Dangerous Memories

Some memory formation involves extracellular structures, DNA damage, and immune component activation / inflammation.

The physical nature of memories in the brain is under intensive scrutiny. The leading general theory is that of positive reinforcement, where neurons that are co-activated strengthen their connections, enhancing their ability to co-fire and thus to express the same pattern again in the future. The nature of these connections has been somewhat nebulous, assumed to just be the size and stability of their synaptic touch-points. But it turns out that there is a great deal more going on.

A recent paper started with a fishing expedition, looking at changes in gene expression in neurons at various time points after the mice were subjected to a fear learning regimen. They took this out to much longer time points (up to a month) than had been contemplated previously. At short times, a bunch of well-known signals and growth-oriented gene expression happened. At the longest time points, organization of a structure called the perineural net (PNN) was read out of the gene expression signals. This is a extracellular matrix sheath that appears to stabilize neuronal connections and play a role in long-term memory and learning. 

But the real shocker came at the intermediate time point of about four days. Here, there was overexpression of TLR9, which is an immune system detector of broken / bacterial DNA, and inducer in turn of inflammatory responses. This led the authors down a long rabbit hole of investigating what kind of DNA fragmentation is activating this signal, how common this is, how influential it is for learning, and what the downstream pathways are. Apparently, neuronal excitation, particularly over-excitation that might be experienced under intense fear conditions, isn't just stressful in a semiotic sense, but is highly stressful to the participating neurons. There are signs of mitochondrial over-activity and oxidative stress, which lead to DNA breakage in the nucleus, and even nuclear perforation. It is a shocking situation for cells that need to survive for the lifetime of the animal. Granted, these are not germ cells that prioritize genomic stability above all else, but getting your DNA broken just for the purpose of signaling a stress response that feeds into memory formation? That is weird.

Some neuronal cell bodies after fear learning. The red dye is against a marker of DNA repair proteins, which form tight dots around broken DNA. The blue is a general DNA stain, and the green is against a component of the nuclear envelope, showing here that nuclear envelopes have broken in many of these cells.

The researchers found that there are classic signs of DNA breakage, which are what is turning on the TLR9 protein, such as seeing concentrated double-strand DNA repair complexes. All this stress also turned on proteases called caspases, though not the cell suicide program that these caspases typically initiate. Many of the DNA break and repair complexes were, thanks to nuclear perforation, located diffusely at the centrosome, not in the nucleus. TLR9 turns on an inflammatory response via NFKB / RELA. This is clearly a huge event for these cells, not sending them into suicide, but all the alarms short of that are going off.

The interesting part was when the researchers asked whether, by deleting the TLR9 or related genes in the pathway, they could affect learning. Yes, indeed- the fear memory was dependent on the expression of this gene in neurons, and on this cell stress pathway, which appears to be the precondition of setting up the perineural net structures and overall stabilization. Additionally, the DNA damage still happened, but was not properly recognized and repaired in the absence of TLR9, creating an even more dangerous situation for the affected neurons- of genomic instability amidst unrepaired DNA.

When TRL9 is knocked out, DNA repair is cancelled. At bottom are wild-type cells, and at top are mouse neurons after fear learning that have had the gene TLR9 deleted. The red dye is against DNA repair proteins, as is the blue dye in the right-most frames. The top row is devoid of these repair activities.

This paper and its antecedent literature are making the case that memory formation (at least under these somewhat traumatic conditions- whether this is true for all kinds of memory formation remains to be seen) has commandeered ancient, diverse, and quite dangerous forms of cell stress response. It is no picnic in the park with madeleines. It is an all-hands-on-deck disaster scene that puts the cell into a permanently altered trajectory, and carries a variety of long-term risks, such as cancer formation from all the DNA breakage and end-joining repair, which is not very accurate. They mention in passing that some drugs have been recently developed against TLR9, which are being used to dampen inflammatory activities in the brain. But this new work indicates that such drugs are likely double-edged swords, that could impair both learning and the long-term health of treated neurons and brains.

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Aging and Death

Our fate was sealed a very long time ago.

Why do we die? It seems like a cruel and wasteful way to run a biosphere, not to mention a human life. After we have accumulated a lifetime of experience and knowledge, we age, decline, and sign off, whether to go to our just reward, or into oblivion. What is the biological rationale and defense for all this, which the biblical writers assigned to the fairy tale of the snake and the apple?

A recent paper ("A unified framework for evolutionary genetic and physiological theories of aging") discusses evolutionary theories of aging, but in typical French fashion, is both turgid and uninteresting. Aging is widely recognized as the consequence of natural selection, or more precisely, the lack thereof after organisms have finished reproducing. Thus we are at our prime in early adulthood, when we seek mates and raise young. Evolutionarily, it is all downhill from there. In professional sports, athletes are generally over the hill at 30, retiring around 35. Natural selection is increasingly irrelevant after we have done the essential tasks of life- surviving to mate and reproduce. We may participate in our communities, and do useful things, but from an evolutionary perspective, genetic problems at this phase of life have much less impact on reproductive success than those that hit earlier. 

All this is embodied in the "disposable soma" theory of aging, which is that our germ cells are the protected jewels of reproduction, while the rest of our bodies are, well, disposable, and thus experience all the indignities of age once their job of passing on the germ cells is done. The current authors try to push another "developmental" theory of aging, which posits that the tradeoffs between youth and age are not so much the resources or selective constraints focused on germ cell propagation vs the soma, but that developmental pathways are, by selection, optimized for the reproductive phase of life, and thus may be out of tune for later phases. Some pathways are over-functional, some under-functional for the aged body, and that imbalance is sadly uncorrected by evolution. Maybe I am not doing justice to these ideas, which maybe feed into therapeutic options against aging, but I find this distinction uncompelling, and won't discuss it further.

A series of unimpressive distinctions in the academic field studying aging from an evolutionary perspective.

Where did the soma arise? Single cell organisms are naturally unitary- the same cell that survives also mates and is the germ cell for the next generation. There are signs of aging in single cell organisms as well, however. In yeast, "mother" cells have a limited lifespan and ability to put out daughter buds. Even bacteria have "new" and "old" poles, the latter of which accumulate inclusion bodies of proteinaceous junk, which apparently doom the older cell to senescence and death. So all cells are faced with processes that fail over time, and the only sure bet is to start as a "fresh" cell, in some sense. Plants have taken a distinct path from animals, by having bodies and death, yes, but being able to generate germ cells from mature tissues instead of segregating them very early in development into stable and distinct gonads.

