Location bias

Location bias:  no this isn’t about real estate or red lining.  It’s about how drugs act differently depending on where they’re able to get.  If this sounds too abstract, location bias may explain why dimethyl tryptamine (DMT) is a hallucinogen (it is the main psychoactive component of ayahuasca) and why serotonin (5 hydroxy tryptamine) is not.

The psychoactive effects of many drugs (LSD, DMT) are explained by their binding to one of the many (> 13) subtypes of serotonin receptors, namely 5HT2AR.

Well serotonin certainly binds to 5HT2AR, so why doesn’t it produce hallucinations?  This is where [ Science vol. 379 pp. 700 – 706 ’23 ] (and local bias) comes in.

We tend to think of receptors for neurotransmitters (like serotonin) as being on the outer membrane of the cell (the plasma membrane).  This makes sense as neurotransmitters are released from neurons into the extracellular space.  However it is now known that some neurotransmitter receptors (such as 5HT2AR) are found inside the cell where they are found on endosomes and the Golgi apparatus.

The article claims that the hallucinogenic effects of DMT, LSD etc. etc. are due to their binding to 5HT2ARs found inside the cell, not those on the plasma membrane. Serotonin with its free OH and NH2 groups is simply too water soluble (hydrophilic) to pass through the lipids of the plasma membrane.   DMT and LSD are not.   Unfortunately we are a long way from understanding how activation of 5HT2ARs inside the cell leads to hallucinations, but if the authors are right, it’s time to look.

We don’t know if animals hallucinate, and use things like head twitch and effects on dendritic branching and size in tissue culture as markers for hallucinations as LSD, DMT produce these things,.

The authors do show that putting a serotonin transporter into neuronal cultures so serotonin gets inside, produces similar effects on dendritic branching and size.  While fascinating, these effects are  pretty far removed from clinical reality.

The authors do raise a fascinating point at the end of their paper.  Perhaps there are endogenous intracellular ligands for intracellular 5HT2AR which differ from serotonin.   Perhaps the hallucinations and mental distortions of schizophrenia and other psychiatric disease are due to too much of them.

Homework assignment

Design a protein to capture cholesterol and triglycerides and insert them between the two leaflets of the standard biological membrane similar but not identical to the plasma membrane. Answer Sunday night 14 March ’21 

I don’t think we fully grasp the chemical ingenuity of Nature when we discover one of its solutions.   Thinking on your homework assignment will give you a chance to appreciate  just how  chemically clever Nature/Evolution/God actually is. 

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.

Not physical organic chemistry but organic physical chemistry

This post is about physical chemistry with organic characteristics in the sense that capitalism in China is called socialism with Chinese characteristics. A lot of cell biology is also involved.

I remember the first time I heard about Irving Langmuir and the two dimensional gas he created. It even followed a modified perfect gas law (PA = nRT where A is area). He did this by making a monolayer of long chain fatty acids on water, with the carboxyl groups binding to the water, and the hydrocarbon side chain sticking up into the air. I thought this was incredibly neat. It was the first example of organic physical chemistry. He published his work in 1917 and won the Nobel in Chemistry for it in 1932.

Fast forward to our understanding of the membrane encasing our cells (the technical term is plasma membrane to distinguish from the myriad other membranes inside our cells. To a first approximation it’s just two Langmuir films back to back with the hydrocarbon chains of the lipids dissolving in each other, and the hydrophilic parts of the membrane lipids binding to the water on either side. This is why it’s called a lipid bilayer.

Most of the signals going into our cells must pass through the plasma membrane, using proteins spanning it. As a neurologist I spent a lot of time throwing drugs at them — examples include every known receptor for neurotransmitters, reuptake proteins for them (think the dopamine transporter), ion channels. The list goes on and on and includes the over 800 G protein coupled receptors (GPCRs) with their 7 transmembrane segments we have in our genome [ Proc. Natl. Acad. Sci. vol. 111 pp. 1825 – 1830 ’14 ].

Glypiated proteins (you heard right) also known as PIGtailed proteins (you heard that right too) don’t follow this pattern. They are proteins anchored in the outer leaflet of the plasma membrane lipid bilayer by covalently linked phosphatidyl inositol. https://en.wikipedia.org/wiki/Phosphatidylinositol — the picture shows you why — inositol is a sugar, hence crawling with hydroxyl groups, while the phosphatidic acid part has two long hydrocarbon chains which can embed in the outer leaflet. We have 150 of them as of 2009 (probably more now). Examples of PIGtailed proteins include alkaline phosphatase, Thy-1 antigen, acetyl cholinesterase, lipoprotein lipase, and decay accelerating factor. So most of them are enzymes working on stuff outside the cell, so they don’t need to signal.

