Ezrin directly interacts with AQP2 and promotes its endocytosis.

The water channel aquaporin-2 (AQP2) is a major regulator of water homeostasis in response to vasopressin (VP). Dynamic trafficking of AQP2 relies on its close interaction with trafficking machinery proteins and the actin cytoskeleton. Here, we report the identification of ezrin, an actin-binding protein from the ezrin/radixin/moesin (ERM) family as an AQP2-interacting protein. Ezrin was first detected in a co-immunoprecipitation (co-IP) complex using an anti-AQP2 antibody in a proteomic analysis. Immunofluorescence staining revealed the co-expression of ezrin and AQP2 in collecting duct principal cells, and VP treatment caused redistribution of both proteins to the apical membrane. The ezrin-AQP2 interaction was confirmed by co-IP experiments with an anti-ezrin antibody, and by pulldown assays using purified full-length and FERM domain-containing recombinant ezrin. By using purified recombinant proteins, we showed that ezrin directly interacts with AQP2 C-terminus through its N-terminal FERM domain. Knocking down ezrin expression with shRNA resulted in increased membrane accumulation of AQP2 and reduced AQP2 endocytosis. Therefore, through direct interaction with AQP2, ezrin facilitates AQP2 endocytosis, thus linking the dynamic actin cytoskeleton network with AQP2 trafficking.

Characterization of the putative phosphorylation sites of the AQP2 C terminus and their role in AQP2 trafficking in LLC-PK1 cells.

Vasopressin (VP) stimulates a signaling cascade that results in phosphorylation and apical membrane accumulation of aquaporin-2 (AQP2), leading to water reabsorption by kidney collecting ducts. However, the roles of most C-terminal phosphorylation events in stimulated and constitutive AQP2 recycling are incompletely understood. Here, we generated LLC-PK1 cells containing point mutations of all potential phosphorylation sites in the AQP2 C terminus: S226, S229, T244, S256, S261, S264, and S269, to determine their impact on AQP2 trafficking. We produced an All Null AQP2 construct in which these serine (S) or threonine (T) residues were mutated to alanine (A) or glycine (G), and we then reintroduced the phosphorylation mimic aspartic acid (D) individually to each site in the All Null mutant. As expected, the All Null mutant does not accumulate at the plasma membrane in response to VP but still undergoes constitutive recycling, as shown by its membrane accumulation when endocytosis is blocked by methyl-ß-cyclodextrin (MßCD), and accumulation in a perinuclear patch at low temperature (20°C). Single phosphorylation mimics S226D, S229D, T244D, S261D, S264D, and S269D were insufficient to cause membrane accumulation of AQP2 alone or after VP treatment. However, AQP2 S256 reintroduced into the All Null mutant maintains its trafficking response to VP. We conclude that 1) constitutive recycling of AQP2 does not require phosphorylation at any C-terminal sites; 2) forced "phosphorylation" of sites in the AQP2 C terminus is insufficient to stimulate membrane accumulation in the absence of S256 phosphorylation; and 3) phosphorylation of S256 alone is necessary and sufficient to cause membrane accumulation of AQP2.

Ethylenedioxy-PIP2 oxalate reduces ganglioside storage in juvenile Sandhoff disease mice.

