The transgenic approach has been used in model systems from yeast to mammals to address basic research questions, and to achieve agricultural, pharmaceutical or industrial objectives. In basic research, transgenic organisms have generated novel observations that could not have been obtained otherwise. This chapter concentrates on the use of transgenics in deciphering the operation of the UPS (ubiquitin–proteasome system) in the yeast, plant, nematode, fly, and mouse model systems, and will touch on ways in which transgenic manipulation of the UPS has been exploited for agricultural, pharmaceutical, and industrial applications.
A transgenic organism is one in which the genome has been altered by the addition of sequences not originally present in the germline DNA of that species. There are two main reasons why one would want to manipulate the UPS (ubiquitin–proteasome system) using transgenic methodology. The first is to gain some insight into the functioning of the UPS itself by perturbing its operation and observing the phenotypic consequences. Much of our understanding of the UPS has been deduced from experiments involving genetic manipulation of UPS components, either adding novel, genetically engineered UPS genes to the genome's existing repertoire or using genetic recombination to alter the endogenous UPS genes. The second rationale for transgenic manipulation of the UPS is to take advantage of its components in the pursuit of some agricultural, industrial, pharmaceutical, or basic research objective not directly associated with UPS function. One could reasonably argue that such approaches have been used to best effect in yeast (where genetic manipulations are straightforward and rapid), but transgenic research has been done with more complex eukaryotes and has been very informative. The summary of the research that follows cannot be considered comprehensive, but is intended to demonstrate what is possible using transgenic approaches, by touching upon some highlights of this powerful research methodology.
Experiments in yeast
As other chapters in this volume make evident, many of the mechanistic details of the UPS were first uncovered in the budding yeast Saccharomyces cerevisiae. It is relatively straightforward to introduce and express transgenes in yeast, and with an efficient system of homologous recombination, the organism is amenable to replacement of endogenous genes with their genetically engineered and often mutant counterparts. Through such experiments it has been possible to elucidate the roles of ubiquitin chains of various topologies (ubiquitin has seven lysine residues, at least six of which are exposed on the surface of the globular domain and could theoretically participate in isopeptide-bond formation with the C-terminal glycine of another ubiquitin molecule). The growth of yeast cells is slowed by overexpression of a number of mutant isoforms, but cells are non-viable if their sole source of ubiquitin is a K48R (substitution of the lysine at position 48 with arginine) or G76A (glycine to alanine) mutant isoform, owing to complete inhibition of proteolysis . Cells expressing only K63R mutant ubiquitin are viable, but sensitive to DNA damage, demonstrating that chains assembled through Lys63 linkages are involved in DNA repair . Indeed the power of yeast genetics and the site-directed mutation approach have been combined in an exhaustive examination of every surface-exposed residue in yeast ubiquitin .
The transgenic approach has also been very fruitful in yeast, in elucidating the existence and functional aspects of the N-end rule. Many proteins are expressed as proproteins that are processed by endoproteolytic cleavage into mature forms; in fact ubiquitin itself is invariably translated as a proprotein composed either of tandemly repeated ubiquitin subunits or of ubiquitin fused to ribosomal subunit peptides . Ubiquitin may serve a chaperone function in such fusions, assisting in the proper folding of the appended polypeptide. This property has been exploited in increasing the yield of poorly translated, transgene-encoded products in the industrial workhorse Escherichia coli . Like any cellular translation product, a proprotein will have methionine at its N-terminus, but the N-terminal residue of the processed protein will be dictated by its primary sequence and the position of the protease cleavage site. It is therefore possible that a processed polypeptide could have any of the twenty amino acids at its N-terminus, and, as it happens, the identity of this residue plays a key role in dictating its stability. This phenomenon has been established in organisms from E. coli to humans, and has been designated the N-end rule by Varshavsky . Transgenes encoding fusions of the structure ubiquitin–Xaa-β-galactosidase (where X is one of the 20 possible amino acid residues) were created. Like the natural ubiquitin–ribosomal-peptide fusions, such test fusions are efficiently cleaved by DUBs (deubiquitinating enzymes) to produce β-galactosidase with various N-terminal residues. It was determined that certain N-terminal residues (for example Gly, Val or Ala) would impart stability to the test protein whereas others (Arg, Leu, Phe) would result in a very short half-life. Using transgenic methodologies, the N-end rule has been demonstrated (and in some cases exploited) in complex metazoans.
