecules. Two SMP domains form a stacked dimer to double the length of the channel. Continuing the theme of lipid composition in organelle identity, Cathy Jackson presented her group's results showing how amphipathic helices (AHs) of distinct chemistries specifically target different organelles through recognition of organelle lipid composition. Regulation of a lipid-binding AH of the Arf guanine nucleotide exchange factor (GEF) GBF1 mediates its localization to either the Golgi or lipid droplets. Looking at a related Arf GEF, yeast Sec7, Brian Richardson (Fromme lab) reported the first crystal structure of a noncatalytic region of these proteins. The N-terminal region is made up of α-helices forming a series of armadillo folds that form an extended platform. This structure provides insight into how this region is involved in dimerization and protein interactions. New vesicle tethering and targeting mechanisms were reported, with Chris Stroupe presenting how the homotypic fusion and protein sorting (HOPS)/class C Vps tethering complex connects specific membrane compartments. This complex binds to a Rab GTPase in one membrane and directly to a second, highly curved membrane through a curvature-sensing amphipathic lipid packing sensor (ALPS) motif. Margaret Heider (Munson lab) provided the most comprehensive view to date of the architecture of the exocyst complex, a multisubunit tether that targets secretory vesicles to the PM. The session ended with a talk by Christine Insinna-Kettenhofen (Westlake group) reporting on a new mechanism for targeting of vesicles to cilia mediated by the epidermal growth factor receptor substrate 15 homology domain–containing (EHD) proteins. These proteins are localized to vesicles, where they cooperate with the Rab8-Rab11 cascade and soluble N-ethylmaleimide–sensitive factor attachment protein receptors (SNAREs) to regulate morphological changes to the vesicle membrane important for fusion.

Autophagy and ubiquitination in cardiovascular diseases.

A main function of the heart is to pump blood to the tissues and organs of the body. Although formed by different types of cells, the cardiomyocytes are the ones responsible for the coordinated and synchronized heart contraction. Given their low mitotic activity, cardiomyocytes largely depend on protein degradation mechanisms to maintain proteostasis and energetic balance. Autophagy, one of the main pathways whereby cells eliminate damaged, nonfunctional, or obsolete proteins, and organelles, is vital to ensure cell function, including in cardiomyocytes, both in rest and stress conditions. However, the impact of autophagy activation in the heart, being either protective or harmful, is not consensual and likely depends upon the severity of the stimuli and consequently the autophagy players involved. One of the signals that direct proteins for autophagy degradation, namely in the context of heart disorders, is ubiquitin. Indeed, the attachment of ubiquitin moieties to a target substrate and further recognition by autophagy adaptors constitute a main regulatory pathway that directs proteins to the lysosome. Therefore, a better understanding of the mechanisms and signals that regulate the autophagy process in the heart, including substrates targeting, is of utmost importance to design new approaches directed to this degradation pathway. We have previously shown that ubiquitination of the gap junction (GJ) protein Connexin43 (Cx43) triggers its degradation by autophagy through a process that requires the ubiquitin adaptors epidermal growth factor receptor substrate 15 (Eps15) and p62. This is particularly relevant in the heart because GJs, that form intercellular channels, are responsible for the rapid and efficient anisotropic propagation of the electrical impulse through the cardiomyocytes, essential for synchronized contraction of the cardiac muscle. In this review, we present recent studies devoted to the involvement of autophagy in heart homeostasis, with a particular focus on ubiquitin and GJs.

Detection of post-translational modifications in single peptides using electron tunnelling currents.

Post-translational modifications alter the properties of proteins through the cleavage of peptide bonds or the addition of a modifying group to one or more amino acids. These modifications allow proteins to perform their primary biological functions, but single-protein studies of post-translational modifications have been hindered by a lack of suitable analysis methods. Here, we show that single amino acids can be identified using electron tunnelling currents measured as the individual molecules pass through a nanoscale gap between electrodes. We identify 12 different amino acids and the post-translational modification phosphotyrosine, which is involved in the process that switches enzymes on and off. Furthermore, we show that the conductance measurements can be used to partially sequence peptides of an epidermal growth factor receptor substrate, and can discriminate a peptide from its phosphorylated variant.

Endocytic Adaptor Epidermal Growth Factor Receptor Substrate 15 (Eps15) Is Involved in the Trafficking of Ubiquitinated α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid Receptors

AMPA-type glutamate receptors (AMPARs) play a critical role in mediating fast excitatory synaptic transmission in the brain. Alterations in receptor expression, distribution, and trafficking have been shown to underlie synaptic plasticity and higher brain functions, including learning and memory, as well as brain dysfunctions such as drug addiction and psychological disorders. Therefore, it is essential to elucidate the molecular mechanisms that regulate AMPAR dynamics. We have shown previously that mammalian AMPARs are subject to posttranslational modification by ubiquitin, with AMPAR ubiquitination enhancing receptor internalization and reducing AMPAR cell surface expression. Here we report a crucial role for epidermal growth factor receptor substrate 15 (Eps15), an endocytic adaptor, in ubiquitination-dependent AMPAR internalization. We find that suppression or overexpression of Eps15 results in changes in AMPAR surface expression. Eps15 interacts with AMPARs, which requires Nedd4-mediated GluA1 ubiquitination and the ubiquitin-interacting motif of Eps15. Importantly, we find that Eps15 plays an important role in AMPAR internalization. Knockdown of Eps15 suppresses the internalization of GluA1 but not the mutant GluA1 that lacks ubiquitination sites, indicating a role of Eps15 for the internalization of ubiquitinated AMPARs. These results reveal a novel molecular mechanism employed specifically for the trafficking of the ubiquitin-modified AMPARs.

