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

Abstract

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.

Nephrotic-syndrome-associated mutation of KANK2 induces pathologic binding competition with physiological interactor KIF21A

“…The protein concentration in the cytosol is likely a determining factor. As a translation initiation factor important for common protein expression, eIF4A1 is highly abundant in cells ( 36 , 37 ). In contrast, the protein level of KIF21A in podocytes is fairly low ( 36 , 37 ).…”

“…As a translation initiation factor important for common protein expression, eIF4A1 is highly abundant in cells ( 36 , 37 ). In contrast, the protein level of KIF21A in podocytes is fairly low ( 36 , 37 ). Furthermore, although our structural data indicate that the KANK1 SF mutant is less likely to interfere with the cellular function of eIF4A1, as discussed above, we cannot rule out the possibility that abnormal binding may interfere with the gene expression regulated by eIF4A1, thus leading to cellular dysfunction and disease progression.…”

Rho GTPase regulatory proteins in podocytes

“…We acknowledge the limitation of the system; many machineries observed in vivo are absent in cultured podocytes and stress fibers observed in vitro do not necessarily correlate with the organized actin bundles in foot processes in vivo. 58 Nonetheless, the cultured podocyte system remains an invaluable tool until better in vitro models will be established. 59 There are additional GEFs and GAPs whose expression has been demonstrated in podocytes and their functions studied only in vitro using cultured podocytes without in vivo correlation or known human gene mutations.…”

The Intertwining of Autophagy and the Ubiquitin Proteasome System in Podocyte (Patho)Physiology

“…In cultured murine and human podocytes, proteomic studies demonstrate a strong proteolytic shift from a predominant proteasomal activity to a lysosomal activity in the course of podocyte differentiation [129,130], which coincides with a globally increased stability of mitochondrial, cytoskeletal, and membrane proteins in differentiated podocytes [130]. How far this finding can be transferred to the physiologic in vivo situation still needs to be determined, as possible skewers such as culture conditions could explain the proteolytic system shift from undifferentiated to differentiated podocytes.…”