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.


References:

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.

Kinetoplastid DNA

It seems to be that time of the year in which we post about something to do with our posts, as these are the topics we are supposed to be reading about now. Therefore, a post on parasites. That’s where the connection with my project finishes, but anyway.

As you might know, tryponosomatids are organisms from the family Troponosomatidae, which include some pretty nasty unicellular parasites such as Trypanosoma brucei, causer for African sleeping disease, and Leishmania, causer of leishmaniasis. As other higher eukaryotes, these parasites are characterised by two genomes: a nuclear genome and a mitochondrion genome, 10-30% of all the cell DNA, situated in the kinetoplast (the name given to the mitochondrion in these organisms).

Fig.1 A- Schematic minicircle organization. B- in vivo network organization, seen sideways.

(Adapted from reference 1)

So far nothing extraordinary. Things get interesting, when one looks at the structure of the kinetoplast DNA (kDNA) in these species. The kDNA is constituted by thousands of small DNA circles, the minicircles, and a few dozen of large DNA circles, the maxicircles. These are interlocked, as in a chain mail of medieval armour. Each circle is interlocked with 3 other neighbouring circles. The minicircles and maxicircles are stretched into a disk-shaped structure, situated near to the flagellar basal body (Fig.1). The maxicircles are those more similar to normal mitochondrion genome. They include rRNAs and genes encoding proteins associated with the respiratory processes that takes place in this organelle. However, the maxicircles mRNA requires extensive editing, namely the introduction or deletion of uridylate. The minicircles encode the guide RNAs used as templates for this editing. Since there are many types of editing required, as well as maxicircles with different sequences, there is the need for the thousand minicircles with different sequences. Seems a bit of a wasteful system, but what do I know!

More interesting is to think of the replication process. The minicircles must stop being interlocked, replicated, interlocked again, and in the end originate two new networks of thousands of minicircles that separate into the two new daughter cells. Let us look at the different steps, following an individual minicircle (Fig.2):

Fig.2 kDNA replication model. (From reference 2)

1- A minicircle must first be released from the network into the so-called kinetoflagellar zone (KTZ), where several proteins involved in the process exist. Here the unidirectional replication of the circle occurs, although the details of this process are not well known.

2- The two daughter minicircles move to opposite antipodal sites of the circle, where there are two protein assemblies. Here a variety of reactions occur: the RNA primers are removed, the gaps between Okazaki fragments are filled by DNA pol, and nick as sealed by DNA ligase. An important characteristic, however, is that the minicircles maintain at least one gap in their structure. This is thought to be a self-check feature of this system, as a way to guarantee that no minicircle is replicated twice.

3- Minicircles are attached by topoisomerase II to the network periphery adjacent to the antipodal sites.

The way the minicircles are attached to the disk varies with the species of trypanosomatid. In T.brucei, for example, the minicircles accumulate at the network poles. In T.cruzi, and C.fasciculata, the minicircles seem to be uniformly attached around the periphery of the disk, creating a ring of new minicircles, of increased thickness. How this uniform attachment is done is not certain, but it is though that either the antipodal protein complexes or the disk itself must rotate! (Fig.3)

Fig.3 Replication in C.fasciculata. New minicircles have been labelled with fluorescent nucleotides. The arrangement of the new minicircles around the periphery of the disk is obvious (from reference 2).

4- Regardless of the method, new minicircles are attached to the disk, and the valence must increase to 4 to 6 attachments per minicircle, in order to ensure twice as many minicircles in the same space (the mitochondrion membrane has not doubled at this point). When the space increases, topoisomerase II ensures that valence returns to 3.

5- Finally, the nicks are repaired and the network splits into 2, although how exactly this division occurs is not well known. Most importantly, it is not known for sure how to guarantee that the two daughter networks have the exactly same minicircles. Considering their importance in maxicircle mRNA editing, the loss of one minicircle could have dramatic consequences.

There is much to be explained in this process, namely identify exactly how the minicircles are moved to the different antipodal sites, or the details of individual minicircle replication, but such understanding will require the identification of the proteins involved. Considering the complexity of the process, many must be involved, but only a few have been identified so far.

This is probably not life-changing research, but it is surely interesting. It is important to remind ourselves once in a while that though we learn the textbook pathways and processes, Nature seems to like using hundreds of alternatives. Just to make scientists lives harder, I assume!

Liu, B., Liu, Y., Motyka, S., Agbo, E., & Englund, P. (2005). Fellowship of the rings: the replication of kinetoplast DNA Trends in Parasitology, 21 (8), 363-369 DOI: 10.1016/j.pt.2005.06.008

(2) Smith D. and Parsons M. (1996). Molecular Biology of Parasitic Protozoa, Kinetoplast DNA: Structure and Replication, Oxford University Press, USA