and what can you say, but…
This week in our Topics in Biotechnology course, we discussed a paper from the laboratory of Karen Vousden of the Francis Crick Institute, TIGAR, a p53-inducible regulator of glycolysis and apoptosis. I wanted to take some time to summarize the conclusions of this paper here in order to prevent any misunderstanding that might have arisen following our in-class discussion of the place of TIGAR with respect to glucose metabolism.
The body is extraordinary in its ability to maintain itself. It keeps a temperature of 37 degrees. It keeps bad stuff out and good stuff in. It has an immune system that attacks and destroys invasive viruses, bacteria, fungi, and other parasites. Cells maintain a constant pH and balance the concentrations of salts, proteins, and nutrients. All these regulatory mechanisms work together to keep us healthy and functioning properly.
One of the ways our body does this is by having our cells monitor their own health and make difficult choices when they are unhealthy. When our cells suffer damage, they work hard to repair the damage, but they also balance this against the greater good of the body as a whole. If they can repair damage, they do so, but when they can’t, cells eliminate themselves by a process called apoptosis. A central protein that controls many of these processes is the ‘guardian of the genome,’ p53.
This paper explores the role of TIGAR (Tp53-induced glycolysis and apoptosis regulator) in modulating the pro-apoptotic effects of p53 and in reducing free radicals. Specifically, TIGAR exerts its effects by rebalancing the normal metabolism of glucose during glycolysis. This prevents further damage and also allows time for repair to occur before making a decision to terminate the cell if repair is unsuccessful.
Under normal conditions, cells take up and process glucose as a fuel for making ATP, which is used directly to power enzymes and carry out all the processes that keep us alive. As part of this process, glucose is broken down stepwise during glycolysis. Some fraction of the products of this reaction gets diverted by an enzyme called PFK-2, which makes Fructose-2,6,-bisphosphate. This sugar goes on to bind to, and activate PFK-1, which keeps the pathway flowing.
Under conditions following DNA damage, p53 will become activated and the cell will arrest glycolysis as well as any cell division while it initiates DNA repair mechanisms and acts to remediate the radical oxygen species (ROSs) that are often associated with this sort of damage.
Among the many genes that are turned on to carry out these operations is TIGAR. Bensaad et al. show that the gene for TIGAR is transcribed and translated into protein, and that protein goes on to act as an enzyme to regulate metabolism.
Specifically, TIGAR functions as an enzyme with a high degree of homology to FBPase-2, which converts Fructose-2,6,-bisphosphate to Fructose-6-phosphate. This has an important regulatory function because, as stated above, Fructose-2,6,-bisphosphate is required to activate the enzyme PFK-1, which is required for glycolysis. In the absence of Fructose-2,6,-bisphosphate, PFK-1 shuts down and the products of glycolysis start backing up.
At first, this results in a backlog of Fructose-6-phosphate. As this accumulates, it will result in the accumulation of Glucose-6-phophate. With nowhere also to go, this will be processed to 6-phosphogluconolactone, making NADPH. NADPH will then oxidize glutathione, which will break down the ROS, H2O2 to water.
With the reduction in the number of ROSs, DNA damage will cease and repair can take place, thus diverting the cell away from a pathway leading to apoptosis.
This paper represents an amazing amount of work and is nearly bulletproof in its findings. I highly recommend it to anyone interested in how DNA damage and p53 interact with metabolic pathways and how this interaction directly leads to a more complete understanding of how p53 does its job.
Here is a link to an Icelandic site that is live-streaming an ongoing volcanic eruption. I just thought it was beautiful to watch geology happening in real-time and wanted to share:
This was published on my other site, 100 films in 100 days.
Neutrophils are the first responding immune cells following exposure to pathogens.
This is due to several characteristics of these cells. Primarily, these cells are very populous in the blood. In fact, they are the most common type of circulating leukocyte, so no matter where in the body there is an insult/injury, there will be neutrophils close by to respond. Secondly, these cells are primed to respond to chemoattractants elicited by complement and from pathogens themselves.
