title: Chloride channel ClC-5 binds to aspartyl aminopeptidase to regulate renal albumin endocytosis.
abstract: ClC-5 is a chloride/proton exchanger that plays an obligate role in albumin uptake by the renal proximal tubule. ClC-5 forms an endocytic complex with the albumin receptor megalin/cubilin. We have identified a novel ClC-5 binding partner, cytosolic aspartyl aminopeptidase (DNPEP; EC 3.4.11.21), that catalyzes the release of N-terminal aspartate/glutamate residues. The physiological role of DNPEP remains largely unresolved. Mass spectrometric analysis of proteins binding to the glutathione-S-transferase (GST)-ClC-5 C terminus identified DNPEP as an interacting partner. Coimmunoprecipitation confirmed that DNPEP and ClC-5 also associated in cells. Further experiments using purified GST-ClC-5 and His-DNPEP proteins demonstrated that the two proteins bound directly to each other. In opossum kidney (OK) cells, confocal immunofluorescence studies revealed that DNPEP colocalized with albumin-containing endocytic vesicles. Overexpression of wild-type DNPEP increased cell-surface levels of ClC-5 and albumin uptake. Analysis of DNPEP-immunoprecipitated products from rat kidney lysate identified β-actin and tubulin, suggesting a role for DNPEP in cytoskeletal maintenance. A DNase I inhibition assay showed a significant decrease in the amount of G actin when DNPEP was overexpressed in OK cells, suggesting a role for DNPEP in stabilizing the cytoskeleton. DNPEP was not present in the urine of healthy rats; however, it was readily detected in the urine in rat models of mild and heavy proteinuria (diabetic nephropathy and anti-glomerular basement membrane disease, respectively). Urinary levels of DNPEP were found to correlate with the severity of proteinuria. Therefore, we have identified another key molecular component of the albumin endocytic machinery in the renal proximal tubule and describe a new role for DNPEP in stabilizing the actin cytoskeleton.
authors.affiliation: UQ Centre for Clinical Research, The University of Queensland, Herston, Brisbane, Queensland, Australia;
authors.forename: Aven
authors.initials: A
authors.lastname: Lee
authors.affiliation: School of Biomolecular and Biomedical Sciences, University College Dublin, Belfield, Dublin, Republic. of Ireland;
authors.forename: Craig
authors.initials: C
authors.lastname: Slattery
authors.affiliation: Department of Nephrology, Monash University Department of Medicine, Monash Medical Centre, Clayton, Victoria, Australia;
authors.forename: David J
authors.initials: DJ
authors.lastname: Nikolic-Paterson
authors.affiliation: Department of Physiology, The University of Melbourne, Parkville, Victoria, Australia;
authors.forename: Deanne H
authors.initials: DH
authors.lastname: Hryciw
authors.affiliation: Department of Pharmacology, Mount Sinai School of Medicine, New York, New York;
authors.forename: Sherwin
authors.initials: S
authors.lastname: Wilk
authors.affiliation: Department of Pharmacology, Mount Sinai School of Medicine, New York, New York;
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Wilk
authors.affiliation: Department of Medicine, Saint Vincent's Hospital, Fitzroy, Victoria, Australia;
authors.forename: Yuan
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Children's Medical Research Institute, The University of Sydney, Westmead New South Wales, Australia; and.
authors.forename: Valentina A
authors.initials: VA
authors.lastname: Valova
authors.affiliation: Children's Medical Research Institute, The University of Sydney, Westmead New South Wales, Australia; and.
authors.forename: Phillip J
authors.initials: PJ
authors.lastname: Robinson
authors.affiliation: Department of Medicine, Saint Vincent's Hospital, Fitzroy, Victoria, Australia;
authors.forename: Darren J
authors.initials: DJ
authors.lastname: Kelly
authors.affiliation: School of Medical Sciences and the Bosch Institute, The University of Sydney, New South Wales, Australia philip.poronnik@sydney.edu.au.
authors.forename: Philip
authors.initials: P
authors.lastname: Poronnik
mesh_terms: D000199:Actins / Q000378:metabolism; D000418:Albumins / Q000378:metabolism*; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D018118:Chloride Channels / Q000378:metabolism*; D004705:Endocytosis / Q000502:physiology*; D043384:Glutamyl Aminopeptidase / Q000378:metabolism*; D007668:Kidney / Q000378:metabolism; D007687:Kidney Tubules, Proximal / Q000378:metabolism*; D026561:Low Density Lipoprotein Receptor-Related Protein-2 / Q000378:metabolism; D051381:Rats
chemical_list: D000199:Actins; D000418:Albumins; C097503:CLC-5 chloride channel; D018118:Chloride Channels; D026561:Low Density Lipoprotein Receptor-Related Protein-2; D043384:Glutamyl Aminopeptidase
keywords: ClC-5; albumin endocytosis; albuminuria; aspartyl aminopeptidase