Epsin-Dependent Ligand Endocytosis Activates Notch by Force.

DSL ligands activate Notch by inducing proteolytic cleavage of the receptor ectodomain, an event that requires ligand to be endocytosed in signal-sending cells by the adaptor protein Epsin. Two classes of explanation for this unusual requirement are (1) recycling models, in which the ligand must be endocytosed to be modified or repositioned before it binds Notch and (2) pulling models, in which the ligand must be endocytosed after it binds Notch to exert force that exposes an otherwise buried site for cleavage. We demonstrate in vivo that ligands that cannot enter the Epsin pathway nevertheless bind Notch but fail to activate the receptor because they cannot exert sufficient force. This argues against recycling models and in favor of pulling models. Our results also suggest that once ligand binds receptor, activation depends on a competition between Epsin-mediated ligand endocytosis, which induces cleavage, and transendocytosis of the ligand by the receptor, which aborts the incipient signal.

Clathrin controls bidirectional communication between T cells and antigen presenting cells.

In circulation, T cells are spherical with selectin enriched dynamic microvilli protruding from the surface. Following extravasation, these microvilli serve another role, continuously surveying their environment for antigen in the form of peptide-MHC (pMHC) expressed on the surface of antigen presenting cells (APCs). Upon recognition of their cognate pMHC, the microvilli are initially stabilized and then flatten into F-actin dependent microclusters as the T cell spreads over the APC. Within 1-5 min, clathrin is recruited by the ESCRT-0 component Hrs to mediate release of T cell receptor (TCR) loaded vesicles directly from the plasma membrane by clathrin and ESCRT-mediated ectocytosis (CEME). After 5-10 min, Hrs is displaced by the endocytic clathrin adaptor epsin-1 to induce clathrin-mediated trans-endocytosis (CMTE) of TCR-pMHC conjugates. Here we discuss some of the functional properties of the clathrin machinery which enables it to control these topologically opposite modes of membrane transfer at the immunological synapse, and how this might be regulated during T cell activation.

Epsin Nanotherapy Regulates Cholesterol Transport to Fortify Atheroma Regression.

BACKGROUND: Excess cholesterol accumulation in lesional macrophages elicits complex responses in atherosclerosis. Epsins, a family of endocytic adaptors, fuel the progression of atherosclerosis; however, the underlying mechanism and therapeutic potential of targeting Epsins remains unknown. In this study, we determined the role of Epsins in macrophage-mediated metabolic regulation. We then developed an innovative method to therapeutically target macrophage Epsins with specially designed S2P-conjugated lipid nanoparticles, which encapsulate small-interfering RNAs to suppress Epsins. METHODS: We used single-cell RNA sequencing with our newly developed algorithm MEBOCOST (Metabolite-mediated Cell Communication Modeling by Single Cell Transcriptome) to study cell-cell communications mediated by metabolites from sender cells and sensor proteins on receiver cells. Biomedical, cellular, and molecular approaches were utilized to investigate the role of macrophage Epsins in regulating lipid metabolism and transport. We performed this study using myeloid-specific Epsin double knockout (LysM-DKO) mice and mice with a genetic reduction of ABCG1 (ATP-binding cassette subfamily G member 1; LysM-DKO-ABCG1fl/+). The nanoparticles targeting lesional macrophages were developed to encapsulate interfering RNAs to treat atherosclerosis. RESULTS: We revealed that Epsins regulate lipid metabolism and transport in atherosclerotic macrophages. Inhibiting Epsins by nanotherapy halts inflammation and accelerates atheroma resolution. Harnessing lesional macrophage-specific nanoparticle delivery of Epsin small-interfering RNAs, we showed that silencing of macrophage Epsins diminished atherosclerotic plaque size and promoted plaque regression. Mechanistically, we demonstrated that Epsins bound to CD36 to facilitate lipid uptake by enhancing CD36 endocytosis and recycling. Conversely, Epsins promoted ABCG1 degradation via lysosomes and hampered ABCG1-mediated cholesterol efflux and reverse cholesterol transport. In a LysM-DKO-ABCG1fl/+ mouse model, enhanced cholesterol efflux and reverse transport due to Epsin deficiency was suppressed by the reduction of ABCG1. CONCLUSIONS: Our findings suggest that targeting Epsins in lesional macrophages may offer therapeutic benefits for advanced atherosclerosis by reducing CD36-mediated lipid uptake and increasing ABCG1-mediated cholesterol efflux.

