Chemiotics IIThe first sequencing of the human genome was ‘completed’ in April 2003 taking 13 years and costing over a billion dollars.
It really wasn’t complete, and even by 2022 10 megaBases (of the 3.2 gigaBases) hadn’t been sequenced
Science vol. 382 1 December p. 980 ’23 The UK biobank just released the WHOLE genome sequences of 500,000 people.
Imagine what we’d know if our understanding of the results had similarly accelerated.
Phase separated droplets of protein, RNA and God knows what else have gotten everyone’s attention. For background see after the ***.
Neurologists know that phase separated TDP43 droplets are involved in a variety of neurologic diseases. For background see after the &&&
Now it’s everyone’s concern because a recent paper [ PNAS vol. 120 e230355120 ’23 ] shows that phase separation is involved in the construction of the pandemic virus SARS-CoV-2.
Cellular Nucleic Acid Binding Protein (CNBP) is part of the interferon generated response to RNA virus infections of our cells. In response to infection CNBP is phosphorylated and translocates from the cytoplasm to the nucleus where it turns on the interferon beta genes.
SARS-CoV-2 evades detection by our RNA sensing pathways (a story in itself), so CNBP is retained in the cytoplasm.
CNBP has another trick up its sleeve, binding to the 3′ and 5′ long terminal repeats (LTRs) of the viral genome, competing with the viral nucleocapsid protein. This prevents the two from liquid liquid phase separation (LLPS — another name for macromolecular phase separation) which is critical for viral replication. Did you know this? I didn’t. The paper gives 4 references in journals I don’t read all appearing in the last few years.
Cells and animals lacking CNBP have higher SARS-CoV-2 viral loads after infection.
****
I’m republishing this old post from 2018, to refresh my memory (and yours) about liquid liquid phase separation before writing a new post on one of the most interesting papers I’ve read in recent years. The field has exploded since this was written.
Until recently, developments in physics basically followed earlier work by mathematicians Think relativity following Riemannian geometry by 40 years. However in the past few decades, physicists have developed mathematical concepts before the mathematicians — think mirror symmetry which came out of string theory — https://en.wikipedia.org/wiki/Mirror_symmetry_(string_theory). You may skip the following paragraph, but here is what it meant to mathematics — from a description of a 400+ page book by Amherst College’s own David A. Cox
Mirror symmetry began when theoretical physicists made some astonishing predictions about rational curves on quintic hypersurfaces in four-dimensional projective space. Understanding the mathematics behind these predictions has been a substantial challenge. This book is the first completely comprehensive monograph on mirror symmetry, covering the original observations by the physicists through the most recent progress made to date. Subjects discussed include toric varieties, Hodge theory, Kahler geometry, moduli of stable maps, Calabi-Yau manifolds, quantum cohomology, Gromov-Witten invariants, and the mirror theorem. This title features: numerous examples worked out in detail; an appendix on mathematical physics; an exposition of the algebraic theory of Gromov-Witten invariants and quantum cohomology; and, a proof of the mirror theorem for the quintic threefold.
Similarly, advances in cellular biology have come from chemistry. Think DNA and protein structure, enzyme analysis. However, cell biology is now beginning to return the favor and instruct chemistry by giving it new objects to study. Think phase transitions in the cell, liquid liquid phase separation, liquid droplets, and many other names (the field is in flux) as chemists begin to explore them. Unlike most chemical objects, they are big, or they wouldn’t have been visible microscopically, so they contain many, many more molecules than chemists are used to dealing with.
These objects do not have any sort of definite stiochiometry and are made of RNA and the proteins which bind them (and sometimes DNA). They go by any number of names (processing bodies, stress granules, nuclear speckles, Cajal bodies, Promyelocytic leukemia bodies, germline P granules. Recent work has shown that DNA may be compacted similarly using the linker histone [ PNAS vol. 115 pp.11964 – 11969 ’18 ]
The objects are defined essentially by looking at them. By golly they look like liquid drops, and they fuse and separate just like drops of water. Once this is done they are analyzed chemically to see what’s in them. I don’t think theory can predict them now, and they were never predicted a priori as far as I know.
No chemist in their right mind would have made them to study. For one thing they contain tens to hundreds of different molecules. Imagine trying to get a grant to see what would happen if you threw that many different RNAs and proteins together in varying concentrations. Physicists have worked for years on phase transitions (but usually with a single molecule — think water). So have chemists — think crystallization.
