MicroExons

MicroExons have been known for a long time.  They are hard to find using the usual software tools because they are short, coding for just 1 (!) to 9 amino acids (3 – 27 nucleotides).  The neurologist is interested in them, because they are enriched in neurons.

 

The evolutionist is interested in them because they are found on the surfaces of proteins, so that adding their amino acids potentially modifies protein protein interactions (example later) since these are largely determined at the protein surface.  The typical protein interface is said to be a surface of 1,000 – 2,000 square Angstroms, so putting a few amino acids in the surface can change things radically.   Not only that, but alanine scanning of the interfaces shows that only a small set of ‘hot spot’ amino acids contribute to the free energy of binding at the protein/protein interface.  (Hot spots are operationally defined as an amino acid, that when mutated to alanine leads to a greater than 10fold drop in the binding constant).  Let’s hear it for the blind watchmaker for figuring out a way to accelerate evolution by moderating protein protein interactions directly.

It is interesting  that the vast majority of microexons contain multiples of 3 nucleotides (this prevents them from producing a frameshift in the mRNA in which they are found).  This implies that natural selection is at work on them.

Most microExons show high inclusion rates at late stages of neuronal differentiation in genes associated with axon formation and synapse function.  One example — a neural specific microExon in Protrudin increases its interaction with Vesicle Associated Membrane protein VAMP) to promote neurite outgrowth.

A protein called SRRM4 controls the inclusion of most neuronal microexons known so far.  Of all known tissue types the human retina has the largest program of tissue enriched microExons .  Some of these microexons are found only in photoreceptor cells. Ectopic expression of SRRM4 is enough to drive the inclusion of most retinal microExons in nonphotorector cells.

For lots of current references on microExons, particularly those in the retina, please see Proc. Natl. Acad. Sci. vol. 119 e2117090119 ’22

I haven’t been posting for a while because of a computer disaster.  All of the notes I’ve taken on the literature and elsewhere were on an old iMac running HyperCard (which just crashed). As my son was told at USC, there are two types of disc drives.  Those that have crashed, and those that haven’t crashed (yet).  I’ve got another one, but I’m busy programming to transfer the data and metadata to Mathematica.  There is plenty to post about, which should be forthcoming.

Brilliant structural work on the Arp2/3 complex with actin filaments and why it makes me depressed

The Arp2/3 complex of 5 proteins forms side branches on existing actin filaments.  The following paper shows its beautiful structure along with movies.  Have a look — it’s open access. https://www.pnas.org/doi/10.1073/pnas.2202723119.

Why should it make me depressed? Because I could spend the next week studying all the ins and outs of the structure and how it works without looking at anything else.  Similar cryoEM studies of other multiprotein machines are coming out which will take similar amounts of time.  Understanding how single enzymes work is much simpler, although similarly elegant — see Cozzarelli’s early work on topoisomerase.

So I’m depressed because I’ll never understand them to the depth I understand enzymes, DNA, RNA etc. etc.

Also the complexity and elegance of these machines brings back my old worries about how they could possibly have arisen simply by chance with selection acting on them.  So I plan to republish a series of old posts about the improbability of our existence, and the possibility of a creator, which was enough to me get thrown off Nature Chemistry as a blogger.

Enough whining.

Here is why the Arp2/3 complex is interesting.  Actin filaments are long (1,000 – 20,000 Angstroms and thin (70 Angstroms).  It you want to move a cell forward by having them grow toward its leading edge, growing actin filaments would puncture the membrane like a bunch of needles, hence the need for side branches, making actin filaments a brush-like mesh which could push the membrane forward as it grows.

The Arp2/3 complex has a molecular mass of 225 kiloDaltons, or probably 2,250 amino acids or 16 thousand atoms.

Arp2 stands for actin related protein 2, something quite similar to the normal actin monomer so it can sneak into the filament. So can Arp3.  The other 5 proteins grab actin monomers and start them polymerizing as a branch.

But even this isn’t enough, as Arp2/3 is intrinsically inactive and multiple classes of nucleation promoting factors (NPFs) are needed to stimulate it.  One such NPF family is the WASP proteins (for Wiskott Aldrich Syndrome Protein) mutations of which cause the syndrome characterized by hereditary thrombocytopenia, eczema and frequent infections.