O-GlcNAc transferase modulates the cellular endocytosis machinery by controlling the formation of clathrin-coated pits.

Clathrin-mediated endocytosis (CME) controls the internalization and function of a wide range of cell surface proteins. CME occurs by the assembly of clathrin and many other proteins on the inner leaflet of the plasma membrane into clathrin-coated pits (CCPs). These structures recruit specific cargo destined for internalization, generate membrane curvature, and in many cases undergo scission from the plasma membrane to yield intracellular vesicles. The diversity of functions of cell surface proteins controlled via internalization by CME may suggest that regulation of CCP formation could be effective to allow cellular adaptation under different contexts. Of interest is how cues derived from cellular metabolism may regulate CME, given the reciprocal role of CME in controlling cellular metabolism. The modification of proteins with O-linked ß-GlcNAc (O-GlcNAc) is sensitive to nutrient availability and may allow cellular adaptation to different metabolic conditions. Here, we examined how the modification of proteins with O-GlcNAc may control CCP formation and thus CME. We used perturbation of key enzymes responsible for protein O-GlcNAc modification, as well as specific mutants of the endocytic regulator AAK1 predicted to be impaired for O-GlcNAc modification. We identify that CCP initiation and the assembly of clathrin and other proteins within CCPs are controlled by O-GlcNAc protein modification. This reveals a new dimension of regulation of CME and highlights the important reciprocal regulation of cellular metabolism and endocytosis.

Targeting Epsins to Inhibit Fibroblast Growth Factor Signaling While Potentiating Transforming Growth Factor-ß Signaling Constrains Endothelial-to-Mesenchymal Transition in Atherosclerosis.

BACKGROUND: Epsin endocytic adaptor proteins are implicated in the progression of atherosclerosis; however, the underlying molecular mechanisms have not yet been fully defined. In this study, we determined how epsins enhance endothelial-to-mesenchymal transition (EndoMT) in atherosclerosis and assessed the efficacy of a therapeutic peptide in a preclinical model of this disease. METHODS: Using single-cell RNA sequencing combined with molecular, cellular, and biochemical analyses, we investigated the role of epsins in stimulating EndoMT using knockout in Apoe-/- and lineage tracing/proprotein convertase subtilisin/kexin type 9 serine protease mutant viral-induced atherosclerotic mouse models. The therapeutic efficacy of a synthetic peptide targeting atherosclerotic plaques was then assessed in Apoe-/- mice. RESULTS: Single-cell RNA sequencing and lineage tracing revealed that epsins 1 and 2 promote EndoMT and that the loss of endothelial epsins inhibits EndoMT marker expression and transforming growth factor-ß signaling in vitro and in atherosclerotic mice, which is associated with smaller lesions in the Apoe-/- mouse model. Mechanistically, the loss of endothelial cell epsins results in increased fibroblast growth factor receptor-1 expression, which inhibits transforming growth factor-ß signaling and EndoMT. Epsins directly bind ubiquitinated fibroblast growth factor receptor-1 through their ubiquitin-interacting motif, which results in endocytosis and degradation of this receptor complex. Consequently, administration of a synthetic ubiquitin-interacting motif-containing peptide atheroma ubiquitin-interacting motif peptide inhibitor significantly attenuates EndoMT and progression of atherosclerosis. CONCLUSIONS: We conclude that epsins potentiate EndoMT during atherogenesis by increasing transforming growth factor-ß signaling through fibroblast growth factor receptor-1 internalization and degradation. Inhibition of EndoMT by reducing epsin-fibroblast growth factor receptor-1 interaction with a therapeutic peptide may represent a novel treatment strategy for atherosclerosis.