Proteins move in and out of these bodies in seconds. Proteins found in them do have low complexity of amino acids (mostly made of only a few of the 20), and unlike enzymes, their sequences are intrinsically disordered, so forget the key and lock and induced fit concepts for enzymes.
Are they a new form of matter? Is there any limit to how big they can be? Are the pathologic precipitates of neurologic disease (neurofibrillary tangles, senile plaques, Lewy bodies) similar. There certainly are plenty of distinct proteins in the senile plaque, but they don’t look like liquid droplets.
It’s a fascinating field to study. Although made of organic molecules, there seems to be little for the organic chemist to say, since the interactions aren’t covalent. Time for physical chemists and polymer chemists to step up to the plate.
It’s been 3 years since AlphaFold blew away the competition in the Critical Assessment of Protein Structure (CASP) coming up with the 3 dimensional structure of proteins given just the amino acid sequence of the protein. The structure was known to the people at CASP, but not to the contestants.
I wrote a post far back in the mists of time (2009 to be exact — https://luysii.wordpress.com/2009/11/29/time-for-the-glass-eye-test-to-be-inserted-into-casp/) saying that this wasn’t fair, as (1) the contestants knew that the protein had a single structure and (2) some proteins didn’t have a structure in all their parts but had intrinsically disordered region, or were completely disordered (like alpha-Synuclein, the main protein component of the Lewy body found in Parkinson’s disease.
I argued that contestants should be given a protein with an intrinsically disordered region (IDR) to see if they came up with a structure that wasn’t there, similar to the glass eye test inflicted on hapless medical students.
What is the glass eye test all about. Here is part of the old post.
One of the hardest things for a fledgling doc to learn, is how to do a decent physical examination (PE). Whole courses are given on the subject. One of the hardest parts of the PE is looking into the back of the eye with an ophthalmoscope. This is important (particularly in neurology) because it’s the only place in the body where you can actually see a nerve (the optic nerve) along with arteries and veins (everything else you do in the PE results in just inference about nerves, rather than direct observation). If the optic nerve appears swollen then you know that pressure inside the head is elevated (always serious).
The temptation to fudge is ever present. This is why the patient with the glass eye is so valuable. If one is present and the unlucky med student attempts to say that they saw the nerve, it’s time for a lecture (hopefully not too sadistically). It didn’t happen to me, but the basic lesson is to report what you see honestly, whether you understand it or not — there’s probably someone smarter or with more experience who will (that’s what I heard when I reported a bunch of findings that just didn’t make sense).
Well AlphaFold just got the glass eye test and failed [Proc. Natl. Acad. Sci. vol. 120 e2304302120 ’23 ] It assigned structures (with high confidence to boot) to 15% of intrinsically disordered regions (IDRs). It is known that some IDRs will fold to a single structure on binding to a target (this is called conditional folding) and here AlphaFold ‘often’ predicts the structure of these states. Of interested is that mutations causing human disease are 5 times more common in conditionally folded IDRs than IDRs in general.
Even more interesting is that 80% of bacterial IDRs conditionally fold, but only 20% of eukaryotic IDRs do.
Ketamine burst like a bombshell on depression research a few years ago. I’ve written two other posts about it (reproduced after the ***).
The drugs we used for depression weren’t great. They didn’t help at least a third of the patients, and they usually took several weeks to work for endogenous depression (e.g. depression not obviously triggered by external events). They seemed to work faster in my MS patients who had a relapse and were quite naturally depressed by an exogenous event completely out of their control.
Because of the weeks of delay an incredible amount of work was done looking for the long term neurochemical and neurophysiologic changes produced by the antidepressants we had (tricyclic antidepressants, selective serotonin reuptake inhibitors — SSRIs)
Enter Ketamine which, when given IV, can transiently lift depression within a few hours. You can find more details and references in an article in Neuron ( vol. 101 pp. 774 – 778 ’19) written by the guys at Yale who did some of the original work. However, here’s the gist of the article.
A single dose of ketamine produced antidepressant effects that began within hours peaked in 24 – 72 hours and dissipated within 2 weeks (if ketamine wasn’t repeated). This occurred in 50 – 75% of people with treatment resistant depression. Remarkably one third of treated patients went into remission.
The incredibly rapid improvement in depression (hours) produced by ketamine is unprecedented and surely is telling us something vitally important about depression. If only we could figure out what it is.
I think that one thing ketamine is telling us, is that depression is in some way an active process, which must be maintained somehow, and that ketamine is breaking up this process.
One problem with trying to figure out what ketamine is doing is that it is gone long before its therapeutic effects end. For instance the elimination half life in man is 3 hours, but the antidepressant activity lasts 3 to 14 days. How can it break up an active process if it’s not around.
