Category Archives: Molecular Biology

You are alive because the lipid bilayer of your plasma membrane is asymmetric

You are an organism with trillions of cells. A mosquito bit you depositing millions of viruses in your tissues. The virus can reproduce only within one of your cells and it has exploited all sorts of protein protein chemistry to get in. Antibodies (if you are fortunate enough to have them) can get rid of the extracellular critters. However, 500,000 have made into the same number of your cells, and are merrily trying to reproduce.

How does the asymmetry of the lipid bilayer of your plasma membrane help you survive. If each virus infected cell killed itself before the virus reproduced, you’d survive. Although 500,000 is a large number is is less than 1 millionth of your cell total.

Well you do have intracellular defenses against viruses, called the innate immune system. One of them is a protein with the ugly name of gasdermin D. The activated innate immune system (in the form of inflammatory caspases) cleaves gasdermin. This breaks up the inhibition of the amino terminal part of gasdermin by the carboxy terminal part giving a fragment which binds to one particular membrane component (phosphatidyl serine) which makes up 20% of the inner leaflet of the cell membrane. It then forms a large diameter (to a cell 140 Angstroms is quite large) pore in the cell membrane. No cell can survive this, so it dies, releasing cellular contents (probably some viral components but not fully formed one). For details see [ Nature vol. 535 pp 111 – 116, 153 – 158 ’16 ]

Wait a minute. The toxic gasdermin fragment is also released. So how come it doesn’t kill everything in sight? Because our cellular membranes keep phosphatidyl serine confined to the inner membrane, normal cells don’t show it on their exterior, so they can be bathed in gasdermin with no ill effect. What is responsible for this asymmetry — believe it or not an ATP consuming enzyme called flippase (about this more later) which takes any phosphatidyl serine it finds on the outer leaflet and schleps it back inside the cell.

There is all sorts of elegant chemistry which explains just how gasdermin binds to phosphatidyl serine and none of the many other phospholipids found on the inner leaflet. There is more elegant chemistry explaining how flippase works (see later).

What chemistry cannot explain, is why organisms would ‘want’ an asymmetric membrane. As soon as you get into the function of a particular compound in an organism, chemistry is powerless to tell you why. Nothing else can explain how a given molecule does what it does on the molecular level but that is not enough for a satisfying explanation.

One further explanation before some hard core cellular biochemistry follows (after ***). Our cells are dying all the time. The lining of your gut is replaced every 5 days. Even the longest lasting element of your blood is gone after half a year, and most other elements are turned over at least once a month. When these cells die, they must be cleaned up, without undue fuss (such as inflammation). The cleaners are cells called macrophages. A dying cell releases chemical signals, actually called ‘eat me’, one of which is phosphatidyl serine found on the membrane fragments of a dead cell. The fact that flippases keep it on the inner leaflet means that macrophages won’t attack a normal cell.

Slick isn’t it?

***

Flippase is a MgATPdependent aminophospholipid translocase. It localizes phosphatidylserine and phosphatidylethanolamine to the inner membrane leaflet by rapidly translocating them from the outer to the inner leaflet against an electrochemical gradient. The stoichiometry between amino phospholipid translocation and ATP hydrolysis is close to one (how will the cell have enough ATP to do anything else?). The flippase is inhibited by high calcium, and by pseudosubstrates such as vanadate, acetylphosphate and para-nitrophenyl phosphate, and by SH reactive reagents such as N-ethylmaleimide and pyridyldithioethylamine (PDA) a specific inhibitor of phospholipid translocation

[ Proc. Natl. Acad. Sci. vol. 109 pp. 1449 – 1454 ’12 ] P4-ATPases are a subfamily of P-type ATPases. They transport aminophospholipids from the exoplasmic to the cytoplasmic leaflet (and are known as flippases). Man has 14 P4-ATPases, expressed in various cell types. They are thought to be similar to the catalytic subunits of the Ca++ ATPase, and the Na, K ATPase, consisting of cytoplasmic, N, P and A domains and a membrane domain made of 10 transmembrane helices (M1 – M10).

[ Proc. Natl. Acad. Sci. vol. 111 pp. E1334 – E1343 ’14 ] The P4-ATPases are thought to resemble the classic P-type ATPase cation pumps — a transmembrane domain of 10 helices and 3 cytoplasmic domains (P for phosphorylation, N for nucleotide binding and A for actuator). ATP8A2 forms an intermediate phosphorylated on aspartic acid (E2P)and undergoes a catalytic cycle similar to the sodium pump (Na+, K+ ATPase). Dephosphorylation of E2P is activated by the transported substrates phosphatidyl serine (PS) and phosphatidyl ethanolamine (PE), similar to the K+ activation of dephosphorylation in the sodium pump.

PE and PS are 10x as large as the cations transported by the sodium pump. This is known as the giant substrate problem. This work shows that isoleucine #364 (mutated in — patients with the ataxia, retardation and dysequilibrium syndrome Eur. J. Hum. Genet. vol. 21 pp. 281 – 285 ’13 aka CAMRQ syndrome ) forms a hydrophobic gate separating the entry and exit sites of PS. I364 likely directs the sequential formation and annihilation of water filled cavities (as shown by molecular dynamics simulations) allowing transport of the hydrophilic phospholipid head group, in a groove outlined by TMs 1, 2, 4 and 6, with the hydrocarbon chains following passively, still in the membrane lipid phase (and presumably outside the channel) — this must disrupt the hell out of the protein as it passes. They call this the credit card model — only the interaction with part of the molecule is important — just as the magnetic stripe is the only important thing about the credit card.

Another fail safe mechanism used by the cell — readthrough

Nothing is perfect in this world, not even the translation of mRNA into protein. The error rate is one amino acid misincorporated into a protein for every 10,000 or so done correctly — but these results are for one celled organisms (E. Coli, yeast). I can’t find a number for mammals, primates etc. etc.

This means that occasionally one of the 3 codons which tell the ribosome to quit (stop codons), will be misread as an amino acid. This is called readthrough, and means that the ribosome will merrily march on producing a much larger protein than coded for by the mRNA until one of two things happens. l. the ribosome reaches the end of the mRNA and stops. 2. the mRNA contains another stop codon (there are 3). The probability of this is 3/64 per codon. If stop codons are randomly distributed (which they are most certainly not in the protein coding segment of an mRNA) the chances of 100 codons in a row not containing a stop codon is under 1% (.822 % to be exact). So any protein containing more than 100 amino acids is a statistical freak in this sense. Since the 3′ untranslated region (3’UTR) of mRNA doesn’t code for protein, they should have stop codons randomly distributed (there being no selective pressure to keep them away).