Epsin2, a novel target for multiple system atrophy therapy via α-synuclein/FABP7 propagation.

Multiple system atrophy (MSA) is a neurodegenerative disease characterized by the accumulation of misfolded α-synuclein (αSyn) and myelin disruption. However, the mechanism underlying αSyn accumulation in MSA brains remains unclear. Here, we aimed to identify epsin-2 as a potential regulator of αSyn propagation in MSA brains. In the MSA mouse model, PLP-hαSyn mice, and FABP7/αSyn hetero-aggregate-injected mice, we initially discovered that fatty acid-binding protein 7 (FABP7) is related to MSA development and forms hetero-aggregates with αSyn, which exhibit stronger toxicity than αSyn aggregates. Moreover, the injected FABP7/αSyn hetero-aggregates in mice selectively accumulated only in oligodendrocytes and Purkinje neurons, causing cerebellar dysfunction. Furthermore, bioinformatic analyses of whole blood from MSA patients and FABP7 knockdown mice revealed that epsin-2, a protein expressed in both oligodendrocytes and Purkinje cells, could potentially regulate FABP7/αSyn hetero-aggregate propagation via clathrin-dependent endocytosis. Lastly, adeno-associated virus type 5-dependent epsin-2 knockdown mice exhibited decreased levels of αSyn aggregate accumulation in Purkinje neurons and oligodendrocytes, as well as improved myelin levels and Purkinje neuron function in the cerebellum and motor performance. These findings suggest that epsin-2 plays a significant role in αSyn accumulation in MSA, and we propose epsin-2 as a novel therapeutic target for MSA.

Epsin1-mediated exosomal sorting of Dll4 modulates the tubular-macrophage crosstalk in diabetic nephropathy.

Tubular epithelial cells (TECs) play critical roles in the development of diabetic nephropathy (DN), and can activate macrophages through the secretion of exosomes. However, the mechanism(s) of TEC-exosomes in macrophage activation under DN remains unknown. By mass spectrometry, 1,644 differentially expressed proteins, especially Dll4, were detected in the urine exosomes of DN patients compared with controls, which was confirmed by western blot assay. Elevated Epsin1 and Dll4/N1ICD expression was observed in kidney tissues in both DN patients and db/db mice and was positively associated with tubulointerstitial damage. Exosomes from high glucose (HG)-treated tubular cells (HK-2) with Epsin1 knockdown (KD) ameliorated macrophage activation, TNF-α, and IL-6 expression, and tubulointerstitial damage in C57BL/6 mice in vivo. In an in vitro study, enriched Dll4 was confirmed in HK-2 cells stimulated with HG, which was captured by THP-1 cells and promoted M1 macrophage activation. In addition, Epsin1 modulated the content of Dll4 in TEC-exosomes stimulated with HG. TEC-exosomes with Epsin1-KD significantly inhibited N1ICD activation and iNOS expression in THP-1 cells compared with incubation with HG alone. These findings suggested that Epsin1 could modulate tubular-macrophage crosstalk in DN by mediating exosomal sorting of Dll4 and Notch1 activation.

Structural Dissection of Epsin-1 N-Terminal Helical Peptide: The Role of Hydrophobic Residues in Modulating Membrane Curvature.

