Apicomplexa micropore: history, function, and formation.

The micropore, a mysterious structure found in apicomplexan species, was recently shown to be essential for nutrient acquisition in Plasmodium falciparum and Toxoplasma gondii. However, the differences between the micropores of these two parasites questions the nature of a general apicomplexan micropore structure and whether the formation process model from Plasmodium can be applied to other apicomplexans. We analyzed the literature on different apicomplexan micropores and found that T. gondii probably harbors a more representative micropore type than the more widely studied ones in Plasmodium. Using recent knowledge of the Kelch 13 (K13) protein interactome and gene depletion phenotypes in the T. gondii micropore, we propose a model of micropore formation, thus enriching our wider understanding of micropore protein function.

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.

CD1d facilitates African swine fever virus entry into the host cells via clathrin-mediated endocytosis.

African swine fever (ASF) is a highly contagious and acute hemorrhagic viral disease with high morbidity and mortality in domestic pigs and wild boars. The disease has become a global threat to the pig production industry and has caused enormous economic losses in many countries in recent years. However, the molecular mechanism underlying ASF virus (ASFV) entry of the host cells is not fully understood, which restricts the development of vaccines and antiviral-drugs of ASFV. In this study, we found that the host protein CD1d acts as a host factor, which mediates ASFV entry into the host cells. As the main capsid protein on the surface of ASFV virions, p72 can mediate viral entry. Using IP-MS assay, CD1d was identified as a binding partner of p72 on surface of ASFV virions. Knockdown of CD1d expression and blocking the cells with anti-pCD1d antibody, or incubating ASFV virions with soluble CD1d protein could significantly inhibit ASFV infection. CD1d is located on the membrane surface of primary porcine alveolar macrophages (PAMs) and mediates the virus entry via binding to p72. CD1d knockout or CD1d knockdown assay showed that CD1d could facilitate ASFV virions internalization via clathrin-mediated endocytosis (CME). Furthermore, CD1d interacts with EPS15 to mediate ASFV entry via clathrin-mediated endocytosis. Overall, our findings revealed that CD1d is a novel host-entry factor involved in ASFV internalization via the EPS15-clathrin endocytosis axis and a potential target for antiviral intervention.

Upregulation of CSNK1A1 induced by ITGB5 confers to hepatocellular carcinoma resistance to sorafenib in vivo by disrupting the EPS15/EGFR complex.

Oral multitarget tyrosine kinase inhibitors (TKIs), such as sorafenib, which suppress tumor cell proliferation and tumor angiogenesis, have been approved to treat patients with hepatocellular carcinoma (HCC). Of note, only approximately 30% of patients can benefit from TKIs, and this population usually acquires drug resistance within 6 months. In this study, we intended to explore the mechanism associated with regulating the sensitivity of HCC to TKIs. We revealed that integrin subunit ß 5 (ITGB5) is abnormally expressed in HCC and contributes to decreased the sensitivity of HCC to sorafenib. Mechanistically, unbiased mass spectrometry analysis using ITGB5 antibodies revealed that ITGB5 interacts with EPS15 to prevent the degradation of EGFR in HCC cells, which activates AKT-mTOR signaling and the MAPK pathway to reduce the sensitivity of HCC cells to sorafenib. In addition, mass spectrometry analysis showed that CSNK1A1 binds to ITGB5 in HCC cells. Further study indicated that ITGB5 increased the protein level of CSNK1A1 through the EGFR-AKT-mTOR pathway in HCC. Upregulated CSNK1A1 phosphorylates ITGB5 to enhance the interaction between ITGB5 and EPS15 and activate EGFR in HCC cells. Thus, we identified a positive feedback loop between ITGB5-EPS15-EGFR-CSNK1A1 in HCC cells. This finding provides a theoretical basis for the future development of therapeutic strategies to improve the anti-HCC efficacy of sorafenib.

YULINK regulates vascular formation in zebrafish and HUVECs.

