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Attaching and effacing (AE) lesion formation on enterocytes by enteropathogenic Escherichia coli (EPEC) requires the EPEC type III secretion system (T3SS). Two T3SS effectors injected into the host cell during infection are the atypical kinases, NleH1 and NleH2. However, the host targets of NleH1 and NleH2 kinase activity during infection have not been reported. Here phosphoproteomics identified Ser775 in the microvillus protein Eps8 as a bona fide target of NleH1 and NleH2 phosphorylation. Both kinases interacted with Eps8 through previously unrecognized, noncanonical “proline-rich” motifs, PxxDY, that bound the Src Homology 3 (SH3) domain of Eps8. Structural analysis of the Eps8 SH3 domain bound to a peptide containing one of the proline-rich motifs from NleH showed that the N-terminal part of the peptide adopts a type II polyproline helix, and its C-terminal “DY” segment makes multiple contacts with the SH3 domain. Ser775 phosphorylation by NleH1 or NleH2 hindered Eps8 bundling activity and drove dispersal of Eps8 from the AE lesion during EPEC infection. This finding suggested that NleH1 and NleH2 altered the cellular localization of Eps8 and the cytoskeletal composition of AE lesions during EPEC infection.

Pathogenic variants in the EPS8 gene result in nonsyndromic hearing loss. This gene encodes the EPS8 protein in cochlear inner hair cells and performs critical roles in stimulating actin polymerization and bundling. Thus far, only four pathogenic variations in EPS8 have been described. In this study, we report the fifth pathogenic variant in the EPS8 gene in an Iranian patient with DFNB102. Furthermore, we review literature cases with EPS8 mutations.

ABSTRACT The aim of this study was to determine new polymorphisms in GDF5 (growth differentiation factor 5 (CDMP-1)) and EPS8 (epidermal growth factor receptor substrate 8). After the PCR analysis of the identified gene regions of 72 head Holstein cattle, samples were separated by the high-resolution melting analysis (HRMA), and sequence analysis was applied to some randomly selected samples. New polymorphisms were identified in the determined genes and gene regions as a result of the sequence analysis. In the 1st exon of the EPS8 gene, 9 polymorphic regions were identified as g.94554132C>T, g.94554252G>C, g.94554348T>G, g.94554354C>G, g.94554372C>G, g.94554389A>G, 94554392C>G, 94554399A>G, and 94554439C>G. Within the region examined in the 2nd exon of the EPS8 gene, only the g.94555920T>G polymorphism was identified. In the 1st exon of the GDF5 gene, the g.65340723G>A and g.65340727T>C polymorphisms were identified. In the 2nd exon of the GDF5 gene, the g.65340902A>G polymorphism was identified. No polymorphism was found in the examined 2nd intron region of the GDF5 gene. As a result, new polymorphic regions were detected on EPS8 and GDF5 genes by HRMA, genotype and allele gene frequencies of polymorphic regions were determined. Hardy-Weinberg equilibrium test generally shown that the distribution of genotype frequencies in polymorphic regions is in genetic equilibrium.

Within the confines of tissues, cancer cells can use blebs to migrate. Eps8 is an actin bundling and capping protein whose capping activity is inhibited by Erk, a key MAP kinase that is activated by oncogenic signaling. We tested the hypothesis that Eps8 acts as an Erk effector to modulate actin cortex mechanics and thereby mediate bleb-based migration of cancer cells. Cells confined in a non-adhesive environment migrate in the direction of a very large ‘leader bleb.’ Eps8 bundling activity promotes cortex tension and intracellular pressure to drive leader bleb formation. Eps8 capping and bundling activities act antagonistically to organize actin within leader blebs, and Erk mediates this effect. An Erk biosensor reveals concentrated kinase activity within leader blebs. Bleb contents are trapped by the narrow neck that separates the leader bleb from the cell body. Thus, Erk activity promotes actin bundling by Eps8 to enhance cortex tension and drive the bleb-based migration of cancer cells under non-adhesive confinement. Cells within an animal have to be able to move both during development and later stages of life. For example, white blood cells have to move around the body and into tissues to fight off infections. Normally, cell movement is heavily controlled and will only happen when it is necessary to keep an animal healthy. However, cancer cells can bypass these controls and ‘metastasize’, or spread to new sites in the body. Cells can move in several different ways: on the one hand, cells can use ‘mesenchymal’ movement, in which the skeleton-like scaffolding of molecules within a cell rearranges to push the cell forward. On the other hand, cells can employ ‘amoeboid’ movement, in which they squeeze their way forward by building up pressure in the cell. Although these different types of movement are only used by some healthy cells and not others, cancer cells can switch between the two. How they do this is still unclear, but now Logue et al. have studied this question using several microscopy techniques. Logue et al. watched skin cancer (or melanoma) cells migrating between a glass plate and a slab of agar, which mimics the confined spaces that cancer cells have to move through within the body. The images showed that the cancer cells formed so-called ‘leader blebs’, finger-like projections that put cells on the right track. The experiments revealed that a protein called Eps8 was responsible for making the skin cancer cells move in this amoeboid fashion. The ‘blebbing’ caused by Eps8 is turned on by another protein called Erk that is often overactive in melanoma cells. Furthermore, Erk can accumulate near and within the cell blebs and this leads to the increased movement of the skin cancer cells. Studying cell movement in melanoma is particularly important because it is the metastatic tumors that kill patients. Therefore, increasing our basic understanding of how cells migrate could eventually lead to better treatment options that stop cancer cells from spreading.

