A Proteomics Strategy for the Identification of FAT10-Modified Sites by Mass Spectrometry

Abstract

The ubiquitin-like protein FAT10 (HLA-F adjacent transcript 10) is uniquely expressed in mammals. The fat10 gene is encoded in the MHC class I locus in the human genome and is related to some specific processes, such as apoptosis, immune response, and cancer. However, biological knowledge of FAT10 is limited, owing to the lack of identification of its conjugates. FAT10 covalently modifies proteins in eukaryotes, but only a few substrates of FAT10 have been reported until now, and no FATylated sites have been identified. Here, we report the proteome-scale identification of FATylated proteins by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). We identified 175 proteins with high confidence as FATylated candidates. A total of 13 modified sites were identified for the first time by a modified search of the raw MS data. The modified sites were highly enriched with hydrophilic amino acids. Furthermore, the FATylation processes of hnRNP C2, PCNA, and PDIA3 were verified by a coimmunoprecipitation assay. We confirmed that most of the substrates were covalently attached to a FAT10 monomer. The functional distribution of the FAT10 targets suggests that FAT10 participates in various biological processes, such as translation, protein folding, RNA processing, and macromolecular complex assembly. These results should be very useful for investigating the biological functions of FAT10.

Extended lifespan and reduced adiposity in mice lacking the FAT10 gene

“…Evidence presented here for important roles of FAT10 in metabolic programming and lifespan determination extends and informs recent identification of numerous FAT10 target proteins and interacting partners with acknowledged roles in energy sensing, nutrient and bile acid metabolism, and insulin-, PI3K/ Akt/mTOR-, and cAMP-dependent signaling as well as NF-κB-dependent gene expression (10,13,14). Previous studies identified p53 and p62/sequestosome1 as FAT10 targets (7,53), and both target proteins are known to modulate energy metabolism, mitochondrial activity, adiposity, glucose-insulin homeostasis, cell stress, and aging (54,55).…”

“…Despite intense recent efforts to identify FAT10 substrates (10,13,14), physiological role(s) of FAT10 remain obscure. FAT10 mRNA is absent or expressed at very low levels in most normal tissues but constitutively expressed during lymphocyte and dendritic cell maturation in some cell culture lines derived from B cells and several types of neoplasms (15).…”

Knockdown of ubiquitin D inhibits adipogenesis during the differentiation of porcine intramuscular and subcutaneous preadipocytes

“…Systemic UBD‐deficient mice exhibited a distinct phenotype of delayed senility accompanied by global alterations in energy metabolism, adiposity, insulin sensitivity and inflammation . In recent years, evidence has indicated that a variety of UBD target proteins and interacting partners that are involved in insulin sensitivity and energy metabolism can be modulated by cAMP‐dependent signalling, PI3K/Akt/mTOR signalling and the NF‐κB pathway . UBD acts as an oncogene that regulates tumourigenesis and tumour metastasis through Akt/GSK3β signalling in hepatocarcinoma .…”

The ubiquitin-like modifier FAT10 in cancer development

“…A mass spectrometry analysis identified 571 putative FAT10 interaction partners, of which 176 proteins were classified as putative conjugation substrates and the remaining 395 proteins as non-covalent interaction partners. Interestingly, these proteins did not belong to distinct cellular pathways but covered a lot of different cellular functions, showing that FAT10 might play a role in numerous cell biological processes Leng et al, 2014). Of importance is that FAT10 apparently is not only functional when covalently conjugated to its substrate proteins, such as the ubiqui-tin activating enzyme UBE1 (Bialas et al, 2015;Rani et al, 2012), but also when interacting non-covalently with interaction partners such as HDAC6 (Kalveram et al, 2008), aryl-hydrocarbon receptor like-1 (AIPL1), an important protein within the retina (Bett et al, 2012), or the autophagosomal receptor p62/SQSTM1 which interacts both, covalently and non-covalently with FAT10 .…”