Eps15 Homology Domain-containing Proteins Research Articles

Identification of drug-like molecules targeting the ATPase activity of dynamin-like EHD4.

Eps15 (epidermal growth factor receptor pathway substrate 15) homology domain-containing proteins (EHDs) comprise a family of eukaryotic dynamin-related ATPases that participate in various endocytic membrane trafficking pathways. Dysregulation of EHDs function has been implicated in various diseases, including cancer. The lack of small molecule inhibitors which acutely target individual EHD members has hampered progress in dissecting their detailed cellular membrane trafficking pathways and their function during disease. Here, we established a Malachite green-based assay compatible with high throughput screening to monitor the liposome-stimulated ATPase of EHD4. In this way, we identified a drug-like molecule that inhibited EHD4's liposome-stimulated ATPase activity. Structure activity relationship (SAR) studies indicated sites of preferred substitutions for more potent inhibitor synthesis. Moreover, the assay optimization in this work can be applied to other dynamin family members showing a weak and liposome-dependent nucleotide hydrolysis activity.

A missense mutation in Ehd1 associated with defective spermatogenesis and male infertility.

Normal function of the C-terminal Eps15 homology domain-containing protein 1 (EHD1) has previously been associated with endocytic vesicle trafficking, shaping of intracellular membranes, and ciliogenesis. We recently identified an autosomal recessive missense mutation c.1192C>T (p.R398W) of EHD1 in patients who had low molecular weight proteinuria (0.7-2.1g/d) and high-frequency hearing loss. It was already known from Ehd1 knockout mice that inactivation of Ehd1 can lead to male infertility. However, the exact role of the EHD1 protein and its p.R398W mutant during spermatogenesis remained still unclear. Here, we report the testicular phenotype of a knockin mouse model carrying the p.R398W mutation in the EHD1 protein. Male homozygous knockin mice were infertile, whereas the mutation had no effect on female fertility. Testes and epididymes were significantly reduced in size and weight. The testicular epithelium appeared profoundly damaged and had a disorganized architecture. The composition of developing cell types was altered. Malformed acrosomes covered underdeveloped and misshaped sperm heads. In the sperm tail, midpieces were largely missing indicating disturbed assembly of the sperm tail. Defective structures, i.e., nuclei, acrosomes, and sperm tail midpieces, were observed in large vacuoles scattered throughout the epithelium. Interestingly, cilia formation itself did not appear to be affected, as the axoneme and other parts of the sperm tails except the midpieces appeared to be intact. In wildtype mice, EHD1 co-localized with acrosomal granules on round spermatids, suggesting a role of the EHD1 protein during acrosomal development. Wildtype EHD1 also co-localized with the VPS35 component of the retromer complex, whereas the p.R398W mutant did not. The testicular pathologies appeared very early during the first spermatogenic wave in young mice (starting at 14 dpp) and tubular destruction worsened with age. Taken together, EHD1 plays an important and probably multifaceted role in spermatogenesis in mice. Therefore, EHD1 may also be a hitherto underestimated infertility gene in humans.

Identification of genomic regions associated with reproductive longevity in the Rubia Gallega beef cattle breed using a censored threshold model

In beef cattle, the economic viability of farms is heavily influenced by the cow's ability to survive subsequent pregnancies. To understand the genetic basis of reproductive longevity in Rubia Gallega beef cattle breed, a ssGWAS was performed by back-solving the output of ssGBLUP under a censored threshold model. It considers the number of parities each cow reaches during its productive life as a phenotypic trait. The results of the study showed that the main source of variation of reproductive longevity is the herd. However, the posterior mean of the heritability of reproductive longevity was 0.173, indicating the potential for an appropriate genetic response to selection. Furthermore, it is shown that four genomic regions in chromosomes 2, 11 and 29 explain a large proportion of the additive genetic variance. The most important signal was detected on chromosome 2 in the vicinity of the MSTN (myostatin) gene that is associated with double muscling, and that it is segregating in the Rubia Gallega population. Some other interesting genes located within these regions encoded for several PAGs (Pregnancy-associated glycoprotein), LHCGR (luteinizing hormone/choriogonadotropin receptor), FSHR (follicle stimulating hormone receptor), PROKR1 (prokineticin receptor 1) and EHD3 (Eps15 homology domain-containing protein 3). This confirms the relevance of the reproductive performance in the reproductive longevity of cows. These findings provide valuable insights for the Rubia Gallega breeding program, as they can be used to define future selection strategies to improve reproductive longevity of the breed.

