EH Domain Binding Protein Research Articles

Abstract 158: The Role Of Ehbp1 In Hypercholesterolemia

Background: Despite aggressive lowering of cholesterol, conventional lipid-lowering therapies only modestly lower cardiovascular risk in the population. Therefore, identifying novel hypercholesterolemia-risk targets is of critical importance. Human genome-wide association studies (GWAS) have revealed that several single-nucleotide polymorphisms (SNPs) in EHBP1 , the gene that encodes EH domain binding protein 1 (EHBP1), an endocytic trafficking regulator, are associated with serum cholesterol. However, the mechanisms underlying this association and the role of EHBP1 in cholesterol metabolism are unknown. We hypothesize that EHBP1 is a novel hypercholesterolemia risk factor. The goal of this study is to explore the role of EHBP1 in hypercholesterolemia and its underlying mechanisms. Methods: Hepatocyte EHBP1 was silenced by intravenous injection (IV) of AAV8-H1-shEhbp1 in wild-type (WT), Apoe-/- and Ldlr-/- mice, followed by Western Diet feeding to induce hypercholesterolemia and atherosclerosis. Primary murine and human hepatocytes were isolated for in vitro mechanistic studies. We also leveraged publicly available human genetic studies and unbiased scRNA datasets to support our study. Results: Individuals with the EHBP1 rs10496099 T>C variant have decreased serum cholesterol and reduced liver EHBP1 expression. Consistent with the human data, our in vivo study with hypercholesterolemic mice shows that EHBP1 silencing in hepatocytes decreases plasma total and LDL/VLDL cholesterol. Our in vitro study with primary hepatocytes reveals that EHBP1 deficiency eliminates sortilin (SORT1)-mediated PCSK9 secretion and induces LDLR-mediated LDL uptake. Mechanistically, we found that EHBP1 silencing abolishes the homeostasis of the retromer complex, a protein complex that helps retrograde and stabilize sortilin. Conclusion: Hepatocyte EHBP1 promotes sortilin-mediated PCSK9 secretion by maintaining retromer homeostasis, leading to LDLR degradation and hypercholesterolemia. Our study elucidates a novel link between EHBP1 and other cholesterol-associated loci (e.g., SORT1 , PCSK9 , and LDLR) and provides new mechanistic insight into the role of EHBP1 in hepatic cholesterol metabolism and hypercholesterolemia.

EHBP1L1 Drives Immune Evasion in Renal Cell Carcinoma through Binding and Stabilizing JAK1.

High lymphocyte infiltration and immunosuppression characterize the tumor microenvironment (TME) in renal cell carcinoma (RCC). There is an urgent need to elucidate how tumor cells escape the immune attack and to develop novel therapeutic targets to enhance the efficacy of immune checkpoint blockade (ICB) in RCC. Overactivated IFN-γ-induced JAK/STAT signaling involves in such TME, but the underlying mechanisms remain elusive. Here, EH domain-binding protein 1-like protein 1 (EHBP1L1) is identified as a crucial mediator of IFN-γ/JAK1/STAT1/PD-L1 signaling in RCC. EHBP1L1 is highly expressed in RCC, and high EHBP1L1 expression levels are correlated with poor prognosis and resistance to ICB. EHBP1L1 depletion significantly inhibits tumor growth, which is attributed to enhanced CD8+ T cell-mediated antitumor immunity. Mechanistically, EHBP1L1 interacts with and stabilizes JAK1. By competing with SOCS1, EHBP1L1 protects JAK1 from proteasomal degradation, which leads to elevated JAK1 protein levels and JAK1/STAT1/PD-L1 signaling activity, thereby forming an immunosuppressive TME. Furthermore, the combination of EHBP1L1 inhibition and ICB reprograms the immunosuppressive TME and prevents tumor immune evasion, thus significantly reinforcing the therapeutic efficacy of ICB in RCC patient-derived xenograft (PDX) models. These findings reveal the vital role of EHBP1L1 in immune evasion in RCC, which may be a potential complement for ICB therapy.

