TY - JOUR
T1 - Calpain activity is negatively regulated by a KCTD7–Cullin-3 complex via non-degradative ubiquitination
AU - Sharma, Jaiprakash
AU - Mulherkar, Shalaka
AU - Chen, Uan I.
AU - Xiong, Yan
AU - Bajaj, Lakshya
AU - Cho, Byoung Kyu
AU - Goo, Young Ah
AU - Leung, Hon Chiu Eastwood
AU - Tolias, Kimberley F.
AU - Sardiello, Marco
N1 - Funding Information:
We thank Hui Zheng, Joanna Jankowsky, Hamed Jafar-Nejad, and Kartik Venkatachalam for critical reading of the manuscript. This work was supported by NIH grant NS079618 (to M.S.) and grants from the Beyond Batten Disease Foundation (to M.S.). The project was supported in part by IDDRC grant number 1U54 HD083092 from the Eunice Kennedy Shriver National Institute of Child Health & Human Development. Cores: BCM RNA In Situ Hybridization Core, Neurobehavioral Core, and The Microscopy Core (IDDRC grant number U54 HD083092 from the Eunice Kennedy Shriver National Institute of Child Health & Human Development). The project was supported in part by Mouse Embryonic Stem Cell Core and Genetically Engineered Mouse Core at Baylor College of Medicine. Mass Spectrometry analyses were performed by the Mass Spectrometry Technology Access Center at McDonnell Genome Institute (MTAC@MGI) at Washington University School of Medicine. We thank Bei Gao for helping with the management of the mouse colony.
Funding Information:
We thank Hui Zheng, Joanna Jankowsky, Hamed Jafar-Nejad, and Kartik Venkatachalam for critical reading of the manuscript. This work was supported by NIH grant NS079618 (to M.S.) and grants from the Beyond Batten Disease Foundation (to M.S.). The project was supported in part by IDDRC grant number 1U54 HD083092 from the Eunice Kennedy Shriver National Institute of Child Health & Human Development. Cores: BCM RNA In Situ Hybridization Core, Neurobehavioral Core, and The Microscopy Core (IDDRC grant number U54 HD083092 from the Eunice Kennedy Shriver National Institute of Child Health & Human Development). The project was supported in part by Mouse Embryonic Stem Cell Core and Genetically Engineered Mouse Core at Baylor College of Medicine. Mass Spectrometry analyses were performed by the Mass Spectrometry Technology Access Center at McDonnell Genome Institute (MTAC@MGI) at Washington University School of Medicine. We thank Bei Gao for helping with the management of the mouse colony.
Publisher Copyright:
© 2023, The Author(s).
PY - 2023/12
Y1 - 2023/12
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=85150951212&partnerID=8YFLogxK
U2 - 10.1038/s41421-023-00533-3
DO - 10.1038/s41421-023-00533-3
M3 - Article
C2 - 36964131
AN - SCOPUS:85150951212
SN - 2056-5968
VL - 9
JO - Cell Discovery
JF - Cell Discovery
IS - 1
M1 - 32
ER -