Nonenzymatic polyubiquitination of expressed proteins

Hosahalli P. Hemantha, Sudhir N. Bavikar, Yifat Herman-Bachinsky, Najat Haj-Yahya, Somasekhar Bondalapati, Aaron Ciechanover, Ashraf Brik

Research output: Contribution to journalArticlepeer-review

83 Scopus citations


Ubiquitination is one of the most ubiquitous posttranslational modifications in eukaryotes and is involved in various cellular events such as proteasomal degradation and DNA repair. The overwhelming majority of studies aiming to understand ubiquitination and deubiquitination have employed unanchored ubiquitin chains and mono-ubiquitinated proteins. To shed light on these processes at the molecular level, it is crucial to have facile access to ubiquitin chains linked to protein substrates. Such conjugates are highly difficult to prepare homogenously and in workable quantities using the enzymatic machinery. To address this formidable challenge we developed new chemical approaches to covalently attach ubiquitin chains to a protein substrate through its Cys residue. A key aspect of this approach is the installation of acyl hydrazide functionality at the C-terminus of the proximal Ub, which allows, after ubiquitin chain assembly, the introduction of various reactive electrophiles for protein conjugation. Employing α-globin as a model substrate, we demonstrate the facile conjugation to K48-linked ubiquitin chains, bearing up to four ubiquitins, through disulfide and thioether linkages. These bioconjugates were examined for their behavior with the USP2 enzyme, which was found to cleave the ubiquitin chain in a similar manner to unanchored ones. Furthermore, proteasomal degradation study showed that di-ubiquitinated α-globin is rapidly degraded in contrast to the mono-ubiquitinated counterpart, highlighting the importance of the chain lengths on proteasomal degradation. The present work opens unprecedented opportunities in studying the ubiquitin signal by enabling access to site-specifically polyubiquitinated proteins with an increased size and complexity.

Original languageEnglish
Pages (from-to)2665-2673
Number of pages9
JournalJournal of the American Chemical Society
Issue number6
StatePublished - 12 Feb 2014

ASJC Scopus subject areas

  • Catalysis
  • Chemistry (all)
  • Biochemistry
  • Colloid and Surface Chemistry


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