One immediate response that will occur following an injury is the cleavage of complement molecules as they react with invading pathogens. Complement may be activated in three ways, spontaneously (i.e., the alternative pathway), by pathogen-associated carbohydrates (i.e., the lectin pathway), and by antibody (i.e., the classical pathway). In all three pathways, C3 convertase is a common element where the complement protein, C3, is cleaved into C3b, which precipitates onto the membrane of the invading cell, and C3a, which diffuses away and acts as a powerful anaphylotoxin.
C3a has a number of effects, notably activating vasculature to dilate (thus slowing blood flow in the area), upregulating adhesion molecules for cells to attach to, and promoting permeability, making it easy to Neutrophils to extravasate (leave circulation and enter the tissue). Neutrophils are quick to respond and to all of these cues and will further be attracted to the C3a gradients themselves.
Once in the vicinity of an infection, neutrophils will become more attracted by more specific attractants, such as those secreted by the pathogens themselves. One beautiful example of this kind of chemoattraction can be seen in the video of a neutrophil pursuing a bacterium in vitro:
Consider the following:
If you were given any (antibody or other) reagents you wished, how might you determine whether the neutrophil pursuing the bacterium was following a trail of fMLF? (what is fMLF? Why might this particular chemoattractant be relevant to this example? How do you determine specificity?)
This was a little hard to bear, but it’s not wrong…
a better, more complete video is this one…
A final video is…
Lastly, some important points are:
-The names and functions of the three pathways
-The initiating molecules for each pathway
-The alternative pathway is often considered a spontaneous pathway, although there are some specific activators
-IgM activates complement better than other classes of antibody
-All three pathways converge at the formation of the C3 convertase
-Complement always results in the assembly of a Membrane Attack Complex (MAC) -lyses bacteria, enveloped viruses, and nucleated cells
-C3a, 35a, and to a lesser extent C4a are anaphylotoxins
Why does it make sense that the antibody most capable of activating complement is IgM?
Dick Hallorann lends his grandmother’s words to describe people like himself and Danny Torrance. She called it “Shining.” The Overlook Hotel is one of those places that shines too.
Here are a few random thoughts, sounds, and images from the many lives of The Shining.
Room 217 of the Stanley Hotel
Before there was an Overlook, there was the Stanley Hotel. Stephen King once stayed in the Presidential Suite of the Stanley, where he was visited by bad dreams, hallucinations, and was inspired to sketch out an outline for his best novel.
But The Shining is not just King’s best novel. It also served as the basis for the film of the same name, as written and directed by Stanley Kubrick. Like the hotel, this Stanley would also haunt King as he took his novel and stretched and twisted it into one of the most influential films ever made. So different from the book, King has said that it is Kubrick’s film, not his.
A reviewer calls the film, “a brilliant, ambitious attempt to shoot a horror film without the Gothic trappings of shadows and cobwebs so often associated with the genre.”
One of the most striking things, aside from masterful performances by Jack Nicholson and Shelley Duvall (heresy!? Yes, I think her performance was nearly as perfect as Nicholson’s – just drawn more from Kubrick’s haranguing rather than her innate talent.) is the spellbinding score and incidental music. From the first intonations of Dies Irae to the frenetic energy of the final chase scene, the music shapes your emotions and pulls you into the haunted world of the Overlook.
Rolling Stone, on the music accompaniment to Stanley Kubrick’s The Shining…
“Kubrick and music editor Gordon Stainforth cherry-picked maximally queasy passages from works by a clutch of Eastern European mavericks: “Lontano” by György Ligeti, the Hungarian genius whose music gave 2001: A Space Odyssey its unearthly atmosphere, and even more importantly Krzysztof Penderecki, the Polish radical whose strangulated strings, barbaric brass yawp, clatter-bone rhythms and hissing choruses in “Utrejna,” “De Natura Sonoris,” “The Awakening of Jacob” and more provided the Overlook Hotel and its denizens with an appropriately unhinged environment.”