Spatiotemporal structural alterations in cellular membranes are the hallmark of many vital processes. In these cellular events, the induction of local changes in membrane curvature often plays a pivotal role. Many amphiphilic peptides are able to modulate membrane curvature, but there is little information on specific structural factors that direct the curvature change. Epsin-1 is a representative protein thought to initiate invagination of the plasma membrane upon clathrin-coated vesicles formation. Its N-terminal helical segment (EpN18) plays a key role in inducing positive membrane curvature. This study aimed to elucidate the essential structural features of EpN18 in order to better understand general curvature-inducing mechanisms, and to design effective tools for rationally controlling membrane curvature. Structural dissection of peptides derived from EpN18 revealed the decisive contribution of hydrophobic residues to (i) enhancing membrane interactions, (ii) helix structuring, (iii) inducing positive membrane curvature, and (iv) loosening lipid packing. The strongest effect was obtained by substitution with leucine residues, as this EpN18 analog showed a marked ability to promote the influx of octa-arginine cell-penetrating peptides into living cells.

Expression of Epsin3 and its interaction with Notch signalling in oral epithelial dysplasia and oral squamous cell carcinoma.

BACKGROUND: Most oral squamous cell carcinoma patients present with late-stage disease. Early detection of the disease is considered to be the most effective way of improving patient outcomes. Several biomarkers have been identified as indicators of oral cancer development and progression; however, none have been translated into clinical practice. In this study, we have investigated the role of Epsin3, an endocytic adaptor protein, and Notch1, a transmembrane signalling protein, in oral carcinogenesis with a view to explore their potential as biomarkers. METHODS: Oral cancer cell lines and a normal oral keratinocyte cell line were used together with tissue samples of normal oral mucosa (n = 21), oral epithelial dysplasia (n = 74) and early stage (Stages I and II) oral squamous cell carcinoma (n = 31). Immunocytochemical staining, immunoblotting and real-time quantitative polymerase chain reaction (PCR) were performed to assess protein as well as gene expression levels. RESULTS: The expression levels of Epsin3 and Notch1 mRNA and protein are variable across different oral squamous cell carcinoma derived cell lines. Epsin3 was upregulated in oral epithelial dysplasia and oral squamous cell carcinoma tissues compared with normal epithelium. Overexpression of Epsin3 resulted in a significant reduction of Notch1 expression in oral squamous cell carcinoma. Notch1 was generally downregulated in the dysplasia and oral squamous cell carcinoma samples. CONCLUSION: Epsin3 is upregulated in oral epithelial dysplasia and oral squamous cell carcinoma and has the potential to be used as a biomarker for oral epithelial dysplasia. Notch signalling is downregulated in oral squamous cell carcinoma, possibly through an Epsin3-induced de-activation pathway.

ESCRT-dependent protein sorting is required for the viability of yeast clathrin-mediated endocytosis mutants.

Endocytosis regulates many processes, including signaling pathways, nutrient uptake, and protein turnover. During clathrin-mediated endocytosis (CME), adaptors bind to cytoplasmic regions of transmembrane cargo proteins, and many endocytic adaptors are also directly involved in the recruitment of clathrin. This clathrin-associated sorting protein family includes the yeast epsins, Ent1/2, and AP180/PICALM homologs, Yap1801/2. Mutant strains lacking these four adaptors, but expressing an epsin N-terminal homology (ENTH) domain necessary for viability (4Δ+ENTH), exhibit endocytic defects, such as cargo accumulation at the plasma membrane (PM). This CME-deficient strain provides a sensitized background ideal for revealing cellular components that interact with clathrin adaptors. We performed a mutagenic screen to identify alleles that are lethal in 4Δ+ENTH cells using a colony-sectoring reporter assay. After isolating candidate synthetic lethal genes by complementation, we confirmed that mutations in VPS4 led to inviability of a 4Δ+ENTH strain. Vps4 mediates the final step of endosomal sorting complex required for transport (ESCRT)-dependent trafficking, and we found that multiple ESCRTs are also essential in 4Δ+ENTH cells, including Snf7, Snf8 and Vps36. Deletion of VPS4 from an end3Δ strain, another CME mutant, similarly resulted in inviability, and upregulation of a clathrin-independent endocytosis pathway rescued 4Δ+ENTH vps4Δ cells. Loss of Vps4 from an otherwise wild-type background caused multiple cargoes to accumulate at the PM because of an increase in Rcy1-dependent recycling of internalized protein to the cell surface. Additionally, vps4Δ rcy1Δ mutants exhibited deleterious growth phenotypes. Together, our findings reveal previously unappreciated effects of disrupted ESCRT-dependent trafficking on endocytic recycling and the PM.

