A Rapid and Versatile Method for Generating Proteins with Defined Ubiquitin Chains

Abstract

Ubiquitin and polyubiquitin chains target proteins for a wide variety of cellular processes. Ubiquitin-mediated targeting is regulated by the lysine through which the ubiquitins are linked as well as the broader ubiquitin landscape on the protein. The mechanisms of this regulation are not fully understood. For example, the canonical proteasome-targeting signal is a lysine 48-linked polyubiquitin chain, and the canonical endocytosis signal is a lysine 63-linked polyubiquitin chain. However, lysine 63-linked polyubiquitin chains can also target substrates for degradation. Biochemical studies of ubiquitinated proteins have been limited by the difficulty of building proteins with well-defined polyubiquitin chains. Here we describe an efficient and versatile method to synthesize ubiquitin chains of defined linkage and length. The synthesized ubiquitin chains are then attached to any protein containing a ubiquitin moiety. These proteins can be used to study ubiquitin targeting in in vitro assays in the tightly controlled manner required for biochemical studies.

Ubiquitin- and ATP-dependent unfoldase activity of P97/VCP•NPLOC4•UFD1L1 is enhanced by a mutation that causes multisystem proteinopathy

“…Although the minimal requirement for recognition of ubiquitylated proteins by p97 is not known, the minimum Ub chain length for recognition by the proteasome is four (75). Therefore, we enzymatically ligated Ub 3 , in which the ubiquitins were joined via K48 linkages and the distal ubiquitin carried a K48R mutation, onto the linearly fused Ub to form pure Ub 3 Ub-GFP (Fig.1A) (76, 77). Second, to create a substrate with longer polyubiquitin chains, we built K48-linked chains directly onto the linearly fused Ub (Fig.…”

“…A plasmid for bacterial expression of Ub-K48R (RDB# 3348) was made from that of Ub (RDB# 2805) by site-directed mutagenesis, and the protein was expressed as previously described (76). Pure K48-linked Ub 3 chains carrying a K48R mutation in the distal Ub were enzymatically synthesized and purified as described previously (76, 77). To form Ub 3 Ub-GFP, 0.5 μM Ube1 (E1), 5 μM gp78RING-Ube2g2, 2.5 μM Ub-GFP, and 5 μM Ub 3 were incubated in 20 mM Hepes pH 7.4, 5 mM ATP, and 5 mM MgCl 2 overnight at 37 °C.…”

An assay for 26S proteasome activity based on fluorescence anisotropy measurements of dye-labeled protein substrates

“…The ubiquitination method used in this paper is efficient, but leads to attachment of one or more heterogeneous polyubiquitin chains to the substrate, which makes it difficult to know the precise concentration of ubiquitinated substrate and can therefore lead to uncertainty in the determined K M and V max values. Other methods allow the synthesis of substrate proteins with precisely defined ubiquitin modifications [24,26,29]. Nevertheless, together, these data demonstrate that the assay described in this paper can easily be used to characterize proteasomal degradation by standard kinetic approaches.…”

“…Incorporating a fluorescent protein such as green fluorescent protein (GFP) into the model protein makes it possible to follow protein degradation by measuring fluorescence using in-gel or high-throughput assays. For example, substrates containing a degradation signal (degron)-fused GFP have been used to monitor proteasome activity by following the decay of fluorescence intensity resulting from degradation of the GFP moiety (e.g., [29,36,37]). Fluorescent proteins often have complicated maturation kinetics and can be difficult to unfold so that they resist degradation by some ATP-dependent proteases [38].…”

“…Fluorescent proteins often have complicated maturation kinetics and can be difficult to unfold so that they resist degradation by some ATP-dependent proteases [38]. However, easier-to-unfold circular permutants of GFP have been developed that make it possible to monitor degradation even by proteases less robust than the proteasome [29,38–41]. …”