To address the differences between the novel and the film, King sought to make a new version of the film that tracked more with the original storyline. In 1997, this version was released as a miniseries staring Steven Weber and Rebecca De Morney as the Torrances and using the Stanley Hotel as the Overlook. This version eschewed the hedge maze that Kubrick had created in place of the technically difficult hedge lions and, most notably stayed faithful to the novel’s end with Jack dying in the fiery explosion sparked by the Overlook’s overpressurized boiler.
Three decades after the original novel, King released Doctor Sleep, following the troubled life of Danny in the years after his ordeal at the Overlook. Fans of the Shining particularly enjoyed the last act of Doctor Sleep which brought us back to the remains of the Overlook, a place that exists only in the world of the original Shining film.
Radiolab did a good piece on Challenge trials for the COVID-19 vaccine which you can find here.
Challenge trials are an expedient, but fraught way to generate data on a vaccine’s efficacy quickly. Briefly, they involve taking a (relatively) small group of people, say 100, and dividing them into two groups of 50 each. One group gets the vaccine and the other group gets the placebo. Then, after waiting some period of time for the immune system to generate a protective response, you expose all 100 people to live virus.
This is essentially the same thing as what is done in the more traditional trial, with the exception of directly exposing subjects.
In the end, it’s a simple matter to calculate the efficacy (or not) of the vaccine by comparing the number of people who got sick in each group. If these numbers are equal, the vaccine is ineffective, etc. Even if all 100 people got sick from this challenge, it would still be less than the number of people who got sick (170) during the Pfizer trial that involved 43,000+ total participants (reported here).
Further, a challenge trial can be completed in as little as two months, while more traditional trials are ongoing over two years.
In the Radiolab piece, trial participants were asked why they participated. Answers ranged from appeals to mathematics (one participant said exposure to COVID did not significantly increase his risk of dying in any given year) to a need to make their experience meaningful (if they caught covid in the trial, at least it would provide important evidence toward approving a drug, whereas if they caught it any other time, it would just be a waste.)
However, the one reason I did not hear was one of financial inducement. Although I could not find actual data on this, challenge trials may pay up to $4000 for the 3-6 weeks of legal quarantine. There are a number of issues associated with these payments – the most obvious being that it may induce those without means to participate because they feel the money if too good to pass up. Jennifer Blumenthal-Barby and Peter Ubel write that, in fact, the payment for these trials may not be enough as it fails to meet the proposed minimum living wage of $15/hr.
I recommend that you listen to the Radiolab piece (it’s less than 30 minutes long) and take a look at the Blumenthal-Barby and Ubel, which is an interesting read.
Often simply called a gel shift assay, these are performed to identify the binding of proteins (or other molecules, conceivably) to DNA. The basic idea is that you have a section of DNA in excess supply that contains a sequence that you suspect may be bound by known or unknown proteins. You first label your DNA by incorporation of labelled bases via PCR or end-labeling. This used to be done using radioactive bases, but there are other alternatives available now. For our purposes, I’m going to assume a radioactive label as it is simple to imagine.
Once you have your radiolabelled DNA, you now need to add some proteins that you suspect might bind. This could be isolated protein from a recombinant source or it could be a cell lysate from cells that have been treated in some way. Again, I’ll imagine that we have stimulated cells with something for a progressively longer time (say, 0 minutes stimulation – 30 minutes of stimulation at 5 minute intervals). Finally, after the get runs, you can blot it onto a membrane and expose it to film where the radioactive DNA will light up. Assuming this, I guess we also have a radioactive DNA ladder too
Here’s an example… (remember, this is entirely fictitious)
We stimulate cultured B cells given the survival cytokine, BLyS. At the timepoints indicated above (0-30 minutes), we harvest cells and lyse them in the cold to obtain nuclear fractions. Our DNA is derived from the promoter region of a protein we are interested in and we are wondering if we will see transcription initiation factors assemble.
