title: Three-layered proteomic characterization of a novel ACTN4 mutation unravels its pathogenic potential in FSGS.
abstract: Genetic diseases constitute the most important cause for end-stage renal disease in children and adolescents. Mutations in the ACTN4 gene, encoding the actin-binding protein α-actinin-4, are a rare cause of autosomal dominant familial focal segmental glomerulosclerosis (FSGS). Here, we report the identification of a novel, disease-causing ACTN4 mutation (p.G195D, de novo) in a sporadic case of childhood FSGS using next generation sequencing. Proteome analysis by quantitative mass spectrometry (MS) of patient-derived urinary epithelial cells indicated that ACTN4 levels were significantly decreased when compared with healthy controls. By resolving the peptide bearing the mutated residue, we could proof that the mutant protein is less abundant when compared with the wild-type protein. Further analyses revealed that the decreased stability of p.G195D is associated with increased ubiquitylation in the vicinity of the mutation site. We next defined the ACTN4 interactome, which was predominantly composed of cytoskeletal modulators and LIM domain-containing proteins. Interestingly, this entire group of proteins, including several highly specific ACTN4 interactors, was globally decreased in the patient-derived cells. Taken together, these data suggest a mechanistic link between ACTN4 instability and proteome perturbations of the ACTN4 interactome. Our findings advance the understanding of dominant effects exerted by ACTN4 mutations in FSGS. This study illustrates the potential of genomics and complementary, high-resolution proteomics analyses to study the pathogenicity of rare gene variants.
authors.affiliation: Department II of Internal Medicine and Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany.
authors.forename: Malte P
authors.initials: MP
authors.lastname: Bartram
authors.affiliation: Department II of Internal Medicine and Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany, Department of Pediatrics.
authors.forename: Sandra
authors.initials: S
authors.lastname: Habbig
authors.affiliation: Department II of Internal Medicine and Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany.
authors.forename: Caroline
authors.initials: C
authors.lastname: Pahmeyer
authors.affiliation: Department II of Internal Medicine and Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany, Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD) and Systems Biology of Ageing Cologne, University of Cologne, Cologne, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Höhne
authors.affiliation: Department of Pediatrics.
authors.forename: Lutz T
authors.initials: LT
authors.lastname: Weber
authors.affiliation: Cologne Center for Genomics (CCG).
authors.forename: Holger
authors.initials: H
authors.lastname: Thiele
authors.affiliation: Institute of Human Genetics, Cologne Center for Genomics (CCG).
authors.forename: Janine
authors.initials: J
authors.lastname: Altmüller
authors.affiliation: Department of Radiology.
authors.forename: Nina
authors.initials: N
authors.lastname: Kottoor
authors.affiliation: Institute of Human Genetics.
authors.forename: Andrea
authors.initials: A
authors.lastname: Wenzel
authors.affiliation: Institute for Genetics, Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD) and.
authors.forename: Marcus
authors.initials: M
authors.lastname: Krueger
authors.affiliation: Department II of Internal Medicine and Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany, Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD) and Systems Biology of Ageing Cologne, University of Cologne, Cologne, Germany.
authors.forename: Bernhard
authors.initials: B
authors.lastname: Schermer
authors.affiliation: Department II of Internal Medicine and Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany, Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD) and Systems Biology of Ageing Cologne, University of Cologne, Cologne, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Benzing
authors.affiliation: Department II of Internal Medicine and Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany, Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD) and Systems Biology of Ageing Cologne, University of Cologne, Cologne, Germany markus.rinschen@uk-koeln.de bodo.beck@uk-koeln.de.
authors.forename: Markus M
authors.initials: MM
authors.lastname: Rinschen
authors.affiliation: Institute of Human Genetics, markus.rinschen@uk-koeln.de bodo.beck@uk-koeln.de.
authors.forename: Bodo B
authors.initials: BB
authors.lastname: Beck
mesh_terms: D000185:Actinin / Q000235:genetics* / Q000378:metabolism; D000199:Actins / Q000378:metabolism; D000293:Adolescent; D000595:Amino Acid Sequence; D003599:Cytoskeleton / Q000378:metabolism; D005260:Female; D005923:Glomerulosclerosis, Focal Segmental / Q000235:genetics* / Q000378:metabolism; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D007676:Kidney Failure, Chronic / Q000235:genetics* / Q000378:metabolism; D008840:Microfilament Proteins / Q000378:metabolism; D040901:Proteomics
chemical_list: C111453:ACTN4 protein, human; D000199:Actins; D008840:Microfilament Proteins; D000185:Actinin