So we turn to our friend the mouse. They don’t talk, so how can you tell if they’re depressed. We do have reasonable animal models of depression (tail suspension test, forced swim test). We can at least get a handle on the anhedonia almost invariably found in depression using the sucrose preference test.
Throw ketamine at an animal and measure the biochemical or the neurophysiologic effect of your choice. There are zillions of them. Throw just about anything at the brain, and all sorts of things change. The problem is showing that the change is relevant. Is the known blockade of NMDA receptors by ketamine how it helps depression. Give enough ketamine to humans and you get out of body experiences and all sorts of craziness, not an antidepressant effect.
Enter Nature vol. 622 pp. 802 – 809 ’23. Ketamine hangs around 13 minutes in mice, but its antidepressant effects (see above) last at least a day. Here the antidepressant effect is measured by suppresion of burst firing in the lateral habenula (which is a tiny structure in man) something a very long way from clinical depression in humans.
So ketamine can’t interrupt an active process if it isn’t there. But it is there according to the paper, which finds ketamine hanging around the NMDA receptor (actually a subtype of glutamic acid receptor) in the lateral habenula for a day, even though you can’t measure it anywhere else.
That’s interesting, but the paper notes that “Currently, there is an intense debate about whether the antidepressant effects of ketamine are mediated by NMDA receptors at all.”
Here are my two other posts on the subject
***
Published almost exactly 4 years ago
The incredibly rapid improvement in depression (hours) produced by ketamine is unprecedented and surely is telling us something vitally important about depression. If only we could figure out what it is. Clinicians were used to waiting weeks for antidepressants of all sorts to work. As a neurologist, I’d see it work in a week or so in my MS patients depressed due a relapse.
Two recent papers show just how hard it is going to be [Neuron vol. 104 pp. 182 – 182, 338 – 352 ’19 ]. First off you have to accept the idea that even though animals (usually mice) can’t tell us how they feel, we still have reasonable animal models of depression (tail suspension test, forced swim test). We can at least get a handle on anhedonia using the sucrose preference test.
Throw ketamine at an animal and measure the biochemical or the neurophysiologic effect of your choice. There are zillions of them. Throw just about anything at the brain, and all sorts of things change. The problem is showing that the change is relevant. Is the known blockade of NMDA receptors by ketamine how it helps depression. Give enough and you get out of body experiences and all sorts of craziness, not an antidepressant effect.
Homer1a is a protein found at the synapse, and like all scaffold proteins, it interacts with a bunch of different proteins. It links another type of glutamic acid receptor (mGluR1 and mGluR5) to inositol 1, 4, 5 trisphosphate receptors (IP3Rs) on the endoplasmic reticulum. It also links mGluR1 and mGluR5 to NMDARs and other ion channels.
So what?
Other work by the authors showed that knockdown of Homer1a (using small interfering RNA – siRNA) in the medial prefrontal cortex (mPFC) abolished the antidepressant effects (in animal models) to ketamine. Well that’s good, but even better is that knockdown also abolished the antidepressant effects of a tricyclic antidepressant (imipramine).
The present work showed that increasing the expression of Homer1a (the protein comes in various isoforms) in the frontal cortex reduced depression in the various models.
Pretty good — all we have to do is increase Homer1a expression to have a treatment of depression.
Don’t get your hopes up, and this is why depression research is so — well depressing.
Increasing Homer1a expression in another brain region (the hippocampus) has exactly the opposite effects.
The drugs we use for depression aren’t great. They don’t help at least a third of the patients, and they usually take several weeks to work for endogenous depression. They seemed to work faster in my MS patients who had a relapse and were quite naturally depressed by an exogenous event completely out of their control.
Enter Ketamine which, when given IV, can transiently lift depression within a few hours. You can find more details and references in an article in Neuron ( vol. 101 pp. 774 – 778 ’19) written by the guys at Yale who did some of the original work. However, here’s the gist of the article. A single dose of ketamine produced antidepressant effects that began within hours peaked in 24 – 72 hours and dissipated within 2 weeks (if ketamine wasn’t repeated). This occurred in 50 – 75% of people with treatment resistant depression. Remarkably one third of treated patients went into remission.
This simply has to be telling us something very important about the neurochemistry of depression.
Naturally there has been a lot of work on the neurochemical changes produced by ketamine, none of which I’ve found convincing ( see https://luysii.wordpress.com/2019/10/27/how-does-ketamine-lift-depression/ ) untilthe following paper [ Neuron vol. 106 pp. 715 – 726 ’20 ].