Influenza is a zoonotic respiratory disease of major public health interest due to its pandemic potential, and a threat to animals and the human population. The influenza A virus genome consists of eight single-stranded RNA segments sequestered within a protein capsid and a lipid bilayer envelope. During host cell entry, cellular cues contribute to viral conformational changes that promote critical events such as fusion with late endosomes, capsid uncoating and viral genome release into the cytosol. In this focused review, we concisely describe the virus infection cycle and highlight the recent findings of host cell pathways and cytosolic proteins that assist influenza uncoating during host cell entry.

Epidermal growth factor receptor pathway substrate number 8 (EPS8) has been reported to be critical in mediating tumor progression. However, the molecular and biological consequences of EPS8 overexpression remain unclear. Here we evaluated whether EPS8 increased DNA damage repair in non-small-cell lung carcinoma (NSCLC) cells and the mechanism of EPS8-mediated DNA damage repair which influenced chemosensitivity. Serial studies of functional experiments revealed that EPS8 knockdown inhibited cell growth, induced cell-cycle arrest and increased cisplatin therapeutic effects on NSCLC. EPS8 was found to induce DNA damage repair via upregulation of phosphorylated-ATM and downregulation of the tumor suppressor p53 and G1 cell kinase inhibitor p21. Moreover, in conjunction with cisplatin, decreasing EPS8 protein levels further increased p53 protein level and inhibited ATM signaling. Transplanted tumor studies were also performed to demonstrate that EPS8 knockdown inhibited tumor growth and sensitized tumors to cisplatin treatment. In conclusion, we have described a novel molecular mechanism through which EPS8 is likely to be involved in cancer progression and chemoresistance via DNA damage repair, indicating that EPS8 expression may influence the response to chemotherapy. Therefore, targeting EPS8 may be a potential therapeutic approach for patients with NSCLC.

Exosomes are small vesicles found in extracellular environments including blood, urine, and cell culture medium. Their contents are cell-type specific, and molecules embedded in exosomes can be useful fluid-based clinical biomarkers. To identify proteins with metastatic marker potential, we conducted a comparative exosomal proteome analysis using human pancreatic cancer cell lines derived from metastasis, ascites, and primary tumors. Metastatic potential of cell lines was assessed by migratory and invasive activities. A pancreatic cancer cell line from metastasis (SU.86.86) revealed 23-fold and 20-fold increases in cell migratory and invasive activities, respectively, compared to the MIA PaCa-2 cell line derived from primary tumor cells. Liquid chromatography-mass spectrometry-based proteome analysis and subsequent validation by immunoblot analysis revealed that epidermal growth factor receptor pathway substrate 8 (Eps8) was highly abundant in exosomes from metastasis-derived SU.86.86 cells. Comparison of 12 pancreatic cancer cell lines derived from different stages of malignancy revealed a strong relationship between exosomal Eps8 protein levels and cell motile activities (migration: r=0.85, P=4.2×10-4; invasion: r=0.60, P=3.2×10-2). Conversely, relationships between intracellular Eps8 protein levels and cell motile activities were moderate (migration: r=0.65, P=2.0×10-2; invasion: r=0.51, P=9.2×10-2). It was therefore concluded that exosomal Eps8 protein levels were correlated with the migratory cell potential of human pancreatic cancer cells, indicating that exosomal Eps8 has the potential to be a metastatic biomarker for human pancreatic cancer.