The Na+ pump Ena1 is a yeast epsin-specific cargo requiring its ubiquitylation and phosphorylation sites for internalization.

It is well known that in addition to its classical role in protein turnover, ubiquitylation is required for a variety of membrane protein sorting events. However, and despite substantial progress in the field, a long-standing question remains: given that all ubiquitin units are identical, how do different elements of the sorting machinery recognize their specific cargoes? Our results indicate that the yeast Na+ pump Ena1 is an epsin (Ent1 and Ent2 in yeast)-specific cargo and that its internalization requires K1090, which likely undergoes Art3-dependent ubiquitylation. In addition, an Ena1 serine and threonine (ST)-rich patch, proposed to be targeted for phosphorylation by casein kinases, was also required for its uptake. Interestingly, our data suggest that this phosphorylation was not needed for cargo ubiquitylation. Furthermore, epsin-mediated internalization of Ena1 required a specific spatial organization of the ST patch with respect to K1090 within the cytoplasmic tail of the pump. We hypothesize that ubiquitylation and phosphorylation of Ena1 are required for epsin-mediated internalization.

Protein-Protein Interactions on Membrane Surfaces Analysed Using Pull-Downs with Supported Bilayers on Silica Beads.

Discovery-based proteomics workflows that identify novel interactors rely on immunoprecipitations or pull-downs with genetically tagged bait proteins immobilized on appropriate matrices. But strategies to analyse protein interactions on a diffusible-membrane surface combined with the practical ease of pull-downs remain unavailable. Such strategies are important to analyse protein complexes that mature in composition and stability because of diffusion-based encounter between participant proteins. Here, we describe a generic pull-down strategy to analyse such complexes using chelating lipid-containing supported bilayers formed on silica beads. These templates can display desired His-tagged bait proteins on a diffusible-membrane surface. Using clathrin-mediated endocytosis as a paradigm, we find that the clathrin-binding adaptor protein epsin1 displayed as bait on these templates pulls down significantly higher amounts of clathrin from brain lysates than when immobilized on conventional matrices. Together, our results establish the potential of such templates as superior matrices for analysing protein-protein interactions and resultant complexes formed on membrane surfaces.

Amphipathic Helices of Cellular Proteins Can Replace the Helix in M2 of Influenza A Virus with Only Small Effects on Virus Replication.

M2 of influenza virus functions as a proton channel during virus entry. In addition, an amphipathic helix in its cytoplasmic tail plays a role during budding. It targets M2 to the assembly site where it inserts into the inner membrane leaflet to induce curvature that causes virus scission. Since vesicularization of membranes can be performed by a variety of amphiphilic peptides, we used reverse genetics to investigate whether the peptides can substitute for M2's helix. Virus could not be generated if M2's helix was deleted or replaced by a peptide predicted not to form an amphiphilic helix. In contrast, viruses could be rescued if the M2 helix was exchanged by helices known to induce membrane curvature. Infectious virus titers were marginally reduced if M2 contains the helix of the amphipathic lipid packing sensor from the Epsin N-terminal homology domain or the nonnatural membrane inducer RW16. Transmission electron microscopy of infected cells did not reveal unequivocal evidence that virus budding or membrane scission was disturbed in any of the mutants. Instead, individual virus mutants exhibit other defects in M2, such as reduced surface expression, incorporation into virus particles, and ion channel activity. The protein composition and specific infectivity were also altered for mutant virions. We conclude that the presence of an amphiphilic helix in M2 is essential for virus replication but that other helices can replace its basic (curvature-inducing) function.IMPORTANCE Influenza virus is unique among enveloped viruses since it does not rely on the cellular ESCRT machinery for budding. Instead, viruses encode their own scission machine, the M2 protein. M2 is targeted to the edge of the viral assembly site, where it inserts an amphiphilic helix into the membrane to induce curvature. Cellular proteins utilize a similar mechanism for scission of vesicles. We show that the helix of M2 can be replaced by helices from cellular proteins with only small effects on virus replication. No evidence was obtained that budding is disturbed, but individual mutants exhibit other defects in M2 that explain the reduced virus titers. In contrast, no virus could be generated if the helix of M2 is deleted or replaced by irrelevant sequences. These experiments support the concept that M2 requires an amphiphilic helix to induce membrane curvature, but its biophysical properties are more important than the amino acid sequence.

