FAT10 (HLA-F locus adjacent transcript 10)

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Figure 1.8Ubiquitin-like protein three-dimensional structures. Structures of (A) Ubiquitin, (B) SUMO-1, (C) NEDD8, (D) ISG15, (E) FAT10 (model structure) and (F) Lys48-linked diubiquitin. The structures show similar folds like ubiquitin(Modified from Dye and Schulman, 2007 and Groettrup et al., 2008).

1.4.1.1 FAT10 (HLA-F locus adjacent transcript 10)

FAT10 was first identified as a gene located in the MHC class I region close to the HLA-F locus (HLA-Fan et al., 1996). It consists of two ubiquitin-like domains, and is hence also called di-ubiquitin or ubiquitin D, with an identity of 29% and 36% to its N- and C-terminal region, respectively (Bates et al., 1997). The lysine residues involved in the formation of polyubiquitin chains, K27, K33, K48 and K63, are conserved in both domains of FAT10. Initially, the mRNA of FAT10 was identified as highly expressed in mature dendritic cells and B cells (Bates et al., 1997) but later, it was found to be expressed in several organs, in particular, lymphatic organs like thymus, spleen and lymph nodes (Liu et al., 1999; Lukasiak et al., 2008). Thus, unlike ubiquitin, its expression is limited to certain organs of the immune system, however, it is inducible by proinflammatory cytokines, IFN-γ and TNF-α in almost every cell type (Raasi et al., 1999). FAT10 has a free diglycine motif at its C-terminus, which is important for its conjugation to target proteins (Chiu et al., 2007; Raasi et al., 2001).

 General Introduction  1.

Ubiquitin and FAT10 share the same E1 enzyme, UBA6, which can be transferred to different E2s (Chiu et al., 2007; Groettrup et al., 2008; Pelzer et al., 2007). Analogous to ubiquitin, FAT10 can target proteins for proteasomal degradation but, on the contrary, FAT10 itself is also degraded together with its substrate by the proteasome. Moreover, the degradation of FAT10 and its conjugates is independent of ubiquitin as implied by the instability of FAT10 lacking lysines (Hipp et al., 2005). In addition, the degradation of FAT10 could be accelerated in the presence of a non-covalent interacting UBL-UBA domain protein NUB1L (Hipp et al., 2004). Further studies revealed that all the three UBA domains of NUB1L bind FAT10, whereas NUB1L lacking the UBL domain does not interact with FAT10. In spite of this fact, NUB1L lacking UBA domains is capable of binding to the proteasome and accelerates the degradation of FAT10 (Schmidtke et al., 2006).

The degradation of FAT10 and its conjugates was also demonstrated in another study where purified 26S proteasome was shown to be able to degrade the model substrate FAT10-dihydrofolate reductase (DHFR) but only in the presence of NUB1L. This study revealed that mere binding of FAT10 to the proteasome is not sufficient for the degradation of this model substrate, in the NUB1L knockdown cells, in vivo (Schmidtke et al., 2009).

Several studies show that FAT10 might be involved in cancer. FAT10 interacts non-covalently with MAD2 (mitotic arrest deficient 2), a spindle checkpoint protein, and hence, may modulate the cell growth during B cell and dendritic cell development (Liu et al., 1999). Ovarian, breast and nasopharyngeal cancers are associated with reduced expression of MAD2 and colorectal and gastric cancers are associated with increased expression of MAD2. Another study reported the interaction of FAT10 with MAD2 during mitosis, which could reduce the efficiency of MAD2 to bind to the unattached kinetochore. Upon treatment with nocodazole, more FAT10-overexpressing cells escaped mitotic arrest and exhibited abnormal and multinuclear morphology compared to the parental cells. This suggested the role of FAT10 in mitosis dysfunction, resulting in aneuploidy and chromosomal instability which is commonly observed in several cancers but no direct evidence was demonstrated (Liu et al., 1999; Ren et al., 2006). FAT10 mRNA expression and promoter activity was significantly reduced in the presence of wild-type p53 (Zhang et al., 2006) but up-regulated by mutant p53 (Ji et al., 2009) and

 General Introduction  1.

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hence, it was proposed that FAT10 might be involved in tumorigenesis. FAT10 overexpression was determined as the epigenetic marker for liver neoplasia (Oliva et al., 2008). Other studies demonstrated the up-regulation of FAT10 in hepatocellular, gastrointestinal and gynecological tumors and it was postulated that FAT10 might be important in cancer development through the dysregulation of MAD2 (Lee et al., 2003;

Lim et al., 2006). However, further study identified that FAT10 has no transforming capability and the expression of FAT10 is regulated in such tumors due to the up-regulation of proinflammatory cytokines (Lukasiak et al., 2008). Recently, it was shown that FAT10 expression began at the serrated adenoma stage and continued in the villous and villotubular stages and the invasive adenocarcinoma stage (Qing et al., 2011).

Knockout of FAT10 in mice causes minimal phenotypic changes with no major pathological or histological differences. Lymphocytes from FAT10 deficient mice are susceptible to apoptosis and sensitive to even low doses of endotoxin, and hence may function as a cellular survival factor (Canaan et al., 2006). The enhanced expression of FAT10 resulted in apoptosis as demonstrated in the stable cell line expressing FAT10 and this is dependent of the caspase activity (Raasi et al., 2001). The down-regulation of FAT10 in renal tubular epithelial cells infected with human immunodeficiency virus, show reduced apoptosis (Ross et al., 2006). FAT10 suppression prevents Vpr-induced apoptosis in human and murine RTEC and these proteins interact non-covalently and co-localize to mitochondria (Snyder et al., 2009). However, it is still unknown whether FAT10-mediated apoptosis is harmful to the host or it is an adaptive response that limits the spread of virus in the host.

Some studies indicate the link of FAT10 to aggregates. FAT10 interacts and co-localizes with histone deacetylase 6 (HDAC6) under conditions of proteasome inhibition. FAT10 as well as a model conjugate, FAT10-GFP, localizes to the aggresome in a microtubule dependent manner, which suggests the possible role of HDAC6 in transporting FAT10 and its conjugates to aggresomes when the proteasomal degradation pathway is non-functional (Kalveram et al., 2008). FAT10 also co-localizes with LMP2 and LMP7 and its expression increases in liver cells forming Mallory-Denk bodies (characterized by accumulation and aggregation of ubiquitylated cytokeratins) (Bardag-Gorce et al., 2010;

Qing et al., 2011). Recently, two proteins have been identified as the covalent conjugates of FAT10: p53 (a role of FAT10 in regulating the transcriptional activity of p53) (Li et

 General Introduction