Sensing Unfolded Proteins

Do cells take joy in tidying? Absolutely! But how can they tell where the messes are?

Any household knows that without cleaning and disposal, nothing else is possible. Things pile up, messes accumulate, everything comes to a standstill. Cells are the same, needing a constant flow of new materials in, trash out, recycling, and cleanups. One of the major kinds of mess in cells is unfolded proteins, which when they accumulate can cause aggregations and diseases like Huntington's, other dementias, atherosclerosis, and inflammation generally. While there are a lot of mechanisms (chaperones, chaperonin cage complexes, proteasomes) dedicated to proper folding, sometimes they are not enough, and excess junk in the form of unfolded proteins builds up. One major response this is the unfolded protein response, or UPR, which is centered at the endoplasmic reticulum (ER).

Why the ER? This is where countless ribosomes dock to feed in nascent proteins destined for secretion, the plasma membrane, and many locations inside the cell other than the cytoplasm. So it is a key organelle for protein synthesis and distribution, and thus for sensing how cellular proteins are doing. This sensing, the UPR, starts with a sensor protein, called IRE1 (also called ERN1), which sits on the inside face of the endoplasmic membrane. IRE1 binds to unfolded proteins inside the ER, and when the concentration rises high enough, it gathers into condensates of its own that trigger a variety of responses that include, in the extreme, cell death. Yes, lack of timely tidying can have serious consequences!

IRE1 is multifunctional, containing a kinase that activates AMPK and JNK, proteins which influence metabolism and inflammation. It also contains an RNAse enzymatic activity, which activates a whole other program by altering the mRNA of XBP1, which then becomes active and produces a transcription regulator that starts building more ER, more chaperones, and more protein folding capacity generally. The RNAse also digests other mRNAs indiscriminately, lightening the load of translation and thus incoming nascent proteins. So IRE1 is a key homeostatic regulator that adjusts the size of the ER to cellular needs, and informs the rest of the cell how things are going in the ER. 

One structure of IRE1, luminal domain only. This is a dimer, with halves shown symmetrically across the middle. How does this bind unfolded proteins?

But how does IRE1 sense unfolded proteins? A recent paper went through a modeling study to show how IRE1 operates. Sitting in the ER membrane, the enzymatic, signaling parts of the protein are outside, while the sensor is inside, called the core luminal domain (cLD). The authors demonstrate that this domain binds unfolded proteins directly, using them as bridging elements to bring multiple IRE1 proteins together and form the glob that nudges aside the inhibitory protein BiP and promotes the phosphorylation activity and thus activation, of IRE1. BiP also binds to unfolded proteins itself, so forms another sensor that indirectly activates IRE1 under stress conditions. 

Firstly, IRE1 is a dimer, and the luminal domain is where it dimerizes. The structure (just of the luminal domain, above) shows a beta ribbon core and peripheral unstructured areas and alpha helices. After running the crystal structure (shown) though very brief molecular dynamics simulations, the unstructured areas flop around dramatically, while the core and dimeric structure remain stable. Intriguingly, the beta stranded center of the dimer resembles (between the blue helices, above) the peptide binding cleft of MHC molecules- those that present random antigens to T-cell receptors of the immune system. Yet the authors show that unfolded proteins bind differently to IRE1. They flop across the whole central region, attracted by negative charge as well as the open hydrophobic surface. The next image shows molecular dynamics before/after shots of various unfolded peptides known to bind IRE1 in vitro, showing how after starting straight across the central region, they each find different comfort zones, mostly over this central region, but some off to a side. 

Collection of IRE1 structures (gray) with unstructured peptides of various kinds bound to the luminal face. t=0 is the starting point for the molecular dynamics simulation, which went for one microsecond. End points are below. In each case, the unfolded peptide stayed bound, and settled into some comfortable position, based on the hydrophobic and negatively charged landscape of the IRE1 face. 

The intriguing thing about this binding is that the structure of IRE1 itself is unaffected. The dimer interface remains stable and open, leading to the hypothesis that these peptides form bridges by becoming the meat of a two-dimer IRE1 sandwich. And it is this that then displaces the inhibitory protein BiP and leads to condensation and activation, by bringing together multiple IRE1 kinase domains (on the outside of the ER), and activating their kinase/signaling functions, turning on the various downstream pathways.

A helpful model from another lab, describing how, once the unfolded proteins are bound inside the ER (by mechanisms unknown at the time), the outside domains would gather, trans-phosphorylate, and activate downstream pathways. 

• Those flock cameras.
• As if charging for previews of presidential posts wasn't corrupt enough.

Revealing the Origin of Eukaryotes

Tracing the breadcrumbs of deep phylogeny through gene duplications. 

Last week, we discussed the jamboree of mutation that is the MHC locus in animals. One part of that story was the persistent duplication and pseudogene-i-zation (which is to say, the birth and death) of genes in that locus through primate evolution, another consequence of the never-ending arms race with our pathogens. Gene duplication has much deeper significance, however, as it has given rise to new genes throughout biological evolution, as it is merely an accident of DNA (or RNA) replication, thus operationally rather frequent. Significant transitions, such as the advent of eukaryotes, and the advent of plants and of angiosperms, all feature rapid gene duplication, even whole-genome duplication, which provide grist for new functions, refinement of old functions, and speciation.

A recent paper did a deep dive into the origin of eukaryotes, perhaps the watershed event in the history of life, second only to the origin of life itself on the early Earth. It is very difficult to reconstruct what happened during these extremely early events, which these authors put as long as three billion years ago. There has been so much mutational churn in our molecules, and no fossil record to speak of, that, again like the origin of life itself, we do not have a great deal to go on. Thankfully, the discovery of the kingdom of archaea, and its especially eukaryotic-like class, the Asgard archaea, has provided a much better platform for speculating about what the originating organisms might have been like.

Eukaryotes differ from bacteria and archaea by numerous characteristics, implying extensive development/evolution before the last common ancestor from which we can trace all the extant descendants. These include the eponymous nucleus, an internal system of membranes and membrane-bound organelles, a complex cytoskeleton of both actin and microtubules, a mitochondrion, meiosis, a microtubule spindle-driven division system, and many other, more obscure molecular differences. The lack of intermediate forms is certainly frustrating from a scientific perspective, making it difficult to go through the catalog of life today to identify the steps involved in this transition. It also implies that the process ended with an evolutionary bang that in essence caused the final iteration to wipe out all the preceding forms, a bit like humans vs the many other Australopithecines and their descendants. 

