Systems, compositions and methods for identifying e3 ligase substrates by ubiquitin biotinylation

A method of identifying a substrate of an E3 ligase, the method comprising:

• (i) expressing in a cell one or more ubiquitin or ubiquitin-like molecules fused to a biotin ligase peptide substrate which comprises a biotinylation site of the biotin ligase;
• (ii) expressing in the cell an E3 ligase fused at its amino (NH2)- or carboxy (COOH)-terminus to a non-promiscuous biotin ligase, wherein the E3 ligase catalyzes the ubiquitination of an E3 ligase substrate bound thereto with the one or more ubiquitin or ubiquitin-like molecules of (i), which are biotinylated by the non-promiscuous biotin ligase fused to the E3 ligase;
• and
• (iii) identifying the substrate of the E3 ligase by detecting and/or selecting the biotinylated and ubiquitinated substrate bound to the E3 ligase.

A system or composition comprising:

• (i) one or more ubiquitin or ubiquitin-like molecules fused to a biotin ligase peptide substrate which comprises a biotinylation site of the biotin ligase, or one or more polynucleotides encoding said one or more ubiquitin or ubiquitin-like molecules;
• and
• (ii) an E3 ligase fused to a biotin ligase, or a polynucleotide encoding said E3 ligase;
• wherein the E3 ligase catalyzes the ubiquitination of an E3 ligase substrate bound thereto with the one or more ubiquitin or ubiquitin-like molecules of (i), and wherein the one or more ubiquitin or ubiquitin-like molecules are biotinylated by the biotin ligase fused to the E3 ligase.

A system or composition comprising:

• (i) one or more ubiquitin proteins fused to a biotin ligase peptide substrate tag which comprises a biotinylation site of the biotin ligase;
• or one or more polynucleotides encoding said one or more ubiquitin proteins;
• (ii) an E3 ligase fused at its amino (NH2)- or carboxy (COOH)-terminus to a biotin ligase, or a polynucleotide encoding said E3 ligase;
• and
• (iii) an agent selected from an IMiD, a molecular glue, a reprogramming molecule, or a proteolysis targeting chimera (PROTAC®), that facilitates an association of a target protein or a target molecule with the E3 ligase, whereby the target protein or target molecule is ubiquitinated with the one or more tagged ubiquitin proteins of (i), which are biotinylated by the biotin ligase fused to the E3 ligase.

A method for identifying a target protein, the method comprising:

• (i) providing one or more ubiquitin proteins fused to a biotin ligase peptide substrate tag which comprises a biotinylation site of the biotin ligase;
• or one or more polynucleotides encoding said one or more ubiquitin proteins;
• (ii) providing an E3 ligase fused at its amino (NH2)- or carboxy (COOH)-terminus to a biotin ligase, or a polynucleotide encoding said E3 ligase;
• (iii) providing an agent selected from an IMiD, a molecular glue, a reprogramming molecule, or a proteolysis targeting chimera (PROTAC®), that facilitates an association of the target protein with the E3 ligase, whereby the target protein is ubiquitinated with the one or more tagged ubiquitin proteins of (i), which are biotinylated by the biotin ligase fused to the E3 ligase;
• and
• (iv) detecting and/or selecting the biotinylated and ubiquitinated target protein associated with the E3 ligase, thereby identifying the target protein.

A method of identifying a substrate of an E3 ubiquitin ligase, the method comprising:

• introducing into a cell a polynucleotide encoding a mutant E3 ubiquitin ligase fused to a biotin ligase and expressing the mutant E3 ubiquitin ligase in the cell;
• wherein the cell expresses one or more ubiquitin or ubiquitin-like molecules fused to a biotin ligase peptide substrate which comprises a biotinylation site of the biotin ligase;
• culturing the cell in biotin-containing medium for a time sufficient for biotinylation of ubiquitinated protein substrates of the E3 ubiquitin ligase;
• enriching the biotinylated protein substrates with streptavidin;
• and
• detecting the levels of biotinylated protein substrates ubiquitinated by the mutant E3 ubiquitin ligase in lysates of the cells compared with the levels of biotinylated protein substrates ubiquitinated by a wildtype E3 ubiquitin ligase to identify substrate proteins differentially enriched by the mutant E3 ubiquitin ligase versus the wildtype E3 ubiquitin ligase.