Acetylation stimulates the epithelial sodium channel by reducing its ubiquitination and degradation.

The epithelial Na(+) channel (ENaC) functions as a pathway for Na(+) absorption in the kidney and lung, where it is crucial for Na(+) homeostasis and blood pressure regulation. ENaC is regulated in part through signaling pathways that control the ubiquitination state of ENaC lysines. A defect in ubiquitination causes Liddle syndrome, an inherited form of hypertension. Here we determined that α-, ß-, and γENaC are also substrates for lysine acetylation. Trichostatin A (TSA), a histone deacetylase inhibitor, enhanced ENaC acetylation and increased ENaC abundance in the total cell lysate and at the cell surface. Moreover, TSA increased ENaC current in Fischer rat thyroid and kidney collecting duct epithelia. We found that HDAC7 is expressed in the kidney collecting duct, supporting a potential role for this histone deacetylase in ENaC regulation. HDAC7 overexpression reduced ENaC abundance and ENaC current, whereas ENaC abundance and current were increased by silencing of HDAC7. ENaC and HDAC7 form a complex, as detected by coimmunoprecipitation. We observed a reciprocal relationship between acetylation and ubiquitination; TSA reduced ENaC ubiquitination, whereas HDAC7 increased ubiquitination. By reducing ENaC ubiquitination, TSA decreased the rate of ENaC degradation. Thus, acetylation increases epithelial Na(+) absorption by antagonizing ENaC ubiquitination. This stabilizes ENaC, and hence, increases its abundance at the cell surface.

Ubiquitin-specific peptidase 8 (USP8) regulates endosomal trafficking of the epithelial Na+ channel.

Ubiquitination plays a key role in trafficking of the epithelial Na(+) channel (ENaC). Previous work indicated that ubiquitination enhances ENaC endocytosis and sorting to lysosomes for degradation. Moreover, a defect in ubiquitination causes Liddle syndrome, an inherited form of hypertension. In this work, we identified a role for USP8 in the control of ENaC ubiquitination and trafficking. USP8 increased ENaC current in Xenopus oocytes and collecting duct epithelia and enhanced ENaC abundance at the cell surface in HEK 293 cells. This resulted from altered endocytic sorting; USP8 abolished ENaC degradation in the endocytic pathway, but it had no effect on ENaC endocytosis. USP8 interacted with ENaC, as detected by co-immunoprecipitation, and it deubiquitinated ENaC. Consistent with a functional role for deubiquitination, mutation of the cytoplasmic lysines of ENaC reduced the effect of USP8 on ENaC cell surface abundance. In contrast to USP8, USP2-45 increased ENaC surface abundance by reducing endocytosis but not degradation. Thus, USP8 and USP2-45 selectively modulate ENaC trafficking at different steps in the endocytic pathway. Together with previous work, the data indicate that the ubiquitination state of ENaC is critical for the regulation of epithelial Na(+) absorption.

LPS impairs phospholipid synthesis by triggering beta-transducin repeat-containing protein (beta-TrCP)-mediated polyubiquitination and degradation of the surfactant enzyme acyl-CoA:lysophosphatidylcholine acyltransferase I (LPCAT1).

Acyl-CoA:lysophosphatidylcholine acyltransferase 1 (LPCAT1) is a relatively newly described and yet indispensable enzyme needed for generation of the bioactive surfactant phospholipid, dipalmitoylphosphatidylcholine (DPPtdCho). Here, we show that lipopolysaccharide (LPS) causes LPCAT1 degradation using the Skp1-Cullin-F-box ubiquitin E3 ligase component, ß-transducin repeat-containing protein (ß-TrCP), that polyubiquitinates LPCAT1, thereby targeting the enzyme for proteasomal degradation. LPCAT1 was identified as a phosphoenzyme as Ser(178) within a phosphodegron was identified as a putative molecular recognition site for glycogen synthase kinase-3ß (GSK-3ß) phosphorylation that recruits ß-TrCP docking within the enzyme. ß-TrCP ubiquitinates LPCAT1 at an acceptor site (Lys(221)), as substitution of Lys(221) with Arg abrogated LPCAT1 polyubiquitination. LPS profoundly reduced immunoreactive LPCAT1 levels and impaired lung surfactant mechanics, effects that were overcome by siRNA to ß-TrCP and GSK-3ß or LPCAT1 gene transfer, respectively. Thus, LPS appears to destabilize the LPCAT1 protein by GSK-3ß-mediated phosphorylation within a canonical phosphodegron for ß-TrCP docking and site-specific ubiquitination. LPCAT1 is the first lipogenic substrate for ß-TrCP, and the results suggest that modulation of the GSK-3ß-SCFß(TrCP) E3 ligase effector pathway might be a unique strategy to optimize dipalmitoylphosphatidylcholine levels in sepsis.

