DUB-le vision: snapshots of the proteasome during substrate processing.

Covalent modification by the small protein ubiquitin can target proteins for destruction by the proteasome, but the ubiquitin signal itself is recycled. Surprisingly, proteasomes contain three different deubiquitinating enzymes (DUBs). Recent work by Zhang and Zou et al. reveals how one of these enzymes, Usp14, regulates, and is regulated by, the proteasome.

A novel recognition site for polyubiquitin and ubiquitin-like signals in an unexpected region of proteasomal subunit Rpn1.

The ubiquitin (Ub)-proteasome system is the primary mechanism for maintaining protein homeostasis in eukaryotes, yet the underlying signaling events and specificities of its components are poorly understood. Proteins destined for degradation are tagged with covalently linked polymeric Ub chains and subsequently delivered to the proteasome, often with the assistance of shuttle proteins that contain Ub-like domains. This degradation pathway is riddled with apparent redundancy-in the form of numerous polyubiquitin chains of various lengths and distinct architectures, multiple shuttle proteins, and at least three proteasomal receptors. Moreover, the largest proteasomal receptor, Rpn1, contains one known binding site for polyubiquitin and shuttle proteins, although several studies have recently proposed the existence of an additional uncharacterized site. Here, using a combination of NMR spectroscopy, photocrosslinking, mass spectrometry, and mutagenesis, we show that Rpn1 does indeed contain another recognition site that exhibits affinities and binding preferences for polyubiquitin and Ub-like signals comparable to those of the known binding site in Rpn1. Surprisingly, this novel site is situated in the N-terminal section of Rpn1, a region previously surmised to be devoid of functionality. We identified a stretch of adjacent helices as the location of this previously uncharacterized binding site, whose spatial proximity and similar properties to the known binding site in Rpn1 suggest the possibility of multivalent signal recognition across the solvent-exposed surface of Rpn1. These findings offer new mechanistic insights into signal recognition processes that are at the core of the Ub-proteasome system.

UBL domain of Usp14 and other proteins stimulates proteasome activities and protein degradation in cells.

The best-known function of ubiquitin-like (UBL) domains in proteins is to enable their binding to 26S proteasomes. The proteasome-associated deubiquitinating enzyme Usp14/UBP6 contains an N-terminal UBL domain and is an important regulator of proteasomal activity. Usp14 by itself represses multiple proteasomal activities but, upon binding a ubiquitin chain, Usp14 stimulates these activities and promotes ubiquitin-conjugate degradation. Here, we demonstrate that Usp14's UBL domain alone mimics this activation of proteasomes by ubiquitin chains. Addition of this UBL domain to purified 26S proteasomes stimulated the same activities inhibited by Usp14: peptide entry and hydrolysis, protein-dependent ATP hydrolysis, deubiquitination by Rpn11, and the degradation of ubiquitinated and nonubiquitinated proteins. Thus, the binding of Usp14's UBL (apparently to Rpn1's T2 site) seems to mediate the activation of proteasomes by ubiquitinated substrates. However, the stimulation of these various activities was greater in proteasomes lacking Usp14 than in wild-type particles and thus is a general response that does not involve some displacement of Usp14. Furthermore, the UBL domains from hHR23 and hPLIC1/UBQLN1 also stimulated peptide hydrolysis, and the expression of hHR23A's UBL domain in HeLa cells stimulated overall protein degradation. Therefore, many UBL-containing proteins that bind to proteasomes may also enhance allosterically its proteolytic activity.

Ubiquitinated proteins promote the association of proteasomes with the deubiquitinating enzyme Usp14 and the ubiquitin ligase Ube3c.

In mammalian cells, the 26S proteasomes vary in composition. In addition to the standard 28 subunits in the 20S core particle and 19 subunits in each 19S regulatory particle, a small fraction (about 10-20% in our preparations) also contains the deubiquitinating enzyme Usp14/Ubp6, which regulates proteasome activity, and the ubiquitin ligase, Ube3c/Hul5, which enhances proteasomal processivity. When degradation of ubiquitinated proteins in cells was inhibited, levels of Usp14 and Ube3c on proteasomes increased within minutes. Conversely, when protein ubiquitination was prevented, or when purified proteasomes hydrolyzed the associated ubiquitin conjugates, Usp14 and Ube3c dissociated rapidly (unlike other 26S subunits), but the inhibitor ubiquitin aldehyde slowed their dissociation. Recombinant Usp14 associated with purified proteasomes preferentially if they contained ubiquitin conjugates. In cells or extracts, adding Usp14 inhibitors (IU-1 or ubiquitin aldehyde) enhanced Usp14 and Ube3c binding further. Thus, in the substrate- or the inhibitor-bound conformations, Usp14 showed higher affinity for proteasomes and surprisingly enhanced Ube3c binding. Moreover, adding ubiquitinated proteins to cell extracts stimulated proteasome binding of both enzymes. Thus, Usp14 and Ube3c cycle together on and off proteasomes, and the presence of ubiquitinated substrates promotes their association. This mechanism enables proteasome activity to adapt to the supply of substrates.

