The Half Decent Pharmaceutical Chemistry Blog

Now that the major attraction of this blog, Saturday Night Synthesis, is over, I face the issue of coming up with something to make this blog worth-reading. A friend of mine has recently come up with a pretty intriguing idea: gossip.
Plainly, he believes I should as most of newspapers these days which, for example, show much more interest in Sarkozy’s love affairs rather than in his opinions on, say, illegal immigration. In fact, focusing my catchy introductions on popular topics would attract readers, so that I could eventually trap them in a labyrinth of scientific facts.

Well, let’s give it a try, by looking at one of favourite countries: France. Now, you all know Monsieur Sarko (who, if you are American, is the French president), 53, divorced from his second wife Cecilia, 50, and almost instantly turned to and got married with Italian, former super-model, Carla Bruni, 40. Can you find in your heart the force to blame him?

This switch might have inspired a French group when focusing their attention on a less glamorous switch not between models but heterochromatin proteins HP1β and HP1γ (doi:10.1038/embor.2008.1).
Despite not being as sexy as Carla Bruni, these histone modifiers are equally very interesting, as their role is far from being understood.
For many years, due to their HMTase activity (which stands for histone methyl transferase) and their huge presence at pericentromeric heterochromatin domains, they have been considered to act as pure silencers of gene expression. Recently, however, many findings has started undermining the basis of this assumption. HP1γ, for instance, has been frequently spotted at active euchromatic genes and a transcription-dependent recruitment has been proposed.

What the article mainly focuses on is the way these proteins act on HIV-1 5’ long terminal repeat, a well-known promoter of retroviruses. Interestingly, both NF-kB and protein kinase signal-transduction pathways stimulate this promoter and both phosphorylate epigenetic-regulation hot spot serine 10 on histone H3.
Obviously, I can’t (and don’t want to) tell you how these French reach their conclusions because that would mean breaking copyright rules. However, going back at the beginning story of Sarko’s love affairs, they postulate a switch between mainly transcription repressing HP1β/transcription stimulating HP1γ due to the aforementioned H3S10 phosphorylation, which they describe using the appealing expression Phospho-Switch. What this names underlines, though, is that not only is the phosphorylation the key aspect of this mechanism, but that it comprehensively overcomes the already existing tri-methylation of lysine 9 on histone H3 (to which HP1β binds), possibly altering HP1β binding site on histone 3 so much that it’s displaced and, after a brief moment while ChIP experiment shows nothing on the 5’-LTR, HP1γ is quickly recruited. Which is basically like displacing an old wife and quickly getting a younger, hotter new one, isn’t it?

In case psi*psi were reading this, happy birthday and, to already answer your question, no: there are no gels in this molecular biology affair.

Well, there’s a chance it’s having a hot threesome with camptothecin and RNA polymerase II (doi:10.1016/j.jmb.2005.12.069). So, it won’t be available when you have problems like this which, I believe, any one who regularly uses an MP3 player faces.

In fact, I now think about topoisomerases any time I have troubles with headphones or (long) wires, in general. For instance, everybody firmly believes to have neatly and carefully wound the earphones around the iPod and put it in your pocket: “God, I always do it four or five times a day: THIS time I’ve finally mastered it!” Sadly what will come out of your pocket is a very accurate model of coiled coil or positively supercoiled DNA duplex which, of course, would desperately needs a topoisomerase.

We all know about camptothecin and topoisomerases: the drug blocks the enzyme right when it cuts a DNA strand. This complex, though, can be reversed. Things worsen (meaning cell death) whether such a damage gets irreversible: this was once believed to be the result of the RNA Polymerase II crashing into the DNA-camptothecin-Topoisomerase complex

Things, however, seem to be more spectacular in vivo. We have to focus on what happens before (and independently from) any DNA-break, which implies a strong relationship between RNA Pol II (and, hence, transcription) and topoisomerases.

A first evidence is found looking at the difference in RNA Pol II distribution after treatment with α-amanitin (a well-known polymerase translocation inhibitor which acts binding directly to this enzyme)  and camptothecin (whose target is, on the other hand, topoisomerase): although both ultimately block transcription, chromatin-immunoprecipitation assays (ChIP) shows a spectacular reduction of the density of polymerases at transcribed regions with camptothecin. Something radically different from what occurs when α-amanitin is added.

Another interesting difference is the chromatin-structure: rather surprisingly, camptothecin enhances histone (H3 and H4) acetylation, which leads to opener chromatin and, generally speaking, means transcription enhancement.

This is aphidicolin, a DNA synthesis inhibitor.

Administering it to cells will stop their growth and, since no DNA duplication will be underway, no topoisomerase will be busy cutting DNA and camptothecin won’t be able to do any harm to the cells. Even so, however, RNA Pol II density was reduced in the way and with the same kinetics observed in the absence of aphidicolin, making absolutely clear that there’s no collision whatsoever behind the transcription block caused by camptothecin.

