Ubiquitin is a highly conserved protein consisting of 76 amino acids which can be reversibly conjugated to lysine residues in a variety of target proteins. This post-translational modification is known to be involved in a number of key processes within cells, such as intracellular trafficking and the marking of proteins for degradation by the proteasome. A paper by Wagner et al. (2011) has recently used mass spectrometry to identify novel sites of ubiquitination within human embryonic kidney (HEK293T) and monocytic leukaemia (MV4-11) cells, and observed that both FLCN and FNIP1 appear to be ubiquitinated at specific lysine residues.
In more detail, proteins from total cell lysates were digested into peptide fragments using trypsin. After this digestion, ubiquitinated proteins contain a mark known as a di-glycine remnant, and this was used to enrich samples using a monoclonal di-glycine-specific antibody. These immuno-enriched samples were then fractionated and analysed using high-resolution mass spectrometry. It should be noted that the trypsin digestion of proteins modified by the ubiquitin-like conjugates NEDD8 and ISG15 also produce an identical di-glycine mark on modified lysines. However, NEDDylation and ISGylation are much less common than ubiquitination.
Using this protocol, the authors identified 11,054 ubiquitination sites on 4,273 human proteins in HEK293T cells (and the ubiquitination of 8 randomly selected proteins was confirmed by western blot). From the mass spectrometry data, it could be seen that FLCN was ubiquitinated at lysine 206 and 559, and that FNIP1 was ubiquitinated at lysine 161. Network analysis of the entire dataset showed that the ubiquitinated proteins were involved in a diverse range of cellular processes, such as DNA replication, DNA repair and receptor endocytosis. In particular, a number of proteins in the MAPK signalling pathway were ubiquitinated, such as AKT1 and mTOR. This signalling pathway is of particular interest as it has been suggested to play a role in the development of BHD syndrome (as illustrated by our signalling diagram).
Moreover, treating MV4-11 cells with the proteasome inhibitor MG-132 also led to changes in the ubiquitination levels of many proteins. For example, an increase in the levels of FNIP1 ubiquitination was observed. In contrast, approximately 40% of total ubiquitination sites did not show a substantial increase in ubiquitination, and approximately 15% saw a reduction after MG-132 treatment (with no data seen for FLCN whatsoever). These results further suggest that ubiquitin is involved in non-proteasomal regulatory pathways. However, it is unclear whether proteins that show an increase in ubiquitination are directly associated with proteasomal degradation.
In summary, this study identified endogenous putative ubiquitination sites in numerous human proteins, including FLCN and FNIP1. Von Hippel-Lindau is an example of a disease caused by a dysfunction in the ubiquitin system. Additionally, a recent paper by Guo et al. (2011) has linked clear cell renal cell carcinoma to a number of other genes involved with the ubiquitin-mediated proteolysis pathway (as discussed in this earlier blog post). Consequently, it would be interesting to discover what factors are involved in the ubiquitination of FLCN and whether this process is associated with renal tumourigenesis.
- Guo G, Gui Y, Gao S, Tang A, Hu X, Huang Y, Jia W, Li Z, He M, Sun L, Song P, Sun X, Zhao X, Yang S, Liang C, Wan S, Zhou F, Chen C, Zhu J, Li X, Jian M, Zhou L, Ye R, Huang P, Chen J, Jiang T, Liu X, Wang Y, Zou J, Jiang Z, Wu R, Wu S, Fan F, Zhang Z, Liu L, Yang R, Liu X, Wu H, Yin W, Zhao X, Liu Y, Peng H, Jiang B, Feng Q, Li C, Xie J, Lu J, Kristiansen K, Li Y, Zhang X, Li S, Wang J, Yang H, Cai Z, & Wang J (2011). Frequent mutations of genes encoding ubiquitin-mediated proteolysis pathway components in clear cell renal cell carcinoma. Nature genetics, 44 (1), 17-9 PMID: 22138691
- Wagner SA, Beli P, Weinert BT, Nielsen ML, Cox J, Mann M, Choudhary C. A proteome-wide, quantitative survey of in vivo ubiquitylation sites reveals widespread regulatory roles. Mol Cell Proteomics. 2011 Oct;10(10):M111.013284. PMID: 21890473
www.bhdsyndrome.org – the primary online resource for anyone interested in BHD Syndrome.