What we are seeing is a 100bp DNA ladder run with our samples. Keep in mind that the ONLY thing we can see here is labeled DNA. In the first several lanes, none of our DNA is being bound and it is running according to its size (~50bp). At 15 minutes, we start to see the shadow of a band that has shifted our DNA up to an apparent ~700bp. IMPORTANT: DNA ladders are not protein ladders! Also, this is a native protein with unknown charge that is bound to our DNA. All we know is that we are getting binding, we can’t assume knowledge of the exact size of the protein (however, in general, we do see shifts going up as more proteins accumulate)
By 30 minutes, we see an appreciable amount of protein binding our DNA. But how can we know what protein(s) are binding?
One way, if we know what we are expecting, is to use an antibody against that suspected protein. Assuming we have extra lysate + DNA mixture to run a second gel, we could spike our antibody into one tube with DNA and lysate and not into the other (or spike an irrelevant antibody).
If we see that the antibody results in a ‘supershift’ where the DNA/protein band has moved up to a larger apparent size, this means that our antibody is binding its target and that target is binding the labeled DNA.
Let’s assume we suspect that Nf-kB is what is binding our DNA, so we use and Nf-kB antibody to do a supershift assay.
These data support our model and we can now go on to ask new questions.
Francis Crick is well known, not only for his work in elucidating the structure of DNA along with James Watson, but also for his hypothesizing about the nature of biology that he thought would be discovered in the wake of the publication of the structure.
Among these hypotheses was the notion of a ‘central dogma’ that described the flow of information from DNA, through RNA, to protein. It was clear how information could flow from DNA to RNA as these were written in the same language of nucleic acids. What was less clear was how this information could then be translated into a protein.
In 1955 Crick proposed the idea of adaptor molecules that would be required to read the information on the nucleic acid and translate this into the language of proteins, comprised of Amino Acids (AA). “In its simplest form there would be 20 different kinds of adaptor molecules, one for each amino acid”
We now understand these adaptor molecules to be transfer RNAs (tRNAs), which bind both messenger RNA (mRNA) and AAs in a way that the AAs can engage in protein synthesis. These tRNAs bind the AAs on one end of the molecule and have three bases, called anticodons, that interact with the codons of the mRNA. This alone would be enough to prove Crick as prescient.
However, that only covers the first part of his statement. He goes on to say, “…and twenty different enzymes to join the amino acid to their adaptor.”
Twenty enzymes to build the adaptor molecules (tRNA with AA).
Yet again, Crick’s mental model was correct, we have a number of aminoacyl tRNA synthetase (aaRS) enzymes that specifically bind the tRNAs and AAs individually and then promote their conjugation. One might expect these synthetase enzymes to be, themselves, at least partially comprised of nucleic acids that can identify the tRNAs by their anticodons and then charge them with their appropriate, cognate, AAs. However, this is not always the case. Each tRNA is, instead, recognized by an identity element that is often discrete from the anticodon, but nevertheless serves to distinguish the correct tRNA that should be bound. These identity elements (aka identity determinants) are often located along the anticodon stem or the acceptor arm of the tRNAs, however they are not restricted to such (see Fig 2 from Giegé et al.)
Figure 2 from Giege et al: Cloverleaf folding of tRNA with location of known identity determinants and its three-dimensional L-shaped organisation.
Although it is conceivable that every individual tRNA would have a specific aaRS to charge it with its AA, “[w]ith some exceptions there is only one aaRS per isoacceptor tRNAfamily (the cognate set of tRNAs).”1 However, I will not go into the specifics of this flexibility here.
Once a tRNA is recognized and bound by its associated aaRS, the appropriate adenylated AA is brought in to the synthetic site where it will become charged to the tRNA. Once charged, the AA will be shifted to a second binding pocket, the editing site, where it is again checked. Together, this is known as the double sieve model, where the first, coarse, sieve (occurring in the synthesis site) allows placement of correctly sized (or smaller) AAs, while the second sieve (occurring in the editing site), allows only AAs of the correct hydrophilicity, mischarged AAs are cleaved by deacetylase enzymes.2