Endophilin A2 interactions detected by BioID2
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens (human)
SUBMITTER: Dessi Malinova
PROVIDER: S-BSST665 | biostudies-other |
REPOSITORIES: biostudies-other
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Project description:Endothelial cell migration, proliferation and survival are triggered by VEGF-A activation of VEGFR2. However, how these cell behaviors are regulated individually is still unknown. Here we identify Endophilin-A2 (ENDOA2), a BAR-domain protein that orchestrates CLATHRIN-independent internalization, as a critical mediator of endothelial cell migration and sprouting angiogenesis. We show that EndoA2 knockout mice exhibit postnatal angiogenesis defects and impaired front-rear polarization of sprouting tip cells. ENDOA2 deficiency reduces VEGFR2 internalization and inhibits downstream activation of the signaling effector PAK but not ERK, thereby affecting front-rear polarity and migration but not proliferation or survival. Mechanistically, VEGFR2 is directed towards ENDOA2-mediated endocytosis by the SLIT2-ROBO pathway via SLIT-ROBO-GAP1 bridging of ENDOA2 and ROBO1. Blocking ENDOA2-mediated endothelial cell migration attenuates pathological angiogenesis in oxygen-induced retinopathy models. This work identifies a specific endocytic pathway controlling a subset of VEGFR2 mediated responses that could be targeted to prevent excessive sprouting angiogenesis in pathological conditions.
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Project description:The crystal structure of the SH3 domain of rat endophilin A2 has been determined by the multiwavelength anomalous dispersion method and refined at a resolution of 1.70 A to R and R(free) values of 0.196 and 0.217, respectively. The structure adheres to the canonical SH3-domain fold and is highly similar to those of the corresponding domains of endophilins A1 and A3. An intermolecular packing interaction between two molecules in the lattice exploits features that are commonly observed in SH3-domain ligand recognition, including the insertion of a proline side chain into the ligand-binding groove of the protein and the recognition of a basic residue by a cluster of acidic side chains on the RT loop.
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Project description:Endophilin, which participates in membrane vesiculation during receptor-mediated endocytosis, is a ?40 kDa SH3 domain-containing protein that binds to the proline/arginine-rich domain of dynamin, a ?100 kDa GTPase that is essential for endocytic membrane scission. It has been suggested that endophilin is monomeric in the cytoplasm and dimerizes only after it binds to membranes (or perhaps to dimers or tetramers of dynamin). To clarify this issue, we studied the oligomeric state of endophilin both in vitro using analytical ultracentrifugation and fluorescence anisotropy, and in living cells using two-photon fluorescence fluctuation spectroscopy. We analyzed the fluctuation data using the Q-analysis method, which allowed us to determine the intrinsic brightness of the labeled protein complexes and hence its aggregation state in the cytoplasmic regions of the cell. Although a relatively high K(d) (?5-15 ?M) was observed in vitro, the cell measurements indicate that endophilin is dimeric in the cytoplasm, even at submicromolar concentrations. We also demonstrate that endophilin significantly enhances the assembly of dynamin, and that this enhancement is proportional to the fraction of dimeric endophilin that is present. Moreover, there is correlation between the concentrations of endophilin that promote dynamin self-assembly and those that stimulate dynamin GTPase activity. These findings support the view that endophilin-dynamin interactions play an important role in endocytosis.
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Project description:Endocytosis of caveolae has previously been implicated in the repair of plasma membrane wounds. Here, we show that caveolin-1-deficient fibroblasts lacking caveolae upregulate a tubular endocytic pathway and have a reduced capacity to reseal after permeabilization with pore-forming toxins compared with wild-type cells. Silencing endophilin-A2 expression inhibited fission of endocytic tubules and further reduced plasma membrane repair in cells lacking caveolin-1, supporting a role for tubular endocytosis as an alternative pathway for the removal of membrane lesions. Endophilin-A2 was visualized in association with cholera toxin B-containing endosomes and was recruited to recently formed intracellular vacuoles containing Trypanosoma cruzi, a parasite that utilizes the plasma membrane wounding repair pathway to invade host cells. Endophilin-A2 deficiency inhibited T. cruzi invasion, and fibroblasts deficient in both caveolin-1 and endophilin-A2 did not survive prolonged exposure to the parasites. These findings reveal a novel crosstalk between caveolin-1 and endophilin-A2 in the regulation of clathrin-independent endocytosis and plasma membrane repair, a process that is subverted by T. cruzi parasites for cell invasion.