Myeloid-Specific Deletion of Epsins 1 and 2 Reduces Atherosclerosis by Preventing LRP-1 Downregulation.

RATIONALE: Atherosclerosis is, in part, caused by immune and inflammatory cell infiltration into the vascular wall, leading to enhanced inflammation and lipid accumulation in the aortic endothelium. Understanding the molecular mechanisms underlying this disease is critical for the development of new therapies. Our recent studies demonstrate that epsins, a family of ubiquitin-binding endocytic adaptors, are critical regulators of atherogenicity. Given the fundamental contribution lesion macrophages make to fuel atherosclerosis, whether and how myeloid-specific epsins promote atherogenesis is an open and significant question. OBJECTIVE: We will determine the role of myeloid-specific epsins in regulating lesion macrophage function during atherosclerosis. METHODS AND RESULTS: We engineered myeloid cell-specific epsins double knockout mice (LysM-DKO) on an ApoE-/- background. On Western diet, these mice exhibited marked decrease in atherosclerotic lesion formation, diminished immune and inflammatory cell content in aortas, and reduced necrotic core content but increased smooth muscle cell content in aortic root sections. Epsins deficiency hindered foam cell formation and suppressed proinflammatory macrophage phenotype but increased efferocytosis and anti-inflammatory macrophage phenotype in primary macrophages. Mechanistically, we show that epsin loss specifically increased total and surface levels of LRP-1 (LDLR [low-density lipoprotein receptor]-related protein 1), an efferocytosis receptor with antiatherosclerotic properties. We further show that epsin and LRP-1 interact via epsin's ubiquitin-interacting motif domain. ox-LDL (oxidized LDL) treatment increased LRP-1 ubiquitination, subsequent binding to epsin, and its internalization from the cell surface, suggesting that epsins promote the ubiquitin-dependent internalization and downregulation of LRP-1. Crossing ApoE-/-/LysM-DKO mice onto an LRP-1 heterozygous background restored, in part, atherosclerosis, suggesting that epsin-mediated LRP-1 downregulation in macrophages plays a pivotal role in propelling atherogenesis. CONCLUSIONS: Myeloid epsins promote atherogenesis by facilitating proinflammatory macrophage recruitment and inhibiting efferocytosis in part by downregulating LRP-1, implicating that targeting epsins in macrophages may serve as a novel therapeutic strategy to treat atherosclerosis.

Enhancing the activity of membrane remodeling epsin-peptide by trimerization.