Disassembly of Lys11 and mixed linkage polyubiquitin conjugates provides insights into function of proteasomal deubiquitinases Rpn11 and Ubp6.

Protein homeostasis is largely dependent on proteolysis by the ubiquitin-proteasome system. Diverse polyubiquitin modifications are reported to target cellular proteins to the proteasome. At the proteasome, deubiquitination is an essential preprocessing event that contributes to degradation efficiency. We characterized the specificities of two proteasome-associated deubiquitinases (DUBs), Rpn11 and Ubp6, and explored their impact on overall proteasome DUB activity. This was accomplished by constructing a panel of well defined ubiquitin (Ub) conjugates, including homogeneous linkages of varying lengths as well as a heterogeneously modified target. Rpn11 and Ubp6 processed Lys(11) and Lys(63) linkages with comparable efficiencies that increased with chain length. In contrast, processing of Lys(48) linkages by proteasome was inversely correlated to chain length. Fluorescently labeled tetra-Ub chains revealed endo-chain preference for Ubp6 acting on Lys(48) and random action for Rpn11. Proteasomes were more efficient at deconjugating identical substrates than their constituent DUBs by roughly 2 orders of magnitude. Incorporation into proteasomes significantly enhanced enzymatic efficiency of Rpn11, due in part to alleviation of the autoinhibitory role of its C terminus. The broad specificity of Rpn11 could explain how proteasomes were more effective at disassembling a heterogeneously modified conjugate compared with homogeneous Lys(48)-linked chains. The reduced ability to disassemble homogeneous Lys(48)-linked chains longer than 4 Ub units may prolong residency time on the proteasome.

Effect of the deubiquitination enzyme gene UBP6 on the stress-responsive transcription factor Msn2-mediated control of the amino acid permease Gnp1 in yeast.

In the yeast Saccharomyces cerevisiae, the transcriptional factor Msn2 plays an essential role in response to a variety of environmental stresses by activating the transcription of many genes that contain the stress-responsive elements in the promoters. We previously reported that overexpression of the MSN2 gene confers tolerance to various stresses in industrial yeast strains. Recently, the overexpression of MSN2 was shown to increase the amount of the amino acid permease Gnp1 on the plasma membrane, leading to the increased uptake of proline into the cell, suggesting a novel link between the Msn2-mediated stress response and amino acid homeostasis in yeast. Here, we found that overexpression of MSN2 increased ubiquitinated protein levels with reduced free ubiquitin. Among deubiquitinating enzymes (DUBs), it was revealed that the loss of Ubp6 depleted the free ubiquitin level and decreased tolerance to the toxic amino acid analogues. The overexpression of UBP6 in MSN2-overexpressing cells clearly complemented the impaired tolerance towards the toxic amino acid analogues. Both the protein level and the plasma-membrane localization of Gnp1 were increased in ubp6-deleted cells, as shown in MSN2-overexpressing cells. These results suggest that an excess level of Msn2 impairs endocytic degradation of Gnp1 through dysfunction of Ubp6 and other DUBs.

Proline Homeostasis in Saccharomyces cerevisiae: How Does the Stress-Responsive Transcription Factor Msn2 Play a Role?

Overexpression of MSN2, which is the transcription factor gene in response to stress, is well-known to increase the tolerance of the yeast Saccharomyces cerevisiae cells to a wide variety of environmental stresses. Recent studies have found that the Msn2 is a feasible potential mediator of proline homeostasis in yeast. This result is based on the finding that overexpression of the MSN2 gene exacerbates the cytotoxicity of yeast to various amino acid analogs whose uptake is increased by the active amino acid permeases localized on the plasma membrane as a result of a dysfunctional deubiquitination process. Increased understanding of the cellular responses induced by the Msn2-mediated proline incorporation will provide better comprehension of how cells respond to and counteract to different kinds of stimuli and will also contribute to the breeding of industrial yeast strains with increased productivity.