Identification of an E3 Ubiquitin Ligase potentially responsible for MAGI-2 degradation in kidney injury.

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Identification of an E3 Ubiquitin Ligase potentially responsible for MAGI-2 degradation in kidney injury.

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Co-author 1

Kazuyuki Ri kazuyuki-ri@chiba-u.jp Chiba university Department of Nephrology Chiba city Japan *

Co-author 2

Atsuhiro Taguchi ataguchi@chiba-u.jp Chiba university Department of Nephrology Chiba city Japan -

Co-author 3

Mariko Ida marikoida.203@chiba-u.jp Chiba university Department of Nephrology Chiba city Japan -

Co-author 4

Issei Okunaga i_okunaga@chiba-u.jp Chiba university Department of Nephrology Chiba city Japan -

Co-author 5

Yasuhiro Yoshimura yyoshimura@chiba-u.jp Chiba university Department of Nephrology Chiba city Japan -

Co-author 6

Katsuhiko Asanuma kasanuma@chiba-u.jp Chiba university Department of Nephrology Chiba city Japan -

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Introduction

Podocytes and their slit diaphragm (SD) prevent the urinary loss of serum proteins, and podocyte apoptosis contributes to chronic kidney disease (CKD) progression. Using murine kidney-injury models, we previously showed that injury induces nuclear translocation of Dendrin, a SD-complex component, thereby promoting podocyte apoptosis. We also found that genetic knock out of Magi2, a Dendrin-binding partner at the SD, triggers Dendrin nuclear translocation and increases apoptosis. Importantly, MAGI-2 expression was diminished, particularly in nephrotic diseases with poor prognosis in patients. These findings led us to hypothesize that MAGI-2 is actively degraded under diseased conditions. In the present study, we aimed to identify MAGI-2-binding proteins potentially responsible for its degradation.

Methods

We screened for MAGI-2-interacting proteins by yeast two-hybrid (Y2H) using a human cDNA library. Direct molecular interaction between the identified protein and MAGI-2 was examined utilizing co-immunoprecipitation (Co-IP). The interaction domains of each protein were further verified by generating genetically engineered mutant constructs.

Results

A yeast two-hybrid screen using a human cDNA library identified the membrane-localized E3 ubiquitin ligase X. A Co-IP assay confirmed a direct physical interaction between MAGI-2 and X. Mutational screening of genetic motifs revealed the respective interaction sites of each protein. An in vitro ubiquitination assay further demonstrated that X ubiquitinates MAGI-2. Importantly, reanalysis of published single-cell RNA-sequencing data of a focal segmental glomerulosclerosis (FSGS) model mouse revealed enhanced expression of X in diseased podocytes, suggesting its potential activation upon injury in vivo.

Conclusion

We identified membrane-localized E3 ubiquitin ligase, X, that directly binds to MAGI-2, promotes its ubiquitination in vitro, and shows enhanced expression in injured podocytes in vivo. These findings suggest that protein X functions as a potential MAGI-2 degrader, actively promoting MAGI-2 degradation during kidney disease progression, and may therefore represent a novel druggable target for CKD.

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