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  • br Acknowledgements The research leading to

    2020-07-25


    Acknowledgements The research leading to these results has received funding from the European Union Seventh Framework Programme (FP7-PEOPLE-2013-COFUND) under grant agreement no 609020 − Scientia Fellows (JB) and the Research Council of Norway (NFR-grant 221444) (JB, JPM, OB).
    Introduction Human leucine-rich repeat and sterile alpha motif-containing protein 1 (LRSAM1), which was previously known as TSG101-associated ligase (Tal), had been identified as an E3 ubiquitin ligase [1]. The predicted full-length LRSAM1 protein, which consists of 723 amino sitagliptin phosphate residues, contains a LRR domain, two coiled-coil (CC1 and CC2) domains, a SAM domain and a RING finger motif. Sequence alignments reveal that LRSAM1 does not share homology with any known E3 ubiquitin ligases. However, studies on activity show that LRSAM1 functions as an E3 ubiquitin ligase based on the fact that it contains a RING-finger motif and can ubiquitinate TSG101 in vitro [1]. Proper function of LRSAM1 is important for cell adhesion [2], self-ubiquitination [3], cell signaling and regulation of cargo sorting [1]. Mutations and deletion of the LRSAM1 gene are involved in axonal Charcot-Marie-Tooth (CMT) diseases [4], [5]. Recently, LRSAM1 was shown to regulate anti-bacterial autophagy, which plays a major role in innate immunity [6]. However, the mechanism of LRSAM1 action in this process still remains elusive. By coordinating 2 zinc ions, a RING-finger domain is one of the remarkable features of E3 ubiquitin ligases, serving as a platform to directly transfer ubiquitin to the substrate in the help of E2-conjugating enzymes. Because most RING E3 proteins facilitate their own auto-ubiquitination, it is a reasonable to define the minimal requirements for their E3 activities using in vitro auto-ubiquitination assays [7], [8]. In addition, LRSAM1 contains other conserved domains (LRR, CC and SAM) (Fig. S1). It is generally accepted that all these domains might functionally involve in regulating molecular interactions [9], [10], [11], [12], [13], [14]. For example, LRR domains were reported to target substrates through intermolecular interactions [6] and inhibit catalytic activities of E3 ligases through intramolecular interactions [14]. The CC domains have been widely implicated in the abilities of E3 ubiquitin ligases to form self-associated oligomers and regulate their activities [9], [12], [13]. The SAM domain is known to form oligomers and is also a protein interaction module for signal transduction [10]. The dimerization of RING domains is essential for E3 ligase activity in some E3 proteins [11]. However, it remains to be determined whether these domains contribute to the regulation of LRSAM1 E3 ligase activity.
    Materials and methods
    Results
    Discussion LRSAM1, a typical RING type E3 ligase, plays important roles in regulating cell functions. However, as one of the identified E3 ligase proteins related to anti-bacterial autophagy, the functionality of the LRSAM1 E3 ligase in vitro remains largely unknown [3], [6]. Parkin, another RING-type E3 ligase required for xenophagy [21], has been studied by a large number of groups to determine its structure [22], self-association [23], modification [23], [24], auto-inhibition [25], function [21], [23], [24], and intermolecular and intramolecular regulation [23], [24]. In contrast, little is known about LRSAM1. It has been shown that LRSAM1 requires the LRR domain to target the intracellular pathogens and the CC2 domain to interact with TSG101 [1], [6]. However, the functions of these domains and their intermolecular or intramolecular regulation are not well characterized. Assays in vitro and in vivo suggest that LRSAM1 likely assembles into oligomers. More importantly, the self-association may contribute to the intermolecular ubiquitination and provides a method to test ubiquitination sites. Site-specific auto-ubiquitination of E3 ligase is one of the critical mediators of functions and signaling processes [26]. In vitro assays describe a potential N-terminal mono-ubiquitination of LRSAM1. One literature [17] showed that it is the UBE2W has contributed to N-terminal mono-ubiquitination with the help of E3 rather than the other E2s. However, other research used UbcH5-type E2 enzymes as the E2 to ubiquitinate the N terminus of substrates [18]. The E2 used in our assays is UbcH5C, so whether UBE2W is the only E2 for this modification deserves to be discussed.