A human polymorphism affects NEDD4L subcellular targeting by leading to two isoforms that contain or lack a C2 domain

<p>Abstract</p> <p>Background</p> <p>Ubiquitination serves multiple cellular functions, including proteasomal degradation and the control of stability, function, and intracellular localization of a wide variety of proteins. NEDD4L is a member of the HECT class of E3 ubiquitin ligases. A defining feature of NEDD4L protein isoforms is the presence or absence of an amino-terminal C2 domain, a class of subcellular, calcium-dependent targeting domains. We previously identified a common variant in human <it>NEDD4L </it>that generates isoforms that contain or lack a C2 domain.</p> <p>Results</p> <p>To address the potential functional significance of the <it>NEDD4L </it>common variant on NEDD4L subcellular localization, NEDD4L isoforms that either contained or lacked a C2 domain were tagged with enhanced green fluorescent protein, transfected into <it>Xenopus laevis </it>kidney epithelial cells, and imaged by performing confocal microscopy on live cells. We report that the presence or absence of this C2 domain exerts differential effects on the subcellular distribution of NEDD4L, the ability of C2 containing and lacking NEDD4L isoforms to mobilize in response to a calcium stimulus, and the intracellular transport of subunits of the NEDD4L substrate, ENaC. Furthermore, the ability of the C2-containing isoform to influence β-ENaC mobilization from intracellular pools involves the NEDD4L active site for ubiquitination. We propose a model to account for the potential impact of this common genetic variant on protein function at the cellular level.</p> <p>Conclusion</p> <p>NEDD4L isoforms that contain or lack a C2 domain target different intracellular locations. Additionally, whereas the C2-containing NEDD4L isoform is capable of shuttling between the plasma membrane and intracellular compartments in response to calcium stimulus the C2-lacking isoform can not. The C2-containing isoform differentially affects the mobilization of ENaC subunits from intracellular pools and this trafficking step requires NEDD4L ubiquitin ligase activity. This observation suggests a new mechanism for the requirement for the PY motif in cAMP-mediated exocytosis of ENaC. We have elucidated how a common genetic variant can underlie significant functional diversity in NEDD4L at the cellular level. We propose a model that describes how that functional variation may influence blood pressure. Moreover, our observations regarding differential function of the NEDD4L isoforms may impact other aspects of physiology that involve this ubiquitin ligase.</p

Disulphide Bridges of Phospholipase C of Chlamydomonas reinhardtii Modulates Lipid Interaction and Dimer Stability

BACKGROUND: Phospholipase C (PLC) is an enzyme that plays pivotal role in a number of signaling cascades. These are active in the plasma membrane and triggers cellular responses by catalyzing the hydrolysis of membrane phospholipids and thereby generating the secondary messengers. Phosphatidylinositol-PLC (PI-PLC) specifically interacts with phosphoinositide and/or phosphoinositol and catalyzes specific cleavage of sn-3- phosphodiester bond. Several isoforms of PLC are known to form and function as dimer but very little is known about the molecular basis of the dimerization and its importance in the lipid interaction. PRINCIPAL FINDINGS: We herein report that, the disruption of disulphide bond of a novel PI-specific PLC of C. reinhardtii (CrPLC) can modulate its interaction affinity with a set of phospholipids and also the stability of its dimer. CrPLC was found to form a mixture of higher oligomeric states with monomer and dimer as major species. Dimer adduct of CrPLC disappeared in the presence of DTT, which suggested the involvement of disulphide bond(s) in CrPLC oligomerization. Dimer-monomer equilibrium studies with the isolated fractions of CrPLC monomer and dimer supported the involvement of covalent forces in the dimerization of CrPLC. A disulphide bridge was found to be responsible for the dimerization and Cys7 seems to be involved in the formation of the disulphide bond. This crucial disulphide bond also modulated the lipid affinity of CrPLC. Oligomers of CrPLC were also captured in in vivo condition. CrPLC was mainly found to be localized in the plasma membrane of the cell. The cell surface localization of CrPLC may have significant implication in the downstream regulatory function of CrPLC. SIGNIFICANCE: This study helps in establishing the role of CrPLC (or similar proteins) in the quaternary structure of the molecule its affinities during lipid interactions

Lysophosphatidic acid is a modulator of cyst growth in autosomal dominant polycystic kidney disease., Lysophosphatidic Acid is a Modulator of Cyst Growth in Autosomal Dominant Polycystic Kidney Disease

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the slow growth of multiple fluid-filled cysts predominately in the kidney tubules and liver bile ducts. Elucidation of mechanisms that control cyst growth will provide the basis for rational therapeutic intervention. We used electrophysiological methods to identify lysophosphatidic acid (LPA) as a component of cyst fluid and serum that stimulates secretory Cl- transport in the epithelial cell type that lines renal cysts. LPA effects are manifested through receptors located on the basolateral membrane of the epithelial cells resulting in stimulation of channel activity in the apical membrane. Concentrations of LPA measured in human ADPKD cyst fluid and in normal serum are sufficient to maximally stimulate ion transport. Thus, cyst fluid seepage and/or leakage of vascular LPA into the interstitial space are capable of stimulating epithelial cell secretion resulting in cyst enlargement. These observations are particularly relevant to the rapid decline in renal function in late-stage disease and to the "third hit" hypothesis that renal injury exacerbates cyst growth

Amino acid status in dogs with protein‐losing nephropathy

BackgroundProteinuria in dogs with kidney disease can contribute to protein-energy wasting and malnutrition. Little is known about amino acid (AA) status in dogs with protein-losing nephropathy (PLN).ObjectivesThe purpose of our study was to further elucidate AA status in PLN dogs, with the hypothesis that PLN dogs would have altered AA status as compared to healthy dogs.AnimalsThirty client-owned PLN dogs were compared to 10 healthy control dogs.MethodsProspective observational study. Dogs with PLN that were presented to the teaching hospital were enrolled. Plasma AA profiles were measured using an automated high-performance liquid chromatography AA analyzer.ResultsCompared to control dogs, PLN dogs had significantly lower concentrations of leucine, threonine, histidine, glycine, proline, asparagine, tyrosine, o-hydroxyproline, and serine, as well as sums of both essential and nonessential AA (P &lt; .05). Dogs with PLN had significantly lower ratios of tyrosine-to-phenylalanine and glycine-to-serine (P &lt; .05), and a significantly greater ratio of valine-to-glycine (P &lt; .05).Conclusions and clinical importanceDogs with PLN have altered AA status compared to healthy dogs. These findings could have therapeutic implications in determining optimal management of PLN dogs, such as providing AA supplementation along with other standard treatment