At this point you’re probably thinking: “Ok, but what if camptothecin directly acts on the polymerase?” Well, first  it has to be stressed all the assays were performed at a concentration of camptothecin lower than that known to lead to degradation of either polymerase or topoisomerase and then, thanks to immunostaining and fluorescence microscopy, no difference with the control test appeared on RNAPolII nuclear foci.

What’s more, camptothecin increases the phosphorylation of polymerases (RNA Pol IIo is the form that carries on the elongation phase during transcription).
This means that, when topoisomerases are inhibited by camptothecin, RNA Pol II density drops as the enzymes are somehow “diluted” over the whole length of the gene (as proved through ChIP) and not highly packed at the 5’-end any more, as the transition (phosphorylation) between RNA Pol IIa and RNA Pol IIo is enhanced by the drug.

This last hypothesis proves to be right when a TFIIH (the enzyme which phosphorylates our mighty polymerase) inhibitor is added and this removes any camptothecin-related effect on the density of RNA polymerases II.

Therefore, at least topoisomerase I-B (the enzyme studied) plays a role in transcription partly through its effects on RNA Pol II, in particular influencing a key step such as promoter clearance.

Back in the seventies, biologist Maurice Sussman must have been pretty fond of that pretty famous British band, The Beatles, to postulate a ”ticket-to-ride” hypothesis to explain the difference in length of the 3’-end between nuclear and cytoplasmic mRNAs.

Although this theory was proved wrong, I’ve decided to dedicate a post to it on my blog for two excellent reasons: first, because a group such as the Beatles deserves at least to be mentioned on my site. Then, because I like Sussman’s hypothesis very much: it's a bright example of creativity and taste for good music. And I think this is all that counts, after all.

Now, the back (3’-end) of a mature mRNA looks pretty much like this picture I drew in a coffee break: with a tail of approximately 250 adenines.

Once this mature transcript exits the nucleus, it gets translated. Then, the transcript remains in the cytoplasm and, little by little, while still ready for further cycles of translation, its tail starts to be shortened by something. Once this structure is entirely gone, the mRNA approaches the conclusion of its task.

Five years after the release of  “Ticket to Ride”, Sussman came up with this: once the yellow submarine…oh, sorry, mRNA has left the nucleus, its polyadenylated 3’-end serves as a (multiple) ticket for being translated: at every translation the ticket is punched, by cutting off a bit of adenines.
So, after a certain amount of cycles, the mRNA hasn’t got any more tickets and has to “get off”.

Sadly, this is not true, as researchers subsequently proved by measuring the rate of shortening of poly(A)s when a translation inhibitor, Emetine, was added: there’s almost no difference with the normal rate, assayed with translation underway, so, there’s no relationship between translating and shortening.

Moreover, after this early work, it became clear that the poly(A) tail isn’t just cut off: instead, a proper turn over is in place, with a cytoplasmic version of the PAP (Poly(A) polymerase) that adds a bit of adenines, whereas RNases degrade this structure.

This mechanism has its limits: after a certain amount of time, in fact, the polymerase succumbs to the efficient RNase, which works faster and, for each cycle, removes more adenines than those the cytoplasmic polymerase can add.

Still, you’ll agree with me that the ticket theory remains fascinating, won’t you?

So, yesterday I finished my life as undergraduate: at least lectures are now over and, to state the obvious, that makes me feel really good.

To conclude the Camptothecin Week, I think it’s finally the time to briefly outline some of the feature of the target of this drug: Topoisomerases.

Not only is this the first time I talk about molecular biology (although somehow indirectly), but this particular enzyme deserve to be mentioned here, since the group where I’m going to work at my graduation thesis, next year, focus their research on this class of enzymes.

Moreover, they have recently got an article about Camptothecin and eukaryotic topoisomerase I published.

Although the use of Camptothecin has to do with the eukaryotic form, the best way to describe the role of this protein is to look at prokaryotes.
In fact,  it’s not that complicated to understand the importance of an enzyme which relaxes the strain in a circular DNA...

As such DNA begins to be duplicated, the replication fork moves forward, another part of the circle predictably coils. As a result, as replication goes on, it gets harder for this procedure to continue, facing a tight, positive supercoil.

To relieve such an intolerable strain (or, in other words, yield negative supercoils), topoisomerases are needed: they cut one (type I) or both (type II topoisomerases) strands of the DNA.

This, however, is only half of the story the nick provides a sort of open gate, which allows an another, unchanged strand to pass through, relaxing the overall structure.

Predictably all this amazingness doesn’t come cheap: there’s a price to pay, in terms of ATP. Still, less than what you may be tempted to say by looking at the picture above.

There’s a tyrosine in the active site of the enzyme which reacts directly with phosphodiester bonds: so, this intermediate guarantees that most of the energy is conserved and ready to power the last step (the reunion of the strand).

The existence of the intermediate DNA-tyrosine not only has been proved, but it is also where Camptothecin plays its function, stabilizing the adduct so much that the pathways can’t reach the last step.
It must be stressed, anyhow, that topoisomerases are involved in transcription and repair too, especially in eukaryotes.