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

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N-Degradomic Analysis Reveals a Proteolytic Network Processing the Podocyte Cytoskeleton BRIEF COMMUNICATION www.jasn.org N-Degradomic Analysis Reveals a Proteolytic Network Processing the Podocyte Cytoskeleton †‡ † | † Markus M. Rinschen,* § Ann-Kathrin Hoppe,* Florian Grahammer, ¶ Martin Kann,* † † † †‡ Linus A. Völker,* Eva-Maria Schurek,* Julie Binz,* Martin Höhne,* § Fatih Demir,** | †† † ‡‡ Milena Malisic,** Tobias B. Huber, ¶ Christine Kurschat,* Jayachandran N. Kizhakkedathu, †‡ †‡ Bernhard Schermer,* § Pitter F. Huesgen,** and Thomas Benzing* § *Department II of Internal Medicine, †Center for Molecular Medicine Cologne (CMMC), ‡Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), and §Systems Biology of Ageing Cologne (Sybacol), BRIEF COMMUNICATION University of Cologne, Cologne, Germany; |Department of Medicine III, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; ¶Department of Medicine IV, Medical Center and Faculty of Medicine – University of Freiburg, Freiburg, Germany; **Central Institute for Engineering, Electronics and Analytics, ZEA-3, Forschungszentrum Jülich, Jülich, Germany; ††BIOSS Centre for Biological Signalling Studies and Center for Biological Systems Analysis (ZBSA), Albert-Ludwigs-University, Freiburg, Germany; and ‡‡Centre for Blood Research, Department of Pathology and Laboratory Medicine, Department of Chemistry, University of British Columbia, Vancouver, Canada ABSTRACT Regulated intracellular proteostasis, controlled in part by proteolysis, is essential in of these genes leads to altered signaling maintaining the integrity of podocytes and the glomerular filtration barrier of the cascades in glomerular diseases and to kidney. We applied a novel proteomics technology that enables proteome-wide FSGS.2 Of critical importance are mem- identification, mapping, and quantification of protein N-termini to comprehensively brane proteins, such as podocin and characterize cleaved podocyte proteins in the glomerulus in vivo.Wefoundevi- nephrin3,4 and cytoskeletal proteins dence that defined proteolytic cleavage results in various proteoforms of important such as a-actinin-4 (ACTN4).5 podocyte proteins, including those of podocin, nephrin, neph1, a-actinin-4, and Proteolytic processing is an irrevers- vimentin. Quantitative mapping of N-termini demonstrated perturbation of protease ible, site-specificproteinmodification action during podocyte injury in vitro, including diminished proteolysis of a-actinin-4. that regulates essential physiologic Differentially regulated protease substrates comprised cytoskeletal proteins as well processes by generating protein isoforms as intermediate filaments. Determination of preferential protease motifs during (proteoforms) and controlling proteo- podocyte damage indicated activation of caspase proteases and inhibition of stasis.6 Deregulated proteolysis is a key arginine-specific proteases. Several proteolytic processes were clearly site-specific, driver for human diseases, and proteases were conserved across species, and could be confirmed by differential migration are promising targets for tailored thera- behavior of protein fragments in gel electrophoresis. Some of the proteolytic peutics.7 Recent studies indicated that changes discovered in vitro also occurred in two in vivo models of podocyte damage maintenance of the renal filtration (WT1 heterozygous knockout mice and puromycin aminonucleoside–treated rats). Thus, we provide direct and systems-level evidence that the slit diaphragm and Received October 21, 2016. Accepted May 8, podocyte cytoskeleton are regulated targets of proteolytic modification, which is 2017. altereduponpodocytedamage. P.F.H. and T.B. shared senior authorship. J Am Soc Nephrol 28: 2867–2878, 2017. doi: https://doi.org/10.1681/ASN.2016101119 Published online ahead of print. Publication date available at www.jasn.org. Correspondence: Dr. Markus M. Rinschen, University Hospital Cologne, Josef-Stelzmann-Str 26, 50931 Köln, 1 The majority of kidney diseases start in membrane, and podocytes. Podocytes Germany, Prof. Thomas Benzing, University Hospital the glomerulus due to the susceptibility are terminally differentiated, interdigi- Cologne, Kerpener Straße 62, 50937 Köln, Germany, of the renal filtration unit to metabolic, tating cells that engulf the glomerular or Dr. Pitter F. Huesgen, ZEA-3 Analytics, For- schungszentrum Jülich,Wilhelm-Johnen-Straße,52425 1 genetic, mechanic, and immunologic capillaries. Damage to these cells results Jülich, Germany. E-mail: [email protected], damage. The glomerular filtration unit in proteinuria.1,2 Genetic studies have [email protected] or [email protected] fi consists of three layers: fenestrated endo- identi ed essential podocyte proteins Copyright © 2017 by the American Society of