Modulating the structural dynamics of biomembranes by inducing bilayer curvature and lipid packing defects has been highlighted as a practical tool to modify membrane-dependent cellular processes. Previously, we have reported on an amphipathic helical peptide derived from the N-terminal segment (residues 1-18, EpN18) of epsin-1, which can promote membrane remodeling including lipid packing defects in cell membranes. However, a high concentration is required to exhibit a pronounced effect. In this study, we demonstrate a significant increase in the membrane-remodeling effect of EpN18 by constructing a branched EpN18 homotrimer. Both monomer and trimer could enhance cell internalization of octaarginine (R8), a cell-penetrating peptide. The EpN18 trimer, however, promoted the uptake of R8 at an 80-fold lower concentration than the monomer. Analysis of the generalized polarization of a polarity-sensitive dye (di-4-ANEPPDHQ) revealed a higher efficacy of trimeric EpN18 in loosening the lipid packing in the cell membrane. Circular dichroism measurements in the presence of lipid vesicles showed that the EpN18 trimer has a higher α-helix content compared with the monomer. The stronger ability of the EpN18 trimer to impede negative bilayer curvature is also corroborated by solid-state 31P NMR spectroscopy. Hence, trimerizing peptides can be considered a promising approach for an exponential enhancement of their membrane-remodeling performance.

Further insights from structural mass spectrometry into endocytosis adaptor protein assemblies.

As a fundament in many biologically relevant processes, endocytosis in its different guises has been arousing interest for decades and still does so. This is true for the actual transport and its initiation alike. In clathrin-mediated endocytosis, a comparatively well understood endocytic pathway, a set of adaptor proteins bind specific lipids in the plasma membrane, subsequently assemble and thus form a crucial bridge from clathrin to actin for the ongoing process. These adaptor proteins are highly interesting themselves and the subject of this manuscript. Using many of the instruments that are available now in the mass spectrometry toolbox, we added some facets to the picture of how these minimal assemblies may look, how they form, and what influences the structure. Especially, lipids in the adaptor protein complexes result in reduced charging of a normal sized complex due to their specific binding position. The results further support our structural model of a double ring structure with interfacial lipids.

Selective Targeting of a Novel Epsin-VEGFR2 Interaction Promotes VEGF-Mediated Angiogenesis.

RATIONALE: We previously reported that vascular endothelial growth factor (VEGF)-induced binding of VEGF receptor 2 (VEGFR2) to epsins 1 and 2 triggers VEGFR2 degradation and attenuates VEGF signaling. The epsin ubiquitin interacting motif (UIM) was shown to be required for the interaction with VEGFR2. However, the molecular determinants that govern how epsin specifically interacts with and regulates VEGFR2 were unknown. OBJECTIVE: The goals for the present study were as follows: (1) to identify critical molecular determinants that drive the specificity of the epsin and VEGFR2 interaction and (2) to ascertain whether such determinants were critical for physiological angiogenesis in vivo. METHODS AND RESULTS: Structural modeling uncovered 2 novel binding surfaces within VEGFR2 that mediate specific interactions with epsin UIM. Three glutamic acid residues in epsin UIM were found to interact with residues in VEGFR2. Furthermore, we found that the VEGF-induced VEGFR2-epsin interaction promoted casitas B-lineage lymphoma-mediated ubiquitination of epsin, and uncovered a previously unappreciated ubiquitin-binding surface within VEGFR2. Mutational analysis revealed that the VEGFR2-epsin interaction is supported by VEGFR2 interacting specifically with the UIM and with ubiquitinated epsin. An epsin UIM peptide, but not a mutant UIM peptide, potentiated endothelial cell proliferation, migration and angiogenic properties in vitro, increased postnatal retinal angiogenesis, and enhanced VEGF-induced physiological angiogenesis and wound healing. CONCLUSIONS: Distinct residues in the epsin UIM and VEGFR2 mediate specific interactions between epsin and VEGFR2, in addition to UIM recognition of ubiquitin moieties on VEGFR2. These novel interactions are critical for pathophysiological angiogenesis, suggesting that these sites could be selectively targeted by therapeutics to modulate angiogenesis.

Therapeutic efficacy of a synthetic epsin mimetic peptide in glioma tumor model: uncovering multiple mechanisms beyond the VEGF-associated tumor angiogenesis.