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Project description:BackgroundTactile and mechanical pain are crucial to our interaction with the environment, yet the underpinning molecular mechanism is still elusive. Endophilin A2 (EndoA2) is an evolutionarily conserved protein that is documented in the endocytosis pathway. However, the role of EndoA2 in the regulation of mechanical sensitivity and its underlying mechanisms are currently unclear.MethodsMale and female C57BL/6 mice (8-12 weeks) and male cynomolgus monkeys (7-10 years old) were used in our experiments. Nerve injury-, inflammatory-, and chemotherapy-induced pathological pain models were established for this study. Behavioral tests of touch, mechanical pain, heat pain, and cold pain were performed in mice and nonhuman primates. Western blotting, immunostaining, co-immunoprecipitation, proximity ligation and patch-clamp recordings were performed to gain insight into the mechanisms.ResultsThe results showed that EndoA2 was primarily distributed in neurofilament-200-positive (NF200+) medium-to-large diameter dorsal root ganglion (DRG) neurons of mice and humans. Loss of EndoA2 in mouse NF200+ DRG neurons selectively impaired the tactile and mechanical allodynia. Furthermore, EndoA2 interacted with the mechanically sensitive ion channel Piezo2 and promoted the membrane trafficking of Piezo2 in DRG neurons. Moreover, as an adaptor protein, EndoA2 also bound to kinesin family member 5B (KIF5B), which was involved in the EndoA2-mediated membrane trafficking process of Piezo2. Loss of EndoA2 in mouse DRG neurons damaged Piezo2-mediated rapidly adapting mechanically activated currents, and re-expression of EndoA2 rescued the MA currents. In addition, interference with EndoA2 also suppressed touch sensitivity and mechanical hypersensitivity in nonhuman primates.ConclusionsOur data reveal that the KIF5B/EndoA2/Piezo2 complex is essential for Piezo2 trafficking and for sustaining transmission of touch and mechanical hypersensitivity signals. EndoA2 regulates touch and mechanical allodynia via kinesin-mediated Piezo2 trafficking in sensory neurons. Our findings identify a potential new target for the treatment of mechanical pain.
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Project description:Decreased autophagy has been reported to contribute to the progression of cardiac hypertrophy. Our previous research has demonstrated that endophilin A2 (EndoA2) attenuates H2O2-induced cardiomyocyte apoptosis by strengthening autophagy. However, the role of EndoA2 in the regulation of autophagy in cardiac hypertrophy is unknown. In this study, we tested the hypothesis that EndoA2 suppresses cardiac hypertrophy induced by isoproterenol (ISO) by activating autophagy. In vivo, we established a cardiac hypertrophy model by subcutaneous injection of ISO and used intramyocardial delivery of adenovirus vector harboring EndoA2 cDNA (Ad-EndoA2) to overexpress EndoA2. The cardiac hypertrophic response and autophagy level were measured. EndoA2 overexpression suppressed pathological cardiac hypertrophy and enhanced autophagy in rat hearts. In addition, the effects of EndoA2 on cardiac hypertrophy and autophagy were observed in cultured neonatal rat cardiomyocytes (NRCMs) with gain- and loss-of-function approaches to regulate EndoA2 expression. The results were consistent with those of the in vivo study. Furthermore, the involvement of EndoA2-mediated autophagy in the attenuation of ISO-induced cardiac hypertrophy was explored by pharmaceutical inhibition of autophagy. Pretreatment with 3-methyladenine (3-MA) clearly diminished the anti-hypertrophic effects of EndoA2 in ISO-treated NRCMs. The results presented here provide the first evidence that EndoA2 is involved in ISO-induced cardiac hypertrophy. The anti-hypertrophic effects of EndoA2 can be partially attributed to its regulation of autophagy.
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Project description:The introduction of the CTLA-4 recombinant fusion protein has demonstrated therapeutic effects by selectively modulating T-cell activation in rheumatoid arthritis. Here we show, using a forward genetic approach, that a mutation in the SH3gl1 gene encoding the endocytic protein Endophilin A2 is associated with the development of arthritis in rodents. Defective expression of SH3gl1 affects T cell effector functions and alters the activation threshold of autoreactive T cells, thereby leading to complete protection from chronic autoimmune inflammatory disease in both mice and rats. We further show that SH3GL1 regulates human T cell signaling and T cell receptor internalization, and its expression is upregulated in rheumatoid arthritis patients. Collectively our data identify SH3GL1 as a key regulator of T cell activation, and as a potential target for treatment of autoimmune diseases.