BACKGROUND: The distinct arterial and venous cell fates are dictated by a combination of various genetic factors which form diverse types of blood vessels such as arteries, veins, and capillaries. We report here that YULINK protein is involved in vasculogenesis, especially venous formation. METHODS: In this manuscript, we employed gene knockdown, yeast two-hybrid, FLIM-FRET, immunoprecipitation, and various imaging technologies to investigate the role of YULINK gene in zebrafish and human umbilical vein endothelial cells (HUVECs). RESULTS: Knockdown of YULINK during the arterial-venous developmental stage of zebrafish embryos led to the defective venous formation and abnormal vascular plexus formation. Knockdown of YULINK in HUVECs impaired their ability to undergo cell migration and differentiation into a capillary-like tube formation. In addition, the phosphorylated EPHB4 was decreased in YULINK knockdown HUVECs. Yeast two-hybrid, FLIM-FRET, immunoprecipitation, as well as imaging technologies showed that YULINK colocalized with endosome related proteins (EPS15, RAB33B or TICAM2) and markers (Clathrin and RHOB). VEGF-induced VEGFR2 internalization was also compromised in YULINK knockdown HUVECs, demonstrating to the involvement of YULINK. CONCLUSION: This study suggests that YULINK regulates vasculogenesis, possibly through endocytosis in zebrafish and HUVECs.

YULINK regulates vascular formation in zebrafish and HUVECs

BACKGROUND: The distinct arterial and venous cell fates are dictated by a combination of various genetic factors which form diverse types of blood vessels such as arteries, veins, and capillaries. We report here that YULINK protein is involved in vasculogenesis, especially venous formation. METHODS: In this manuscript, we employed gene knockdown, yeast two-hybrid, FLIM-FRET, immunoprecipitation, and various imaging technologies to investigate the role of YULINK gene in zebrafish and human umbilical vein endothelial cells (HUVECs). RESULTS: Knockdown of YULINK during the arterial-venous developmental stage of zebrafish embryos led to the defective venous formation and abnormal vascular plexus formation. Knockdown of YULINK in HUVECs impaired their ability to undergo cell migration and differentiation into a capillary-like tube formation. In addition, the phosphorylated EPHB4 was decreased in YULINK knockdown HUVECs. Yeast two-hybrid, FLIM-FRET, immunoprecipitation, as well as imaging technologies showed that YULINK colocalized with endosome related proteins (EPS15, RAB33B or TICAM2) and markers (Clathrin and RHOB). VEGF-induced VEGFR2 internalization was also compromised in YULINK knockdown HUVECs, demonstrating to the involvement of YULINK. CONCLUSION: This study suggests that YULINK regulates vasculogenesis, possibly through endocytosis in zebrafish and HUVECs. Key points Knockdown of YULINK with morpholino in embryos of double transgenic zebrafish exhibited abnormal venous formation. Tube formation and phosphorylated EPHB4 were decreased in YULINK knockdown HUVECs. FLIM-FRET, immunoprecipitation, as well as other imaging technologies showed that YULINK colocalized with endosome related proteins (EPS15, RAB33B and TICAM2) and endosome markers (Clathrin and RHOB). Knockdown of YULINK decreased the internalization of VEGF and VEGFR2 in HUVECs.

lncRNA EPS15-AS1 affects the biological behavior of liver cancer stem cells by regulating EPS15 expression.

OBJECTIVE: To investigate whether long non-coding RNA (lncRNA) EPS15-antisense RNA (EPS15-AS1) affects the biological behavior of liver cancer stem cells (LCSCs) by targeting EPS15. METHODS: The expression of EPS15 in liver cancer was analyzed based on TCGA database. The expression of EPS15-AS1 and EPS15 in LCSCs was detected by real-time quantitative PCR (RT-qPCR). MTT method, flow cytometry, and Transwell assay were used to detect the effects of EPS15-AS1 and EPS15 expression on the biological behavior of LCSCs. RESULTS: EPS15 was highly expressed in liver cancer tissues in TCGA, and EPS15 was closely related to the survival and prognosis of liver cancer patients (P<0.05). EPS15 was highly expressed in LCSCs, while lncRNA EPS15-AS1 was lowly expressed in LCSCs (P<0.05). After silencing lncRNA EPS15-AS1, the proliferation, invasion, and EPS15 protein expression of LCSCs were promoted (P<0.05) while apoptosis was suppressed (P<0.05). After overexpression of lncRNA EPS15-AS1, the proliferation, invasion, and EPS15 protein expression of LCSCs were suppressed while the apoptosis ability was promoted. However, simultaneous overexpression of lncRNA EPS15-AS1 and EPS15 attenuated the effect of lncRNA EPS15-AS1 overexpression alone on proliferation and apoptosis of LCSCs. CONCLUSION: lncRNA EPS15-AS1 overexpression can inhibit proliferation and invasion but promote apoptosis of LCSCs by down-regulating the expression of EPS15.

