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

Abstract

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.