A proof-of-concept study in engineering synthetic protein for selective recognition of substrate-free polyubiquitin.
Similar to substrate-conjugated polyubiquitin, unanchored polyubiquitin chains are emerging as important regulators for diverse biological processes. The affinity purification of unanchored polyubiquitin from various organisms has been reported, however, tools able to distinguish unanchored polyubiquitin chains with different isopeptide linkages have not yet been described. Toward the goal of selectively identifying and purifying unanchored polyubiquitin chains linked through different Lysines, Scott et al. developed a novel strategy in their study [Proteomics 2016, 16, 1961-1969]. They designed a linker-optimized ubiquitin-binding domain hybrid (t-UBD) containing two UBDs, a ZnFCUBP domain, and a linkage-selective UBA domain, to specifically recognize unanchored Lys48-linked polyubiquitin chains. Subsequently, a series of assays has proved the feasibility of this novel strategy for the purification of endogenous substrate-free Lys48-linked polyubiquitin chains from mammalian cell extracts. Their research not only provides a tool for purifying unanchored polyubiquitin with different isopeptide linkages, but also paves the way for generating reagents to study the function of unanchored polyubiquitin chains of different linkages in the future. The design of UBD hybrids for defined unanchored polyubiquitin (Lys48-polyubiquitin) in this study also set an excellent example for future methodology studies regarding monitoring in vivo dynamic changes in the patterns of ubiquitination.
State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, P. R. China.
Yehui
Y
Xiong
Plant Pathology department, Center for Plant Science Innovation, University of Nebraska, Lincoln, NE, USA.
Lirong
L
Zeng
State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, P. R. China.
Wende
W
Liu
D001665:Binding Sites; D045424:Complex Mixtures / Q000737:chemistry; D015870:Gene Expression; D057809:HEK293 Cells; D006801:Humans; D008239:Lysine / Q000737:chemistry / Q000378:metabolism*; D025821:Polyubiquitin / Q000737:chemistry / Q000302:isolation & purification*; D011485:Protein Binding; D000072417:Protein Domains; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination
D045424:Complex Mixtures; D011993:Recombinant Fusion Proteins; D025821:Polyubiquitin; D008239:Lysine
Mass spectrometry; Ubiquitin-binding domain; Unanchored polyubiquitin chain