Multicellularity began innocently enough. Take slime molds, for example. They live as independent amoebae most of the time, but come together to put out spores, when they have used up the local food. They form a small slug-like body, which then grows a spore-bearing head. Some cells form the spores and get to reproduce, but most don't, being part of the body. The same thing happens with mushrooms, which leave a decaying mushroom body behind after releasing their spores. 

We don't shed alot of tears for the mushrooms of the world, which represent the death-throes of their once-youthful mycelia. But that was the pattern set at the beginning- that bodies are cells differentiated from the germ cells, that provide some useful, competitive function, at the cost of being terminal, and not reproducing. Bodies are forms of both lost energy and material, and lost reproductive potential from all those extra cells. Who could have imagined that they would become so ornate as to totally overwhelm, in mass and complexity, the germ cells that are the point of the whole exercise? Who could have imagined that they would gain feelings, purposes, and memories, and rage against the fate that evolution had in store for them?

On a more mechanistic level, aging appears to arise from many defects. One is the accumulation of mutations, which in soma cells lead to defective proteins being made and defective regulation of cell processes. An extreme form is cancer, as is progeria. Bad proteins and other junk like odd chemicals and chemically modified cell components can accumulate, which is another cause of aging. Cataracts are one example, where the proteins in our lenses wear out from UV exposure. We have quite intricate trash disposal processes, but they can't keep with everything, as we have learned from the advent of modern chemistry and its many toxins. Another cause is more programmatic: senescent cells, which are aged-out and have the virtue that they are blocked from dividing, but have the defect that they put out harmful signals to the immune system that promote inflammation, another general cause of aging.

Aging research has not found a single magic bullet, which makes sense from the evolutionary theory behind it. A few things may be fixable, but mostly the breakdowns were never meant to be remedied or fixed, nor can they be. In fact, our germ cells are not completely immune from aging either, as we learn from older fathers whose children have higher rates of autism. We as somatic bodies are as disposable as any form of packaging, getting those germ cells through a complicated, competitive world, and on to their destination.

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Nominee for Most Amazing Protein: RAD51

On the repair and resurrection of DNA, which gets a lot of help from a family of proteins including RAD51, DMC1, and RecA.

Proteins do all sorts of amazing things, from composing pores that can select a single kind of ion- even just a proton- to allow across a membrane, to massive polymerizing enzymes that synthesize other proteins, DNA, and RNA. There is really no end to it. But one of the most amazing, even incredible, things that happens in a cell is the hunt for DNA homology. Even over a genome of billions of base pairs, it is possible for one DNA segment to find the single other DNA segment that matches it. This hunt is executed for several reasons. One is to line up the homologous chromosomes at meiosis, and carry out the genetic cross-overs between them (when they are lined up precisely) that help scramble our genetic lineages for optimal mix-and-matching during reproduction. Another is for DNA repair, which is best done with a good copy for reference, especially when a full double-strand break has happened. Just this week, a fascinating article showed that memories in our brains depend in some weird way on DNA breaks occurring in neurons, some of which then use the homologous repair process, including homology search, to patch things up.

The protein that facilitates this DNA homology search is deeply conserved in evolution. It is called RecA in bacteria, radA and radB in archaea, and the RAD51 family in eukaryotes. Naturally, the eukaryotic family is most closely related to the archaeal versions (RAD51 and DMC1 evolving from radA, and a series of other, and poorly understood family members, from radB). In this post, I will mostly just call them all RAD51, unless I am referring to DMC1 specifically. The name comes from genetic screens for radiation-sensitive mutants in human and other eukaryotes, since RAD51 plays a crucial role in DNA repair, as noted above. RAD51 is not a huge protein, but it is an ATPase. It binds to itself, forming linear filaments with ATP at the junction points between units. It binds to a single strand of DNA, which is going to be what does the hunting. And it binds, in a complicated way, to another double-stranded DNA, which it helps to open briefly to allow its quality as a target to be evaluated. 

This diagram describes the repair of double strand breaks (DSB) in DNA. First the ends are covered with a bunch of proteins that signal far and wide that something terrible has happened- the cell cycle has to stop.. fire engines need to be called. One of these proteins is RPA, which simply binds all over single-stranded DNA and protects it. Then the RAD51 protein comes in, displaces RPA, and begins the homology search process. The second DNA shown, in dark black, doesn't just happen, but is hunted for high and low throughout the nucleus to find the exact homolog of the broken end. When that exact match is found, the repair process can proceed, with continued DNA synthesis through the lesion, and resolution of the newly repaired double strands, either to copy up the homolog version, or exchange versions (GC, for gene conversion). 

This diagram shows how the notorious (when mutated) oncogene BRCA2 (in green) works. It binds RAD51 (in blue) and brings it, chain-gang style, to the breakpoints of DNA damage to speed up and specify repair.

There have been several structural studies by this point that clarify how RAD51 does its thing. ATP is simply required to form filaments on single-stranded DNA. When a match has been found and RAD51 is no longer needed, ATP is cleaved, and RAD51 falls off, back to reserve status. The magic starts with how RAD51 binds the single stranded DNA. One RAD51 binds for every ~3 bases in the DNA, and the it binds the phosphate backbone, so that the bases are nicely exposed in front, and all stretched out, ready to hunt for matching DNA.

A series of RAD51 molecules (in this case, RecA from bacteria) bound sequentially to single-stranded DNA (red). Note the ATP homolog chemicals in yellow, positioned between each protein unit. One can see that the DNA is stretched out a bit and the bases point outwards.

A closeup view of one of the RAD51 units from above, showing how the bases of the DNA (yellow) are splayed out into the medium, ready to find their partners. They are arranged in orientations similar to how they sit in normal (B-form) DNA, further enhancing their ability to find partners.

The second, and more mysterious part of the operation is how RAD51 scans double-stranded DNA throughout the genome. It has binding sites for double-stranded DNA, away from the single-stranded DNA, and then it also has a little finger that splits open the double-stranded DNA, encouraging separation and allowing one strand to face up to the single stranded DNA that is held firmly by the RAD51 polymer. The transient search happens in eight-base increments, with tighter capture of the double-strand DNA happening when nine bases are matched, and committment to recombination or repair happening when a match of fifteen bases is found.  

These structures show an intermediate where a double-stranded DNA (ends in teal and lavender, and separated DNA segments in green and red) has been captured, making a twelve base match with the stable single-stranded DNA (brown). Note how the double-stranded DNA ends are held by outside portions of the RAD51 protein. Closeup on the right shows the dangling, non-paired DNA strand in red, and the newly matched duplex DNA with green-brown colored base interactions.