Cell type-specific expression of Eps8 in the mouse hippocampus

BackgroundEpidermal growth factor receptor substrate 8 (Eps8) is a multifunctional protein that regulates actin cytoskeleton dynamics and architecture through its barbed-end capping and bundling activities. In cultured hippocampal neurons, Eps8 is enriched at dendritic spine heads and is required for spine morphogenesis; however, the detailed expression pattern of Eps8 in the hippocampus has not yet been explored.ResultsHere, we demonstrate that endogenous Eps8 protein is restrictively expressed in neurons (NeuN-positive), but not in glial cells (glial fibrillary acidic protein-positive) in area CA1 of the mouse hippocampus. Surprisingly, Eps8 immunoreactivity is rarely found in pyramidal cell somata, but is expressed predominantly in the somata and dendrites of 67 kDa isoform of glutamic acid decarboxylase-expressing GABAergic interneurons in the stratum radiatum and at the border of stratum radiatum and lacunosum-moleculare of area CA1. On the basis of co-localizing markers, we found that Eps8 is not present in perisomatic inhibitory parvalbumin-expressing cells or calretinin-expressing interneurons. However, Eps8 is richly expressed in calbindin-expressing interneurons. Furthermore, Eps8 is also present in cholecystokinin-expressing interneurons, but not in somatostatin-expressing interneurons in area CA1 stratum pyramidale and stratum radiatum.ConclusionsThese results reveal a previously unknown cell type-specific expression pattern of endogenous Eps8 protein in the mouse hippocampus and speculate that the role of Eps8 in controlling and orchestrating neuronal morphogenesis and structural plasticity might be more prominent in interneurons than in pyramidal cells of the hippocampus.

Eps8 controls Src- and FAK-dependent phenotypes in squamous carcinoma cells.

ABSTRACTEps8 is an actin regulatory scaffold protein whose expression is increased in squamous cell carcinoma (SCC) cells. It forms a complex with both focal adhesion kinase (FAK, also known as PTK2) and Src in SCC cells derived from skin carcinomas induced by administration of the chemical DMBA followed by TPA (the DMBA/TPA model). Here, we describe two new roles for Eps8. Firstly, it controls the spatial distribution of active Src in a FAK-dependent manner. Specifically, Eps8 participates in, and regulates, a biochemical complex with Src and drives trafficking of Src to autophagic structures that SCC cells use to cope with high levels of active Src when FAK is absent. Secondly, when FAK is expressed in SCC cells, thereby meaning active Src becomes tethered at focal adhesion complexes, Eps8 is also recruited to focal adhesions and is required for FAK-dependent polarization and invasion. Therefore, Eps8 is a crucial mediator of Src- and FAK-regulated processes; it participates in specific biochemical complexes and promotes actin re-arrangements that determine the spatial localization of Src, and modulates the functions of Src and FAK during invasive migration.

Abstract 3814: Exome sequencing for identification of causative genes for mosaic variegated aneuploidy.

Abstract Mosaic Variegated Aneuploidy (MVA: OMIM 257300) is a rare autosomal recessive syndrome associated with growth deficiency of prenatal onset, microcephaly, intellectual disability, anomalies of the central nervous system, mild physical dysmorphic features and predisposition to cancer. Of the 37 reported cases of MVA, the cancers reported include Wilms tumor, rhabdomyosarcoma and leukemia. The cells from MVA patients demonstrate variable whole chromosome loss and gains suggesting an underlying chromosome segregation dysfunction. Previous studies have demonstrated biallelic loss-of-function mutations in mitotic spindle-assembly checkpoint gene BUB1B or a centrosomal microtubule stabilizing gene CEP57 in some MVA patients. Therefore, the goal of this project is to identify novel causative genes that contribute to the development of MVA. We have identified one patient diagnosed with clinical features of MVA associated with myelodysplasia and acute myeloid leukemia. Cytogenetic analysis of peripheral blood karyotype demonstrated low-level mosaicism with multiple clones of various chromosomal gains such as trisomy 1, 5, 6, 8 and 18. Molecular sequence analysis confirmed normal BUB1B and CEP57 in this patient. Therefore, whole-exome sequencing is being used as an approach to identify disease-causing genes in our patient. Exome data analysis assuming an autosomal recessive model and Sanger sequencing confirmed four putative genes with biallelic mutations in AIM1 (absent in melanoma 1), EPS15 (epidermal growth factor receptor substrate 15), MICALL2 (molecule interacting with CasL-like protein 2) and ZSCAN12 (zinc finger and SCAN domain containing 12). Review of literature reveals potential roles of these genes on cytoskeleton, mitotic spindle formation and cytokinesis. To further investigate these potential loci in our MVA patient, we designed cellular and functional assays using patient skin fibroblasts. Using interphase-FISH analysis, we have recapitulated the aneuploidy phenotype with abnormal chromosome numbers involving trisomy 8 in the patient skin fibroblasts. Moreover, immunocytochemistry with cytoskeletal and mitotic markers display aberrant anaphase with dysfunctional spindle mid-zone and cytokinesis failure. In addition, the patient fibroblasts also exhibited increased percentage of binucleated and micronucleated progeny due to abnormal chromosome segregation and cytokinesis. In summary, exome sequencing revealed a small number of potential causative genes as the etiology of MVA in our patient. Our early results demonstrate biomarkers for chromosome instability and aneuploidy such as non-disjunction, cytokinesis failure, micronuclei and binucleation in patient fibroblasts. Further work will determine whether dysregulation of these candidate genes can recapitulate this instability phenotype leading to mosaic aneuploidy in our patient. Citation Format: Sirisha Peddibhotla, Bradford Powell, Meha P. Patel, Pulivarthi H. Rao, David A. Wheeler, Richard A. Gibbs, Sharon E. Plon. Exome sequencing for identification of causative genes for mosaic variegated aneuploidy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3814. doi:10.1158/1538-7445.AM2013-3814

