UbiQ Probes, activity-based DUB probes

Target identification and validation

 

 

단백질 탈유비퀴틴화에 관련된 효소의 crystallization, identification, validation에 유용합니다.

프로브는 많은 DUB가 가지는 활성 부위 시스테인 잔기에 반응하도록 설계되어 있으며, C 말단에는 반응기가

결합하고, N 말단에 검출용 태그가 부가되어 있습니다. 반응기는 친전자체(electrophile)로 작용하고 친핵

(nucleophile) 활성 부위인 시스테인 잔기와 반응합니다.

 

 

Activity-based probes for deubiquitinases (DUBs) are based on ubiquitin with a C-terminal warhead designed

to react with the active site cysteine residue present in most DUBs.

UbiQ offers a selection of activity-based probes (ABPs) with various N-terminal tags. They enable the

researcher to crystallize, identify and validate dozens of DUBs and ubiquitin like proteases enzymes as

potential targets involved in protein deubiquitination.

 

 

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Three generations of DUB probes:

1. 1st generation of DUB probes target the S1 pocket of a DUB and have contributed greatly to our understanding

   of DUBs.

2. 2nd generation of DUB probes target the S1 and S1’ pockets. These are di-ubiquitin-based probes with an

   electrophilic group between the two ubiquitins.

3. 3rd generation of DUB probes target the S1 and S2 pocket and are useful tools to identify DUBs that rely on

   S2 site interactions to fine tune their activity.

 

 

Activity-based DUB probes

 

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UbiQ’s wide selection of activity-based DUB probes consists of various electrophiles, substrates and

N- terminal tags. In addition there is the option to order tailor-made activity based probes.

 

 

Electrophiles

UbiQ offers three different electrophiles: propargylamide (PA or Prg), vinylmethyl ester (VME) and vinylsulfone

(VS). All are irriversibe DUB inhibitors and together these target all major DUB families.

    

Electrophiles

PA/Prg

VME

VS

Target DUBs

OTU, USP, UCH, MINDY* and Machado-Josephin Domain1**

 USP & UCH

USP & UCH

Linkage

Irreversible, covalent linkage can be cleaved by acid treatment

Irreversible

Irreversible

 *  A recently discovered DUB family see References 7

** According to a proteomics study the PA probes also target DUBs of the Machado-Josephin Domain family.1

 

 

N-terminal tags

TAMRA2a

Cy52a

Biotin2

HA2

Fluorescence detection

Affinity tag

 Fast (in-gel), sensitive, and distinct read-out

 No background labelling by cross reactivity as seen in

 immunoblots

 Allows pull-down

 Detection by western-blotting

  

 Exc 550 nm, abs 590 nm

 Exc 625 nm, abs 670 nm

 Strongest known non-

 covalent interaction

 Influenza epitope tag

      

 

Applications

• Structural studies, target crystallization

• Phenotypic protein profiling on cell lysates for target validation

• Chemical proteomics for target identification (i.e., pull down, purification, western blotting, MS)

 

 

Experience

• In addition to various activity-based probes, UbiQ has developed more than15 custom probes for a range

  of customers

• Numerous successfully crystallized DUBs using UbiQ’s activity-based probes, including: USP4, UCH-L5,

  and the viral ovarian tumor DUB (vOTU), and Ub-PA probes.

• Target validation and identification of DUBs in infectious diseases (University Glasgow / GSK)

 

 

References

1 (a) Ekkebus et al. J Am Chem Soc 2013, 135, 2867. (b) Sommer et al. Bioorg Med Chem 2013, 21, 2511.

2 (a) de Jong et al. ChemBioChem 2012, 13, 2251. (b) Altun et al. Chem Biol 2011, 18, 1401.

3 (a) Mulder & El Oualid et al. ChemBioChem 2014, 15, 946. (b) El Oualid et al. Angew. Chem. Int. Ed.

   2010, 49, 10149.

4 (a) Misaghi et al. J. Biol. Chem. 2005, 280, 1512. (b) Galardy et al. Methods in Enzymology 2005, 399, 120.

5 (a) Borodovsky et al. Chemistry and Biology 2002, 9, 1149. (b) Borodovsky et al. EMBO J. 2001, 20, 5187.

6 (a) Clerici, M., et al. Nat.Commun. 2014, 5, 5399. (b) Sahtoe, D.D. et al.Mol.Cell 2015, 57, 887

7 (a) Rehman, A., et al. Mol Cell. 2016, 63, 1. (b) Bekes, M. et al. Mol.Cell 2016, 62, 572 For a complete list of

   references please we refer to the product group overview document.

 

 

  Brochure (PDF)

 

 

 

Ordering informations

    

1st generation DUB probes: target the S1 pocket

Catalog No.

Product Name

Electrophile

Tag

UbiQ-108

Ub-VS

VS

-

UbiQ-057

Ub-PA

PA (Prg)

-

UbiQ-078

HA-Ahx-Ahx-Ub-PA

PA (Prg)

HA

UbiQ-076

Biotin-Ahx-Ub-PA

PA (Prg)

Biotin

UbiQ-058

TAMRA-Ub-PA

PA (Prg)

TAMRA

UbiQ-072

Cy5-Ub-PA

PA (Prg)

Cy5

UbiQ-005

Ub-VME

VME

-

UbiQ-035

HA-Ahx-Ahx-Ub-VME

VME

HA

UbiQ-054

Biotin-Ahx-Ub-VME

VME

Biotin

UbiQ-050

TAMRA-Ub-VME

VME

TAMRA

UbiQ-071

Cy5-Ub-VME

VME

HA

* Size ; 50ug

 

2nd generation DUB probes: target the S1 and S1’ pockets

Catalog No.

Product Name

Electrophile

Tag

UbiQ-081

K6 diUb-VME

VME

-

UbiQ-082

K11 diUb-VME

VME

-

UbiQ-083

K27 diUb-VME

VME

-

UbiQ-084

UbiQ-084

VME

-

UbiQ-085

UbiQ-085

VME

-

UbiQ-086

UbiQ-086

VME

-

UbiQ-087

UbiQ-087

VME

-