Vaults are the largest (13 megadalton) cytoplasmic ribonucleoprotein particles known to exist in eukaryotic cells. They have a unique barrel-shaped structure with 8-fold symmetry. Although the precise function of vaults is unknown, their wide distribution and highly conserved morphology in eukaryotes suggests that their function is essential and that their structure must be important for their function. The 100-kDa major vault protein (MVP) constitutes ϳ75% of the particle mass and is predicted to form the central barrel portion of the vault. To gain insight into the mechanisms for vault assembly, we have expressed rat MVP in the Sf9 insect cell line using a baculovirus vector. Our results show that the expression of the rat MVP alone can direct the formation of particles that have biochemical characteristics similar to endogenous rat vaults and display the distinct vault-like morphology when negatively stained and examined by electron microscopy. These particles are the first example of a single protein polymerizing into a non-spherically, non-cylindrically symmetrical structure. Understanding vault assembly will enable us to design agents that disrupt vault formation and hence aid in elucidating vault function in vivo.Vaults are predominantly cytoplasmic ribonucleoprotein particles that have been conserved throughout evolution and are found in phylogenies as diverse as those of mammals, avians, amphibians, sea urchins, and slime molds (1). Many different roles including nucleocytoplasmic transport have been proposed for vaults since their first description in 1986 (2). However, their normal cellular function remains nebulous. Mammalian vaults comprise three proteins, the major vault protein (MVP) 1 (3), the vault poly(A)DP-ribose polymerase (VPARP) (4), and the telomerase-associated protein 1 (TEP1) (5) and one or more small untranslated RNAs (6). Vaults have been implicated in the phenomenon of multidrug resistance and as prognostic markers for cancer chemotherapy failure (7,8). One recent study has shown that the major vault protein is involved directly in the efflux of drugs from the nucleus (9). Although the majority of vaults are found in the cytoplasm, small amounts have been localized to the nuclear pore complexes (10). Recently a 31-Å resolution structure of the vault has been published indicating that vaults have a hollow interior consistent with a transport or sequestration function. Scanning transmission electron microscopic analysis has shown that the molecular mass of the vault is 12.9 Ϯ 1 MDa, and cryo-EM single-particle reconstruction has provided overall dimensions of 42 ϫ 75 nm (11). Freeze-etch images of the vault on polylysine-coated mica show that each half of the vault midsection can open into eight distinct "petals" (3), which has lead to the proposal that vaults may open and close in vivo. The MVP is presumed to be present in 96 copies/vault, based on the observed symmetry of the particle and the estimate that MVP accounts for ϳ75% of the total protein mass in the particle. In many way...
Epithelial-to-mesenchymal transition (EMT) is an important developmental process, participates in tissue repair, and occurs during pathologic processes of tumor invasiveness, metastasis, and tissue fibrosis. The molecular mechanisms leading to EMT are poorly understood. Although it is well documented that transforming growth factor (TGF)-β plays a central role in the induction of EMT, the targets of TGF-β signaling are poorly defined. We have shown earlier that Na,K-ATPase β 1 -subunit levels are highly reduced in poorly differentiated kidney carcinoma cells in culture and in patients' tumor samples. In this study, we provide evidence that Na,K-ATPase is a new target of TGF-β 1 -mediated EMT in renal epithelial cells, a model system used in studies of both cancer progression and fibrosis. We show that following treatment with TGF-β 1 , the surface expression of the β 1 -subunit of Na,K-ATPase is reduced, before well-characterized EMT markers, and is associated with the acquisition of a mesenchymal phenotype. RNAi-mediated knockdown confirmed the specific involvement of the Na,K-ATPase β 1 -subunit in the loss of the epithelial phenotype and exogenous overexpression of the Na,K-ATPase β 1 -subunit attenuated TGF-β 1 -mediated EMT. We further show that both Na,K-ATPase α-and β-subunit levels are highly reduced in renal fibrotic tissues. These findings reveal for the first time that Na,K-ATPase is a target of TGF-β 1 -mediated EMT and is associated with the progression of EMT in cancer and fibrosis. Mol Cancer Ther; 9(6); 1515-24. ©2010 AACR.
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