EHD2 shuttles to the nucleus and represses transcription

EHD {EH [Eps15 (epidermal growth factor receptor substrate 15) homology]-domain-containing} proteins participate in several endocytic events, such as the internalization and the recycling processes. There are four EHD proteins in mammalian cells, EHD1-EHD4, each with diverse roles in the recycling pathway of endocytosis. EHD2 is a plasma-membrane-associated member of the EHD family that regulates internalization. Since several endocytic proteins have been shown to undergo nucleocytoplasmic shuttling and have been assigned roles in regulation of gene expression, we tested the possibility that EHD proteins also shuttle to the nucleus. Our results showed that, among the three EHD proteins (EHD1-EHD3) that were tested, only EHD2 accumulates in the nucleus under nuclear export inhibition treatment. Moreover, the presence of a NLS (nuclear localization signal) was essential for its entry into the nucleus. Nuclear exit of EHD2 depended partially on its NES (nuclear export signal). Elimination of a potential SUMOylation site in EHD2 resulted in a major accumulation of the protein in the nucleus, indicating the involvement of SUMOylation in the nuclear exit of EHD2. We confirmed the SUMOylation of EHD2 by employing co-immunoprecipitation and the yeast two-hybrid system. Using GAL4-based transactivation assay as well as a KLF7 (Krüppel-like factor 7)-dependent transcription assay of the p21WAF1/Cip1 [CDKN1A (cyclin-dependent kinase inhibitor 1A)] gene, we showed that EHD2 represses transcription. qRT-PCR (quantitative real-time PCR) of RNA from cells overexpressing EHD2 or of RNA from cells knocked down for EHD2 confirmed that EHD2 represses transcription of the p21WAF1/Cip1 gene.

Abstract B148: The miRNA-200 family regulates cell migration and metastasis in lung adenocarcinoma.

Abstract miRNAs are post-transcriptional regulators of genes involved in such canonical signaling pathways as the TGF-β and EGF pathways, both of which are frequently altered in tumor cells. miRNA downregulation results in upregulation of its target genes and ultimately aberrant signaling of pathways involved in invasion and migration, for example. To better understand how alterations in miRNA expression could lead to metastasis, we used a lung adenocarcinoma mouse model in which a lentivirus expressing the Cre recombinase activates oncogenic K-Ras and deletes p53, giving rise to primary lung tumors (T) and metastases (M). We performed miRNA expression profiling on 30 T and M derived cell lines and identified three members of the miR-200 family - miR-200b, miR-200c, miR-429 - to be significantly downregulated in the M lines compared to their related T line. To explore the functional contribution of the miR-200s to metastasis, we designed a miRNA sponge to stably inhibit endogenous miR-200 levels in T cell lines. We found miR-200 depletion promoted wound healing and cell migration towards either serum or EGF. Transplantation of the sponge-expressing lines via tail vein led to in vivo formation of more invasive, metastatic tumors compared to controls. Previously, the miR-200 family has been shown to inhibit the TGF-β target gene and EMT mediator, Zeb1. We have identified another miR-200 target, Epidermal Growth Factor Receptor Substrate 8 (Eps8), which mediates actin based cell motility. In miR-200 low (M) cells, Eps8 is expressed three-fold higher than in corresponding miR-200 high (T) cells. Additionally, exogenous expression of Eps8 in T lines promotes wound healing and cell migration, relative to control cells. Thus, our findings implicate the miR-200s as a class of metastasis regulators that modulate expression of genes functionally relevant in tumor cell dissemination and provide new targets for therapeutic intervention of late stage lung adenocarcinoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B148.