In what follows you have to have some basic knowledge of synaptic structure, but here’s a probably inadequate elevator pitch. Synapses have two sides, pre- and post-. On the presynaptic side neurotransmitters are enclosed in synaptic vesicles. Their contents are released into the synaptic cleft when an action potential arrives from elsewhere in the neuron. The neurotransmitters flow across the very narrow synapse to bind to receptors on the postsynaptic side, triggering (or not) a response of the postsynaptic neuron. Presynaptic terminals vary in the number vesicles they contain.
Synapses are able to change their strength (how likely an action potential is to produce a postsynaptic response). Otherwise our brains wouldn’t be able to change and learn anything. This is called synaptic plasticity.
One way to change the strength of a synapse is to adjust the number of synaptic vesicles found on the presynaptic side. Presynaptic neurons form synapses with many different neurons. The average neuron in the cerebral cortex is post-synaptic to thousands of neurons.
We think that synaptic plasticity involves changes at particular synapses but not at all of them.
Not so with ketamine according to the paper. It changes the number of presynaptic vesicles at all synapses of a given neuron by the same percentage — this is called synaptic scaling. Given 3 synapses containing 60 50 and 40 vesicles, upward synaptic scaling by 20% would add 12 vesicles to the first 10 to the second and 8 to the third. The paper states that this is exactly what ketamine does to neurons using glutamic acid (the major excitatory neurotransmitter found in brain). Even more interesting, is the fact that lithium which treats mania has the opposite effects decreasing the number of vesicles in each synapse by the same percentage.
I found this rather depressing when I first read it, as I realized that there was no chemical process intrinsic to a neuron which could possibly work quickly enough to change all the synapses at once. To do this you need a drug which goes everywhere at once.
But you don’t. There are certain brain nuclei which send their processes everywhere in the brain. Not only that but their processes contain varicosities which release their neurotransmitter even where there is no post-synaptic apparatus. One such nucleus (the pars compacta of the substantia nigra) has extensively ramified processes so much so that “Individual neurons of the pars compact are calculated to give rise to 4.5 meters of axons once all the branches are summed” — [ Neuron vol. 96 p. 651 ’17 ]. So when that single neuron fires, dopamine is likely to bathe every neuron in the brain. We think that something similar occurs in the locus coeruleus of the lower brain which has only 15,000 neurons and releases norepinephrine, and also in the raphe nuclei of the brainstem which release serotonin.
It should be less than a surprise that drugs which alter neurotransmission by these neurotransmitters are used to treat various psychiatric diseases. Some drugs of abuse alter them as well (Cocaine and speed release norepinephrine, LSD binds one of the serotonin receptors etc, etc.)
The substantia nigra contains only 450,000 neurons at birth, so you don’t need a big nucleus to affect our 80 billion neuron brains.
So the question before the house, is have we missed other nuclei in the brain which control volume neurotransmission by glutamic acid? If they exist, could their malfunction be a cause of mania and/or depression? There is plenty of room for 10,000 to 100,000 neurons to hide in an 80 billion neuron brain.
Time to think outside the box people. Here is an example: Since ketamine blocks activation of one receptor for glutamic acid, could there be a system using volume neurotransmission which releases a receptor inhibitor?
Addendum 7 July — I sent a copy of the post to the authors and received this back from one of them. “Thank you very much for your kind words and interest in our work. Your explanation is quite accurate (my only suggestion would be to replace “vesicles” with “receptors”, as the changes we propose are postsynaptic). Reading your blog reassures us that our review article accomplished its main goal of reaching beyond the immediate neuroscience community to a wider audience like yourself.”
Cassava Sciences has been under attack since 8/21 reaching a crescendo recently — https://www.science.org/content/article/co-developer-cassava-s-potential-alzheimer-s-drug-cited-egregious-misconduct and https://www.science.org/content/blog-post/saga-cassava. Both contain demands that their current double blind placebo controlled studies on Simufilam, their Alzheimer drug be stopped.
Full disclosure. I do not own any Cassava stock and have not received anything from them for writing about them (aside from a free meal from Lindsay Burns 11/21 at the CTAD (Clinical Trials in Alzheimer’s Disease) in Boston. There was and is no quid pro quo about anything I’ve written.
I do know Derek Lowe and have spent several pleasant afternoons discussing organic chemistry and drug development with him at my cousin’s New Year’s Day bash, before COVID put a stop to the affair.