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

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.

Circular RNA Eps15-homology domain containing protein 2 motivates proliferation, glycolysis but refrains autophagy in non-small cell lung cancer via crosstalk with microRNA-3186-3p and forkhead box K1.

Numerous studies have clarified the involvement of circular RNAs (circRNAs) in modulating malignant behavior of non-small cell lung cancer (NSCLC), while the concrete mechanism is not completely elucidated. The aim of the study was to figure out the latent functions and molecular mechanisms of circRNA Eps15-homology domain containing protein 2 (EHD2) on NSCLC proliferation, glycolysis and autophagy. The results clarified in NSCLC elevated expression of circEHD2 and declined expression of microRNA (miR)-3186-3p. Repressive circEHD2 or enhancive miR-3186-3p facilitated cell apoptosis rate and autophagy substrates LC3BII and Beclin-1, but curbed the colony-formation and DNA replication ability of NSCLC, glucose consumption, lactic acid production, glycolytic rate-limiting enzyme HK-2 and glutamine hydrolase GLS1 and P62, while overexpressed circEHD2 was adverse. Meanwhile, the impacts of repressive and elevated circEHD2 on NSCLC were turned around via reduced miR-3186-3p or forkhead box k1 (FOXK1) separately. Mechanically, FOXK1 was augmented via circEHD2's competitive integration of miR-3186-3p. Depressive circEHD2 refrained NSCLC tumor growth, which was accelerated via enhancive one. All in all, circEHD2 accelerates the proliferation and glycolysis of NSCLC, but refrains autophagy and apoptosis via strengthening FOXK1 via the adsorption of miR-3186-3p, which is supposed to be a latent molecular target for NSCLC therapy later.

circEPS15 Overexpression in Hepatocellular Carcinoma Modulates Tumor Invasion and Migration.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths worldwide. Recent evidence has shown that circular RNAs (circRNAs) play important roles in tissue development, gene transcription, signal regulation and tumorigenesis. However, whether circRNAs are involved in HCC progression and encode functional proteins remains largely unknown. In the present study, we aimed to explore the function and molecular mechanism of circRNAs in HCC. First, many circRNAs were found to be differentially expressed in HCC samples and paired adjacent normal liver tissues. The validation of dysregulated circRNAs by qRT-PCR revealed that circEPS15 expression was downregulated in HCC tissues, and the survival curves showed that low circEPS15 levels were associated with poor overall survival in HCC patients. Then, the overexpression of circEPS15 suppressed tumor cell invasion and migration by inhibiting the TJP1/CDH2/VIM signaling pathway and retarded cell cycle progression, which was confirmed by the Transwell culture system, wound healing assays, flow cytometry and western blot assays. After that, the spanning junction open reading frame in circEPS15 driven by IRES was shown to encode a novel protein, which was verified by western blotting with full-length, mutated, and truncated sequences of circEPS15 with a FLAG tag. Moreover, ceRNA analysis and qRT-PCR results suggest a possible circRNA (circEPS15)-miRNA-mRNA network in HCC. Collectively, our study reveals that endogenous circEPS15 plays a novel role in repressing HCC through the ceRNA network and encodes a functional protein.

A Founder Mutation in EHD1 Presents with Tubular Proteinuria and Deafness.