Enter Nature vol. 534 pp. 719 – 723 ’16 — if you attach a 3′ UTR section of an mRNA to a normal protein sequence (mimicking readthrough) you get much less protein. The authors think the 3’UTRs code for peptide sequences destabilizing the attached protein. They don’t know what this might be, so it’s terra incognita for researchers, and a worthwhile PhD project to figure it out. Another example of ‘coding’ by a presumably nonCoding sequence in the genome. It may also tell us something about protein structure.

Why you do and don’t need chemistry to understand why we have big brains

You need some serious molecular biological chops to understand why primates such as ourselves have large brains. For this you need organic chemistry. Or do you? Yes and no. Yes to understand how the players are built and how they interact. No because it can be explained without any chemistry at all. In fact, the mechanism is even clearer that way.

It’s an exercise in pure logic. David Hilbert, one of the major mathematicians at the dawn of the 20th century famously said about geometry — “One must be able to say at all times–instead of points, straight lines, and planes–tables, chairs, and beer mugs”. The relationships between the objects of geometry were far more crucial to him than the objects themselves. We’ll take the same tack here.

So instead of the nucleotides Uridine (U), Adenine (A), Guanine (G), Cytosine (C), we’re going to talk about lock and key and hook and eye.

We’re going to talk about long chains of these four items. The order is crucial Two long chains of them can pair up only only if there are segments on each where the locks on one pair with the keys on the other and the hooks with the eyes. How many possible combinations of the four are there on a chain of 20 — just 4^20 or 2^40 = 1,099,511,621,776. So to get two randomly chosen chains to pair up exactly is pretty unlikely, unless in some way you or the blind Watchmaker chose them to do so.

Now you need a Turing machine to take a long string of these 4 items and turn it into a protein. In the case of the crucial Notch protein the string of locks, keys, hooks and eyes contains at least 5,000 of them, and their order is important, just as the order of letters in a word is crucial for its meaning (consider united and untied).

The cell has tons of such Turing machines (called ribosomes) and lots of copies of strings coding for Notch (called Notch mRNAs).

The more Notch protein around in the developing brain, the more the proliferating precursors to neurons proliferate before differentiating into neurons, resulting in a bigger brain.

The Notch string doesn’t all code for protein, at one end is a stretch of locks, keys, hooks and eyes which bind other strings, which when bound cause the Notch string to be degraded, mean less Notch and a smaller brain. The other strings are about 20 long and are called microRNAs.

So to get more Notch and a bigger brain, you need to decrease the number of microRNAs specifically binding to the Notch string. One particular microRNA (called miR-143-3p) has it in for the Notch string. So how did primates get rid of miR-143-3p they have an insert (unique to them) in another string which contains 16 binding sites for miR-143-3p. So this string called lincND essentially acts as a sponge for miR-143-3p meaning it can’t get to the Notch string, meaning that neuronal precursor cells proliferate more, and primate brains get bigger.

So can you forget organic chemistry if you want to understand why we have big brains? In the above sense you can. Your understanding won’t be particularly rich, but it will be at a level where chemical explanation is powerless.

No amount of understanding of polyribonucleotide double helices will tell you why a particular choice out of the 1,099,511,621,776 possible strings of 20 will be important. Literally we have moved from physicality to the realm of pure ideas, crossing the Cartesian dichotomy in the process.

Here’s a copy of the original post with lots of chemistry in it and all the references you need to get the molecular biological chops you’ll need.

Why our brains are large: the elegance of its molecular biology

Primates have much larger brains in proportion to their body size than other mammals. Here’s why. The mechanism is incredibly elegant. Unfortunately, you must put a sizable chunk of recent molecular biology under your belt before you can comprehend it. Anyone can listen to Mozart without knowing how to read or write music. Not so here.

I doubt that anyone can start from ground zero and climb all the way up, but here is all the background you need to comprehend what follows. Start here — https://luysii.wordpress.com/2010/07/07/molecular-biology-survival-guide-for-chemists-i-dna-and-protein-coding-gene-structure/
and follow the links (there are 5 more articles).

Also you should be conversant with competitive endogenous RNA (ceRNA) — here’s a link — https://luysii.wordpress.com/2014/01/20/why-drug-discovery-is-so-hard-reason-24-is-the-3-untranslated-region-of-every-protein-a-cerna/

Also you should understand what microRNAs are — we’re still discovering all the things they do — here’s the background you need — https://luysii.wordpress.com/2015/03/22/why-drug-discovery-is-so-hard-reason-26-were-discovering-new-players-all-the-time/weith.

Still game?

Now we must delve into the embryology of the brain, something few chemists or nonbiological type scientists have dealt with.

You’ve probably heard of the term ‘water on the brain’. This refers to enlargement of the ventricular system, a series of cavities in all our brains. In the fetus, all nearly all our neurons are formed from cells called neuronal precursor cells (NPCs) lining the fetal ventricle. Once formed they migrate to their final positions.

Each NPC has two choices — Choice #1 –divide into two NPCs, or Choice #2 — divide into an NPC and a daughter cell which will divide no further, but which will mature, migrate and become an adult neuron. So to get a big brain make NPCs adopt choice #1.

This is essentially a choice between proliferation and maturation. It doesn’t take many doublings of a NPC to eventually make a lot of neurons. Naturally cancer biologists are very interested in the mechanism of this choice.

Well to make a long story short, there is a protein called NOTCH — vitally important in embryology and in cancer biology which, when present, causes NPCs to make choice #1. So to make a big brain keep Notch around.

Well we know that some microRNAs bind to the mRNA for NOTCH which helps speed its degradation, meaning less NOTCH protein. One such microRNA is called miR-143-3p.

We also know that the brain contains a lncRNA called lncND (ND for Neural Development). The incredible elegance is that there is a primate specific insert in lncND which contains 16 (yes 16) binding sites for miR-143-3p. So lncND acts as a sponge for miR-143-3p meaning it can’t bind to the mRNA for NOTCH, meaning that there is more NOTCH around. Is this elegant or what. Let’s hear it for the Blind Watchmaker, assuming you have the faith to believe in such things.