I’ve known Lindsay since she was a teenager, when I was practicing neurology in Montana. This was primarily through her parents who were sheep ranchers and good friends of my wife and me.
The basic position of the two articles above, is that some of the protein electrophoreses backing up Cassava’s model of Simufilam were either fudged or missing.
For details about Cassava’s model and the science behind it please see — https://luysii.wordpress.com/2023/04/23/the-science-behind-cassava-sciences-sava-the-latest-as-of-19-april-23/
But now it’s time to talk about models of disease causation and how important and reliable they are. I’ll start with an impeccable model of Alzheimer’s disease and the exquisite chemistry, biochemistry and genetics backing it up. It’s technical, but so are the protein electrophoreses which have been criticized.
Basically the model says that the accumulation of Abeta peptides, the major component of the senile plaque, causes Alzheimer’s disease, and removing them should help the disease.
The following is pretty long and if you want to skip the details for the rest of the argument — fast forward to *****
First (and probably the best evidence) is the mutation that protects against Alzheimer’s disease. As most of you know, the aBeta peptide (39 to 42 amino acids) is part of a much larger protein the Amyloid Precursor Protein (APP) which contains 639 to 770 amino acids. This means that enzymes must cut it out. Such enzymes (called proteases) are finicky, cutting only between certain amino acids. In what follows A673T stands for the 673rd position which normally has amino acid Alanine (A) there. Instead there is amino acid Threonine (T). The enzyme cleaving at 673 is Beta Secretase 1 (BACE1).
Recall that in amyloid fibrils the peptide backbone is flat as a flounder (well in a box 4.8 Angstroms high) with the amino acid side chains confined to this plane. The backbone winds around in this plane like a snake. The area in the leftmost loop is particularly crowded with bulky side chains of glutamic acid (single letter E) at position 22 and aspartic acid (single letter D) at position 23 crowding each other. If that wasn’t enough, at the physiologic pH of 7 both acids are ionized, hence negatively charged. Putting two negative charges next to each other costs energy and makes the sheet making up the fibril less stable.
To make an amyloid fiber just stack 1000 or more of these flounder aBeta peptides on top of each other.
The marvelous paper (the source for much of this) Cell vol. 184 pp. 4857 – 4873 ’21 notes that there are 3 types of amyloid — pathological, artificial, and functional, and that the pathological amyloids are the most stable.
In 2007 there were 7 mutations associated with familial Alzheimer’s disease (10 years later there were 11). Here are 5 of them.
Glutamic Acid at 22 to Glycine (Arctic)
Glutamic Acid at 22 to Glutamine (Dutch)
Glutamic Acid at 22 to Lysine (Italian)
Aspartic Acid at 23 to Asparagine (Iowa)
Alanine at 21 to Glycine (Flemish)
All of them lower the energy of the amyloid fiber making them more stable
Here’s why
Glutamic Acid at 22 to Glycine (Arctic) — glycine is the smallest amino acid (side chain hydrogen) so this relieves crowding. It also removes a negatively charged amino acid next to the aspartic acid (which is also negatively charged). Putting two negative charges next to each other costs energy — because like charges repel. Both effects lower the energy of the amyloid fiber
Glutamic Acid at 22 to Glutamine (Dutch) — really no change in crowding, but it removes a negative charge next to the negatively charged Aspartic acid
Glutamic Acid at 22 to Lysine (Italian)– no change in crowding, but the lysine is positively charged at physiologic pH, so we have a positive charge next to the negatively charged Aspartic acid, lowering the energy because like charges attract.
Aspartic Acid at 23 to Asparagine (Iowa) –really no change in crowding, but it removes a negative charge next to the negatively charged Glutamic acid next door
Alanine at 21 to Glycine (Flemish) — no change in charge, but a reduction in crowding as alanine has a methyl group and glycine a hydrogen.
As a chemist, I find this immensely satisfying. The structure explains why the mutations in the 42 amino acid aBeta peptide are where they are, and the chemistry explains why the mutations are what they are.
****
It doesn’t get any better than this, yet therapy based on the model (monoclonal antibodies to remove the aBeta peptide) have produced minimal benefit (a less than 30% decrease in the rate of decline) and serious side effects namely brain hemorrhage, because the aBeta peptides don’t confine themselves to the senile plaque but can be found in the walls of blood vessels. This is called cerebral amyloid angiopathy (CAA).
What about wildly successful therapies which were found without ‘benefit’ of any theory, with theoreticians scrambling to explain them post hoc.