These structures can only give a hint of what is going on, since the whole process relies so clearly on the brownian motion that allows super-rapid diffusion of the stablized single-strand DNA+RAD51 over the genome, which it scans efficiently in one-dimensional fashion, despite all the chromatin and other proteins parked all over the place. And while the structures provide insight into how the process happens, it remains incredible that this search can happen, on what is clearly a quite reliable basis, day and day out, as our genomes get hit by whatever the environment throws at us.

"Unfortunately, most RAD51 and RAD51 paralog point mutations that have been clinically identified are classified as variants of unknown significance (VUSs). Future studies to reclassify these RAD51 gene family VUSs as pathogenic or benign are desperately needed, as many of these genes are now included on hereditary breast and ovarian cancer screening panels. Reclassification of HR-deficient VUSs would enable these patients to benefit from therapies that specifically target HR deficiency, as do poly(ADP)-ribose polymerase (PARP) inhibitors in BRCA1/2-deficient cells."

Lastly, one paper made the point that clinicians need better understanding of the various mutations that can affect RAD51 itself. Genetic testing now is able to find all of our mutations, but we don't always know what each mutation is capable of doing. Thus deeper studies of RAD51 will have beneficial effects on clinical diagnosis, when particular mutations can be assigned as disease-causing, thus justifying specific therapies that would otherwise not be attempted.

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Getting Cancer Cells to Shoot Themselves

New chemicals that make novel linkages among cellular components can be powerful drugs.

One theme that has become common in molecular biology over the years is the prevalence of proteins whose only job is to bring other proteins together. Many proteins lack any of the usual jazzy functions, like catalytic enzyme, or ion channel, or signaling kinase, but just serve as "conveners", bringing other proteins together. Typically they are regulated in some way, by phosphorylation, expression, or localization, and some of these proteins serve as key "scaffolds" for the activation of some process, like G-protein activation, or cell cycle control, or cell growth. 

Well, the drug industry has caught on, and is starting to think about chemicals that can do similar things, resulting in occasionally powerful results. Conventional drug design has aimed to bind to whatever protein is responsible for some ill, and inhibit it. Such as an oncogene, or an over-active component of the immune system. This has led to many great drugs, but has significant limitations. The chemical has to bind not just anywhere on the target, but at the particular spot (the active site) that is its business end, where its action happens. And it has to bind really well, since binding and inhibiting only half the target proteins in a cell (or the body) will typically only have a modest effect. These requirements are quite stringent and result in many protein targets being deemed difficult to drug, or "undruggable".

A paradigm for a new kind of chemical drug, which links two functions, is the PROTAC class, which combines binding with a target on one end, with another end that binds to the cell's protein destruction machinery, thereby not just inhibiting the target, but destroying it. A new paper describes an even more nuclear option along this line of drug development, linking an oncogene with a second part that activates the cellular suicide machinery. One can imagine that this approach can have far more dramatic effects.

These researchers synthesize and demonstrate a chemical that binds on one end the oncogene BCL6, mutations of which can cause B cell lymphoma. This gene is a transcription repressor, and orchestrates the development of particular immunologic T cells called T follicular helper cells. One of its roles is to prevent the suicide of these cells when an antigen is present, which is when the cells are most needed. If over-expressed in cancer, these cells think they really need to protect the body and proliferate wildly.

The other end of this chemical, called TCIP1, binds to BRD4, which is another transcription regulator, but this one activates the cell suicide genes, instead of turning them off. Both ends of this molecule were based on previously known structures. The innovation was solely in linking them together. I should say parenthetically that BRD4 is itself recognized as an oncogene, as it can promote cell growth and prevent cell suicide in many settings. So it has ambivalent roles, (inviting a lot of vague writing), and it is somewhat curious that these researchers focused on BRD4 as an apoptosis driver.

"TCIP1 kills diffuse large B cell lymphoma cell lines, including chemotherapy-resistant, TP53-mutant lines, at EC50 of 1–10 nM in 72 h" 

Here EC50 means the effective concentration where the effect is 50% of maximal. This value of 1.3 nano molar is a very low concentration for a drug, meaning it is highly effective. TP53 is another cancer-driving mutation, common in treatment-resistant cancers. The drug has a characteristic and curious dosage behavior, as its effect decreases at higher concentrations. This is because each individual end of the molecule starts to bind and saturate targets independently, reducing the rate of linkage between the two target proteins, and thus the intended effect.

Chemical structure of TCIP1. The left side binds to BRD4, a regulator of cell suicide, while the right side binds to BCL6, an oncogene.

The authors did numerous controls with related chemicals, and tracked genes that were targeted by the novel chemical, all to show that the dramatic effects they were seeing were specifically caused by the linkage of the two chemical functions. Indeed, BCL6 represses its own transcription in the natural course of affairs, and the new drug reverses this behavior as well, inducing more of its own synthesis, which now potentiates the drug's lethal effect. While the authors did not show effectiveness in animals, they did show that TCIP1 is not toxic in mice. Neither did they show that TCIP1 is orally available, but administered it by injection. But even by this mode, it would, if effective, be a very exciting therapy. Not surprisingly, the authors report a long series of biotech industry ties (rooted at Stanford) and indicate that this technology is under license for drug development.

This approach is highly promising, and a significant advance in the field. It should allow increased flexibility in targeting all kinds of proteins that may or not cause disease, but are specific to or over-expressed in disease states, in order to address those diseases. It will allow increased flexibility in targeting apoptosis (cell suicide) pathways through numerous entry points, to have the same ultimate (and highly effective) therapeutic endpoint. It allows drugs to work at low concentrations, not needing to fully occupy or inhibit their targets. Many possible areas of therapy can be envisioned, but one is aging. By targeting and killing senescent cells, which are notorious for promoting aging, significant increases in lifespan and health are conceivable. 

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A Hard Road to a Cancer Drug

The long and winding story of the oncogene KRAS and its new drug, sotorasib.

After half a century of the "War on Cancer", new treatments are finally straggling into the clinic. It has been an extremely hard and frustrating road to study cancer, let alone treat it. We have learned amazing things, but mostly we have learned how convoluted a few billion years of evolution can make things. The regulatory landscape within our cells is undoubtedly the equal of any recalcitrant bureaucracy, full of redundant offices, multiple veto points, and stakeholders with obscure agendas. I recently watched a seminar in the field, which discussed one of the major genes mutated in cancer and what it has taken to develop a treatment against it. 