Raft platforms highly enriched in cholesterol: major scaffolds for IL-6 signalling assembly with implications in inflammation and cancer.

Interleukin-6 (IL-6) is a pleiotropic cytokine with complex and major roles in inflammation, which could be linked to the different ways IL-6 signals at the plasma membrane. In this issue of FEBS Journal, Woo and co-workers present evidence for the involvement of Eps15 homology domain-containing protein 1 (EHD1)-mediated lipid raft platforms, highly enriched in cholesterol, in the IL-6 signalling pathway. Because of the strong connection between IL-6, inflammation and cancer, one implication of this report is that agents or approaches targeting cholesterol-rich raft platforms may assist the development of novel strategies to treat inflammatory and malignant diseases. Comment on: https://doi.org/10.1111/febs.16458.

Abstract 87: EHD1 is required for IGF1R-mediated oncogenic signaling in Ewing Sarcoma

Abstract Background: Ewing Sarcoma (EWS) is the second most common malignant bone tumor of children and adolescents. Patients with metastatic or recurrent disease have very poor outcomes. The receptor tyrosine kinase (RTK) insulin-like growth factor 1 receptor (IGF1R) has been implicated in EWS tumorigenesis and development of metastatic disease, with anti-IGF1R antibodies and kinase inhibitors in clinical studies. However, with only ~10% of patients achieving objective responses, delineation of novel pathways that facilitate IGF1R-driven oncogenesis in EWS could provide avenues for more effective therapy. Intracellular trafficking plays a central role in regulating RTK expression and signaling, and this pathway is frequently deregulated in cancer cells. The EPS15 homology domain-containing (EHD) proteins regulate intracellular traffic of cell surface receptors, including RTKs. We observed high frequency (67%) of EHD1 overexpression in 266 primary EWS patient tumor tissues and Kaplan-Meier survival analysis of publicly available mRNA expression data showed that high EHD1 expression was associated with shorter patient survival. Objective: This study aims to comprehend the underlying role of EHD1 in EWS oncogenesis. Study design: Three EHD1-expressing EWS cell lines, TC71, SKES1, and A673, were engineered with doxycycline-inducible EHD1 shRNAs to assess the impact of EHD1 knockdown (KD) on in vitro oncogenic properties. Stable EHD1-Crispr-KO and mouse EHD1-rescue TC71 and A673 cell line models were developed and characterized. Further, non-targeting control or EHD1-KD TC71 cells engineered with RFP-luciferase reporter were implanted in the tibia of nude mice in an orthotopic xenograft model and tumor growth was monitored by IVIS imaging. Results: EHD1-KD led to a significant impairment of EWS cell proliferation, migration, invasion, soft-agar colony formation, and tumor-sphere formation in vitro and reduced tumor growth in nude mice. Using a phospho-RTK profiling antibody array, we found reduced phospho-IGF1R levels upon EHD1-KD, identifying IGF1R as a potential target of regulation by EHD1. Western blotting showed a reduction in total IGF1R levels, and flow cytometric and immunofluorescence analyses revealed a pronounced decrease in the cell surface IGF1R levels upon EHD1 KD/KO in EWS cell lines. EHD1-KO EWS cell lines also exhibited a defect in IGF1-dependent migration and proliferation, and the phenotypes were restored by mouse EHD1 rescue. IGF1R and EHD1 were found to colocalize intracellularly and to co-immunoprecipitate after IGF1 stimulation. Finally, EHD1 depletion was found to impair the IGF-1R-mediated activation of downstream AKT and MAPK pathways. Conclusion: Our studies indicate a novel requirement of the intracellular traffic regulator EHD1 in sustaining IGF1R mediated oncogenesis in EWS. Future studies will aim to evaluate dual targeting of EHD1 and IGF1R in EWS. Citation Format: Sukanya Chakraborty, Bhopal C. Mohapatra, Sameer Mirza, Aaqib M. Bhat, Matthew D. Storck, Isidro Machado, José Antonio López, Antonio Llomart Bosch, Donald W. Coulter, Gargi Ghosal, Vimla Band, Hamid Band. EHD1 is required for IGF1R-mediated oncogenic signaling in Ewing Sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 87.