Here are 3:
l. Lithium for mania– the story is particularly fascinating — https://www.nature.com/articles/d41586-019-02480-0. We still don’t know how it works but theories abound, despite the original discovery reaching the ripe old age of 70.
2. Ketamine analogs for depression. For decades we were taught that antidepressants take a few weeks to work, and this was true of the drugs we had (tricyclics, selective serotonin reuptake inhibitors). Hell, I taught it myself back in the day at Montana State University. This was until ketamine analogs (esketamine) lifted depression within hours. We are still trying to figure out how this works, and what it tells us about depression (which is almost certainly a lot).
3. Wegovy, Ozempic for weight loss. I’ve been reading about glucagon like peptide (GLP) for years, because it is a peptide neurotransmitter. People have been studying it for years. Only when GLP agonists used to treat diabetes produced weight loss did theorists scramble to figure out why.
As an old med school friend, warped by his experiences at the University of Chicago used to say. “That’s how it works in practice, but how does it work in theory?”
So here we have a superb model for Alzheimer’s disease which hasn’t produced a terribly useful therapy, and three incredibly useful therapies for which we presently have no satisfying model.
So dumping on Cassava and wishing to stop an ongoing study because the model underneath it is based on flawed data just doesn’t make sense.
In particular, it doesn’t make sense given the data Cassava has already released, but that’s for part II.
One of the bad things about making it to 85.5 later this month is losing old friends such as Don Voet. We met as first year chemistry grad students almost exactly 63 years ago. Another grad student described him as amiable, but he was more than that with a very dry sense of humor. The first lecture of every course seemed to be about units. Voet said he preferred the hand stone fortnight system. We weren’t that far from World War II and the place was filled with emigrees. Voet said that the universal scientific language was broken English.
Later on, he and his wife Judy wrote a fabulously successful textbook of Biochemistry which sold over 250,000 copies. The illustrations were terrific. If you want to see how dull Biochemistry books were in the 50’s and 60’s, go to the library and look at Fruton and Simmonds.
Typical of his unusual thought patterns is the introduction to water (p. 39 third edition of their book). “Water is so familiar, we generally consider it to be a rather bland fluid of simple character. It is, however, a chemically reactive liquid with such extraordinary physical properties that, if chemists had discovered it in recent times, it would undoubtedly have been classified as an exotic substance.”
After our first year, Don wanted to see some old friends from Cal Tech in LA and Berkeley. So we saved up some money from our NSF grants and money made by acting as lab teaching assistants and drove across the country in the summer of 1961 on 25 cent a gallon gas and 5 or 10 cent Hershey bars. What do you think our yearly income was? Answer at the end.
It was the first time I really saw the country as it should be seen. In Rapid City South Dakota, I went into a fairly scummy looking motel, asking for a room for two. The clerk had two questions. Is he drunk? Is he Indian? My answers were satisfactory so he rented us a room. (For the nonUSA readers; Indian at that time and place meant Native American, a term invented and used only much later.)
We pretty much got up with the sun and went to bed with it not wishing to miss anything, and saw people lined up outside bars in little Wyoming towns at 7AM waiting for to open so they could have a drink. We swam in the Great Salt Lake and drove across the desert at night to avoid the heat (car air conditioning didn’t exist). We arrived in Reno at 2 AM. The place was jumping and all the motels were full. Finally we found a place with a vacancy sign, but when we went in the clerk asked us how long we wanted the room for. So we slept in the car out in the desert that night.
One of Don’s old Cal Tech profs had us out to his house in Malibu (something I think would be impossible for a Cal Tech prof today).
On they way back, I dropped Don off at his parent’s house in Borger Texas, the carbon black capital of the world, about 50 miles north of Amarillo. It’s hard to believe but I had a conversation with his parents which is incredibly relevant today.
Voet is a Dutch name, and his parents (a chemist and an ophthalmologist) were Dutch Jews. They got out before WWII. I asked them why they left and they said that there had been some sort of incident at the German embassy, and the Germans went crazy about it. “This is not the act of a friendly country” and they left.
Fast forward 62 years
The response of various political and cultural figures to the massacre in Israel is a moment of clarity, showing who is and who is not a friend of the Jews.
Virtue signaling is easy, virtue acting is not.
The following paper [ Proc. Natl. Acad. Sci. vol. 120 e2220026120n ’23 ] wasn’t as spectacular as it first appeared to me. The authors found a peptide (sequence CGSPGWVRC — or Cys Gly Ser Pro Gly Trp Val Arg Cys ) which binds to lung endothelial cells (and no other kind). Moreover the receptor for CGSPGWVRC wasn’t another protein, but ceramide, a membrane lipid.
