Quantitative deep mapping of the cultured podocyte proteome uncovers shifts in proteostatic mechanisms during differentiation

Abstract

The renal filtration barrier is maintained by the renal podocyte, an epithelial postmitotic cell. Immortalized mouse podocyte cell lines-both in the differentiated and undifferentiated state-are widely utilized tools to estimate podocyte injury and cytoskeletal rearrangement processes in vitro. Here, we mapped the cultured podocyte proteome at a depth of more than 8,800 proteins and quantified 7,240 proteins. Copy numbers of proteins mutated in forms of hereditary nephrotic syndrome or focal segmental glomerulosclerosis (FSGS) were assessed. We found that cultured podocytes express abundant copy numbers of endogenous receptors, such as tyrosine kinase membrane receptors, the G protein-coupled receptor (GPCR), NPR3 (ANP receptor), and several poorly characterized GPCRs. The data set was correlated with deep mapping mRNA sequencing ("mRNAseq") data from the native mouse podocyte, the native mouse podocyte proteome and staining intensities from the human protein atlas. The generated data set was similar to these previously published resources, but several native and high-abundant podocyte-specific proteins were not identified in the data set. Notably, this data set detected general perturbations in proteostatic mechanisms as a dominant alteration during podocyte differentiation, with high proteasome activity in the undifferentiated state and markedly increased expression of lysosomal proteins in the differentiated state. Phosphoproteomics analysis of mouse podocytes at a resolution of more than 3,000 sites suggested a preference of phosphorylation of actin filament-associated proteins in the differentiated state. The data set obtained here provides a resource and provides the means for deep mapping of the native podocyte proteome and phosphoproteome in a similar manner.

Angiotensin II regulates phosphorylation of actin‐associated proteins in human podocytes

“…With this technique, we identified 6323 independent phosphopeptides that corresponded to 2081 different proteins. More than three fourths of proteins identified in our screen have been previously found in a non-phospho-specific characterization of the mouse podocyte proteome (34), which validated the outcome of this SILAC approach. Of interest, one fourth of proteins identified in our approach are new and had not been identified in the mouse podocyte proteome, likely as a result of the phospho-enrichment of proteins of our podocyte cell line.…”

“…The ratio of cell areas is shown (n $ 3, mean 6 SD, Student's t test). *P , 0.05, **P , 0.01, ***P , 0.001. podocyte proteome (34), which validated the outcome of this SILAC approach. Of interest, one fourth of proteins identified in our approach are new and had not been identified in the mouse podocyte proteome, likely as a result of the phospho-enrichment of proteins of our podocyte cell line.…”

“…In comparison to other studies, it is interesting to overlap the phosphoproteins identified in the podocyte cell line in our study with native glomerular podocytes, and to evaluate parallels with studies that characterized AngII-induced phosphoproteomic changes in different cell types. Of proteins, 1595 from our screen have previously been proven to be expressed in podocytes by a non-phospho-enriched proteome characterization of primary cultured murine podocytes (34) (Fig. 1D).…”

“…D) Venn diagram of all proteins identified in this study (AngII-PhosProt) compared with a non-phospho-enriched proteome characterization of primary murine podocytes (cPodoProt) from Rinschen et al(33). E ) Venn diagram comparing the phosphoproteins that were increased in this study (AngII-PhosProt) compared with phosphoproteins that were increased in a study of AT1R signaling in HEK293 cells (HEK-AT1R) from Christensen et al(34).…”

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

“…In culture, podocytes do not form secondary processes with slit diaphragms in between neighboring ABBREVIATIONS: 2D, 2-dimensional; ACTN4, a-actinin-4; ANLN, anillin; ARHGDIA, r-GDP-dissociation inhibitor a; BM, basement membrane; BNIP3, BCL2/adenovirus E1B 19 kDa protein-interacting protein 3; BNIP3L, BCL2/adenovirus E1B 19 kDa protein-interacting protein 3-like; BSA, bovine serum albumin; CBX1, chromobox protein homolog 1; CD2AP, CD2-associated protein; CD80, T lymphocyte-activation antigen CD80; CHX, cycloheximide; CNN1, calponin-1; COBLL1, cordon-bleu protein-like 1; CTSD, cathepsin D; DAPK1/3, death-associated protein kinase 1; DAPK3, death-associated protein kinase 3; DQ-BSA, dequenched bovine serum albumin; ER, endoplasmic reticulum; ESI, electrospray ionization; FA, formic acid; F-actin, filamentous actin; FBS, fetal bovine serum; FDR, false-discovery rate; FPKM, fragments per kilobase million; FSGS, focal segmental glomerulosclerosis; GO, gene ontology; GO-CC, gene ontology cellular component; GSN, gelsolin; iBAQ, intensity-based absolute quantification; ITGA3, integrin a-3; LAMB2, laminin subunit b-2; LAMP1/2, lysosomal-associated membrane protein 1/2; LFQ, label-free quantification; LMNA, lamin A/C; MS/MS, tandem mass spectrometry; MYH9, myosin-9; NCK1/2, cytoplasmic protein NCK1/2; nLC, nano liquid chromatography; pSILAC, pulsed stable isotope labeling by amino acids in cell culture; PSM, peptide spectrum match; RBBP4, retinoblastoma-binding protein 4; RPMI, Roswell Park Memorial Institute; SYNPO, synaptopodin; t 1/2 , half-life; TRPC6, short transient receptor potential cation channel 6; WT1, Wilms tumor protein 1 cells. Hence, podocyte-derived cells show only a very limited expression of podocyte-specific marker proteins, including slit diaphragm-associated proteins like nephrin (7), podocin (8), or transient receptor potential cation channel 6 (9)(10)(11).…”