Mutant or wild-type: 7 effective means to screen and profile Human EZH2 Methyltransferase

Histone-lysine N-methyltransferase EZH2  is a methyltransferase involved in transcriptional gene regulation. Together with with EED, RbAP48, SUZ12 and AEBP2, EZH2 forms a 5-member complex that adds 3 methyl groups to Lysine 27 of Histone H3 (H3K27). EZH2 acts on chromatin condensation and gene transcription activity.

EZH2 belongs to the Polycomb-group protein complex family (PcG). PcG family members are involved in maintaining the transcriptional repressive state of genes over successive cell generations.

EZH2 – a valuable druggable target

Because of its implication in epigenetics but also its interactions with intracellular protein partners (including histones), multiple lines of evidence make EZH2 a valuable target for drug discovery studies:

  • Some data have shown that abnormal EZH2 expression is observed during various types of cancer (breast, prostate, endometrial cancers…)
  • EZH2 mutations (Tyr641, Ala677 or Ala687), which hypermethylate H3K27, are associated with non-Hodgkins lymphomas and leukemias
  • Other EZH2 gene alterations lead to reduced methylation activity (Tyr153deletion, His689 and Pro132 mutations) and are associated to the Weaver Syndrome (also called Weaver-Smith Syndrome, a rare congenital disorder characterized by skeletal abnormalities and developmental delay) but not to the Sotos Syndrome (another rare genetic disease with physical overgrowth),  which is caused by mutations in the NSD1 gene.

Source of EZH2 complexes for research and drug discovery

Obtaining access to wild-type and active complexes has often been quite challenging for researchers who have worked on the first on EZH1 and EZH2 methyltransferase complexes.

BPS Bioscience was the first company to offer active wild-type EZH1 and EZH2 complexes for Methyltransferase inhibitor screening and profiling. BPS is now producing EZH2 complexes with “Weaver mutations”, as well as the inactive Phe 667 mutation, the recently published Arg 69 and Glu740 mutations, and also 5 different versions of the Tyr641 mutation.

All these complexes are compatible for identifying specific inhibitors or running enzyme profiling. Below is an overview of BPS’s EZH2 wild-type or mutant portfolio:

  • EZH2 wild-type multi-protein complexes – full 5-member or partial complexes
  • 5 Tyr641 mutants and 8 other EZH2 mutants
  • EZH2 inhibitor screening kits, plus two different Y641-specific kits.
  • EZH2 screening and profiling services, as well as Y641 mutant screening services.

The 2 latter kits and services are also available for EZH1.

7 Human EZH2 complexes – from wild-type to mutantsEZH2/EED/SUZ12/RbAp48/AEBP2 wild-type active Complex Human, recombinant (cat. nr 51004, BPS tebu-bio)

Here is tebu-bio’s selection of  recombinant complexes, co-expressed in baculovirus systems and ideal for EZH2 inhibitor screening and EZH2 profiling:

#1 – Wild-type EZH2/EED/SUZ12/RbAp48/AEBP2 Complex Human, recombinant (cat. nr 51004)

Complex of human wild-type EZH2 (GenBank Accession No. NM_004456), (a.a. 2-end) with N-terminal His tag, MW= 86 kDa, human EED (NM_003797) (a.a. 2-end) with N-terminal Flag  tag, MW= 51 kDa, human SUZ12 (NM_015355) (a.a. 2-end) with N-terminal His tag, MW = 87 kDa, Human AEBP2 (NM_153207) (a.a. 2-end) with N-terminal His tag, MW= 53 kDa, and human RbAp48 (NM_005610) (a.a. 2-end) with N-terminal His tag,MW = 48 kDa.

#2 – EZH2 (del153Y)/EED/SUZ12/Rb Ap48/AEBP2 Recombinant Human Protein (cat. nr 51027)

EZH2 5 Member Mutant del153Y – Mutant version of the BPS’ regular EZH2 5-member complex, but with a Tyr deletion on a.a. 153 of the EZH2 protein. Complex of human EZH2 (GenBank #NM_004456), (a.a. 2-end; del153Y) with N-terminal His-tag, MW= 86 kDa, human EED (NM_003797) (a.a.2-end) with N-terminal FLAG-tag, MW= 51 kDa, human SUZ12 (NM_015355) (a.a. 2-end) with N-terminal His-tag, MW = 87 kDa, Human AEBP2 (NM_153207) (a.a. 2-end) with N-terminal His-tag, MW= 53 kDa, and human RbAp48 (NM_005610) (a.a. 2-end) with N-terminal His-tag, MW = 48 kDa.

#3 – EZH2 (R679C)/EED/SUZ12/Rb Ap48/AEBP2Recombinant Human Protein (cat. nr 51033)

EZH2 5 Member Mutant R679C – EZH2 5 Member Mutant R679C – Mutant version of the BPS’ regular EZH2 5-member complex, but with an Arg-to-Cys mutation on a.a. 684 of the EZH2 protein. This mutation is equivalent to R679C in variant 3 (GenBank #NM_001203247). Complex of human EZH2 (GenBank #NM_004456), (a.a. 2-end ; R684C) with N-terminal His-tag, MW= 86kDa, human EED (NM_003797) (a.a. 2-end) with N-terminal FLAG-tag, MW= 51 kDa, human SUZ12 (NM_015355) (a.a. 2-end) with N-terminal His-tag, MW = 87 kDa, Human AEBP2 (NM_153207) (a.a. 2-end with N-terminal His-tag, MW= 53 kDa, and human RbAp48 (NM_005610) (a.a. 2-end) with N-terminal His-tag, MW = 48 kDa.

