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

Rinschen, Markus M.; Hoppe, Ann-Kathrin; Grahammer, Florian; Kann, Martin; Völker, Linus A.; Schurek, Eva-Maria; Binz, Julie; Höhne, Martin; Demir, Fatih; Malisic, Milena; Huber, Tobias B.; Kurschat, Christine; Kizhakkedathu, Jayachandran N.; Schermer, Bernhard; Huesgen, Pitter F.; Benzing, Thomas

doi:10.1681/asn.2016101119

Cited by 41 publications

(52 citation statements)

References 51 publications

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“…Podocytes in culture, especially human podocyte cell lines, are important tools for understanding glomerular biology [reviewed in Brinkkoetter et al (2)]. They have been used to elucidate the pathophysiology of glomerular diseases (66) as well as to examine various physiologic mechanisms of podocyte biology. Investigated biologic aspects include cytoskeleton (67), cell cycle (68), cell death (69), signaling (70,71), and protein degradation (70).…”

Section: Discussionmentioning

confidence: 99%

“…MS-based proteomics allows the development of comprehensive and detailed maps of protein expression by various cell types (74,75), including podocytes (76). In fact, these analyses revealed that podocyte proteins are strongly subjected to posttranslational modifications, including phosphorylation (9,(77)(78)(79), ubiquitination (70), and proteolytic processing (66), and undergo rapid proteome remodeling when injured (5). Previously, we reported that podocyte differentiation markedly reduces proteasomal activity (9), and podocyte injury increases proteasome expression and activity (80).…”

Section: Discussionmentioning

confidence: 99%

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Protein half‐life determines expression of proteostatic networks in podocyte differentiation

et al. 2018

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Podocytes are highly specialized, epithelial, postmitotic cells, which maintain the renal filtration barrier. When adapting to considerable metabolic and mechanical stress, podocytes need to accurately maintain their proteome. Immortalized podocyte cell lines are a widely used model for studying podocyte biology in health and disease in vitro. In this study, we performed a comprehensive proteomic analysis of the cultured human podocyte proteome in both proliferative and differentiated conditions at a depth of >7000 proteins. Similar to mouse podocytes, human podocyte differentiation involved a shift in proteostasis: undifferentiated podocytes have high expression of proteasomal proteins, whereas differentiated podocytes have high expression of lysosomal proteins. Additional analyses with pulsed stable-isotope labeling by amino acids in cell culture and protein degradation assays determined protein dynamics and half-lives. These studies unraveled a globally increased stability of proteins in differentiated podocytes. Mitochondrial, cytoskeletal, and membrane proteins were stabilized, particularly in differentiated podocytes. Importantly, protein half-lives strongly contributed to protein abundance in each state. These data suggest that regulation of protein turnover of particular cellular functions determines podocyte differentiation, a paradigm involving mitophagy and, potentially, of importance in conditions of increased podocyte stress and damage.-Schroeter, C. B., Koehler, S., Kann, M., Schermer, B., Benzing, T., Brinkkoetter, P. T., Rinschen, M. M. Protein half-life determines expression of proteostatic networks in podocyte differentiation.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Protein half‐life determines expression of proteostatic networks in podocyte differentiation

et al. 2018

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“…Multiple studies have indicated that cathepsin proteases can inactivate CD2AP and other proteins essential for podocyte structure and function(39). Proteolytic processing of α-actinin-4, podocin, and other proteins by cathepsins has recently been suggested to facilitate maintenance of podocyte structure and filtration barrier integrity(40). It is thus interesting to note that INF2 is also a target of cathepsin proteases.…”

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confidence: 99%

Proteolytic program-dependent functions are impaired in INF2-mediated focal segmental glomerulosclerosis

