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A new activity-based probe for E1-E2-E3 enzymes involved in autophagy: UbiQ-158

In honor of Prof. Yoshinori Ohsumi being awarded the Nobel prize in Medicine for his discoveries of mechanisms for autophagy, UbiQ has developed the activity-based probe UbiQ-158 (His6-3C-MAP1LC3a-Dha). UbiQ-158 is based on the MAP1LC3a protein sequence in which the C-terminal Gly has been replaced by a dehydroalanine residue (Dha). The N-terminus is labeled with an His6 affinity tag and a 3C protease cleavage site (QG), Cys17 has been mutated to a Ser residue (S). UbiQ-158 is designed to target E1-E2-E3 enzymes involved in autophagy (such as the E1 ATG7 and E2 ATG3) and is a new addition to our family of Triple E probes (see below), based on the first activity-based probe design that allows monitoring of full E1-E2-E3 cascade activity (Figure 1, Mulder et al. Nat Chem Biol 2016, 12, 523).

 

figure showing triple E probe mechanism

Figure 1. Mode of action Triple E probes. At physiological pH the Dha group is relatively inert, but upon activation by the E1 enzyme the electrophilicity increases significantly. As the probe is processed in a native manner by the E1-E2-E3 cascade, the Dha group can react with an active site cysteine residue in an irreversible way, thereby trapping the E1-E2-E3 enzymes.
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FAQ.

  1. Which of the E1-E2-E3 enzymes is targeted by the Triple E probe? The probe targets active site cysteine based E1-E2-E3 enzymes.
  2. Is the probe transferred to substrates of the E1-E2-E3 enzymes? As far as we know the Triple E Probe is not transferred to substrates.
  3. How specific is the probe labelling? The ubiquitin(-like) substrate context of the probe in combination with the required activation by the E1, makes Triple E probes highly specific for E1-E2-E3 enzymes. For a complete background of probe reactivity please see Mulder et al.