Calpain Activity Is Negatively Regulated by a KCTD7-Cullin-3 Complex via Non-degradative Ubiquitination

Authors

Jaiprakash Sharma
Shalaka Mulherkar
Uan-I Chen
Yan Xiong
Lakshya Bajaj
Byoung-Kyu Cho
Young Ah Goo
Hon-Chiu Eastwood Leung
Kimberley F Tolias
Marco Sardiello

Publication Date

3-24-2023

Journal

Cell Discovery

DOI

10.1038/s41421-023-00533-3

PMID

36964131

PMCID

PMC10038992

PubMedCentral® Posted Date

3-24-2023

PubMedCentral® Full Text Version

Post-print

Published Open-Access

yes

Keywords

Ubiquitylation, Ubiquitin ligases, Ubiquitylation, Mechanisms of disease

Abstract

Calpains are a class of non-lysosomal cysteine proteases that exert their regulatory functions via limited proteolysis of their substrates. Similar to the lysosomal and proteasomal systems, calpain dysregulation is implicated in the pathogenesis of neurodegenerative disease and cancer. Despite intensive efforts placed on the identification of mechanisms that regulate calpains, however, calpain protein modifications that regulate calpain activity are incompletely understood. Here we show that calpains are regulated by KCTD7, a cytosolic protein of previously uncharacterized function whose pathogenic mutations result in epilepsy, progressive ataxia, and severe neurocognitive deterioration. We show that KCTD7 works in complex with Cullin-3 and Rbx1 to execute atypical, non-degradative ubiquitination of calpains at specific sites (K398 of calpain 1, and K280 and K674 of calpain 2). Experiments based on single-lysine mutants of ubiquitin determined that KCTD7 mediates ubiquitination of calpain 1 via K6-, K27-, K29-, and K63-linked chains, whereas it uses K6-mediated ubiquitination to modify calpain 2. Loss of KCTD7-mediated ubiquitination of calpains led to calpain hyperactivation, aberrant cleavage of downstream targets, and caspase-3 activation. CRISPR/Cas9-mediated knockout of Kctd7 in mice phenotypically recapitulated human KCTD7 deficiency and resulted in calpain hyperactivation, behavioral impairments, and neurodegeneration. These phenotypes were largely prevented by pharmacological inhibition of calpains, thus demonstrating a major role of calpain dysregulation in KCTD7-associated disease. Finally, we determined that Cullin-3-KCTD7 mediates ubiquitination of all ubiquitous calpains. These results unveil a novel mechanism and potential target to restrain calpain activity in human disease and shed light on the molecular pathogenesis of KCTD7-associated disease.

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