EHBP1, TUBB, and WWOX SNPs, Gene-Gene and Gene-Environment Interactions on Coronary Artery Disease and Ischemic Stroke.

The associations among the EH domain-binding protein 1 (EHBP1), tubulin beta class I (TUBB), and WW domain-containing oxidoreductase (WWOX) single nucleotide polymorphisms (SNPs) and coronary artery disease (CAD) and ischemic stroke (IS) are not yet understood. This study aimed to detect the associations of these SNPs, gene-gene and gene-environment interactions and CAD and IS in the Guangxi Han population. A total of 1853 unrelated subjects were recruited into normal control (n = 638), CAD (n = 622), and IS (n = 593) groups. Related genotypes were determined by high-throughput sequencing. The genotypic and minor allelic frequencies of rs2278075 were different between the CAD and control groups, and those of rs2710642, rs3130685, and rs2278075 were also different between the IS and control groups. The rs2278075T allele, rs3130685-rs2222896-rs2278075, rs3130685-rs2222896-diabetes, rs3130685-rs2222896-drinking, and haplotype rs2710642A-rs10496099C-diabetes interactions were associated with increased risk, while G-T-G-C-G-A and G-T-T-T-G-T-drinking were associated with reduced risk of CAD. The rs2278075T and rs2710642G alleles, rs2710642G-rs10496099C haplotype, rs3130685-rs2278075-rs2222896, and rs2710642-rs2278075-hypertension interactions aggravated the association with IS, whereas the rs3130685T allele, rs2710642A-rs10496099C haplotype and the interactions of H1 (s2710642A-rs10496099C)-H2 (rs2710642G-rs10496099C)-drinking and I1 (A-C-G-C-A-A)-I3 (A-C-G-T-A-A)-I4 (A-C-G-T-G-A)-I5 (G-T-G-C-G-A) diminished the association with IS. Carrying WWOX rs2278075T was strongly associated with CAD or IS, while EHBP1 rs2710642 and TUBB rs3130685 might alter the association of IS by modifying the serum lipid profile. This study demonstrates that the EHBP1, TUBB, and WWOX SNPs, gene-gene and gene-environment interactions are associated with the risk of CAD and IS in the Guangxi Han population.

Genome-Wide Association Study of Alzheimer's Disease Brain Imaging Biomarkers and Neuropsychological Phenotypes in the European Medical Information Framework for Alzheimer's Disease Multimodal Biomarker Discovery Dataset.

Alzheimer’s disease (AD) is the most frequent neurodegenerative disease with an increasing prevalence in industrialized, aging populations. AD susceptibility has an established genetic basis which has been the focus of a large number of genome-wide association studies (GWAS) published over the last decade. Most of these GWAS used dichotomized clinical diagnostic status, i.e., case vs. control classification, as outcome phenotypes, without the use of biomarkers. An alternative and potentially more powerful study design is afforded by using quantitative AD-related phenotypes as GWAS outcome traits, an analysis paradigm that we followed in this work. Specifically, we utilized genotype and phenotype data from n = 931 individuals collected under the auspices of the European Medical Information Framework for Alzheimer’s Disease Multimodal Biomarker Discovery (EMIF-AD MBD) study to perform a total of 19 separate GWAS analyses. As outcomes we used five magnetic resonance imaging (MRI) traits and seven cognitive performance traits. For the latter, longitudinal data from at least two timepoints were available in addition to cross-sectional assessments at baseline. Our GWAS analyses revealed several genome-wide significant associations for the neuropsychological performance measures, in particular those assayed longitudinally. Among the most noteworthy signals were associations in or near EHBP1 (EH domain binding protein 1; on chromosome 2p15) and CEP112 (centrosomal protein 112; 17q24.1) with delayed recall as well as SMOC2 (SPARC related modular calcium binding 2; 6p27) with immediate recall in a memory performance test. On the X chromosome, which is often excluded in other GWAS, we identified a genome-wide significant signal near IL1RAPL1 (interleukin 1 receptor accessory protein like 1; Xp21.3). While polygenic score (PGS) analyses showed the expected strong associations with SNPs highlighted in relevant previous GWAS on hippocampal volume and cognitive function, they did not show noteworthy associations with recent AD risk GWAS findings. In summary, our study highlights the power of using quantitative endophenotypes as outcome traits in AD-related GWAS analyses and nominates several new loci not previously implicated in cognitive decline.