Binding of epsin ubiquitin-interacting motif (UIM) with ubiquitylated VEGFR2 is a critical mechanism for epsin-dependent VEGFR2 endocytosis and physiological angiogenesis. Deletion of epsins in vessel endothelium produces uncontrolled tumor angiogenesis and retards tumor growth in animal models. The aim of this study is to test the therapeutic efficacy and targeting specificity of a chemically-synthesized peptide, UPI, which compete for epsin binding sites in VEGFR2 and potentially inhibits Epsin-VEGFR2 interaction in vivo, in an attempt to reproduce an epsin-deficient phenotype in tumor angiogenesis. Our data show that UPI treatment significantly inhibits and shrinks tumor growth in GL261 glioma tumor model. UPI peptide specifically targets VEGFR2 signaling pathway revealed by genetic and biochemical approaches. Furthermore, we demonstrated that UPI peptide treatment caused serious thrombosis in tumor vessels and damages tumor cells after a long-term UPI peptide administration. Besides, we revealed that UPI peptides were unexpectedly targeted cancer cells and induced apoptosis. We conclude that UPI peptide is a potent inhibitor to glioma tumor growth through specific targeting of VEGFR2 signaling in the tumor vasculature and cancer cells, which may offer a potentially novel treatment for cancer patients who are resistant to current anti-VEGF therapies.

Effect of clathrin light chains on the stiffness of clathrin lattices and membrane budding.

Clathrin-dependent transport processes require the polymerization of clathrin triskelia into polygonal scaffolds. Together with adapter proteins, clathrin collects cargo and induces membrane bud formation. It is not known to what extent clathrin light chains affect the structural and functional properties of clathrin lattices and the ability of clathrin to deform membranes. To address these issues, we have developed a novel procedure for analyzing clathrin lattice formation on rigid surfaces. We found that lattices can form on adaptor-coated convex-, planar- and even shallow concave surfaces, but the rate of formation and resistance to thermal dissociation of the lattice are greatly enhanced on convex surfaces. Atomic force microscopy on planar clathrin lattices demonstrates that the stiffness of the clathrin lattice is strictly dependent on light chains. The reduced stiffness of the lattice also compromised the ability of clathrin to generate coated buds on the surface of rigid liposomal membranes.

Interaction with epsin 1 regulates the constitutive clathrin-dependent internalization of ErbB3.

BACKGROUND: In contrast to other members of the EGF receptor family, ErbB3 is constitutively internalized in a clathrin-dependent manner. Previous studies have shown that ErbB3 does not interact with the coated pit localized adaptor complex 2 (AP-2), and that ErbB3 lacks two AP-2 interacting internalization signals identified in the EGF receptor. Several other clathrin-associated sorting proteins which may recruit cargo into coated pits have, however, been identified, and the study was performed to identify adaptors needed for constitutive internalization of ErbB3. METHODS: A high-throughput siRNA screen was used to identify adaptor proteins needed for internalization of ErbB3. Upon knock-down of candidate proteins internalization of ErbB3 was identified using an antibody-based internalization assay combined with automatic fluorescence microscopy. RESULTS: Among 29 candidates only knock-down of epsin 1 turned out to inhibit ErbB3. Epsin 1 has ubiquitin interacting motifs (UIMs) and we show that ErbB3 interacts with an epsin 1 deletion mutant containing these UIMs. In support of an ErbB3-epsin 1 UIM dependent interaction, we show that ErbB3 is constitutively ubiquitinated, but that both ubiquitination and the ErbB3-epsin 1 interaction increase upon ligand binding. CONCLUSION: Altogether the results are consistent with a model whereby both constitutive and ligand-induced internalization of ErbB3 are regulated through interaction with epsin 1. GENERAL SIGNIFICANCE: Internalization is an important regulator of growth factor receptor mediated signaling and the current study identify mechanisms regulating plasma membrane turnover of ErbB3.