Cancer is caused by DNA mutations, and several different types need to occur in succession. There are driver mutations, which are the first step in the loss of normal cellular control. But additional mutations have to happen for such cells to progress through regulatory blocks, like escape from local environmental controls on cell type and cell division, past surveillance by the immune system, and past the reluctance of differentiated cells to migrate away from their resident organ. By the end, cancer cells typically have huge numbers of mutations, having incurred mutations in their DNA repair machinery in an adaptive effort to evade all these different controls.

While this means that many different targets exist that can treat some cancers, it also means that any single cancer requires a precisely tailored treatment, specific to its mutated genes. And that resistance is virtually inevitable given the highly mutable nature of these cells. 

One of the most common genes to be mutated to drive cancer (in roughly 20% of all cases) is KRAS, part of the RAS family of NRAS, KRAS, and HRAS. These were originally discovered through viruses that cause cancer in rats. These viruses (such as Kirsten rat sarcoma virus) had a copy of a rat gene in it, which it overpoduces and uses to overcome normal proliferation controls during infection. The viral gene was called an oncogene, and the original rat (or human) version was called a proto-oncogene, named KRAS. The RAS proteins occupy a central part of the signaling path that external events and stresses turn on to activate cell growth and proliferation, called the MAP kinase cascade. For instance, epidermal growth factor comes along in the blood, binds to a receptor on the outside of a cell, and turns on RAS, then MEK, MAPK, and finally transcription regulators that turn on genes in the nucleus, resulting in new proteins being expressed. "Turning on" means different things at each step in this cascade. The transcription regulators typically get phosphorylated by their upstream kinases like MAPK, which tag them for physical transport into the nucleus, where they can then activate genes. MAPK is turned on by being itself phosphorylated by MEK, and MEK is phosphorylated by RAF. RAF is turned on by binding to RAS, whose binding activity in turn is regulated by the state of a nucleotide (GTP) bound by RAS. When binding GTP, RAS is on, but if binding GDP, it is off.

A schematic of the RAS pathway, whereby extracellular growth signals are interpreted and amplified inside our cells, resulting in new gene expression as well as other more immediate effects. The cell surface receptor, activated by its ligand, activates associated SOS which activates RAS to the active (GTP) state. This leads to a kinase cascade through RAF, MEK, and MAPK and finally to gene regulators like MYC.

This whole system seems rather ornate, but it accomplishes one important thing, which is amplification. One turned-on RAF molecule or MEK molecule can turn on / phosphorylate many targets, so this cascade, though it appears linear in a diagram, is acutally a chain reaction of sorts, amplifying as it goes along. And what governs the state of RAS and its bound GTP? The state of the EGFR receptor, of course. When KRAS is activated, the resident GDP leaves, and GTP comes to take its place. RAS is a weak GTPase enzyme itself, slowly converting itself from the active back to the inactive state with GDP. 

Given all this, one would think that RAS, and KRAS in particular, might be "druggable", by sticking some well-designed molecule into the GTP/GDP binding pocket and freezing it in an inactive state. But the sad fact of the matter is that the affinity KRAS has to GTP is incredibly high- so high it is hard to measure, with a binding constant of about 20 pM. That is, half the KRAS-bound GTP comes off when the ambient concentration of GTP is infinitesimal, 0.02 nano molar. This means that nothing else is likely to be designed that can displace GTP or GDP from the KRAS protein, which means that in traditional terms, it is "undruggable". What is the biological logic of this? Well, it turns out that the RAS enzymes are managed by yet other proteins, which have the specific roles of prying GDP off (GTP exchange factor, or GEF) and of activating the GTP-ase activity of RAS to convert GTP to GDP (GTPase activating protein, or GAP). It is the GEF protein that is stimulated by the receptors like EGFR that induce RAS activity. 

So we have to be cleverer in finding ways to attack this protein. Incidentally, most of the oncogenic mutations of KRAS are at the twelfth residue, glycine, which occupies a key part of the GAP binding site. As glycine is the smallest amino acid, any other amino acid here is bulkier, and blocks GAP binding, which means that KRAS with any of these mutations can not be turned off. It just keeps on signaling and signaling, driving the cell to think it needs to grow all the time. This property of gain of function and the ability of any mutation to fit the bill is why this particular defect in KRAS is such a common cancer-driving mutation. It accounts for ~90% of pancreatic cancers, for instance. 

The seminar went on a long tangent, which occupied the field (of those looking for ways to inhibit KRAS with drugs) for roughly a decade. RAS proteins are not intrinsically membrane proteins, but they are covalently modified with a farnesyl fatty tail, which keeps them stuck in the cell's plasma membrane. Indeed, if this modification is prevented, RAS proteins don't work. So great- how to prevent that? Several groups developed inhibitors of the farnesyl transferase enzyme that carries out this modification. The inhibitors worked great, since the farnesyl transferase has a nice big pocket for its large substrate to bind, and doesn't bind it too tightly. But they didn't inhibit the RAS proteins, because there was a backup system- geranygeranyl transferase that steps into the breach as a backup, which can attach an even bigger fatty tail to RAS proteins. Arghhh!

While some are working on inhibiting both enzymes, the presenter, Kevan Shokat of UCSF, went in another direction. As a chemist, he figured that for the fraction of the KRAS mutants at position 12 that transform from glycine to cysteine, some very specific chemistry (that is, easy methods of cross-linking), can be brought to bear. Given the nature of the genetic code, the fraction of mutations that go from glycine to cysteine are small, there being eight amino acids that are within a one-base change of glycine, coded by GGT. So at best, this approach is going to have a modest impact. Nevertheless, there was little choice, so they forged ahead with a complicated chemical scheme to make a small molecule that could chemically crosslink to that cysteine, with selectivity determined by a modest shape fit to the surface of the KRAS protein near this GEF binding site. 

A structural model of KRAS, with its extremely tightly-bound substrate GDP in orange. The drug sotorasib is below in teal, bound in another pocket, with a tail extending upwards to the (mutant) cysteine 12, which is not differentiated by color, but sits over a magnesium ion (green) being coordinated by GDP. The main job of sotorasib is to interfere with the binding of the guanine exchange factor (GEF) which happens on the surface to its left, and would reset KRAS to an active state.

This approach worked surprisingly well, as the KRAS protein obligingly offfered a cryptic nook that the chemists took advantage of to make this hybrid compound, now called the drug sotorasib. This is an FDA-approved treatment for cancers which are specifically driven by this particular KRAS mutation of position 12 from glycine to cysteine. That research group is currently trying to extend their method to other mutant forms, with modest success. 

