Shoshiro Hirayama

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. DOI: http://dx.doi.org/10.7554/eLife.18357.001

Redundant Roles of Rpn10 and Rpn13 in Recognition of Ubiquitinated Proteins and Cellular Homeostasis

Intracellular proteins tagged with ubiquitin chains are targeted to the 26S proteasome for degradation. The two subunits, Rpn10 and Rpn13, function as ubiquitin receptors of the proteasome. However, differences in roles between Rpn10 and Rpn13 in mammals remains to be understood. We analyzed mice deficient for Rpn13 and Rpn10. Liver-specific deletion of either Rpn10 or Rpn13 showed only modest impairment, but simultaneous loss of both caused severe liver injury accompanied by massive accumulation of ubiquitin conjugates, which was recovered by re-expression of either Rpn10 or Rpn13. We also found that mHR23B and ubiquilin/Plic-1 and -4 failed to bind to the proteasome in the absence of both Rpn10 and Rpn13, suggesting that these two subunits are the main receptors for these UBL-UBA proteins that deliver ubiquitinated proteins to the proteasome. Our results indicate that Rpn13 mostly plays a redundant role with Rpn10 in recognition of ubiquitinated proteins and maintaining homeostasis in Mus musculus.

Sirt1-deficiency causes defective protein quality control.

Protein quality control is an important mechanism to maintain cellular homeostasis. Damaged proteins have to be restored or eliminated by degradation, which is mainly achieved by molecular chaperones and the ubiquitin-proteasome system. The NAD+-dependent deacetylase Sirt1 has been reported to play positive roles in the regulation of cellular homeostasis in response to various stresses. However, its contribution to protein quality control remains unexplored. Here we show that Sirt1 is involved in protein quality control in both an Hsp70-dependent and an Hsp70-independent manner. Loss of Sirt1 led to the accumulation of ubiquitinated proteins in cells and tissues, especially upon heat stress, without affecting proteasome activities. This was partly due to decreased basal expression of Hsp70. However, this accumulation was only partially alleviated by overexpression of Hsp70 or induction of Hsp70 upon heat shock in Sirt1-deficient cells and tissues. These results suggest that Sirt1 mediates both Hsp70-dependent and Hsp70-independent protein quality control. Our findings cast new light on understanding the role of Sirt1 in maintaining cellular homeostasis.

Nuclear export of ubiquitinated proteins via the UBIN-POST system

Significance It is commonly observed that proteasome impairment results in accumulation of ubiquitinated proteins in the cytosol. Even proteins originally located in the nucleus show similar cytosolic accumulation, suggesting that unidentified machinery proactively transports them to the cytosol. Here, we report that a protein complex, UBIN–polyubiquitinated substrate transporter, harboring ubiquitin binding domain and nuclear export signal specifically mediates this process. In addition, their worm homologues showing similar transportation activity are important to maintain the lifespan of worms under natural condition. Our findings provide an answer to the long-standing question of why ubiquitinated proteins are deposited in the cytosol by proteasome impairment; they provide definite identification of underlying molecular machinery and show its essential involvement in the proteostasis in animal cells. Although mechanisms for protein homeostasis in the cytosol have been studied extensively, those in the nucleus remain largely unknown. Here, we identified that a protein complex mediates export of polyubiquitinated proteins from the nucleus to the cytosol. UBIN, a ubiquitin-associated (UBA) domain-containing protein, shuttled between the nucleus and the cytosol in a CRM1-dependent manner, despite the lack of intrinsic nuclear export signal (NES). Instead, the UBIN binding protein polyubiquitinated substrate transporter (POST) harboring an NES shuttled UBIN through nuclear pores. UBIN bound to polyubiquitin chain through its UBA domain, and the UBIN-POST complex exported them from the nucleus to the cytosol. Ubiquitinated proteins accumulated in the cytosol in response to proteasome inhibition, whereas cotreatment with CRM1 inhibitor led to their accumulation in the nucleus. Our results suggest that ubiquitinated proteins are exported from the nucleus to the cytosol in the UBIN-POST complex-dependent manner for the maintenance of nuclear protein homeostasis.

PAC1-PAC2 proteasome assembly chaperone retains the core α4-α7 assembly intermediates in the cytoplasm

The proteasome core particle (CP) is a cytoplasmic and nuclear protease complex and is comprised of two α‐rings and two β‐rings stacked in order of αββα. The assembly of CP proceeds by ordered recruitment of β‐subunits on an α‐ring with help of assembly chaperones PAC1‐PAC2, PAC3‐PAC4, and UMP1. However, the mechanism of α‐ring formation remains unsolved. Here, we show that α4, α5, α6, and α7 form a core intermediate as the initial process of α‐ring assembly, which requires PAC3‐PAC4. α1 and α3 can be incorporated independently into the core α4–α7 intermediate, whereas α2 incorporation is dependent on preceding incorporation of α1. Through these processes, PAC1‐PAC2 prevents nonproductive dimerization of α‐ring assembly intermediates. We also found that PAC1‐PAC2 overrides the effect of nuclear localization signals of α‐subunits and retains α‐ring assembly intermediates in the cytoplasm. Our results first show a detailed assembly pathway of proteasomal α‐ring and explain the mechanism by which CP assembly occurs in the cytoplasm.