α-Hemolysin promotes uropathogenic E. coli persistence in bladder epithelial cells via abrogating bacteria-harboring lysosome acidification.

There is a growing consensus that a significant proportion of recurrent urinary tract infections are linked to the persistence of uropathogens within the urinary tract and their re-emergence upon the conclusion of antibiotic treatment. Studies in mice and human have revealed that uropathogenic Escherichia coli (UPEC) can persist in bladder epithelial cells (BECs) even after the apparent resolution of the infection. Here, we found that, following the entry of UPEC into RAB27b+ fusiform vesicles in BECs, some bacteria escaped into the cytoplasmic compartment via a mechanism involving hemolysin A (HlyA). However, these UPEC were immediately recaptured within LC3A/B+ autophagosomes that matured into LAMP1+ autolysosomes. Thereafter, HlyA+ UPEC-containing lysosomes failed to acidify, which is an essential step for bacterial elimination. This lack of acidification was related to the inability of bacteria-harboring compartments to recruit V-ATPase proton pumps, which was attributed to the defragmentation of cytosolic microtubules by HlyA. The persistence of UPEC within LAMP1+ compartments in BECs appears to be directly linked to HlyA. Thus, through intravesicular instillation of microtubule stabilizer, this host defense response can be co-opted to reduce intracellular bacterial burden following UTIs in the bladder potentially preventing recurrence.

Apolipoprotein E-mediated regulation of selenoprotein P transportation via exosomes.

Selenoprotein P (SELENOP), secreted from the liver, functions as a selenium (Se) supplier to other tissues. In the brain, Se homeostasis is critical for physiological function. Previous studies have reported that SELENOP co-localizes with the apolipoprotein E receptor 2 (ApoER2) along the blood-brain barrier (BBB). However, the mechanism underlying SELENOP transportation from hepatocytes to neuronal cells remains unclear. Here, we found that SELENOP was secreted from hepatocytes as an exosomal component protected from plasma kallikrein-mediated cleavage. SELENOP was interacted with apolipoprotein E (ApoE) through heparin-binding sites of SELENOP, and the interaction regulated the secretion of exosomal SELENOP. Using in vitro BBB model of transwell cell culture, exosomal SELENOP was found to supply Se to brain endothelial cells and neuronal cells, which synthesized selenoproteins by a process regulated by ApoE and ApoER2. The regulatory role of ApoE in SELENOP transport was also observed in vivo using ApoE-/- mice. Exosomal SELENOP transport protected neuronal cells from amyloid ß (Aß)-induced cell death. Taken together, our results suggest a new delivery mechanism for Se to neuronal cells by exosomal SELENOP.

Identification of a redox-modulatory interaction between selenoprotein W and 14-3-3 protein.

Selenoprotein W (SelW) contains a selenocysteine (Sec, U) in a conserved CXXU motif corresponding to the CXXC redox motif of thioredoxin, suggesting a putative redox function of SelW. We have previously reported that the binding of 14-3-3 protein to its target proteins, including CDC25B, Rictor and TAZ, is inhibited by the interaction of 14-3-3 protein with SelW. However, the binding mechanism is unclear. In this study, we sought to determine the binding site of SelW to understand the regulatory mechanism of the interaction between SelW and 14-3-3 and its biological effects. Phosphorylated Ser(pS) or Thr(pT) residues in RSXpSXP or RXXXp(S/T)XP motifs are well-known common 14-3-3-binding sites, but Thr41, Ser59, and T69 of SelW, which are computationally predicted to serve are phosphorylation sites, were neither phosphorylation sites nor sites involved in the interaction. A mutant SelW in which Sec13 is changed to Ser (U13S) was unable to interact with 14-3-3 protein and thus did not inhibit the interaction of 14-3-3 to other target proteins. However, other Cys mutants of SelW(C10S, C33S and C37S) normally interacted with 14-3-3 protein. The interaction of SelW to 14-3-3 protein was enhanced by diamide or H2O2 and decreased by dithiothreitol (DTT). Taken together, these findings demonstrate that the Sec of SelW is involved in its interaction with 14-3-3 protein and that this interaction is increased under oxidative stress conditions. Thus, SelW may have a regulatory function in redox cell signaling by interacting with 14-3-3 protein.