These differences are enormous and momentous, making eukaryotic cells far larger, energetically capable, and complex than their antecedents. Another key characteristic is a prevalence of gene duplication and specialization, leading to much larger genomes along with all the other complexity. It has long been theorized that it was the union of the proto-mitochondrion, which was a bacterium, with the archaeal founding cell, that set the whole process off, particularly providing the massive amount of energy required for all this rise in complexity. 

However, the current authors claim that this definitively is not the case- that the mitochondrial symbiosis was a rather late event, coincident with the oxygenation of the atmosphere about two billion years ago. They argue that, since gene duplication and specialization is in any case a clear characteristic of eukaryotes, using molecular clocks to time these many gene duplications can give us relatively specific and data-rich insight into the epochs at which each of the processes that they participate in first developed. 

The authors present estimated times of origin for key duplications involved in RNA synthesis. The RNA polymerases I, II, and III were duplicated from one polymerase in archaea, and are composed of numerous gene products, as well as ancillary regulatory proteins. While some subunits are still shared, the ones that duplicated did so in the range of 2.3 to 2.8 billion years ago, before the symbiosis with the mitochondrial precursor, timed at "mFECA", or the mitochondrial first eukaryotic common ancestor, about 2.2 billion years ago. "nFECA" is nuclear first eukaryotic common ancestor, while "LECA" is last eukaryotic common ancestor. The axis at the top is time before present, in giga-years ago, or Ga.

For example, eukaryotes all have three RNA polymerases, while bacteria and archaea only have one. These RNA polymerases specialize on mRNA synthesis (RNA pol II), ribosomal RNA synthesis (RNA pol I), and tRNA, 5S RNA and U6 snRNA (RNA pol III). Why this specialization exists remains a bit mysterious, though once it started there was no going back. In any case, it happened and is totally characteristic of eukaryotes, so these duplications (which involve the duplication of numerous genes encoding proteins of the polymerase complex itself and its regulators and way-finding helpers) can be used to date eukaryogenesis, using a very carefully calibrated and species-sampled molecular clock. And what they find is that all of these duplications, given as ranges in the graphs above, with green dots at the likeliest time points, all lie prior to the point of "mFECA", which is the mitochondrial first eukaryotic common ancestor, at roughly 2.2 billion years ago. Tabulated over studies of almost a hundred other genes that are similarly duplicated and characteristic of eukaryotes, the authors place the "nFECA", or nuclear eukaryotic common ancestor way back at almost 3 billion years ago. 

That is a very long time! It is a very long time ago that the characteristics of eukaryotes started to come together, and it is also a very long time (well over a billion years) over which that development spanned before culminating in the last common ancestor of all extant eukaryotes, which the authors put at about 1.7 billion years ago. And note that that still comes a billion years before the Cambrian explosion of animal life. We are talking about really deep time here. 

If mitochondrial symbiosis came later, (causing another bout of genomic change as hundreds of bacterial genes moved from the symbiont to the nucleus), just as the atmosphere was getting oxygenated, what was going on in the preceding billion years? One can speculate that these evolving proto-eukaryotes were the top predators of their world, a bit like eukaryotic protists in microbial environments today. The competition would as always have been intense, but there were no higher life forms (i.e. animals or protists) to worry about. Size was not a problem, but rather an advantage. The focus was on quality and effectiveness in finding, disabling, and digesting prey. Thus there was little downside to complexity, unlike the constraints on prey species, which had to optimize for rapid growth and reproduction under both nutrient and predation constraints. New and costly systems for protein secretion, for prey digestion, and intelligent, responsive regulation of all cell processes might have been consistently advantageous.

I take this work as being definitive, at least in terms of settling the debate about early vs late symbiosis. It benefits from a clear theoretical basis and new, voluminous sequence data from appropriately related species in its molecular comparisons. I recommend it highly, as I can only scratch the surface of its analysis here. The advent of mitochondria was then a later step change in complexity, but brought incredible metabolic productivity that, at least in oxygen-rich environments, would have definitively superseded all the prior forms of the proto-eukaryote, and (unfortunately for us) erased that record of evolution. A later innovation, placed between mFECA and the last eukaryotic common ancestor "LECA" was sexual meiosis, another fascinating and characteristic innovation of eukaryotes. These authors thus provide a true revolution in our understanding of eukaryogenesis, extending it across well over a billion years.

One more sample from the author's presentation of molecular duplications that help time eukaryogenesis. Here, DNA repair proteins are emphasized, including some involved in meiosis (light blue dots, see legend). Some of these duplications involve genes from the mitochondrial endosymbiont, (MSH4), and some (such as EME1/2 and MUS81) come from older repair processes, but later specialized for meiosis. 

• Critique of our court.
• A vision .. "in which elections are hollow exercises"
• The end of structure, the end of truth.
• A confluence of corruption, bad faith, and poop.

Strides in Cancer Treatment

A new paper shows that CART therapies can be unleashed against solid tumors.

We are finally in the payoff period in the decades-long war on cancer. Slowly, painfully, precision approaches are being developed to treat specific molecular lesions in ways that are superior to the old blunderbuss kill-everything approaches. At first, these treatments had only marginal effects, at astounding costs. But increasingly, the effects are lengthening and cures are in sight in some forms of cancer. One unexpected area of revolutionary progress has been immunotherapies, which in various ways help our immune systems attack cancers. It turns out that many cancers have tricks to hide from the immune system, and once those tricky dampening molecules are circumvented, dramatic reductions are possible. One paper recently described an anticancer vaccine made up of a witch's brew of targeting molecules, cancer antigens, and adjuvants, that achieves strong anti-melanoma action.

