Biopunks help solve structure of key viral enzyme

I've been meaning to blog about the program FoldIt for some time.  It's a program out of David Baker's lab (link to University of Washington) where users can manipulate protein structures to improve folding but in a unique twist, the improvement is 'scored', just like a game.  A problem is posed on the website and thousands of players compete to see who can find the best solution. They also have regular competitions to see how well players can predict the structure of a protein from just the amino acid sequence (the Holy Grail of the protein folding world).  I've played on and off for a couple of years and it's pretty fun... but it's a lot like my day job, so if I take the time to play a video game it is usually something like Rock Band.  Still, FoldIt is a pretty easy game to play (the number of rules is limited and the GUI is very intuitive) so the real challenge is in the player's ability to use logic and their skill in 3D visualization. I'll have a more detailed post about it and its less-familiar cousin EteRNA (the RNA equivalent of FoldIt) later. When you are ready to play, click here (it's free, easy, and fun... really!)

This week in the journal Nature Structural and Molecular Biology (I have had several rejection letters from this fine journal) is an article (abstract) describing the use of FoldIt to solve a scientific problem at least ten years old!  Crystallographers have been trying to determine the structure of the monomeric form of MPV protease. MPV stands for Mason Pfizer Virus, a virus that causes an AIDS-like syndrome in monkeys.  Although it is not HIV per se, the protease is a key enzyme in the production of a mature virus and so developing drugs using this structure could be useful in developing an HIV therapy.  This protein was one of those rare cases where good crystals could be grown but interpreting the x-ray data back into the final 3D picture of the protein could not be done. So, researchers looked to crowd-sourcing as a means to solve this problem.  For three weeks, FoldIt players had the chance to optimize a 3D model, based on an NMR structure of the monomer.  Teams competed to see who could come up with the best solution (based on how well the different structural pieces of the protein fit together).  After all that tweaking and optimizing, more than a million different models were created.  Crystallographers used the best of these models as a starting point and one of them successfully generated a solution to the x-ray data using molecular replacement.

Who were the people who provided the key to success?  The top groups were listed as co-authors by their team name (FoldIt Contenders Group and FoldIt Void Crushers Group) and by a few handles listed in the article (spvincent, grabhorn, mimi) so not much is known about these folks, but I'll bet many of them had no knowledge of crystallography or biochemistry and probably had no clue what this enzyme did.  However, the fact that thousands of players worked together on this problem and were ultimately successful highlights the power of crowd-sourcing and that top-flight science can be accomplished through a "game".  I think this paper will also motivate others, both players and scientists, to leverage the power of biopunks!

How about a GFP cat to go with your GFP beagle?



The GFP cat:
From Figure 2 of Wongsrikeao et al.



Well, that didn't take long.  I blogged last month about a paper describing GFP beagles that were able to glow under the control of a tetracycline promotor.  Now, a new Nature Methods paper describes a GFP cat!  The basic point of the paper (here's the abstract) was to demonstrate gamate-targeted transgenesis in cats and to use this ability to make a transgenic feline model for HIV research.  The HIV part was interesting, as they introduced the gene for a protein from the rhesus macaque known as TRIM5.  I studied TRIM5 pretty extensively when I was doing HIV research as it is a species-specific restriction factor that is effective at stopping HIV replication.  Cats don't have an analogous TRIM protein (that we know of) so by introducing this protein into the cat, researchers can study the transmission of FIV (the cat version of HIV).