Cross-talk between remodeling and de novo pathways maintains phospholipid balance through ubiquitination.

Phosphatidylcholine (PtdCho), the major phospholipid of animal membranes, is generated by its remodeling and de novo synthesis. Overexpression of the remodeling enzyme, LPCAT1 (acyl-CoA:lysophosphatidylcholine acyltransferase) in epithelia decreased de novo PtdCho synthesis without significantly altering cellular PtdCho mass. Overexpression of LPCAT1 increased degradation of CPT1 (cholinephosphotransferase), a resident Golgi enzyme that catalyzes the terminal step for de novo PtdCho synthesis. CPT1 degradation involved its multiubiquitination and processing via the lysosomal pathway. CPT1 mutants harboring arginine substitutions at multiple carboxyl-terminal lysines exhibited proteolytic resistance to effects of LPCAT1 overexpression in cells and restored de novo PtdCho synthesis. Thus, cross-talk between phospholipid remodeling and de novo pathways involves ubiquitin-lysosomal processing of a key molecular target that mechanistically provides homeostatic control of cellular PtdCho content.

Anion and carboxylic acid binding to monotopic and ditopic amidopyridine macrocycles.

Binding of inorganic anions, carboxylic acids, and tetraalkylammonium carboxylates by macrocyclic compounds of different size was studied by NMR in DMSO-d6. It has been shown that at least a 15-membered ring is necessary for successful recognition of fluoride. Larger macrocycles were shown to bind HSO4(-), H2PO4(-), Cl(-), and carboxylic acid salts. Effects of binding topicity are discussed. The 30-membered macrocycles 4 and 4m selectively bind substrates that are size- and shape-complementary: maximum binding is observed for dicarboxylic acids and dicarboxylates with four-carbon chains, and the binding constant for association of fumaric acid and 4 is ca. 5 orders of magnitude higher than that of maleic acid. The 30-membered macrocycle 4m showed selectivity toward alpha-ketocarboxylic acids. Secondary amino groups were not crucial for binding of fluoride to the macrocycles; however, they proved to be very important for selectivity and strength of carboxylic acid binding. The X-ray structure of the adduct of 4 and nitrobenzoic acid confirmed the guest H-bonding with both the amide and the secondary amino groups of the 30-membered macrocyclic host.

Anion binding to monotopic and ditopic macrocyclic amides.

[Structure: see text] Binding of fluoride anion as well as carboxylic acid tetraalkylammonium salts by macrocyclic compounds of different size was studied by NMR in DMSO-d6. It has been found that at least a 15-membered ring is necessary for successful recognition of fluoride. Larger macrocycles obtained in a [2+2] cyclization were shown to bind dicarboxylic acid salts. Effects of binding topicity are discussed.

Dynamic regulation of cardiolipin by the lipid pump Atp8b1 determines the severity of lung injury in experimental pneumonia.

Pneumonia remains the leading cause of death from infection in the US, yet fundamentally new conceptual models underlying its pathogenesis have not emerged. We show that humans and mice with bacterial pneumonia have markedly elevated amounts of cardiolipin, a rare, mitochondrial-specific phospholipid, in lung fluid and find that it potently disrupts surfactant function. Intratracheal cardiolipin administration in mice recapitulates the clinical phenotype of pneumonia, including impaired lung mechanics, modulation of cell survival and cytokine networks and lung consolidation. We have identified and characterized the activity of a unique cardiolipin transporter, the P-type ATPase transmembrane lipid pump Atp8b1, a mutant version of which is associated with severe pneumonia in humans and mice. Atp8b1 bound and internalized cardiolipin from extracellular fluid via a basic residue-enriched motif. Administration of a peptide encompassing the cardiolipin binding motif or Atp8b1 gene transfer in mice lessened bacteria-induced lung injury and improved survival. The results unveil a new paradigm whereby Atp8b1 is a cardiolipin importer whose capacity to remove cardiolipin from lung fluid is exceeded during inflammation or when Atp8b1 is defective. This discovery opens the door for new therapeutic strategies directed at modulating the abundance or molecular interactions of cardiolipin in pneumonia.