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The Translation Post Vol.2 Issue 1

Review Article - The Ubiquitin Proteasome Pathway: a ubiquitous drug target?

 

By Herve Calvez, Ph.D., Director Business Development

 

When Ubiquitous Means Interesting

Back to my years as a postdoc, I remember my PI repeating at lab meetings: "Controls, controls, controls! Always use a control in your experiment! Have a negative and a positive control. Use a normal cell line to back up what you see in a cancer cell line. And don't disturb me if the staining is all over the tissue sample!". This story could illustrate the ubiquitin proteasome pathway, once considered so ubiquitous that likely no pharmaceutical company would qualify ubiquitin as a drug target. A Nobel prize and an FDA-approved drug later, this is one of the hottest fields of the moment. This brief review will highlight some of the latest advances in the ubiquitin proteasome pathway research and its potential outcome for clinicians.

Introduction to the Ubiquitin Proteasome Pathway

In 2004, Avram Hershko, Aaron Ciechanover and Irwin Rose won the Nobel Prize for Chemistry for their contribution to the understanding of the ubiquitin proteasome role in cell biology. The ubiquitin pathway is the principal mechanism for protein catabolism in the mammalian cytosol and nucleus. The tightly regulated pathway affects a wide variety of cellular processes, and defects in the system can result in multiple important human diseases. The ubiquitin pathway is central to:

• Antigen processing
• Apoptosis
• Biogenesis of organelles
• Cell cycle and division
• DNA transcription and repair
• Differentiation and development
• Immune response and inflammation
• Neural and muscular degeneration
• Morphogenesis of neural networks
• Modulation of cell surface receptors, ion channels and the secretory pathway
• Response to stress and extracellular modulators
• Ribosome biogenesis
• Viral infection

Pathologies that result directly or indirectly from aberrations in the ubiquitin pathway are:

• Malignancies (brain, breast, cervix, colorectal, kidney, prostate and retina cancers)
• Liddle's syndrome (kidney osmosis deregulation)
• Angelman syndrome (brain development abnormality)
• Neurodegenerative diseases (Alzheimer's disease, Parkinson's disease, Huntington's disease, Kennedy's syndrome)
• Cystic fibrosis
• Autoimmune disease (inflammation)
• Viral infection (EBV and Herpes viruses)
• Muscle wasting

Degradation of a protein via the ubiquitin pathway involves two successive steps: tagging of the substrate protein by the covalent attachment of multiple ubiquitin molecules (conjugation); and the subsequent degradation of the tagged protein by the 26S proteasome. Therefore, the primary functions of ubiquitin have been associated first with protein housekeeping and turnover as well as antigenic-peptide generation. More recently, a significant regulative function of protein modification by ubiquitin has been unveiled in DNA repair and endocytosis. These functions are controlled by the number of ubiquitin units attached to proteins (mono- or poly-ubiquitination) and the type of ubiquitin chain linkage. The reversible linkage of one molecule of ubiquitin or ubiquitin-like molecules such as SUMO and NEDD8 to proteins is involved in critical biological processes, including protein localization, transcriptional activity, nucleocytoplasmic transport and genomic replication (see The Translation Post vol 1, issue 2 for review).

The Ubiquitin Conjugation Mechanism

Ubiquitin, an essential protein of 76 amino acids, represents one of the most conserved polypeptides in eukaryotes, and only four of its amino acids differ among yeast, plants and animals [1]. Ubiquitin is expressed as three different precursors: a polymeric head-to-tail concatemer of identical units (polyubiquitin); and two N-terminal ubiquitin moieties, Ub_L40 and Ub_S27, that are fused to the ribosomal polypeptides L40 and S27, respectively [2]. Specific endopeptidases cleave these precursor molecules to release ubiquitin moieties that are identical in sequence [3]. In budding yeast, the ribosomal fusion proteins are responsible for the bulk of the free ubiquitin pool [4]. Before its proteolytic separation from L40 and S27, the ubiquitin moiety acts as a chaperone and thus facilitates ribosome assembly. Only a few amino acids in ubiquitin are vital for its function, however it seems that the highly conserved ubiquitin gene sequence is driven not only by functional properties of the ubiquitin protein, but also by the propensity of the polyubiquitin locus to act as donor or substrate for recombination to maintain the non-allelic ubiquitin genes [5]. 