So let's take a step back. This new treatment requires, obviously, the patient's tumor to be sequenced to figure out its molecular nature. That is pretty standard these days. And then, only a small fraction of patients will get the good news that this drug may help them. Lung cancers are the principal candidates currently, (of which about 15% have this mutation), while only about 1-2% of other cancers have this mutation. This drug has some toxicity- while it is a magic bullet, its magic is far from perfect, (which is odd given the exquisite selectivity it has for the mutated form of KRAS, which should only exist in cancer tissues). And lastly, it gives, on average, under six months of reprieve from cancer progression, compared to four and a half months with a more generic drug. As mentioned above, tumors at this stage are riven with other mutations and evolve resistence to this treatment with appalling relentlessness.

While it is great to have developed a new class of drugs like this one against a very recalcitrant target, and done so on a highly rational basis driven by our growing molecular knowlege of cancer biology, this result seems like a bit of a let-down. And note also that this achievement required decades of publicly funded research, and doubtless a billion dollars or more of corporate investment to get to this point. Costs are about twenty five thousand dollars per patient, and overall sales are maybe two hundred million dollars per year, expected to increase steadily.

Does this all make sense? I am not sure, but perhaps the important part is that things can not get worse. The patent on this drug will eventually expire and its costs will come down. And the research community will keep looking for other, better ways to attack hard targets like KRAS, and will someday succeed.

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Senescent, Cancerous Cannibals

Tumor cells not only escape normal cell proliferation controls, but some of them eat nearby cells.

Our cells live in an uneasy truce. Cooperation is prized and specialization into different cell types, tissues, and organs is pervasive. But deep down, each cell wants to grow and survive, prompting many mechanisms of control, such as cell suicide (apoptosis) and immunological surveillance (macrophages, killer T-cells). Cancer is the ultimate betrayal, not only showing disregard for the ruling order, but in its worst forms killing the whole organism in a pointless drive for growth.

A fascinating control mechanism that has come to prominence recently is cellular senescence. In petri dishes, cells can only be goosed along for a few dozen cycles of division until they give out, and become senescent. Which is to say, they cease replicating but remain alive. It was first thought that this was another mechanism to keep cancer under control, restricting replication to "stem" cells and their recent progeny. But a lot of confusing and interesting observations indicate that the deeper meaning of senescence lies in development, where it appears to function as an alternate form of cell suicide, delayed so that tissues are less disrupted. 

Apoptosis is used very widely during development to reshape tissues, and senescence is used extensively as well in these programs. Senescent cells are far from quiescent, however. They have high metabolic activity and are particularly notorious for secreting a witches' brew of inflammatory cytokines and other proteins- the senescence-associated secretory phenotype, or SASP. in the normal course of events, this attracts immune system cells which initiate repair and clearance operations that remove the senescent cells and make sure the tissue remains on track to fulfill its program. These SASP products can turn nearby cells to senescence as well, and form an inflammatory micro-environment that, if resolved rapidly, is harmless, but if persistent, can lead to bad, even cancerous local outcomes. 

The significance of senescent cells has been highlighted in aging, where they are found to be immensely influential. To quote the wiki site:

"Transplantation of only a few (1 per 10,000) senescent cells into lean middle-aged mice was shown to be sufficient to induce frailty, early onset of aging-associated diseases, and premature death."

The logic behind all this seems to be another curse of aging, which is that while we are young, senescent cells are cleared with very high efficiency. But as the immune system ages, a very small proportion of senescent cells are missed, which are, evolutionarily speaking, an afterthought, but gradually accumulate with age, and powerfully push the aging process along. We are, after all, anxious to reduce chronic inflammation, for example. A quest for "senolytic" therapies to clear senescent cells is becoming a big theme in academia and the drug industry and may eventually have very significant benefits. 

Another odd property of senescent cells is that their program, and the effects they have on nearby cells, resemble to some partial degree those of stem cells. That is, the prevention of cell death is a common property, as is the prevention of certain controls preventing differentiation. This brings us to tumor cells, which frequently enter senescence under stress, like that of chemotherapy. This fate is highly ambivalent. It would have been better for such cells to die outright, of course. Most senescent tumor cells stay in senescence, which is bad enough for their SASP effects in the local environment. But a few tumor cells emerge from senescence, (whether due to further mutations or other sporadic properties is as yet unknown), and they do so with more stem-like character that makes them more proliferative and malignant.

A recent paper offered a new wrinkle on this situation, finding that senescent tumor cells have a novel property- that of eating neighboring cells. As mentioned above, senescent cells have high metabolic demands, as do tumor cells, so finding enough food is always an issue. But in the normal body, only very few cells are empowered to eat other cells- i.e. those of the immune system. To find other cells doing this is highly unusual, interesting, and disturbing. It is one more item in the list of bad things that happen when senescence and cancer combine forces.

A senescent tumor cell (green) phagocytoses and digests a normal cell (red).

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DNA Damage Domain Declines to Bind DNA

How one protein domain changed through time.

The BRCA1 and BRCA2 genes are notorious for harboring mutations that increase susceptibility to breast cancer (thus their name, breast cancer type 1 (or 2) susceptibility protein). They have therefore been intensively studied for what they do in the normal course of our cellular lives. Their common naming does not mean they are similar- their structures are completely different. They play related, but distinct, roles in DNA repair, which is naturally influential in our susceptibility to cancer caused by DNA mutations.

An article some time back delved into the history of one domain of the BRCA1 protein, tracing how its functions have changed significantly over evolutionary time. BRCA1 is a large gene encoding a large protein, (1863 amino acids long), composed of several domains. Proteins frequently possess several domains in order to integrate several functions in an orderly way, such as binding a few different partners that together form a complex and carry out some function. Modular protein domains facilitate evolution by being easily duplicated, transferred, and generally being able to be passed around, thanks to rearrangement mutations. BRCA1 has domains that bind to at least 11 other proteins,  most of which play some role in DNA damage responses. So it is a key protein, and damage to it has correspondingly bad effects. 

The domains of BRCA1. Each one has some role in the protein's function, which integrates responses to DNA damage. The BRCT domains are on the very end, right side. NLS is nuclear localization (import) sequence, and NES is the nuclear export signal. These would be typically regulated by other interacting proteins or phosphorylation, to control the access of BRCA1 to the nucleus.

