Structure of hRpn10 Bound to UBQLN2 UBL Illustrates Basis for Complementarity between Shuttle Factors and Substrates at the Proteasome

Abstract

The 26S proteasome is a highly complex 2.5 MDa molecular machine responsible for regulated protein degradation. Proteasome substrates are typically marked by ubiquitination for recognition at receptor sites contributed by Rpn1/S2/PSMD2, Rpn10/S5a, and Rpn13/Adrm1. Each receptor site can bind substrates directly by engaging conjugated ubiquitin chains or indirectly by binding to shuttle factors Rad23/HR23, Dsk2/PLIC/UBQLN, or Ddi1, which contain a ubiquitin-like domain (UBL) that adopts the ubiquitin fold. Previous structural studies have defined how each of the proteasome receptor sites bind to ubiquitin chains as well as some of the interactions that occur with the shuttle factors. Here, we define how hRpn10 binds to the UBQLN2 UBL domain, solving the structure of this complex by NMR, and determine affinities for each UIM region by a titration experiment. UBQLN2 UBL exhibits 25-fold stronger affinity for the N-terminal UIM-1 over UIM-2 of hRpn10. Moreover, we discover that UBQLN2 UBL is fine-tuned for the hRpn10 UIM-1 site over the UIM-2 site by taking advantage of the additional contacts made available through the longer UIM-1 helix. We also test hRpn10 versatility for the various ubiquitin chains to find less specificity for any particular linkage type compared to hRpn1 and hRpn13, as expected from the flexible linker region that connects the two UIMs; nonetheless, hRpn10 does exhibit some preference for K48 and K11 linkages. Altogether, these results provide new insights into the highly complex and complementary roles of the proteasome receptor sites and shuttle factors.

Dimer Interface Organization is a Main Determinant of Intermonomeric Interactions and Correlates with Evolutionary Relationships of Retroviral and Retroviral-Like Ddi1 and Ddi2 Proteases

“…Our results imply that non-viral retroviral-like PRs potentially have relatively lower dimer stability compared to retroviral counterparts. We assume that getting better insight into the structural requirements for the dimer formation through the protease domain may help understanding the roles of Ddi-like proteins in proteasomal shuttles and ubiquitination pathways [6,7,63,64,67,68,84,85], even in physiological or pathological conditions. Furthermore, future studies are needed to prove our findings by determining the in vitro dimer stabilities, and correlating the features described in our study with those of additional eukaryotic retroviral-like or endogenous retrovirus PRs.…”

Impact of Losing hRpn13 Pru or UCHL5 on Proteasome Clearance of Ubiquitinated Proteins and RA190 Cytotoxicity

“…Ubiquitinated substrates are recognized by the 19S regulatory particle (RP) of the proteasome, which abuts the 20S core particle (CP) where proteolysis occurs (1,4). Ubiquitin receptor sites in RP subunits hRpn1/S2/PSMD2 (5), hRpn10/S5a (6), and hRpn13/ADRM1 (7,8) bind directly to substrate-attached ubiquitin chains or ubiquitin folds of substrate shuttle factors (5,(7)(8)(9)(10)(11)(12)(13), which also activate enzymatic activities of the 26S proteasome (14). Prior to proteolysis, ubiquitin chains are deconjugated and removed from substrates by the activity of RP deubiquitinating enzymes (DUBs) UCHL5/Uch37 (15), USP14/Ubp6 (16)(17)(18), and hRpn11 (19,20).…”

Global proteomics ofUbqln2-based murine models of ALS

“…fALS can similarly be caused by mutation of aggregation-prone proteins, such as TDP-43, SOD1, and C9ORF72, or of genes involved in protein degradation, such as VCP, Optineurin, and Ubiquilin2 (UBQLN2; Kiernan et al, 2011). UBQLN2 belongs to a large family of proteins that link proteasomes with ubiquitinated proteins through a ubiquitin-like domain (UBL) that binds to the proteasome, and a ubiquitin-binding domain (UBA) that binds to a variety of mono-or polyubiquitinated proteins (Chen et al, 2019;Kleijnen et al, 2003;Zhang et al, 2008). As such, Ubqlns and other UBL/UBA proteins facilitate proteasomal degradation of ubiquitinated proteins.…”