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  • Predictions of enzyme topology for DGAT are more

    2021-02-27

    Predictions of enzyme topology for DGAT2 are more difficult since the DGAT2 polypeptide sequences are more diverse across species than those of DGAT1. The length and function of the hydrophilic N-terminus preceding the first hydrophobic segment varies with species – for example, segments in fungi are much larger than in higher plants and animals [79]. The removal of the N-terminus from DGAT2 of Saccharomyces cerevisiae leads to a substantial decrease in DGAT activity, but it is dispensable in the murine enzyme [80]. The cDNAs of mouse and human DGAT2 are predicted to encode proteins consisting of 388 and 387 amino acids, respectively, with a number of potential N-linked glycosylation and phosphorylation sites [42]. The protein sequence analysis of human DGAT2 (UniProt_Q96PD7) suggests that the protein has 34% hydrophobic amino Octopamine HCl mg residues (Fig. 2c and d) and the TMHMM model predicts a single TMD spanning amino acid residues 73 to 95 (Fig. 2f). However, this hydrophobic region (residues 73–112) could have enough residues to form two TMDs with a short connecting segment in the ER lumen or a hemi-loop embedding the protein in the ER membrane but not traversing it. The presence of two TMDs traversing the ER membrane or otherwise would orient both termini towards the cytosol. Experimental verification of the tertiary structures of the human DGAT proteins would be required to confirm the exact orientation of the protein termini and the number of TMDs. A detailed topological model for murine DGAT2 proposed by Stone et al. [81] and McFie et al. [[82], [83], [84]] describes such an arrangement (Fig. 2h). These authors have shown that the murine DGAT2 has a hydrophobic region near the N-terminus with two TMDs connected by a short luminal loop orienting both the N- and C-termini towards the cytosol and a C-terminus distal to the TMDs contributing to the bulk of the protein that bears two helical domains embedded in the lipid bilayer of the ER membrane - a membrane binding domain (MBD) [83] and an LD targeting domain [84]. The two TMDs (residues 93–115) function in retaining DGAT2 in the ER membrane. The first TMD (residues 66–88) serves as a signal sequence for targeting DGAT2 to the ER [77]. In addition, the first TMD contains a sequence FLVLGVAC (residues 80–87) which is thought to be a neutral lipid binding domain [85]. This domain may function in binding DAG, a substrate of DGAT2, owing to its proximity to the ER lumen, or it may transiently bind the TAG product of the reaction and facilitate its transfer to proteins that are involved in its trafficking [81]. It has been reported that DGAT2 contains a conserved sequence, FLXLXXXn (n is a non-polar residue and X is any amino acid except proline), in the putative first TMD, with exceptions in only a few species [68]. The FLXLXXXn sequence is vital to full enzyme function in either binding to or metabolizing neutral lipids [81,86]. The MBD (residues 156–199) in the C-terminus interacts with the ER membrane and contains the highly conserved sequence HPHG (residues 161–164) in all DGAT2 family members that is thought to be part of the active site vital to DGAT2 activity. Moreover, the tandem helices contained in the MBD function in facilitating the stable interaction of DGAT2 with lipid droplets for droplet expansion [84]. The LD targeting domain (residues 293–309) has been demonstrated to be necessary and sufficient to target DAGT2 to LDs [84]. This domain could exist as an amphipathic α-helix which is normally embedded in the ER membrane. As DGAT2 synthesizes TAG, lipids accumulate in the region of the ER membrane where the LD targeting domain is embedded, and nascent droplets form and bud off from the ER. The LD targeting region becomes incorporated into the droplet surface with the hydrophobic face embedded in the phospholipid monolayer of the droplets and the charged face towards the cytosol [84]. Consequently, DGAT2 forms a bridge between the ER and the LD, tethering the two organelles. This arrangement would allow the efficient channelling of TAG from DGAT2 as it is synthesized to LDs for their expansion. Despite this encouraging experimentally-elucidated membrane topology of DGAT2, it still only predicts the surface of the folded segments of the protein that interact with the membrane. The fully folded three-dimensional structure remains unclear. Structural studies would help our understanding of the mechanisms of action of DGAT2 enzyme.