BACKGROUND: The endocytic reabsorption of proteins in the proximal tubule requires a complex machinery and defects can lead to tubular proteinuria. The precise mechanisms of endocytosis and processing of receptors and cargo are incompletely understood. EHD1 belongs to a family of proteins presumably involved in the scission of intracellular vesicles and in ciliogenesis. However, the relevance of EHD1 in human tissues, in particular in the kidney, was unknown. METHODS: Genetic techniques were used in patients with tubular proteinuria and deafness to identify the disease-causing gene. Diagnostic and functional studies were performed in patients and disease models to investigate the pathophysiology. RESULTS: We identified six individuals (5-33 years) with proteinuria and a high-frequency hearing deficit associated with the homozygous missense variant c.1192C>T (p.R398W) in EHD1. Proteinuria (0.7-2.1 g/d) consisted predominantly of low molecular weight proteins, reflecting impaired renal proximal tubular endocytosis of filtered proteins. Ehd1 knockout and Ehd1R398W/R398W knockin mice also showed a high-frequency hearing deficit and impaired receptor-mediated endocytosis in proximal tubules, and a zebrafish model showed impaired ability to reabsorb low molecular weight dextran. Interestingly, ciliogenesis appeared unaffected in patients and mouse models. In silico structural analysis predicted a destabilizing effect of the R398W variant and possible inference with nucleotide binding leading to impaired EHD1 oligomerization and membrane remodeling ability. CONCLUSIONS: A homozygous missense variant of EHD1 causes a previously unrecognized autosomal recessive disorder characterized by sensorineural deafness and tubular proteinuria. Recessive EHD1 variants should be considered in individuals with hearing impairment, especially if tubular proteinuria is noted.

LncRNA ABHD11-AS1 promotes tumor progression in papillary thyroid carcinoma by regulating EPS15L1/EGFR signaling pathway.

OBJECTIVES: lncRNA ABHD11 antisense RNA 1 (ABHD11-AS1) acts as an oncogene involved in papillary thyroid carcinoma (PTC) occurrence and progression. ABHD11-AS1 exerts biologic functions by some miRNAs and proteins to regulate multiple targets. Identification of novel mechanism of ABHD11-AS1 could be helpful in therapeutic targeting for PTC treatment. METHODS: Differentially expressed lncRNAs were selected from TCGA database. qRT-PCR analysis was applied to examine the expression of ABHD11-AS1 in PTC cell lines and tissues. The relationship of ABHD11-AS1 expression and clinicopathological features was analyzed by Kaplan-Meier analysis. Two PTC cell lines (TPC-1 and KTC-1) were transfected with pcDNA 3.1, pcDNA3.1-ABHD11-AS1, si-NC and si-ABHD11-AS1, respectively, to verify the ABHD11-AS1 oncogene-regulating capacity to promote tumor progression. The cell metastasis and proliferation had been evaluated both in vitro and in vivo. RESULTS: High expression of ABHD11-AS1 was found in PTC tissues (P < 0.01), which was significantly correlated with lymph node metastasis (P < 0.05). ABHD11-AS1 overexpression noticeably promoted cell proliferation, migration, and invasion capabilities, which were obviously decreased upon ABHD11-AS1 knockdown. ABHD11-AS1 positively regulated EGFR/EPS15L1 pathway, as EGFR, EPS15L1, STAT3, and p-STAT3 were activated. CONCLUSION: ABHD11-AS1 promotes tumor progression in PTC by regulating EPS15L1/EGFR pathway.

Completing the family of human Eps15 homology domains: Solution structure of the internal Eps15 homology domain of γ-synergin.

Eps15 homology (EH) domains are universal interaction domains to establish networks of protein-protein interactions in the cell. These networks mainly coordinate cellular functions including endocytosis, actin remodeling, and other intracellular signaling pathways. They are well characterized in structural terms, except for the internal EH domain from human γ-synergin (EHγ). Here, we complete the family of EH domain structures by determining the solution structure of the EHγ domain. The structural ensemble follows the canonical EH domain fold and the identified binding site is similar to other known EH domains. But EHγ differs significantly in the N- and C-terminal regions. The N-terminal α-helix is shortened compared to known homologues, while the C-terminal one is fully formed. A significant proportion of the remaining N- and C-terminal regions are well structured, a feature not seen in other EH domains. Single mutations in both the N-terminal and the C-terminal structured extensions lead to the loss of the distinct three-dimensional fold and turn EHγ into a molten globule like state. Therefore, we propose that the structural extensions in EHγ function as a clamp and are undoubtedly required to maintain its tertiary fold.

Lineage Switch in an Infant B-Lymphoblastic Leukemia With t(1;11)(p32;q23); KMT2A/EPS15, Following Blinatumomab Therapy.