The actin cytoskeleton usually lies beneath the plasma membrane. When the membrane-associated actin cytoskeleton is transiently disrupted or the intracellular pressure is increased, the plasma membrane detaches from the cortex and protrudes. Such protruded membrane regions are called blebs. However, the molecular mechanisms underlying membrane blebbing are poorly understood. This study revealed that epidermal growth factor receptor kinase substrate 8 (Eps8) and ezrin are important regulators of rapid actin reassembly for the initiation and retraction of protruded blebs. Live-cell imaging of membrane blebbing revealed that local reassembly of actin filaments occurred at Eps8- and activated ezrin-positive foci of membrane blebs. Furthermore, we found that a RhoA–ROCK–Rnd3 feedback loop determined the local reassembly sites of the actin cortex during membrane blebbing.

Oral squamous cell carcinoma (OSCC) is a lethal disease and early death usually occurs as a result of local invasion and regional lymph node metastases. Current treatment regimens are, to a certain degree, inadequate, with a 5-year mortality rate of around 50% and novel therapeutic targets are urgently required. Using expression microarrays, we identified the eps8 gene as being overexpressed in OSCC cell lines relative to normal oral keratinocytes, and confirmed these findings using RT–PCR and western blotting. In human tissues, we found that Eps8 was upregulated in OSCC (32% of primary tumors) compared with normal oral mucosa, and that expression correlated significantly with lymph node metastasis ( P =0.032), suggesting a disease-promoting effect. Using OSCC cell lines, we assessed the functional role of Eps8 in tumor cells. Although suppression of Eps8 produced no effect on cell proliferation, both cell spreading and migration were markedly inhibited. The latter cell functions may be modulated through the small GTP-ase, Rac1 and we used pull-down assays to investigate the role of Eps8 in Rac1 signaling. We found that αvβ6- and α5β1-integrin-dependent activation of Rac1 was mediated through Eps8. Knockdown of either Eps8 or Rac1, inhibited integrin-dependent cell migration similarly and transient expression of constitutively active Rac1 restored migration of cells in which Eps8 expression had been suppressed. We also showed that knockdown of Eps8 inhibited tumor cell invasion in an organotypic model of OSCC. These data suggest that Eps8 and Rac1 are part of an integrated signaling pathway modulating integrin-dependent tumour cell motility and identify Eps8 as a possible therapeutic target.

Background Polarity is necessary for epithelial cells to perform distinct functions at their apical and basal surfaces. Oral epithelial cell-derived ameloblasts at secretory stage (SABs) synthesize large amounts of enamel matrix proteins (EMPs), largely amelogenins. EMPs are unidirectionally secreted into the enamel space through their apical cytoplasmic protrusions, or Tomes’ processes (TPs), to guide the enamel formation. Little is known about the transcriptional regulation underlying the establishment of cell polarity and unidirectional secretion of SABs. Results The higher-order chromatin architecture of eukaryotic genome plays important roles in cell- and stage-specific transcriptional programming. A genome organizer, special AT-rich sequence-binding protein 1 (SATB1), was discovered to be significantly upregulated in ameloblasts compared to oral epithelial cells using a whole-transcript microarray analysis. The Satb1 −/− mice possessed deformed ameloblasts and a thin layer of hypomineralized and non-prismatic enamel. Remarkably, Satb1 −/− ameloblasts at the secretory stage lost many morphological characteristics found at the apical surface of wild-type ( wt) SABs, including the loss of Tomes’ processes, defective inter-ameloblastic adhesion, and filamentous actin architecture. As expected, the secretory function of Satb1 −/− SABs was compromised as amelogenins were largely retained in cells. We found the expression of epidermal growth factor receptor pathway substrate 8 ( Eps8 ), a known regulator for actin filament assembly and small intestinal epithelial cytoplasmic protrusion formation, to be SATB1 dependent. In contrast to wt SABs, EPS8 could not be detected at the apical surface of Satb1 −/− SABs. Eps8 expression was greatly reduced in small intestinal epithelial cells in Satb1 −/− mice as well, displaying defective intestinal microvilli. Conclusions Our data show that SATB1 is essential for establishing secretory ameloblast cell polarity and for EMP secretion. In line with the deformed apical architecture, amelogenin transport to the apical secretory front and secretion into enamel space were impeded in Satb1 −/− SABs resulting in a massive cytoplasmic accumulation of amelogenins and a thin layer of hypomineralized enamel. Our studies strongly suggest that SATB1-dependent Eps8 expression plays a critical role in cytoplasmic protrusion formation in both SABs and in small intestines. This study demonstrates the role of SATB1 in the regulation of amelogenesis and the potential application of SATB1 in ameloblast/enamel regeneration.