Moonlighting molecules

Just when you thought you knew what your protein did, it goes off and does something completely different (and unexpected). This is called moonlighting, and is yet another reason drug discovery is hard. You can never be sure that your target is doing only what you think it’s doing.

Today’s example is PACAP, a neuromodulator/neurotransmitter made by neurons. Who knew that PACAP can and does act as an antibiotic when the brain is infected. [ Proc. Natl. Acad. Sci. vol. 118 e1917623117 ’21 ] does (PNAS no longer pages its journals, as last year’s total was over 33,000 !).   PACAP is a member of the vasoactive intestinal polypeptide, secretin, glucagon family of neuropeptides (mammals have over 100 neuropeptides according to the paper).

PACAP stands for Pituitary Adenylate Cyclase Activating Polypeptide. It comes in two forms containing 27 or 38 amino acids, both cleaved from a 176 amino acid precursor. There are 3 receptors for PACAP, all G Protein Coupled Receptors (GPCRs). A zillion functions have been ascribed to it, setting the circadian clock, protecting granule cells of the cerebellum. Outside the nervous system it is produced by immune cells in response to inflammatory conditions and antigenic stimulation. It is one of the most conserved neuropeptides throughout the course of evolution. Now we probably know why.

Showing how hard protein chemistry really is, PACAP is structurally similar to cathelicidin LL-37 an antimicrobial peptide, despite having less than 5% amino acid sequences in common. PACAP is cationic. Different sides of the protein have different characteristics, with one side being highly positively charged, and the other being hydrophobic (e.g. the protein is amphipathic). This is typical of antimicrobial peptides, and perturbation of microbial membranes by inducing negative Gaussian curvature probably explains its antibacterial activity.

In mouse models of Staph Aureus or Candida infections, PACAP is induced ‘up to’ 50 fold in the brain (or spleen or kidney) where it kills the bugs. Yet another reason drug discovery is so hard. We are mucking about in a system we barely understand.

There are many other examples of moonlighting proteins. Probably the best known is cytochrome c which is is a heme protein localized in the compartment between the inner and outer mitochondrial membranes where it functions to transfer electrons between complex III and complex IV of the respiratory chain. Oxidation and reduction of the iron atom in the heme along with movement along the mitochondrial intermembrane space allows it to schlep electrons between complexes of the respiratory chain.

All well and good. But cytochrome c also can tell a cell to commit suicide (apoptosis) when mitochondria are sufficiently damaged that cytochrome c can escape the intermembrane space. Who’d a thunk it?

How many more players are there in the cell (whose function we think we know) that are sneaking around — doing more things in heaven and Earth, Horatio, than are dreamt of in your philosophy?

The Dr. Jekyll and Mr. Hyde protein

If there ever was a protein with a Dr. Jekyll and Mr. Hyde character (https://en.wikipedia.org/wiki/Dr._Jekyll_and_Mr._Hyde_(character))it is EZH2, a protein whose function we thought we knew.

EZH2 is an enzyme which puts methyl groups on lysine #27 of histone H3 (forming H3K27Me3).     So as good chemists that tells you that it removes the positive charge usually found on the amine group lysine at physiological pH.  But chemistry is helpless here unless you know what histone H3 does.  Addendum 27 July — clearly I’m not a good chemist when I write late at night.  Ashutosh points out that the positive charge on nitrogen at physiologic pH remains even when one, two or all the hydrogens are replaced by methyl groups.  Acetylation of the amino group of lysine removes the positive charge.  The methylation of lysine #27 is just one part of the histone code allowing other proteins to specifically bind here. As of 2013 some 130 different post-translational histone modifications were known.  

The DNA in each of our cells is just over a yard long.  To fit inside it must be compacted down.  4 different histone proteins get together to form an octomer around which DNA wraps in nearly two complete turns, compacting DNA down by a factor of ten.  The details of the further compaction have been studied for 50 years and are still under debate.

You’d think that the methylation of lysine #27 and loss of the positive charge would make it less likely to bind to the negative phosphates of the DNA chain.  This should free up the DNA so it can be transcribed to RNA and make proteins.

That’s not at all what happens. There are other proteins which bind H3K27Me3 and compact the DNA down so it becomes inactive (unable to be transcribed into mRNA).

Well that was the state of play until Proc. Natl. Acad. Sci. vol. 117 pp. 16992 – 17002 ’20.  So much for Dr. Jekyll.

Some forms of cancer activate kinase enzymes (AKT, JAK3) which place phosphates on serine #21 or tyrosine #244 of EZH2 respectively.   This causes a giant structural arrangement of amino acids #135  to #195.  Now the protein interacts with another enzyme p300 (a histone acetyl transferase) whose net effect is to UNcompact DNA and activate gene transcription.  Even worse, the transcription products of the genes help the cancer along.  Definitely Mr. Hyde.

This is a radically different function for EZH2 and you have to wonder how many other proteins lead double lives like EZH2.

The classic examples of a huge structural shift in a protein complex are the spike proteins of viruses (notably SARS-CoV-2), which unfold to form a needle piercing a cell allowing injection of the viral genetic material. Here are some nice pictures of the fusion protein of influenza virus in action — http://faculty.washington.edu/kklee/Influenza_SAXS.html.  But this is structural change in pursuit of a known physiological effect.

The double life of EZH2 is remarkable.   Some proteins have more than one effect in the cell — this is called moonlighting.  One example is cytochrome c, which is normally found in the intermembrane space of the mitochondrion where it is involved in electron transport.  When it is released into the cell cytoplasm due to mitochondrial damage, the cell quietly kills itself (apoptosis) — definitely a moonlighting function.  But the structure of cytochrome c doesn’t change to accomplish this, just its location.

Fascinating stuff, and the paper should be read to see just how profound the shift in structure that EZH2 undergoes actually is.

This is just a small window into the intricacy (and beauty if you will) of the cellular and biochemical events underlying our existence.  There is far more to discover, so stay tuned.

For some further musings on this point — https://luysii.wordpress.com/2009/09/17/the-solace-of-molecular-biology/