EH domain binding protein 1-like 1 (EHBP1L1), a protein with calponin homology domain, is expressed in the rat testis.

In this paper, with the aim to find new genes involved in mammalian spermatogenesis, we isolated, for the first time in the rat testis, a partial cDNA clone that encoded EH domain binding protein 1-like 1 (Ehbp1l1), a protein that has a single calponin homology domain (CH). Bioinformatic analysis showed that EHBP1l1 contains three domains: the N-terminal C2-like, the CH and the C-terminal bivalent Mical/EHBP Rab binding (bMERB) domains, which are evolutionarily conserved in vertebrates. We found that Ehbp1l1 mRNA was expressed in several rat tissues, including the liver, intestine, kidney and also in the testis during its development, with a higher level in testis from 12-month-old animals. Interestingly, in situ hybridization experiments revealed that Ehbp1l1 is specifically expressed by types I and II spermatocytes, this result was validated by RT-PCR performed on total RNA obtained from enriched fractions of different testicular cell types. As EHBP1l1 has been described as linked to vesicular transport to the actin cytoskeleton and as an effector of the small GTPase Rab8, we hypothesized that it could participate both in cytoskeletal remodelling and in the regulation of vesicle sorting from the trans-Golgi network to the apical plasma membrane. Our findings provide a better understand of the molecular mechanisms of the differentiation process of spermatogenesis; Ehbp1l1 may also be used as a new marker of testicular activity.

EHBP1 SNPs, Their Haplotypes, and Gene-Environment Interactive Effects on Serum Lipid Levels.

The associations between single nucleotide polymorphisms (SNPs) rs2710642 and rs10496099 and their effect on the EH domain-binding protein 1 (EHBP1) gene and serum lipid profiles remain uncertain. This study was performed to investigate the two EHBP1 SNPs in Han and Maonan populations, including their association, haplotypes, and effects on serum lipid levels. Two EHBP1 SNPs in 564 Han and 796 Maonan participants were genotyped by high-throughput sequencing, and then the genotype and haplotype distributions of two EHBP1 SNPs were analyzed. Moreover, risk factors and their effects on serum lipid levels were analyzed using multivariable linear regression and logistic regression analyses. In Han and Maonan populations, a significant difference was found in the allelic and genotypic frequencies of the EHBP1 rs2710642 and rs10496099 SNPs and the alternate alleles of rs2710642A and rs10496099C might be potentially beneficial for healthy lipid levels. Medium linkage disequilibrium between the two SNPs was noted in each ethnic group, and four main haplotypes were detected. The rs2710642G–rs10496099C haplotype was associated with high triglycerides (TGs) and low high-density lipoprotein cholesterol, and the rs2710642A–rs10496099C haplotype was associated with low TGs and high apolipoprotein A1. The rs2710642G–rs10496099C haplotype was a high-risk factor for hyperlipidemia, and it interacted with smoking, fasting blood glucose, and hypertension to increase but with the female factor to decrease the prevalence of hyperlipidemia in Han individuals. The EHBP1 rs2710642 and rs10496099 SNPs and gene–environment interactions were associated with serum lipid profiles and hyperlipidemia, which is of ethnic specificity to our study populations.

Integrated analysis of gene expression and copy number variations in MET proto‑oncogene‑transformed human primary osteoblasts.

