SEB Bulletin March 2008
Ubiquitination: labelling the proteins
In the late 70’s and early 80’s, ground-breaking research led to the discovery of a highly-specific, energy-dependent process enabling the cell to target specific proteins for degradation. In 2004, Aaron Ciechanover, Avram Hershko and Irwin Rose were awarded the Nobel Prize in Chemistry for their discovery, which enabled the understanding of protein degradation at the molecular level as well as its implication in biological processes as diverse as control of the cell cycle, gene transcription and immunity (1, 2).
While proteins are built-up to cater for the structural and biochemical requirements of the cell, they are also broken-down in a highly-regulated process serving more purposes than just destruction and space management. Proteins have different half-lives, determined by the nature of the amino acids present at their N-termini. Some will be long-lived, while other will rapidly be degraded. Proteolysis not only enables the cell to dispose of misfolded or damaged proteins, but also to fine-tune the concentration of essential proteins within the cell, such as the proteins involved in the cell cycle. This rapid, highly specific degradation can be achieved through the addition of one to several ubiquitin molecules to a target protein. The process is called ubiquitination.
Ubiquitins are small regulatory proteins found in all eukaryotic cells from plants to mammals. Their addition onto a protein has occasionally been called “kiss of death” because it often commits the labelled protein to degradation in the proteasome, a barrel-shaped protein complex, where proteins are disassembled by proteases (4).
Ubiquitination is a post-translational modification carried out by a set of three enzymes, E1, E2 and E3. Ubiquitin is first activated by ubiquitin-activating enzyme E1, before being transferred to its active site, the amino acid cystein. This transfer requires ATP, making the process energy-dependent. The ubiquitin molecule is then passed on to the second enzyme of the complex, E2 (ubiquitin-conjugating enzyme), before reaching the final enzyme, E3, the ubiquitin protein ligase, which recognises, binds the target substrate and labels it with the ubiquitin. The process can be repeated until a short chain is formed, with three or more ubiquitin molecules usually targeting the protein to the proteasome (3, 4).
Substrate specificity is mainly defined by the multiple E2 and E3 combinations possible. E2 and E3 belong to large protein families, but while E2 share many well-conserved catalytic domains, E3 ligases only share a few conserved motifs and are, therefore, very specific. As the three-steps process advances, specificity increases: E1 interacts with all E2s, which interact with a more limited subset of E3s, which in turn target a limited array of protein substrates, based on shared recognition motif within the proteins to be labelled. This enables the ubiquitination-proteasome pathway to be highly specific in the selection of the proteins to be labelled (2).
Ubiquitin labelling is however not always fatal for the protein, with several non-proteolytic functions associated with the addition of a single ubiquitin molecule (mono-ubiquitination) or specific cases of polyubiquitination. Mono-ubiquitination can alter the fate of the protein in a less terminal fashion, potentially affecting its cellular sub-location, function or its degradation though lysosomes. The process is also reversible with enzymes (deubiquitinases) able to cleave ubiquitin from its target (4). “Ubiquitin is the founding member of a family of ubiquitin-like proteins (such as the SUMO protein) and unlike ubiquitin many of the other family members have a host of little understood non-degradative functions in the cell”, says Professor John Mayer (University of Nottingham). “Modification of proteins with SUMO (or Small Ubiquitin-related MOdifier), known as SUMOylation, often increases the protein lifespan and stability. It is also linked to nuclear-cytosolic transport, regulation and transcription”.
Thirty years on since the original studies led by Ciechanover, Hershko and Irwin and ubiquitination research is a thriving field, eliciting interest amongst plant, cell and animal researchers alike. Throughout the years, a wealth of substrates has been identified for the ubiquitination pathway, progressively revealing its crucial importance in cell cycle regulation, apoptosis and immunity alike. Amongst these substrates are cyclins, involved in the control of the cell cycle, p53, the tumour suppressor protein and NF-kB, the transcription factor involved in inflammation and the immune response. To each substrate corresponds an E3 ligase, such as the anaphase-promoting complex for cyclins, or the oncogenes mdm2 for p53. However, the task is still only in its infancy, and challenges ahead include unravelling the mechanisms by which the ubiquitination pathway “chooses” appropriate substrates for degradation.
