Structural insights into the human PA28–20S proteasome enabled by efficient tagging and purification of endogenous proteins

Abstract

The ability to produce folded and functional proteins is a necessity for structural biology and many other biological sciences. This task is particularly challenging for numerous biomedically important targets in human cells, including membrane proteins and large macromolecular assemblies, hampering mechanistic studies and drug development efforts. Here we describe a method combining CRISPR-Cas gene editing and fluorescence-activated cell sorting to rapidly tag and purify endogenous proteins in HEK cells for structural characterization. We applied this approach to study the human proteasome from HEK cells and rapidly determined cryogenic electron microscopy structures of major proteasomal complexes, including a high-resolution structure of intact human PA28αβ–20S. Our structures reveal that PA28 with a subunit stoichiometry of 3α/4β engages tightly with the 20S proteasome. Addition of a hydrophilic peptide shows that polypeptides entering through PA28 are held in the antechamber of 20S prior to degradation in the proteolytic chamber. This study provides critical insights into an important proteasome complex and demonstrates key methodologies for the tagging of proteins from endogenous sources.

Structure, Function, and Allosteric Regulation of the 20S Proteasome by the 11S/PA28 Family of Proteasome Activators

“…However, as PA28αβ and PA26 are heteromeric and homomeric, respectively, there is some variability in their ability to open the gate. PA26 has perfect seven-fold symmetry of its activation loops due to its seven identical subunits, which PA28αβ does not; in humans, recent research showed PA28 stoichiometry of 3α/4β, which is different from the mice stoichiometry 4α/3β reported, as the preferred conformation to engage the 20S proteasome [ 71 ]. Therefore, PA26 can fully open the 20S CP gate, whereas PA28αβ only partially opens the gate due to asymmetrically binding to the 20S CP [ 71 , 72 ].…”

“…PA26 has perfect seven-fold symmetry of its activation loops due to its seven identical subunits, which PA28αβ does not; in humans, recent research showed PA28 stoichiometry of 3α/4β, which is different from the mice stoichiometry 4α/3β reported, as the preferred conformation to engage the 20S proteasome [ 71 ]. Therefore, PA26 can fully open the 20S CP gate, whereas PA28αβ only partially opens the gate due to asymmetrically binding to the 20S CP [ 71 , 72 ]. Nevertheless, the gate-opening mechanism of these 11S regulators remains the same, just to different extents.…”

Comprehensive Analysis of PSME2 and Its Significant Role in Non- small cell lung cancer

“…Among them, PA28 consists of three subunits: α, β and γ. PA28α and β are 50% homologous, and almost all cell types exhibit their expression (Murata, Udono et al 2001). PSME1/2 is comprised of two homologous genes that, respectively, code for the PA28α and PA28β proteins (Cascio 2014, Zhao, Makhija et al 2022. It primarily does this by interacting with the 20S proteasome in a non-ATP-dependent manner, which boosts the activity of protein degradation(Lesne, Locard-Paulet et al…”

PSME2 offers value as a biomarker of M1 macrophage infiltration in pan-cancer and inhibits osteosarcoma malignant phenotypes

“…As a key regulator of 20S proteasomal activity, proteasome activator 28 (PA28) plays vital roles in the control of transcriptional activity, antigen presentation, cell cycle progression, learning, memory, and the suppression of depressive behaviors [1][2][3]. PA28 is composed of three protein subunits known as PA28α, PA28β (also known as PSME2), and PA28γ.…”