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  • Studies have focused on a hydrophobic

    2019-07-10

    Studies have focused on a hydrophobic cage at the CULT domain, conserved from bacteria to mammals, as it binds to substrates and IMiDs (Chamberlain et al., 2014, Lupas et al., 2015). In addition, CRBN ligand-mediated protein degradation was highly conserved, which was confirmed in vertebrates by focusing on residues near the cage (Akuffo et al., 2018). However, divergence of the CRBN sequence around the cage has not been analyzed to determine if it contributes to functional Itraconazole weight in vertebrates. Since protein-protein interaction tends to occur on a large scale, adaptive evolution can also be expected on a broad area of the protein’s surface. To understand the fundamental function of CRBN, comparative analysis extended to the mammalian level is required, as current studies in mammals mainly focus only on humans or mice (Kim et al., 2016). Furthermore, mammalian evolution of CRBN is worth analyzing, as it is involved in cellular energy homeostasis and neuronal development, both exhibiting various patterns among mammals (Herculano-Houzel et al., 2014, Price and Hopkins, 2015). As CRBN is a multifunctional protein, it is also important to find out which function was under selective pressure in mammals. Revealing the link between mammalian evolution and molecular evolution of CRBN will help in understanding the central role of CRBN.
    Materials and methods
    Results
    Discussion Auto-ubiquitination of the E3 ubiquitin ligase is a self-catalyzed ubiquitination reaction observed in E3 ubiquitin ligases, including CRBN (de Bie and Ciechanover, 2011). It occurs when the substrate receptor subunit of CRL complexes cannot recognize and bind specific substrates. They, therefore, may operate in a negative feedback pathway to prevent excessive degradation. In the results, C366S CRBN expression increased ubiquitination levels as determined by immunoprecipitated HA-CRBN. This indicates more auto-ubiquitination. Thus, E3 ubiquitin ligase activity may also be elevated in C366S CRBN and possibly more generally mammals with the Ser366 variant. AMPK is one of the binding partners of CRBN, which controls homeostasis of metabolism by monitoring the AMP/ATP ratio level (Steinberg and Kemp, 2009). In the results, AMPK displayed enhanced binding with CRBN when cysteine 366 was mutated to serine. This result suggests that substitution of residue 366 resulted in enhanced binding of AMPK, since residue 366 is on the protein surface and can serve as a location for protein-protein interaction (Meitzler et al., 2013). After binding to CRBN, metabolic cascades initiated by AMPK are inhibited (Lee et al., 2011, Lee et al., 2013, Bavley et al., 2018, Sawamura et al., 2018). Therefore, mammals with serine at position 366 may control metabolism more acutely for its augmented interaction between AMPK and CRBN. Based on Kleiber’s Law, the basal metabolic rate scales to 3/4 power of the animal mass. Therefore, continuous and high-quality food intake is significant for small mammal energy maintenance (Kleiber, 1932). Cellular metabolism including biosynthesis and mitochondrial oxidation are one of the important factors of the basal metabolic rates and are reported to be regulated by the AMPK-mTOR cascade (Konarzewski and Ksiazek, 2013). As such, small mammals are likely to meet extra chances of activation of AMPK for its feeding behavior. One possibility is that serine 366 is preferred in relatively small sized mammals, such as rodents and chiroptera, selective pressure may have occurred to optimize CRBN’s interaction AMPK to adapt to intense AMPK fluctuation. However, large animals such as polar bears and giant pandas also have a serine at 366, suggesting the selective pressure on this residue is relatively weak in some species, regardless of how prone it is to pressure in other species for their functional requirement. It should be noted that the performed selection tests are site tests, therefore they show position 366 to be under positive selection, but do not support lineage specific selection. Further investigation is required, since the binding of CRBN and AMPK may change the binding role in distant species.