Interleukin-6 signaling requires EHD1-mediated alteration of membrane rafts.

Interleukin-6 (IL-6) is involved in many inflammatory diseases. IL-6 binds to membrane-bound IL-6 receptor α (IL-6Rα) (classic signaling) or soluble IL-6Rα (trans-signaling); this complex then associates with the signal-transducing membrane protein gp130. IL-6Rα and gp130 float on membrane (i.e., lipid) rafts; however, how membrane rafts regulate IL-6 signaling remains unclear. Here, we demonstrate that both IL-6 classic signaling and trans-signaling depend on membrane cholesterol, an essential raft component. Super-resolution fluorescence imaging using perfringolysin O D4 fragments that selectively bind to high cholesterol concentrations revealed that IL-6 and hyper-IL-6, a fusion protein of IL-6 and soluble IL-6Rα, induce the alteration of membrane rafts. IL-6 and hyper-IL-6 induced D4-positive raft (D4 raft) formation without affecting cholera toxin subunit B (CTB)-positive rafts (CTB rafts). Receptor clustering of IL-6Rα and gp130 and STAT3 phosphorylation occurred in D4 rafts. These results indicate that D4 rafts serve as platforms for the assembly of functional IL-6 receptor complexes. We found that Eps15 homology domain-containing protein 1 (EHD1) mediates the formation of functional IL-6 receptor complexes through D4 rafts. Overall, we uncover a novel regulatory mechanism of the EHD1-mediated alteration of membrane raft in IL-6 signaling.

Circular RNA Eps15-homology domain-containing protein 2 induce resistance of renal cell carcinoma to sunitinib via microRNA-4731-5p/ABCF2 axis

ABSTRACT Circular RNAs (circRNAs) are linked with the occurrence and progression of renal cell carcinoma (RCC). However, circRNAs’ mechanism in developing resistance to RCC has not been clarified. This research assessed the role and mechanism of circular RNA circ Eps15-homology domain-containing protein 2 (EHD2) in the resistance of sunitinib (SU) to RCC. ACHN, 786-O, 769P, and HEK-293 T cells and RCC tissue samples were used for the investigations. The circEHD2 expression in RCC cells and tissues was determined through RT-qPCR. Association of circEHD2 with RCC histological grade of RCC was done through Chi-square. MiR-4731-5p, ABCF2, and circEHD2 were transfected into RCC cell lines. A dual-luciferase reporter assay was used to determine the interaction between miR-4731-5p, circEHD2, and ABCF2. MTT assay was used to analyze cell viability, while apoptosis was studied using flow cytometry. Colony-formation and transwell experiments were used to assess migration and invasion. The ATP Binding Cassette Subfamily F Member 2 (ABCF2) expression was analyzed through western blot. The results showed increased circEHD2 in SU-resistant RCC tissues and cell lines and implicated in RCC histological grade and SU resistance. Knock-down of circEHD2 down-regulated the resistance of RCC to SU in vitro and vivo; circEHD2 bound to miR-4731-5p to mediate ABCF2 in RCC; ABCF2 rescued the inhibitory effect of circEHD2 knock-down on SU resistance of RCC. In conclusion, circEHD2 enhances RCC resistance to SU via acting as a miR-4731-5p sponge to mediate ABCF2. MiR-4731-5p can target circEHD2 and ABCF2, thus providing a novel and effective therapeutic against renal cell carcinoma.

EHD3 positively regulated by NR5A1 participates in testosterone synthesis via endocytosis