Another one of these immunotherapies is CART, or chimeric antigen receptor T-cell therapy. T cells have a receptor repertoire, just as B-cells do, which target things to be attacked- foreign pathogens, diseased states, etc. at molecules called antigens. One problem in cancer is that the cells are, originally at least, our own, so they mostly evade immune detection by having few "foreign" antigens. But there are nevertheless some antigens, comprised of normal molecules that are out of place (such as DNA found outside the cell) and "neoantigens" that are proteins expressed from the mutations in cancer cells. Additionally, as mentioned above, cancer cells express additional molecules (PD-L1) that can dampen even the immune response that does get generated by these few cancer antigens. So, the chimeric part of CART is taking the patient's own T-cells and engineering some of them to express new anti-antigen receptors that are relevant to the patient's cancer. Perhaps there is a mutant fusion protein that the cancer depends on. Perhaps the cancer displays an unusual surface molecule. Perhaps the tables need to be turned and PD-L1 targeted. There are many possible targets. 

CART therapies have, to date, been mostly directed at blood tumors. Solid tumors have extra protection in their micro-environments, and have not been good targets, though they necessarily have blood supplies and thus exposure to systemic T-cells. A recent paper blows open this field by revealing a magic molecule that plays a very significant role in the structure of solid tumors- the urokinase receptor. The urokinase plasminogen activator receptor (uPAR) is heavily expressed on senescent cells and many solid tumors, but rarely expressed elsewhere. Indeed, its expression correlates with tumor aggressiveness. Plasminogen is a protease that is sort of a cleanup crew for the circulatory system and body generally. It breaks up blood clots, and digests follicle tissues allowing ovulation. It encourages wound healing and discourages fibrosis- the buildup of scar tissue. However, in the cancer setting, the same activity seems to encourage fibrosis in a sort of constant wound healing state. Reviews in this field are rather confused about the direction of action. But one thing is clear- uPAR has myriad signaling activities relevant to tissue repair and immune activation that are not all dependent on the uPA (plasminogen activator) and plasmin activation system. Indeed, it is expressed not just in cancers, but in many other fibrotic settings.

A wide array of proteins are assessed here for their expression in a cancer tissue sample. uPAR is in red at the upper left. The matrix on the right shows the correlation of expression in a wide variety of cell types and tissues, like cancer-associated fibroblasts (CAFs), monocytes/macrophages (Mo/Mac), and with the protein fibroblast activation protein alpha (FAP).

The authors sought to target CART cells against uPAR, principally as a targeting device, since this marks many solid tumors and correlates with metastasis and rapid cancer progression, in addition to inflammation and fibrosis. While only tested in mice, the results were remarkable. 

"CAR T cells targeting the D2-D3 domain of uPAR display broad antitumor activity in xenograft, syngeneic, and patient-derived models, including in adjuvant and combination settings, supporting the concept that targeting conserved malignant cell states can enable therapeutic strategies that transcend tumor type. ... our prior work shows that uPAR CAR T cells targeting senescent cells remodel fibrotic tissues, and, as shown herein, this remodeling is associated with CAR T cell infiltration and cytotoxic activity. Similarly, parallel work demonstrates that uPAR CAR T cells exhibit potent efficacy in glioblastoma models and can co-target supportive stromal cells."

This is to say that these CART cells target not only tumor cells, but the surrounding solid tissues (stromal cells) that they rely on. That is the key to defeating solid tumors. It also indicates that other autoimmune and fibrotic conditions may be addressable with this therapy as well. 

Treatment effects from the CART therapy in mice, against several tumors. The red graphs are controls, and the blue graphs are treatments. Top is the tumor volume over time, while at bottom is survival of the mice over time.  Lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), high-grade serous ovarian carcinoma (HGSOC), and pancreatic ductal adenocarcinoma (PDAC).

The results of treatment of xenografted human ovarian tumors into susceptible mice, at 3 weeks, bottom. On the left is the control, while the other two sets were treated with CART cells against uPAR.

The authors note that relapses were seen occasionally, but that in these cases, the uPAR target was still highly expressed. That suggests firstly that it is difficult for tumors of these targeted types to do without uPAR, and secondly that something else went wrong with the tailored CART therapy, other than that its target went away. Perhaps future work can enhance its penetration or activity. The researchers also strained to make their model systems as human-relevant as possible, using cancer tissue transplanted (xenografted) from human cell lines, human CART cells, and mice with transplanted immune systems from humans. This work is thus not only a scientific breakthrough of the highest order, but is a technical tour de force as well. It also ends up with a variety of patent declarations and commercial ties, indicating that this breakthrough is being fully milked by its inventors and commercialized at breakneck speed.

One major problem with this mode of therapy is that CART cells require a great deal of engineering. First, antibodies against uPAR were developed in mice or other species. Then the genes from those immune systems were recovered from those mice, to get the precisely recombined gene that expressed the antibody with highest binding activity against uPAR. Then that gene, hooked up to new transmembrane and intracellular domains, (specially selected to activate the T cell they will be put into), was introduced into a transformation vector and put into the T cells collected from the diseased mice. In humans, this treatment routinely runs a half a million dollars. It is incredibly ornate, and one expects that gene therapy will someday allow the patient's T cells to be directly modified in the body, without all the collection and laboratory work, (which takes months), given a high-quality gene encoding the antibody fragment that is generally applicable- not tailored to a specific patient.

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• Renewables and batteries have stabilized California's grid and made electricity cheaper.
• Map of where electricity has gotten more expensive in the last year.
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• Electric cars are good for everyone.

Pumping Calcium

An ornate ion pump manages rapid outflow of calcium.

In the beginning, the egg cell experienced a wave of calcium release, triggered by union with a sperm cell. This blocked other sperm from entering, and prepared the egg to become a zygote and embark on embryogenesis. It is but one example of the pervasive role of calcium signaling among animals. Another is the muscle activation cycle, which relies on calcium release from the specialized sarcoplasmic reticulum (in response to a nerve activation) to get the cell as a whole contracting. Generally, calcium is kept very low in the cytoplasm, and high in the endoplasmic reticulum and outside the cell. Thus, channels gated by electrical activation or other signals can cause rapid cytoplasmic calcium spikes and signal widely within a cell. 

