Proteolysisubiquitin Proteins


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 Proteolysisubiquitin Proteins Background

Proteolysis

Metabolic homeostasis of various cellular constituents is essential for proper cell functioning. One major driving force of various cellular processes is proteins. While the production and regulation of the proteins have been extensively studied in many pathways, the disposal of proteins remains poorly understood. In eukaryotes, there are two major protein degradation machineries: lysosome and 26S proteasome. Whereas lysosome mainly deals with long-lived proteins in a non-selective manner, proteasome degrades abnormal or short-lived proteins in a tightly regulated fashion.

Proteasomes and lysosomes are two major proteolytic sites in eukaryotic cells. They both degrade a variety of proteins, but most of their substrates have very different attributes, without much overlap between the two systems. Proteasomal substrates are usually short-lived proteins, including cell cycle regulators, tumor suppressors, MHC class I molecules, and misfolded proteins. Notably, many studies have implicated the role of the proteasome in many pathologic disease processes, such as cancer and Alzheimer’s disease.

 

Ubiquitin and ubiquitylation

Many proteins targeted to the proteasome for degradation are modified by ubiquitin (Ub) in an ATP-dependant process called ubiquitylation. This abundant and highly conserved 76-residue protein, Ub, is covalently attached to the target protein by an isopeptide linkage between the carboxyl terminal glycine of Ub and, usually, the ε-amino group of lysine in the target protein. The lysines on an Ub can then be used to form an Ub chain through the same mechanism. The Ub-proteasome-mediated proteolysis system can be generally separated in two phases: Ub conjugation and substrate delivery for degradation. In the Ub conjugation phase, Ub is activated and transferred to the substrate through several enzymes including an Ub-activating enzyme (E1), an Ub-conjugating enzyme (E2), and an Ub-protein ligase (E3). For substrates targeted for degradation, successive Ub molecules are added, thereby forming an Ub chain on the substrates. In some cases, chain elongation requires an E4 enzyme, which is involved in Ub chain elongation. In the second phase, the ubiquitylated substrate is delivered to the 26S proteasome, often with the help of adaptor molecules, and then degraded.

 

The enzymes in the ubiquitylation cascade

For the Ub/proteasome system, the human genome encodes two E1s, UBE1 (or Uba1) and UBE1L1 (or Uba6), at least 38 E2s and more than 600 E3s; while yeast have only one E1, Uba1; 13 E2s and about 50 E3s. Each E2 interacts with its cognate E1. All active E2s possess a core Ub-conjugating (UBC) domain, which interacts with E1 and contains an active Cys residue. In the ubiquitylation step, E1 first forms a thioester bond between its active Cys and the C-terminus of Ub using ATP. The Ub is then transferred to the active Cys of an E2, and finally the Ub is transferred and covalently attached to its substrate mediated by an E3 enzyme. There are two major classes of E3s defined by two distinct sequence motifs, a RING domain and a HECT (homologous to E6-AP carboxy terminal) domain. Ligases of these two classes mediate the transfer of Ub differently, in that Ub on the E2 is transferred onto the active Cys of the HECT domain to form an intermediate product before being attached to the substrate; while a RING-type E3 lacking the active Cys residue serves as a bridge to facilitate the transfer of Ub directly from E2 to the substrate. In the RING domain’s core, conserved Cys and His residues help to maintain the overall structure by binding to two Zinc atoms. Another class of E3 possesses a U-box domain, which has a similar structure to the RING domain. However, the U-box domain does not have the metal-chelating residues; instead, its structure is maintained by the hydrogen bonding of conserved charged and polar residues.

 

Reference

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4. Ciechanover A, Brundin P. The ubiquitin proteasome system in neurodegenerative diseases: sometimes the chicken, sometimes the egg[J]. Neuron, 2003, 40(2): 427-446.

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7. Schwartz D C, Hochstrasser M. A superfamily of protein tags: ubiquitin, SUMO and related modifiers[J]. Trends in biochemical sciences, 2003, 28(6): 321-328.