FAT10 is a relatively new member of the ubiquitin-like protein family, since it is only expressed in mammals. FAT10 consists of two ubiquitin-like domains arranged in tandem and bears the typical di-glycine motif in its C-terminus, which is necessary for the conjugation to so far unidentified target proteins. The fat10 gene is located in the major histocompatibility complex class I (MHC I), adjacent to other genes that play a role in the immune system. Its expression was originally found to be restricted to mature B-cells and mature dendritic cells (DCs) but it has been shown that it is synergistically inducible with the proinflammatory cytokines TNF-a and IFN-g; in almost all tissues. One study reported that the fat10 promotor was negatively regulated by p53. Over expression of wild type FAT10 in some human cell lines and also from mice led to the induction of caspase-dependent apoptosis. This was not the case when a di-glycine mutant (deltaGG) was used instead.
A recent study reported that FAT10 is highly over expressed in 90% of human Hepatocellular carcinomas (HCC) and also in other gastrointestinal and gynaecological carcinoma (Lee et al. 2003). With the finding of a different study that FAT10 non-covalently interacted with the spindle assembly checkpoint protein MAD2 that prevents premature entry into anaphase, the authors concluded that FAT10 may play a role in tumour development.
One of the main aims of this thesis was to re-evaluate this startling finding more thoroughly with more HCC samples and with the use of quantitative RT-PCR since the antecedent study measured FAT10 mRNA levels using semi-quantitative northern blots.
A SYBR Green I based assay was designed and validated on cDNA samples from seven common laboratory cell lines, including cell lines derived from HCC. Experiments in this thesis show that FAT10 was up regulated by an IFN-g and TNF-a treatment in all cell lines, suggesting that no cell line, including the ones derived from HCC, has lost its responsiveness to both cytokines. Furthermore, FAT10 mRNA levels were determined in 51 tissue samples from patients with HCC and in 15 samples derived from Colon carcinoma (CC). Additionally, levels of LMP2 mRNA, an IFN-g and TNF-a inducible proteasome subunit, were measured in all samples. This thesis shows that significant up regulation of FAT10 was found only in 37/51 (72%) HCC samples and 8/15 (53%) CC samples. Moreover, the expression of FAT10 correlated in both cancer types in most cases with the expression of LMP2, suggesting that an ongoing immune response against the cancerous tissue is responsible for the up regulation of FAT10. A soft agar assay also showed that FAT10 on its own has no transforming capabilities. Therefore, this thesis shows evidence that FAT10 qualifies as a marker for an ongoing immune response, mediated by proinflammatory cytokines, in HCC and in CC.
The second aim of this thesis was to find further enzymes involved in the fatylation cascade, since only the E1 enzyme for FAT10, Uba6 has been identified so far and furthermore, to find substrates that are modified with FAT10.
The E2 enzyme USE1 (Uba6-specific E2 enzyme 1) was identified as a new interaction partner in a yeast two-hybrid screen, using a human thymus cDNA library. Furthermore, this thesis shows that USE1 is an E2 enzyme for FAT10, since FAT10 was covalently linked to USE1 in vitro and in vivo. The interaction was not present when a catalytic inactive USE1 was used instead. This covalent conjugate could be reduced with Beta-mercaptoethanol, suggesting a thioester linkage. SiRNA mediated knock-down of USE1 strongly reduced FAT10 conjugate formation in a cell line stably transfected with Flag-FAT10. Subsequent experiments also show that USE1 itself is the first physiological substrate of fatylation, since it still formed a conjugate under reducing conditions. USE1 can be fatylated in cis but not in trans and co-expression of NUB1L, a linker protein that facilitates the degradation of FAT10 and its conjugates, lead to proteolytic down regulation of USE1. These results suggest that USE1 can auto modify itself with FAT10 thereby negatively regulating the FAT10 conjugation pathway.

FAT10 is a relatively new member of the ubiquitin-like protein family, since it is only expressed in mammals. FAT10 consists of two ubiquitin-like domains arranged in tandem and bears the typical di-glycine motif in its C-terminus, which is necessary for the conjugation to so far unidentified target proteins. The fat10 gene is located in the major histocompatibility complex class I (MHC I), adjacent to other genes that play a role in the immune system. Its expression was originally found to be restricted to mature B-cells and mature dendritic cells (DCs) but it has been shown that it is synergistically inducible with the proinflammatory cytokines TNF-a and IFN-g; in almost all tissues. One study reported that the fat10 promotor was negatively regulated by p53. Over expression of wild type FAT10 in some human cell lines and also from mice led to the induction of caspase-dependent apoptosis. This was not the case when a di-glycine mutant (deltaGG) was used instead.
A recent study reported that FAT10 is highly over expressed in 90% of human Hepatocellular carcinomas (HCC) and also in other gastrointestinal and gynaecological carcinoma (Lee et al. 2003). With the finding of a different study that FAT10 non-covalently interacted with the spindle assembly checkpoint protein MAD2 that prevents premature entry into anaphase, the authors concluded that FAT10 may play a role in tumour development.
One of the main aims of this thesis was to re-evaluate this startling finding more thoroughly with more HCC samples and with the use of quantitative RT-PCR since the antecedent study measured FAT10 mRNA levels using semi-quantitative northern blots.
A SYBR Green I based assay was designed and validated on cDNA samples from seven common laboratory cell lines, including cell lines derived from HCC. Experiments in this thesis show that FAT10 was up regulated by an IFN-g and TNF-a treatment in all cell lines, suggesting that no cell line, including the ones derived from HCC, has lost its responsiveness to both cytokines. Furthermore, FAT10 mRNA levels were determined in 51 tissue samples from patients with HCC and in 15 samples derived from Colon carcinoma (CC). Additionally, levels of LMP2 mRNA, an IFN-g and TNF-a inducible proteasome subunit, were measured in all samples. This thesis shows that significant up regulation of FAT10 was found only in 37/51 (72%) HCC samples and 8/15 (53%) CC samples. Moreover, the expression of FAT10 correlated in both cancer types in most cases with the expression of LMP2, suggesting that an ongoing immune response against the cancerous tissue is responsible for the up regulation of FAT10. A soft agar assay also showed that FAT10 on its own has no transforming capabilities. Therefore, this thesis shows evidence that FAT10 qualifies as a marker for an ongoing immune response, mediated by proinflammatory cytokines, in HCC and in CC.
The second aim of this thesis was to find further enzymes involved in the fatylation cascade, since only the E1 enzyme for FAT10, Uba6 has been identified so far and furthermore, to find substrates that are modified with FAT10.
The E2 enzyme USE1 (Uba6-specific E2 enzyme 1) was identified as a new interaction partner in a yeast two-hybrid screen, using a human thymus cDNA library. Furthermore, this thesis shows that USE1 is an E2 enzyme for FAT10, since FAT10 was covalently linked to USE1 in vitro and in vivo. The interaction was not present when a catalytic inactive USE1 was used instead. This covalent conjugate could be reduced with Beta-mercaptoethanol, suggesting a thioester linkage. SiRNA mediated knock-down of USE1 strongly reduced FAT10 conjugate formation in a cell line stably transfected with Flag-FAT10. Subsequent experiments also show that USE1 itself is the first physiological substrate of fatylation, since it still formed a conjugate under reducing conditions. USE1 can be fatylated in cis but not in trans and co-expression of NUB1L, a linker protein that facilitates the degradation of FAT10 and its conjugates, lead to proteolytic down regulation of USE1. These results suggest that USE1 can auto modify itself with FAT10 thereby negatively regulating the FAT10 conjugation pathway.