Specific Modification of Aged Proteasomes Revealed by Tag-Exchangeable Knock-In Mice.

The proteasome is the proteolytic machinery at the center of regulated intracellular protein degradation and participates in various cellular processes. Maintaining the quality of the proteasome is therefore important for proper cell function. It is unclear, however, how proteasomes change over time and how aged proteasomes are disposed. Here, we show that the proteasome undergoes specific biochemical alterations as it ages. We generated Rpn11-Flag/enhanced green fluorescent protein (EGFP) tag-exchangeable knock-in mice and established a method for selective purification of old proteasomes in terms of their molecular age at the time after synthesis. The half-life of proteasomes in mouse embryonic fibroblasts isolated from these knock-in mice was about 16 h. Using this tool, we found increased association of Txnl1, Usp14, and actin with the proteasome and specific phosphorylation of Rpn3 at Ser 6 in 3-day-old proteasomes. We also identified CSNK2A2 encoding the catalytic α' subunit of casein kinase II (CK2α') as a responsible gene that regulates the phosphorylation and turnover of old proteasomes. These findings will provide a basis for understanding the mechanism of molecular aging of the proteasome.

The aspartyl protease DDI2 activates Nrf1 to compensate for proteasome dysfunction.

In response to proteasome dysfunction, mammalian cells upregulate proteasome gene expression by activating Nrf1. Nrf1 is an endoplasmic reticulum-resident transcription factor that is continually retrotranslocated and degraded by the proteasome. Upon proteasome inhibition, Nrf1 escapes degradation and is cleaved to become active. However, the processing enzyme for Nrf1 remains obscure. Here we show that the aspartyl protease DNA-damage inducible 1 homolog 2 (DDI2) is required to cleave and activate Nrf1. Deletion of DDI2 reduced the cleaved form of Nrf1 and increased the full-length cytosolic form of Nrf1, resulting in poor upregulation of proteasomes in response to proteasome inhibition. These defects were restored by adding back wild-type DDI2 but not protease-defective DDI2. Our results provide a clue for blocking compensatory proteasome synthesis to improve cancer therapies targeting proteasomes.

A primary role for disulfide formation in the productive folding of prokaryotic Cu,Zn-superoxide dismutase.

Enzymatic activation of Cu,Zn-superoxide dismutase (SOD1) requires not only binding of a catalytic copper ion but also formation of an intramolecular disulfide bond. Indeed, the disulfide bond is completely conserved among all species possessing SOD1; however, it remains obscure how disulfide formation controls the enzymatic activity of SOD1. Here, we show that disulfide formation is a primary event in the folding process of prokaryotic SOD1 (SodC) localized to the periplasmic space. Escherichia coli SodC was found to attain ß-sheet structure upon formation of the disulfide bond, whereas disulfide-reduced SodC assumed little secondary structure even in the presence of copper and zinc ions. Moreover, reduction of the disulfide bond made SodC highly susceptible to proteolytic degradation. We thus propose that the thiol-disulfide status in SodC controls the intracellular stability of this antioxidant enzyme and that the oxidizing environment of the periplasm is required for the enzymatic activation of SodC.