The paper’s pictures do not include WASP, just the 7 proteins of the complex snuggling up to an actin filament.

In the complex the Arps are in a twisted conformation, in which they resemble actin monomers rather than filamentous actin subunits which have a flattened conformation.  After activation arp2 and arp3 mimic the arrangement of two consecutive subunits along the short pitch helical axis of an actin filament and each arp transitions from a twisted (monomerLike) to a flattened (filamentLike) conformation.

So look at the pictures and the movies and enjoy the elegance of the work of the Blind Watchmaker (if such a thing exists).

Barking up the wrong therapeutic tree in Alzheimer’s disease

Billions have been spent by big pharma (and lost) trying to get rid of the senile plaque of Alzheimer’s disease.  The assumption has always been that the plaque is the smoking gun killing neurons.  But it’s just an assumption which a recent paper has turned on its ear [ Proc. Natl. Acad. Sci. vol. 116 23040 – 23049 ’19 ]

It involves a protein, likely to be a new face even to Alzheimer’s cognoscenti.  The protein is called SERF1A (in man) and MOAG-4 in yeast. It enhances amyloid formation, the major component of the senile plaque.  SERF1A is clearly doing something important as it has changed little from the humble single yeast cell to man.

The major component of the senile plaque is the aBeta peptide of 40 and/or 42 amino acids.  It polymerizes to form the amyloid of the plaque.  The initial step of amyloid formation is the hardest, getting a bunch of Abeta peptides into the right conformation (e.g. the nucleus) so others can latch on to it and form the amyloid fiber.   It is likely that the monomers and oligomers of Abeta are what is killing neurons, not the plaques, otherwise why would natural selection/evolution keep SERF1A around?

So, billions of dollars later, getting rid of the senile plaque turns out to be a bad idea. What we want to do is increase SERF1A activity, to sop up the monomers and oligomers. It is a 180 degree shift in our thinking. That’s the executive summary, now for the fascinating chemistry involved.

First the structure of SERF1A — that is to say its amino acid sequence.  (For the nonChemists — proteins are linear string of amino acids, just as a word is a linear string of characters — the order is quite important — just as united and untied mean two very different things). There are only 68 amino acids in SERF1A of which 14 are lysine 9 are arginine 5 Glutamic acid and 5 Aspartic acid.  That’s interesting in itself, as we have 20 different amino acids, and if they occurred randomly you’d expect about 3 -4 of each.  The mathematicians among you should enjoy figuring out just how improbable this compared to random assortment. So just four amino acids account for 33 of the 68 in SERF1A  Even more interesting is the fact that all 4 are charged at body pH — lysine and arginine are positively charged because their nitrogen picks up protons, and glutamic and aspartic acid are negatively charged  giving up the proton.

This means that positive and negative can bind to each other (something energetically quite favorable).  How many ways are there for the 10 acids to bind to the 23 bases?  Just 23 x 22 x 21 X 20 X 19 X 18 x 17 x 16 x 15 x 14 or roughly 20^10 ways.  This means that SERF1A doesn’t have a single structure, but many of them.  It is basically a disordered protein.

The paper shows exactly this, that several conformations of SERF1 are seen in solution, and that it binds to Abeta forming a ‘fuzzy complex’, in which the number of Abetas and SERF1s are not fixed — e.g. there is no fixed stoichiometry — something chemists are going to have to learn to deal with — see — https://luysii.wordpress.com/2018/12/16/bye-bye-stoichiometry/.  Also different conformations of SERF1A are present in the fuzzy complex, explaining why it has such a peculiar amino acid composition.  Clever no?  Let’s hear it for the blind watchmaker or whatever you want to call it.

The paper shows that SERF1 increases the rate at which Abeta forms the nucleus of the amyloid fiber.  It does not help the fiber grow.  This means that the fiber is good and the monomers and oligomers are bad.  Not only that but SERF1 has exactly the same effect with alpha-synuclein, the main protein of the Lewy body of Parkinsonism.

So the paper represents a huge paradigm shift in our understanding of the cause of at least 2 bad neurological diseases.   Even more importantly, the paper suggests a completely new way to attack them.

Does gamma-secretase have sex with its substrates?

