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| Dr Arno Alpi |
| E: a.f.alpi@dundee.ac.uk |
| T: 44 1382 384999 |
| F: 44 1382 388500 |
Dr Arno Alpi
Research
My laboratory aims to elucidate ubiquitin-signaling pathways that regulate cellular responses to DNA damage. Particularly, we focus on the identification of novel ubiquitylated substrates in the Fanconi anaemia and BRCA1/BARD1 tumour suppressor pathways. We use a biochemical strategy that combines the genetically tractable cell line, DT40, with 2D-Dimensional Differential Gel Electrophoreses (2D-DIGE) and Stable Isotope Labeling with Amino acids in Cell culture (SILAC) proteomics. Our goal is to functionally dissect ubiquitin signalling pathways and their potential substrates to gain a better understanding of how these pathways protect cells from cellular transformation and carcinogenesis.
DNA damage and genome stability
Every form of life is strictly dependent on the maintenance of its genome and on the correct transmission of its genetic information from one generation to the next. It is therefore quite surprising that DNA – the molecular basis of genetic information - is in fact chemically unstable and vulnerable to many different insults that alter the information in the genome. Environmental factors (including UV light, ionizing radiation, oxygen) and also cell-endogenous factors including metabolic biproducts (e.g. Reactive Oxygen Species, ROS) constantly cause chemical changes to the DNA that can lead to a vast number of DNA lesions resulting in alternations to the genetic information. These changes in genetic information (mutations) can affect all biological processes and are often the basis for human disease. The most prominent is the development of cancer, where mutations change cellular homoeostasis, inducing cell proliferation and blocking the cell death response.
Cells face a staggering number of genotoxic insults and activate a variety of DNA damage responses. Highly specialized DNA repair mechanisms recognize DNA lesions and remove them to protect and retain the genetic information. DNA repair is usually accompanied by DNA damage checkpoint mechanisms. These mechanisms ensure that damaged DNA does not impede DNA metabolism (DNA replication, DNA transcription) and that potentially mutagenic lesions are not transmitted to the next generation. Both DNA repair and checkpoint mechanisms have to be functionally linked in order to maintain the genomic stability of the cell (Fig. 1). This has been highlighted by recent research that recognized the important roles that posttranslational modifications by ubiquitylation play in orchestrating DNA damage response pathways to protect the cell in an efficient manner.
Ubiquitylation, a process that attaches an ubiquitin molecule to a substrate protein, is achieved by an enzymatic cascade that consists of three enzymes called E1 activating enzyme, E2 conjugating enzyme and E3 ubiquitin ligase. The interplay of these enzymes defines substrate specificity and the kind of ubiquitylation needed (e.g. monoubiquitin versus polyubiquitin chain formation). Ubiquitin (and polyubiquitin chains) on target proteins can be disassembled by deubiquitylating enzymes (DUBs). Therefore, ubiquitylation is a dynamic and reversible modification and provides an efficient tool acting as a molecular switch in signal transduction pathways.
Ubiquitin signalling in DNA damage response pathways
Fanconi Anaemia is a rare recessive, genetically heterogeneous disorder of progressive bone marrow failure, spontaneous chromosomal instability and a severe predisposition to the development of leukaemia. Cells derived from FA patients are characterized by a very high frequency of chromosomal abnormalities. Moreover, these cells are hypersensitive to agents that cause interstrand DNA crosslinks. FA is therefore considered to be a genome instability syndrome.
A key step in the FA tumour suppressor pathway is the site-specific monoubiquitylation of the protein FANCD2. Genetic studies show that this crucial modification requires a multisubunit E3 ligase (composed of eight FA proteins) and the E2 conjugating enzyme, Ube2t. Recently we successfully reconstituted this monoubiquitylation reaction in vitro with Ube2t and FANCL, revealing that monoubiquitylation is stimulated by a newly identified and evolutionary conserved RWD-like domain in FANCL. Furthermore, we uncovered a potential function of FANCI in restricting the monoubiquitylation to the in vivo substrate lysine residue (K563) on FANCD2 (Fig. 2). We are exploring this in vitro system to gain further insight into how FANCD2 ubiquitylation is regulated in response to DNA damage.
Each E2 conjugating enzyme can interact with a number of different E3 ligases, extending their range of specific substrates and revealing the complexity of ubiquitin signalling. Conversely, it has recently been reported that an E3 ligase can select different E2s in order to define the kind of ubiquitin modification. This has been shown for the E3 RING ubiquitin ligase BRCA1. BRCA1 can functionally interact with two different groups of E2s, which then perform either mono- or polyubiquitin chain formation, respectively. Surprisingly, we recently discovered that FANCL can also interact with two different E2s, Ube2t and Ube2w, thereby increasing the numbers of possible substrates that are targeted within the FA pathway (Fig. 3).
It will be an exciting challenge for our laboratory to study the novel E2/E3 pairs and to identify their potential substrates in DNA repair and pathways of genome maintenance.
References
1. Alpi, A.F. and Patel, K.J. (2009). Monoubiquitylation in the Fanconi anemia DNA damage response pathway. Review in press, DNA repair doi:10.1016/j.dnarep.2009.01.019
2. Alpi, A.F., Pace. P., Babu, M.M. and Patel, K.J. (2008). Mechanistic insight into site-restricted monoubiquitylation of FANCD2 by Ube2t, FANCL, and FANCI. Mol Cell 32, pp. 767-777.
3. Alpi, A., Langevin, F., Mosedale, G., Machida, Y.J., Dutta, A. and Patel, K.J. (2007). UBE2T, the Fanconi anemia core complex, and FANCD2 are recruited independently to chromatin: a basis for the regulation of FANCD2 monoubiquitination. Mol Cell Biol. 27, pp. 8421-8430.
4. Mosedale, G., Niedzwiedz, W., Alpi, A., Perrina, F., Pereira-Leal, J.B., Johnson, M., Langevin, F., Pace, P. and Patel, K.J. (2005). The vertebrate Hef ortholog is a component of the Fanconi anemia tumor-suppressor pathway. Nat Struct Mol Biol. 12, pp. 763-771.
The underlined authors have contributed equally to the work
University of Dundee