DCAF11 Supports Targeted Protein Degradation by Electrophilic Proteolysis-Targeting Chimeras.

Ligand-induced protein degradation has emerged as a compelling approach to promote the targeted elimination of proteins from cells by directing these proteins to the ubiquitin-proteasome machinery. So far, only a limited number of E3 ligases have been found to support ligand-induced protein degradation, reflecting a dearth of E3-binding compounds for proteolysis-targeting chimera (PROTAC) design. Here, we describe a functional screening strategy performed with a focused library of candidate electrophilic PROTACs to discover bifunctional compounds that degrade proteins in human cells by covalently engaging E3 ligases. Mechanistic studies revealed that the electrophilic PROTACs act through modifying specific cysteines in DCAF11, a poorly characterized E3 ligase substrate adaptor. We further show that DCAF11-directed electrophilic PROTACs can degrade multiple endogenous proteins, including FBKP12 and the androgen receptor, in human prostate cancer cells. Our findings designate DCAF11 as an E3 ligase capable of supporting ligand-induced protein degradation via electrophilic PROTACs.

Electrophilic PROTACs that degrade nuclear proteins by engaging DCAF16.

Ligand-dependent protein degradation has emerged as a compelling strategy to pharmacologically control the protein content of cells. So far, however, only a limited number of E3 ligases have been found to support this process. Here, we use a chemical proteomic strategy that leverages broadly reactive, cysteine-directed electrophilic fragments coupled to selective ligands for intracellular proteins (for example, SLF for FKBP12, JQ1 for BRD4) to screen for heterobifunctional degrader compounds (or proteolysis targeting chimeras, PROTACs) that operate by covalent adduction of E3 ligases. This approach identified DCAF16-a poorly characterized substrate recognition component of CUL4-DDB1 E3 ubiquitin ligases-as a target of electrophilic PROTACs that promote the nuclear-restricted degradation of proteins. We find that only a modest fraction (~10-40%) of DCAF16 needs to be modified to support protein degradation, pointing to the potential for electrophilic PROTACs to induce neosubstrate degradation without substantially perturbing the function of the participating E3 ligase.

Chemical Protein Synthesis with the KAHA Ligation.

Since the first report of the chemoselective amide bond forming reaction between α-ketoacids and hydroxylamines in 2006, the KAHA (α-ketoacid-hydroxylamine) ligation has advanced to a useful tool for the routine synthesis of small to medium sized proteins and cyclic peptides. In this chapter we introduce the concept of KAHA ligation starting with the synthesis and properties of hydroxylamines and α-ketoacids, methods for their incorporation into peptides, and give an insight into the mechanism of the KAHA ligation. We cover important improvements including sequential ligations with 5-oxaproline, traceless synthesis of peptide α-ketoacids and show their application in chemical protein synthesis and cyclic peptide synthesis. Recent developments of the KAT (potassium acyl trifluoroborate) ligation and its application as fast and chemoselective bioconjugation method are described and an outlook on ongoing work and possible future developments is given at the end of the chapter.

Formation and rearrangement of homoserine depsipeptides and depsiproteins in the α-ketoacid-hydroxylamine ligation with 5-oxaproline.

The primary products of the chemical ligation of α-ketoacids and 5-oxaproline peptides are esters, rather than the previously reported amides. The depsipeptide product rapidly rearranges to the amide in basic buffers. The formation of esters sheds light on possible mechanisms for the type II KAHA ligations and opens an avenue for the chemical synthesis of depsiproteins.

Traceless preparation of C-terminal α-ketoacids for chemical protein synthesis by α-ketoacid-hydroxylamine ligation: synthesis of SUMO2/3.

A novel protecting group for enantiopure α-ketoacids delivers C-terminal peptide α-ketoacids directly upon resin cleavage and allows the inclusion of all canonical amino acids, including cysteine and methionine. By using this approach, SUMO2 and SUMO3 proteins were prepared by KAHA ligation with 5-oxaproline. The synthetic proteins containing homoserine residues were recognized by and conjugated to RanGAP1 by SUMOylation enzymes.

Lysine acylation using conjugating enzymes for site-specific modification and ubiquitination of recombinant proteins.

Enzymes are powerful tools for protein labelling due to their specificity and mild reaction conditions. Many protocols, however, are restricted to modifications at protein termini, rely on non-peptidic metabolites or require large recognition domains. Here we report a chemoenzymatic method, which we call lysine acylation using conjugating enzymes (LACE), to site-specifically modify folded proteins at internal lysine residues. LACE relies on a minimal genetically encoded tag (four residues) recognized by the E2 small ubiquitin-like modifier-conjugating enzyme Ubc9, and peptide or protein thioesters. Together, this approach obviates the need for E1 and E3 enzymes, enabling isopeptide formation with just Ubc9 in a programmable manner. We demonstrate the utility of LACE by the site-specific attachment of biochemical probes, one-pot dual-labelling in combination with sortase, and the conjugation of wild-type ubiquitin and ISG15 to recombinant proteins.