Biology subjects

Fushman, D.

Publications and source records attributed to Fushman, D..

3 recordsLinked to original sources

bioRxiv2021

Dual domain recognition determines SARS-CoV-2 PLpro selectivity for human ISG15 and K48-linked di-ubiquitin

The Papain-like protease (PLpro) is a domain of a multi-functional, non-structural protein 3 of coronaviruses. PLpro cleaves viral polyproteins and posttranslational conjugates with poly-ubiquitin and protective ISG15, composed of two ubiquitin-like (UBL) domains. Across coronaviruses, PLpro showed divergent selectivity for recognition and cleavage of posttranslational conjugates despite sequence conservation. We show that SARS-CoV-2 PLpro binds human ISG15 and K48-linked di-ubiquitin (K48-Ub2) with nanomolar affinity and detect alternate weaker-binding modes. Crystal structures of untethered PLpro complexes with ISG15 and K48-Ub2 combined with solution NMR and cross-linking mass spectrometry revealed how the two domains of ISG15 or K48-Ub2 are differently utilized in interactions with PLpro. Analysis of protein interface energetics predicted differential binding stabilities of the two UBL/Ub domains that were validated experimentally. We emphasize how substrate recognition can be tuned to cleave specifically ISG15 or K48-Ub2 modifications while retaining capacity to cleave mono-Ub conjugates. These results highlight alternative druggable surfaces that would inhibit PLpro function.

biophysics↗

bioRxiv2021

Branched ubiquitin chain binding and deubiquitination by UCH37 facilitate proteasome clearance of stress-induced inclusions

UCH37, also known as UCHL5, is a highly conserved deubiquitinating enzyme (DUB) that associates with the 26S proteasome. Recently it was reported that UCH37 activity is stimulated by branched ubiquitin chain architectures. To understand how UCH37 achieves its unique debranching specificity, we performed biochemical and NMR structural analyses and found that UCH37 is activated by contacts with the hydrophobic patches of both distal ubiquitins that emanate from a branched ubiquitin. In addition, RPN13, which recruits UCH37 to the proteasome, further enhances branched-chain specificity by restricting linear ubiquitin chains from having access to the UCH37 active site. In cultured human cells under conditions of proteolytic stress, we show that substrate clearance by the proteasome is promoted by both binding and deubiquitination of branched polyubiquitin by UCH37. Proteasomes containing UCH37(C88A), which is catalytically inactive, aberrantly retain polyubiquitinated species as well as the RAD23B substrate shuttle factor, suggesting a defect in recycling of the proteasome. These findings provide a foundation to understand how proteasome degradation of substrates modified by a unique ubiquitin chain architecture is aided by a DUB.

biochemistry↗

bioRxiv2020

Rub1/NEDD8, a ubiquitin-like modifier, is also a ubiquitin modifier

Of all ubiquitin-like small protein modifiers, Rub1/NEDD8 is the closest kin of ubiquitin in sequence and in structure. Despite their profound similarities, prevalence of ubiquitin and of Rub1 is starkly different: targets of ubiquitin modification reach into the thousands, whereas unique targets of Rub1/NEDD8 appear limited to one family of proteins, Cullins. This distinction is likely due to dedicated E1 activating enzymes that select either one or the other and relay the modifier until it is covalently attached to a target. To convert typical neddylation targets for modification by ubiquitin, and vice versa, we designed reciprocal substitutions at position 72 of Rub1 and of ubiquitin to render them substrates for activation by their non-cognate E1 activating enzymes. We found that this single amino acid is sufficient to distinguish between Ub and Rub1 in living cells, and determine their targets. Thus, modification of Cullins by UbR72T could compensate for loss of Rub1, even as it maintained its ability to polymerize and direct conjugates for degradation. Conversely, Rub1T72R activated by ubiquitin-activating enzyme entered into the ubiquitination cascade, however was not efficiently polymerized, essentially capping polyubiquitin chains. Upon shortage of free ubiquitin under stress, even native Rub1 spilled-over into the ubiquitinome suppressing polyubiquitination. By contrast, the need to maintain monomeric modifications on unique targets is a likely explanation for why the Rub1-activating enzyme strictly discriminates against ubiquitin. Swapping Rub1 and ubiquitin signals uncovered a reason for maintaining two separate pathways across eukaryotic kingdom.

biochemistry↗