Experiments in plants
The technology for introducing foreign genes into plant genomes is well advanced; indeed this technology is the basis for a large and often controversial industry (genetically modified foods). The ubiquitin promoter has been utilized to obtain constitutive expression of transgenes in plants, as has the strategy of expressing proteins as ubiquitin fusions (see above) to be processed by DUBs . Expression of epitope-tagged ubiquitin has served as an effective means of isolating ubiquitinated substrates from plant cells for identification and further analysis . Plants have also provided us with an important insight into a recurrent structural arrangement in the UPS. The most commonly used model system in plants is Arabidopsis thaliana, which like most higher plants uses different developmental programmes in light compared with dark. Mutants that inappropriately use the light programme when growing in the dark are designated COP (constitutive photomorphogenesis). The gene mutated in one such mutant (COP9) was found to encode a novel protein of 197 amino acids that could restore the functional light response when expressed from a transgene . COP9 protein functions in a multimeric complex designated the CSN (COP9 signalosome), a component of a light-sensitive signalling cascade in plants. The CSN is conserved in other species including mammals, where it functions in other pathways including cell-cycle checkpoints. The CSN has striking structural similarity to the lid of the proteasome, with a proteasome counterpart for each CSN subunit. The similarity of CSN and the 19 S lid may reflect divergent (or convergent) evolution, but there are intriguing connections in function that require further investigation (reviewed in ).
Experiments in nematodes
It would be fair to say that the nematode model system has not been exploited to the same extent as the yeast or plant systems in fleshing out our understanding of ubiquitin–proteasome biology, but transgenic manipulation of the UPS in worms has provided some penetrating insights within specialized fields, most notably neuroscience. Caenorhabditis elegans is a favourite of neuroscientists because it has a nervous system that is of sufficient complexity to endow the nematode worm with simple patterns of behaviour, but is not so complex so as to thwart to the construction of wiring diagrams (greatly facilitated by the transparency of the organism and a complete understanding of its cell lineages). C. elegans is a useful model system for the study of synapses, the junctions through which neuronal transmission of impulses occurs. There is an emerging body of evidence from a variety of model systems to the effect that the plasticity of synapses (modulation of their activity, which is thought to play a role in establishing memory, for example) involves alterations in protein abundance through proteasome-mediated degradation. Burbea et al.  demonstrated that in transgenic worms engineered to overexpress epitope-tagged ubiquitin in a specific subset of neurons, synapses were modified by ubiquitination and degradation of glutamate receptors (involved in fast excitatory neurotransmission). More recently, the same group has shown that the regulation of glutamate receptors in this population of neurons is mediated by the APC (anaphase-promoting complex) . This finding comes as a surprise; the APC (also known as the cyclosome) is a multi-subunit E3 (ubiquitin ligase) complex whose well-established function is in the regulation of cell-cycle transitions. Neurons are post-mitotic cells that apparently make use of the APC machinery for this unrelated and wholly unexpected function. The appropriation of the APC for a synaptic role (the cell-cycle function of the APC predates the evolution of nervous systems) probably occurred early in metazoan evolution; the synaptic function of the APC has also been documented in Drosophila melanogaster .
