The E3 ubiquitin ligase RNF216 contains a linear ubiquitin chain-determining-like domain that functions to regulate dendritic arborization and dendritic spine type in hippocampal neurons.

Of the hundreds of E3 ligases found in the human genome, the RING-between RING (RBR) E3 ligase in the LUBAC (linear ubiquitin chain assembly complex) complex HOIP (HOIL-1-interacting protein or RNF31), contains a unique domain called LDD (linear ubiquitin chain determining domain). HOIP is the only E3 ligase known to form linear ubiquitin chains, which regulate inflammatory responses and cell death via activation of the NF-κB pathway. We identified an amino acid sequence within the RNF216 E3 ligase that shares homology to the LDD domain found in HOIP (R2-C). Here, we show that the R2-C domain of RNF216 promotes self-assembly of all ubiquitin chains, with a dominance for those assembled via K63-linkages. Deletion of the R2-C domain altered RNF216 localization, reduced dendritic complexity and changed the distribution of apical dendritic spine morphology types in primary hippocampal neurons. These changes were independent of the RNF216 RBR catalytic activity as expression of a catalytic inactive version of RNF216 had no effect. These data show that the R2-C domain of RNF216 diverges in ubiquitin assembly function from the LDD of HOIP and and functions independently of RNF216 catalytic activity to regulate dendrite development in neurons.

Profiling and verifying the substrates of E3 ubiquitin ligase Rsp5 in yeast cells.

Yeast is an essential model organism for studying protein ubiquitination pathways; however, identifying the direct substrates of E3 in the cell presents a challenge. Here, we present a protocol for using the orthogonal ubiquitin transfer (OUT) cascade to profile the substrate specificity of yeast E3 Rsp5. We describe steps for OUT profiling, proteomics analysis, in vitro and in cell ubiquitination, and stability assay. The protocol can be adapted for identifying and verifying the ubiquitination targets of other E3s in yeast. For complete details on the use and execution of this protocol, please refer to Wang et al.1.

Synthesis and Evaluation of Cereblon-Recruiting HaloPROTACs.

Target validation is key to the development of protein degrading molecules such as proteolysis-targeting chimeras (PROTACs) to identify cellular proteins amenable for induced degradation by the ubiquitin-proteasome system (UPS). Previously the HaloPROTAC system was developed to screen targets of PROTACs by linking the chlorohexyl group with the ligands of E3 ubiquitin ligases VHL and cIAP1 to recruit target proteins fused to the HaloTag for E3-catalyzed ubiquitination. Reported here are HaloPROTACs that engage the cereblon (CRBN) E3 to ubiquitinate and degrade HaloTagged proteins. A focused library of CRBN-pairing HaloPROTACs was synthesized and screened to identify efficient degraders of EGFP-HaloTag fusion with higher activities than VHL-engaging HaloPROTACs at sub-micromolar concentrations of the compound. The CRBN-engaging HaloPROTACs broadens the scope of the E3 ubiquitin ligases that can be utilized to screen suitable targets for induced protein degradation in the cell.

Linkage-Specific Synthesis of Di-ubiquitin Probes Enabled by the Incorporation of Unnatural Amino Acid ThzK.

Di-ubiquitin (diUB) conjugates of defined linkages are useful tools for probing the functions of UB ligases, UB-binding proteins and deubiquitinating enzymes (DUBs) in coding, decoding and editing the signals carried by the UB chains. Here we developed an efficient method for linkage-specific synthesis of diUB probes based on the incorporation of the unnatural amino acid (UAA) Nϵ -L-thiaprolyl-L-Lys (L-ThzK) into UB for ligation with another UB at a defined Lys position. The diUB formed by the UAA-mediated ligation reaction has a G76C mutation on the side of donor UB for conjugation with E2 and E3 enzymes or undergoing dethiolation to generate a covalent trap for DUBs. The development of UAA mutagenesis for diUB synthesis provides an easy route for preparing linkage-specific UB-based probes to decipher the biological signals mediated by protein ubiquitination.

Enhancing the incorporation of lysine derivatives into proteins with methylester forms of unnatural amino acids.

We have improved the incorporation of l- and d-forms of unnatural amino acid (UAA) Nε-thiaprolyl-l-lysine (ThzK) into ubiquitin (UB) and green fluorescent protein (GFP) by 2-6 folds with the use of the methylester forms of the UAAs in E coli cell culture. We also improved the yields of UAA-incorporated UB and GFP with the methylester forms of Nε-Boc-l-Lysine (BocK) and Nε-propargyl-l-Lysine (PrK) by 2-5 folds compared to their free acid forms. Our work demonstrated that using methylester-capped UAAs for protein expression is a useful strategy to enhance the yields of UAA-incorporated proteins.