Ubiquitin Proteasome Pathway
The ubiquitin proteasome pathway is required for the targeted degradation of most shortlived proteins in the eukaryotic cell. Unlike most regulatory mechanisms, protein degradation is inherently irreversible. This indiscriminate proteolytic step provides directionality for a signaling pathway - destruction of a protein can lead to a complete, rapid, and sustained termination of the process involving the protein as well as a change in cell composition. The rapid degradation of specific proteins permits adaptation to new physiologic conditions.
The post-translational addition of ubiquitin to is called ubiquitination. Only polyubiquitination on defined lysines, mostly on K48 and K29, is related to degradation by the proteasome, while other mon- and polyubiquitinations may regulate processes such as endocytic trafficking, inflammation, translation and DNA repair. Tagged proteins are recognized by the 26S proteasome, unfolded, and threaded into the 20S proteolytic chamber in an ATP-dependent manner.
Ubiquitination involves three main steps: activation, conjugation, and ligation, performed by ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s), and ubiquitin ligases (E3s). Only the E1 family is higly conserved. The variance in E2 and E3 and their combinations allow selective tagging and degradation of specific intracellular proteins.
Ubiquitin-conjugating enzyme E2 N (UBE2N) belongs to the E2 family that receives Ub from E1 enzyme. Once the protein substrate is mono-ubiquitinated, a poly-ubiquitin chain is formed through the same ubiquitination conjugation cascade. TNF α induces receptor-interacting protein kinase (RIP) poly-ubiquitination, and ubiquitinated RIP associates with NEMO (NF-κB essential modulator) to activate inhibitors of nuclear factor kappa B (IκB) kinase complex which is formed by IKKα and IKKβ. This leads to ubiquitination of IκB which is separated from NF-κB and directed to the 26S proteasome. Valosin-containing peptide (VCP) facilitates the separation. The released NF-κB activates transcription of target genes in the nucleus. Ectodysplasin (EDA-A1) is able to bind to the TNF-receptor EDAR which results in the formation of a complex containing EDAD, TRAF6, TAB2, and TAK1. TAK1 can also activate the IKK, which in turn phosphorylates IKK.
Downregulation of NF-κB activation by the T-cell receptor (TCR) involves the TCR-induced ubiquitination and degradation of Bcl10. Bcl10 can stimulate K63 poly-ubiquitination of NEMO, and TRAF6 can catalyze mono-ubiquitination of NEMO or K63 poly-ubiquitination of itself. Furthermore, K63 poly-ubiquitination of RIP is recognized by NEMO as a binding and activation signal. SUMO and NEDD8 are two examples for ubiquitin-like proteins (UBLs). SUMO modification often acts antagonistically to that of ubiquitination and serves to stabilize protein substrates. Proteins conjugated to UBLs are typically not targeted for degradation by the proteasome, but rather function in diverse regulatory activities.
Ubiquitin in Drug-Discovery
A proteolysis targeting chimera (PROTAC) is a heterobifunctional small molecule composed of two active domains and a linker. It is an effective endogenous protein degradation tool. Rather than acting as a conventional enzyme inhibitor, a PROTAC works by inducing selective intracellular proteolysis. PROTAC can bind the protein of interest and recruit E3 ligase for ubiquitination and subsequent degradation of the entire protein. By removing target proteins directly rather than merely blocking them, PROTACs can provide multiple advantages over small molecule inhibitors, which can require high systemic exposure to achieve sufficient inhibition, often resulting in toxic side effects and eventual drug resistance. Targeted protein degradation using the PROTAC technology is emerging as a novel therapeutic method to address diseases, such as cancer, driven by the aberrant expression of a disease-causing protein.
References
- Cermakova K, Hodges HC Next-Generation Drugs and Probes for Chromatin Biology: From Targeted Protein Degradation to Phase Separation. Cell (2018).
- Ciechanover A et al. The ubiquitin–proteasome pathway: on protein death and cell life. The EMBO Journal. (1998)
- Nalepa G et al. Drug discovery in the ubiquitin–proteasome system. Nature Reviews Drug Discovery (2006).
- Li X, Song Y Proteolysis-targeting chimera (PROTAC) for targeted protein degradation and cancer therapy. Journal of Hematology & Oncology (2020).