On the flip side, there have to be pumps that keep the cytoplasmic concentration low, and a recent paper elucidates the structure of one such pump that is remarkably fast, while also closely regulated. It is an impressive machine. PMCA2 is an ATP-using calcium pump that sits in the plasma membrane and carries out what is called the Post-Albers cycle. This is a flip-switch mechanism for pumping ions, where ATP drives conformational switches alternately exposing ion binding sites to each side of the membrane. When the pore is open to the cytoplasm, there is no competition from higher concentrations outside, so the active site can bind one internal calcium, given a high-affinity site. Then, after the conformational switch, the pore is exposed to the outside, and at the same time the site is reconfigured to be lower-affinity, releasing the calcium ion into a high concentration environment. Neurons especially use calcium signaling extensively to operate synapses and regulate growth and development. Their rapid and frequent signaling requires a pump that has especially high capacity. PMCA2 operates at a maximal rate of several thousands of Ca2+ ions pumped per second.

Cartoon of the Post-Albers cycle, which is shared by a large family of active ATP-using pumps that transfer ions against their chemical concentration gradient. M is the main transmembrane domain of the pump, where the ions traverse the membrane. The N, P, and A domains are regulatory, especially binding and cleaving ATP  at an interface between the N, P, and A domain. The cycle links power steps (1,2) with conformational changes that carefully gate the pumping process.

And that is not all. Since calcium has a charge of 2+ and this pump does not intend to alter charge across the membrane, the pump simultaneously has binding sites for counter-ions (generally two OH-) that are transferred in the opposite direction from the calcium. Not only that, but every pump of this kind requires regulation of various kinds. PMAC2 is activated by phosphatidyl inositol 4,5 bisphosphate (PIP2), which is another important signaling molecule generated by specific PI kinases in response to activation of G-protein coupled receptors or protein kinase C, which may respond to external signals. In very general terms, these tend to be pro-growth or stress-induced pathways. These regulatory processes can tune the overall rate of recovery from rapid Ca2+ signaling events, by adjusting the level and activity of pumps like PMAC2. 

ATP binds at the N/P/A domain interface, and its hydrolysis (and loss of ADP) generates extensive shape changes, including into the transmembrane M domain. At the very bottom, the calcium ion is shown in green, bound inside the M domain pumping channel. The motions here are subtle, but enough to dramatically reshape the calcium channel.

The authors, using various substrate variants and other tricks, were able to develop structures of PMAC2 in several steps of the pumping cycle, using cryo-electron microscopy. The ATPase site in the N domain (red) is far from the channel that conducts the calcium ion (brown, far bottom). They show extensive shape changes from binding or losing the ATP molecule, though they mostly concern the intracellular domains (red, blue, yellow). The effects on the transmembrane pore domain are rather subtle, shown on right. The authors claim that, compared to other pumps of this large family, the structural changes are significantly less, suggesting that evolution for speed has caused the mechanism to become more efficient, with less wasted motion per conduction event, at least in the channel region itself.

Relation of the PIP2 binding domain (orange/red stick figures) to the calcium core binding site. PIP2 appears to be essential for rapid pump operation. At bottom is shown some schematics of the gating provided by PIP2 in bound and unbound states, especially via the D873 side chain (negatively charged aspartic acid).

They also find that the activating molecule PIP2 is neatly parked right next to the main calcium binding and conduction region, and is more or less essential for enzyme activity. In the graph above (e), they show that several single mutations made in the calcium binding high affinity site, for example switching the negatively charged D873 for the positively charged K (lysine), kills ion pumping activity. Mutation of the PIP2 binding pocket (KKQ->TLL, around position 347) likewise kills enzyme activity.

Relation of the counter-ion channel (red dots) with the calcium channel. Both are essential parts of the mechanism. Closeups with the coordinating protein side chains shown on the right.

The whole mechanism is alluded to in the last figure, where the central calcium binding site is shown, with the general direction of calcium pumping. The counter-ion transport area is shown nearby as a flurry of red dots (standing for water molecules, which at this scale are interchangeable with OH ions). Specific single mutations in either area, either changing negatively charged E412 to positively charged lysine at the calcium binding pocket, or changing polar S877 in the water/hydroxy binding area to the bulky and hydrophobic F (phenylalanine), each kill pumping activity (graph). 

While it would be ideal to have a more dynamic representation of what is going on, the new structures give tremendous detail, including the associated ATP, PIP2, calcium, and water molecules. The mutations also nail down several functional points. Obviously a rather intricate and well-oiled machine that keeps its bit of cellular calcium homeostasis on an even keel. It is hard to believe that the sum of thousands of machines like this one is life, but the deeper we look the more true that appears to be.

• Gullibility-maxxing.
• There is a serious bifurcation in Catholic thought.
• Reality has a well-known liberal bias.
• Well, that didn't work out so great.
• Is the internet going to collapse?

Not Every Transcript is Golden

 Reflections on junk DNA, and junk transcripts.

Some time ago, a large project in molecular biology determined that most regions of the genome are transcribed. The authors and most observers took this to mean that most regions are functional, quite in contrast to the reigning theory up to that point, that our genomes host a smattering of genes floating in a sea of "junk" DNA. That theory was based on the now-ancient observations of reannealing curves for bulk DNA from humans and other species which found that most of our DNA re-anneals very quickly, due to the fact that it is repetitive. Most of our genomes (60%) are taken up with LINE repeats, SINE repeats, old retro-transposons, stray duplications, and other repetitive material that, at a first glance, seems like junk. There has been a battle ever since, between proponents of junk DNA and those who see function around every corner. As we learn more about the genome, many more functions have indeed come to light, like distant enhancers and regulatory RNAs of many flavors. But overall, there still seems to be a lot of junk. 

A recent paper took an oblique shot at this field, looking at the profusion of alternative gene transcripts, which can number into the hundreds for a single gene. (This was also reviewed.) These are generally called isoforms, and arise due to variable ways one gene's RNA products can be initiated, terminated, and spliced. So not only are most regions of the genome transcribed in some form, actively transcribed regions can be transcribed and processed in myriad ways to lead to different RNA products. Here again, there has been an analogous argument, about whether every such isoform has a function, or whether isoforms might arise from more or less sporadic processes, often as unintended and non-functional sparks coming out of the machinery. The importance of isoforms is very well documented in many cases, so the possibility of function, sometimes highly conserved, is not in question. Only the importance of every last variation in combinatorial collections of isoforms that can number into the hundreds.

