Compositions and methods are provided for the cell-mediated targeted killing of diseased cells based on the presence of an intracellular antigen, rather than a surface-bound marker. The targeting cells are modified to express a cytotoxic protein that is delivered into a targeted cell, and after delivery is selectively activated by the presence of a cytoplasmic protein of interest. In one embodiment of the invention, the cytotoxic molecule is a Granzyme B (GrB) polypeptide. In the compositions of the invention, GrB is modified to render its cytotoxic enzymatic functions inactive, until the presence of an intracellular antigen unlocks the GrB molecule to enable enzymatic activities.

The invention provides compositions including a fusion polypeptide and methods for making a fusion polypeptide that includes a degradation polypeptide and a heterologous polypeptide of interest.

The present disclosure provides a compound screening method comprising screening a test compound that binds a target protein of interest and another ligand protein that is not previously known to form a complex, comprising, (i) in the presence and absence of a test compound, incubating an immobilized ligand protein with a target protein of interest, comprising a nucleic acid tag, wherein the protein of interest and the nucleic acid tag differ from each other; (ii) removing unbound target protein of interest; and (iii) detecting the presence or absence of complex between the immobilized ligand protein, test compound, and the target protein of interest, wherein an increase in the amount of target protein of interest bound to the immobilized ligand protein in the presence of test compound as compared to the absence of test compound indicates the test compound binds to and enables protein-protein complex formation between the immobilized ligand protein and the target protein of interest.

The present invention provides a means to modulate gene expression in vivo in a manner that avoids problems associated with CRISPR endogenous protein knock-out or knock-in strategies and strategies that provide for correction, or alteration, of single nucleotides. The invention includes inserting into the genome a nucleotide encoding a heterobifunctional compound targeting protein (dTAG) in-frame with the nucleotide sequence of a gene encoding an endogenously expressed protein of interest which, upon expression, produces an endogenous protein-dTAG hybrid protein. This allows for targeted protein degradation of the dTAG and the fused endogenous protein using a heterobifunctional compound.

Provided herein are methods and compositions for targeted protein degradation. In one aspect, a protein degradation chimera is provided, comprising: a first moiety that is capable of binding to a chaperone complex component; a second moiety that is capable of binding to a target protein or proteins, wherein the target protein (s) is targeted for degradation; and a tether configured to covalently couple the first moiety and the second moiety.

The present invention relates to a poxvirus vector comprising a nucleic acid sequence encoding a fusion protein comprising: (i) a peanut allergen selected from list consisting of at least two peanut allergens from ara h 1, ara h 2, ara h 3, ara h 4, ara h 5, ara h 6, ara h 7, ara h 8, ara h 9, ara h 8, ara h 9, ara h 10 and ara h 11 or a derivative or part thereof having at least 70% sequence identity thereto, and (ii) a proteasome degradation tag to enhance intracellular degradation of the fusion protein.

The present invention provides a means to modulate gene expression in vivo in a manner that avoids problems associated with CRISPR endogenous protein knock-out or knock-in strategies and strategies that provide for correction, or alteration, of single nucleotides. The invention includes inserting into the genome a nucleotide encoding a heterobifunctional compound targeting protein (dTAG) in-frame with the nucleotide sequence of a gene encoding an endogenously expressed protein of interest which, upon expression, produces an endogenous protein-dTAG hybrid protein. This allows for targeted protein degradation of the dTAG and the fused endogenous protein using a heterobifunctional compound.

Described herein are compositions and methods for modulating protein abundance in a target-specific manner via degron tags.

Disclosed is a dTAG system comprising a small molecule degraders of FKBP12-tagged proteins via recruitment of Von Hippel-Lindau E3 ubiquitin ligase (VHL) E3 ubiquitin ligase and uses thereof.

The present disclosure provides methods to identify peptides and small molecule moieties that are able to functionally bridge an interaction between a target protein and an E3 ubiquitin ligase to mediate the degradation of the target protein. Some moieties can degrade specific target variants, but not others. The moieties create a neosubstrate for an E3 ligase of interest. The methods described enable generation of compounds able to selectively degrade specific targets within cells with implications for drug development for pathological conditions. The disclosure also describes the generation of modified peptides using post-translational modification enzymes, such as N-methyltransferases, prolyloligopeptidases, lactamases, hydroxylases, and dehydratases, along with methods of using the same.