Sandhoff disease is an incurable neurodegenerative disorder caused by mutations in the lysosomal hydrolase ß-hexosaminidase. Deficiency in this enzyme leads to excessive accumulation of ganglioside GM2 and its asialo derivative, GA2, in brain and visceral tissues. Small molecule inhibitors of ceramide-specific glucosyltransferase, the first committed step in ganglioside biosynthesis, reduce storage of GM2 and GA2. Limited brain access or adverse effects have hampered the therapeutic efficacy of the clinically approved substrate reduction molecules, eliglustat tartrate and the imino sugar NB-DNJ (Miglustat). The novel eliglustat tartrate analog, 2-(2,3-dihydro-1H-inden-2-yl)-N-((1R,2R)-1-(2,3-dihydrobenzo[b][1, 4]dioxin-6-yl)-1-hydroxy-3-(pyrrolidin-1-yl)propan-2-yl)acetamide (EtDO-PIP2, CCG-203586 or "3h"), was recently reported to reduce glucosylceramide in murine brain. Here we assessed the therapeutic efficacy of 3h in juvenile Sandhoff (Hexb-/-) mice. Sandhoff mice received intraperitoneal injections of phosphate buffered saline (PBS) or 3h (60 mg/kg/day) from postnatal day 9 (p-9) to postnatal day 15 (p-15). Brain weight and brain water content was similar in 3h and PBS-treated mice. 3h significantly reduced total ganglioside sialic acid, GM2, and GA2 content in cerebrum, cerebellum and liver of Sandhoff mice. Data from the liver showed that 3h reduced the key upstream ganglioside precursor (glucosylceramide), providing evidence for an on target mechanism of action. No significant differences were seen in the distribution of cholesterol or of neutral and acidic phospholipids. These data suggest that 3h can be an effective alternative to existing substrate reduction molecules for ganglioside storage diseases.

Filipin recognizes both GM1 and cholesterol in GM1 gangliosidosis mouse brain.

Filipin is an antibiotic polyene widely used as a histochemical marker for cholesterol. We previously reported cholesterol/filipin-positive staining in brain of ß-galactosidase (ß-gal) knockout ((-/-)) mice (GM1 gangliosidosis). The content and distribution of cholesterol and gangliosides was analyzed in plasma membrane (PM) and microsomal (MS) fractions from whole-brain tissue of 15 week-old control (ß-gal(+/-)) and GM1 gangliosidosis (ß-gal(-/-)) mice. Total ganglioside content (µg sialic acid/mg protein) was 3-fold and 7-fold greater in the PM and MS fractions, respectively, in ßgal(-/-) mice than in ßgal(+/-) mice. GM1 content was 30-fold and 50-fold greater in the PM and MS fractions, respectively. In contrast, unesterified cholesterol content (µg/mg protein) was similar in the PM and the MS fractions of the ßgal(-/-) and ßgal(+/-) mice. Filipin is known to bind to various sterol derivatives and phospholipids on thin-layer chromatograms. Biochemical evidence is presented showing that filipin also binds to GM1 with an affinity similar to that for cholesterol, with a corresponding fluorescent reaction. Our data suggest that the GM1 storage seen in the ß-gal(-/-) mouse contributes to the filipin ultraviolet fluorescence observed in GM1 gangliosidosis brain. The data indicate that in addition to cholesterol, filipin can also be useful for detecting GM1.

Ku can contribute to telomere lengthening in yeast at multiple positions in the telomerase RNP.

Unlike ribonucleoprotein complexes that have a highly ordered overall architecture, such as the ribosome, yeast telomerase appears to be much more loosely constrained. Here, we investigate the importance of positioning of the Ku subunit within the 1157-nt yeast telomerase RNA (TLC1). Deletion of the 48-nt Ku-binding hairpin in TLC1 RNA (tlc1Δ48) reduces telomere length, survival of cells with gross chromosomal rearrangements, and de novo telomere addition at a broken chromosome end. To test the function of Ku at novel positions in the telomerase RNP, we reintroduced its binding site into tlc1Δ48 RNA at position 446 or 1029. We found that Ku bound to these repositioned sites in vivo and telomere length increased slightly, but statistically significantly. The ability of telomerase to promote survival of cells with gross chromosomal rearrangements by healing damaged chromosome arms was also partially restored, whereas the kinetics of DNA addition to a specific chromosome break was delayed. Having two Ku sites in TLC1 caused progressive hyperelongation of a variable subset of telomeres, consistent with Ku's role in telomerase recruitment to chromosome ends. The number of Ku-binding sites in TLC1 contributed to telomerase RNA abundance in vivo but was only partially responsible for telomere length phenotypes. Thus, telomerase RNA levels and telomere length regulation can be modulated by the number of Ku sites in telomerase RNA. Furthermore, there is substantial flexibility in the relative positioning of Ku in the telomerase RNP for native telomere length maintenance, although not as much flexibility as for the essential Est1p subunit.