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Project description:Endophilin is a key protein involved in clathrin-mediated endocytosis. Previous computational and experimental work suggested that the N-terminal helix is embedded into the membrane to induce curvature; however, the role of the SH3 domain remains controversial. To address this issue, we performed computer simulations of the endophilin dimer in solution to understand the interaction between the N-BAR and SH3 domains and its effect on biological function. We predict that the helix binds to the SH3 domain through hydrophobic and salt-bridge interactions. This protects the hydrophobic residues on both domains and keeps the SH3 domain near the end of the N-BAR domain, in agreement with previous experimental results. The complex has a binding strength similar to a few hydrogen bonds (13.0 ± 0.6 kcal/mol), and the SH3 domain stabilizes the structure of the N-terminal helix in solution. Electrostatic calculations show a large region of strongly positive electrostatic potential near the N-terminal that can orient the helix toward the membrane and likely embed the helix into the membrane surface. This predicted mechanism suggests that endophilin can select for both curvature and electrostatic potential when interacting with membranes, highlighting the importance of the SH3 domain in regulating the function of endophilin.
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Project description:TMEM16A Ca2+-activated chloride channel (CaCC) plays an essential role in vascular homeostasis. In this study we investigated the molecular mechanisms underlying downregulation of TMEM16A CaCC activity during hypertension. In cultured basilar artery smooth muscle cells (BASMCs) isolated from 2k2c renohypertesive rats, treatment with angiotensin II (0.125-1 μM) dose-dependently increased endophilin A2 levels and decreased TMEM16A expression. Similar phenomenon was observed in basilar artery isolated from 2k2c rats. We then used whole-cell recording to examine whether endophilin A2 could regulate TMEM16A CaCC activity in BASMCs and found that knockdown of endophilin A2 significantly enhanced CaCC activity, whereas overexpression of endophilin A2 produced the opposite effect. Overexpression of endophilin A2 did not affect the TMEM16A mRNA level, but markedly decreased TMEM16A protein level in BASMCs by inducing ubiquitination and autophagy of TMEM16A. Ubiquitin-binding receptor p62 (SQSTM1) could bind to ubiquitinated TMEM16A and resulted in a process of TMEM16A proteolysis in autophagosome/lysosome. These data provide new insights into the regulation of TMEM16A CaCC activity by endophilin A2 in BASMCs, which partly explains the mechanism of angiotensin-II-induced TMEM16A inhibition during hypertension-induced vascular remodeling.
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Project description:Endophilin A1 is a homodimeric membrane-binding endocytic accessory protein with a high dimerization affinity. Its function has been hypothesized to involve autoinhibition. However, the autoinhibition mechanism, as well as the physicochemical basis for the high dimerization affinity of endophilin in solution, have remained unclear. In this contribution, we use a Förster resonance energy transfer (FRET) method to investigate the homodimerization mechanism and intradimer molecular interactions in endophilin. For the endophilin N-BAR domain (which lacks the SH3 domain including a linker region of the full length protein), we observe a large temperature dependence of the dimerization affinity and dimer dissociation kinetics, implying large dimerization enthalpy and dissociation activation enthalpy, respectively. Our evaluation of the protein concentration dependence of dimer dissociation kinetics implies that endophilin reversibly forms monomers via a dissociation/reassociation mechanism. Furthermore, we use a kinetic method that allows us to compare the dissociation kinetics of full-length endophilin to that of truncated mutants. We find that mutants that lack either H0 helix or SH3 domain show significantly faster dissociation kinetics relative to full-length endophilin. This observation supports the presence of an intradimer, intermonomer cross-interaction between H0 helix and SH3 domain from different subunits within a homodimer. Because the H0 helix is known to play a significant role in endophilin's membrane interactions, our measurements support a syngergistic model where these interactions are inhibited in the absence of SH3 domain binding ligands such as dynamin's prolin rich domains, and where the binding of these ligands may be suppressed for non-membrane-bound endophilin.
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