Fluorogenic ATP analogues for online monitoring of ATP consumption: observing ubiquitin activation in real time.

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 1a) 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 ubiquitin conjugating enzyme (E2) initiates the conjugation of ubiquitin to a target protein (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 E1 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. So far, only a few assays that directly measure E1 activity have been described. However, these are laborious and not suitable for the continuous monitoring of E1 activity. In fact, until now no assay is available that allows the direct detection of ubiquitin activation in real time and that, for example, can be easily used to screen for E1 effectors. We, therefore, decided to elaborate on a conceptually novel assay: real time detection of ubiquitin activation by monitoring the cleavage of an ATP analogue (Figure 1b) that harbors two different fluorophores with the potential to undergo Fçrster resonance energy transfer (FRET). In the envisioned doubly labeled ATP analogue, excitation of the fluorescence donor (D) leads to transfer of the excitation energy to the fluorescence acceptor (A) whose fluorescence is monitored. Upon cleavage of the a/b anhydride bond of ATP, FRET is no longer possible and, thus, direct emission of the fluorescence donor can be detected. In this way, E1 activity results in a large change of fluorescence characteristics of the ATP analogue. Similar approaches based on the cleavage of FRET cassettes have been used for studying other hydro lyzing enzymes like proteases. For the envisaged time resolved ATPase sensor (TRASE) approach two fluorophores have to be attached to ATP. As the N6 position of ATP has been proven to be an attractive site for modifications without compromising UBA1 activity, the second modification has to be placed at the phosphoanhydride chain so that the two fluorophores are spatially separated upon cleavage. As earlier studies revealed that phosphate esters of ATP are stable, we tested g modified triphosphate II and d modified tetraphosphate III in promoting the autoubiquity lation of E6AP, a reaction that involves UBA1 and the E2 enzyme UbcH5b (Figure 2a,b). E6AP is an E3 ligase that has been causally associated with the development of three different human disorders: cervical cancer, Angelman syn drome, and autism spectrum disorders. As only the UBA1 reaction is ATP dependent, the appearance of polyubiquity lated E6AP (E6AP Ub), which does not significantly migrate in the applied gel electrophoresis, and consequently Figure 1. a) Mechanism of the activation of ubiquitin by UBA1. Ubiquitin is loaded onto UBA1 through the formation of a thioester bond with consumption of ATP. AMP: adenosine monophosphate, PP: pyrophosphate. b) Concept of signaling ATP consumption. The intact ATP analogue shows fluorescence of the acceptor (A) upon excitation of the donor (D) due to FRET. Upon cleavage by UBA1 direct donor fluorescence can be observed.