Subramanian¹,

Chun²,

Pérez³

et al. 2019

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Regulation of the actin cytoskeleton is critical for normal glomerular podocyte structure and function. Altered regulation of the podocyte cytoskeleton can lead to proteinuria, reduced kidney filtration function and focal segmental glomerulosclerosis (FSGS). Mutations in inverted formin 2 (INF2), a member of the formin family of actin regulatory proteins, are the most common cause of autosomal dominant FSGS. INF2 is a multi-domain protein regulated by interaction between its N-terminal Diaphanous Inhibitory Domain (DID) and its C-terminal Diaphanous Auto-regulatory Domain (DAD). Although many aspects of the INF2 DID-DAD interaction are understood, it remains unclear why disease-causing mutations are restricted to the DID and how these mutations cause human disease. Here we report a proteolytic cleavage in INF2 that liberates the INF2 N-terminal DID to function independently of the INF2 C-terminal fragment containing the DAD domain. N-terminal DID region epitopes are differentially localized to podocyte foot process structures in normal glomeruli. This Nterminal fragment localization is lost in INF2-mediated FSGS, whereas INF2 C-terminal fragment epitopes localize to the podocyte cell body in both normal and disease conditions. INF2 cleavage is mediated by cathepsin proteases. In cultured podocytes, the wild-type INF2 N-terminal fragment localizes to membrane regions and promotes cell spreading, while these functions are impaired in a disease-associated INF2 mutant R218Q in the DID. These features are dependent on INF2cleavage, with accompanying interaction of INF2 N-fragment with mDIA1. Our data suggest a unique cellular function of the DID dependent on INF2 cleavage and help explain the altered localization of FSGS-associated INF2 mutant polypeptides.

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“…Therefore, we focused on components of the lysosomes of higher accessibility which could be better druggable targets. Cathepsin L but also other cathepsin proteases are expressed in cultured podocytes and possibly functional in podocytes based on degradomics data (27)(28)(29)(30). Since cathepsins can also act outside of lysosomes (31), we investigated whether lysosomal (LAMP1) abundance is linked to cathepsin abundance.…”

Section: Resultsmentioning

confidence: 99%

Individual nephron proteomes connect morphology and function in proteinuric kidney disease

Hoehne

Frese

Grahammer³

et al. 2017

Preprint

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In diseases of many parenchymatous organs, heterogenous detoriation of individual functional units determines the clinical prognosis. However, the molecular characterization of these subunits remains a technological challenge that needs to be addressed in order to better understand pathological mechanisms. Sclerotic and proteinuric glomerular kidney disease is a frequent and heterogeneous disease which affects a fraction of nephrons, glomeruli and draining tubules, to variable extents, and for which no treatment exists. Here, we developed and applied an antibody-independent methodology to investigate heterogeneity of individual nephron segment proteomes from mice with proteinuric kidney disease. This "one-segment-one-proteome-approach" defines mechanistic connections between upstream (glomerular) and downstream (tubular) nephron segment populations. In single glomeruli from two different mouse models of sclerotic glomerular disease, we identified a coherent protein expression module consisting of extracellular matrix protein deposition (reflecting glomerular sclerosis), glomerular albumin (reflecting proteinuria) and LAMP1, a lysosomal protein. This module was associated with a loss of podocyte marker proteins. In an attempt to target this protein co-expression module, genetic ablation of LAMP1-correlated lysosomal proteases in mice could ameliorate glomerular damage. Furthermore, individual glomeruli from patients with genetic sclerotic and non-sclerotic proteinuric diseases demonstrated increased abundance of lysosomal proteins, in combination with a decreased abundance of the mutated gene products. Therefore, increased glomerular lysosomal load is a conserved key mechanism in proteinuric kidney diseases, and the technology applied here can be implemented to address heterogeneous pathophysiology in a variety of diseases at a sub-biopsy scale.

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

Cited by 41 publications

References 51 publications

Protein half‐life determines expression of proteostatic networks in podocyte differentiation

Protein half‐life determines expression of proteostatic networks in podocyte differentiation

Proteolytic program-dependent functions are impaired in INF2-mediated focal segmental glomerulosclerosis

Individual nephron proteomes connect morphology and function in proteinuric kidney disease

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