AimsIncreasing evidence has shown that hormone secretion is regulated by endocytosis. Eps15 homology domain-containing protein 3 (EHD3) is an endocytic-trafficking regulatory protein, but whether EHD3 is associated with testosterone secretion is not clear. This work aims to explore the role of EHD3 in testosterone synthesis. Main methodsTestosterone concentration was determined by ELISA. The effects of EHD3 on endocytosis were assessed by exosomes tracing assay and Immunofluorescence. Targeting relationship between EHD3 and NR5A1 was verified by chromatin immunoprecipitation (ChIP) and dual luciferase reporter gene assay in Leydig cells. For in vivo assessments, conditional NR5A1 knockout mouse model was established with CRISPR/Cas9 gene targeting technology. Key findingsEHD3 overexpression significantly increased the concentration of testosterone. EHD3 knockdown markedly decreased testosterone synthesis by reducing endocytosis. The activity of the EHD3 promoter was positively regulated by NR5A1, which occupied the conserved sequence “AGGTCA” in the EHD3 promoter. Furthermore, mice with a Leydig cell-specific conditional NR5A1 knockout displayed the blunted levels of EHD3 and clathrin (a key factor for endocytosis), and serum testosterone concentration compared with NR5A1f/f mice. SignificanceThis study suggests a potential molecular mechanism of testosterone synthesis to fully understand male reproductive health.

Eps15 Homology Domain-Containing Protein 3 Hypermethylation as a Prognostic and Predictive Marker for Colorectal Cancer

Colorectal cancer (CRC) arises from chromosomal instability, resulting from aberrant hypermethylation in tumor suppressor genes. This study identified hypermethylated genes in CRC and investigated how they affect clinical outcomes. Methylation levels of specific genes were analyzed from The Cancer Genome Atlas dataset and 20 breast cancer, 16 esophageal cancer, 33 lung cancer, 15 uterine cancer, 504 CRC, and 9 colon polyp tissues and 102 CRC plasma samples from a Taiwanese cohort. In the Asian cohort, Eps15 homology domain-containing protein 3 (EHD3) had twofold higher methylation in 44.4% of patients with colonic polyps, 37.3% of plasma from CRC patients, and 72.6% of CRC tissues, which was connected to vascular invasion and high microsatellite instability. Furthermore, EHD3 hypermethylation was detected in other gastrointestinal cancers. In the Asian CRC cohort, low EHD3 mRNA expression was found in 45.1% of patients and was connected to lymph node metastasis. Multivariate Cox proportional-hazards survival analysis revealed that hypermethylation in women and low mRNA expression were associated with overall survival. In the Western CRC cohort, EHD3 hypermethylation was also connected to overall survival and lower chemotherapy and antimetabolite response rates. In conclusion, EHD3 hypermethylation contributes to the development of CRC in both Asian and Western populations.

CLEM Characterization of Rab8 and Associated Membrane Trafficking Regulators at Primary Cilium Structures.

Correlative light and electron microscopy (CLEM) enables determination of high-resolution structural information for proteins of interest within their biological context through the combination of electron and fluorescence microscopies. Numerous electron microscopy (EM) studies of primary cilia have provided ultrastructural details about these antennal-like organelles that extend from the surface of the cell. The core structure of the cilium includes a microtubule-based axoneme, a basal body derived from the mother centriole, and the ciliary membrane, which is connected to the plasma membrane. The small GTPase Rab8 localizes to the ciliary membrane and is important for ciliogenesis, and Rab11 transports the Rab8 guanine nucleotide exchange factor (GEF) Rabin8 to the mother centriole to activate Rab8-dependent ciliary membrane growth. Some primary cilia have a ciliary pocket membrane (CPM) which is observed as an involution from the plasma membrane to the base of the cilia membrane. The Rab11- and Rab8-assocaited membrane trafficking regulator Eps15 Homology Domain-containing protein 1 (EHD1) and EHD3 also function in early stages of ciliogenesis; however, they localize to the CPM. These ciliary localizations of Rab8 and EHD1 can be resolved using CLEM with conventional fluorescence microscopy and transmission electron microscopy (TEM) imaging. Here, we describe in detail the protocol for this CLEM method applicable for ciliary proteins and proteins in other cellular organelles.

The mechanism of activation of the actin binding protein EHBP1 by Rab8 family members

EHBP1 is an adaptor protein that regulates vesicular trafficking by recruiting Rab8 family members and Eps15-homology domain-containing proteins 1/2 (EHD1/2). It also links endosomes to the actin cytoskeleton. However, the underlying molecular mechanism of activation of EHBP1 actin-binding activity is unclear. Here, we show that both termini of EHBP1 have membrane targeting potential. EHBP1 associates with PI(3)P, PI(5)P, and phosphatidylserine via its N-terminal C2 domain. We show that in the absence of Rab8 family members, the C-terminal bivalent Mical/EHBP Rab binding (bMERB) domain forms an intramolecular complex with its central calponin homology (CH) domain and auto-inhibits actin binding. Rab8 binding to the bMERB domain relieves this inhibition. We have analyzed the CH:bMERB auto-inhibited complex and the active bMERB:Rab8 complex biochemically and structurally. Together with structure-based mutational studies, this explains how binding of Rab8 frees the CH domain and allows it to interact with the actin cytoskeleton, leading to membrane tubulation.

