Evaluating enzyme activities and structures of DUBs

Abstract

Ubiquitin signaling requires tight control of all aspects of protein ubiquitination, including the timing, locale, extent, and type of modification. Dysregulation of any of these signaling features can lead to severe human disease. One key mode of regulation is through the controlled removal of the ubiquitin signal by dedicated families of proteases, termed deubiquitinases. In light of their key roles in signal regulation, deubiquitinases have become a recent focus for therapeutic intervention as a means to regulate protein abundance. This work and recent discoveries of novel deubiquitinases in humans, viruses, and bacteria, provide the impetus for this chapter on methods for evaluating the activities and structures of deubiquitinases. An array of available deubiquitinase substrates for biochemical characterization are presented and their limitations as standalone tools are discussed. Methods for the determination and analysis of deubiquitinase structure are also presented, with a focus on visualizing recognition of the ubiquitin substrate.

Identification and characterization of diverse OTU deubiquitinases in bacteria

“…The Ub‐PA activity‐based probe was prepared using intein chemistry as described previously (Wilkinson et al , ). Activity‐based probe reactions were performed as described (Pruneda & Komander, ). Bacterial OTUs were prepared at 5 μM concentration in 25 mM Tris, 150 mM NaCl, 10 mM DTT, pH 7.4 and incubated at room temperature for 15 min.…”

“…Fluorescent Ub‐ and Ub‐like‐KG(TAMRA) substrates were prepared as described previously (Geurink et al , ; Basters et al , ). Cleavage was monitored by fluorescence polarization as previously described (Pruneda & Komander, ). Bacterial OTUs were prepared at twice the desired enzyme concentration in 25 mM Tris, 100 mM NaCl, 5 mM β‐mercaptoethanol, 0.1 mg/ml BSA, pH 7.4 (FP buffer) and incubated at room temperature for 15 min.…”

“…K27‐linked diUb was prepared chemically (van der Heden van Noort et al , ), M1‐linked diUb was expressed and purified as a gene fusion, and the six other linkages were prepared enzymatically (Michel et al , ). Ub chain cleavage assays were performed as described (Pruneda & Komander, ). Bacterial OTUs were prepared at twice the desired concentration in 25 mM Tris, 150 mM NaCl, 10 mM DTT, pH 7.4 and incubated at room temperature for 15 min.…”

Identification and characterization of diverse OTU deubiquitinases in bacteria

“…Fluorescent Ub-and Ub-like-KG(TAMRA) substrates were prepared as described previously 488 diUb was expressed and purified as a gene fusion, and the six other linkages were prepared 505 enzymatically (Michel et al, 2018). Ub chain cleavage assays were performed as described 506 (Pruneda & Komander, 2019). Bacterial OTUs were prepared at twice the desired concentration 507 in 25 mM Tris, 150 mM NaCl, 10 mM DTT, pH 7.4 and incubated at room temperature for 15 508 min.…”

“…Geurink et al, 2012; Basters et al, 2014). Cleavage was monitored by fluorescence polarization 489 as previously described(Pruneda & Komander, 2019). Bacterial OTUs were prepared at twice 490 the desired enzyme concentration in 25 mM Tris, 100 mM NaCl, 5 mM b-mercaptoethanol, 0.1 491 mg/mL BSA, pH 7.4 (FP buffer) and incubated at room temperature for 15 min.Fluorescent 492 Ub/Ub-like substrates were prepared at 20 nM concentration in FP buffer.…”