How Ya Gonna Keep ’em Down on the Farm (After They’ve Seen Paree) is a song of 100+ years ago when World War I had just ended. In 1920, for the first time America was 50/50 urban/rural. Now it’s 82%.
What does this have to do with cellular biology? A lot. One of the first second messengers to be discovered was cyclic adenosine monophosphate (CAMP). It binds to an enzyme complex called protein kinase A (PKA), activating it, making it phosphorylate all sorts of proteins changing their activity. But PKA doesn’t float free in the cell. We have some 47 genes for proteins (called AKAPs for protein A Kinase Anchoring Protein) which bind PKA and localize it to various places in the cell. CAMP is made by an enzyme called adenyl cyclase of which we have 10 types, each localized to various places in the cell (because most of them are membrane embedded). We also have hundreds of G Protein Coupled Receptors (GPCRs) localized in various parts of the cell (apical, basal, primary cilia, adhesion structures etc. etc.) many of which when activated stimulate (by yet another complicated mechanism) adenyl cyclase to make CAMP.
So the cell tries to keep CAMP when it is formed relatively localized (down on the farm if you will). Why have all these ways of making it if its going to run all over the cell after all.
Actually the existence of localized signaling by CAMP is rather controversial, particularly when you can measure how fast it is moving around. All studies previous to Cell vol. 182 pp. 1379 – 1381, 1519 – 1530 ’20 found free diffusion of CAMP.
This study, found that CAMP (in low concentrations) was essentially immobile, remaining down on the farm where it was formed.
The authors used a fluorescent analog of CAMP which allowed them to use fluorescence fluctuation spectroscopy which gives the probability distribution function of an individual molecule occupying a given position in space and time (SpatioTemporal Image correlation Spectroscopy — STICS).
Fascinating as the study is, it is ligh tyears away from physiologic — the fluorescent CAMP analog was not formed by anything resembling a physiologic mechanism (e.g. by adenyl cyclase). A precursor to the fluorescent CAMP was injected into the cell and broken down by ‘intracellular esterases’ to form the fluorescent CAMP analog.
Then the authors constructed a protein made of three parts (1) a phosphodiesterase (PDE) which broke down the fluorescent CAMP analog and (2) another protein — the signaler — which fluoresced when it bound the CAMP analog. The two were connected by (3) a flexible protein linker e.g. the ‘ruler’ of the previous post. The ruler could be made of various lengths.
Then levels of fluorescent CAMP were obtained by injecting it into the cell, or stimulating a receptor.
If the sensor was 100 Angstroms away from the PDE, it never showed signs of CAMP, implying the the PDE was destroying it before it could get to the linker implying that diffusion was quite slow. This was at low concentrations of the fluorescent CAMP analog. At high injection concentrations the CAMP overcame the sites which were binding it in the cell and moved past the signaler.
It was a lot of work but it convincingly (to me) showed that CAMP doesn’t move freely in the cell unless it is of such high concentration that it overcomes the binding sites available to it.
They made another molecule containing (1) protein kinase A (2) a ruler (3) a phophodiesterase. If the kinase and phosphodiesterase were close enough together, CAMP never got to PKA at all.
Another proof that phosphodiesterase enzymes can create a zone where there is no free CAMP (although there is still some bound to proteins).
Hard stuff (to explain) but nonetheless impressive, and shows why we must consider the cell a bunch of tiny principalities jealously guarding their turf like medieval city states.
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A molecular ruler
Time to cleanse your mind by leaving the contentious world of social issues and entering the realm of pure thought with some elegant chemistry.
You are asked to construct a molecular ruler with a persistence length of 150 Angstroms.
Hint #1: use a protein
Hint #2; use alpha helices
Spoiler alert — nature got there first.
The ruler was constructed and used in an interesting paper on CAMP nanoDomains (about which more on the next post).
It’s been around since 2011 [ Proc. Natl. Acad. Sci. vol. 108 pp. 20467 – 20472 ’11 ] and I’m embarrassed to admit I’d never heard of it.
It’s basically a run of 4 negatively charged amino acids (glutamic acid or aspartic acid) followed by a run of 4 positively charged amino acids (lysine, arginine). This is a naturally occurring motif found in a variety of species.
My initial (incorrect) thought was that this couldn’t work as the 4 positively charged amino acids would bend at the end and bind to the 4 negatively charged ones. This can’t work even if you make the peptide chain planar, as the positive charges would alternate sides on the planar peptide backbone.
Recall that there are 3.5 amino acids/turn of the alpha helix, meaning that between a run of 4 Glutamic acid/Aspartic acids and an adjacent run of 4 lysines/arginines, an ionic bond is certain to form between the side chains (and not between adjacent amino acids on the backbone, but probably one 3 or 4 amino acids away)
Since a complete turn of the alpha helix is only 5.4 Angstroms, a persistence length of 150 means about 28 turns of the helix using 28 * 3.5 = 98 amino acids or about 12 blocks of ++++—- charged amino acids.
The beauty of the technique is that by starting with an 8 amino acid ++++—- block, you can add length to your ruler in 12 Angstrom increments. This is exactly what Cell vol. 182 pp. 1519 – 1530 ’20 did. But that’s for the next post.
Chemiotics II 