thelial cells, the glomerular basement which maintain the barrier. Mutation Nephrology J Am Soc Nephrol 28: 2867–2878, 2017 ISSN : 1046-6673/2810-2867 2867 BRIEF COMMUNICATION www.jasn.org barrier largely relies on proteases as key such as acetylation, or in vitro by Ala59 (Table 1, Figure 1D). The 34-kD regulators of podocyte function, includ- dimethylation (Figure 1A). Next, tryptic band was observed in both human and ing Cathepsin D, Cathepsin L, and ma- digest was performed and N-terminal mouse glomeruli (Figure 1E) and is trix metalloproteases.8–11 High copy peptides were enriched (see Concise therefore not the previously described numbers of intracellular proteases with Methods for details). Proteomic analysis short podocin isoform specific to hu- broad specificity are found in the podo- of two such enriched samples from mans (and not expressed in mice) cyte transcriptome and proteome, and mouse glomeruli identified a total of with a similar molecular size.20 Interest- they accumulate in disease states.8,9,12–14 3815 annotated N-termini, of which ingly, this podocin cleavage site is asso- Although essential proteases and a few 2976 were modified by dimethylation in ciated with a polymorphism in the of their targets have been identified, it vitro whereas 784 were endogenously human sequence with unknown patho- is largely unclear which particular po- acetylated (Figure 1B, Supplemental genicity21 (Figure 1F). Three of the docyte proteins are cleaved, which pro- Table 1; 55 were both acetylated and di- cleavage sites observed in nephrin and teoforms are generated at which site(s), methylated). We mapped these to their ACTN4 were located in close vicinity to and how proteolytic cleavage affects topology and classified them depending 12 described mutations leading to he- their physiologic function in health and on their positional annotation19: 1343 reditary nephrotic syndrome and/or disease. Distinction of processed and were “expected” termini, of which 720 FSGS (Figure 1F). precursorproteinsischallengingby started with intact or removed initiation Totest whether proteolytic processing standard proteomics because many methionine (P1, P2), six were termini is altered in podocyte damage, we chal- tryptic peptides are shared by both pro- mapping to known alternative transcrip- lenged cultured human podocytes with teoforms, whereas the informative pro- tion start sites, and 521 were termini puromycin aminonucleoside (PAN; tease-generated neo-terminal peptides matching to known or predicted sites 50 mg/ml, 24 hours), an artificial yet constitute only a very minor frac- for signal- or propetide removal (Figure well established model of podocyte in- tion that is rarely identified.15 Here, we 1B). Ninety-six termini were annotated jury,22,23 and performed quantitative overcome this limitation using the inno- as previously described cleavage sites TAILS analysis. Immunofluorescence vative Terminal Amine–based Iso- associated with known proteases. Ca- analysis demonstrated the expected typ- tope Labeling of Substrates (TAILS) thepsin D cleavage sites were markedly ical rearrangement of actin stress fibers technique, a strategy enabling site- overrepresented in the dataset as com- but also partial rearrangement of the in- specific mapping of protease-generated pared with all other known cleavage termediate filament vimentin (Supple- novel protein termini (“degrado- sites (Figure 1C). Two thousand four mental Figure 2A).24 Podocyte viability mics”).16–18 The aim of this first unbi- hundred forty-eight termini mapped as measured by 2,3-Bis-(2-Methoxy-4- ased, systems-level study of podocyte to “unexpected” positions, indicative of Nitro-5-Sulfophenyl)-2H-Tetrazolium- degradomics was to delineate proteolytic extensive proteolysis in podocytes in vivo. 5-Carboxanilide (XTT) assay was not protein modifications and demonstrate The vast majority (91%) of unexpected altered by PAN at this time point (Sup- dynamic proteolytic regulation of cyto- termini were localized outside known plemental Figure 2B). The PAN-treated skeletal proteins in injury. protein domains. and vehicle-treated proteomes were iso- To obtain an atlas of proteolytically We found novel termini in the slit di- lated and labeled with two distinct stable cleaved proteins in podocytes of living aphragm proteins podocin (Figure 1D), isotope variants of formaldehyde to allow animals we applied the TAILS technology nephrin, and neph1 (Kirrel) (Supple- quantification of protease-generated combined with mass spectrometry– mental Figure 1A), and sites in podocyte neo-termini (Supplemental Figure 2C, based protein identification. Technical cytoskeleton proteins (Supplemental n=4). After TAILS enrichment we precautions were taken to minimize ex Figure 1B). Immunoblot analysis with identified a total of 3085 N-termini (Fig-
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