Fortunately lncND is confined to the brain, otherwise we’d all be dead of cancer.

Should you want to read about this, here’s the reference [ Neuron vol. 90 pp. 1141 – 1143, 1255 – 1262 ’16 ] where there’s a lot more.

Historically, this was one of the criticisms of the Star Wars Missile Defense — the Russians wouldn’t send over a few missles, they’d send hundreds which would act as sponges to our defense. Whether or not attempting to put Star Wars in place led to Russia’s demise is debatable, but a society where it was a crime to own a copying machine, could never compete technically to produce such a thing.

Why our brains are large: the elegance of its molecular biology

Primates have much larger brains in proportion to their body size than other mammals. Here’s why. The mechanism is incredibly elegant. Unfortunately, you must put a sizable chunk of recent molecular biology under your belt before you can comprehend it. Anyone can listen to Mozart without knowing how to read or write music. Not so here.

I doubt that anyone can start from ground zero and climb all the way up, but here is all the background you need to comprehend what follows. Start here — https://luysii.wordpress.com/2010/07/07/molecular-biology-survival-guide-for-chemists-i-dna-and-protein-coding-gene-structure/
and follow the links (there are 5 more articles).

Also you should be conversant with competitive endogenous RNA (ceRNA) — here’s a link — https://luysii.wordpress.com/2014/01/20/why-drug-discovery-is-so-hard-reason-24-is-the-3-untranslated-region-of-every-protein-a-cerna/

Also you should understand what microRNAs are — we’re still discovering all the things they do — here’s the background you need — https://luysii.wordpress.com/2015/03/22/why-drug-discovery-is-so-hard-reason-26-were-discovering-new-players-all-the-time/weith.

Still game?

Now we must delve into the embryology of the brain, something few chemists or nonbiological type scientists have dealt with.

You’ve probably heard of the term ‘water on the brain’. This refers to enlargement of the ventricular system, a series of cavities in all our brains. In the fetus, all nearly all our neurons are formed from cells called neuronal precursor cells (NPCs) lining the fetal ventricle. Once formed they migrate to their final positions.

Each NPC has two choices — Choice #1 –divide into two NPCs, or Choice #2 — divide into an NPC and a daughter cell which will divide no further, but which will mature, migrate and become an adult neuron. So to get a big brain make NPCs adopt choice #1.

This is essentially a choice between proliferation and maturation. It doesn’t take many doublings of a NPC to eventually make a lot of neurons. Naturally cancer biologists are very interested in the mechanism of this choice.

Well to make a long story short, there is a protein called NOTCH — vitally important in embryology and in cancer biology which, when present, causes NPCs to make choice #1. So to make a big brain keep Notch around.

Well we know that some microRNAs bind to the mRNA for NOTCH which helps speed its degradation, meaning less NOTCH protein. One such microRNA is called miR-143-3p.

We also know that the brain contains a lncRNA called lncND (ND for Neural Development). The incredible elegance is that there is a primate specific insert in lncND which contains 16 (yes 16) binding sites for miR-143-3p. So lncND acts as a sponge for miR-143-3p meaning it can’t bind to the mRNA for NOTCH, meaning that there is more NOTCH around. Is this elegant or what. Let’s hear it for the Blind Watchmaker, assuming you have the faith to believe in such things.

Fortunately lncND is confined to the brain, otherwise we’d all be dead of cancer.

Should you want to read about this, here’s the reference [ Neuron vol. 90 pp. 1141 – 1143, 1255 – 1262 ’16 ] where there’s a lot more.

Historically, this was one of the criticisms of the Star Wars Missile Defense — the Russians wouldn’t send over a few missles, they’d send hundreds which would act as sponges to our defense. Whether or not attempting to put Star Wars in place led to Russia’s demise is debatable, but a society where it was a crime to own a copying machine, could never compete technically to produce such a thing.

ONTX Good news and semibad news.

The stock I recommended 1 June (ONTX) was up 11% today on a fourfold increase in volume. The rationale based on a Cell paper (vol. 165 pp. 643 – 655 ’16 ) will be found in a copy of the entire post below the ****

It is worth looking at the chart — https://finance.yahoo.com/echarts?s=ONTX+Interactive#{“range”:”1d”,”allowChartStacking”:true}

After a delay in opening, it exploded most of the way up on high volume (for it). Why? My guess is that people looked at the poster of the study in progress using their Ras blocking drug Rigosertib. Who looked? Why some of the 30,000 attendees at the 2016 American Society of Clinical Oncology Annual Meeting in Chicago, Illinois.

Why is this good (aside from the rise)? Assuming the people who bought ONTX were attendees at the convention, these are very informed buyers (e.g. professional oncologists) laying down their long green (e.g. very smart money).   In one of the many books I read about the Bernie Madoff Ponzi scheme, the people who invested with him were described as ‘dumb money’. They’d made their pile elsewhere and were babes in the woods when it came to investing.

Why is this also bad for what I predicted? Have a look at the abstract of one of the posters. Here’s a link to it —
http://meetinglibrary.asco.org/content/165681-176

The skinny is that the phase III study I was so excited about began only last December. It likely will be years before the results will be in. So goodbye 10x – 100x pop in the stock right away. Possibly big pharma will be impressed with their work and buy out the company which should also mean a significant gain.

Now 30,000 people can’t crowd around a single poster presentation. The stock is likely to continue moving up on volume this week as word spreads from the people who’ve already bought it and more people see the possibilities.

Here’s the post of 1 June — note that I didn’t own ONTX when I wrote the first post 3 May ’16, but did when I wrote the 1 June post.

*****

In a gambling mood?

If a pair of posters to be presented Monday 6 June at the 2016 American Society of Clinical Oncology Annual Meeting in Chicago, Illinois, contains the results of a phase III clinical trial of rigosertib, and if the results are as good as a paper discussed below the stock Onconova Therapeutics (ONTX) will jump by a factor of 10 to 100.

Full disclosure: I own some. The posters may just describe the clinical trial rather than report the results in which case all bets are off. In that case, I’ll just hold the stock until the results are in. This isn’t the ‘pump and dump’ beloved of boiler room operators everywhere. The rationale for the drug and my take on the original paper (3 May ’16) are reproduced below.