Here is an image from the first page (of about six pages) of RNA transcripts coming off the notorious BRCA1 gene, which is intensely studied for its role in breast cancer. Each line is a distinct mRNA transcript. Each darker bar is an exon, which are separated by introns. The darker colored exons are in the protein coding region, while the lighter exons signify the untranslated upstream and downstream ends. I count about 315 transcripts described for this genetic locus. The idea that each of these has some evolutionarily constrained and important function is, on the face of it, absurd.

The authors took an interesting evolutionary approach, reasoning that species with larger population sizes experience more stringent purifying selection, and thus should (in theory) show tighter control over stray genomic products such as isoforms, if most transcript isoforms are neutral (or even deleterious) accidents, rather than intentional and functional forms. Thankfully, animals come in a wide range of population sizes, from insects to crocodiles and primates; very large to very small. While population size is hard to calculate, several convenient proxies are known, like lifespan, body size, etc. When they totted everything up, they saw clear correlations between these proxies and the number of alternative RNA products per gene- also termed transcript diversity. They sliced up the data by organ where the RNA was expressed, and by the source of the RNA variation- either different initiation, different termination, different splicing. In all cases the trend was the same. In species with larger population sizes, the diversity of transcripts was lower, agreeing with their hypothesis that when greater selecive force is available, the slop from the transcription and transcript processing machinery declines.

The authors draw correlations between alternative splicing (AI) diversity in an organism's cells and its population size. 

The authors additionally note that there is a similar relationship between alternative splice site usage and expression level of a gene. That is, the higher the gene expression, the less likely that minor splice sites are used, indicating that here again, higher selective pressure helps to clear out non-functional off-products of the transcription apparatus.

The correlations found here are only that- correlations. While significant, they are not terribly strong, let alone stark. So it is evident that our gene expression machinery has a lot of play in it, and this falls on a spectrum from deleterious to critically functional. It is, after all, machinery, not divine. It is also grist for evolution itself- it is useful to have some slop so that there is always some diversity in the gearing to accommodate new selective pressures. But the idea that just because a distinct transcript exists, it is biologically functional, or that, similarly, because a genomic region is transcribed, it is a "gene" rather than junk DNA.. that does not hold water. Every nucleotide in the genome has its own unique selective constraints, and for many of them, that constraint is zero.

• The world order, and our position in it, is crumbling.
• Whence Hungary?
• Another AI tax, as if gobbling up power wasn't bad enough.
• Mindless.

We Have Rocks in Our Heads ... And Everywhere Else, Too

On the evolution and role of iron-sulfur complexes.

Some of the more persuasive ideas about the origin of life have it beginning in the rocks of hydrothermal vents. Here was a place with plenty of energy, interesting chemistry, and proto-cellular structures available to host it. Some kind of metabolism would by this theory have come first, followed by other critical elements like membranes and RNA coding/catalysis. This early earth lacked oxygen, so iron was easily available, not prone to oxidation as now. Thus life at this early time used many minerals in its metabolic processes, as they were available for free. Now, on today's earth, they are not so free, and we have complex processes to acquire and manage them. One of the major minerals we use is the iron-sulfur complex, (similar to pyrite), which comes in a variety of forms and is used by innumerable enzymes in our cells. 

The more common iron-sulfur complexes, with sulfur in yellow, iron in orange.

The principle virtue of the iron-sulfur complex is its redox flexibility. With the relatively electronically "soft" sulfur, iron forms semi-covalent-style bonds, while being able to absorb or give up an electron safely, without destroying nearby chemicals as iron alone typically does. Depending on the structure and liganding, the voltage potential of such complexes can be tuned all over the (reduction potential) map, from -600 to +400 mV. Many other cofactors and metals are used in redox reactions, but iron-sulfur is the most common by far.

Reduction potentials (ability to take up an electron, given an electrical push) of various iron-sulfur complexes.

Researchers had assumed that, given the abundance of these elements, iron-sulfur complexes were essentially freely acquired until the great oxidation event, about two to three billion years ago, when free oxygen started rising and free iron (and sulfur) disappeared, salted away into vast geological deposits. Life faced a dilemma- how to reliably construct minerals that were now getting scarce. The most common solution was a three enzyme system in mitochondria that 1) strips a sulfur from the amino acid cysteine, a convenient source inside cells, 2) scaffolds the construction of the iron-sulfur complex, with iron coming from carrier proteins such as frataxin, and 3) employs several carrier proteins to transfer the resulting complexes to enzymes that need them. 

But a recent paper described work that alters this story, finding archaeal microbes that live anaerobically and make do with only the second of these enzymes. A deep phylogenetic analysis shows that the (#2) assembly/scaffold enzymes are the core of this process, and have existed since the last common ancestor of all life. So they are incredibly ancient, and it turns out to that iron-sulfur complexes can not just be gobbled up from the environment, at least not by any reasonably advanced life form. Rather, these complexes need to be built and managed under the care of an enzyme.

The presented structures of the dimer of SmsB (orange) and SmsC (blue) that dimerize again to make up a full iron-sulfur scaffolding and production enzyme in the archaean Methanocaldococcus jannaschii. Note the reaction scheme where ATP comes in and evicts the iron-sulfur cluster. On right is shown how ATP fits into the structure, and how it nudges the iron-sulfur binding area (blue vs green tracing).

A recent paper from this group extended their analysis to the structure of the assembly/scaffold enzyme. They find that, though it is a symmetrical dimer of a complex of two proteins, it only deals with one iron-sulfur complex at at time. It also binds and cleaves ATP. But ATP seems to have more of an inhibitory role than one that stimulates assembly directly. The authors suggest that high levels of ATP signal that less iron-sulfur complex is needed to sustain the core electron transport chains of metabolism, making this ATP inhibition an allosteric feedback control mechanism in these archaeal cells. I might add, however, that ATP binding may well also have a role in extricating the assembled iron-sulfur cluster from the enzyme, as that complex is quite well coordinated, and could use a push to pop out into the waiting arms of target enzymes.

"These ancestral systems were kept in archaea whereas they went through stepwise complexification in bacteria to incorporate additional functions for higher Fe-S cluster synthesis efficiency leading to SUF, ISC and NIF." - That is, the three-component systems present in eukaryotes, which come in three types.

