Several cytosolic and nuclear protein aggregates are characterized and differ for example in composition, mobility and localization. Aggresomes and aggresome like inducible structures (ALIS) for example contain ubiquitinated, aggregated proteins, but they differ in several ways. The existence of ALIS was first described during maturation of dendritic cells (DCs) and they were therefore termed dendritic ALIS (DALIS) (Lelouard et al., 2002). However, ALIS can be observed in several cell types and are inducible in response to divers stress stimuli (Pankiv et al., 2007; Szeto et al., 2006). The formation of these structures is typically dependent on translation and induced by puromycin treatment, indicating that misfolded proteins, also known as defective ribosomal products (DRiPs) account for the majority of proteins stored in ALIS. Furthermore, ALIS are not localized to the MTOC and they are not static like aggresomes, although the actual way of movement is unclear, as they circulate within cells independently of microtubules and actin cytoskeleton (Lelouard et al., 2002;
Lelouard et al., 2004; Szeto et al., 2006). Interestingly, the regulation of (D)ALIS elimination is still a matter of debate and was likewise suggested to be proteasomal (Lelouard et al., 2002;
Seibenhener et al., 2004; Szeto et al., 2006) as well as autophagy dependent (Bjørkøy et al., 2005; Fujita et al., 2011; Pankiv et al., 2007; Szeto et al., 2006). The two major constituent of ALIS, NBR1 and p62, mediate the selective degradation of misfolded proteins by autophagy (Kirkin et al., 2009a; Pankiv et al., 2007). Their role in aggregate formation and protein clearance has been studied intensively leading to the following model proposed by Kirkin et al. (Kirkin et al., 2009b). Misfolded proteins are ubiquitinated and preferentially destroyed by the 26S proteasome. However, under stress conditions, which eventually increase the production of misfolded proteins, these soluble but potentially toxic oligomeric proteins accumulate and are polyubiquitinated. In a next step, these substrates, designated for degradation, are recognized by NBR1 and p62 and delivered to the forming autophagosome.
If the production of misfolded proteins further increases and degradation of these soluble complexes is incomplete, NBR1 and p62 oligomerize and form ALIS (Pankiv et al., 2007;
Szeto et al., 2006).
Histone deacetylase 6 (HDAC6) can bind dynein molecular motors, and is thereby involved in the microtubuli dependent transport of ubiquitin and FAT10 conjugated substrates to aggresomes (Kalveram et al., 2008; Kawaguchi et al., 2003). Additionally, it was likewise suggested to be involved in other stages of aggrephagy, like recruitment of autophagy components and the facilitation of autophagosome lysosome fusion (Figure 8) (Iwata et al., 2005; Lee et al., 2010). Besides direct substrate and dynein binding, the main function of HDAC6 in these processes is to ensure microtubule dynamics and F-actin assembly by its deacetylation activity. Interestingly, p62 and HDAC6 are likewise phosphorylated by casein kinase 2 (CK2). Both modifications have been shown to facilitate efficient clearance of ubiquitinated cargo in autophagosomes (Matsumoto et al., 2011; Watabe and Nakaki, 2011).
Of interest is the proposed function of DALIS as a source of MHC class I peptides via direct presentation. Philippe Pierre suggested that antigens in form of DRiPs are stored in DALIS and are subsequently degraded via the proteasome in matured DCs (Pierre, 2005). The main substrates for DALIS formation and subsequent proteasomal MHC class I peptide generation are DRiPs. This pool of proteins represents the major part of rapidly degraded proteins (RDPs) in contrast to fully translated and properly folded, long-lived proteins (Yewdell and Nicchitta, 2006). However, it has to be stressed that this model was previously challenged by Kenneth Rock and colleagues (Farfán-Arribas et al., 2012; Rock et al., 2014). They argue in favor of stable, slowly degraded proteins to represent a source for MHC class I peptides with equal efficiency than DRiPs.
Figure 8: The multiple roles of HDAC6 in proteostasis. HDAC6 is involved in several stages of aggrephagy. Aggregate formation is regulated via the direct interaction with dyneine motor protein complexes and ubiquitinated cargo. A further function of HDAC6 in this process is to ensure microtubule dynamics and F-actin assembly by its deacetylation activity. Prior to aggresome formation, HDAC6 also influences aggregation of proteasome substrates via its interaction with valosin-containing protein (VCP). In a later step of aggregation prone protein degradation HDAC6 facilitates the autophagosome lysosome fusion by recruitment of autophagy components. The image is adapted from (d’Ydewalle et al., 2012)
Aim of this study
FAT10 was shown to localize to aggresomes and to interact with p62. The aim of this study was to further investigate functional aspects of the covalent and non-covalent interaction between FAT10 and p62. Both interaction partners, HDAC6 and p62, are involved in aggregation of ubiquitinated proteins designated for proteasomal as well as autophagosomal degradation. The role of FAT10ylation in targeting substrates for proteasomal degradation was already described. However, the question arose, whether FAT10ylation might additionally mediate the targeting of substrates for autophagosomal elimination via binding to p62. Thereby the interesting question, whether FAT10 might be relevant for the immunological function of autophagy to eliminate cytosolic pathogens should be investigated.
Several aspects within the process of xenophagy are thereby of special interest. First of all, the question whether FAT10 is covalently or non-covalently associated with xenophagy substrates should be addressed. Furthermore, the functional relevance in vitro and in vivo should be investigated. Additionally, since p62 represents not only an autophagy adapter but is also involved in autophagosomal regulation, the potential impact of FAT10 on the p62 regulator function should be addressed.
In the second part, the recently proposed immunoproteasome function, that describes an increased proteolytic capacity compared to the standard proteasome, should be reinvestigated.
Therefore, two different aspects should be addressed. On the one hand, the polyubiquitin conjugate degradation and secondly, the elimination of cytosolic aggregates, both in dependence of the immunoproteasome subunit LMP7.
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