Structural Biochemistry/Enzyme Regulation/Ubiquitination

Ubiquitin (Ub) is a 76 amino acid protein that tags proteins for destruction by proteasomes. It is found in all eukaryotic cells and is involved in various signaling pathways including cell cycle, endocytosis, transcription, DNA repair, signal transduction, apoptosis, and the immune response. The proteins degraded through ubiquitin are regulatory proteins. Ub-activating enzymes (E1), Ub-conjugating enzymes (E2), and Ub ligases (E3) are essential in protein ubiquitylation, which is most commonly known for its ability to execute controlled protein degradation by the 26S proteosome. However, ubiquitylation can not only induce protein degradation, but it can also control protein behavior, modulate subcellular localization, and moderate protein-protein interactions. Ubiquitin medications can signal irreversible proteolytic events (such as degradation through Lys⁴⁸) and reversible nonproteolytic events. The nonproteolytic events utilize mono-ubiquitin and poly-ubiquitin chains connected through Lys⁶ or Lys⁶³.

Ubiquitylation Pathway

E1 activates ubiquitin through a thiol-ester bond between the cysteine found in its active site and ubiquitin's carboxyl-terminal glycine. The now activated ubiquitin is transferred to E2 through transesterification. E3 brings the ubiquitin-charged E2 and relevant substrate to facilitate the formation of an isopeptide linkage between ubiquitin's carboxyl-terminal glycine and the ε-amino group on the substrate or an ubiquitin attached to the protein.

E3s are the ones responsible for substrate specificity, which correlates to the relatively large amount of E3s in respect to E1s and E2s.

Mechanism

Ubiquitin attaches to a protein by covalently forming isopeptide bonds, using energy from ATP. Ubiquitin is normally found inactive, but a Ub activating enzyme (E1) links the carboxyl group of Ub to its sulfhydryl group. The enzyme binds an ATP-activated Ub complex where a transfer of the Ub to the a cysteine residue on E1 forms the thioester bond and releases AMP. Ub is then transferred to a Ub-conjugating protein (E2) and finally that protein complex is recognized by a Ub-protein ligase (E3). E3 enzymes recognize N-terminal residues that signal the protein for degradation. The complex then binds the particular protein and the E2-Ub complex, facilitating the transfer of Ub to tag the protein.

A protein marked with a Lys⁴⁸-linked poly-ubitquitin chain is recognized by the 19S cap of the 26S proteasome and is ultimately degraded. These proteases are composed of a 20S catalytic subunit and a 19S regulatory subunit. The 19S regulatory subunit normally blocks access to the 20S subunit's active site. 19S subunits recognize and bind only ubiquinated molecules, thus moderating the degradation by the 20S catalytic core. Six ATPases are required for the regulatory complex to function: ATP hydrolysis likely causes a conformational change in the 19S subunit which is transferred to the 20S subunit, allowing the active site to become available for substrate binding.

The 20S subunit is composed of two outer 7 subunit rings (alpha rings), and two inner 7 subunit beta rings. The beta subunits contain N-terminal threonine whose hydroxyl group is activated to attack the carbonyl groups of peptides. Reminiscent of serine proteases, these threonine residues also form acyl-enzymes. The degradation is completed by removal of the Ub molecules by an isopeptidase of the 19S regulatory unit. These ubiquitin molecules are then released to tag more proteins.

Regulation

Post-translational modifications are a method for regulating ubiquitylation. These modification consist of phosphorylation, oxidation, sumoylation, acetylation, and neddylation. Phosphorylation creates binding sites for E3s on substrates, oxidation is associated with ubiquitylation targeting, sumoylation blocks ubiquitylation sites, acetylation competes with ubiquitylation in modification of ubiquitylation sites, and neddylation increases E3 activity by increasing ubiquitin-loaded E2 affinity.

Deubiquitylating enzymes (DUBs) also play a role in ubiquitylation regulation. These enzymes essentially undo the work of the E2s and E3s. Examples of DUBs are ubiquitin C-terminal hydrolases (UCHs) and ubiquitin-specific processing proteases (UBPs). UCHs hydrolyze the carboxyl-terminal ester and amide bonds of ubiquitin. UBPs take apart the polyubiquitin chains.

Mono and Poly Ubiquitylation

Mono-ubiquitylation is a regulatory modification that is a contributing factor in transcription, histone function, endocytosis, and membrane trafficking. It acts as a signal for endocytosis receptors and for lysosomal targeting. These are all proteasome-independent mechanisms. Lys⁶³-linked poly-ubiquitylation is engaged in signalling DNA repair, the stress response, endocytosis, and signal transduction. Poly-ubiquitin chains with varying linkages signal different effects.

Disease

Because ubiquitin is an enzyme and protein regulatory system, errors in tagging lead to various diseases. Several neurological diseases such as juvenile and early onset Parkinsons, Huntingtons, and other chronic neurological diseases have been linked to errors or decreased activity of the ubiquitin-protein ligase (E3) and its consequences: inability of the ubiquitin and proteasome duo to remove damaged or malfunctioning proteins leads to aggregation and Lewy bodies - aggregates of protein inside neurons that displace functioning cellular components.

Viruses and other invasive pathogens may inappropriately activate the ubiquitin system to destroy immune response such as in HPV, leading to the formation of tumors or other diseases.

Examples E3s

APC- anaphase-promoting complex/cyclosome

SCF- Skp1-Cdc53/Cu11-F-box protein

References

Biochemistry 6th Edition, Berg et al.

The ubiquitin system: pathogenesis of human diseases and drug targeting, Ciechanover, Schwartz; http://www.elsevier.com/framework_aboutus/pdfs/ciechanover01.pdf

Getting into position: the catalytic mechanisms of protein ubiquitylation, Passmore, Barford; http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1224133/pdf/14998368.pdf

A field guide to ubiquitylation, Fang, Weissman; http://springerlink.metapress.com/content/tgl62jj9uyt19d2f/fulltext.pdf