Ubiquitin E3 ligase atrogin-1 (Fbox-32) in Atlantic salmon (Salmo salar): Sequence analysis, genomic structure and modulation of expression Luca Tacchi a , Ralph Bickerdike b , Christopher J. Secombes a , Nicholas J. Pooley a , Katy L. Urquhart c , Bertrand Collet c , Samuel A.M. Martin a, ⁎ a Institute of Biological and Environmental Sciences, University of Aberdeen, Tillydrone Avenue, Aberdeen, AB24 2TZ, UK b BioMar Ltd, Grangemouth Docks, Grangemouth FK3 8UL, UK c Marine Scotland, Marine Laboratory, Aberdeen AB11 9DB, UK abstract article info Article history: Received 22 April 2010 Received in revised form 5 August 2010 Accepted 15 August 2010 Available online 20 August 2010 Keywords: Atlantic salmon Proteasome Atrogin-1 Protein degradation E3 ubiquitin ligase E3 ubiquitin ligases are central for the selection of proteins targeted for degradation by the ubiquitin proteasome pathway. In this study atrogin-1 (Fbox-32), a major E3 ligase in muscle, has been characterized in Atlantic salmon (Salmo salar). The protein sequence is highly conserved between teleosts and mammals and is characterized by the presence of five conserved motifs related to the identification of protein targets. The genomic structure is conserved between teleosts and mammals and contains 9 exon and 8 introns. The phylogenetic relationship between atrogin-1 and two other closely related ubiquitin E3 ligases FBXO25 and MuRF1 showed atrogin-1 and FBXO25 grouped together with MuRF1 being more distant. The mRNAs were expressed in multiple tissues, atrogin-1 and MuRF1 were most abundant in white muscle and heart whereas FBXO25 had greatest expression in brain, white muscle and heart. The transcriptional modulation of these E3 ligases was examined in starved fish and fish following different immune stimulations. Expression of atrogin-1 and MuRF1 was increased following food deprivation, implementing these two genes in degradation of muscle protein during starvation. During viral infection atrogin-1 expression was not altered, whereas it was increased following stimulation with LPS, indicating an onset of catabolic processes during inflammatory responses. © 2010 Elsevier Inc. All rights reserved. 1. Introduction Fish are highly efficient at depositing consumed protein as growth in comparison to endothermic terrestrial animals (Houlihan et al., 1995). The control of protein deposition is a tightly controlled balance between protein growth (anabolism) and protein degradation (catabolism), the greater the difference between synthesis and degradation maximizes the efficiency of conversion of protein feed to deposition as flesh in the growing animals (Boonyarom and Inui, 2006). As there is no body store of free amino acids, these processes are continually active and modulated to respond to physiological needs resulting in a continual turnover of proteins, both processes being energy demanding (Houlihan et al., 1995). Fish have lower whole body protein turnover rates than mammals (Fraser and Rogers, 2007) which likely reflects a lower metabolic rate of ectothermic animals. Protein synthesis rates are comparable between individuals when protein consumption is accounted for, but the retention of synthesized proteins varies due to differing rates of protein degradation (McCarthy et al., 1994). Even though most of the biochemical pathways are evolution- arily conserved, the control of protein metabolism in fish is different to that in mammals. The efficiency with which fish achieve protein growth depends on a multitude of factors including nutrition (Gomez-Requeni et al., 2005) and health status (Johansen et al., 2006), both influenced by genotype (Tobin et al., 2006; Kause et al., 2007). To fully understand the mechanisms with which protein deposition is regulated the key genes relating to control of deposition need investigation. Protein degradation is a highly regulated process with three main outcomes, 1) release of free amino acids for oxidation and energy production, 2) destruction of incorrectly or damaged proteins, 3) release of amino acids for synthesis of new proteins (Attaix et al., 2005). Protein degradation is orchestrated by three major enzyme systems, the energy dependant ubiquitin proteasome (UbP) pathway, membrane bound lysosomal enzymes and calpain proteases (Seiliez et al., 2008). In mammalian muscle UbP is responsible for the bulk of cellular proteolysis (up to 90%) (Lecker and Goldberg, 2002; Lecker et al., 2004). The UbP pathway operates through a multisubunit proteolytic complex, termed the proteasome, and is characterized by specific targeting of proteins for destruction. In addition to its role in muscle protein turnover, the UbP pathway is involved in many other cellular processes including antigen presentation (Driscoll et al., 1993) and regulation of the cell cycle. Proteins targeted for destruction are ligated to a chain of ubiquitin molecules by a series of ubiquitin ligases (Glickman and Ciechanover, 2002). These ubiquitinated proteins are Comparative Biochemistry and Physiology, Part B 157 (2010) 364–373 ⁎ Corresponding author. Institute of Biological and Environmental Sciences, University of Aberdeen, Tillydrone Avenue, Aberdeen, AB24 2TZ, UK. Tel.: +44 1224 272867; fax: +44 1224 272396. E-mail address: sam.martin@abdn.ac.uk (S.A.M. Martin). 1096-4959/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpb.2010.08.004 Contents lists available at ScienceDirect Comparative Biochemistry and Physiology, Part B journal homepage: www.elsevier.com/locate/cbpb