Has the great white whale of oncology finally been harpooned?

The ras oncogene is the great white whale of oncology. Mutations in 20 – 40% of cancer turn its activity on so that nothing can turn it off, resulting in cellular proliferation. People have been trying to turn mutated ras off for years with no success.

A current paper [ Cell vol. 165 pp. 643 – 655 ’16 ] describes a new and different way to attack it. Once ras is turned on (either naturally or by mutation) many other proteins must bind to it, to produce their effects — they are called RAS effectors, among which are the uneuphoniously named RAF, RalGDS and PI3K. They bind to activated ras by the cleverly named Ras Binding Domain (RBD) which has 78 amino acids.

The paper describes rigosertib, a not that complicated molecule to the chemist, which inhibits the binding (by resembling the site on ras that the RBD binds to). It is a styryl benzyl sulfone and you can see the structure here — https://en.wikipedia.org/wiki/Rigosertib.

What’s good about it? Well it is in phase III trials for a fairly uncommon form of cancer (myelodysplastic syndrome). That means it isn’t horribly toxic or it wouldn’t have made it out of phase I.

Given the mechanism described, it is possible that Rigosertib will be useful in 20 – 40% of all cancer. Can you say blockbuster drug?

Do you have a speculative bent? Buy the company testing the drug and owning the patent — Onconova Therapeutics. It’s quite cheap — trading at $.40 (yes 40 cents !). It once traded as high as $30.00 — symbol ONTX. I don’t own any (yet), but for the price of a movie with a beer and some wings afterwards you could be the proud owner of 100 shares. If Rigosertib works, the stock will certainly increase more than a hundredfold.

Enough kidding around. This is serious business. In what follows you will find some hardcore molecular biology and cellular physiology showing just what we’re up against. Some of the following is quite old, and probably out of date (like yours truly), but it does give you the broad outlines of what is involved.

The pathway from Ras to the nucleus

The components of the pathway had been found in isolation (primarily because mutations in them were associated with malignancy). Ras was discovered as an oncogene in various sarcoma viruses. Mutations in ras found in tumors left it in a ‘turned on’ state, but just how ras (and everything else) fit into the chain of binding of a growth factor (such as platelet derived growth factor, epidermal growth factor, insulin, etc. etc.) to its receptor on the cell surface to alterations in gene expression wasn’t clear. It is certain to become more complicated, because anything as important as cellular proliferation is very likely to have a wide variety of control mechanisms superimposed on it. Although all sorts of protein kinases are involved in the pathway it is important to remember that ras is NOT a protein kinase.

l. The first step is binding of a growth factor to its receptor on the cell surface. The receptor is usually a tyrosine kinase. Binding of the factor to the receptor causes ‘activation’ of the receptor. Activation usually means increasing the enzymatic activity of the receptor in the tyrosine kinase reaction (most growth factor receptors are tyrosine kinases). The increase in activity is usually brought about by dimerization of the receptor (so it phosphorylates itself on tyrosine).

2. Most activated growth factor receptors phosphorylate themselves (as well as other proteins) on tyrosine. A variety of other proteins have domains known as SH2 (for src homology 2) which bind to phosphorylated tyrosine.

3. A protein called grb2 binds via its SH2 domain to a phosphorylated tyrosine on the receptor. Grb2 binds to the polyproline domain of another protein called sos1 via its SH3 domain. At this point, the unintiated must find the proceedings pretty hokey, but the pathway is so general (and fundamental) that proteins from yeast may be substituted into the human pathway and still have it work.

4. At last we get to ras. This protein is ‘active’ when it binds GTP, and inactive when it binds GDP. Ras is a GTPase (it can hydrolyze GTP to GDP). Most mutations which make ras an oncogene decrease the GTPase activity of RAS leaving it in a permanently ‘turned on’ state. It is important for the neurologist to know that the defective gene in type I neurofibromatosis activates the GTPase activity of ras, turning ras off. Deficiencies (in ras inactivation) lead to a variety of unusual tumors familiar to neurologists.

Once RAS has hydrolyzed GTP to GDP, the GDP remains bound to RAS inactivating it. This is the function of sos1. It catalyzes the exchange of GDP for GTP on ras, thus activating ras.

5. What does activated ras do? It activates Raf-1 silly. Raf-1 is another oncogene. How does activated ras activate Raf-1 ? Ras appears to activate raf by causing raf to bind to the cell membrane (this doesn’t happen in vitro as there is no membrane). Once ras has done its job of localizing raf to the plasma membrane, it is no longer required. How membrane localization activates raf is less than crystal clear. [ Proc. Natl. Acad. Sci. vol. 93 pp. 6924 – 6928 ’96 ] There is increasing evidence that Ras may mediate its actions by stimulating multiple downstream targets of which Raf-1 is only one.

6. Raf-1 is a protein kinase. Protein kinases work by adding phosphate groups to serine, threonine or tyrosine. In general protein kinases fall into two classes those phosphorylating on serine or threonine and those phosphorylating on tyrosine. Biochemistry has a well documented series of examples of enzymes being activated (or inhibited) by phosphorylation. The best worked out is the pathway from the binding of epinephrine to its cell surface receptor to glycogen breakdown. There is a whole sequence of one enzyme phosphorylating another which then phosphorylates a third. Something similar goes on between Raf-1 and a collection of protein kinases called MAPKs (mitogen activated protein kinases). These were discovered as kinases activated when mitogens bound to their extracellular receptors.There may be a kinase lurking about which activates Raf (it isn’t Ras which has no kinase activity). Removal of phosphate from Raf (by phosphatases) inactivates it.

7. Raf-1 activates members of the MAPK family by phosphorylating them. There may be several kinases in a row phosphorylating each other. [ Science vol. 262 pp. 1065 – 1067 ’93 ] There are at least three kinase reactions at present at this point. It isn’t known if some can be sidestepped. Raf-1 activates mitogen activated protein kinase kinase (MAPK-K) by phosphorylation (it is called MEK in the ras pathway). MAPK-K activates mitogen activation protein kinase (MAPK) by phosphorylation. Thus Raf-1 is actually mitogen activated protein kinase kinase kinase (sort of like the character in Catch-22 named Junior Junior Junior). (1/06 — I think that Raf-1 is now called BRAF)

8. The final step in the pathway is activation of transcription factors (which turn genes off or on) by MAP kinases by (what else) phosphorylation. Thus the pathway from cell surface is complete.