Experiments in flies
As in the nematode worm, transgenesis has been of more use in studying particular roles of the UPS in the fly than in studying the UPS itself, and (as in the worm) one area that has benefited greatly from transgenic manipulation of the UPS in this model system is neuroscience. The nervous system of flies is rather more complex than that of worms, and has a crowning feature of supreme use to neuroscientists: the compound eye. Fly eyes are made up of repeated elements of the nervous system and are macroscopic. One can use them as a ‘read out’ of neuronal health, and they have been heavily exploited in studies of neurodegeneration. There are many neurodegenerative diseases for which the fly eye has been useful, but the polyglutamine disorders (diseases in which an abnormally long stretch of glutamine residues occurs in the causative protein) are illustrative. If one expresses in the eyes of transgenic flies the expanded polyglutamine protein (the mutant huntingtin protein of Huntington's disease, for example, or one of the ataxin proteins associated with the spinocerebellar ataxias) the eyes will have a roughened appearance indicating neuronal degeneration. It is possible to conduct a genetic screen to identify modifiers of the severity of the eye degeneration, and such a screen will identify components of the UPS . A second approach is to identify components of the UPS that physically associate with the polyglutamine protein, then to assess the contribution of these components in flies that are transgenic for both the polyglutamine protein and the UPS component. Through such an approach it has been demonstrated that an E4 (polyubiquitin chain conjugation factor) protein that associates with ataxin-3 and promotes its degradation has therapeutic potential in limiting the neurodegeneration induced by this toxic entity . Remarkably, normal ataxin-3 is itself a ubiquitin-specific protease whose activity is involved in the suppression of neurodegeneration in vivo, as demonstrated in transgenic flies .
Experiments in mice
Given the slow reproductive rate and the greater genomic complexity of the mammalian system it is not surprising that experiments involving manipulation of the mammalian UPS have not been as numerous as in other model systems. The manipulation of the mammalian germline is a daunting task (relative to the yeast germline, for example), but there are questions involving the mammalian UPS that simply cannot be answered in any other way. Clearly one cannot address the role of the UPS in signalling pathways [for example the NF-κB (nuclear factor κB) pathway], DNA repair systems [for example involving BRCA1 (breast-cancer susceptibility gene 1)] or disease states in simple model systems lacking some or all of the relevant components. The mammalian model system of choice for genetic manipulation is the mouse, and methodologies for germline manipulation of the mouse are continuously being refined. As in other systems, it is possible to introduce expression cassettes containing a gene of interest into the mouse germline in order to accomplish constitutive, inducible or tissue-specific expression. Using gene targeting one can also manipulate genes in the mouse germline to generate mice in which all cells receive a mutated copy of the gene of interest, or mice in which a mutation-generating recombination event will occur in specific cells or at specific times (conditional mutations). All of these strategies have been used or are currently in use to exploit known aspects of the UPS that can improve expression of transgenes, or to reveal novel aspects of the functioning of the UPS in the mouse model system.
In the production of transgenic mice it is sometimes desirable to have the gene of interest expressed widely and at high levels (rather than in a tissue-specific manner). For ubiquitous expression it is hardly surprising that a ubiquitin promoter might serve the purpose, and the human ubiquitin C promoter has been shown to function admirably. The ubiquitin C promoter drives expression of transgenes in most if not all mouse tissues , with expression detectable as early as the morula stage of embryogenesis . The utility of the ubiquitin C promoter is exemplified by a body of work modelling reproductive disorders arising from abnormally high levels of serum oestradiol. Sustained elevation of oestradiol has been orchestrated by placing expression of the human aromatase p450 cDNA under the control of the ubiquitin C promoter. In male transgenic mice overexpressing aromatase, the development of the male reproductive tissues is inhibited, and functional mammary glands develop .
In mammalian cells (as in prokaryotes and yeast) one can take advantage of the efficient translation and folding of ubiquitin to enhance the production of proteins that may otherwise have lower abundance. In eukaryotic cells and in transgenic mice, a ubiquitin–EGFP (enhanced green fluorescent protein) fusion product is efficiently processed by DUBs, resulting in abundant expression of the fluorescent protein marker [18,20]. The strategy of expressing proteins as ubiquitin fusions also allows for the production of proteins that, once processed by DUBs, will have an amino acid other than methionine at the N-terminus (described in more detail in the yeast section above). This strategy can be used to circumvent the requirement for processing of proproteins to generate active, mature polypeptides. As an example, the active form of the pro-apoptotic protein Smac/DIABLO has an absolute requirement for an alanine residue at the N-terminus, and processing of the proprotein is not efficient in cells transfected with plasmids encoding the full length protein. Cells expressing a fusion of ubiquitin with the active Smac/DIABLO polypeptide efficiently cleave the ubiquitin moiety, bypassing the requirement for mitochondrial processing of the proprotein, and generating biologically active Smac/DIABLO .
