Endofin is an endosomal protein implicated in regulating membrane trafficking. It is characterized by the presence of a phosphatidylinositol 3-phosphate-binding FYVE domain positioned in the middle of the molecule. To determine its potential effectors or binding partners, we used the carboxyl-terminal half of endofin as bait to screen a human brain cDNA library in a yeast two-hybrid system. Three clones that encode TOM1 were recovered. TOM1 is a protein closely related to the VHS (VPS-27, Hrs, and STAM) domain-containing GGA family. Although the function of the GGAs in mediating Golgi to lysosomal trafficking is well established, the subcellular localization and function of TOM1 remain unknown. Glutathione S-transferase pull-down assays as well as co-immunoprecipitation experiments confirmed that the carboxyl-terminal half of endofin binds specifically to the carboxyl-terminal region of TOM1. Neither SARA nor Hrs, two other FYVE domain proteins, interact with this region of TOM1. Moreover, endofin does not interact with the analogous region of two other members of the TOM1 protein family, namely, TOM1-like 1 (TOM1-L1) or TOM1-like 2 (TOM1-L2). The carboxyl-terminal region of TOM1 was used as immunogen to generate TOM1-specific antibody. This antibody can distinguish TOM1 from the other family members as well as coimmunoprecipitate endogenous endofin. It also revealed the primarily cytosolic distribution of TOM1 in a variety of cell types by immunofluorescence analyses. In addition, sucrose density gradient analysis showed that both TOM1 and endofin can be detected in cellular compartments marked by the early endosomal marker EEA1. A marked recruitment of TOM1 to endosomes was observed in cells overexpressing endofin or its carboxylterminal fragment, indicating TOM1 to be an effector for endofin and suggesting a possible role for TOM1 in endosomal trafficking.Endofin is a member of the FYVE domain family of proteins. The FYVE finger domain (1) is involved in interaction with the phospholipid phosphatidylinositol 3-phosphate, which is found in significant levels on endosomal membranes (2). In many instances, therefore, the FYVE domain serves to localize the protein containing it to endosomes. In endofin, the FYVE domain appears to be necessary and sufficient to localize the protein to early endosomes (3). The FYVE domain is a modular motif that can function independent of its position on the molecular structure. In the case of endofin, the FYVE domain sits in the middle of the molecule, thereby effectively dividing the molecule into two halves.Apart from a few exceptions like the proteins FGD1 (faciogenital dysplasia gene product 1) (4), Frabin (5), and DFCP1 (double FYVE-containing protein 1) (6), members of the mammalian FYVE domain family have, in general, two characteristics in common: the presence of the FYVE domain and the consequential location of the proteins on endosomal membranes. The presence of these proteins on endosomal structures imply that they have roles in trafficking and/or signaling involving e...
BackgroundLong QT syndrome type 2 (LQT2) is the second most common type of all long QT syndromes. It is well-known that trafficking deficient mutant human ether-a-go-go-related gene (hERG) proteins are often involved in LQT2. Cells respond to misfolded and trafficking-deficient proteins by eliciting the unfolded protein response (UPR) and Activating Transcription Factor (ATF6) has been identified as a key regulator of the mammalian UPR. In this study, we investigated the role of ER chaperone proteins (Calnexin and Calreticulin) in the processing of G572R-hERG and E637K-hERG mutant proteins.MethodspcDNA3-WT-hERG, pcDNA3-G572R-hERG and pcDNA3-E637K-hERG plasmids were transfected into U2OS and HEK293 cells. Confocal microscopy and western blotting were used to analyze subcellular localization and protein expression. Interaction between WT or mutant hERGs and Calnexin/Calreticulin was tested by coimmunoprecipitation. To assess the role of the ubiquitin proteasome pathway in the degradation of mutant hERG proteins, transfected HEK293 cells were treated with proteasome inhibitors and their effects on the steady state protein levels of WT and mutant hERGs were examined.ConclusionOur results showed that levels of core-glycosylated immature forms of G572R-hERG and E637K-hERG in association with Calnexin and Calreticulin were higher than that in WT-hERG. Both mutant hERG proteins could activate the UPR by upregulating levels of active ATF6. Furthermore, proteasome inhibition increased the levels of core-glycosylated immature forms of WT and mutant hERGs. In addition, interaction between mutant hERGs and Calnexin/Calreticulin was stronger after proteasome inhibition, compared to WT-hERG. These results suggest that trafficking-deficient G572R-hERG and E637K-hERG mutant proteins can activate ER stress pathways and are targeted to the proteasome for degradation. Calnexin and Calreticulin play important roles in these processes.
Our studies demonstrate two miRNAs (miR-206 and miR-574-5p) that are significantly up-regulated in coronary artery disease (CAD) patients as compared with healthy controls, and the two miRNAs can be potential non-invasive biomarkers for early detection of CAD.
We compared the miRNA expression profiles of spermatozoa from adult men living in an environment contaminated with electronic waste with those living in a normal environment, using a miRNA microarray. There were 73 significantly upregulated and 109 downregulated miRNAs in spermatozoa from men in the polluted environment compared with controls. Ten miRNAs were expressed significantly differently between the control and polluted samples and this result were further confirmed through quantitative real-time polymerase chain reaction amplification. A bioinformatics pipeline was developed to distinguish relationship with spermatogenesis. Our data indicated specific miRNAs expression of spermatozoa in men living in environmentally polluted sites and suggested that miRNAs have a role in regulating spermatogenesis. We also propose a subclassification scheme for miRNAs for assisting future experimental and computational functional analyses.
Thymic stromal lymphopoietin (TSLP) is an IL-7-like cytokine that has been reported to be associated with several malignant tumors. The present study aimed to evaluate its role in epithelial ovarian carcinoma (EOC). The mRNA levels of TSLP in human EOC samples and EOC cell lines were determined. Then, the expression of TSLP was examined in 144 clinical tissue microarray samples and correlated with clinicopathological factors. Finally, the correlation between TSLP overexpression and prognosis of EOC patients was analyzed. Our data show that mRNA levels of TSLP were significantly higher in EOC tissues and cell lines. Chi-square tests revealed that TSLP overexpression in EOC was significantly associated with age, histological type, Federation of Gynecology and Obstetrics (FIGO) stage, histological differentiation, pelvic involvement, and lymphatic metastasis. Kaplan–Meier survival analysis revealed that poor prognosis was significantly correlated with older age, advanced FIGO stage, poor histological differentiation, pelvic involvement, lymphatic involvement, or TSLP overexpression (P<0.05). Additionally, multivariate Cox regression analysis confirmed pelvic involvement and TSLP overexpression as independent prognostic factors for both overall and disease-free survival. Taken altogether, TSLP overexpression reflects a more malignant phenotype and TSLP may be a novel biomarker for EOC.
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