High-affinity free ubiquitin sensors for quantifying ubiquitin homeostasis and deubiquitination

Abstract

Ubiquitin (Ub) conjugation is an essential post-translational modification that affects nearly all proteins in eukaryotes. The functions and mechanisms of ubiquitination are areas of extensive study, and yet the dynamics and regulation of even free (i.e., unconjugated) Ub are poorly understood. A major impediment has been the lack of simple and robust techniques to quantify Ub levels in cells and to monitor Ub release from conjugates. Here we describe avidity-based fluorescent sensors that address this need. The sensors bind specifically to free Ub, have K d values down to 60 pM, and, in concert with a newly developed workflow, allow us to distinguish and quantify the pools of free, protein-conjugated, and thioesterified forms of Ub from cell lysates. Alternatively, free Ub in fixed cells can be visualized microscopically by staining with a sensor. Real-time assays using the sensors afford unprecedented flexibility and precision to measure deubiquitination of virtually any (poly)Ub conjugate.

Comparative profiling of stress granule clearance reveals differential contributions of the ubiquitin system

“…To further characterize the Ub species present in SGs induced by arsenite, VER/Puro and heat shock, we performed immunofluorescence microscopy using monoclonal antibodies specific for K48-and K63-linked Ub chains, respectively, as well as a recently developed, avidity-based sensor for the detection of free Ub (HA-tUI) (Figs 3 and 4). HA-tUI binds to the free C-termini of mono-Ub and unanchored Ub chains with sub-nanomolar affinities and exhibits >10 6 -fold selectivity for free over conjugated Ub (Choi et al, 2019). The analysis with the linkage-specific antibodies revealed the presence of both K48-and K63-linked chains in all three types of SGs tested (Fig 3A and B), with relative frequencies similar to those determined with the FK2 antibody (Fig 4B).…”

“…Protein-conjugated and unanchored Ub chains, in turn, can be discriminated by virtue of the absence and presence, respectively, of a free Ub C-terminus. Our confocal and SIM analysis using the recombinant sensor protein HA-tUI, which has been shown before to stain endogenous free Ub in immunofluorescence microscopy (Choi et al, 2019), demonstrated a very low frequency (Fig 4B ) and a distinct appearance (Fig 4G) of HA-tUI-positive structures at SGs, strongly suggesting that most of the SG-associated Ub chains exists in protein conjugates, not as unanchored chains.…”

“…Whereas this is consistent with a previous report for heat-and VER/Puro-induced SGs (Xie et al, 2018), the association of K48-and K63-linked chains with arsenite-induced SGs had remained undetected in that study, perhaps because of their lower frequency. The recombinantly produced free Ub sensor HA-tUI was added to fixed and permeabilized cells and detected by anti-HA immunofluorescence microscopy (Choi et al, 2019). It showed a mostly diffuse cytoplasmic staining under control conditions, whereas additional cytoplasmic puncta were detectable under stress conditions (Fig 4A).…”

Ubiquitin Homeostasis Is Disrupted in TDP-43 and FUS Cell Models of ALS

“…A significant decrease in monomeric Ub levels was also observed in cells expressing FUS R495X -GFP in comparison with cells expressing FUS WT -GFP and tGFP (Figure 4C). To determine whether TDP-43-GFP and FUS-GFP expression also alters the endogenous monomeric Ub pool, we used the probe tUI-HA, which is designed to bind strongly and specifically to free Ub (Choi et al, 2019). Using confocal microscopy, we confirmed the specificity and sensitivity of this probe for free Ub by measuring tUI-HA levels in NSC-34 cells treated with the E1/UBA1 inhibitor TAK-243 (Hyer et al, 2018).…”

A deubiquitylase with an unusually high-affinity ubiquitin-binding domain from the scrub typhus pathogen Orientia tsutsugamushi

“…The OtDUB UBD will be useful as a tool for the enrichment and detection of ubiquitin and ubiquitylated proteins. Related to this, avidity-based, high-affinity protein binders specific for free ubiquitin have been engineered that are useful as sensors for free mono-ubiquitin levels 53 . Recent work has also shown that Cys-to-Ala mutation within some DUB catalytic triads results in highaffinity ubiquitin binding that could be used similarly 54 .…”