The 3D-structure of ubiquitin solved by X-ray diffraction and NMR has shown that its structure is highly compact and rigid (figure 1) [6,7]. Only the last four C-terminal amino acids are protuberant and unstructured. Within these four residues, Gly-76 is activated in an ATP-dependent manner for subsequent ubiquitin ligation with the ε-amino group of an internal Lys residue of the protein substrate. Another important residues, Lys-48, provides an ε-amino group to form an additional peptide bond to generate multi-ubiquitin chains.

The ubiquitin conjugation cascade starts with the activation of the carboxyl group of Gly-76 of ubiquitin by the ubiquitin-activating enzyme E1 (UBA1), the only E1 enzyme coded by the yeast genome. This ATP-dependent reaction generates a ubiquitinyl adenylate intermediate bound to the E1 enzyme. The activated ubiquitin is then transferred to a thiol group of an active site Cys of a ubiquitin-conjugating enzyme E2 [8]. Eleven E2 enzymes (Ubc1-8, 10, 11, 13) have been identified in the yeast genome and many more in higher organisms. They all share an active-site ubiquitin-binding Cys residue and are distinguished by the presence of a UBC domain required for binding of distinct E3 enzymes. Finally, E2 enzymes transfer the ubiquitin moiety, either directly to a protein substrate or indirectly in cooperation with a ubiquitin-protein ligase enzyme E3, to form a bond between the carboxyl group of Gly-76 of ubiquitin and the protein substrate™s internal Lys residue. The first case is catalyzed by E3 ligases encompassing a RING finger domain, which serve as scaffolds to bring together E2 enzymes and substrates. The second case is catalyzed by HECT domain E3 ligases. Each E2 enzyme interacts with numerous E3 ligases, thus expanding the number of substrates targeted. Once the protein substrate is mono-ubiquitinated, a polyubiquitin chain is formed through the same ubiquitination conjugation cascade (figure 2, [9]).

The 26S Proteasome: a Multi-subunit Complex for Protein Degradation

The 26S proteasome is a large 25,000 kDa complex made of two copies of at least 32 different subunits. The overall structure can be divided into two major subcomplexes: the 20S CP that contains the protease subunits, and the 19S RP regulatory particle that regulates the function of the 20S CP (figure 3, [10]). The structure of the 20S proteasome is well conserved in all organisms. The barrel-shaped complex consists of four stacked heptameric rings that form a channel in which the unfolded substrate goes through. Each ring is composed of seven distinct subunits. The alpha-subunits form the two outer rings, which have a regulatory role with the 19S particle. The beta-subunits form the two inner rings, which contain the proteolytic active sites within a sequestered chamber [11]. The 19S RP particle is made of two subunits, a lid and a base [12]. While the lid recognizes ubiquitinated substrates, the base unfolds protein substrates and threads them into the catalytic chamber of the 20S particle. The base contains six ATPases that cap the 20S particle by interacting with the alpha-subunits. Altogether with ancillary proteins, the 19S complex regulates the opening of a gated channel into the 20S core to free access to the proteolytic chamber otherwise inactive in the 20S particle alone [13].