#4 – EZH2 (F667I)/EED/SUZ12/Rb Ap48/AEBP2 Recombinant Human Protein (cat. nr 51035)

EZH2 5 Member Mutant F667I – Mutant version of the BPS’ regular EZH2 5-member complex, but with a Phe-to-Ile mutation on a.a. 672 of the EZH2 protein. This mutation is equivalent to F667I in variant 3 (GenBank#NM_001203247). Complex of human EZH2 (GenBank #NM_004456), (a.a. 2-end ; F672I) with N-terminal His-tag, MW=86 kDa, human EED (NM_003797) (a.a. 2-end) with N-terminal FLAG-tag, MW= 51 kDa, human SUZ12 (NM_015355) (a.a. 2-end) with N-terminal His-tag, MW = 87 kDa, Human AEBP2 (NM_153207) (a.a. 2-end) with N-terminal His-tag, MW= 53 kDa, and human RbAp48 (NM_005610) (a.a. 2-end) with N-terminal His-tag, MW = 48 kDa (baculovirus co-expression system).

#5 – EZH2 (E740K)/EED/SUZ12/Rb Ap48/AEBP2 Recombinant Human Protein (cat nr. 51032)

EZH2 5 Member Mutant E740K – Mutant version of the BPS’ regular EZH2 5-member complex, but with a Glu-to-Lys mutation on a.a. 745 of the EZH2 protein. This mutation is equivalent to E740K in variant 3 (GenBank #NM_001203247). Complex of human EZH2 (GenBank #NM_004456), (a.a. 2-end ; E745K) with N-terminal His-tag, MW= 86 kDa, human EED (NM_003797) (a.a. 2-end) with N-terminal FLAG-tag, MW= 51 kDa, human SUZ12 (NM_015355) (a.a. 2-end) with N-terminal His-tag, MW = 87 kDa, Human AEBP2 (NM_153207) (a.a. 2-end) with N-terminal His-tag, MW=53 kDa, and human RbAp48 (NM_005610) (a.a. 2-end) with N-terminal His-tag, MW = 48 kDa, co-expressed in a baculovirus expression system. *This mutation is equivalent to E740K in variant 3 (GenBank #NM_001203247).

#6 – EZH2 (H689Y)/EED/SUZ12/Rb Ap48/AEBP2 Recombinant Human Protein (cat. nr 51023)

EZH2 5 Member Mutant H689Y – Mutant version of the BPS’ regular EZH2 5-member complex, but with a His-to-Tyr mutation on a.a. 694 of the EZH2 protein. This mutation is equivalent to variant 3 (GenBank #NM_001203247). Complex of human EZH2 (GenBank #NM_004456), (a.a. 2-end; H694Y) with N-terminal His-tag, MW= 86 kDa, human EED (NM_003797) (a.a. 2-end) with N-terminal FLAG-tag, MW= 51 kDa, human SUZ12 (NM_015355) (a.a. 2-end) with N-terminal His-tag, MW = 87 kDa, Human AEBP2 (NM_153207) (a.a. 2-end) with N-terminal His-tag, MW= 53 kDa, and human RbAp48 (NM_005610) (a.a. 2-end) with N-terminal His-tag, MW= 48 kDa.

#7 – EZH2 (P132S)/EED/SUZ12/Rb Ap48/AEBP2 Recombinant Human Protein (cat. nr 51019)

EZH2 5 Member Mutant P132S – Mutant version of the BPS’ regular EZH2 5-member complex, but with a Pro-to-Ser mutation on a.a. 132 of the EZH2 protein. Complex of human EZH2 (GenBank #NM_004456), (a.a. 2-end ; P132S) with N-terminal His-tag, MW= 86 kDa, human EED (NM_003797) (a.a. 2-end) with N-terminal FLAG-tag, MW= 51 kDa, human SUZ12 (NM_015355) (a.a. 2-end) with N-terminal His-tag, MW = 87 kDa, Human AEBP2 (NM_153207) (a.a. 2-end) with N-terminal His-tag, MW= 53 kDa, and human RbAp48 (NM_005610) (a.a. 2-end) with N-terminal His-tag, MW = 48 kDa.

Small molecules – a source of Methyltransferase effectors

To conclude, I’d like to mention that catalogued small molecules are effective alternatives for researchers looking for well-characterized and pure Methyltransferase modulators to perform and develop their in vitro assays. A few examples:

3-Deazaneplanocin (cat nr 27223)

Lysine Methyltransferase EZH2 (KMT6) inhibitor. Potent S-adenosylhomocysteine hydrolase (SAHH) and Histone MethylTransferase (HMT) inhibitor.

Chaetocin (Cat. nr 10-2404-200)

Lysine Methyltransferase inhibitor. Antimicrobial fungal metabolite, selective inhibitor of lysine specific Histone MethylTransferase (HMT).

BIX-01294 (cat. nr 10-1335-5)

Histone Methyl transferase inhibitor (HMT).  Selective inhibitor of G9a histone methyltransferase and GLP HMTase leading to a decrease in H3K9me2 (Histone H3 Lysine 9 Methylation) in vitro.1 BIX-01294 facilitates the reactivation of pluripotency genes and induces passive demethylation, thus promoting reprogramming.

Trackbacks

  1. […] cancers, making H3K27 a key druggable target. The new  H3K27 site is tri- methylated by the HMTs (EZH2, or by NSD2/3). Alternately, H3K27 can be demethylated by specific HMDTs like UTX (KDM6A) and […]

  2. […] or mutant enzymes and small compounds (ex. EZH2 Methyltransferase) for drug discovery […]

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