Mind the gap (junction that is)

Gap junctions don’t get much play in pharmacology, or even in neurology, where they are widespread in the central nervous system, linking neurons to neurons, astrocytes to astrocytes. They may get quite a bit more if blocking them is a way of treating metastatic disease (see later).

A bit of background if you’re unfamiliar with them. This is from my notes Molecular Biology of the Cell 4th Edition p. 1074

The gap junction is a cylindrical oligomer composed of 6 identical rod shaped subunits (called connexins). They have 4 transmembrane segments and two extracellular loops which contain a beta-strand structure (and which are an essential structural basis for the docking of the two connexons). Multiple connexons in a membrane tend to form hexagonal arrays.

The gap junction spans the lipid bilayer creating a channel along the central axis. The pore is made of two such protein hexamers one from each cell (called a hemichannel or a connexon) arranged end to end. Different tissues have different specific gap junction proteins (connexins). Man has 14 distinct connexins each encoded by a separate gene (20 homologous proteins in man PNAS 103 pp. 5213 – 5218 ’06). Most cell types express more than one. Connexins are capable of assembling into a heteromeric connexon Adjacent cells expressing different connexins can form intercellular channels in which the two aligned dihalf-channels are different. Each gap junction can contain a cluster of a few to MANY THOUSANDS of CONNEXONs.

Neuroscientists should be interested in them as they form a functional ‘synapse’ between cells, e.g. a way of transferring information between them. For the afficienado there will be much more at the end. To flog a nearly dead horse, this is yet another way a wiring diagram of the brain won’t help you understand it — gap junctions don’t show up when you’re looking at classic synapses. For details see https://luysii.wordpress.com/2011/04/10/would-a-wiring-diagram-of-the-brain-help-you-understand-it/

A recent paper in Nature implied that cancer cells can form gap junctions with astrocytes (a glial cell of the brain). Usually we think of gap junctions being of the same cell type, but not here apparently.

Then they describe a mechanism for the cancer cell tweak the astrocyte so it produces something enabling the cancer cell to survive. Here’s whqt they claim

[ Nature vol. 533 pp. 493 – 498 ’16 ] Human and mouse breast and lung cancer cells express protocadherin7 (PCDH7) whicboth promotes (how?) the assembly of carcinoma – astrocyte gap junctions made of connexin43. PCDH7 normally is only expressed in brain. It joints the stialyl transferase ST6GALNAC5 and neuroserpin as brain restricted proteins which metastastic cells from breast and lung cancer use to colonize the brain.

Metastastic cells then uswe the channels to transfer cGAMP to astrocytes activating the STING pathway, which results in InterferonAlpha (IFNalpha) and Tumor Necrosis Factor (TNF), paracrine signals. These activate STAT1 and NFkappaB in the metastatic cells, supporting tumor growth and chemoresistance.

Meclofenamate and tonabersat are ‘modulators’ of gap junctions, breaking the loop between metastatic cancer cell and the astrocyte. Adding them to the tissue culture studied in the paper, inhibited tumor growth. So here might be a way treat metastatic cancer — particularly since meclofenamate is an FDA approved generic drug available without a prescription.

I think the mechanism described above is incomplete — why should a tumor cell transfer something to another cell to have it secrete something which makes the original cell use something it already had.

Now for a few of the things gap junctions are doing in the brain.
****

[ Neuron vol. 90 pp. 810 – 823 ’16 ] ManhyGABAeric interneurons (are there other kinds?) IN VITRO are coupled by gap junctions. This work used dual patch clamp recordings of interneurons IN VIVO. They studied coupled cerebellar Golgi cells, and showed that, in the presence of spontaneous background synaptic activity, electrically coupled cerebellar Golgi cells showed robust milliSecond precision correlated activity. This was further enhanced by sensory stimulation.

The electrical coupling equlized membrane potential fluctuations, so that coupled neurons approach action potential threshold together. They say that something called spike triggered spikelets transmitted through gap junctions conditionally triggered postJunctional spikes, if both neurons were close to threshold.

Spikelets are brief low amplitude potentials which look like action potentials but which are much smaller. A spike cannot be generated without a much larger potential change than provided by a spikelet, because the spikelet voltage is too small to activate the ion channels of electrically excitable membranes.

So gap junctions controls the temporal precision and degree of both spontaneous and sensory evoked correlated activity betwen interneurons, by the cooperative effects of shared synaptic depolarization and spikelet transmission.

[ Neuron vol. 90 pp. 912 – 913, 1043 – 1056 ’16 ] It has been found that the strength of electrical coupling between neurons in a network is highly variable (even in the same neuron, so it could be coupled at different strengths with each of its partners). Site specific modulation of electrical coupling quickly reconfigures networks of electrically coupled neurons in the retina. Phosphorylation of connexin36 alters its conductivity.

The number of gap junctions determines the strength of ele tical coupling between cerebellar Golgi cells. Ultrastructural analysis shows that gap junctions vary widely in size, which also influences coupling strength (according to a computer simulation). These are dendro-dendritic electrical synapses (widespread in the brain between inhibitory interneurons).

Only 18% or so of the channels present at the gap junctions account for the boserved strength of electrical transmission between cerebellar golgi cells.

Somato-somatic junctions occur in the mammalian trigeminal mesencephalic nucleus. Could the excess junctions be acting as adhesion molecules.

In one system, the turnover of gap junction channel proteins is rapid and comparable with that of glutamic acid receptors.

Gap junctions are ‘low pass filters’ (they pass slow fluctuations of membrane potential better than they pass rapid fluctuations). This is why the electrical synapses are inhibitory — each action potential from a Golgi cell consists of a rapid (but brief) depolarizing spike followed by a relatively deep and protracted afterhyperpolarization — which is 200 times longer than the spike — and transmitted much more effectively.

Inhibition by sparse excitatory input breaks up Golgi network synchronization, because the coupling to adjacent cells is different for each one, causing dispersion of the spikes.

In quietly attentive animals cerebellar Golgi cells generate rhythmic synchronous activity at 8 Hertz. The same behavior is seen in cerebellar slices. The hyperpolarizing electrical post-synaptic potentials (PSPs) are the only synchronizing force. This is the default state, but it can be disrupted by a variety of sensory stimuli (or by movements) which reduce spiking frequency and rhythmicity.