We report a 6 month-old infant girl with t(1;11)(p32;q23), KMT2A/EPS15-rearranged B-acute lymphoblastic leukemia (B-ALL) that was refractory to traditional ALL-directed chemotherapy. Following administration of blinatumomab, she experienced lineage switch from B-ALL to acute myeloid leukemia (AML). Myeloid-directed chemotherapy resulted in clearance of AML by flow cytometry, though a residual CD19+ B-ALL population persisted (0.14%). Following bridging blinatumomab, the patient achieved B-ALL and AML remission, as measured by flow cytometry. The patient subsequently underwent allogeneic hematopoietic stem cell transplant. Unfortunately, she relapsed with CD19+ B-ALL one-month post-transplantation. Next generation sequencing study of IGH/IGL using ClonoSEQ® analysis detected 3 dominant sequences all present in her original B-ALL, lineage switched AML, and post-transplant relapsed B-ALL, though the latter showed an additional 4 sequences, three of which were present at low abundance in the original diagnostic sample. The presence of the same clones throughout her disease course suggests cellular reprogramming and differentiation following chemotherapy and immunotherapy. This is the first reported case of lineage switch of B-ALL with t(1;11) and also the first report of a lineage switch case that used ClonoSEQ® to define the clonality of the original B-ALL, lineage switched AML, and relapsed B-ALL.

Expression of a novel type of KMT2A/EPS15 fusion transcript in FLT3 mutation-positive B-lymphoblastic leukemia with t(1;11)(p32;q23).

The t(1;11)(p32;q23) translocation is a rare but recurrent cytogenetic aberration in acute myeloid leukemia (AML) and B-cell acute lymphoblastic leukemia (B-ALL). This translocation was initially shown to form a fusion gene between KMT2A exon 8 at 11q23 and EPS15 exon 2 at 1p32 in AML. Activating mutations of FLT3 are frequently found in AML but are very rare in ALL. Here, we describe a 75-year-old woman who was diagnosed with B-ALL since her bone marrow was made up of 98.2% lymphoblasts. These blasts were positive for CD19, CD22, CD79a, CD13, and CD33 but negative for CD10 and myeloperoxidase. The karyotype by G-banding and spectral karyotyping was 46,XX,t(1;11)(p32;q23). Expression of KMT2A/EPS15 and reciprocal EPS15/KMT2A fusion transcripts were shown: KMT2A exon 8 was in-frame fused to EPS15 exon 12, indicating that this fusion transcript was a novel type. Considering three reported B-ALL cases, EPS15 breakpoints were markedly different between AML (exon 2) and B-ALL (exons 10-12). Furthermore, an uncommon type of FLT3 mutation in the juxtamembrane domain was detected: in-frame 4-bp deletion and 10-bp insertion. Accordingly, our results indicate that the novel type of KMT2A/EPS15 fusion transcript and FLT3 mutation may cooperate in the pathogenesis of adult B-ALL as class II and class I mutations, respectively.

ALS-causing SOD1 mutants regulate occludin phosphorylation/ubiquitination and endocytic trafficking via the ITCH/Eps15/Rab5 axis.

It is increasingly recognized that blood-spinal cord barrier (BSCB) breakdown is a hallmark of amyotrophic lateral sclerosis (ALS). BSCB integrity is disrupted prior to disease onset. Occludin, as the functional component of the endothelial barrier, is downregulated in mouse models expressing ALS-linked superoxide dismutase-1 (SOD1) mutants. However, the molecular mechanisms underlying the regulation of occludin expression remain elusive. Here, using SOD1G93A transgenic mice and endothelial cells expressing SOD1 mutants of different biochemical characteristics, we found that the SOD1 mutation disrupted endothelial barrier integrity and that the occludin expression level was downregulated with disease progression. Our mechanistic studies revealed that abnormal reactive oxygen species (ROS) in mutant SOD1-expressing cells induced occludin phosphorylation, which facilitated the subsequent occludin ubiquitination mediated by the E3 ligase ITCH. Moreover, ubiquitinated occludin interacted with Eps15 to initiate its internalization, then trafficked to Rab5-positive vesicles and be degraded by proteasomes, resulting in a reduction in cell surface localization and total abundance. Notably, either ITCH or Eps15 knockdown was sufficient to rescue occludin degradation and ameliorate endothelial barrier disruption. In conclusion, our study reveals a novel mechanism of occludin degradation mediated by ALS-causing SOD1 mutants and demonstrates a role for occludin in regulating BSCB integrity.