This is a family blog (for the most part), so discretion is advised in reading further.   Billions have been spent trying to inhibit gamma-secretase.  Over 150 different mutations have been associated with familial Alzheimer’s disease.  The more we know about the way it works, the better.

A recent very impressive paper from China did just that [ Science vol. 363 pp. 690- 691, 701 eaaw0930 pp. 1 –> 8 ’19 ].

Gamma secretase is actually a combination of 4 proteins (presenilin1, nicastrin, APH1 (anterior pharynx defect) and PEN-2 (presenilin enhancer 2). It is embedded in membranes and has at least 19 transmembrane segments.  It cleaves a variety of proteins spanning membranes (e.g it hydrolyzes a peptide bond — which is just an amide).  The big deal is that cleavage occurs in the hydrophobic interior of the membrane rather than in the cytoplasm where there is plenty of water around.

Gamma secretase cleaves at least 20 different proteins this way, not just the amyloid precursor protein, one of whose cleavage products is the Abeta peptide making up a large component of the senile plaque of Alzheimer’s disease.

To get near gamma secretase, another enzyme must first cleave APP in another place so one extramembrane fragment is short.  Why so the rest of the protein can fit under a loop between two transmembrane helices of nicastrin.  This is elegance itself, so the gamma secretase doesn’t go around chopping up the myriad of extracellular proteins we have.

The 19 or so transmembrane helices of the 4 gamma secretase proteins form a horseshoe, into which migrates the transmembrane segment of the protein to be cleaved (once it can fit under the nicastrin loop).

So why is discretion advised before reading further?  Because the actual mechanism of cleavage involves intimate coupling of the proteins.    One of the transmembrane helices of presenilin1 unfolds to form two beta strands, and the transmembrane helix of the target protein unfolds to form one beta strand, the two strands pair up forming a beta sheet, and then the aspartic acid at the active site of gamma secretase cleaves the target (deflowers it if you will).  Is this sexual or what?

All in all another tribute to ingenuity (and possibly the prurience) of the blind watchmaker. What an elegant mechanism.

Have a look at the pictures in the Science article, but I think it is under a paywall.

Goodbye to the blind watchmaker — take I

The Michelson and Morley experiment destroyed the ether paradigm in 1887, but its replacement didn’t occur until Einstein’s special relativity in 1905.  One can disagree with a paradigm without being required to come up with something to replace it. Unfortunately, we tend to think in dichotomies, so disagreeing with the blind watchmaker hypothesis for life itself tends to place you in the life was created by some sort of conscious entity.  “Hypotheses non fingo”  (Latin for “I feign no hypotheses”) which is what  Newton famously said  when discussing action at a distance which his theory of gravity entailed (which he thought was pretty crazy).

Here are  summaries of four previous posts (with links) showing why I have problems accepting the blind watchmaker hypothesis.  These are not arguments from faith which nowhere appears, but deduction from experimental facts about the structures and processes which make life possible. Be warned.  This is hard core chemistry, biochemistry and molecular biology.

First the 20,000 or so proteins which make us up, a nearly vanishing fraction of the possible proteins.  For how vanishing see — https://luysii.wordpress.com/2009/12/20/how-many-proteins-can-be-made-using-the-entire-earth-mass-to-do-so/.  Just start with 20 amino acids, 400 dipeptides, 8000 tripeptides.  Make one molecule of each and see how long a protein you wind up with making all possibilities along the way.  The answer will surprise you.

Next the improbability of a protein having a single shape (or a few shapes) for some chemical arguments about this — see https://luysii.wordpress.com/2010/08/04/why-should-a-protein-have-just-one-shape-or-any-shape-for-that-matter/

After that — have a look at https://luysii.wordpress.com/2010/10/24/the-essential-strangeness-of-the-proteins-that-make-us-up/.

The following quote is from an old book on LISP programming (Let’s Talk LISP) by Laurent Siklossy.“Remember, if you don’t understand it right away, don’t worry. You never learn anything, you only get used to it.”   Basically I think biochemists got used to thinking of proteins have ‘a’ shape or a few shapes because that’s what they found when they studied them.

If you think of amino acids as letters, then proteins are paragraphs of them, but to have biochemical utility they must have ‘meaning’ e.g. a constant shape.

Obviously the ones making us do have shapes, but how common is this in the large universe of possible proteins.  Here is an experiment which might show us (or not)– https://luysii.wordpress.com/2010/08/08/a-chemical-gedanken-experiment/.