Enter the lipid raft. [ Cell vol. 161 pp. 433 – 434, 581 – 594 ’15 ] It’s been 18 years since rafts were first proposed, and their existence is still controversial (with zillions of papers saying they exist and more zillions saying they don’t). What are they — definitions vary (particularly about how large they are). Here’s what Molecular Biology of the Cell 4th edition p. 589 had to say about them — Rafts are small (700 Angstroms in diameter). Rafts are rich in sphingolipids, glycolipids and cholesterol. The hydrocarbon chains are longer and straighter than those of most membrane lipids, rafts are thicker than other parts of bilayer. This allows them to better accomodate ‘certain’ membrane proteins, which accumulate there. [ Proc. Natl. Acad. Sci. vol. 100 p. 8055 – 7’03 ] These include glycosylphosphatidylinositol anchored proteins (glypiated proteins), cholesterol linked and palmitoylated proteins such as Hedgehog, Src family kinases and the alpha subunits of G proteins, cytokine receptors and integrins.

Biochemical analysis shows that rafts consist of cholesterol and sphingolipids in the exoplasmic leaflet (outer layer of the plasma membrane) of the lipid bilayer and cholesterol and phospholipids with saturated fatty acids in the endoplasmic leaflet (layer facing the cytoplasm). The raft is less fluid than surrounding areas of the membrane. So if they in fact exist, rafts contain a lot of important cellular players.

The Cell paper introduced synthetic fluorescent glypiated proteins into the outer plasma membrane leaflet of Chinese hamster ovary cells and was able to demonstrate nanoClustering on scales under 1,000 Angstroms (way too small to see with visible light, accounting for a lot of the controversy concerning their existence).

How can the authors make such a statement? The evidence was a decrease in fluorescence anisotropy due to Forster resonance energy transfer effects. Forster energy transfer is interesting in that it doesn’t involve molecule #1 losing energy by emitting a photon which is absorbed by molecule #2 increasing its energy. It works by molecule #1 inducing a dipole in molecule #2 (by a Van der Waals effect). Obviously, to do this, the molecules must be fairly close, and transfer efficiency falls off as the inverse 6th power of the distance between the two molecules.

In Fluorescence Resonance Energy Transfer (FRET), one fluorophore (the donor) transfers its excited state energy to a different fluorophore (the acceptor) which emits fluorescence of a different color. For more details see — https://en.wikipedia.org/wiki/Förster_resonance_energy_transfer — its interesting stuff. Again an example of physical chemistry with organic characteristics (and pretty good evidence for the existence of lipid rafts to boot).

Now it gets even more interesting. Nanoclustering is dependent on the length of the acyl chain forming the GPI anchor (at least 18 carbons must be present). NanoClustering diminishes on cholesterol depletion in actin depleted cell blebs and mutant cell lines deficient in the inner leaflet lipid — phosphatidylserine (PS) — which has two long chain fatty acids hanging off the glycerol. So it looks as if the saturated acyl chains of the glypiated proteins of the outer leaflet interdigitate with those of PS in inner leaflet. The effect is also enhanced on expression of proteins specifically linking PS to the actin cytoskeleton of the cortex. Binding of PS to the cortical actin cytoskeleton determines where and when the clusters will be stabilized. The coupling can work both ways — if something immobilizes and stabilizes the glypiated proteins extracellularly, than PS lipids can form correlated patches.

This might be a mechanism for information transfer across the plasma membrane (and acrossother membranes to boot). This could also serve as a way to couple many outer leaflet membrane lipids such as gangliosides and other sphingolipids with events internal to the cell. Cholesterol can stabilize the local liquid ordered domain over a length scale that is large than the size of the immobilized cluster. A variety of membrane associated proteins inside the cell (spectrin, talin, caldesmon) are able to bind actin. “The formation of the contractile actin clusters then determine when and where the domains may be stabilized, bringing the generation of membrane domains in live cells under control of the actomyosin signaling network.’

So just like the integrins which can signal from outside the cell to inside and from inside the cell to outside, glypiated proteins and the actin cytoskeleton may form a two way network for signaling. No one should have to tell you how important the actomyosin cytoskeleton is in just about everything the cell does. Truly fascinating stuff. Stay tuned.