In the author's structure, the iron-sulfur complex, liganded by three cysteines within the SmsC protein. But note how, facing the viewer, the complex is quite exposed, ready to be taken up by some other enzyme that has a nice empty spot for it.

Additionally, these archaea, with this simple one-step iron cluster formation pathway, get their sulfur not from cysteine, but from ambient elemental sulfur. Which is possible, as they live only in anaerobic environments, such as deep sea hydrothermal vents. So they represent a primitive condition for the whole system as may have occurred in the last common ancestor of all life. This ancestor is located at the split between bacteria and archaea, so was a fully fledged and advanced cell, far beyond the earlier glimmers of abiogenesis, the iron sulfur world, and the RNA world.

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Jonathan Singer and the Cranky Book

An eminent scientist at the end of his career writes out his thoughts and preoccupations.

Jonathan Singer was a famous scientist at my graduate school. I did not interact with him, but he played a role in attracting me to the program, as I was interested in biological membranes at the time. Singer himself studied with Linus Pauling, and they were the first to identify a human mutation in a specific gene as a cause for a specific disease- sickle cell disease. After further notable work in electron microscopy, he reached a career triumph by developing, in 1972, the fluid mosaic model of biological membranes. This revolutionized and clarified the field, showing that cells are bounded by something incredibly simple- a bilayer of phospholipids that naturally order themselves into a remarkably stable sheet, (a bubble, one might say), all organized by their charged headgroups and hydrophobic fatty tails. This model also showed that proteins would be swimming around freely in this membrane, and could be integrated in various ways, ether lightly attached on one side, or spanning it completely, thereby enabling complex channel and transporter functions. The model implied the typical length of a protein alpha helix that, by virtue of its hydrophobic side chains, would naturally be able to do this spanning function- a prediction that was spot-on. He could have easily won a Nobel for this work.

I was intrigued when I learned recently that Singer had written a book near the end of his career. It is just the kind of thing that a retired professor loves to do in the sunset of his career, sharing the wisdom and staving off the darkness by taking a stab at the book biz. And Singer's is a classic of the form- highly personal, a bit stilted, and ultimately meandering. I will review some of its high points, and then take a stab of my own at knitting together some of the interesting themes he grapples with.

For at base, Singer turns out to be a spiritual compadre of this blog. He claims to be a rationalist, in a world where, as he has it, no more than 9% of people are rational. Definition? It is the poll question of whether one believes that god created man, rather than the other way around. Singer recognizes that the world around him is crazy, and that the communities he has been a part of have been precious oases amid the general indifference and grasping of the world. But changing it? He is rather fatalistic about that, recognizing that reason is up against overwhelming forces.

His specific themes cover a great deal of biology, and then some more mystical reflections on balance and diversity in biology, and later, in capitalism and politics. He points out that the nature/nurture debate has been settled by twin studies. Nature, which is to say, genetics, is the dominant influence on human characteristics, including a wide variety of psychological traits, including intelligence. Environment and nurture is critical for reaching one's highest potential, and for using it in socially constructive ways, but the limits of that potential are largely set by one's genes. Singer does not, however, draw the inevitable conclusion from these observations, which is that some kind of long-term eugenic approach would be beneficial to our collective future, assuming machines do not replace us forthwith. Biologists know that very small selective coefficients can have big effects, so nothing drastic is needed. But what criteria to use- that is the sticky part. Just as success in the capitalist system hardly signals high moral or personal qualities, nor does incarceration by the justice system always show low ones. It is virtually an insoluble problem, so we muddle along, destined probably for continued cycles of Spenglerian civilizational collapse.

Turning to social affairs, Singer settles on "structural chaos" as his description of how the scientific enterprise works, and how capitalism at large works. With a great deal of waste, and misdirected effort, it nevertheless ends up providing good results- better than those that top-down direction can provide. He seems a sigh a little that "scientific" methods of social organization, such as those in Soviet Russia, were so ineffective, and that the best we can do is to muddle along with the spontaneous entrepreneurship and occasional flashes of innovation that push the process along. Not to mention the "monstrous vulgarity" of advertising, etc. Likewise, democracy is a mess, with most people totally incapable of making the reasoned decisions needed to maintain it. Again, the chaos of democracy is sadly the best we can do, and the duty of rational people, in Singer's view, is to keep alive the flame of intellectual freedom while outside pressures constantly threaten.

Art, and science.

What can we do with this? I think that the unifying thread that Singer was groping for was competition. One can frame competition as a universal principle that shapes the physical, biological, and social worlds. Put two children on a teeter-totter, and you can see how physical forces (e.g. gravitation) compete all the time, subtly producing equilibria that characterize the universe. Chemical equilibria are likewise a product of constant competition, even including the perpetual jostling of phospholipids to find their lowest energy configuration amidst the biological membrane bilayer, which has the side-effect of creating such a stable, yet highly flexible, structure. With Darwin, competition reaches its apotheosis- the endless proliferation, diversification, and selection of organisms. Singer marvels at the fragility of individual life, at the same time that life writ large is so incredibly durable and prolific. Well, the mechanism behind that is competition. And naturally, economics of any free kind, including capitalism and grant-making in science, are based on competition as well- the natural principle that selects which products are useful, which employees are productive, and which technologies are helpful. Waste is part of the process, as diversity amidst excess production is the essential ingredient for subsequent selection. 

And yet.. something is missing. The earth's biosphere would still be a mere bacterial soup if competition were the only principle at work. Bacteria (and their viruses) are the most streamlined competition machines- battlebots of the living world. It took cooperation between a bacterial cell and an archaeal cell to make a revolutionary new entity- the eukaryotic cell. It then took some more cooperation for eukaryotic cells to band together into bodies, making plants and animals. And among animals, cooperation in modest amounts provides for reproduction, family structure, flock structures, and even complex insect societies. It is with humans that cooperation and competition reach their most complex heights, for we are able to regulate ourselves, rationally. We make rules. 

Without rules, human society is anarchic mayhem- a trumpian, dystopian and corrupt nightmare. With them, it (ideally) balances competition with cooperation to harness the benefits of each. Our devotion to sports can be seen as a form of rule worship, and explicit management of the competitive landscape. Can there be too many rules? Absolutely, there are dangers on both sides. Take China as an example. In the last half-century, it revamped its system of rules to lower the instability of political competition, harness the power of economic competition, and completely transform its society. 