The aim of the present study was to screen the potential osteosarcoma (OS)‑associated genes and to obtain additional insight into the pathogenesis of OS. Transcriptional profile (ID: GSE28256) and copy number variations (CNV) profile were downloaded from Gene Expression Omnibus database. Differentially expressed genes (DEGs) between MET proto‑oncogene‑transformed human primary osteoblast (MET‑HOB) samples and the control samples were identified using the Linear Models for Microarray Data package. Subsequently, CNV areas and CNVs were identified using cut‑off criterion of >30%‑overlap within the cases using detect_cnv.pl in PennCNV. Genes shared in DEGs and CNVs were obtained and discussed. Additionally, the Database for Annotation, Visualization and Integrated Discovery was used to identify significant Gene Ontology (GO) functions and pathways in DEGs with P<0.05. A total of 1,601 DEGs were screened out in MET‑HOBs and compared with control samples, including 784 upregulated genes, such as E2F transcription factor 1 (E2F1) and 2 (E2F2) and 817 downregulated genes, such as retinoblastoma 1 (RB1) and cyclin D1 (CCND1). DEGs were enriched in 344 GO terms, such as extracellular region part and extracellular matrix and 14 pathways, including pathways in cancer and extracellular matrix‑receptor interaction. Additionally, 239 duplications and 439 deletions in 678 genes from 1,313 chromosome regions were detected. A total of 12 genes were identified to be CNV‑driven genes, including cadherin 18, laminin subunit α 1, spectrin β, erythrocytic, ciliary rootlet coiled‑coil, rootletin pseudogene2, β‑1,4-N-acetyl-galactosaminyltransferase 1, G protein regulated inducer of neurite outgrowth 1, EH domain binding protein 1‑like 1, growth factor independent 1, cathepsin Z, WNK lysine deficient protein kinase 1, glutathione S‑transferase mu 2 and microsomal glutathione S‑transferase1. Therefore, cell cycle‑associated genes including E2F1, E2F2, RB1 and CCND1, and cell adhesion‑associated genes, such as CDH18 and LAMA1 may be used as diagnosis and/or therapeutic markers for patients with OS.

EHBP1L1 coordinates Rab8 and Bin1 to regulate apical-directed transport in polarized epithelial cells.

The highly conserved Rab guanosine triphosphatase (GTPase) Rab8 plays a role in exocytosis toward the polarized plasma membrane in eukaryotic cells. In murine Rab8-deficient small intestine cells, apical proteins are missorted into lysosomes. In this study, we identified a novel Rab8-interacting protein complex containing an EH domain-binding protein 1-like 1 (EHBP1L1), Bin1/amphiphysin II, and dynamin. Biochemical analyses showed that EHBP1L1 directly bound to GTP-loaded Rab8 and Bin1. The spatial dependency of these complexes at the endocytic recycling compartment (ERC) was demonstrated through overexpression and knockdown experiments. EHBP1L1- or Bin1-depleted or dynamin-inhibited small intestine organoids significantly accumulated apical membrane proteins but not basolateral membrane proteins in lysosomes. Furthermore, in EHBP1L1-deficient mice, small intestine cells displayed truncated and sparse microvilli, suggesting that EHBP1L1 maintains the apical plasma membrane by regulating apical transport. In summary, our data demonstrate that EHBP1L1 links Rab8 and the Bin1-dynamin complex, which generates membrane curvature and excises the vesicle at the ERC for apical transport.

Atorvastatin prevents ATP-driven invasiveness via P2X7 and EHBP1 signaling in PTEN-expressing prostate cancer cells