Recycling Ubiquitin and Deubiquitination Enzymes

Ubiquitin moieties attached to a protein substrate degraded by the 20S complex are released from the proteasome and recycled back into the ubiquitin pathway. The deubiquitination activity specifically associated with the proteasome complex has not been yet identified but could be attributed to p37a protein in Drosophila [14]. Deubiquitinating enzymes (DUBs) serve to edit the ubiquitin chains and remove ubiquitin from protein substrates before degradation. More than 17 DUB enzymes have been identified in eukaryotes. Based on their molecular size, sequence homology and active site residues, DUBs are categorized as UCHs (ubiquitin COOH-terminal hydrolyses) or UBPs (ubiquitin-specific proteases). UCHs are generally small enzymes (20-30 kDa) that remove short and flexible peptide chains from the COOH terminus of ubiquitin. UBPs on the other hand belong to a more diverse group of enzymes with a larger molecular mass (100 kDa). UBPs cleave the isopeptide bond linking Ub-Ub or Ub-protein and also biosynthetic linear fusions of ubiquitin. DUBs have several roles in maintaining the steady-state levels of free ubiquitin and in affecting the stability of ubiquitin-conjugated proteins [15]. DUBs generate and recycle ubiquitin, edit poly-ubiquitin chains, and aid in the proteasome-dependent degradation of proteins. 

Drug development for the ubiquitin proteasome pathway

Most currently available inhibitors of the ubiquitin proteasome pathway directly target and inhibit the 20S proteasome, the core of the proteolysis machinery, rather than the upstream ubiquitination mechanism. The proteasome inhibitors are broadly categorized into two groups: synthetic analogs and natural products (see [16] for review). Synthetic proteasome inhibitors target serine and cysteine proteases, therefore inhibiting the proteolytic activity within the proteasome. From well-known peptide aldehyde inhibitors (leupeptide, calpain inhibitor) with a broad spectrum, refinement of the specificity using peptide vinyl sulfones and later peptide-boronic acid derivatives, has led to highly potent and specific drugs like PS-341 (bortezomib/Velcade™) (figure 4). Like other proteasome inhibitors, PS-341 blocks cell cycle progression, eventually leading to apoptosis [17]. PS-341 blocks NF-kB activation by inhibiting IkBα degradation by the proteasome [18]. Natural products are also selective and potent proteasome inhibitors. Lactacystin, a Streptomyces metabolite, inhibits cell cycle progression by modifying irreversibly β-subunits of the 20S proteasome [19]. Gliotoxin, a fungal compound with a heterobicyclic core containing a disulfide bridge, displays an inhibitory activity against NF-kB [20]. The NF-kB pathway is intrinsically linked to the ubiquitin proteasome pathway. Ubiquitin not only targets IkBα for degradation, but also the NF-kB precursors, p105 and p100, for processing into mature forms by the proteasome. Moreover, upstream kinases in the NF-kB pathway are activated by ubiquitination independently of protein degradation [21]. Because of the central role of NF-kB in the inflammation response, proteasome inhibitors have been under heavy investigation for any effect on tumor growth and therapeutic implications in general.

Velcade® (bortezomib) from Millenium Pharmaceuticals, was approved by the FDA in 2003 for refractory multiple myeloma. Bortezomib demonstrated a superior response rate to dexamethasone in patients with relapsed/refractory disease [22]. Development of bortezomib for other indications has not yet advanced to the same degree, but additional studies are underway to further define the potency of bortezumib in hematological malignancies and solid tumors as a single agent as well as in combination with other agents. Combination of bortezumib with dexamethasone, thalidomide or doxorubicin is an active regimen on patients with multiple myeloma [23]. A combination with chemotherapeutic agents such as geldanamycin could reduce chemoresistance developed in myeloma and NHL upon single regimen treatment [24,25]. Hundreds of clinical trials have been launched now for testing multiple combinations with bortezomib, including: with trastuzumab (Herceptin®) for metastatic breast cancer (Jules Bordet Institute, phase I), with radiation therapy for head and neck cancer (NIH, phase I), with bevacizumab (Avastin®) for kidney cancer (Norris Comprehensive Cancer Center, phase I/II), and with docetaxel in hormone-refractory prostate cancer (Norris Comprehensive Cancer Center and Millenium Pharmaceuticals, phase II). 