The domain of interest here is the BRCT, or BRCA1 C-terminal domain. It is ~90 amino acids long and BRCA1 has two of them, side by side. Other work has shown that it binds to other proteins, but only after they have been modified by phosphate addition. The DNA damage sensor ATM is one such kinase that adds phosphates to BRCA1 targets such as Abraxis. Thus the BRCT domain plays the key role of bringing this DNA damage repair integrating protein to the right sites, where there is DNA damage to repair. 

Structure of the BRCT double domains in BRCA1 (E). The pocket that binds a phosphorylated serine residue on a partner protein such as abraxis is shown in teal, and in (C), close up. (B) shows a single BRCT domain.

This paper did a sensitive computer search for all possible versions of this domain in all available species and proteins, finding it in 23 human proteins, and in species all the way back to bacteria, so is quite ancient. And the phylogeny they reconstruct indicates that the original versions of these domains had a different function, which was to bind DNA directly, at sites of DNA damage! Such frayed ends also have phosphate groups, so it isn't a huge leap from one function to another. Additionally, other examples of BRCT domains have dispensed with phosphate-dependent binding altogether, but simply bind other proteins regardless. This transition may have happened after phosphorylation became the central way to alert the cell, and key proteins, to the existence of DNA damage, instead of dealing with it solely through enzymes that find & fix such damage directly. This transition allowed a much more robust response by cells, which now includes halting the cell division cycle and activating other stress responses to help the cell recover.

Some of the BRCT domains (along with many others) found in various species and their proteins.

The BRCT domain is mostly used among proteins involved in DNA repair, and even in humans some versions bind DNA directly (PARP1, RFC1). So through the long path of evolution, this single domain has stuck generally to its original role, while it also- along with the organisms and proteins it acts within- diversified and ramified in its functions. From an initial role in direct DNA damage and end recognition, it has become a card-carrying member of the bureaucracy of the cell, playing regulatory and organizing roles within numerous actors important to DNA handling and repair. It is a classic story of how eukaryotes used their surfeit of energy and material resources to develop whole orders of novel molecular, and concomitant outward, complexity.

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The RNAs Shall Protect Us

The humble skin mole has at least one oncogenic mutation. But it is not cancer- why not?

We know that mutations cause cancer. But we also know that it takes multiple mutations, not just one, in virtually all cases. This is one reason why age is such a strong risk factor, providing the time to accumulate multiple "hits". One place where this is particularly apparent is the skin. Most people have moles (nevi) and other imperfections, which are no cause for alarm. We are also on the lookout for the unusual signs and forms that indicate melanoma- which truly is a cause for alarm. Moles typically have one of the key oncogenic mutations for melanoma, however: BRAF V600E (which means the 600th amino acid in its protein chain has been changed from valine to glutamic acid). So what is behind the difference? What systems do cells and organs have to keep this train on the tracks, despite a wheel or two coming off?

A recent paper (review) explored this issue, and tells a complicated technical and scientific story. But the bottom line is that certain miRNAs- a novel form a gene regulator discovered just in the last couple of decades- form a firewall against further proliferation. The BRAF mutation is an activating change, which disrupts the normal "off" state of this protein kinase. BRAF is a protein kinase that attaches phosphate groups to serines and threonines on other proteins. And some those other proteins are specifically other (MAP) protein kinases that form cascades promoting cell proliferation and differentiation. In the case of melanocytes in the skin, the BRAF mutation promotes just that: proliferation, mole formation, and, in some cases, progression to full blown melanoma. 

What is a skin mole? Well, it clearly is composed of lots of cells, so whatever is arresting the mutant BRAF-activated proliferation is taking its sweet time. Proliferation goes for a while, but then stops for an unknown reason. It had been thought in the field (and by these researchers as well) that mole cells had gone into senescence- an irreversible division arrest that is frequently activated in cancer cells and is similar to age-dependent cell cycle arrest. But they show now that senescence is not the explanation. If the BRAF mutation state is reversed, the cells resume dividing. And they also have other hallmarks of a different form of (G2/M) cell division arrest. So something more dynamic is going on.

They do a few technical tours de force of modern DNA sequencing and large-scale molecular biology to find what unusual genes are being expressed in these cells, and find two:  MIR211-5p and MIR328-3p. These are miRNAs, which are short RNA pieces that repress the expression of other genes. We have thousands of them, and each can repress hundreds of other genes, forming a somewhat crazy interdigitated regulatory network. They evolved from an immune function of repressing the expression of viruses and other foreign DNA, but have been repurposed to have broad regulatory effects, often in development and disease.

In BRAF-activated skin mole cells, these miRNAs have one effective target, which is AURKB (Aurora B kinase), another protein kinase that is needed for cell division. No AURKB, no cell division. Indeed, skin mole cells have a high rate of cells stuck in the last phase of cell division, with 4 genome equivalents. They found that AURKB has low expression in skin mole cells, but high expression, as expected, in melanoma cells, while the miRNAs had the reverse pattern. And tellingly, artificial inhibition of these miRNAs released mole cells from their proliferation arrest and allowed the BRAF mutation to have its way with them.

Model of this paper's findings about melanocytes. Starting with stem-like melanocytes, mutated BRAF can cause oncogenenic or pre-oncogenic proliferation. Separately, TPA, or some local tissue factor like TPA, can encourage stem melanocytes to grow and differentiate properly into mature melanocytes. But those same activators (TPA and its natural analog) increase miRNA expression of particularly MIR211-5p, which (by inhibiting AURKB) arrests growth as part of the differentiation program, and also shuts down proliferation caused by mutated BRAF, (at late mitosis / G2 arrest), at least most of the time.

But there was still a problem- what activates the miRNA gene expression in the natural setting? It isn't the mutated BRAF protein, since it routinely drives cells through several replication cycles to form moles, and didn't have any regulatory effect on the miRNAs. The researchers focused on the kinds of local secreted hormones, like endothelin, that might locally inhibit overgrowth of cells, and logically lead to a mole-like pattern. What they hit on was TPA, an artificial analog of diacylglycerol, which is an activator of yet another protein kinase, PKC. TPA is paradoxically a tumor promoter, and is routinely used in cell culture systems to goose the proliferation of melanocytes. But for the mutated BRAF- driven cells from moles, TPA arrests their growth, and it does so because PKC activates the expression of MIR211-5p. They showed that taking TPA out of their cell culture mixes dramatically restarted the growth of mole-derived and other BRAF mutation-driven cells. So this closes the circle in some degree, explaining how it is that skin moles form as sort of arrested mini-cancers.

