Identification of Cell Cycle Regulators Targeted by CAPN3 and Their Role in the Development and Function of Digestive Organs

CAPN3 is a member of the calcium-dependent cysteine protease family, calpains. Studies in zebrafish have revealed that Capn3b (a homolog of CAPN3) can be recruited by the nucleolar protein Def to the nu cleolus, where it cleaves cell cycle regulatory proteins such as p53, Chk1, and Wee1, thereby regulating liver devel opment and regeneration. This suggests that the DEF-CAPN3/Def-Capn3b proteolytic pathway plays a critical role in cell cycle regulation. However, whether CAPN3 targets other cell cycle regulators remains unknown. This study employed an in vitro enzymatic activity assay system to examine the cleavage of 29 human cyclins and 13 cell cycle-related factors by hCAPN3 (human CAPN3). This study identified four proteins—CCAR2, CCNL1, RBL1, and RBL2—as novel substrates of hCAPN3. Subsequently, this study used CRISPR-Cas9 technology to generate zebrafish mutants for the homologous genes encoding these four proteins. In situ hybridization analysis with liver-, pancreas-, and intestine-specific probes revealed that knockout of rbl1 significantly delayed liver development in zebrafish, while knockout of rbl2 showed a potential impact on intestinal development. Although the rbl1 mutants were viable and fertile, RNA-seq data analysis indicated significant upregulation of genes related to hepatocyte metabolic activity and significant downregulation of genes associated with circadian rhythm and immune response in adult fish, suggesting disrupted liver function in the rbl1 mutants. These findings enhance the understanding of the functional roles and mechanisms of the DEF-CAPN3/Def-Capn3b complex in cell cycle regulation.

CSTR: 32200.14.cjcb.2026.02.0005