The cool part was the GFP expression.  Wongsrikeao et al. wanted to see if they could introduce multiple genes, and since GFP is a convenient marker, they could also study the presence of the transgenes in progeny cats.  As I mentioned in the Beagle post, fluorescent proteins have been introduced in animals previously (see here and here for cats) but the efficiency in the present work is better.  I'm certainly no expert on transgenics, but the general trend over the last few years is clear... we are moving from the realm of the nearly impossible to the land of the challenging but doable.  Glowing cats, dogs, hamsters, bunnies, you name it are going to be popping up in your pet store relatively soon.  Lost your dog?  No problem... just look for the glowing blob in the woods.  How about a government program to develop a GFP opossum?  Think of how much road kill would be eliminated if you could see these animals before they got up close and personal with your tire.  Think about how cool the forest would be if all of the little woodland creatures glowed bright green. Think about how easy it would be to hunt.  I wonder how screwed up the food chain would get. I guess we would have to make GFP plants for the little GFP bunnies to hide in. Man, this would make a really cool dystopian/biopunk story!

Can gut bacteria make zombies?



L. rhamnosus: Not a zombie-
producing bacteria...yet.



If T. gondii weren't enough to worry about, now there is evidence that bacteria in our gut can influence brain function.  Lactobacillus and other probiotic organisms have long been speculated to have beneficial in vivo effects, and are most commonly known for improving the health of the digestive tract.  L. acidophilus is probably the most widely known, since it is used to make yogurt, but there are many other types of lactobacilli with alleged health benefits ranging from lowered cholesterol to improved mood.  Some of these benefits are speculative, and for many years any benefit (such as improved gut health) was thought to be due to local effects or secreted chemicals.  However, the impact of these bacterial colonies may be much more far-reaching.

A Proceeding of the National Academy of Sciences (PNAS) paper published by Heujtz et al. last January (abstract) showed that microbial colonization in the mouse gut led to the activation of signaling pathways involved with motor control and emotional response.  This was the latest in a growing body of literature that suggests gut bacteria could influence how we think and act.  Now, in the August edition of PNAS, Bravo and colleagues take this one step further (abstract).  They show that Lactobacillus rhamnosus can directly influence the expression of GABA receptors in the brain.  GABA is the primary neurotransmitter for regulating many physiological and psychological activities in humans.  For example, caffeine inhibits GABA and results in an overall increase in neurotransmitter activity.  In contrast, alcohol and sedatives tend to increase GABA activity, leading to reduced neurotransmitter activity. Bravo et al. showed that feeding mice L. rhamnosus reduced GABA expression in some areas of the brain, while increasing it in others.  The overall effect was to make the mice more calm.  Here in the Dark Lab, we would test this by subjecting the rats to endless episodes of Jersey Shore, Barney and Friends, and The Jonas Brothers, and then asking how long it takes before they fall into convulsions.  Bravo measured stress-induced hyperthermia (rise in core body temperature from stress) after a battery of different tests, including  forced swimming and mazes (less barbaric than listening to the Barney jingle, but presumably effective at producing stress) and showed that the L. rhamnosus-fed animals exhibited less stress during these activities.

The final point of the paper was what I found most interesting.  Some of the animals had the vagus nerve cut prior to the start of the experiment.  This nerve is a direct link between the gut and the brain and is responsible for transmitting signals about hunger and satiation.  In these animals,there was absolutely no effect from consuming L. rhamnosus.  No changes in GABA expression and no behavior changes versus the control animals.  This means that the probiotic bacteria that colonize in the gut might actually use this nerve to signal directly to the brain.  Although these bacteria appear to provide a health benefit, I can certainly imagine other strains that are more nefarious.  Yeah, I'm talking about zombies again.  This phenomenon is not all that different from what is seen in T. gondii or the zombie ants... microbial agents that manipulate the brain function of the host.  If a probiotic strain can mimic the effect of caffeine-overstimulation or, even worse, caffeine-deprivation, then that would be a whole new kind of scary.  Anyone who has seen me before that first cup of coffee knows what I'm talking about... it's bad enough to give a zombie nightmares!

Zombie rats are horny!

