Fluorogenic ATP Analogues for Online Monitoring of ATP Consumption: Observing Ubiquitin Activation in Real Time

摘要

The conjugation of ubiquitin to proteins plays an important role in the regulation of numerous cellular processes. Deregulation of this pathway has been associated with different human disorders including cancer and neurodege- nerative diseases. For ubiquitylation, ubiquitin is initially activated by a ubiquitin-activating enzyme (E1) at the expense of adenosine triphosphate (ATP; see Figure la) to form a thioester bond between the C-terminal glycine of ubiquitin and the catalytic cysteine residue of E1.Subse- quent transfer to a cysteine residue of a ubiquitm-conjugating enzyme (E2) initiates the conjugation of ubiquitin to a target em (mostly to a lysine residue by means of an isopeptide bond), a process which in many cases requires the help of a ubiquitin ligase (E3). As UBA1 is one of only two known human El enzymes for ubiquitin, modulation of its activity may prove beneficial in the treatment of certain disorders. Hence, assays for studying the activation of ubiquitin by UBA1 directly and without the interfering effects of the downstream enzymatic cascade are important tools to analyze UBA1 activity and identify modulators of this-enzyme.