Yuko Hirano

A heterodimeric complex that promotes the assembly of mammalian 20S proteasomes

The 26S proteasome is a multisubunit protease responsible for regulated proteolysis in eukaryotic cells. It comprises one catalytic 20S proteasome and two axially positioned 19S regulatory complexes. The 20S proteasome is composed of 28 subunits arranged in a cylindrical particle as four heteroheptameric rings, α1–7β1–7β1–7α1–7 (refs 4, 5), but the mechanism responsible for the assembly of such a complex structure remains elusive. Here we report two chaperones, designated proteasome assembling chaperone-1 (PAC1) and PAC2, that are involved in the maturation of mammalian 20S proteasomes. PAC1 and PAC2 associate as heterodimers with proteasome precursors and are degraded after formation of the 20S proteasome is completed. Overexpression of PAC1 or PAC2 accelerates the formation of precursor proteasomes, whereas knockdown by short interfering RNA impairs it, resulting in poor maturation of 20S proteasomes. Furthermore, the PAC complex provides a scaffold for α-ring formation and keeps the α-rings competent for the subsequent formation of half-proteasomes. Thus, our results identify a mechanism for the correct assembly of 20S proteasomes.

Dissecting β-ring assembly pathway of the mammalian 20S proteasome

The 20S proteasome is the catalytic core of the 26S proteasome. It comprises four stacked rings of seven subunits each, α1–7β1–7β1–7α1–7. Recent studies indicated that proteasome‐specific chaperones and β‐subunit appendages assist in the formation of α‐rings and dimerization of half‐proteasomes, but the process involved in the assembly of β‐rings is poorly understood. Here, we clarify the mechanism of β‐ring formation on α‐rings by characterizing assembly intermediates accumulated in cells depleted of each β‐subunit. Starting from β2, incorporation of β‐subunits occurs in an orderly manner dependent on the propeptides of β2 and β5, and the C‐terminal tail of β2. Unexpectedly, hUmp1, a chaperone functioning at the final assembly step, is incorporated as early as β2 and is required for the structural integrity of early assembly intermediates. We propose a model in which β‐ring formation is assisted by both intramolecular and extrinsic chaperones, whose roles are partially different between yeast and mammals.

Crystal structure of a chaperone complex that contributes to the assembly of yeast 20S proteasomes

Eukaryotic 20S proteasomes are composed of two α-rings and two β-rings, which form an αββα stacked structure. Here we describe a proteasome-specific chaperone complex, designated Dmp1–Dmp2, in budding yeast. Dmp1–Dmp2 directly bound to the α5 subunit to facilitate α-ring formation. In Δdmp1 cells, α-rings lacking α4 and decreased formation of 20S proteasomes were observed. Dmp1–Dmp2 interacted with proteasome precursors early during proteasome assembly and dissociated from the precursors before the formation of half-proteasomes. Notably, the crystallographic structures of Dmp1 and Dmp2 closely resemble that of PAC3—a mammalian proteasome-assembling chaperone; nonetheless, neither Dmp1 nor Dmp2 showed obvious sequence similarity to PAC3. The structure of the Dmp1–Dmp2–α5 complex reveals how this chaperone functions in proteasome assembly and why it dissociates from proteasome precursors before the β-rings are assembled.

PAC1 Gene Knockout Reveals an Essential Role of Chaperone-Mediated 20S Proteasome Biogenesis and Latent 20S Proteasomes in Cellular Homeostasis

ABSTRACT The 26S proteasome, a central enzyme for ubiquitin-dependent proteolysis, is a highly complex structure comprising 33 distinct subunits. Recent studies have revealed multiple dedicated chaperones involved in proteasome assembly both in yeast and in mammals. However, none of these chaperones is essential for yeast viability. PAC1 is a mammalian proteasome assembly chaperone that plays a role in the initial assembly of the 20S proteasome, the catalytic core of the 26S proteasome, but does not cause a complete loss of the 20S proteasome when knocked down. Thus, both chaperone-dependent and -independent assembly pathways exist in cells, but the contribution of the chaperone-dependent pathway remains unclear. To elucidate its biological significance in mammals, we generated PAC1 conditional knockout mice. PAC1-null mice exhibited early embryonic lethality, demonstrating that PAC1 is essential for mammalian development, especially for explosive cell proliferation. In quiescent adult hepatocytes, PAC1 is responsible for producing the majority of the 20S proteasome. PAC1-deficient hepatocytes contained normal amounts of the 26S proteasome, but they completely lost the free latent 20S proteasome. They also accumulated ubiquitinated proteins and exhibited premature senescence. Our results demonstrate the importance of the PAC1-dependent assembly pathway and of the latent 20S proteasomes for maintaining cellular integrity.