From a philosophical point of view, the experiment is quite specific.  From a practical point of view quite possible to start, but impossible to carry to completion.

Well this is a lot of reading to do (assuming anyone does it) and I’ll stop now (although there is more to come).

Why do this at all?  Because I’ve been around long enough to see authoritative statements (by very authoritative figures) crash and burn.  Most of them I didn’t believe at the time — here are a few

l. The club of Rome’s predictions

2. The population bomb of Ehrlich

3. Junk DNA

4. We are 98% Chimpanzee because our proteins are that similar.

5. Gunther Stent, very distinguished molecular biologist, writing that we were close to the end of our understanding of genetic biology.  This in 1969.

The links elaborate several reasons why I find the Blind Watchmaker hypothesis difficult to accept.  There is more to come.

“Hypotheses non fingo”

Let’s hear it for the blind watchmaker

The blind watchmaker had a lot of foresight in choosing to use a rather  funky looking amino acid (proline) resembling none of the others.  A lot of kindness was also shown to structural molecular biologists by two of the watchmaker’s henchmen – Burkholderia gladioli and the common daisy.

All appear in a fascinating paper [ Cell vol. 176 pp. 435 – 447  ’19 ] in which the structure and better the mechanism of action of the mitochondrial ADP/ATP translocase, a molecule of some interest since our mitochondria make our body weight of ATP each day and need some way to get it out into the cytoplasm where it is used.

The molecule has quite a job to do, getting the rather large ATP molecule out to the intermembrane space (and thence out to the cytoplasm) without allowing protons to sneak out with it, since it is the proton gradient which is used to power ATP synthase the exquisite machine which makes ATP.   This is quite a trick as no chemical moiety is as small as a proton.

The translocase has two states — one in which it is open to the mitochondrial matrix (called the m-state) and another in which it is open (eventually) to the cytoplasm — called the c-state. In the m-state the cytoplasmic portion is shut, and in the c-state the membrane portion is shut.

The rather wierd looking molecule bongkrekic acid  made by Burkholderia gladioli  https://en.wikipedia.org/wiki/Bongkrek_acid binds to the translocase fixing it in the m-state.  Atractyloside, made by daisies binds to the molecule fixing it in the c-state.  They made life much easier for the structural biologist and cryoEMographers who wrote the paper.

Proline comes in because when placed in an alpha helix, proline’s 5 membered ring structure fixes the alpha carbon so that it is essentially inflexible, meaning that it can’t get into the conformation that the other 19 amino acids can get into when an alpha helix is formed.  Translation — proline is a helix breaker, forming a kink in the helix.

The translocase contains 3 modules of 100 amino acids each of which has 2 alpha helices, one of them containing a proline causing a kink in the helix.  The prolines are in the middle of the helix.  The ATP channel is formed by the 6 helices.

Essentially in the middle of the membrane, the kinked alpha helices form a pivot (fulcrum), so the helices rock back and forth, opening one side while simultaneously shutting the other, permitting ATP to bind near the fulcrum without letting anything else through, when the pivot shifts   — out goes the ATP (without letting protons sneak past).

There is far more beautiful protein chemistry on display.  There is a conserved signature motif Proline x Aspartic acid/Glutamic acid X X Lysine/Arginine at the carboxy terminal end of one of the helices of each other 3 modules — this forms a salt bridge shutting the channel on the matrix side.  Glycine and other small amino acids (alanine) allow close packing of the helices on the cytoplasmic side.

It is unfortunate that the most of humanity doesn’t have the background to appreciate the elegance and beauty of Nature’s solution to the problem.  I say Nature rather than God to be scientifically correct, but it’s elegant chemistry like this that makes it hard for me to accept that it arose by the machinations of a blind watchmaker.

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.

Are you as smart as the (inanimate) blind watchmaker

Here’s a problem the cell has solved. Can you? Figure out a way to send a protein to two different membranes in the cell (the membrane encoding it { aka the plasma membrane }, and the endoplasmic reticulum) in the proportions you wish.