You may remember a very early post on zombies, where I talked about a parasite known as Toxoplasma gondii.  I just read a new article published in PLoS that is really mind-blowing.  It also should fall in the category of science known as what-kind-of-PhD-do-I-need-to-study-this-shit.  Very interesting, very strange, and very cool.  Here's the abstract to check out for yourself (abstract).  As you know, rats that are infected with T. gondii lose their fear of cats.  This is important for the parasite because part of its life-cycle occurs in the gut of the cat.  This report takes that one step further.  House et al. show that as part of this shift in rat behavior, the rats are actually attracted to cat urine.  Not attracted like "this smells like roses" but rather "yowsa, hot babes" (rats do say 'yowsa'... I've heard them).  Yes, the parasite changes the way the rat brain responds to the smell of cat urine.  Neurons in the ventromedial hypothalmus, dorsomedial part (mercifully abbreviated as VMHdm), which normally are active in response to fear, are silent when infected rats are exposed to urine.  In contrast, the area in the brain the DOES light up is the posterodorsal medial amygdala.  These are the same neurons that light up when rats watch porn (or are exposed to estrous females, but somehow that doesn't sound as sexy).  Their conclusion is that T. gondii makes rats think that if they follow the cat pee, they will get laid.  Although that may work in some dive bars, for these rats it ultimately leads to just a single dinner date.

These results are also consistent with other findings that show an increased level of dopamine in infected rats.  Dopamine is, of course, the primary molecule in behavioral reward so it seems reasonable that this pathway would also be effective at shifting rat behaviors.  They cite a 2006 paper by Webster et al. (abstract) where it was shown that dopamine receptor antagonists prevent the attraction to cat urine.  Could this receptor be the first therapeutic target for treating zombies?  I'll have to propose that at the next New Target meeting. 

The ability of T. gondii to alter brain function and behavioral response is incredibly cool and a little bit scary.  Why scary?  Because it is estimated that at least one third of the human population has been exposed to the zombie-inducing T. gondii (and no, it's not just pop stars and politicians).  Even subtle changes in behavior on that scale can lead to massive changes in society.  Although it is unclear how well this study translates to humans, it does suggest that in the event of a zombie apocalypse, it's probably not a good idea to hide out in the girls' bathroom.

Highlights from the Protein Society Symposium

A week ago, I attended the 25th annual symposium of the Protein Society in Boston.  I've gone to this meeting three times and this was the best yet.  Very, very cool stuff.  I also got to see a lot of old friends from grad school, including my graduate advisor.  It was great to hear him talk fondly about the 'good ol' days' (ie, when I was his student) as I distinctly remember them being less fond and more frantic.  I think finishing a PhD thesis is the mental equivalent of giving birth to an elephant while running a marathon, but for several obvious reasons I will never be able to test the hypothesis.  I also met some cool new people.  I talked politics with a girl from Poland in a loud Irish pub and met another girl who is some kind of flute prodigy from a well-known coffee empire.  At the reception, I met a guy from, well, some European country who had done a postdoc in San Diego and I knew many of the trails he had hiked.  We tried watching the Red Sox game from the 50th floor of the Prudential building... great view but when the outfielders look like fleas on a green dog, it's really hard to see what is happening.  We kind of made it up as we went along and since the Polish chick didn't know the game, it was all good.  He still owes me pictures of Fenway.  The poster session was crazy, with two overlapping sessions and the very friendly (but bored) vendor who bribed me with chocolate every time I passed her booth.  I was also a poster judge this year, so I missed most of that session, but tracked the presenters down later to ask questions.  It's a little awkward at the coffee breaks, since everyone is staring at the nametags trying to find people they want to talk to.  I would try and catch a glimpse of their badge over the rim of my coffee and hope they were not offended when I simply walked away.  It's a very unusual hierarchy at conferences.  There is absolutely no guessing about where you stand in the pecking order.