Selenoprotein S is required for clearance of C99 through endoplasmic reticulum-associated degradation.

Amyloid beta precursor protein (APP) is normally cleaved by α-secretase, but can also be cleaved by ß-secretase (BACE1) to produce C99 fragments in the endoplasmic reticulum (ER) membrane. C99 is subsequently cleaved to amyloid ß (Aß), the aggregation of which is known to cause Alzheimer's disease. Therefore, C99 removing is for preventing the disease. Selenoprotein S (SelS) is an ER membrane protein participating in endoplasmic reticulum-associated degradation (ERAD), one of the stages in resolving ER stress of misfolded proteins accumulated in the ER. ERAD has been postulated as one of the processes to degrade C99; however, it remains unclear if the degradation depends on SelS. In this study, we investigated the effect of SelS on C99 degradation. We observed that both SelS and C99 were colocalized in the membrane fraction of mouse neuroblastoma Neuro2a (N2a) cells. While the level of SelS was increased by ER stress, the level of C99 was decreased. However, despite the induction of ER stress, there was no change in the amount of C99 in SelS knock-down cells. The interaction of C99 with p97(VCP), an essential component of the ERAD complex, did not occur in SelS knock-down cells. The ubiquitination of C99 was decreased in SelS knock-down cells. We also found that the extracellular amount of Aß1-42 was relatively higher in SelS knock-down cells than in control cells. These results suggest that SelS is required for C99 degradation through ERAD, resulting in inhibition of Aß production.

Selenoprotein S-dependent Selenoprotein K Binding to p97(VCP) Protein Is Essential for Endoplasmic Reticulum-associated Degradation.

Cytosolic valosin-containing protein (p97(VCP)) is translocated to the ER membrane by binding to selenoprotein S (SelS), which is an ER membrane protein, during endoplasmic reticulum-associated degradation (ERAD). Selenoprotein K (SelK) is another known p97(VCP)-binding selenoprotein, and the expression of both SelS and SelK is increased under ER stress. To understand the regulatory mechanisms of SelS, SelK, and p97(VCP) during ERAD, the interaction of the selenoproteins with p97(VCP) was investigated using N2a cells and HEK293 cells. Both SelS and SelK co-precipitated with p97(VCP). However, the association between SelS and SelK did not occur in the absence of p97(VCP). SelS had the ability to recruit p97(VCP) to the ER membrane but SelK did not. The interaction between SelK and p97(VCP) did not occur in SelS knockdown cells, whereas SelS interacted with p97(VCP) in the presence or absence of SelK. These results suggest that p97(VCP) is first translocated to the ER membrane via its interaction with SelS, and then SelK associates with the complex on the ER membrane. Therefore, the interaction between SelK and p97(VCP) is SelS-dependent, and the resulting ERAD complex (SelS-p97(VCP)-SelK) plays an important role in ERAD and ER stress.

Pro178 and Pro183 of selenoprotein S are essential residues for interaction with p97(VCP) during endoplasmic reticulum-associated degradation.