Epidemiological studies indicate that statins, cholesterol-lowering drugs, prevent aggressive prostate cancer and other types of cancer. Employing essentially non-prostate cell lines, we previously showed that statins rapidly downregulate nuclear levels of phosphorylated Akt via P2X7, a purinergic receptor recently implicated in invasive growth. Here, we present studies on phosphatase and tensin homolog deleted on chromosome 10 (PTEN)-positive prostatic cells. We document an involvement of EH domain-binding protein 1 (EHBP1), previously associated with aggressive prostate cancer and insulin-stimulated trafficking and cell migration, in P2X7 signaling. We also show that EHBP1 is essential for an anti-invasive effect of atorvastatin. Furthermore, EHBP1 interacted with P-Rex1, a guanine nucleotide exchange factor previously implicated in invasive growth. Mevalonate did not prevent this anti-invasive effect of atorvastatin. These data indicate that atorvastatin modulates invasiveness via P2X7, EHBP1 and P-Rex1. Interestingly, the interaction between EHBP1 and P-Rex1 was not induced by extracellular adenosine triphosphate (ATP), the endogenous P2X7 ligand, and statins counteracted invasiveness stimulated by extracellular ATP. In support of these experimental data, a population-based genetic analysis showed that a loss of function allele in the P2X7 gene (rs3751143) associated with non-aggressive cancer, and the common allele with aggressive cancer. Our data indicate a novel signaling pathway that inhibits invasiveness and that is druggable. Statins may reduce the risk of aggressive prostate cancer via P2X7 and by counteracting invasive effects of extracellular ATP.

Genetic Susceptibility Loci, Pesticide Exposure and Prostate Cancer Risk

Uncovering SNP (single nucleotide polymorphisms)-environment interactions can generate new hypotheses about the function of poorly characterized genetic variants and environmental factors, like pesticides. We evaluated SNP-environment interactions between 30 confirmed prostate cancer susceptibility loci and 45 pesticides and prostate cancer risk in 776 cases and 1,444 controls in the Agricultural Health Study. We used unconditional logistic regression to estimate odds ratios (ORs) and 95% confidence intervals (CIs). Multiplicative SNP-pesticide interactions were calculated using a likelihood ratio test. After correction for multiple tests using the False Discovery Rate method, two interactions remained noteworthy. Among men carrying two T alleles at rs2710647 in EH domain binding protein 1 (EHBP1) SNP, the risk of prostate cancer in those with high malathion use was 3.43 times those with no use (95% CI: 1.44–8.15) (P-interaction = 0.003). Among men carrying two A alleles at rs7679673 in TET2, the risk of prostate cancer associated with high aldrin use was 3.67 times those with no use (95% CI: 1.43, 9.41) (P-interaction = 0.006). In contrast, associations were null for other genotypes. Although additional studies are needed and the exact mechanisms are unknown, this study suggests known genetic susceptibility loci may modify the risk between pesticide use and prostate cancer.

Quantitative Proteomic Analysis of PCSK9 Gain of Function in Human Hepatic HuH7 Cells

Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays an important role in cholesterol homeostasis, mediating degradation of the liver low-density lipoprotein receptor (LDLR). In fact, gain- and loss-of-function PCSK9 variations in human populations associate with hyper- or hypo- cholesterolemia, respectively. Exactly how PCSK9 promotes degradation of the LDLR, the identity of the other biomolecules involved in this process, and the global effect of PCSK9 on other proteins has not been thoroughly studied. Here we employ stable isotope labeling with amino acids in cell culture (SILAC) to present the first quantitative, subcellular proteomic study of proteins affected by the stable overexpression of a gain-of-function PCSK9 membrane-bound chimera (PCSK9-V5-ACE2) in comparison to control, empty vector transfections in a human hepatocyte (HuH7) cell line. The expression level of 327 of 5790 peptides was modified by PCSK9-V5-ACE2 overexpression. Immunoblotting was carried out for the control transferrin receptor, shown to be unaffected in cells overexpressing PCSK9-V5-ACE2, thus validating our SILAC results. We also used immunoblotting to confirm the novel SILAC results of up- and down-regulation of several proteins in cells overexpressing PCSK9-V5-ACE2. Moreover, we documented the novel down-regulation of the EH domain binding protein-1 (EHBP1) in a transgenic PCSK9 mouse model and its up-regulation in a PCSK9 knockout mouse model.