Cellular functions and intrinsic attributes of the ATP-binding Eps15 homology domain-containing proteins.

Several cellular processes rely on a cohort of dedicated proteins that manage tubulation, fission, and fusion of membranes. A notably large number of them belong to the dynamin superfamily of proteins. Among them is the evolutionarily conserved group of ATP-binding Eps15-homology domain-containing proteins (EHDs). In the two decades since their discovery, EHDs have been linked to a range of cellular processes that require remodeling or maintenance of specific membrane shapes such as during endocytic recycling, caveolar biogenesis, ciliogenesis, formation of T-tubules in skeletal muscles, and membrane resealing after rupture. Recent work has shed light on their structure and the unique attributes they possess in linking ATP hydrolysis to membrane remodeling. This review summarizes some of these recent developments and reconciles intrinsic protein functions to their cellular roles.

Isolation and Characterization of a CD34+ Sub-Clone in B-Cell Lymphoma

Non-Hodgkin's lymphoma (NHL) is the most common hematological malignancy in the US. Many types remain incurable despite response to initial therapy and achievement of complete remission (CR). Advanced laboratory techniques like multicolor flow cytometry (MCF) and polymerase chain reaction (PCR) have demonstrated persistence of rare malignant cell population post therapy referred to as minimal residual disease (MRD). However, the functional and biological characteristics of this population have not been fully elucidated. Established B-lymphoma cell lines (B-NHL) and patient-derived samples (PDS) were analyzed using 8-color FCM of leukemia and lymphoma antibody panels (28 antibodies). The CD34+ sub-population was enriched using in vitro exposure to 2-chlorodeoxyadenosine (2-CdA), and a CD34-coated magnetic beads isolation procedure (Miltenyi Biotech). Genetic analysis of CD34+ and CD34-/parent cell fractions was done by karyotyping, and by chromosomal microarray (CMA) using the oligonucleotide-single nucleotide polymorphism (Oligo-SNP), whole genome Agilent 180K GGXChip+SNP (Agilent Technologies, Inc). Sensitivity to chemotherapy was assayed by short-term in vitro exposure to drugs. Clonogenicity was determined by soft agar colony formation assay, and proliferation was determined using DNA staining with propidium iodide and flow cytometry. The side population was determined using the fluorescent vital dye Hoechst 33342 and flow cytometry. Analysis of three B-NHL cell lines revealed the presence of a minute sub-clone (<1%) of monotypic B-cells that expresses CD34. This sub-population enriched several folds in response to exposure of the cells to 2-CdA. Enrichment was highest in the follicular lymphoma cell line, WSU-FSCCL reaching more than 50 folds compared with control (13.2% vs 0.26%), followed by WSU-WM, 10-fold (2.36% vs 0.23%), and least in WSU-DLCL2 (1.25% vs 0.71%). Using a CD34 microbead positive selection technique, we were able to enrich the CD34+ population up to 80% purity. Western blots confirmed higher expression of CD34 protein in the enriched population compared with parent cells. Using StemPro media (Gibco by Life Technologies), CD34+ cell fraction of WSU-WM showed more sustained growth and viability in culture over 9-day period compared with parent cells. This technique allowed the generation of large number of CD34+ cells for further characterization. Except for CD34 expression, this cell fraction expressed identical phenotype, karyotype, SNP and MCA profile to parent cells. However, the CD34+ fraction was more proliferative with an increase in S-phase cells (~60% vs 20% in control cells) and Hoechst 33342-positive cells (~40%). These cells were clonogenic even in presence of the cytotoxic agents Doxorubicin and 2-CdA that completely inhibited colony formation in parent cells. Resistance to chemotherapy was also noticed in liquid culture. A CD34+ population within clonal B-cells was also detected in 8 of 8 PDS. The lowest number of CD34+ cells was detected in chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) and the highest (5.35%) was seen in Richter's transformation of CLL. The isolated CD34+ cells in this study show many features of what is known as Lymphoma Stem Cells implicated in resistance to therapy and disease recurrence. Its relatedness to MRD cells is uncertain, however, since CD34 is not routinely included in MRD antibody panel. The observation that CD34+ cells are genetically identical to parent cells suggests an epigenetic modification. This conclusion is supported by our finding that Ehd3 expression is dramatically reduced in the CD34+ cell population of WSU-DLCL2 compared with parent cells. Ehd3 was recently identified as an epigenetically-silenced gene in solid tumors and in hematological malignancies including leukemia and lymphoma. The Ehd3-encoded protein belongs to the family of C-terminal Eps15 homology domain-containing (EHD) proteins, implicated in intracellular trafficking of tubular recycling endosomes (TRE), apoptosis, and cell cycle arrest. Further studies are underway to determine the contribution of Ehd3 to the biological and functional characteristics of the CD34+ cells in B-NHL. Utilizing the CD34+ monotypic B-cells in B-NHL as a model for screening new therapeutic agents and to identify potential new therapeutic strategies may lead to a more effective treatment of B-NHL. Disclosures No relevant conflicts of interest to declare.

