Capn3 depletion causes Chk1 and Wee1 accumulation and disrupts synchronization of cell cycle reentry during liver regeneration after partial hepatectomy

Recovery of liver mass to a healthy liver donor by compensatory regeneration after partial hepatectomy(PH)is a prerequisite for liver transplantation.Synchronized cell cycle reentry of the existing hepatocytes after PH is seemingly a hallmark of liver compensatory regeneration.Although the molecular control of the PH-triggered cell cycle reentry has been extensively studied,little is known about how the synchronization is achieved after PH.The nucleolus-localized protein cleavage complex formed by the nucleolar protein Digestive-organ expansion factor(Def)and cysteine proteinase Calpain 3(Capn3)has been implicated to control wounding healing during liver regeneration through selectively cleaving the tumor suppressor p53 in the nucleolus.However,whether the Def-Capn3 complex participates in regulating the synchronization of cell cycle reentry after PH is unknown.In this report,we generated a zebrafish capn3b null mutant(capn3b^(Δ19Δ14)).The homozygous mutant was viable and fertile,but suffered from a delayed liver regeneration after PH.Delayed liver regeneration in capn3b^(Δ19Δ14)was due to disruption of synchronized cell proliferation after PH.Mass spectrometry(MS)analysis of nuclear proteins revealed that a number of negative regulators of cell cycle are accumulated in the capn3b^(Δ19Δ14)liver after PH.Moreover,we demonstrated that Check-point kinase 1(Chk1)and Wee1,two key negative regulators of G2 to M transition,are substrates of Capn3.We also demonstrated that Chk1 and Wee1 were abnormally accumulated in the nucleoli of amputated capn3bΔ19Δ14 liver.In conclusion,our findings suggest that the nucleolar-localized Def-Capn3 complex acts as a novel regulatory pathway for the synchronization of cell cycle reentry,at least partially,through inactivating Chk1 and Wee1 during liver regeneration after PH.

Nucleolus-localized Def-CAPN3 protein degradation pathway and its role in cell cycle control and ribosome biogenesis

The nucleolus,as the‘nucleus of the nucleus’,is a prominent subcellular organelle in a eukaryocyte.The nucleolus serves as the centre for ribosome biogenesis,as well as an important site for cell-cycle regulation,cellular senescence,and stress response.The protein composition of the nucleolus changes dynamically through protein turnover to meet the needs of cellular activities or stress responses.Recent studies have identified a nucleolus-localized protein degradation pathway in zebrafish and humans,namely the Def-CAPN3 pathway,which is essential to ribosome production and cell-cycle progression,by controlling the turnover of multiple substrates(e.g.,ribosomal small-subunit[SSU]processome component Mpp10,transcription factor p53,check-point proteins Chk1 and Wee1).This pathway relies on the Ca2þ-dependent cysteine proteinase CAPN3 and is independent of the ubiquitin-mediated proteasome pathway.CAPN3 is recruited by nucleolar protein Def from cytoplasm to nucleolus,where it proteolyzes its substrates which harbor a CAPN3 recognition-motif.Def depletion leads to the exclusion of CAPN3 and accumulation of p53,Wee1,Chk1,and Mpp10 in the nucleolus that result in cell-cycle arrest and rRNA processing abnormality.Here,we summarize the discovery of the Def-CAPN3 pathway and propose its biological role in cell-cycle control and ribosome biogenesis.