Mol. Cell. doi:10.1016/j.molcel.2017.08.020 (2017)

Nat. Struct. Mol. Biol. doi:10.1038/nsmb.3475 (2017)

Credit: ELSEVIER

The formation of covalent chains of ubiquitin on target proteins regulates a large variety of signaling events, and different chain types, linked through ubiquitin's primary amines at M1, K6, K11, K27, K29, K33, K48 and K63, are present in cells in varying amounts. For example, most linkages (>75%) are formed via K48- or K63-linked chains, whereas K6-linked chains are rare (<1%). Phage-display technology has enabled the development of antibodies for the more abundant linkage types; however, the detection of K6 chains remains challenging because of selectivity and technical issues. Michel et al. utilized phage-display-derived affimer reagents based on a non-antibody, cystatin-fold protein scaffold, in which a series of randomized loops produces high-affinity binders. Structure-guided optimization of a candidate K6-specific affimer improved binding affinity, enabled detection of endogenous K6 linkages, and revealed by proteomic analysis the E3 ubiquitin ligase HUWE1 as a major source for cellular K6 chains. K6 affimers label damaged mitochondria upon activation of another E3 ligase, Parkin. Gersch et al. used affimers to reveal Parkin substrates that were modified with K6 chains upon depolarization. Together, these studies show that the development of the K6 affimer provides exciting potential for revealing new insights into ubiquitin biology.