Synaptic vesicle generation from activity-dependent bulk endosomes requires a dephosphorylation-dependent dynamin-syndapin interaction.

Activity‐dependent bulk endocytosis generates synaptic vesicles (SVs) during intense neuronal activity via a two‐step process. First, bulk endosomes are formed direct from the plasma membrane from which SVs are then generated. SV generation from bulk endosomes requires the efflux of previously accumulated calcium and activation of the protein phosphatase calcineurin. However, it is still unknown how calcineurin mediates SV generation. We addressed this question using a series of acute interventions that decoupled the generation of SVs from bulk endosomes in rat primary neuronal culture. This was achieved by either disruption of protein–protein interactions via delivery of competitive peptides, or inhibition of enzyme activity by known inhibitors. SV generation was monitored using either a morphological horseradish peroxidase assay or an optical assay that monitors the replenishment of the reserve SV pool. We found that SV generation was inhibited by, (i) peptides that disrupt calcineurin interactions, (ii) an inhibitor of dynamin I GTPase activity and (iii) peptides that disrupt the phosphorylation‐dependent dynamin I–syndapin I interaction. Peptides that disrupted syndapin I interactions with eps15 homology domain‐containing proteins had no effect. This revealed that (i) calcineurin must be localized at bulk endosomes to mediate its effect, (ii) dynamin I GTPase activity is essential for SV fission and (iii) the calcineurin‐dependent interaction between dynamin I and syndapin I is essential for SV generation. We therefore propose that a calcineurin‐dependent dephosphorylation cascade that requires both dynamin I GTPase and syndapin I lipid‐deforming activity is essential for SV generation from bulk endosomes.

WIP-1 and DBN-1 promote scission of endocytic vesicles by bridging actin and Dynamin-1 in the C. elegans intestine.

There has been a consensus that actin plays an important role in scission of the clathrin-coated pits (CCPs) together with large GTPases of the dynamin family in metazoan cells. However, the recruitment, regulation and functional interdependence of actin and dynamin during this process remain inadequately understood. Here, based on small-scale screening and in vivo live-imaging techniques, we identified a novel set of molecules underlying CCP scission in the multicellular organism Caenorhabditis elegans We found that loss of Wiskott-Aldrich syndrome protein (WASP)-interacting protein (WIP-1) impaired CCP scission in a manner that is independent of the C. elegans homolog of WASP/N-WASP (WSP-1) and is mediated by direct binding to G-actin. Moreover, the cortactin-binding domain of WIP-1 serves as the binding interface for DBN-1 (also known in other organisms as Abp1), another actin-binding protein. We demonstrate that the interaction between DBN-1 and F-actin is essential for Dynamin-1 (DYN-1) recruitment at endocytic sites. In addition, the recycling regulator RME-1, a homolog of mammalian Eps15 homology (EH) domain-containing proteins, is increasingly recruited at the arrested endocytic intermediates induced by F-actin loss or DYN-1 inactivation, which further stabilizes the tubular endocytic intermediates. Our study provides new insights into the molecular network underlying F-actin participation in the scission of CCPs.This article has an associated First Person interview with the first author of the paper.