EHDS are serine phosphoproteins: EHD1 phosphorylation is enhanced by serum stimulation

Endocytic processes are mediated by multiple protein-protein interacting modules and regulated by phosphorylation and dephosphorylation. The Eps15 homology domain containing protein 1 (EHD1) has been implicated in regulating recycling of proteins, internalized both in clathrin-dependent and clathrin-independent endocytic pathways, from the recycling compartment to the plasma membrane. EHD1 was found in a complex with clathrin, adaptor protein complex-2 (AP-2) and insulin-like growth factor-1 receptor (IGF-1R), and was shown to interact with Rabenosyn-5, SNAP29, EHBP1 (EH domain binding protein 1) and syndapin I and II. In this study, we show that EHD1, like the other human EHDs, undergoes serine-phosphorylation. Our results also indicate that EHD1 is a serum-inducible serine-phosphoprotein and that PKC (protein kinase C) is one of its kinases. In addition, we show that inhibitors of clathrin-mediated endocytosis decrease EHD1 phosphorylation, while inhibitors of caveolinmediated endocytosis do not affect EHD1 phosphorylation. The results of experiments in which inhibitors of endocytosis were employed strongly suggest that EHD1 phosphorylation occurs between early endosomes and the endocytic recycling compartment.

EHD2 and the Novel EH Domain Binding Protein EHBP1 Couple Endocytosis to the Actin Cytoskeleton

Here we identified two novel proteins denoted EH domain protein 2 (EHD2) and EHD2-binding protein 1 (EHBP1) that link clathrin-mediated endocytosis to the actin cytoskeleton. EHD2 contains an N-terminal P-loop and a C-terminal EH domain that interacts with NPF repeats in EHBP1. Disruption of EHD2 or EHBP1 function by small interfering RNA-mediated gene silencing inhibits endocytosis of transferrin into EEA1-positive endosomes as well as GLUT4 endocytosis into cultured adipocytes. EHD2 localizes with cortical actin filaments, whereas EHBP1 contains a putative actin-binding calponin homology domain. High expression of EHD2 or EHBP1 in intact cells mediates extensive actin reorganization. Thus EHD2 appears to connect endocytosis to the actin cytoskeleton through interactions of its N-terminal domain with membranes and its C-terminal EH domain with the novel EHBP1 protein.

Purification of an EH Domain-binding Protein from Rat Brain That Modulates the Gating of the Rat ether-à-go-goChannel

Mutations in the gene encoding ether-à-go-go (EAG) potassium channel impair the function of several classes of potassium currents, synaptic transmission, and learning in Drosophila. Absence of EAG abolishes the modulation of a broad group of potassium currents. EAG has been proposed to be a regulatory subunit of different potassium channels. To further explore this regulatory role we searched for signaling molecules that associate with EAG protein. We have purified a approximately 95-kDa protein from rat brain membranes that binds to EAG. When co-expressed in mammalian cells this protein coimmunoprecipites with EAG and alters the gating of EAG channels. Expression of this protein is regulated during neuronal differentiation. The protein is identical to the recently reported rat protein epsin, which is an EH domain-binding protein similar to the Xenopus mitotic phosphoprotein MP90. These results show that proteins of the epsin family are modulators of channel activity that may link signaling pathways, or the cell cycle, to EAG and thus to various potassium channel functions.

EH domain-dependent interactions between Eps15 and clathrin-coated vesicle protein p95.

The endocytic protein Eps15 contains three copies of the EH domain, a protein module thought to function in protein-protein interactions. Using overlay assays with an Eps15 EH domain fusion protein, we have now identified a protein of 95 kDa (p95) as a major EH domain-binding partner in a wide variety of tissues. The amino acids asparagine-proline-phenylalanine (NPF) form the core of an EH domain-binding motif and three NPF repeats are found in the endocytic protein synaptojanin-170. We have confirmed previous studies indicating that synaptojanin-170 is an EH domain-binding protein, and have used peptide blocking experiments to demonstrate that the interaction is mediated through the NPF repeats. Interestingly, the same peptide also blocks EH domain-binding to p95. Finally, we have shown that p95 is enriched on clathrin-coated vesicles, suggesting an endocytic role for the protein. These data support an important role for EH domain-NPF motif interactions in endocytosis.