The level of protein within cells is determined by both the rate of synthesis and the rate of degradation. The difference in protein degradation rates is an important aspect of cell regulation. Many rapidly degraded proteins have regulatory molecular functions. The rapid turnover of these proteins allows their levels to change in response quickly to external stimuli. Faulty or damaged proteins are recognized and rapidly degraded within the cells, thereby eliminating the consequences of errors in protein synthesis. In eukaryotic cells, the ubiquitin/proteasome pathway and lysosomal proteolysis are the two main pathways of protein degradation. The ubiquitin/proteasome pathway that degrades ubiquitin-tagged cell target proteins through the proteasome is a common regulatory modification system involved in the regulation of cell cycle, signal transduction, DNA damage response, apoptosis, and immune response.

Ubiquitin binds reversibly to the target protein via an enzyme cascade including ubiquitin-activating enzyme E1, ubiquitin-conjugation enzyme E2, and ubiquitin-protein ligase E3. Subsequently, the ubiquitin-labeled target protein is recognized by the proteasome and eventually degraded into small peptides.

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Ubiquitin and Ubiquitin-like Proteins

Ubiquitin is an 8.5-kDa polypeptide that exists in all eukaryotic cells and acts as a tag in post-translational modification of proteins. The ubiquitin consists of 76 amino acids, of which 7 lysine residues are LYS-6, LYS-11, LYS-27, LYS-29, LYS-33, LYS-48, and lys-63 (K6, K11, K27, K29, K33, K48, and K63). Any one of the seven lysine residues or N-terminal Met1 residues is likely to be involved in ubiquitin chain formation. Ubiquitin is a β-grasp fold protein consisting of a 3.5-turn α-helix, a short 310 helix against a five-strand mixed β-sheet and seven reverse turns.

In addition to ubiquitin, there are several ubiquitin-like proteins (UBLs) that can also be conjugated to and alter the function of substrate proteins. UBLs are a family of proteins that have similar three-dimensional structures and related sequences to ubiquitin. With the discovery of ubiquitin, more than a dozen UBLs have been identified, such as small ubiquitin-like modifier (SUMO), neural precursor cell expressed and developmentally down-regulated 8 (NEDD8), interferon-stimulated gene 15 (ISG15), human leukocyte antigen F locus adjacent transcription 10 (FAT10), ubiquitin-fold modifier 1 (UFM1), ubiquitin-related modifier 1 (URM1), autophagy-related protein 12 (ATG12), autophagy-related protein 8 (ATG8), Finkel-Biskis-Reilly murine sarcoma virus (FBR-MuSV) ubiquitously expressed (FUBI) and ubiquitin-like protein 5 (UBL5). UBLs such as SUMOs, ISG15, NEDD8 and ATG8 act as key regulators of many cellular processes, including transcription, DNA repair, signal transduction, autophagy and cell cycle control.

Ubiquitin Chains

Ubiquitin chains occur when ubiquitin proteins are linked by the carboxy terminus of one ubiquitin to one of lysine residues (K6, K11, K27, K29, K33, K48, and K63) or the N-terminal methionine residue (M1) on the other ubiquitin. Ubiquitin chains that comprise only a single linkage type are called homotypic. In contrast, heterotypic chains contain mixed linkages within the same polymer. The biology role of homotypic ubiquitin chains:

M1 chains - regulating the activation of the transcription factor NF-κB
K6 chains - associated with DNA damage response
K11 chains – involved in endoplasmic reticulum associated degradation (ERAD), proteasomal degradation, and cell cycle regulation
K27 chains – involved in the DNA damage response and innate immunity
K29 chains – an inhibitor of Wnt signaling
K33 chains – negatively regulating T-cell antigen receptor and the activity of AMP-activated protein kinase (AMPK), and implicated in post-Golgi protein trafficking
K48 chains – targeting proteins for degradation by the proteasome
K63 chains - regulating vital processes such as DNA repair, NF-κB signaling, activation of protein kinases, and protein trafficking

Enzymes of Ubiquitination and Deubiquitination

Three types of enzymes are required to ubiquitination process of proteins. E1 enzymes (ubiquitin-activating enzymes), E2 enzymes (ubiquitin-conjugating enzymes), and E3 enzymes (ubiquitin ligases). The C terminus of ubiquitin must be activated before it can form isopeptide bonds with other proteins. Initially, ubiquitin is conjugated in an ATP-requiring reaction to E1 enzymes via a thioester bond. The activated ubiquitin is then transferred via a transesterification reaction to a cysteine residue in the active site of the E2 enzymes, which finally transfers the activated ubiquitin moiety to an amino group of the target protein. The E3 enzyme is responsible for substrate recognition and it serves to bring together the E2 enzymes and the substrate in a single complex, thereby allowing ubiquitination to occur. Ubiquitinated proteins are in a dynamic state, subject to either further rounds of ubiquitin addition, ubiquitin removal by deubiquitinating enzymes, or degradation by a complex multicatalytic proteinase called the 26S proteasome.

In general, the different ubiquitins have their own discrete E1-E2-E3 cascades, and they impart distinct functions to their targets.

Ubiquitin/Ubiquitin-like Proteins E1 Enzymes E2 Enzymes E3 Enzymes
Ubiquitin UBA1 UBC1-8, UBC10,
UBC-11, UBC13-MMS2
Many
SUMO (SUMO1, SUMO2, SUMO3, SUMO4) UBA2/SAE1 UBC9 SIZ1, SIZ2, MMS21
NEDD8 UBA3/NAE1 UBC12, UBE2F DCN1
ATG8 (LC3A, LC3B, LC3B2, LC3C, GABARAP, GABARAPL1, GATE-16) ATG7 ATG3 -
ATG12 ATG7 ATG10 -
URM1 UBA4 - -
UFM1 UBA5 UFC1 -
FAT 10 UBA6 UBE2Z -
ISG15 UBA7 UBCH8 HERC5, EFP

Annu. Rev. Cell Dev. Biol. 2006.22:159-180.

Proteasomes

Proteasomes are cylindrical, multisubunit proteases found in eukaryotes, eubacteria, and archaebacteria. Eukaryotic proteasomes come in two sizes, the 20S proteasome and the larger adenosine triphosphate (ATP)-dependent 26S proteasome formed by the 20S proteasome and a regulatory complex. The most important proteasome related component is the 19S regulatory complex, which binds to the 20S proteasome to form the 26S proteasome. In addition to 19S complex, there are at least two other intracellular regulatory complexes, PA28 and PA200, that can also use certain types of peptide substrates as models to activate the 20S proteasome.

Both 20S and 26S proteasomes can associate with protein complexes that activate peptide hydrolysis and may serve to localize the enzymes within cells. The 26S proteasome is a core protease in the ubiquitin-mediated proteolysis pathway and is essential for a range of cellular processes such as transcription control, enzyme level regulation, and antigen presentation.

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