Extracellular Vesicle Subtypes Released From Activated or Apoptotic T-Lymphocytes Carry a Specific and Stimulus-Dependent Protein Cargo.

Extracellular vesicles (EVs) are released from nearly all mammalian cells and different EV populations have been described. Microvesicles represent large EVs (LEVs) released from the cellular surface, while exosomes are small EVs (SEVs) released from an intracellular compartment. As it is likely that different stimuli promote the release of distinct EV populations, we analyzed EVs from human lymphocytes considering the respective release stimuli (activation Vs. apoptosis induction). We could clearly separate two EV populations, namely SEVs (average diameter <200 nm) and LEVs (diameter range between 200 and 1000 nm). Morphology and size were analyzed by electron microscopy and nanoparticle tracking analysis. Apoptosis induction caused a massive release of LEVs, while activated T-cells released SEVs and LEVs in considerably lower amounts. The release of SEVs from apoptotic T-cells was comparable with LEV release from activated ones. LEVs contained signaling proteins and proteins of the actin-myosin cytoskeleton. SEVs carried cytoplasmic/endosomal proteins like the 70-kDa heat shock protein 70 (HSP70) or tumor susceptibility 101 (TSG101), microtubule-associated proteins, and ubiquitinated proteins. The protein expression profile of SEVs and LEVs changed substantially after the induction of apoptosis. After apoptosis induction, HSP70 and TSG101 (often used as exosome markers) were highly expressed within LEVs. Interestingly, in contrast to HSP70 and TSG101, gelsolin and eps15 homology domain-containing protein 3 (EHD3) turned out to be specific for SEVs irrespective of the stimulus causing the EV release. Finally, we detected several subunits of the proteasome (PSMB9, PSMB10) as well as the danger signal HMGB1 exclusively within apoptotic cell-released LEVs. Thus, we were able to identify new marker proteins that can be useful to discriminate between distinct LEV subpopulations. The mass spectrometry proteomics data are available via ProteomeXchange with identifier PXD009074.

The trafficking protein, EHD2, positively regulates cardiac sarcolemmal KATP channel surface expression: role in cardioprotection.

ATP-sensitive K+ (KATP) channels uniquely link cellular energy metabolism to membrane excitability and are expressed in diverse cell types that range from the endocrine pancreas to neurons and smooth, skeletal, and cardiac muscle. A decrease in the surface expression of KATP channels has been linked to various disorders, including dysregulated insulin secretion, abnormal blood pressure, and impaired resistance to cardiac injury. In contrast, up-regulation of KATP channel surface expression may be protective, for example, by mediating the beneficial effect of ischemic preconditioning. Molecular mechanisms that regulate KATP channel trafficking are poorly understood. Here, we used cellular assays with immunofluorescence, surface biotinylation, and patch clamping to demonstrate that Eps15 homology domain-containing protein 2 (EHD2) is a novel positive regulator of KATP channel trafficking to increase surface KATP channel density. EHD2 had no effect on cardiac Na+ channels (Nav1.5). The effect is specific to EHD2 as other members of the EHD family-EHD1, EHD3, and EHD4-had no effect on KATP channel surface expression. EHD2 did not directly affect KATP channel properties as unitary conductance and ATP sensitivity were unchanged. Instead, we observed that the mechanism by which EHD2 increases surface expression is by stabilizing KATP channel-containing caveolar structures, which results in a reduced rate of endocytosis. EHD2 also regulated KATP channel trafficking in isolated cardiomyocytes, which validated the physiologic relevance of these observations. Pathophysiologically, EHD2 may be cardioprotective as a dominant-negative EHD2 mutant sensitized cardiomyocytes to ischemic damage. Our findings highlight EHD2 as a potential pharmacologic target in the treatment of diseases with KATP channel trafficking defects.-Yang, H. Q., Jana, K., Rindler, M. J., Coetzee, W. A. The trafficking protein, EHD2, positively regulates cardiac sarcolemmal KATP channel surface expression: role in cardioprotection.

EHD4 is a novel regulator of urinary water homeostasis.