I was excited to think that something in our genome was making a peptide to bind to ceramide. It didn’t make much sense because there is so much ceramide around. It turns out that the peptide was found by phage display looking at zillions of peptides for those which bind specifically to lung endothelium. It wasn’t an antibody and we don’t have a gene for it.
Such peptides have potential use, to target drugs to specific organs, and attaching a proapoptotic peptide to CGSPGWVRC produced apoptosis of lung endothelium. Such peptides clearly would be worth exploring for cancer chemotherapy, if they can be found for other organs.
When you think of what happens to our DNA during mitosis, it’s remarkable that the two daughter cells produced look anything like their mother. Our 3.2 billion positions in DNA when stretched out (as they are in cells not in mitosis { in interphase for those who like terminology } ) are about 1 meter long. You can’t see them with a light microscope as they’re only 2 nanoMeters wide, and the shortest wavelength of visible light is 400 nanoMeters. To scrunch our chromosomes down so they are visible and more importantly, so they don’t get tangled up with each other as they migrate to the daughter cells, they are compacted 100,000 fold. Unsurprisingly, DNA transcription into mRNA and protein synthesis pretty much stops during mitosis [ Cell vol. 150 pp. 725 – 737 ’12 ], as the transcription machinery can’t get into compacted DNA, and even if it did couldn’t unwind the DNA of a gene enough to transcribe it.
Well, we’ve got 20,000 protein genes, and what distinguishes the wildly different cell types in our body is the collection of proteins they make, and the way they organize the membranes of the cell. Each cell type has a different collection of proteins. After mitosis how do they make the correct collection. What keeps a blood cell from turning into a neuron (or vice versa). The answer is bookmarking, some sort of way to distinguish an interphase gene making mRNA (e.g. an active gene) from an inactive one, so that only the previously active genes start up again.
We’re just beginning to find out what those bookmarks are. One mechanism has long been known, keep the DNA of an active gene from being compacted [ Science vol. 307 pp. 421 – 423 ’05 ] and is particularly true for heat shock proteins.
TATA binding protein (TBP) is an essential component of transcription factor complexes which remains bound to promoters of active genes during mitosis [ Nature Cell Biology vol. 10 pp. 1318 –> ’08 ] forming yet another bookmark.
The latest bookmark found is the chromatin remodeler SWI/SNF which moves nucleosomes around so the transcription machinery can get to DNA. Some of its core subunits remain bound to gene promoters during mitosis [ Nature vol. 618 pp. 180 – 187 ’23 ]
I’m sure more bookmarks will be found
I think we’ve become far too blase about mitosis and our DNA and how it sits in out cells. So I’m going to republish a series of posts, that puts the goings on in the nucleus on a humanly comprehensible scale — a (US) football field and enclosing stadium.
So relax and enjoy (and hopefully be amazed). Here’s the first post — more will be coming or you can follow the link at the bottom
The nucleus is a very crowded place, filled with DNA, proteins packing up DNA, proteins patching up DNA, proteins opening up DNA to transcribe it etc. Statements like this produce no physical intuition of the sizes of the various players (to me at least). How do you go from the 1 Angstrom hydrogen atom, the 3.4 Angstrom thickness per nucleotide (base) of DNA, the roughly 20 Angstrom diameter of the DNA double helix, to any intuition of what it’s like inside a spherical nucleus with a diameter of 10 microns?
How many bases are in the human genome? It depends on who you read — but 3 billion (3 * 10^9) is a lowball estimate — Wikipedia has 3.08, some sources have 3.4 billion. 3 billion is a nice round number. How physically long is the genome? Put the DNA into the form seen in most textbooks — e.g. the double helix. Well, an Angstrom is one ten billionth (10^-10) of a meter, and multiplying it out we get
3 * 10^9 (bases/genome) * 3.4 * 10^-10 (meters/base) = 1 (meter).
The diameter of a typical nucleus is 10 microns (10 one millionths of a meter == 10 * 10^-6 = 10^-5 meter. So we’ve got fit the textbook picture of our genome into something 1/100,000 smaller. We’ll definitely have to bend it like Beckham.
As a chemist I think in Angstroms, as a biologist in microns and millimeters, but as an American I think in feet and inches. To make this stuff comprehensible, think of driving from New York City to Seattle. It’s 2840 miles or 14,995,200 feet (according to one source on the internet). Now we’re getting somewhere. I know what a foot is, and I’ve driven most of those miles at one time or other. Call it 15 million feet, and pack this length down by a factor of 100,000. It’s 150 feet, half the size of a (US) football field.