Golgi cells can inhibit thousands of granule cells, and every granule cell gets inhibitory input from 4 – 8 Golgi cells. The transient nature of network desynchronization ‘could’ allow the cerebellar input layer to act as a timing device over the 10 milliSecond to 1 second timescale.

In a gambling mood?

If a pair of posters to be presented Monday 6 June at the 2016 American Society of Clinical Oncology Annual Meeting in Chicago, Illinois, contains the results of a phase III clinical trial of rigosertib, and if the results are as good as a paper discussed below the stock Onconova Therapeutics (ONTX) will jump by a factor of 10 to 100.

Full disclosure: I own some. The posters may just describe the clinical trial rather than report the results in which case all bets are off. In that case, I’ll just hold the stock until the results are in. This isn’t the ‘pump and dump’ beloved of boiler room operators everywhere. The rationale for the drug and my take on the original paper (3 May ’16) are reproduced below.

Has the great white whale of oncology finally been harpooned?

The ras oncogene is the great white whale of oncology. Mutations in 20 – 40% of cancer turn its activity on so that nothing can turn it off, resulting in cellular proliferation. People have been trying to turn mutated ras off for years with no success.

A current paper [ Cell vol. 165 pp. 643 – 655 ’16 ] describes a new and different way to attack it. Once ras is turned on (either naturally or by mutation) many other proteins must bind to it, to produce their effects — they are called RAS effectors, among which are the uneuphoniously named RAF, RalGDS and PI3K. They bind to activated ras by the cleverly named Ras Binding Domain (RBD) which has 78 amino acids.

The paper describes rigosertib, a not that complicated molecule to the chemist, which inhibits the binding (by resembling the site on ras that the RBD binds to). It is a styryl benzyl sulfone and you can see the structure here — https://en.wikipedia.org/wiki/Rigosertib.

What’s good about it? Well it is in phase III trials for a fairly uncommon form of cancer (myelodysplastic syndrome). That means it isn’t horribly toxic or it wouldn’t have made it out of phase I.

Given the mechanism described, it is possible that Rigosertib will be useful in 20 – 40% of all cancer. Can you say blockbuster drug?

Do you have a speculative bent? Buy the company testing the drug and owning the patent — Onconova Therapeutics. It’s quite cheap — trading at $.40 (yes 40 cents !). It once traded as high as $30.00 — symbol ONTX. I don’t own any (yet), but for the price of a movie with a beer and some wings afterwards you could be the proud owner of 100 shares. If Rigosertib works, the stock will certainly increase more than a hundredfold.

Enough kidding around. This is serious business. In what follows you will find some hardcore molecular biology and cellular physiology showing just what we’re up against. Some of the following is quite old, and probably out of date (like yours truly), but it does give you the broad outlines of what is involved.

The pathway from Ras to the nucleus

The components of the pathway had been found in isolation (primarily because mutations in them were associated with malignancy). Ras was discovered as an oncogene in various sarcoma viruses. Mutations in ras found in tumors left it in a ‘turned on’ state, but just how ras (and everything else) fit into the chain of binding of a growth factor (such as platelet derived growth factor, epidermal growth factor, insulin, etc. etc.) to its receptor on the cell surface to alterations in gene expression wasn’t clear. It is certain to become more complicated, because anything as important as cellular proliferation is very likely to have a wide variety of control mechanisms superimposed on it. Although all sorts of protein kinases are involved in the pathway it is important to remember that ras is NOT a protein kinase.

l. The first step is binding of a growth factor to its receptor on the cell surface. The receptor is usually a tyrosine kinase. Binding of the factor to the receptor causes ‘activation’ of the receptor. Activation usually means increasing the enzymatic activity of the receptor in the tyrosine kinase reaction (most growth factor receptors are tyrosine kinases). The increase in activity is usually brought about by dimerization of the receptor (so it phosphorylates itself on tyrosine).

2. Most activated growth factor receptors phosphorylate themselves (as well as other proteins) on tyrosine. A variety of other proteins have domains known as SH2 (for src homology 2) which bind to phosphorylated tyrosine.

3. A protein called grb2 binds via its SH2 domain to a phosphorylated tyrosine on the receptor. Grb2 binds to the polyproline domain of another protein called sos1 via its SH3 domain. At this point, the unintiated must find the proceedings pretty hokey, but the pathway is so general (and fundamental) that proteins from yeast may be substituted into the human pathway and still have it work.

4. At last we get to ras. This protein is ‘active’ when it binds GTP, and inactive when it binds GDP. Ras is a GTPase (it can hydrolyze GTP to GDP). Most mutations which make ras an oncogene decrease the GTPase activity of RAS leaving it in a permanently ‘turned on’ state. It is important for the neurologist to know that the defective gene in type I neurofibromatosis activates the GTPase activity of ras, turning ras off. Deficiencies (in ras inactivation) lead to a variety of unusual tumors familiar to neurologists.

Once RAS has hydrolyzed GTP to GDP, the GDP remains bound to RAS inactivating it. This is the function of sos1. It catalyzes the exchange of GDP for GTP on ras, thus activating ras.

5. What does activated ras do? It activates Raf-1 silly. Raf-1 is another oncogene. How does activated ras activate Raf-1 ? Ras appears to activate raf by causing raf to bind to the cell membrane (this doesn’t happen in vitro as there is no membrane). Once ras has done its job of localizing raf to the plasma membrane, it is no longer required. How membrane localization activates raf is less than crystal clear. [ Proc. Natl. Acad. Sci. vol. 93 pp. 6924 – 6928 ’96 ] There is increasing evidence that Ras may mediate its actions by stimulating multiple downstream targets of which Raf-1 is only one.

6. Raf-1 is a protein kinase. Protein kinases work by adding phosphate groups to serine, threonine or tyrosine. In general protein kinases fall into two classes those phosphorylating on serine or threonine and those phosphorylating on tyrosine. Biochemistry has a well documented series of examples of enzymes being activated (or inhibited) by phosphorylation. The best worked out is the pathway from the binding of epinephrine to its cell surface receptor to glycogen breakdown. There is a whole sequence of one enzyme phosphorylating another which then phosphorylates a third. Something similar goes on between Raf-1 and a collection of protein kinases called MAPKs (mitogen activated protein kinases). These were discovered as kinases activated when mitogens bound to their extracellular receptors.There may be a kinase lurking about which activates Raf (it isn’t Ras which has no kinase activity). Removal of phosphate from Raf (by phosphatases) inactivates it.

