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UbiQ-news: launch of new reagents


Farid El Oualid

New reagents

Ub photoLeu73 (UbiQ-154)
a new and powerful crosslinkable Ub 
UbiQ-154 is a new crosslinking reagent based on ubiquitin in which Leu73 is replaced by the photoreactive analog photoleucine (photoLeu, Fig 1). Upon irradiation of the diazirine with UV light, a very reactive carbene species is formed (Chojnack et al, Zhou et al, Liang et al). Because carbenes are quenched quickly by water, unspecific binding is minimal for carbene based crosslinking (Dubinsky et al). This means that only residues that are very nearby (and contribute to tight binding) will react covalently. Thus any unbound UbiQ-154 will react with water before being able to undergo non-specific reactions with other proteins.

Figure 1
Chojnacki et al. Cell Chem Biol 2017, 24, 443
Liang et al. Angew. Chem. Int. Ed. 2017, 56, 2744
Zhou et al. Nat Comm 2016, 7, article 10589
Dubinsky et al. Bioorg Med Chem 2012, 20, 554

Ac-ISG15prox-Rh110MP (UbiQ-127)
a new activity assay reagent for ISG15 proteases

UbiQ-127 is a quenched, fluorescent substrate for ISG15 proteases. Hereby cleavage of the amide bond between the C-terminal Gly and rhodamine110 moiety releases the highly fluorescent Rh110-morpholinecarbonyl (Rh110MP, Terentyeva et al). UbiQ-127 is based on the proximal part of mouse ISG15 (aa 77-165), in line with recent data (Basters et al) that showed that only the C-terminal ubiquitin-like domain of ISG15 is recognized and essential for USP18 activity. 

Basters et al. Nat Struct Mol Biol 2017, 24, 270
Terentyeva et al. Bioconj Chem 2011, 22, 1932.

cRh110-SUMO2 (UbiQ-134)
fluorescent SUMO2 protein
UbiQ-134 is based on the human SUMO2 sequence (C48S). It is functionalized on the N-terminus with a 5-carboxyrhodamine110 dye (λex/emi = 480/520 nm), allowing sensitive and fast (in-gel fluorescence) detection of SUMO2 processing.

Phosphorylated ubiquitin
Although proteomics studies have shown that ubiquitin is phosphorylated at various sites, it remains unclear how phosphorylation affects Ub signaling in general. Access to well-defined phosphorylated Ub reagents is important for elucidating this and therefore UbiQ offers the below set of (site-selective* and biotin labeled)** phosphorylated Ub proteins.
 *  because our phosphorylated Ub proteins are made by total chemical synthesis (El Oualid et al. Angew      
     Chem Int Ed 2010, 49, 10149), incorporation of the phosphorylated residue is 100%.
**  an aminohexanoic acid (Ahx) linker creates extra space for efficient access of biotin binding entities.

Ub pSer57 (UbiQ-113)
Ub pSer65 (UbiQ-089)
Biotin-Ahx-Ub pThr7 (UbiQ-092)

Biotin-Ahx-Ub pThr12 (UbiQ-093)
Biotin-Ahx-Ub pSer57 (UbiQ-094)
Biotin-Ahx-Ub pTyr59 (UbiQ-095)
Biotin-Ahx-Ub pSer65 (UbiQ-091)

(1) Kane et al. J Cell Biol 2014, 205, 143. (2) Kazlauskaite et al. Biochem J 2014, 460, 127. (3) Kondapalli et al. Open Biol 2012, 2, 120080. (4) Koyano et al. Nature 2014, 510, 162. (5) Sauve and Gehring Cell Res 2014, 24, 1025. (6) Spratt et al. Nat Commun 2013, 4, 1983. (7) Trempe et al. Science 2013, 340, 1451. (8) Wauer and Komander EMBO J 2013, 32, 2099. (9) Yamamoto et al. J Biol Chem 2005, 280, 3390

Biotin-ANP-Ub-PA (UbiQ-077)
DUB activity based probe with UV cleavable biotin tag
UbiQ-077 is an activity based probe for deubiquitinating enzymes (DUBs), in which the N-terminal biotin tag is linked to the Ub protein through a UV cleavable linker (ANP= 3-amino-3-(2-nitrophenyl)propanoic acid, Fig 2). UbiQ-077 allows one to use the powerful biotin affinity tag but also to release any covalently bound Ub-DUB complexes under milder (photolysis) conditions. Note that the propargylamide (PA) electrophile forms an acid labile bond with the active site cysteine of a DUB (Fig 2), allowing deconjugation of the Ub-DUB complex if required.

Figure 2
(1) Aoki et al. Bioorg Med Chem 2009, 17, 3405. 
(2) Rodenko et al. Nat Prot 2006, 1, 1120. 
(3) Ekkebus et al. J Am Chem Soc 2013, 135, 2867.

Biotin-Ahx-SUMO2-VME (UbiQ-156)
activity based probe for SUMO proteases
UbiQ-156 is an activity based probe for SUMO proteases. It is based on the human SUMO2 sequence (C48S) in which the C-terminus is functionalized with the electrophilic vinyl methyl ester (VME) group. The N-terminus is labeled with biotin*.

*  an aminohexanoic acid (Ahx) linker creates extra space for efficient access of biotin binding entities.

Mendes et al. Biochim Biophys Acta - Mol Cell Res 2016, 1863, 139

Ub Lys-to-Cys mutants (UbiQ-174 - UbiQ-180) 
Ub Lys-to-Cys mutants are convenient reagents for the preparation of well-defined Ub-chains or site-selective modified Ub (by using thiol-selective reagents).
Ub K6C (UbiQ-174)
Ub K11C (UbiQ-175)
Ub K27C (UbiQ-176) 
Ub K29C (UbiQ-177) 
Ub K33C (UbiQ-178) 
Ub K48C (UbiQ-179) 
Ub K63C (UbiQ-180) 

(1) Raasi and Pickart, Methods Mol Biol 2005, 301, 47.
(2) Valkevich et al. J Am Chem Soc 2012, 134, 6919. 

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