The proteins must have exactly the same sequence and content of amino acids, ruling out alternative splicing of exons in the mRNA (if this is Greek to you have a look at the following post — https://luysii.wordpress.com/2012/01/09/molecular-biology-survival-guide-for-chemists-v-the-ribosome/ and the others collected under — https://luysii.wordpress.com/category/molecular-biology-survival-guide/).

The following article tells you how the cell does it. Recall that not all of the messenger RNA (mRNA) is translated into protein. The ribosome latches on to the 5′ end of the mRNA,  subsequently moving toward the 3′ end until it finds the initiator codon (AUG which codes for methionine). This means that there is a 5′ untranslated region (5′ UTR). It then continues moving 3′ ward stitching amino acids together.  Similarly after the ribosome reaches the last codon (one of 3 stop codons) there is a 3′ untranslated region (3′ UTR) of the mRNA. The 3′ UTR isn’t left alone but is cleaved and a polyAdenine tail added to it. The 3′ UTR is where most microRNAs bind controlling mRNA stability (hence the amount of protein produced from a given mRNA).

The trick used by the cell is described in [ Nature vol. 522 pp. 363 – 367 ’15 ]. The 3’UTR is alternatively processed producing a variety of short and long 3’UTRs. One such protein where this happens is CD47 — which is found on the surface of most cells where it stops the cell from being eaten by scavenger cells such as macrophages. The long 3′ UTR of CD47 allows efficient cell surface expression, while the short 3′ UTR localizes it to the endoplasmic reticulum.

How could this possibly work? Once the protein is translated by the ribosome, it leaves the ribosome and the mRNA doesn’t it? Not quite.

They say that the long 3′ UTR of CD47 acts as a scaffold to recruit a protein complex which contains HuR (aka ELAVL1), an RNA binding protein and SET to the site of translation. The allows interaction of SET with the newly translated cytoplasmic domains of CD47, resulting in subsequent translocation of CD47 to the plasma membrane via activated RAC1.

The short 3′ UTR of CD47 doesn’t have the sequence binding HuR and SET, so CD47 doesn’t get to the plasma membrane, rather to the endoplasmic reticulum.

The mechanism may be quite general as HuR binds to thousands of mRNAs. The paper gives two more examples of proteins where this happens.

It’s also worth noting that all this exquisite control, does NOT involve covalent bond formation and breakage (e.g. not what we consider classic chemical reactions). Instead it’s the dance of one large molecular object binding to another in other ways. The classic chemist isn’t smiling. The physical chemist is.

Making an enzyme do a carbene reaction

The P450 cytochrome enzymes are the chemical workhorses of biosynthesis and xenobiotic detoxification.  As a neurologist I always had to worry about the way adding a new anticonvulsant to a an existing set would alter the body levels of all of them.  The new anticonvulsant would cause the P450 cytochrome enzyme metabolizing it to increase, and the new P450 might also chew up some of the others.  Phenobarbital was a particular problem this way.

Over 14,500 P450 genes have been found in various organisms. FYI the 450 in P450 comes from the absorption at 4500 Angstroms of the Fe++ in the heme group when complexed to carbon monoxide.  This is called the Soret band.  The P450 cytochromes are a superfamily comprising 36 known gene families, each consisting of 2 -20 discrete cytochromes.

The P450 cytochromes take 2 electrons from a reduced cofactor (NADPH) which itself gets the electrons from another molecule.  The cytochrome then binds an oxygen molecule splitting it so an oxygen atom is bound to the Fe++  (the other oxygen goes to water).  The oxygen is then used by the various enzymes to insert into C-H bonds with an incredible specificity (this produced by the conformation of the protein around the heme-iron-oxygen).

In Science vol. 339 pp. 283 – 284, 307 – 310 ’13 a group at Caltech which has been using direct evolution (randomly mutate a protein and select for the reaction you want) used a P450 cytochrome to accept diazo esters as reagents.  This formed a carbene linked to the Fe.  They found that it would add to olefins.  Further engineering (random selection actually) produced variants of the P450 to produce diastereoselective and enantioselective cyclopropanation of styrene.

It would be nice to see the crystal structures of the engineered enzymes carrying out the reactions, to see how they constrain the styrene to react so specifically.

So here we have chemists acting like the blind watchmaker of Dawkins.  It would be nice if theory was good enough to predict and design the P450 variants produced here a prioi, but it isn’t.  The editorialist calls it biomimicry in reverse.