7. Raf-1 activates members of the MAPK family by phosphorylating them. There may be several kinases in a row phosphorylating each other. [ Science vol. 262 pp. 1065 – 1067 ’93 ] There are at least three kinase reactions at present at this point. It isn’t known if some can be sidestepped. Raf-1 activates mitogen activated protein kinase kinase (MAPK-K) by phosphorylation (it is called MEK in the ras pathway). MAPK-K activates mitogen activation protein kinase (MAPK) by phosphorylation. Thus Raf-1 is actually mitogen activated protein kinase kinase kinase (sort of like the character in Catch-22 named Junior Junior Junior). (1/06 — I think that Raf-1 is now called BRAF)

8. The final step in the pathway is activation of transcription factors (which turn genes off or on) by MAP kinases by (what else) phosphorylation. Thus the pathway from cell surface is complete.

Has the great white whale of oncology finally been harpooned?

The ras oncogene is the great white whale of oncology. Mutations in 20 – 40% of cancer turn its activity on so that nothing can turn it off, resulting in cellular proliferation. People have been trying to turn mutated ras off for years with no success.

A current paper [ Cell vol. 165 pp. 643 – 655 ’16 ] describes a new and different way to attack it. Once  ras is turned on (either naturally or by mutation) many other proteins must bind to it, to produce their effects — they are called RAS effectors, among which are the uneuphoniously named RAF, RalGDS and PI3K. They bind to activated ras by the cleverly named Ras Binding Domain (RBD) which has 78 amino acids.

The paper describes rigosertib, a not that complicated molecule to the chemist, which inhibits the binding (by resembling the site on ras that the RBD binds to). It is a styryl benzyl sulfone and you can see the structure here — https://en.wikipedia.org/wiki/Rigosertib.

What’s good about it? Well it is in phase III trials for a fairly uncommon form of cancer (myelodysplastic syndrome). That means it isn’t horribly toxic or it wouldn’t have made it out of phase I.

Given the mechanism described, it is possible that Rigosertib will be useful in 20 – 40% of all cancer. Can you say blockbuster drug?

Do you have a speculative bent? Buy the company testing the drug and owning the patent — Oncova Therapeutics. It’s quite cheap — trading at $.40 (yes 40 cents !). It once traded as high as $30.00 — symbol ONTX. I don’t own any (yet), but for the price of a movie with a beer and some wings afterwards you could be the proud owner of 100 shares. If Rigosertib works, the stock will certainly increase more than a hundredfold.

Enough kidding around. This is serious business. In what follows you will find some hardcore molecular biology and cellular physiology showing just what we’re up against. Some of the following is quite old, and probably out of date (like yours truly), but it does give you the broad outlines of what is involved.

The pathway from Ras to the nucleus

The components of the pathway had been found in isolation (primarily because mutations in them were associated with malignancy). Ras was discovered as an oncogene in various sarcoma viruses. Mutations in ras found in tumors left it in a ‘turned on’ state, but just how ras (and everything else) fit into the chain of binding of a growth factor (such as platelet derived growth factor, epidermal growth factor, insulin, etc. etc.) to its receptor on the cell surface to alterations in gene expression wasn’t clear. It is certain to become more complicated, because anything as important as cellular proliferation is very likely to have a wide variety of control mechanisms superimposed on it. Although all sorts of protein kinases are involved in the pathway it is important to remember that ras is NOT a protein kinase.

l. The first step is binding of a growth factor to its receptor on the cell surface. The receptor is usually a tyrosine kinase. Binding of the factor to the receptor causes ‘activation’ of the receptor. Activation usually means increasing the enzymatic activity of the receptor in the tyrosine kinase reaction (most growth factor receptors are tyrosine kinases). The increase in activity is usually brought about by dimerization of the receptor (so it phosphorylates itself on tyrosine).

2. Most activated growth factor receptors phosphorylate themselves (as well as other proteins) on tyrosine. A variety of other proteins have domains known as SH2 (for src homology 2) which bind to phosphorylated tyrosine.

3. A protein called grb2 binds via its SH2 domain to a phosphorylated tyrosine on the receptor. Grb2 binds to the polyproline domain of another protein called sos1 via its SH3 domain. At this point, the unintiated must find the proceedings pretty hokey, but the pathway is so general (and fundamental) that proteins from yeast may be substituted into the human pathway and still have it work.

4. At last we get to ras. This protein is ‘active’ when it binds GTP, and inactive when it binds GDP. Ras is a GTPase (it can hydrolyze GTP to GDP). Most mutations which make ras an oncogene decrease the GTPase activity of RAS leaving it in a permanently ‘turned on’ state. It is important for the neurologist to know that the defective gene in type I neurofibromatosis activates the GTPase activity of ras, turning ras off. Deficiencies (in ras inactivation) lead to a variety of unusual tumors familiar to neurologists.

Once RAS has hydrolyzed GTP to GDP, the GDP remains bound to RAS inactivating it. This is the function of sos1. It catalyzes the exchange of GDP for GTP on ras, thus activating ras.

5. What does activated ras do? It activates Raf-1 silly. Raf-1 is another oncogene. How does activated ras activate Raf-1 ?  Ras appears to activate raf by causing raf to bind to the cell membrane (this doesn’t happen in vitro as there is no membrane). Once ras has done its job of localizing raf to the plasma membrane, it is no longer required. How membrane localization activates raf is less than crystal clear. [ Proc. Natl. Acad. Sci. vol. 93 pp. 6924 – 6928 ’96 ] There is increasing evidence that Ras may mediate its actions by stimulating multiple downstream targets of which Raf-1 is only one.

6. Raf-1 is a protein kinase. Protein kinases work by adding phosphate groups to serine, threonine or tyrosine. In general protein kinases fall into two classes those phosphorylating on serine or threonine and those phosphorylating on tyrosine. Biochemistry has a well documented series of examples of enzymes being activated (or inhibited) by phosphorylation. The best worked out is the pathway from the binding of epinephrine to its cell surface receptor to glycogen breakdown. There is a whole sequence of one enzyme phosphorylating another which then phosphorylates a third. Something similar goes on between Raf-1 and a collection of protein kinases called MAPKs (mitogen activated protein kinases). These were discovered as kinases activated when mitogens bound to their extracellular receptors.There may be a kinase lurking about which activates Raf (it isn’t Ras which has no kinase activity). Removal of phosphate from Raf (by phosphatases) inactivates it.