Large‐ and Small‐Scale Purification of Mammalian 26S Proteasomes

The 26S proteasome is an ATP-dependent protease known to collaborate with ubiquitin, whose polymerization acts as a marker for regulated and enforced destruction of unnecessary proteins in eukaryotic cells. It is an unusually large multi-subunit protein complex, consisting of a central catalytic machine (called the 20S proteasome or CP/core particle) and two terminal regulatory subcomplexes, termed PA700 or RP/regulatory particle, that are attached to both ends of the central portion in opposite orientations to form an enzymatically active proteasome. To date, proteolysis driven by the ubiquitin-proteasome system has been shown to be involved in a diverse array of biologically important processes, such as the cell cycle, immune response, signaling cascades, and developmental programs; and the field continues to expand rapidly. Whereas the proteasome complex has been highly conserved during evolution because of its fundamental roles in cells, it has also acquired considerable diversity in multicellular organisms, particularly in mammals, such as immunoproteasomes, PA28, S5b, and various alternative splicing forms of S5a (Rpm 10). However, the details of the ultimate pathophysiological roles of mammalian proteasomes have remained elusive. This article focuses on methods for assay and purification of 26S proteasomes from mammalian cells and tissues.

Ethical issues in invasive mechanical ventilation for amyotrophic lateral sclerosis

Currently in Japan, discontinuing an invasive mechanical ventilator (IMV) is illegal; therefore IMV-related decision making is a crucial issue. This study examined IMV decision-making factors and psychological conflict in 50 patients with amyotrophic lateral sclerosis. The Herth Hope Index was used for the assessment of pre- and post-IMV conflict. Interviews identified some decision-making factors: patient’s decision, patient’s and family’s mutual decision, family’s decision, and emergency-induced without patient’s or family’s consent. Participants who experienced no IMV-related regret received sufficient prior IMV education from physicians and nurses, and time for reflection and family consultation. Their hope was similar to their pre-onset levels. Patients who received no prior IMV education accepted treatment as a natural progression. Their hope levels were lower than pre-onset. Those who received only a brief prior IMV explanation rejected the ventilator, experiencing regret if they were given an emergency IMV. Their hope levels were among the lowest. However, some of these patients managed to overcome their regret through being helped by nurses. Sufficient physician explanation and nursing advocacy for autonomous patient decision making are critical for improving hope in this patient group.

Assembly Mechanisms of Specialized Core Particles of the Proteasome

The 26S proteasome has a highly complicated structure comprising the 20S core particle (CP) and the 19S regulatory particle (RP). Along with the standard CP in all eukaryotes, vertebrates have two more subtypes of CP called the immunoproteasome and the thymoproteasome. The immunoproteasome has catalytic subunits β1i, β2i, and β5i replacing β1, β2, and β5 and enhances production of major histocompatibility complex I ligands. The thymoproteasome contains thymus-specific subunit β5t in place of β5 or β5i and plays a pivotal role in positive selection of CD8+ T cells. Here we investigate the assembly pathways of the specialized CPs and show that β1i and β2i are incorporated ahead of all the other β-subunits and that both β5i and β5t can be incorporated immediately after the assembly of β3 in the absence of β4, distinct from the assembly of the standard CP in which β-subunits are incorporated in the order of β2, β3, β4, β5, β6, β1, and β7. The propeptide of β5t is a key factor for this earlier incorporation, whereas the body sequence seems to be important for the earlier incorporation of β5i. This unique feature of β5t and β5i may account for preferential assembly of the immunoproteasome and the thymoproteasome over the standard type even when both the standard and specialized subunits are co-expressed.