With more than three thousands E3 ligases (against approximately one thousand in humans), plants have a rich ubiquitination machinery enabling in-depth studies, including protein-substrate interactions. An emerging theme in plant science is the link between ubiquitination and plant immunity, and the mechanisms by which pathogens manipulate the plant defences. Plants recognise potential pathogens using resistance (R) genes, which recognise the pathogen avirulence (avr) genes. R-avr recognition leads to the mobilisation of defences, including accumulation of salicylic acid and production of oxygen intermediates. Dr Ari Sadanandom, from the University of Glasgow, is one of the leaders in this research area (5). Using techniques such as the yeast two-hybrid system and gene silencing, his studies enabled to link SGT1, a regulator of the cell cycle, to the hypersensitive response as well as the ubiquitination pathway. Current challenges involve identifying the proteins ubiquitinated by pathogens in a bid to undermine the plant defences. The SEB’s Annual Meeting held in Marseille in April this year will provide an ideal forum to discuss new findings, in a session chaired by Dr Sadanandom (6). “Given its importance in many aspects of plant growth and development, ubiquitination represents an important avenue with which to improve crop productivity”, says Professor Vierstra (Michigan State University). “Trying to find ways to modify the system to degrade proteins that are problematic (for example coming from a plant or human pathogen) or stop degradation when the stability of a protein has obvious benefits is amongst the challenges ahead”. Dr Sadanandom also highlights that the knowledge gathered in the field of plant ubiquitination is often transferable to mammalian systems, where alterations of the ubiquitination pathway are at the heart of the aetiology of many malignancies, such as cancers, neurodegenerative diseases or genetic disorders.
In certain cancers, oncogenic targets (such as the adenovirus E1 A or the oncogene c-myc) are mutated so that they are no longer subject to ubiquitination, and therefore escape degradation and accumulate in the cell. The human papilloma virus (HPV), responsible for certain forms of cervical cancer, relies on its own viral E6 protein to promote ubiquitin-mediated degradation of the tumour suppressor p53. Other cancers may promote the over-expression of E3 ligases such as mdm2 leading enhanced degradation of p53 (7).
Ubiquitination also plays a part in diseases involving membrane proteins, such as Cystic Fibrosis, where ubiquitination is responsible for the degradation of the mis-folded CFTR chloride ion channel, or Liddle’s syndrome, where a mutation in a E3 ligase (NEDD4) prevents the efficient ubiquitin-mediated degradation of the ENaC epithelial sodium channel, leading to hypertension through excessive sodium and water re-absorption.
Professor Mayer has a special interest in the importance of the ubiquitin-proteasome system for human chronic neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, and dementia with Lewy bodies (the second most common cause of cognitive decline in the elderly, discovered in Nottingham), motor neurone disease and all the other diseases including Huntington’s disease and frontotemporal dementias. “All these diseases are chacterised neuropathologically by the occurrence of inclusion bodies (Lewy bodies) in neurones, containing ubiquitinated proteins”, says Professor Mayer, whose current research involves gene targeting in mice to study the role and function of proteasomal genes in Parkinson’s disease and dementia with Lewy bodies. Animal models are currently used to help shed light on a range of neurodegenerative diseases thought to involve alteration of the ubiquitination pathway (8).
And the reach of ubiquitination goes beyond cancer and neurodegenerative diseases. With dramatic stakes such as drug development, the relevance of these studies is tremendous for the public (9, 10). “Research in the ubiquitin field has unravelled the mechanisms of many diseases, immune disorders, neurodegenerative diseases and malignancies among them”, says Professor Aaron Ciechanover (Israel Institute of Technology). As a result, one successful anti-cancer drug has been developed and is already in use (Bortezomib, Velcade), while many othesr are in the pipeline. Future research will be focused on specific recognition of novel target proteins by the system, with the hope to develop specific modulators that will be able to control the level of key regulatory proteins involved in basic cellular processes in health and disease”.
University of Glasgow
(7) Ciechanover & Schwartz, 2004, Biochimica et Biophysica Acta 1695, pp 3-17