Anyhow, I can't talk about the things I found most exciting because I was there for work, and work stuff has to stay off the radar.  However, let me briefly describe two (not work-related) things that were pretty cool.  One was a talk by Della David at UCSF on protein aggregation as a part of aging.  I don't know a lot about this field, but one of her early slides really caught my attention.  She was discussing the role of protein "aging" in inducing aggregation using C. elegans (a worm) as a model.  As the worm aged, she showed that the concentration of over 400 different proteins increased in the insoluble fraction.  In simpler terms, if you take all of the proteins out of the worm, many of them are soluble but some fraction are in an aggregated form, which is not soluble.  Although the total amount of protein seemed fairly constant with age, the proportion of aggregated protein increased and seemed to disrupt the natural process of homeostasis.  Then came the kicker... to show that this was an active process (that is, controlled by a cellular system) she used a C. elegans that had been engineered to have a specific mutation in the Daf-2 receptor.  These worms had twice the life span of a normal worm.  Whoa!  Sign me up for that mutation! Imagine living 160 years!  It turns out that the fraction of aggregates is independent of the lifespan, suggesting that the process is controlled.  Two things here... Daf2, which is part of the insulin/IGF-1 signaling pathway, can regulate lifespan (possibly related to the observation that mice that eat less live longer?) and that protein aggregation as a result of aging could also be controlled.  Here is a review on insulin/IGF-1 signaling in aging (abstract) and here is David's recent open-access paper covering some of this story (PLOS paper).  Listen folks, please hurry up with this important work... I'm not getting any younger.

The other talk I liked was by Ken Dill (a long-time favorite of mine and also from UCSF).  I'm used to him talking about transfer free energies and lattice models for proteins but this time he was talking about the stability of the proteome.  He (and others) have shown pretty convincingly that on a macro scale, protein stability is roughly dependent on the length of the protein.  (Seems simple but it has taken decades to model it in a way that makes physical sense).  Armed with this model, he determined the stability of the entire proteome and found that it is only marginally stable.  Over 500 proteins have stabilities less than 3 kcal/mol, which means they are barely folded and functional.  The implication of this result is that even slight increases in temperature can cause many of these proteins to unfold.  The resulting denaturation catastrophe overwhelms the cell and causes cell death.  This is the most plausible explanation yet for why slight increases in temperatures cause such problems (even for humans, an increase in body temp of 7-8 degrees can be fatal).  I asked him about the proteome of thermophilic bacteria and whether it might explain their ability to survive extreme temperatures and he said he is working on that now.  I'm guessing that might explain some of the adaptability, although the detailed mechanism is still a mystery.  For you DIYbio people out there, this model provides a pretty simple way to do this type of analysis yourself. The simplicity of the model, and the fact that minor ensemble changes can be magnified into major improvements for the organism tell me that life might be lurking everywhere there is an energy gradient (I'm looking at you, Titan).  On the flip side, it shows how sensitive life can be to slight changes in the environment. Here are the links to the articles (proteome stability and Dill's model)

  Dill also started off with a joke: "There are three kinds of mathematicians... those that can count, and those that can't."  Nothing like a geek joke to start off a talk... but hopefully his material will be better in San Diego next year.

GFP Beagles: Disease model or designer pet?

A recent paper in the journal genesis describes the production of transgenic beagles that glow when exposed to UV light (abstract).  The eGFP transgene was introduced into a beagle embryo using a similar (but much improved) technique that was used to clone Dolly the sheep.  Making a puppy that glows is not really new, as it has been demonstrated by the same group in 2009 (abstract) and others (in dogs, as well as other small mammals) but there are two cool things about the recent work.  First of all, they put the transgene under a promotor.  This means that the puppy does not glow green until the gene gets switched on, in this case by the drug doxycycline.  Feeding the dog low levels of doxycycline induced the expression of the GFP protein (green glow) and the effect could be turned off simply by removing the drug from the dog's diet.  This is pretty easy to do in small animals (like rats and mice) but pretty complicated in large animals. 

