Adaptive evolution in two large families of ubiquitin-ligase adapters in nematodes and plants

Host genes encoding proteins directly involved in recognizing pathogens are expected to be subject to unusual patterns of molecular evolution, driven by an arms race with the pathogens. One expected pattern, typified by mammalian MHC genes, includes site-specific adaptive evolution (positive selection) and a high degree of population polymorphism (Hughes and Nei 1988, 1989; Hughes et al. 1990; Swanson et al. 2001). Positive selection is often detected by a rate of nonsynonymous codon change higher than synonymous codon change, a pattern the reverse of that produced by the more common purifying (negative) selection. Such positive selection in MHC proteins results in regions of rapidly evolving amino acid sequence that interact with foreign proteins, interspersed with regions of highly conserved amino acid sequence that form the structural core of the protein (Hughes and Nei 1988, 1989; Hughes et al. 1990). To identify genes that are candidates for pathogen interaction in Caenorhabditis elegans, I conducted a systematic test for positive selection. Lack of sufficient population sequence data and the absence of close sibling species eliminate two of the methods used to detect positive selection. However, recent paralogous gene duplicates can be analyzed for evidence of positive selection acting on the paralogs relative to each other (Thomas et al. 2005). To apply this method systematically, I clustered the entire gene complement of C. elegans to define 544 paralog groups and analyzed each paralog group for positive selection by the maximum-likelihood method of Yang and Nielsen (Yang 1997; Yang and Nielsen 2000). The most prominent novel gene classes identified in this search were the MATH-BTB family and the F-box superfamily (PFAM domains PF00917, PF00651, and PF00646, respectively). F-box and MATH-BTB proteins function as adapters that target substrate proteins for poly-ubiquitination and proteolysis. Ubiquitin-dependent protein degradation is initiated by the transfer of ubiquitin to substrate proteins by E3 ubiquitin ligases. Ubiquitinated substrate proteins are then targeted to the 26S proteasome for degradation (Moon et al. 2004; van den Heuvel 2004; Varshavsky 2005). Substrates for ubiquitination are recruited by large Cullin complexes (also called SCF complexes), which include the E3 ligase, regulatory subunits, a Cullin scaffold protein, and an adapter protein that binds specific substrate proteins. There are several distinct Cullin complexes, which differ primarily in the Cullin scaffold protein and adapter proteins (Fig. ​(Fig.1).1). Each specific Cullin protein uses a distinct class of adapter protein. Figure 1. Schematic of ubiquitin-targeting system. The top panel shows the SCF1 (Cullin1) complex, which uses Skp-related and F-box proteins as substrate adapters. The domain marked “FTH etc.” varies depending on the specific adapter. The bottom ... Several members of the F-box superfamily are known adapters for Cullin1 complexes (Bai et al. 1996; Winston et al. 1999; Zheng et al. 2002; Jin et al. 2004). The F-box domain binds to Cullin1 via Skp1-related (Skr) proteins (Bai et al. 1996; Zheng et al. 2002); diverse regions outside the F-box domain bind to specific substrate proteins (Winston et al. 1999; Hsiung et al. 2001; Brunson et al. 2005; Nayak et al. 2005). In these adapter proteins, the F-box is near the N terminus, and the remainder of the protein falls into several families, including kelch repeat, WD-40 repeat, LRR, FTH, FBA, FBA1, and FBA2 domain-containing families (http://www.sanger.ac.uk/Software/Pfam/2005) (Jiang and Struhl 1998; Ilyin et al. 1999; Winston et al. 1999; Clifford et al. 2000; Andrade et al. 2001; Gagne et al. 2002). Studies in this paper focus mostly on the two largest F-box families in C. elegans, the F-box-FTH and the F-box-FBA2 families. Very little is known about the FTH and the FBA2 domains; both are classified as sequence domains of unknown function that are present in large numbers of nematode proteins (http://www.sanger.ac.uk/Software/Pfam/2005). They have no known sequence relationship to each other or to any other known protein domain. Many BTB proteins are adapters for Cullin3 complexes (Furukawa et al. 2003; Pintard et al. 2003; Xu et al. 2003; Figueroa et al. 2005). The BTB domain binds directly to Cullin3, and other domains in the BTB protein confer substrate specificity. Like F-box proteins, BTB-containing adapters have a variety of substrate-binding domains, including MATH, WD-40 repeat, Zn-finger repeat, and kelch repeat domains (Winston et al. 1999; Pintard et al. 2003; Prag and Adams 2003; Xu et al. 2003; van den Heuvel 2004; Brunson et al. 2005; Figueroa et al. 2005; Stogios et al. 2005). In C. elegans, most of these adapters have a BTB domain near their C terminus and an N-terminal MATH domain, which is responsible for substrate binding (Pintard et al. 2003; Xu et al. 2003). Taken together, the ∼520 F-box and 50 MATH-BTB genes in C. elegans account for ∼2.5% of total coding potential. Given their number, remarkably little is known about these genes; for example, only a few have been identified in forward genetic screens, and the vast majority of the genes tested by RNAi have no observed phenotype (Kamath et al. 2003). Based on results presented in this paper, I propose that most of the genes function to target foreign proteins for degradation as part of the innate immune system.