The Eps15-homology domain-containing (EHD) protein family comprises 4 members that regulate endocytic recycling. Although the kidney expresses all 4 EHD proteins, their physiologic roles are largely unknown. This study focused on EHD4, which we found to be expressed differentially across nephron segments with the highest expression in the inner medullary collecting duct. Under baseline conditions, Ehd4-/- [EHD4-knockout (KO)] mice on a C57Bl/6 background excreted a higher volume of more dilute urine than control C57Bl/6 wild-type (WT) mice while maintaining a similar plasma osmolality. Urine excretion after an acute intraperitoneal water load was significantly increased in EHD4-KO mice compared to WT mice, and although EHD4-KO mice concentrated their urine during 24-h water restriction, urinary osmolality remained significantly lower than in WT mice, suggesting that EHD4 plays a role in renal water handling. Total aquaporin 2 (AQP2) and phospho-S256-AQP2 (pAQP2) protein expression in the inner medulla was similar in the two groups in baseline conditions. However, localization of both AQP2 and pAQP2 in the renal inner medullary principal cells appeared more dispersed, and the intensity of apical membrane staining for AQP2 was reduced significantly (by ∼20%) in EHD4-KO mice compared to WT mice in baseline conditions, suggesting an important role of EHD4 in trafficking of AQP2. Together, these data indicate that EHD4 play important roles in the regulation of water homeostasis.-Rahman, S. S., Moffitt, A. E. J., Trease, A. J., Foster, K. W., Storck, M. D., Band, H., Boesen, E. I. EHD4 is a novel regulator of urinary water homeostasis.

Spatio-temporal regulation of EGFR signaling by the Eps15 homology domain-containing protein 3 (EHD3).

The epidermal growth factor (EGF) receptor EGFR is a major receptor tyrosine kinase whose role in gliomagenesis is well established. We have recently identified EHD3 [Eps15 homology (EH) domain-containing protein 3], an endocytic trafficking regulatory protein, as a putative brain tumor suppressor. Here, we investigate the underlying mechanisms, by establishing a novel mechanistic and functional connection between EHD3 and the EGFR signaling pathway. We show that, in response to stimulation with the EGF ligand, EHD3 accelerates the rate of EGFR degradation by dramatically increasing its ubiquitination. As part of this process, EHD3 also regulates EGFR endosomal trafficking by diverting it away from the recycling route into the degradative pathway. Moreover, we found that upon EGF activation, rather than affecting the total MAPK and AKT downstream signaling, EHD3 decreases endosome-based signaling of these two pathways, thus suggesting the contribution of EHD3 in the spatial regulation of EGFR signaling. This function explains the higher sensitivity of EHD3-expressing cells to the growth-inhibitory effects of EGF. In summary, this is the first report supporting a mechanism of EHD3-mediated tumor suppression that involves the attenuation of endosomal signaling of the EGFR oncogene.

CSF-1 receptor signalling is governed by pre-requisite EHD1 mediated receptor display on the macrophage cell surface

Colony stimulating factor-1 receptor (CSF-1R), a receptor tyrosine kinase (RTK), is the master regulator of macrophage biology. CSF-1 can bind CSF-1R resulting in receptor activation and signalling essential for macrophage functions such as proliferation, differentiation, survival, polarization, phagocytosis, cytokine secretion, and motility. CSF-1R activation can only occur after the receptor is presented on the macrophage cell surface. This process is reliant upon the underlying macrophage receptor trafficking machinery. However, the mechanistic details governing this process are incompletely understood. C-terminal Eps15 Homology Domain-containing (EHD) proteins have recently emerged as key regulators of receptor trafficking but have not yet been studied in the context of macrophage CSF-1R signalling. In this manuscript, we utilize primary bone-marrow derived macrophages (BMDMs) to reveal a novel function of EHD1 as a regulator of CSF-1R abundance on the cell surface. We report that EHD1-knockout (EHD1-KO) macrophages cell surface and total CSF-1R levels are significantly decreased. The decline in CSF-1R levels corresponds with reduced downstream macrophage functions such as cell proliferation, migration, and spreading. In EHD1-KO macrophages, transport of newly synthesized CSF-1R to the macrophage cell surface was reduced and was associated with the shunting of the receptor to the lysosome, which resulted in receptor degradation. These findings reveal a novel and functionally important role for EHD1 in governing CSF-1R signalling via regulation of anterograde transport of CSF-1R to the macrophage cell surface.