Unfortunately, TPA is not a natural chemical, and diacylglycerol is not hormone, though many hormones, such as thyroid hormone and oxytocin, do affect PKC activity. So the natural PKC and miRNA activator, and inhibitor of excess proliferation in these BRAF mutation-driven melanocytes remains unknown. I am sure that this research group will be hunting diligently for it, since it is an extremely interesting issue not just in oncology, but in skin and tissue development generally.

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Tale of an Oncogene

Research on a key oncogene of melanoma, MITF, moves from seeing it as a rheostat to seeing it as a supercomputer.

The war on cancer was declared fifty years ago, yet effective therapies are only now trickling in. And very few of them can be characterized as cures. What has been going on, and why is the fight so slow? Here I discuss one example, of melanoma and one of its drivers and central players, the gene MITF.

Melanocytes are not really skin cells, but neural crest cells, i.e. originating in the the embryonic neural tube and giving rise to various peripheral neural structures in the spine, gut, and head. One sub-population migrates off into the epidermis to become melanocytes, which generate skin pigment in melanosome packets, which they distribute around to local keratinocytes. Evolutionarily, these cells are apparently afterthoughts, after originally having developed as part of photoreceptor systems. This history, of unusual evolution and extensive developmental migration and eventual invasion into foreign tissues, has obvious implications for their capacity to form cancers later in life, if mutations re-activate their youthful propensities.

 

Above is shown a sketch of some genes known to play roles in melanoma, and key pathways in which they act. In red are oncogenes known to suffer activating mutations that promote cancer progression. In grey are shown additional oncogenes, ones whose oncogenic mutations are simpler loss-of function, not gain of function, events. And green marks ancillary proteins in these pathways that have not (yet) been found as oncogenes of any sort. MITF is a transcription regulator that drives many genes needed for  melanocyte development and melanosome formation. It also influences cell cycle control and cytoskeletal and cell surface features relevant to migration and invasion of other tissues. This post is based mostly on reviews of the molecules active in melanoma, and the more focused story of MITF.

MITF binds to DNA near target genes, often in concert with other proteins, and activates transcription of the local gene (in most cases, though it represses some targets as well). The evidence linking MITF with melanoma and melanocytes is mostly genetic. It is an essential gene, so complete deletions are lethal. But a wide variety of "mi" mutations in mice and in humans lead to unusual phenotypes like white hair color, loss of hearing, large head formation, small blue eyes, osteopetrosis, and much else. Originally researchers thought there were several different genes involved, but they all resolved down to one complex locus, now called MITF, for mi transcription factor. Certain hereditary mutations also predispose to melanoma, as do some spontaneous mutations. That the dose of MITF also correlates with how active and aggressive a melanoma is also contributes to the recognition that MITF is central to the melanocyte fate and behavior, and also one of the most central players in the disease of melanoma.

The MITF gene spreads over 229,000 base pairs, though it codes for a protein of only 419 amino acids. The gene contains nine alternate transcription start sites, 18 exons (coding regions), and five alternate translation start sites, as sketched above. This structure allows dozens of different forms of the protein to be produced in different tissues and settings, via alternative splicing. The 1M form (above, bottom) is the main one made in melanocytes. Since the gene is essential, mutations that have the phenotypes mentioned above tend to be very small, affecting one amino acid or one splice site, or perhaps truncating translation near the end of the protein. Upstream of the MITF gene and in some of its introns, there are dozens of DNA sites that bind other regulators, which either activate or repress MITF transcription in response to developmental or environmental cues. For example, a LEF1/TCF site binds the protein LEF1, which receives signals from WNT1, which is a central developmental regulator, driving proliferation and differentiation of melanocytes from the stem neural crest cells.

That is just the beginning of MITF's complexity, however. The protein contains in its sequence codes for a wide array of modifications, by regulatory protein kinases (that attach phosphate groups), and other modifiers like SUMO-ylation and ubiquitination. Key cellular regulators like GSK3, AKT, RSK, ERK2, and TAK kinases each attach phosphates that affect MITF's activity. Additionally, MITF interacts with at least a dozen proteins, some of which also bind DNA and alter its target gene specificity, and others that cooperate to activate or repress transcription. One of the better-known signaling inputs is indirectly from the kinase BRAF1, which is a target of the first precision melanoma-fighting drugs. BRAF1 is mutated in half of melanoma cases, to a hyper-active form. It is a kinase responsive to growth factors, generally, and activates a core growth-inducing (MAP) kinase cascade (as shown above), among other pathways. BRAF1 has several effects on MITF by these pathways, but the dominant one seems to be its phosphorylation and activation of PAX3, which is a DNA-binding regulator that activates the MITF gene (and is, notably, absent from the summary figure above, showing how dynamic this field remains). Thus inhibition of BRAF1, which these precision drugs do, effectively reduces MITF expression, most of the time.

Then there are the gene targets of MITF, of which there are thousands, including dozens known to have significant developmental, cell cycle, pigment synthesis, cytoskeletal, and metabolic effects. All this is to say that this one gene participates in a bewilderingly complex network of activities only some of which are recognized to date, and none of which are understood at the kind of quantitative level that would allow for critical modeling and computation of the system. What has been found to date has led to a "switch", or rheostat hypothesis. One of the maddening aspects of melanoma is its resistance to therapy. This is thought in part to be due to this dynamic rheostat, which allows levels of MITF to vary widely and send individual cancer cells reversibly into several different states. At high levels of MITF, cancer cells are pigmented and proliferative (and sensitive to BRAF1 inhibition). But at medium levels of MITF, they revert more to their early migratory behavior, and become metastatic and invasive. So melanoma benefits from a diversity of cell types and states, dynamically switching between states that are both variable in their susceptibility to therapies like anti-BRAF1, and also maximally damaging in their proliferation and ranging activities (diagrammed below).

The theme that comes out of all this is enormous complexity, a complexity that only deepens the more one studies this field. It is a typical example in biology, however, and can be explained by the fact that we are a product of 4 billion years of evolution. The resulting design is far from intelligent- rather, it is a compendium of messy contraptions, historical compromises, and accreted mechanisms. We are very far from having the data to construct proper models that would critically analyze these systems and provide accurate predictions of their behavior. It is not really a computational issue, but a data issue, given the vast complexity we are faced with. Scientists in these fields are still thinking in cartoons, not in equations. 

