The ubiquitin-proteasome pathway (UPP) is the major mechanism for protein processing within cells, responsible for the selective proteolytic degradation of about 90% of cellular proteins. Proteins degraded by the UPP play roles in a variety of biological processes including development, differentiation, proliferation, signal transduction and apoptosis. While its major role is regulation of protein turnover, the proteasome also functions in several non-proteolytic processes, such as transcription-coupled nucleotide excision repair, transcription initiation and elongation, and regulation of gene ex
UPP-mediated protein degradation is carried out via two critical steps: 1) conjugation of multiple ubiquitin molecules to the protein substrate, and 2) degradation of the ubiquitin-tagged substrate by the 26S proteasome. The 26S proteasome is a large (2.5 MDa), multi-subunit complex, comprised of a catalytic 20S core, and one or two 19S regulatory caps, that is localized both in the cytosol and nucleus of cells.
The 20S core was initially referred to as the conjugate-degrading factor-3 (CF-3), and the 19S regulatory cap as CF-1 and inhibitory CF-2. The 20S core consists of 28 subunits that form a barrellike structure of four alternately stacked rings: two α rings surrounding two β rings, containing seven subunits each. The α subunits block direct access to the catalytic sites by allowing access only to unfolded proteins, while the role of the β subunits is to carry out the proteolytic activities of the proteasome, which are dependent on an amino-terminal nucleophilic Thr1 residue. There are three active β subunits: β1, β2 and β5, responsible for caspase or peptidyl-glutamyl peptide-hydrolyzing (PGPH)-like, trypsin-like and chymotrypsin (CT)-like activities, respectively.
The 19S regulatory cap(s) (700 kDa) can be divided into base and lid components: with the base responsible for recognition and unfolding of ubiquitinated protein substrates, as well as opening of the 20S core and transport of substrates into the core, and the lid deubiquitinating substrates prior to degradation. The base is comprised of six ATPase subunits, Rpt1-6, which form a hexameric ring, as well as two non-ATPase subunits Rpn-1 and -2 and the lid consists of at least six non-ATPases, including Rpn-10/S5a and Rpn-13/Adrm1, which contain ubiquitin interacting motifs (UIMs). Rpn-10/S5a has two UIMs that bind preferentially to poly-ubiquitinated substrates and Rpn-13/Adrm1 binds to the non-ATPase Rpn-2, promoting recruitment of deubiquitinating enzymes (DUBs) to the proteasome. Like the rest of the pathway, deubiquitination is highly regulated and very important for recycling of ubiquitin molecules and controlling the rate of degradation.
The ubiquitination step of the UPP is executed by three distinct types of enzymes, E1, E2s and E3s. The pathway is initiated by ATP-dependent E1-mediated activation of ubiquitin, a small 76 amino acid protein that is expressed ubiquitously throughout cells and serves as a tag for protein substrates destined for UPP-mediated degradation as well as various other fates, including membrane-trafficking, protein kinase activation, DNA repair and chromatin remodeling. Activated ubiquitin is then transferred from E1 to one of several ubiquitin-conjugating E2 enzymes, and then to an E3 ubiquitin-ligating enzyme, which aid in the transfer of active ubiquitin to lysine residues within the target protein. Following the conjugation of a sufficiently sized ubiquitin chain, usually four, except in the case of proteins like mODC and HIF-1α, which require no ubiquitination for proteasome-mediated degradation, the ubiquitinated protein substrate is then recognized, deubiquitinated and translocated to the 26S proteasome by components of the 19S regulatory cap. Finally, the substrate is degraded into short peptide fragments and the ubiquitin is recycled. This process is tightly regulated and critical to the regulation of a number of cellular processes, including those involved in tumorigenesis, making it a promising target for anti-cancer agents.
The essential role of unbalanced protein homeostasis in development, growth and survival of various cancers, has led to intensive investigation into the targeting of factors involved in the synthesis and degradation of proteins, including the UPP, as a potential anticancer strategy. Increased proteasome activity has been reported in several types of human cancer, including colon, prostate and leukemia, suggesting that malignant cells are more dependent on the UPP than non-malignant cells and indicating that targeting the UPP is a viable strategy in the treatment of cancer. Indeed, inhibition of the β5 subunit (chymotrypsin-like activity) by as little as 25% has been shown to be associated with cell cycle arrest and apoptosis induction. Thus, proteasome inhibition may selectively induce apoptosis in cancer cells with minimal effects on healthy cells, but may also effectively sensitize resistant cancer cells to chemo- and/or radiotherapeutics. Furthermore, the clinical use of proteasome inhibitors was validated by the USFDA approval of bortezomib for the treatment of relapsed/refractory multiple myeloma and mantle cell lymphoma.
One major pathway that is an established target of the UPP is the apoptosis cascade. There are several checkpoints for which ubiquitination-dependent degradation is responsible, including degradation of transcription factors and enzymes necessary for cell proliferation, such as NF-κB and ODC, cell cycle regulators like p27 and pro- and anti-apoptotic proteins like caspases and IAPs. The IAPs (inhibitor of apoptosis) are perhaps the most important of these apoptosis-related proteasome substrates. The IAP family of proteins, including c-IAP and XIAP, serve as endogenous inhibitors of apoptosis, whose major role is binding and inhibiting caspase proteins. All members of the IAP family contain 1-3 BIR (baculoviral IAP repeat) domains, which are critical to their function. Importantly, many IAPs also contain RING finger domains, which, in addition to their E3 ligase activity, activate and recruit caspases through their CARD domains, ultimately leading to caspase degradation. Additionally, IAPs, in response to apoptotic stimuli, are auto-ubiquitinated and degraded by the proteasome. Therefore, in addition to targeting the UPP, targeting inhibitory factors, like XIAP, within the apoptosis cascade, is also a promising strategy in the treatment of human cancers.