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J Am Soc Nephrol. 2017 Oct;28(10):2867-2878. doi: 10.1681/ASN.2016101119. Epub 2017 Jul 19.

N-Degradomic Analysis Reveals a Proteolytic Network Processing the Podocyte Cytoskeleton.

Author information

1
Department II of Internal Medicine.
2
Center for Molecular Medicine Cologne (CMMC).
3
Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), and.
4
Systems Biology of Ageing Cologne (Sybacol), University of Cologne, Cologne, Germany.
5
Department of Medicine III, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
6
Department of Medicine IV, Medical Center and Faculty of Medicine - University of Freiburg, Freiburg, Germany.
7
Central Institute for Engineering, Electronics and Analytics, ZEA-3, Forschungszentrum Jülich, Jülich, Germany.
8
BIOSS Centre for Biological Signalling Studies and Center for Biological Systems Analysis (ZBSA), Albert-Ludwigs-University, Freiburg, Germany; and.
9
Centre for Blood Research, Department of Pathology and Laboratory Medicine, Department of Chemistry, University of British Columbia, Vancouver, Canada.
10
Central Institute for Engineering, Electronics and Analytics, ZEA-3, Forschungszentrum Jülich, Jülich, Germany; markus.rinschen@uk-koeln.de thomas.benzing@uk-koeln.de p.huesgen@fz-juelich.de.
11
Department II of Internal Medicine, markus.rinschen@uk-koeln.de thomas.benzing@uk-koeln.de p.huesgen@fz-juelich.de.

Abstract

Regulated intracellular proteostasis, controlled in part by proteolysis, is essential in maintaining the integrity of podocytes and the glomerular filtration barrier of the kidney. We applied a novel proteomics technology that enables proteome-wide identification, mapping, and quantification of protein N-termini to comprehensively characterize cleaved podocyte proteins in the glomerulus in vivo We found evidence that defined proteolytic cleavage results in various proteoforms of important podocyte proteins, including those of podocin, nephrin, neph1, α-actinin-4, and vimentin. Quantitative mapping of N-termini demonstrated perturbation of protease action during podocyte injury in vitro, including diminished proteolysis of α-actinin-4. Differentially regulated protease substrates comprised cytoskeletal proteins as well as intermediate filaments. Determination of preferential protease motifs during podocyte damage indicated activation of caspase proteases and inhibition of arginine-specific proteases. Several proteolytic processes were clearly site-specific, were conserved across species, and could be confirmed by differential migration behavior of protein fragments in gel electrophoresis. Some of the proteolytic changes discovered in vitro also occurred in two in vivo models of podocyte damage (WT1 heterozygous knockout mice and puromycin aminonucleoside-treated rats). Thus, we provide direct and systems-level evidence that the slit diaphragm and podocyte cytoskeleton are regulated targets of proteolytic modification, which is altered upon podocyte damage.

KEYWORDS:

Cell Signaling; Pathophysiology of Renal Disease and Progression; cell biology and structure; intracellular signal; podocyte; signaling

PMID:
28724775
PMCID:
PMC5619959
DOI:
10.1681/ASN.2016101119
[Indexed for MEDLINE]
Free PMC Article

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