But there are shortcuts of various kinds. One promising method is to analyze those patients who respond unusually well to one of the new precision treatments. They typically carry some hereditary alteration in some other pathway that in most people generates resistance or backup activity to the one that was drug-treated. If their genomes are fully sequenced and analyzed in depth, they can provide insight into what other pathway(s) may need to be targeted to achieve effective combination treatment. This is a lesson from the HIV and tuberculosis treatment experiences- that the redundancy and responsiveness of biological systems calls for multiple targets and multiple treatments to meet complex disease challenges.

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Why Are Cells So Small?

Or, why are they one size, and not another?

One significant conundrum in biology is how cells know what size they are, and what size they are supposed to be. Bacteria are tiny, while eukaryotic cells are huge in comparison. And eukaryotic cells vary tremendously in size, from small yeast cells to peripheral nerves that span much of your body, even on to ostrich eggs. Outside of yeasts, not much is known about how these cells judge what size is right and when to divide. A recent paper proposed that the protein Rb plays an important role in setting cell size, at least for some eukaryotic cell types.

Rb is named for retinoblastoma, the form of cancer it is most directly responsible for, and is a well known gene. Many other cancers also have mutations in Rb, since it is what is called a "tumor suppressor gene". That is, it is the opposite of an oncogene. Rb interacts with hundreds of proteins in our cells, but its most important partner is transcription activator E2F1, an activator of cell cycle progression. Rb binds to and inhibits the activity of E2F1, (and a family of related proteins), halting cell division until some alteration takes place, like a regulatory phosphorylation that shuts Rb off, or an insufficient amount remaining in the cell.

The researchers took a clue from yeast, whose gene Whi5 accomplishes similar inhibition of the cell cycle as Rb, and is known to regulate the size of cells at division. So this work was not a big surprise. The interesting aspect is that Rb now has one more role, which logically integrates with its other known roles in the cell cycle. The authors used cells that over or under-express Rb to show that the copy number of Rb has a significant, if not overwhelming, effect on cell size. 

Amount of Rb correlates with the size of cell. The authors set up an inducible genetic construct to drive Rb expression, from zero to four times normal amounts.

So how do they imagine this mechanism working? Rb is a durable, stable protein, with a half-life almost twice as long (29 hours) as the cell division cycle in the conditions the experimenters used. Secondly, all Rb is pretty much in the nucleus, attached to DNA. So at cell division, roughly equal amounts necessarily partition to each daughter cell, even if their cell volumes are very different. Thereafter, each cell synthesizes Rb at a low rate, which does not keep up with cell growth, especially during the G1 phase of the cell cycle- that period prior to DNA replication and commitment to division. In fact, very little Rb is made in that period, allowing it to serve as a limiting factor through dilution as the cell grows. And when it is sufficiently dilute, it then contributes to the decision to have new cell cycle, by letting go of its repression of E2F1.

How several proteins accumulate during the cell cycle. Rb is shown in dark blue, and hardly accumulates at all in G1, the growth phase of the cell cycle before DNA replication (S phase) and division (M phase). For comparison, nuclear volume and a generic translation protein (EF1) rise monotonically with cell growth. Cdt1 is a key licensing factor for DNA replication. It accumulates during G1, and after the DNA replication origins fire, is destroyed by the end of S phase. Conversely, Geminin is a protein that binds to and represses Cdt1, preventing re-replication of DNA that has already replicated once. It accumulates during S phase and stays high until after division. After S phase, more Rb is made, partially catching up to the current cell size. 

That is the theory, at least, backed by pretty good evidence. But its effect is not proportional, and not uniform among cell types. There are clearly other controls over cell size in play- this is only one. Indeed, there are a couple of siblings of Rb (in a family termed "pocket proteins") which also regulate the cell cycle, and a vast network of other controls and stimuli that impinge on it. So finding even one regulator of this kind, and finding conditions where it has strong effects on cell size, is quite significant. As for the ultimate rationale of cell size in these or other instances, Rb regulation is only a mechanism that enforces logic that has been arrived at over evolutionary time, about the practical limits and ideal proportions of cells in, in this case, the human body, in response to various situations. Smaller cells have one virtue, that they are more easily disposable- such as the countless skin and gut epithelial cells that are sacrificed daily. Our long peripheral nerves are much more difficult to replace.

Conversely, Rb has many other roles in the cell, as suggested by the vast number of its interaction partners, diagrammed below by functional classification.

Functional classification of the many proteins that interact directly with Rb. It also has about 15 phosphorylation sites that can be regulated by various kinases.

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RB: Short Name For a Complicated Protein

A key cancer protein operates in a huge network of regulatory protein interactions.

RB stands for retinoblastoma, one of the first diseases tied to a causal oncogene, now also called RB. For lack of time, this will be a very short post about a very lengthy story- how complicated one protein can be. The RB protein doesn't really do much on its own. It isn't an enzyme, or bind DNA, or do other dramatic things. But it binds to a lot of other proteins- 322 have been documented to date. And one protein that it binds to and represses, the transcription factor (family) E2F, is a key activator of cell division, promoting transcription of many other genes including cyclins and cyclin-dependent kinases that run the cell cycle. So RB is typically a key actor that keeps cells quiescent in G1 phase, the normal non-dividing state most of our cells are in. And this is how mutations in RB promote cancer, by removing this brake.

A recent paper expanded this story by investigating some of the regulation of the RB protein, which has at least 15 sites where it gets a phosphate group added (phosphorylated) by regulatory proteins called kinases. The most prominent regulatory kinases are the cell cycle dependent kinases, or CDK. Naturally when a cell does really want to divide, these function to turn RB off, via certain of these phosphorylations. The authors erased each of these phosphorylation sites, and then restored one at a time, asking what binds to them and their effect is. The upshot is that each site turned out to show a distinct pattern of downstream effects, indicating that different proteins bind more or less well to each phosphorylated form. These proteins include transcriptional regulators of a wide variety of kinds, and affect differentially the expression of key genes like BRCA1, 2, and MSH2, and processes ranging from DNA repair to oxidative phosphorylation to protein secretion.

Diagram of the sites of phosphorylation of RB by other proteins. The amino acid sequence goes from left to right, and functional regions of RB that bind to other proteins are colored.

"Collectively, this mass spectrometric analysis identified 438 proteins with a statistically significant enrichment in complexes with at least one of the 16 forms of RB examined. The 22 proteins significantly enriched with all forms of RB included multiple E2F and DP [E2F partner] proteins."

Evolution has had several billion years to tinker with these systems. So while the solution sometimes has been elegance incarnate, (like the DNA molecule), other times it is a messy network of sprawling and mystifying scope. It is one reason why biologists will remain tied to their benches for decades to come.

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