The most characteristic and powerful human institution may be the legislature, which is our ongoing effort to make rational rules regulating how the incredibly powerful motive force of competition shapes our lives. Our rules, in the US, were authored, at the outset, by the founders, who were- drumroll please- rationalists. To read the Federalist Papers is to see exquisite reasoning drawing on wide historical precedent, and particularly on the inspirations of the rationalist enlightenment, to formulate a new set of rules mediating between cooperation and competition. Not only were they more fair than the old rules, but they were designed for perpetual improvement and adjustment. The founding was, at base, a rationlist moment, when characters like Franklin, Hamilton, Madison, and Jefferson- deists at best and rationalists through and through, led the new country into a hopeful, constitutional future. At the current moment, two hundred and fifty years on, as our institutions are being wantonly destroyed and anything resembling reason, civility, and truth is under particularly vengeful attack, we should appreciate and own that heritage, which informs a true patriotism against the forces of darkness.

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The Fire Inside: Eukaryotic Locomotion

The GTP-based Rho/Rac system of actin regulation runs in unseen waves of activation.

One of the amazing capabilities of eukaryotic cells, inherited in part from their archaeal parents, is free movement and phagocytosis. These cells have an internal cytoskeleton, plus methods to anchor to a substrate, (via focal adhesions), which allows them to manipulate their membrane, their shape, and their locomotion. The cytoskeleton is composed of two main types of fibers, actin and microtubules. Microtubules are much larger than actin and organize major trackways of organelle movement around the cell (including the movement of chromosomes in mitosis), and also form the core of cilia and flagella. But it is actin that does most of the work of moving cells around, with dynamic networks that generate the forces behind spiky to ruffly protrusions, that power things like the adventuresome pathfinding of neurons as they extend their axons into distant locations.

Schematic of the actin cytoskeleton of a typical eukaryotic cell.

Actin is an ATPase all by itself. ATP promotes its stability, and also its polymerization into filaments. So, cell edges can grow just by adding actin to filament ends. Actin cross-linking proteins also exist, that create the meshwork that supports extended filopodia. But obviously, actin all by itself is not a regulated solution to cell movement. There is an ornately complex system of control, not nearly understood, that revolves around GTPase and binding proteins. These proteins (mainly RhoA, Rac1, and Cdc42, though there are twenty related family members in humans) have knife-edge regulation, being on when binding GTP, and off after they cleave off the phosphate and are left binding GDP (the typical, default, state). Yet other proteins regulate these regulators- GTPase exchange factors (GEFs) encourage release of GDP and binding of GTP, while GTPase activating proteins (GAPs) encourage the cleavage of GTP to GDP. The GTP binding proteins interact (depending on their GTP status) with a variety of effector proteins. One example is a family of formins, which chaperone the polymerization of actin. At the head of the pathway, signals coming from external or internal conditions regulate the GTPases, creating (in extremely simplified terms) a pathway that gets the cell to respond by moving toward things it wants, and away from things it does not want. 

This is a very brief post, just touching on one experiment done on this system. Exploring its full complexity is way beyond my current expertise, though we may return to aspects of this fascinating biological pathway periodically in the future. An important paper in the field hooked up fluorescent dyes to one of the effector protein domains that binds only GTP/active RhoA. They tethered this to the (inside) membrane of their cultured cells, and took movies of what the cell looked like, using a microscopy method that looks at very thin sections- only the membrane, essentially not the rest of the cell. RhoA, though graced with a small lipid tail, is typically cytoplasmic when inactive, and travels to the membrane when activated. They were shocked to find that in resting cells, without much locomotion going on, there were recurring waves of activation of RhoA that swept hither and yon across the cell membranes. 

Four examples of RhoA getting bound in its active state in a wave-like way, over 7 1/2 minutes in a resting cell. GEF-H1 is ARHGEF2, one of the regulators that can turn RhoA on. The first three panels have ARHGEF2 versions that are operational, but the fourth (bottom right) is of a cell with an anti-RNA to ARHGEF2, turning its expression level down. In this cell, the waves of RhoA activation and recruitment to the membrane are substantially dampened.

These pulses were made even more intense if the cells were treated with nocodazole, which disrupts microtubules, destabilizes the cytoskeleton, and makes the actin regulatory / structure system work harder. They found that myosin (the motor protein that moves cargoes over actin filaments) was also rapidly relocalized, mirroring some of what happened with RhoA. They also found that ARHGEF2 contained two RhoA binding domains, (one binding active RhoA, one binding inactive RhoA), enabling it to feedback-amplify the positive activation of RhoA, thereby explaining some of the extremely dynamic activity seen here. 

And they also found that the arrival of negative regulators such as ARHGAP35 was delayed by a couple of seconds vs the activation of RhoA, providing the time window needed to see wave formation out of a mechanism of positive feedback followed by squelching by a negative regulator. Lastly, they found that these dynamics were significantly different if the cells were grown on stiffer vs softer substrates. Stiffer substrates allowed the formation of stronger surface attachments, concentrating RhoA and myosin at these adhesion locations. 

These researchers are clearly only scratching the surface of this system, as there are endless complexities left to investigate. The upshot of this one set of observations is that neurons are not the only excitable cells. With a bit of molecular / experimental magic, heretofore unseen intracellular dynamics can be visualized to show that eukaryotic cells have an exquisitely regulated internal excitation system that is part of what drives their shape-shifting capabilities, including processes like phagocytosis and neuronal growth / path-finding. 

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Lipid Pumps and Fatty Shields- Asymmetry in the Plasma Membrane

The two faces constituting eukaryotic cell membranes are asymmetric.

Membranes are one of those incredibly elegant things in biology. Simple chemicals forces are harnessed to create a stable envelope for the cell, with no need to encode the structure in complicated ways. Rather, it self-assembles, using the oil-vs-water forces of surface tension to form a huge structure with virtually no instruction. Eukaryotes decided to take membranes to the max, growing huge cells with an army of internal membrane-bound organelles, individually managed- each with its own life cycle and purposes.