A lactacystin-related compound, NPI-0052 dicovered by Nereus Pharmaceuticals, has a broader specificity than bortezomib, i.e. inhibiting both chymotrypsin-like (as bortezomib does) and trypsin-like activities of the proteasome [26]. NPI-0052 is more potent and selective than bortezomib as well as overcoming drug resistance seen with the latter. Investigations are still at the preclinical stage for treating multiple myeloma and NHL [27,28]. 

With the success of bortezomib to control protein degradation and subsequently tumor growth, the other enzymes of the ubiquitin proteasome pathway are now potential targets for drug development. The E1, E2, and E3 enzymes, as well as DUBs, are now being investigated by numerous drug companies for different indications, such as cancers and cachexia, a muscle atrophy observed in 60-90% of cancer patients. Other potential therapeutic indications for effectors of the ubiquitin pathway enzymes include diabetes, cardiovascular diseases, metabolic diseases, and inflammation and neurodegenerative diseases. Most of the product development is still in discovery or preclinical phase, therefore little is known on these efforts (reviewed in [29,30]). E2 and E3 ligase inhibitor programs are ongoing at Celgene, Genentech, Meso Scale, Novartis, Progenra, Proteolix, Regeneron, Roche, and Rigel Pharmaceuticals. Meso Scale Discovery has identified compounds inhibiting Mdm2, an E3 ubiquitin ligase that degrades p53 [31]. Roche has identified inhibitors, called nutlins, that block protein-protein interaction between Mdm2 and p53 [32]. A recent study of nutlin-3 on cancer models showed a good efficacy against tumors, suggesting applications to wild-type p53 tumors with normal Mdm2 expression [33]. Rigel Pharmaceuticals, in collaboration with Johnson & Johnson Pharmaceutical Research and Development, identified UHRF1 as an E3 ligase involved in tumor proliferation. Johnson & Johnson will develop drug compounds against UHRF1 that will be tested for inhibiting tumor growth or sensitizing tumor cells to chemotherapeutic agents [34].  

Concluding remarks

The ubiquitin conjugation mechanism includes about 50 E1 and E2 enzymes, 500 to 700 E3 enzymes, and 70 to 100 DUB proteins. From our current knowledge of the protein degradation field, numerous ligases have emerged as tempting anticancer targets. Subsequent target validation and drug screening have been initiated by pharmaceutical companies. Although preliminary studies have shown exciting results on cancer cell models, there are still many unknown effects of the potential drug candidates to investigate. With each ligase degrading multiple proteins, it is difficult to predict every secondary effect a drug may trigger by inhibiting only one ligase. In this aspect, E3 enzymes may be better targets than E1 or E2 enzymes, which are likely less specific than E3s due to their lower number. DUB enzymes are considered as the next set of enzymes to target in the ubiquitin pathway, with Millenium Pharmaceuticals and Progenra being among the first companies to initiate new validation programs. Another family of enzymes of high interest for drug development is the ubiquitin-like molecules. Numerous substrates for SUMO and NEDD8 have been identified by proteomics approaches as druggable targets. Nonetheless, it will take a serious commitment to unveil the detailed mechanism of ubiquitin-mediated protein modification and to understand the therapeutic ramifications of pharmaceutical intervention.  

Abgent and the Ubiquitin Proteasome Pathway

In the past four years, Abgent has produced more than 100 antibodies against components of the ubiquitin proteasome pathway, including: E2 and E3 ligases, and DUBs antibodies. Additionally, Abgent is a leader in the SUMO research field, providing scientists with the newest antibodies targeting SUMO-1 through SUMO-4, as well as a literature-cited bioinformatics tool, SUMOplot^TM, to predict sumoylation sites in protein sequences.  

SUMOplot^TM is a trademark of Abgent. 

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