Next, consider how thick DNA is relative to its length. 20 Angstroms is 20 * 10^-10 meters or 2 nanoMeters (2 * 10^-9 meters), so our DNA is 500 million times longer than it is thick. What is 1/500,000,000 of 15,000,000 feet? Well, it’s 3% of a foot which is .36 of an inch, very close to 3/8 of an inch. At least in my refrigerator that’s a pair of cooked linguini twisted around each other (the double helix in edible form). The twisting is pretty tight, a complete turn of the two strands every 35.36 angstroms, or about 1 complete turn every 1.5 thicknesses, more reminiscent of fusilli than linguini, but fusilli is too thick. Well, no analogy is perfect. If it were, it would be a description. One more thing before moving on.
How thinly should the linguini be sliced to split it apart into the constituent bases? There are roughly 6 bases/thickness, and since the thickness is 3/8 of an inch, about 1/16 of an inch. So relative to driving from NYC to Seattle, just throw a base out the window every 1/16th of an inch, and you’ll be up to 3 billion before you know it.
You’ve been so good following to this point that you get tickets for 50 yardline seats in the superdome. You’re sitting far enough back so that you’re 75 feet above the field, placing you right at the equator of our 150 foot sphere. The north and south poles of the sphere are over the 50 yard line. halfway between the two sides. You are about to the watch the grounds crew pump 15,000,000 feet of linguini into the sphere. Will it burst? We know it won’t (or we wouldn’t exist). But how much of the sphere will the linguini take up?
The volume of any sphere is 4/3 * pi * radius^3. So the volume of our sphere of 10 microns diameter is 4/3 * 3.14 * 5 * 5 * 5 * = 523 cubic microns. There are 10^18 cubic microns in a meter. So our spherical nucleus has a volume of 523 * 10^-18 cubic meters. What is the volume of the DNA cylinder? Its radius is 10 Angstroms or 1 nanoMeter. So its volume is 1 meter (length of the stretched out DNA) * pi * 10^-9 * 10^-9 meters 3.14 * 10^-18 cubic meters (or 3.14 cubic microns == 3.14 * 10^-6 * 10^-6 * 10^-6
Even though it’s 15,000,000 feet long, the volume of the linguini is only 3.14/523 of the sphere. Plenty of room for the grounds crew who begin reeling it in at 60 miles an hour. Since they have 2840 miles of the stuff to reel in, we’ll have to come back in a few days to watch the show. While we’re waiting, we might think of how anything can be accurately located in 2840 miles of linguini in a 150 foot sphere.
Did you ever wonder why most mRNAs reaching the cytoplasm, have polyAdenine tacked on to their 3′ end in the nucleus. Why do it at all? Why not something else (poly C, poly G, polyT) or just some random string of nucleosides? For the answer see the end of the post. It takes work (and a lot of ATP) to cleave the mRNA in the nucleus and then tack on a string of adenines to the 3′ end of the mRNA.
Early work showed that the polyA tail kept the mRNA from being degraded. Eventually the tail gets munched on by poly(A) ribonuclease which shortens the tail. So early on we regarded the polyA tail as a sort of timer keeping the mRNA alive. The longer the tail the longer lived the mRNA.
But there’s another reason. The polyA is a fail safe mechanism. If for some reason the machinery for translation of the mRNA into protein (e.g. the ribosome) reads through a termination codon (well nobody’s perfect), it will translate the AAAA … into a long string of the same amino acid. What amino acid is coded for by AAA ?? Why it’s lysine. So AAAA … becomes the highly positively charged polyLysine (because of its epsilon amino group), which clogs the exit tunnel of the ribosome, and calls forth the ribosome quality control complex, which tears apart the ribosome allowing escape of the mRNA and its eventual destruction. Clever no?
So why not polyT — e.g. TTTTT . . .. — because it codes for an amino acid without any charges (phenylalanine), but still plausible as phenylalanine is fairly bulky and might gum up the exit tunnel
So why not polyG — e.g. GGGGG … — because it codes for another amino acid with charges (glycine) but not plausible because glycine is the smallest amino acid we have and couldn’t possibly gum up the exit tunnel
So why not polyC – e.g. CCCCCC . . . — this actually is a possibility because it codes for proline, and polyProline does form an unusual helix, and one that is left handed, unlike the right handed alpha helix of our proteins.
Chemiotics II 