WW domain-containing E3 ubiquitin protein ligase 1 (WWP1) plays an important role in the proliferation of tumor cells and the lifespan of Caenorhabditis elegans. However, the role of WWP1 in cellular senescence is still unknown. Here, we show that the expression patterns of p27 Kip1 and WWP1 are inversely correlated during cellular senescence. Moreover, the overexpression of WWP1 delayed senescence, whereas the knockdown of WWP1 led to premature senescence in human fibroblasts. Furthermore, we demonstrate that WWP1 repressed endogenous p27Kip1 expression through ubiquitin-proteasome-mediated degradation. Additionally, WWP1 had a strong preference for catalyzing the Lys-48-linked polyubiquitination of p27 Kip1 in vitro. Finally, we demonstrate that WWP1 markedly inhibited the replicative senescence induced by p27Kip1 by promoting p27 Kip1 degradation. Therefore, our study provides a new molecular mechanism for the regulation of cellular senescence.The concept of replicative senescence is based on the inability of cells to divide indefinitely in culture. Instead, the cells enter an irreversible state of proliferative arrest with specific associated changes in cellular morphology and gene expression. The senescent phenotype is characterized by growth inhibition with enlarged and flattened cellular morphology (1), highly active senescence-associated ␤-galactosidase (SA-␤-gal) 2 (2), and the accumulation of senescence-associated heterochromatin foci (SAHF) (3, 4). In a sense, cellular senescence is a reflection of organism aging, and investigation into replicative senescence may thus provide information about the molecular mechanisms of organism senescence and aging-related diseases. In recent years, increasing numbers of molecular mechanisms of cellular senescence have been studied. It is well accepted that there are three pathways that play key roles in replicative senescence, namely the p16INK4a pathway, the p53/ p21Cip1 pathway, and the p27 Kip1 pathway (5). However, in comparison with the first two pathways, relatively few studies have focused on the p27 Kip1 pathway. The majority of proteins involved in replicative senescence are modified by polyubiquitin, which directs proteins for degradation by the 26 S proteasome. For example, cyclin-dependent kinase inhibitors, which are of special relevance to senescence, are mainly regulated by the ubiquitin-proteasome system. The ubiquitination cascade operates by the sequential action of the E1, E2, and E3 enzymes. E3 ubiquitin ligases are significant in cellular regulation because E3 enzymes specifically recognize the substrate to be modified. It is well established that the E3 enzymes control substrate specificity. WW domain-containing E3 ubiquitin protein ligase 1 (WWP1) is an E3 ubiquitin ligase first identified by its WW domain (6). WWP1 belongs to the Nedd4-like family of E3 ubiquitin ligases, a subfamily of HECT, which contains Nedd4, Itch, WWP2, AIP4, and RPF1 (7, 8). Members of this family have been shown to be involved in the regulation of cellular signaling...

Characterization of the bacterial composition and functional repertoires of microbiome samples is the most common application of metagenomics. Although deep whole-metagenome shotgun sequencing (WMS) provides high taxonomic resolution, it is generally cost-prohibitive for large longitudinal investigations. Until now, 16S rRNA gene amplicon sequencing (16S) has been the most widely used approach and usually cooperates with WMS to achieve cost-efficiency. However, the accuracy of 16S results and its consistency with WMS data have not been fully elaborated, especially by complicated microbiomes with defined compositional information. Here, we constructed two complex artificial microbiomes, which comprised more than 60 human gut bacterial species with even or varied abundance. Utilizing real fecal samples and mock communities, we provided solid evidence demonstrating that 16S results were of poor consistency with WMS data, and its accuracy was not satisfactory. In contrast, shallow whole-metagenome shotgun sequencing (shallow WMS, S-WMS) with a sequencing depth of 1 Gb provided outputs that highly resembled WMS data at both genus and species levels and presented much higher accuracy taxonomic assignments and functional predictions than 16S, thereby representing a better and cost-efficient alternative to 16S for large-scale microbiome studies.

Cellular senescence-inhibited gene (CSIG) protein significantly prolongs the progression of replicative senescence, but its role in tumorigenesis is unclear. To reveal the role of CSIG in HCC, we determined its expression in HCC tissues and surrounding tissues and its functions in tumor cell proliferation in vitro and in vivo. CSIG protein was overexpressed in 86.4% of the human HCC cancerous tissues as compared with matched surrounding tissues, and its protein expression was greater in HCC cells than the non-transformed hepatic cell line L02. Furthermore, upregulation of CSIG significantly increased the colony formation of SMMC7721 and HepG2 cells, and silencing CSIG could induce cell cycle arrest and cell apoptosis. The tumorigenic ability of CSIG was confirmed in vivo in a mouse xenograft model. Our results showed that CSIG promoted the proliferation of HepG2 and SMMC7721 cells in vivo. Finally, CSIG protein directly interacted with c-MYC protein and increased c-MYC protein levels; the ubiquitination and degradation of c-MYC protein was increased with knockdown of CSIG. CSIG could also increase the expression of c-MYC protein in SMMC7721 cells in vivo, and it was noted that the level of c-MYC protein was also elevated in most human cancerous tissues with high level of CSIG.

Background: Transcription factor 4 (TCF4) is found to be associated with schizophrenia. TCF4 mutations also cause Pitt-Hopkins Syndrome, a neurodevelopmental disorder associated with severe mental retardation. However, the function of TCF4 during brain development remains unclear.Results: Here, we report that Tcf4 is expressed in the developing cerebral cortex. In utero suppression of Tcf4 arrested neuronal migration, leading to accumulation of ectopic neurons in the intermediate zone. Knockdown of Tcf4 impaired leading process formation. Furthermore, Bone Morphogenetic Protein 7 (Bmp7) is upregulated in Tcf4-deficient neurons. In vivo gain of function and rescue experiments demonstrated that Bmp7 is the major downstream effector of Tcf4 required for neuronal migration.Conclusion: Thus, we have uncovered a new Tcf4/Bmp7-dependent mechanism underlying neuronal migration, and provide insights into the pathogenesis of neurodevelopmental disorders.

BackgroundLoss of function mutations in RAB18, has been identified in patients with the human neurological and developmental disorder Warburg Micro syndrome. However, the function of RAB18 in brain remains unknown.ResultsIn this study, we report that RAB18 is a critical regulator of neuronal migration and morphogenesis. Using in utero electroporation suppression of RAB18 in the mouse brain impairs radial migration. Overexpression of dominant negative RAB18 or disruption of RAB3GAP (RAB18GEF) also results in delayed neuronal migration in the developing mouse cortex and inhibition of neurite growth in vitro. Moreover, loss of RAB18 induces an acceleration of N-cadherin degradation by lysosomal pathway resulting in the decrease of surface level of N-cadherin on neurons.ConclusionsRAB18 regulates neuronal migration and morphogenesis during development. Our findings highlight the critical role of RAB3GAP-RAB18 pathway in the developing cerebral cortex and might explain some of clinical features observed in patients with Warburg Micro syndrome.Electronic supplementary materialThe online version of this article (doi:10.1186/s13041-016-0198-2) contains supplementary material, which is available to authorized users.