Yet, there are complexities. How do proteins get into this membrane? How do they orient themselves? Does it need to be buttressed against rough physical insult, with some kind of outer wall? How do nutrients get across, while the internal chemistry is maintained as different from the outside? How does it choose which other cells to interact with, preventing fusion with some, but pursuing fusion with others? For all the simplicity of the basic structure, the early history of life had to come up with a lot of solutions to tough problems, before membrane management became such a snap that eukaryotes became possible.

The authors present their model (in atomic simulation) of a plasma membrane. Constituents are cholesterol, sphingomyelin (SM), phosphatidyl choline (PC) phosphatidyl serine (PS), phosphatidyl ethanolamine (PE), and phosphotidyl ethanolamine plasmalogen. Note how in this portrayal, there is far more cholesterol in the outer leaflet (top), facing the outside world, than there is in the inner leaflet (bottom).

The major constituents of the lipid bilayer are cholesterol, phospholipids, and sphingomyelin. The latter two have charged head groups and long lipid (fatty) tails. The head groups keep that side of the molecule (and the bilayer) facing water. The tails hate water and like to arrange themselves in the facing sheets that make up the inner part of the bilayer. Cholesterol, on the other hand, has only a mildly polar hydroxyl group at one end, and a very hydrophobic, stiff, and flat multi-ring body, which keeps strictly with the lipid tails. The lack of a charged head group means that cholesterol can easily flip between the bilayer leaflets- something that the other molecules with charged headgroups find very difficult. It has long been known that our genomes code for flippases and floppases: ATP-driven enzymes that can flip the charged phospholipids and sphingomyelin from one leaflet to the other. Why these enzymes exist, however, has been a conundrum.

Pumps that drive phospholipids against their natural equilibrium distribution, into one or the other leaflet.

It is not immediately apparent why it would be helpful to give up the natural symmetry and fluidity of the natural bilayer, and invest a lot of energy in keeping the compositions of each leaflet different. But that is the way it is. The outer leaflet of the plasma membrane tends to have more sphingomyelin and cholesterol, and the inner leaflet has more phospholipids. Additionally, those phospholipids tend to have unsaturated tails- that is, they have double bonds that break up the straight fatty tails that are typical in sphingomyelin. Membrane asymmetry has a variety of biological effects, especially when it is missing. Cells that lose their asymmetry are marked for cell suicide, intervention of the immune system, and also trigger coagulation in the blood. It is a signal that they have broken open or died. But these are doubtless later (maybe convenient) organismal consequences of universal membrane asymmetry. They do not explain its origin. 

A recent paper delved into the question of how and why this asymmetry happens, particularly in regard to cholesterol. Whether cholesterol even is asymmetric is controversial in the field, since measuring their location is very difficult. Yet these authors carefully show that, by direct measurement, and also by computer simulation, cholesterol, which makes up roughly forty percent of the membrane (its most significant single constituent, actually), is highly asymmetric in human erythrocyte membranes- about three fold more abundant in the outer leaflet than in the cytoplasmic leaflet. 

Cholesterol migrates to the more saturated leaflet. B shows a simulation where a poly-unstaturated (DAPC) phospholipid with 4 double bonds (blue) is contrasted with a saturated phospholipid (DPPC) with staight lipid tails (orange). In this simulation, cholesterol naturally migrates to the DPPC side as more DAPC is loaded, relieving the tension (and extra space) on the inner leaflet. Panel D shows that in real cells, scrambling the leaflet composition leads to greater cholesterol migration to the inner leaflet. This is a complex experiment, where the fluorescent signal (on the right-side graph) comes from a dye in an introduced cholesterol analog, which is FRET-quenched by a second dye that the experimenters introduced which is confined to the outer membrane. In the natural case (purple), signal is more quenched, since more cholesterol is in the outer leaflet, while after phospholipid scrambling, less quenching of the cholesterol signal is seen. Scrambling is verified (left side) by fluorescently marking the erythrocytes for Annexin 5, which binds to phosphatidylcholine, which is generally restricted to the inner leaflet. 

But no cholesterol flippase is known. Indeed, such a thing would be futile, since cholesterol equilibrates between the leaflets so rapidly. (The rate is estimated at milliseconds, in very rough terms.) So what is going on? These authors argue via experiment and chemical simulation that it is the pumped phospholipids that drive the other asymmetries. It is the straight lipid tails of sphingomyelin that attract the cholesterol, as a much more congenial environment than the broken/bent tails of the other phospholipids that are concentrated in the cytoplasmic leaflet. In turn, the cholesterol also facilitates the extreme phospholipid asymmetry. The authors show that without the extra cholesterol in the outer leaflet, bilayers of that extreme phospholipid composition break down into lipid globs.

When treated (time course) with a chemical that scrambles the plasma membrane leaflet lipid compositions, a test protein (top series) that normally (0 minutes) attaches to the inner leaflet floats off and distributes all over the cell. The bottom series shows binding of a protein (from outside these cells) that only binds phosphatidylcholine, showing that scrambling is taking place.

This sets up the major compositional asymmetry between the leaflets that creates marked differences in their properties. For example, the outer leaflet, due to the straight sphingomyelin tails and the cholesterol, is much stiffer, and packed much tighter, than the cytoplasmic leaflet. It forms a kind of shield against the outside world, which goes some way to explain the whole phenomenon. It is also almost twice as impermeable to water. Conversely, the cytoplasmic leaflet is more loosely packed, and indeed frequently suffers gaps (or defects) in its lipid integrity. This has significant consequences because many cellular proteins, especially those involved in signaling from the surface into the rest of the cytoplasm, have small lipid tails or similar anchors that direct them (temporarily) to the plasma membrane. The authors show that such proteins localize to the inner leaflet precisely because that leaflet has this loose, accepting structure, and are bound less well if the leaflets are scrambled / homogenized.

When the fluid mosaic model of biological membranes was first conceived, it didn't enter into anyone's head that the two leaflets could be so different, or that cells would have an interest in making them so. Sure, proteins in those membranes are rigorously oriented, so that they point in the right direction. But the lipids themselves? What for? Well, they do and now there are some glimmerings of reasons why. Whether this has implications for human disease and health is unknown, but just as a matter of understanding biology, it is deeply interesting.

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