Molecular and Structural Basis of the Proteasome α Subunit Assembly Mechanism Mediated by the Proteasome-Assembling Chaperone PAC3-PAC4 Heterodimer.

Tadashi Satoh,Kenta Okamoto,Eiji Kurimoto,Koichi Kato,Maho Yagi-Utsumi,Keiji Tanaka

doi:10.3390/ijms20092231

Abstract

The 26S proteasome is critical for the selective degradation of proteins in eukaryotic cells. This enzyme complex is composed of approximately 70 subunits, including the structurally homologous proteins α1–α7, which combine to form heptameric rings. The correct arrangement of these α subunits is essential for the function of the proteasome, but their assembly does not occur autonomously. Assembly of the α subunit is assisted by several chaperones, including the PAC3-PAC4 heterodimer. In this study we showed that the PAC3-PAC4 heterodimer functions as a molecular matchmaker, stabilizing the α4-α5-α6 subcomplex during the assembly of the α-ring. We solved a 0.96-Å atomic resolution crystal structure for a PAC3 homodimer which, in conjunction with nuclear magnetic resonance (NMR) data, highlighted the mobility of the loop comprised of residues 51 to 61. Based on these structural and dynamic data, we created a three-dimensional model of the PAC3-4/α4/α5/α6 quintet complex, and used this model to investigate the molecular and structural basis of the mechanism of proteasome α subunit assembly, as mediated by the PAC3-PAC4 heterodimeric chaperone. Our results provide a potential basis for the development of selective inhibitors against proteasome biogenesis.

Highlights

The selective degradation of proteins in eukaryotic cells is essential for the maintenance of physiological homeostasis
To study the biochemical processes involved in proteasome α-subunit assembly mediated by the PAC3-PAC4 heterodimer, we prepared all of the human proteasome α subunits as recombinant proteins
We designed and prepared a PAC3-PAC4 heterodimer as a single-chain form, termed scPAC3/4, in which the C-terminus of PAC4 is connected to the N-terminus of PAC3 via a (GGGS)4 liner

Summary

Introduction

The selective degradation of proteins in eukaryotic cells is essential for the maintenance of physiological homeostasis. As with CP-assembly, yeast Pba and Pba have structural resemblance, and form a heterodimer [16] which functions as a matchmaker mediating the association between α4 and α5 [17] It remains unclear, how the human PAC3-PAC4 complex functions in α-ring assembly through specific, direct interactions with cognate proteasomal subunits, the crystal structures of human PAC3 and PAC4 have been solved for their homodimeric forms [16,18]. Structural insights into the chaperone-mediated formation of the human proteasome are important for the design and development of low-toxicity anticancer drugs which can inhibit the protein-protein interactions involved in the proteasome-assembly process. We performed a biochemical and biophysical study of the human PAC3-PAC4 heterodimer in order to understand the functional and structural mechanisms of α-ring formation mediated by the proteasome-assembling chaperones

The PAC3-PAC4 Heterodimer Interacts Primarily with α5

Sample Preparation

Pull-Down Experiments

Computer-Aided Model Building

NMR Spectroscopy

Accession Numbers