Non-SUMOylated CRMP2 decreases NaV1.7 currents via the endocytic proteins Numb, Nedd4-2 and Eps15.

Voltage-gated sodium channels are key players in neuronal excitability and pain signaling. Functional expression of the voltage-gated sodium channel NaV1.7 is under the control of SUMOylated collapsin response mediator protein 2 (CRMP2). When not SUMOylated, CRMP2 forms a complex with the endocytic proteins Numb, the epidermal growth factor receptor pathway substrate 15 (Eps15), and the E3 ubiquitin ligase Nedd4-2 to promote clathrin-mediated endocytosis of NaV1.7. We recently reported that CRMP2 SUMO-null knock-in (CRMP2K374A/K374A) female mice have reduced NaV1.7 membrane localization and currents in their sensory neurons. Preventing CRMP2 SUMOylation was sufficient to reverse mechanical allodynia in CRMP2K374A/K374A female mice with neuropathic pain. Here we report that inhibiting clathrin assembly in nerve-injured male CRMP2K374A/K374A mice precipitated mechanical allodynia in mice otherwise resistant to developing persistent pain. Furthermore, Numb, Nedd4-2 and Eps15 expression was not modified in basal conditions in the dorsal root ganglia (DRG) of male and female CRMP2K374A/K374A mice. Finally, silencing these proteins in DRG neurons from female CRMP2K374A/K374A mice, restored the loss of sodium currents. Our study shows that the endocytic complex composed of Numb, Nedd4-2 and Eps15, is necessary for non-SUMOylated CRMP2-mediated internalization of sodium channels in vivo.

Identification of phostensin in association with Eps 15 homology domain-containing protein 1 (EHD1) and EHD4.

Phostensin (PTS) encoded by KIAA1949 is a protein phosphatase 1 (PP1)-binding protein. In order to explore the cellular functions of PTS, we have searched PTS-binding proteins by using co-immunoprecipitation in combination with shotgun proteomics. Here, we report two novel PTS-binding proteins, Eps 15 homology domain-containing protein 1 (EHD1) and EHD4. PTS associated with EHD proteins was also observed in GST pull-down assays. Immunofluorescence microscopy demonstrated that the complex was co-localized at the endocytic vesicles. EHD proteins have been known to play a critical role in regulation of endocytic transport. Overexpression of PTS-ß can attenuate the endocytic trafficking of transferrin.

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.

Sorting nexin 17 (SNX17) links endosomal sorting to Eps15 homology domain protein 1 (EHD1)-mediated fission machinery.

Following endocytosis, receptors that are internalized to sorting endosomes are sorted to different pathways, in part by sorting nexin (SNX) proteins. Notably, SNX17 interacts with a multitude of receptors in a sequence-specific manner to regulate their recycling. However, the mechanisms by which SNX17-labeled vesicles that contain sorted receptors bud and undergo vesicular fission from the sorting endosomes remain elusive. Recent studies suggest that a dynamin-homolog, Eps15 homology domain protein 1, catalyzes fission and releases endosome-derived vesicles for recycling to the plasma membrane. However, the mechanism by which EHD1 is coupled to various receptors and regulates their recycling remains unknown. Here we sought to characterize the mechanism by which EHD1 couples with SNX17 to regulate recycling of SNX17-interacting receptors. We hypothesized that SNX17 couples receptors to the EHD1 fission machinery in mammalian cells. Coimmunoprecipitation experiments and in vitro assays provided evidence that EHD1 and SNX17 directly interact. We also found that inducing internalization of a SNX17 cargo receptor, low-density lipoprotein receptor-related protein 1 (LRP1), led to recruitment of cytoplasmic EHD1 to endosomal membranes. Moreover, surface rendering and quantification of overlap volumes indicated that SNX17 and EHD1 partially colocalize on endosomes and that this overlap further increases upon LRP1 internalization. Additionally, SNX17-containing endosomes were larger in EHD1-depleted cells than in WT cells, suggesting that EHD1 depletion impairs SNX17-mediated endosomal fission. Our findings help clarify our current understanding of endocytic trafficking, providing significant additional insight into the process of endosomal fission and connecting the sorting and fission machineries.