Tuesday, 7 April 2009
When a bit more fat is not that bad
Nothing is as simple as it looks in Biology. We can’t even rely on gene or protein sequences to tell us what is going to happen. At the chromosomal level, the new field of epigenetics is showing how the nucleotide sequence does not define everything. At protein level we have to consider post-translational modifications. My final year project, now at the final write up stages, concentrated on an aspect of Trypanosoma brucei molecular biology which I very pompously described as a potential drug target and the cure for African sleeping disease. This bit was probably ‘paper talk’ though. In any case, the project focused on protein palmitoylation.
Palmitoylation is part of a group of several lipid modifications that can occur in proteins. Examples include N-myristoylation, prenylation and GPI-anchors. N-myristoylation is the reason why I thought it was not pushing it to much to say that we can kill parasites by targeting these modifications. N-myristoylation corresponds to the co-translational addition of myristate to an N-terminus glycine, and is catalyzed by N-myristoyltransferases (NMT). It so happens that NMTs were shown to be essential for parasite viability, and several antifungal NMT inhibitors apparently work on reducing NMT activity in T.brucei as well(1).
But back to palmitoylation, the post-translational addition of palmitic acid to a cysteine residue (reviewed in 2 and 3). It can occur in any place in the protein, and so far no consensus palmitoylation motif has been identified. Often it occurs in an N-terminus cysteine, just next to a myristoylation site. These proteins are said to be dually acylated. Palmitoylation can also happen in close proximity to prenylation sites. Palmitoylation can perform many functions in the cell, the most obvious being protein tethering to membranes and cellular localization. It has also been shown to interfere with protein-protein interactions and even with protein degradation. For example, the yeast SNARE Tgl1, when is not palmitoylated, can interact with the ubiquitin ligase Tul1(4).
Those paying attention (or still reading) will think that it is a bit strange that palmitoylation can regulate protein degradation like that. In fact, most of these lipid modifications, N-myristoylation for example, last for the life time of the protein. Well, palmitoylation is characterized by a unique feature: it is reversible. This means that it can determine processes that other lipidations cannot. Dynamic trafficking of proteins of proteins, for example. This is the case of Ras protein, which through an deacylation/reacylation cycle is able to exchange between the plasma membrane and the Golgi apparatus (5). Reversibility can also be important for signalling. Regulator of G-protein signalling 2 (RGS2) was shown to increase or decrease its GTPase-activating ability depending on which of its cysteine residues are palmitoylated(6).
The process of palmitoylation in itself is catalyzed by the enzymes palmitoyl-acyltransferases (PATs). These were pretty hard to identify, apparently. Not only they were difficult to purify, but the reaction itself can occur spontaneously with biological significance(7), which made many sceptic on whether these enzymes existed at all. However, they were identified, first in yeast, now in mammals and in my dear T.brucei.
And what was supposed to be a short post if already long enough. I finish by adding that my project was about finding proteins that were palmitoylated and that would change their localization in the absence of this modification, in an attempt to identify substrates for RNAi PAT studies in T.brucei. I must say that the project was not particularly successful, but I developed the important skill of writing long discussions based on few results, which I bet will come handy in the future.
I now challenge Mel to continue in this post-translational modification topic and tell us about his project on glycosylation.
1. Price, H. (2002). Myristoyl-CoA:Protein N-Myristoyltransferase, an Essential Enzyme and Potential Drug Target in Kinetoplastid Parasites Journal of Biological Chemistry, 278 (9), 7206-7214 DOI: 10.1074/jbc.M211391200
2. Nadolski MJ, Linder ME. Protein lipidation. FEBS J 2007;274:5202-10.
3. Greaves J, Chamberlain LH. Palmitoylation-dependent protein sorting. J Cell Biol 2007;176:249-54.
4. Valdez-Taubas J, Pelham H. Swf1-dependent palmitoylation of the SNARE Tlg1 prevents its ubiquitination and degradation. EMBO J 2005;24:2524-32.
5. Rocks O, Peyker A, Kahms M, et al. An acylation cycle regulates localization and activity of palmitoylated Ras isoforms. Science 2005;307:1746-52.
6. Ni J, Qu L, Yang H, et al. Palmitoylation and its effect on the GTPase-activating activity and conformation of RGS2. Int J Biochem Cell Biol 2006;38:2209-18.
7. Kummel D, Heinemann U, Veit M. Unique self-palmitoylation activity of the transport protein particle component Bet3: a mechanism required for protein stability. Proc Natl Acad Sci U S A 2006;103:12701-6.