7. Raf-1 activates members of the MAPK family by phosphorylating them. There may be several kinases in a row phosphorylating each other. [ Science vol. 262 pp. 1065 – 1067 ’93 ] There are at least three kinase reactions at present at this point. It isn’t known if some can be sidestepped. Raf-1 activates mitogen activated protein kinase kinase (MAPK-K) by phosphorylation (it is called MEK in the ras pathway). MAPK-K activates mitogen activation protein kinase (MAPK) by phosphorylation. Thus Raf-1 is actually mitogen activated protein kinase kinase kinase (sort of like the character in Catch-22 named Junior Junior Junior). (1/06 — I think that Raf-1 is now called BRAF)

8. The final step in the pathway is activation of transcription factors (which turn genes off or on) by MAP kinases by (what else) phosphorylation. Thus the pathway from cell surface is complete.

Is that mutation significant?

Face it, our genomes are a real mess. A study of just the parts of the genome coding for amino acids (2% at most) in about 2,500 people found an average of 205 variants which change the amino acid coded for IN EACH PERSON. Each person also had an average of 3 termination codons in the 15,000+ protein coding sequences they studied. So they are wandering around with 3 abnormally short proteins. You can read more about it in this old post –https://luysii.wordpress.com/2012/07/31/how-badly-are-thy-genomes-oh-humanity/

Here’s the problem — these people were healthy. Obviously, not a problem for them, but a big problem for physicians attempting to do genetic counseling. For how it affected epilepsy counseling see — https://luysii.wordpress.com/2011/07/17/weve-found-the-mutation-causing-your-disease-not-so-fast-says-this-paper/.

This brings us to Lynch syndrome (aka Hereditary NonPolyposis Colorectal Cancer — HNPCC). It is a familial cancer syndrome, and we now know what the problem is — mutations in any of four genes involved in a type of DNA mutation repair (there are many). The genes are called MSH2, MSH6, MLH1 and PMS2 (acronyms all whose names you don’t need to know) and the type of repair is called MisMatch Repair (MMR).

This isn’t academic at all. Suppose your aunt comes down with colon cancer and you get tested for mutations in one of the four, and a mutation is found. You’re fine now. The question before the house is — should you have your colon out? Colonoscopy won’t help because this kind of colon cancer doesn’t arise from polyps (which is what colonoscopy is looking for).

The problem is that the 4 genes are ‘peppered’ with missense variants (change the amino acid coded for). They are called VUS (Variants of Unknown Significance). The following paper [ Proc. Natl. Acad. Sci. vol. 113 pp. 3918 – 3820, 4128 – 4133 ’16 ] used a clever way to test a VUS for significance. This would have been impossible 5 years ago. What they did was use CRISPR to introduce the variant into the appropriate protein in mouse Embryonic Stem cells. Then they tested the manipulated stem cells for defects in MisMatch Repair. They tested 59 (yes fifty-nine) such VUSs and found that about 1/3 (19) produced MMR defects.

Fascinating time to be alive and reading about all this stuff.

Activating a proto-oncogene without mutating it

Many proto-oncogenes have to be mutated to cause cancer. Not so the TAL1, LMO2 genes. They drive blood formation, and are aberrantly activated (e.g. more proteins made from them is expressed) in T cell Acute Lymphoblastic Leukemia (TALL). [ Science vol. 351 pp. 1298- 1299, 1454 – 1458 ’16 ] activated them experimentally using the CRISPR technique, and therein hangs a tale.

Addendum 11 April — LMO2 is well known to gene therapists as early work (2002) using retroviruses inserted randomly in the genome to cure SCID (Severe Combined Immunodeficiency) resulted in TALL in 4kids.  The problem was that the vector integrated in multiple sites all over the genome and one such random site  turned on expression of LMO2.

I’ve written a series of six posts trying to imagine the incredible mass of DNA in a 10 micron nucleus on a human scale — we take it for granted, but it’s far from obvious how this is accomplished — here’s the link to the first — https://luysii.wordpress.com/2010/03/22/the-cell-nucleus-and-its-dna-on-a-human-scale-i/. — just follow the links to the rest.

[ Cell vol. 153 pp. 1187 – 1189, 1281 – 1295 ’13 ] Hi-C and 5C (Carbon Copy Chromosome Conformation Capture) allow determination of chromatin organization and long range chromatin interactions in an unbiased genome wide manner at the megaBase scale. Topologically associated domains (TADs) are the way the genome in the nucleus is organized into megabase to submegaBase sized interacting domains. TADs are conserved between species and are invariant across cell types. [ Call vol. 156 p. 19 ’14 ] They average 700 – 800 kiloBases and are said to contain 5 – 10 protein coding genes and a few hundred enhancers. The expression of genes within a TAD is ‘somewhat correlated’. Some TADs have active genes, while others have repressed genes. Genomic interactions are strong within a domain, but are sharply depleted on crossing the boundary between two TADs.

Well TADs have to be separated from each other. The current thinking is that the boundaries are formed by sites in the DNA which bind the CTCF protein, and possibly cohesin proteins as well. CTCF is a large protein (although maddeningly I can’t seem to find out how many amino acids it has) with a molecular mass of 80 kiloDaltons. It’s DNA binding is quite specific as it contains 11 zinc fingers (each of which can specifically bind a 3 nucleotide stretch of DNA). In addition to binding to DNA it can bind to itself, forming a perfect way to form loops of DNA.

All the Science paper did was to delete a few CTCF binding sites using the CRISPR technique around the two oncogenes and bang — expression increased. Why?  Because the insulation between the TAD containing the genes and adjacent TADs was broken, allowing control of the genes by enhancers in the new and larger TAD that had been previously sequestered in an adjacent TAD.  The deletions were thousands of basepairs away from the coding sequence of the genes themselves.  All very nice, but it’s fairly artificial.

However the paper notes that across a large pan-cancer cohort, there was a 2 fold enrichment for boundary CTCF site mutations.

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