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  • Both the kK and kK mediated ubiquitination leads to rapid

    2021-02-20

    Both, the kK3 and kK5-mediated ubiquitination leads to rapid internalisation of target proteins followed by lysosomal degradation, similar to that seen for MARCH-1/8 [77]. Ubiquitination activity of kK3 and kK5 depends on the positioning of the targeted lysine (or cysteine) residues [22]. The positioning of the lysine residue on the substrate is in turn determined by the interaction site of the substrate with the respective E3 ligase. Although mK3 targets its substrates for proteasomal degradation, it is noteworthy that mK3-mediated ubiquitination seems to be independent of any secondary structure or the amino LDC000067 australia sequence in the cytosolic tail of an MHC-I heavy chain, as long as two lysine, serine or threonine residues are present at the C-terminus of an engineered glycine-rich tail [20]. This suggests that the cytosolic tail of the substrate is not involved in mK3-binding and in turn indicates that the interface in mK3 is located within the TM helices or the luminal L1-loop. However, it was also suggested that mK3 binds MHC-I heavy chains indirectly via heterodimer TAP-1 and TAP-2, peptide transporters that are part of the MHC-I peptide loading complex [78]. Experimentally validated interaction partners of membrane-embedded MARCHs are mostly membrane proteins themselves (Table 1). Interestingly, these MARCH-E3 substrates vary in their number of TM segments and the sizes of their cytosolic and luminal regions. It is therefore tempting to speculate that the MARCH TM helices fulfil two functions: to provide interfaces for both oligomerisation and substrate recognition. Thoroughly characterised substrates for human MARCH-1/8 are the MHC-II molecules, such as HLA-DRα1/β1, which become ubiquitinated on their ∼15 amino acid long cytosolic tails of the α- and β-chains [36], [76]. HLA-DRα/β are heterodimeric, mainly luminal proteins with one TM-helix each and a short cytosolic tail. In contrast, the E2-binding RINGv domains of MARCH-1/8 are located in the cytoplasm (Fig. 2). Thus, interaction between HLA-DRα/β and MARCH-1/8 can in principle be mediated by the TM regions, the luminal loop, and/or the cytosolic linker between RINGv and TM1 of MARCH-1/8. Formation of MHC-II heterodimers is mediated by GxxxG motifs in their transmembrane helices [79], [80]. The functional importance of the GxxxG mediated HLA-DRα/β interface was recently highlighted, when it was pointed out that the two GxxxG motifs in the TM region of HLA-DRα allow for two positions that HLA-DRβ can recognise with its single GxxxG motif, also located in the TM region. Importantly, the two different vertical positions of TM helices forming the HLA-DRα/β interface likely have long-range implications for binding of antigens and their presentation to T-cells [81], [82]. Interestingly, GxxxG motifs are also found in MARCH TM helices and might therefore also serve as recognition points for the MHC-molecules and other substrate proteins (Fig. 2). In soluble proteins, the GxxxG-motif is mainly found in helix-helix interactions and, in a few cases, also in helix-β-strand interactions [83]. Furthermore, in amyloid fibrils GxxxG motifs occur in parallel-stranded β-sheets forming grooves on the β-sheet surface [84]. Interestingly, GxxxG-motifs are most often found in TM helices of integral membrane proteins where they can mediate oligomerisation, intramolecular helix-helix interactions, and/or protein-protein interactions [85], [86], [87]. Although GxxxG is one of the most abundant identified motifs in TM helix-helix interactions, its presence per se is only a weak predictor for a physical interaction [88], [89]. The key to a specific interaction likely includes the sequence context of the GxxxG-motif [90], [91]. However, it is unclear whether the surrounding amino acid composition in such a structural motif can be used to predict involvement in dimerisation or protein-protein interactions [88]. However, a central placement of the GxxxG motif within a TM helix does result in a stronger dimerisation interface [92].