Biology subjects

DeMartino, G. N.

Publications and source records attributed to DeMartino, G. N..

3 recordsLinked to original sources

bioRxiv2025

PI31 is a positive regulator of 20S immunoproteasome assembly

PI31 (Proteasome Inhibitor of 31,000 Da) is a 20S proteasome-binding protein originally identified as an inhibitor of in vitro 20S proteasome activity. Although recent studies have provided a detailed structural basis for this activity, the physiologic significance of PI31-mediated proteasome inhibition remains uncertain and alternative cellular roles for PI31 have been described. Here we report a role for PI31 as a positive regulator for the assembly of the 20S immuno-proteasome (20Si), a compositionally and functionally distinct isoform of the proteasome that is poorly inhibited by PI31. Genetic ablation of PI31 in mammalian cells had no effect on the cellular content or activity of constitutively expressed proteasomes but reduced the cellular content and activity of interferon-{gamma}-induced immuno-proteasomes. This selective effect is a consequence of defective 20Si assembly, as indicated by the accumulation of 20Si assembly intermediates. Our results highlight a distinction in the assembly pathways of constitutive and immuno-proteasomes and indicate that PI31 plays a chaperone-like role for the selective assembly of 20S immunoproteasomes.

cell biology↗

bioRxiv2023

Differential interactions of the proteasome inhibitor PI31 with constitutive and immuno-20S proteasomes

PI31 (Proteasome Inhibitor of 31,000 Daltons) is a 20S proteasome binding protein originally identified as an in vitro inhibitor of 20S proteasome proteolytic activity. Recently reported cryo-electron microscopy structures of 20S-PI31 complexes reveal a surprising structural basis for proteasome inhibition. The natively disordered proline-rich C-terminus of PI31 enters the central chamber in the interior of the cylindrical 20S proteasome and interacts directly with the proteasomes multiple catalytic threonine residues a manner predicted to inhibit their enzymatic function while evading its own proteolysis. Higher eukaryotes express an alternative form of 20S proteasome featuring genetically and functionally distinct catalytic subunits. This proteasome is expressed in tissues involved in immune function or in response to certain cytokines such as interferon-{gamma} and has been termed "immuno-proteasome." We examined the relative effects of PI31 on constitutive and immuno-20S proteasomes and show that PI31 inhibits the immuno-20S proteasome (20Si) to a significantly lesser degree than it inhibits constitutive 20S proteasome (20Sc). Unlike 20Sc, 20Si hydrolyzes the carboxyl-terminus of PI31 and this effect contributes to the reduced inhibitory activity of PI31 towards 20Si. These results demonstrate unexpected differential interactions of PI31 with 20Sc and 20Si and document their functional consequences.

biochemistry↗

bioRxiv2023

High resolution structure of mammalian PI31 20S proteasome complex reveals mechanism of proteasome inhibition.

Proteasome-catalyzed protein degradation mediates and regulates critical aspects of many cellular functions and is an important element of proteostasis in health and disease. Proteasome function is determined in part by the types of proteasome holoenzymes formed between the 20S core particle that catalyzes peptide bond hydrolysis and any of multiple regulatory proteins to which it binds. One of these regulators, PI31, was previously identified as an in vitro 20S proteasome inhibitor, but neither the molecular mechanism nor the possible physiologic significance of PI31-mediated proteasome inhibition has been clear. Here we report a high- resolution cryo-EM structure of the mammalian 20S proteasome in complex with PI31. The structure shows that two copies of the intrinsically-disordered carboxyl-terminus of PI31 are present in the central cavity of the closed-gate conformation of the proteasome and interact with proteasome catalytic sites in a manner that blocks proteolysis of substrates but resists their own degradation. The two inhibitory polypeptide chains appear to originate from PI31 monomers that enter the catalytic chamber from opposite ends of the 20S cylinder. We present evidence that PI31 can inhibit proteasome activity in mammalian cells and may serve regulatory functions for the control of cellular proteostasis.

molecular biology↗