Arguably the simplest manipulations of the UPS in the mouse involve addition of transgenes encoding ubiquitin itself to the mouse germline. The objectives of such manipulations might be to increase the ubiquitin pools by supplying additional wild-type ubiquitin, or to perturb the functioning of the system by incorporating mutations in ubiquitin that would interfere with chain assembly in a dominant negative fashion. One might also incorporate an epitope tag to facilitate the detection of ubiquitin and ubiquitin conjugates, as well as some form of reporter system to monitor the expression of the transgene in living animals. These were the strategies employed in the author's laboratory in the generation of transgenic mice expressing wild-type or mutant ubiquitin (the source of the ubiquitin DNA was human, but this is of no functional significance given the perfect identity of mouse and human ubiquitin amino acid sequences). A hexahistidine epitope tag was incorporated at the N-terminus of ubiquitin to allow detection of transgene-derived ubiquitin by Western blotting and enrichment of ubiquitin conjugates by nickel affinity chromatography. EGFP was appended to the C-terminus of the ubiquitin to serve as a reporter of gene expression, with the expectation that cellular DUBs would efficiently cleave the fluorescent protein from ubiquitin and free its C-terminus for conjugation. The human ubiquitin C promoter was used to drive expression in most, if not all, tissues. The strategy is shown schematically in Figure 1. As expected, fluorescence could easily be detected in the resulting mice ; in living mice the retinas demonstrated intense fluorescence when illuminated with blue light. Mice expressing wild-type human ubiquitin or the mutant isoforms K48R or K63R were viable and fertile, with no obvious phenotype. Although the analysis of these transgenic lines is ongoing, it is apparent that the K48R mice are resistant to neurotoxic insults  and show features of delayed aging . The molecular basis of the hardiness of K48R mice is not yet understood — one might expect such mice to be sickly as a consequence of impaired proteolysis. At least some of the positive in vivo effects of K48R mutant ubiquitin may be attributable to stabilization of transcriptional mediators whose degradation is associated with disease or aging .
The UPS is impaired or overwhelmed in many disease conditions and declines in efficiency with age . Recently it has been demonstrated that the subunits of the proteasome are co-ordinately regulated, and production of proteasomes can be stimulated by forced expression of just one subunit . It may therefore be possible to restore flagging proteasome activity through a relatively simple genetic intervention. One can monitor the efficiency of the UPS in vivo using a transgenic mouse in which a destabilized version of a fluorescent protein is expressed constitutively. In this transgenic reporter mouse, proteasome inhibition results in accumulation of the otherwise unstable protein and its fluorescence becomes detectable . Such reagents should prove very valuable in future evaluation of UPS-based therapeutics.
Manipulation of the germline through insertion of genes and/or regulatory elements derived from the UPS has enormous use not only in expanding our understanding of the UPS itself, but also in practical applications where constitutive, high-level expression is desirable. In the future, such transgenic animals will continue to be a mainstay of biomedical research, but it is expected that new applications will emerge through genetic crosses of transgenic animals with the growing repertoire of ‘knock-out’ animals, or through ‘knockdown’ approaches [(small interfering siRNA) etc. RNA] that can mine new information from transgenic strains. Ubiquitin transgenics will also figure prominently in the ‘omics’ era, for example in cataloguing the substrates of the UPS in complex metazoans through proteomic strategies.
Transgenic manipulation of the UPS has been pivotal in determining how its components function in yeast and other model systems.
Because ubiquitin is expressed in all eukaryotic cells, regulatory elements from ubiquitin genes can be used to direct constitutive expression of transgenes in metazoans, from plants to mice.
Transgenic approaches have revealed cellular pathways governed by the UPS in a variety of organisms, including yeast, plants, nematode worms, fruit flies and mice.
Interference with proteolytic function through transgenic manipulation of the UPS has been informative with regard to the role of the UPS in diseases, most notably the neurodegenerative diseases.
- © 2005 The Biochemical Society