During endoplasmic reticulum (ER)-associated degradation, p97(VCP) is recruited to the ER membrane through interactions with transmembrane proteins, such as selenoprotein S (SelS), selenoprotein K (SelK), hrd1, and gp78. SelS has a single-spanning transmembrane domain and protects cells from ER stress-induced apoptosis through interaction with p97(VCP). The cytosolic tail of SelS consists of a coiled-coil domain, a putative VCP-interacting motif (VIM), and an unpronounced glycine- and proline-rich secondary structure. To understand the regulatory mechanism of SelS during ER stress, we investigated the interaction of the protein with p97(VCP) using mouse neuroblastoma cells and human embryonic kidney 293 cells. The SelS expression level increased when ER stress was induced. In addition, the effect of ER stress was enhanced, and recruitment of p97(VCP) to the ER membrane was inhibited in SelS knockdown cells. The effect of SelS knockdown was rescued by ectopic expression of SelS U188C. p97(VCP) interacted with SelS U188C and was recruited to the ER membrane. The expression of SelS[ΔVIM], which is a VIM deletion mutant of SelS, also showed both a recovery effect and an interaction with p97(VCP) in cells. However, mutants in which the proline residue positions 178 or 183 of SelS were changed to alanine or were deleted did not interact with p97(VCP). The proline mutants did not rescue ER stress in SelS knockdown cells. These results suggest that both Pro(178) and Pro(183) of SelS play important roles in the translocation of p97(VCP) to the ER membrane and protect cells from ER stress.

S-Glutathionylation of mouse selenoprotein W prevents oxidative stress-induced cell death by blocking the formation of an intramolecular disulfide bond.

Mouse selenoprotein W (SELENOW) is a small protein containing a selenocysteine (Sec, U) and four cysteine (Cys, C) residues. The Sec residue in SELENOW is located within the conserved CXXU motif corresponding to the CXXC redox motif of thioredoxin (Trx). It is known that glutathione (GSH) binds to SELENOW and that this binding is involved in protecting cells from oxidative stress. However, the regulatory mechanisms controlling the glutathionylation of SELENOW in oxidative stress are unclear. In this study, using purified recombinant SELENOW in which Sec13 was changed to Cys, we found that SELENOW was glutathionylated at Cys33 and that this S-glutathionylation was enhanced by oxidative stress. We also found that the S-glutathionylation of SELENOW at Cys33 in HEK293 cells was due to glutathione S-transferase Pi (GSTpi) and that this modification was reversed by glutaredoxin1 (Grx1). In addition to the disulfide bond between the Cys10 and Cys13 of SELENOW, a second disulfide bond was formed between Cys33 and Cys87 under oxidative stress conditions. The second disulfide bond was reduced by Trx1, but the disulfide bond between Cys10 and Cys13 was not. The second disulfide bond was also reduced by glutathione, but the disulfide bond in the CXXC motif was not. The second disulfide bond of the mutant SELENOW, in which Cys37 was replaced with Ser, was formed at a much lower concentration of hydrogen peroxide than the wild type. We also observed that Cys37 was required for S-glutathionylation, and that S-glutathionylated SELENOW containing Cys37 protected the cells from oxidative stress. Furthermore, the SELENOW (C33, 87S) mutant, which could not form the second disulfide bond, also showed antioxidant activity. Taken together, these results indicate that GSTpi-mediated S-glutathionylation of mouse SELENOW at Cys33 is required for the protection of cells in conditions of oxidative stress, through inhibition of the formation of the second disulfide bond.

Degradation of selenoprotein S and selenoprotein K through PPARγ-mediated ubiquitination is required for adipocyte differentiation.

Adipocyte differentiation is known to be related with endoplasmic reticulum (ER) stress. We have reported that selenoprotein S (SelS) and selenoprotein K (SelK) have a function in the regulation of ER stress and ER-associated degradation. However, the association between adipocyte differentiation and the ER-resident selenoproteins, SelS and SelK, is unclear. In this study, we found that the levels of SelS and SelK were decreased during adipocyte differentiation and were inversely related to the levels of peroxisome proliferator-activated receptor γ (PPARγ), a central regulator of adipogenesis. It has been recently reported that PPARγ has E3 ubiquitin ligase activity. Here, we report that PPARγ directly interacts with both SelS and SelK via its ligand-binding domain to induce ubiquitination and degradation of the selenoproteins. Lysine residues at the 150th position of SelS and the 47th and 48th positions of SelK were the target sites for ubiquitination by PPARγ. We also found that adipocyte differentiation was inhibited when either SelS or SelK was not degraded by PPARγ. Thus, these data indicate that PPARγ-mediated ubiquitination and degradation of SelS and SelK is required for adipocyte differentiation.