  In a separate article, also in genesis, they report the transmission of the transgene to offspring (abstract).  This was also interesting, as the GFP-containing females were totally fertile and had normal pregnancies and births.  The dads were wild type beagles so roughly 50% of the offspring carried the transgene.  This is consistent with stable germline transmission.  This result suggests that we are really not far off from having designer dogs.  The initial cloning will be hard (efficiency is still in the 1-5% range) but if the transgene is stable, simple breeding techniques should be enough to make zillions of glowing puppies!  Although this is a crude example, one can envision a vastly different world where hair color, facial and body features, and perhaps even personality traits could be engineered.  Genetic defects and disease determinants could be engineered out.  One could build the perfect dog.  You did realize I was still talking about dogs... right?

Foxfire: Chemistry of the undead



Ghostly mushrooms



I am currently approaching the half way mark in my current work-in-progress (WIP-2) and have been making particularly heavy use of foxfire.  I didn't start out to write about that... in fact, this book started out as a humorous middle grade adventure and quickly turned into a dark, YA biopunk.  Write what you know, I suppose.  One of the emerging themes is the struggle between the protagonists and the technologically-advanced fungi that exists in their (slightly dystopian) world.  So, I found it a little coincidental that a report came out a week ago by Marina Capelari and colleagues about a type of ghost mushroom that had been re-discovered in a Brazilian rainforest after being extinct for over 150 years (abstract in the journal Mycologia).  The mushroom, formerly known as Agaricus gardneri, is notable for its bright and constant bioluminescence.  To understand why this is unusual, here is a brief description of how foxfire comes to be:

Bioluminescence is generally accepted to come from a 2-step reaction.  A chemical called luciferin (L) is first reduced (to LH₂) and this reaction is catalyzed by an enzyme called reductase.  NADH is a molecule (di-nucleotide, actually) that is a cofactor in many redox reactions.  Its basic function is to move protons around (you're a geek if you noticed the chemistry pun).

L + 2NADH <--> LH₂ + 2NAD+

Reduced luciferin is then oxidized (to LO) by an enzyme called luciferase.  This process also produces a photon of light and is the source of the creepy glow.

LH₂ + O₂ <--> LO + H₂O+ LIGHT



Illudin S: Potential substrate for ghost fungi

Why am I cryptically showing fungal luciferin as L, instead of showing the chemical structure?  Could it be that my chemistry skills are so bad, I couldn't tell the difference between L and LH if my life depended on it?  Well, yes -- but it is also true that the luciferase substrate in fungi is not well characterized. The luciferin for A.gardneri is probably a member of the sesquiterpene family, most likely an illudin.  Some of these compounds have been studied as anticancer agents but the illudins tend to be extremely toxic (possibly another reason they are called ghost mushrooms!).  Interestingly, other luciferins (such as those found in fireflies, shrimp, etc) have totally different chemical structures, which gives them different biological properties and unique spectral characteristics (ie, different colors, brightness, etc).  Changes in the luciferin structure, amino acid substitutions in the active site of luciferase, and varying levels of oxygen or water can each contribute to changes in the emission of light.  What is unusual about A. gardneri is that unlike other species, the bioluminescence is almost constant.  In fireflies, the luciferin is released when they want to blink and in the case of other species, they light up only after contact (probably a means of self-defence).  So why does this mushroom glow all the time? No one knows yet.  The biochemistry of these things is almost as mysterious as seeing their eerie ghostly glow on some rotting tree stump at midnight.

However, it provides a great real-world example of the potential technology for my story.  It doesn't take much imagination to think that these mushrooms could be engineered to be very bright or to respond in controlled ways depending on environmental input.  A basic example from my WIP is that these types of fungi are used for lighting underground.  No electricity required, no pollution, and little maintenance.  They are almost the perfect type of lighting... or are they?  Anyhow, I thought it was a pretty clever idea early on until I found out that Ben Franklin used foxfire from mushrooms to light the inside of one of the first submarines.  Was there anything this guy didn't know about?  I guess he's going to have to go on my list of card-carrying biopunks.