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  • Autophagy is a degradation pathway existing in

    2020-11-20

    Autophagy is a degradation pathway existing in all eukaryotic organisms. It is the main system for the degradation of bulk cytoplasmic components in cells (Feng et al., 2014), but it also plays a crucial role in other processes, as immunity (Wileman, 2013), mitochondrial (Dengjel and Abeliovich, 2014) and peroxisomal (Till et al., 2012) degradation and clearance of misfolded protein HKI-272 (Lamark and Johansen, 2012). These pathways of selective autophagy, in contrast to the more or less random sequestration of cytosol components produced by the bulk autophagy, are regulated by the participation of specific adaptors. The first protein reported to have this adaptor function was p62 (also named sequestosome-1, SQSTM1) (Pankiv et al., 2007), which, in addition to its implication in cell growth and proliferation, was detected in ubiquitinated protein aggregates (Moscat and Diaz-Meco, 2009). p62 possesses, among other interacting motifs, a C-terminal ubiquitin-binding domain (UBA) which allows its interaction with ubiquitinated substrates [containing mainly K63-linked polyubiquitin chains (Seibenhener et al., 2004)] and a short LIR (LC3-interacting region) sequence (Ciani et al., 2003) which allows its interaction with LC3 (Pankiv et al., 2007), a protein anchored in the autophagosomal membrane (Kirisako et al., 1999). In addition, p62 contains an N-terminal PB1 domain that allows homopolymerization of the protein (Johansen and Lamark, 2011). It has been reported that once p62 binds to ubiquitinated proteins, it oligomerizes serving these oligomers as a nucleating factor for autophagosome formation (Lippai and Low, 2014). In this work, we aimed to deepen our knowledge on the functional regulation of the malin–laforin complex. First, we focused our attention on the E2-conjugase that participates in the ubiquitination reaction. We found that the ubiquitin conjugating enzyme E2-N (UBE2N; Ubc13), one of the more than 38 different E2 conjugating enzymes which are encoded in the human genome (Ye and Rape, 2009), interacts with the malin–laforin complex and modulates its function. Second, we have also found a physical and functional interaction between p62 and the malin–laforin complex, highlighting the relationship between this complex and autophagy.
    Materials and methods
    Results
    Discussion Pathophysiology of LD involves alterations in many cellular processes, among which glycogen metabolism and protein homeostasis (proteostasis) mechanisms seem to play a central role. In the last years, growing evidence of numerous and particularly diverse molecular pathways such as the unfolded protein and heat-shock responses (Sengupta et al., 2011), antioxidant defense systems (Romá-Mateo et al., 2015a) and autophagy (Aguado et al., 2010, Criado et al., 2012) have been reported to be altered as a consequence of mutations in either malin and laforin, the two proteins related to the disease. Most of these alterations correlate with a dysfunction of the E3-ubiquitin ligase complex formed by malin and laforin; however, there is scarce evidence reporting the specific molecular determinants that participate in the ubiquitination reaction mediated by the complex and how it takes place. Ubiquitination is a versatile modification that can lead substrates to a wide variety of destinations. Knowing the modulators that participate in the malin–laforin mediated ubiquitination will allow us to better understand the multiple physiological roles of these proteins, providing novel insights and deeper knowledge of the pathophysiology of LD as well as widening our understanding of the complex interactions that drive specific intracellular signaling pathways. In the present work, we have identified novel partners that affect the E3-ubiquitin ligase activity of the malin–laforin complex. First, we have studied which E2-conjugase interacts with the complex. Since there are more than 38 genes encoding for E2 enzymes in humans, and our group previously found evidence showing that the malin–laforin complex incorporates K63-linked ubiquitin chains (Moreno et al., 2010, Romá-Mateo et al., 2011, Rubio-Villena et al., 2013), we centered our study in UBE2N, the only E2 with a known specificity for the incorporation of these type of chains (Eddins et al., 2006, Petroski et al., 2007). Two complementary approaches were performed to assess the relevance of UBE2N on the malin–laforin complex mediated ubiquitination. First, it is known that mutation of the catalytic cysteine of an E2 enzyme to alanine provides a stable form which can no longer bind ubiquitin, but its overexpression has a dominant negative effect, impeding endogenous functional E2s to bind their putative E3s and transfer the ubiquitin. This approach has been successfully employed to study the interaction of UBE2N with other E3s (Slotman et al., 2012), and for other E2 enzymes such as UBE2D2 (Gonen et al., 1999). Our results show that overexpression of the catalytically inactive version of UBE2N (UBE2N-C87A) drastically impairs the ubiquitination of different substrates by the malin–laforin complex. However, taking into account that the overexpression of this dominant negative mutant could affect the whole cellular ubiquitination machinery, we performed a complementary approach based on the specific silencing of the expression of the endogenous UBE2N. Again, when HEK293 cells were silenced with specific siRNA against UBE2N, the activity of the malin–laforin complex was diminished. Therefore, both approaches confirmed the participation of UBE2N in the malin–laforin complex mediated ubiquitination of different substrates.