Advillin acts upstream of phospholipase C ϵ1 in steroid-resistant nephrotic syndrome
Jia Rao, Shazia Ashraf, Weizhen Tan, Amelie T. van der Ven, Heon Yung Gee, Daniela A. Braun, Krisztina Fehér, Sudeep P. George, Amin Esmaeilniakooshkghazi, Won-Il Choi, Tilman Jobst-Schwan, Ronen Schneider, Johanna Magdalena Schmidt, Eugen Widmeier, Jillian K. Warejko, Tobias Hermle, David Schapiro, Svjetlana Lovric, Shirlee Shril, Ankana Daga, Ahmet Nayir, Mohan Shenoy, Yincent Tse, Martin Bald, Udo Helmchen, Sevgi Mir, Afig Berdeli, Jameela A. Kari, Sherif El Desoky, Neveen A. Soliman, Arvind Bagga, Shrikant Mane, Mohamad A. Jairajpuri, Richard P. Lifton, Seema Khurana, Jose C. Martins, Friedhelm Hildebrandt
Jia Rao, Shazia Ashraf, Weizhen Tan, Amelie T. van der Ven, Heon Yung Gee, Daniela A. Braun, Krisztina Fehér, Sudeep P. George, Amin Esmaeilniakooshkghazi, Won-Il Choi, Tilman Jobst-Schwan, Ronen Schneider, Johanna Magdalena Schmidt, Eugen Widmeier, Jillian K. Warejko, Tobias Hermle, David Schapiro, Svjetlana Lovric, Shirlee Shril, Ankana Daga, Ahmet Nayir, Mohan Shenoy, Yincent Tse, Martin Bald, Udo Helmchen, Sevgi Mir, Afig Berdeli, Jameela A. Kari, Sherif El Desoky, Neveen A. Soliman, Arvind Bagga, Shrikant Mane, Mohamad A. Jairajpuri, Richard P. Lifton, Seema Khurana, Jose C. Martins, Friedhelm Hildebrandt
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Research Article Nephrology

Advillin acts upstream of phospholipase C ϵ1 in steroid-resistant nephrotic syndrome

Abstract

Steroid-resistant nephrotic syndrome (SRNS) is a frequent cause of chronic kidney disease. Here, we identified recessive mutations in the gene encoding the actin-binding protein advillin (AVIL) in 3 unrelated families with SRNS. While all AVIL mutations resulted in a marked loss of its actin-bundling ability, truncation of AVIL also disrupted colocalization with F-actin, thereby leading to impaired actin binding and severing. Additionally, AVIL colocalized and interacted with the phospholipase enzyme PLCE1 and with the ARP2/3 actin-modulating complex. Knockdown of AVIL in human podocytes reduced actin stress fibers at the cell periphery, prevented recruitment of PLCE1 to the ARP3-rich lamellipodia, blocked EGF-induced generation of diacylglycerol (DAG) by PLCE1, and attenuated the podocyte migration rate (PMR). These effects were reversed by overexpression of WT AVIL but not by overexpression of any of the 3 patient-derived AVIL mutants. The PMR was increased by overexpression of WT Avil or PLCE1, or by EGF stimulation; however, this increased PMR was ameliorated by inhibition of the ARP2/3 complex, indicating that ARP-dependent lamellipodia formation occurs downstream of AVIL and PLCE1 function. Together, these results delineate a comprehensive pathogenic axis of SRNS that integrates loss of AVIL function with alterations in the action of PLCE1, an established SRNS protein.

Authors

Jia Rao, Shazia Ashraf, Weizhen Tan, Amelie T. van der Ven, Heon Yung Gee, Daniela A. Braun, Krisztina Fehér, Sudeep P. George, Amin Esmaeilniakooshkghazi, Won-Il Choi, Tilman Jobst-Schwan, Ronen Schneider, Johanna Magdalena Schmidt, Eugen Widmeier, Jillian K. Warejko, Tobias Hermle, David Schapiro, Svjetlana Lovric, Shirlee Shril, Ankana Daga, Ahmet Nayir, Mohan Shenoy, Yincent Tse, Martin Bald, Udo Helmchen, Sevgi Mir, Afig Berdeli, Jameela A. Kari, Sherif El Desoky, Neveen A. Soliman, Arvind Bagga, Shrikant Mane, Mohamad A. Jairajpuri, Richard P. Lifton, Seema Khurana, Jose C. Martins, Friedhelm Hildebrandt

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Figure 3

Mutations in AVIL affect podocyte cytoskeleton architecture and inhibit the actin-bundling function of AVIL.

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Mutations in AVIL affect podocyte cytoskeleton architecture and inhibit ...
(A) Locations of mutations in gelsolin homology domains of advillin and their effect on conformational flexibility. Overlay of averaged structures of WT (dark gray) and mutated GH2 (blue) and GH4 (red) domains from MD studies of human AVIL (PDB references: 1RGI and 1H1V). Averaging was carried out over the whole scaled MD trajectory. A homology model for hAvil-1 was built using gelsolin as a template based on the crystal structures of actin-bound gelsolin 1H1V4 (S4–S6 or GH4–GH6 domains) (57) and 1RGI (S1–S3 or GH1–GH3 domains) (58). The models built served as starting structures for all MD studies. Structural elements with increased conformational flexibility are highlighted in teal and magenta for the GH2 domain and in yellow for the GH4 domain. Mutated residues are depicted with sticks, and spheres denote calcium ions. (B) Colocalization of AVIL with F-actin using cDNA clones representing WT and mutations detected in individuals with SRNS (Table 1). Human podocytes were transfected with GFP-tagged WT or the mutant AVIL (Arg135Gln, Leu425Met, or Phe656Valfs*7) and were stained for F-actin with phalloidin (red). Colocalization of AVIL and phalloidin-labeled F-actin resulted in yellow fluorescence. Note that podocytes with overexpression of the truncating mutant Phe656Valfs*7 lost the costaining pattern with phalloidin-labeled F-actin at the cell periphery (white arrowheads). Cell nuclei were stained with DAPI (blue). Scale bars: 10 μm. (C) For the actin-bundling assay, WT or mutant (Arg135Gln, Leu425Met, or Phe656Valfs*7) Avil GST proteins (1 μM) were incubated with F-actin. To determine actin bundling, samples were centrifuged at 10,000 g for 15 minutes, and actin distribution in supernatant (S) and pellet (P) fractions was analyzed by 10% SDS-PAGE and GelCode Blue staining. Control refers to F-actin filaments in the absence of Avil GST protein.