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UbiQ news: Launching a new proteasome activity assay reagent


Wieger Rupert


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September 19, 2016

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

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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 His6-3C-MAP1LC3a-Dha (UbiQ-158). 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), which are based on the first activity-based probe design that allows monitoring of full E1-E2-E3 cascade activity. 

figure showing triple E probe mechanism
Figure 1
As outlined in Figure 1, Triple E probes are based on a ubiquitin(-like) protein in which the native C-terminal glycine is replaced by a dehydroalanine (Dha) group. 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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1) MPC Mulder et al. A cascading activity-based probe sequentially targets E1–E2–E3 ubiquitin enzymes. Nat. Chem. Biol. 2016, 12, 523.
2) MPC Mulder, F. El Oualid and H. Ovaa. Adenylation enzyme inhibitors. 
Application WO/2016/032332 and NL2015/050596
DOI: 10.1038/NCHEMBIO.2084
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UbiQ is offering free samples of UbiQ-158. All you have to do is contact us:
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Triple E probes

Available Triple E probes

UbiQ-101 : Ub-Dha
UbiQ-102 : Biotin-Ahx-Ub-Dha
UbiQ-103 : His6-Ahx-Ahx-Ub-Dha
UbiQ-104 : Cy5-Ub-Dha
UbiQ-131 : 5-carboxyRh110-Ub-Dha
UbiQ-105 : Nedd8-Dha
UbiQ-106 : Biotin-Ahx-Nedd8-Dha
UbiQ-122 : 5-carboxyRh110-Nedd8-Dha

UbiQ-116 : SUMO1-Dha
UbiQ-130 : 5-carboxyRh110-SUMO1-Dha

UbiQ-117 : SUMO2-Dha
UbiQ-159 : Biotin-Ahx-SUMO2-Dha

UbiQ-118 : SUMO3-Dha
UbiQ-124 : His10-SUMO3-Dha
UbiQ-158 : His6-3C-MAP1LC3a-Dha New
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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.

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.

How specific is the probe labelling?
The ubiquitine(-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 reference 1.
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