Wednesday, April 15, 2015

Assignment #5

Final Update

I have attempted to add in text citations as much as I could but it has been a while since I wrote assignment #3, so I have not been able to add everything.

Following another recommendation, here are some additional pictures of my protein with some new angles and colour schemes:




All images made with PyMol Viewer.

Sunday, March 22, 2015

Assignment #3

δ-Atracotoxin


δ-Atracotoxin is a rather obscure protein that many people have not heard of. Why then, does an obscure protein from the bottom of the world deserve the illustrious title Protein of the Year? Please allow this riveting blog post to convince you.

 

 Introduction

 The protein δ-Atracotoxin is a beautiful mistake of evolution born out of the diverse ecological deathtrap known as Australia. The Australian continent is home to many natural serial killers. One of the most under-appreciated, and my personal favorite, is this hellish thing:
1. Male Sydney-Funnel Web Spider Atrax robustus

 The unholy arachnid you now feast your eyes on is the Sydney-Funnel Web Spider. All species of Australian Funnel Web produce a form of δ-ACTX, but the Sydney Funnel Web's is by far the most deadly. It has fangs larger than a brown snake's, and is fond of hiding in tennis shoes after a rainstorm. This highly aggressive spider claimed at least 13 lives before the creation of the anti-venom became available in 1981. Although no one has died of a bite since 1981, they are still quite serious. A human victim will begin experiencing symptoms with 10 minutes of a bite. Why the emphasis on human? (1, 12)

One of the most interesting things about δ-ACTX is that it is harmless to virtually all mammals except primates (and baby mice.) What about this protein makes it toxic only to primates? I hope this caught your attention. If you keep reading, you might find out why.

 

Structure

  For such a deadly protein, δ-ACTX has a elegantly simple structure. It contains only 42 amino acid residues, which is really quite astounding when you consider the havoc it wreaks on a primate nervous system. δ-ACTX's tertiary structure is also quite simple, containing two beta-sheets and no alpha helices. (5)

2. δ-ACTX-Ar1
The most key element of δ-ACTX's structure is the disulphide bonds between it's cysteine residues. There are four total: Cys1-Cys15, Cys8-Cys20, Cys14-Cys31, and Cys16-Cys42. The first three create what is known as a cystine knot motif. Cystine knots are common in the venom of many arachnids, and can also be found in the proteins of mollusks and plants. Cystine knots create very stable proteins that are resistant to denaturation by heat and proteolysis. The cystine knot motif also appears to be key to δ-ACTX's toxicity as the removal of the disulphide bonds renders the protein non-toxic.
3. Cystine Knot
I've included a schematic of a basic cytine knot, but it is important to note that it does not represent δ-ACTX. (8)

Beyond that, the structure of δ-ACTX-Ar1 is remarkably simple. This is another one of my favorite things about this protein. There is no fancy quaternary structure, no intricate bouquet of alpha helices and beta sheets. On the surface it appears to be an incredibly boring protein, like the boring neighbor you would never suspect of being a serial killer. Until the police find 13 bodies in his basement. Not only does this protein kill people, it does it in a highly interesting and unique way.

Mechanism

Why is δ-ACTX's Modus operandi so interesting? Well first lets see what we are comparing it to.
Snakes make up the majority of venomous animals that pose a threat to humans. Snake's evolved to kill warm blooded vertebrates, and their venom reflects this. Snake venom contains multiple toxic components. A single snake can produce venom that contains multiple neurotoxins, cytotoxins, and hemotoxins. If a rattlesnake bites you, your death will be the result of many different proteins and molecules working in unison. However, if a Sydney Funnel-Web Spider bites you, your death will be the result of one protein: δ-ACTX.

δ-ACTX acts by attacking voltage-gated sodium ion channels. (5, 6) These channels are key to creating action potential in a cell, allowing it to depolarize and propagate signals to adjacent cells. Voltage-gated channels open and close by sensing voltage differences inside and outside the cell. Normally sites III and IV will sense the difference in voltage created by the release of Na+, and cause the channel to inactivate itself (11).

4. A Voltage Gated Na+ Channel, showing binding sites I-IV
In primates, δ-ACTX binds to site III of the channel. This prevents the channel from inactivating, and the affected cell starts to rapidly fire signals. The physiological results of this one protein interaction are devastating. Symptoms onset within 10 minutes and death can occur in as little as 15. Repetitive firing of motor neurons causes severe muscle fasciculation (twitching), which in turn causes the body temperature to increase. Autonomic neurons are also affected, causing salivation, lachrymation (tears), sweating, vomiting, diarrhea, blood pressure abnormalities, and eventually cardiopulmonary collapse. (1, 7, 12) Cool right?

In invertebrates (the spider's natural prey),  δ-ACTX has a similar effect on calcium and potassium channels. So why doesn't it have the same effect on other vertebrates?

 It appears that the immune systems of most vertebrates are able to protect them from δ-ACTX, in a way that primate immune systems aren't. That being said we still do not have a clear picture of exactly how δ-ACTX binds to the channel. It is entirely possible that primate sodium channels have a unique feature that gives δ-ACTX a particularly high binding affinity. In either scenario, it appears that δ-ACTX's toxic effects on primates are simply an unfortunate result of evolutionary chance.

We are not likely to get a definitive answer anytime soon. With an effective anti-venom on the market, research interest in δ-ACTX has declined. This makes me sad, and it should make you sad too.

 

 Why  δ-ACTX deserves to be Protein of the Year

Now that you know more about δ-ACTX you should be able to see that despite its obscurity, it is a fascinating protein that deserves to be in the running POTY. Now I am going to give you a couple reasons why it should win.

 

1. Everybody loves a Villain

Everybody loves a good movie villain: the Joker, Darth Vader, Hannibal Lecter, and HAL 9000. Villains can kill as many people as they want and as long as they do it in a cool way people still love them. I think δ-ACTX definitely fits the bill.

 

2. Anti-venom sucks

Most people think that because anti-venom is so "widely available", research into venom can fall by the wayside. After all, why continue to research something you have a cure for? This is a big mistake.

Anti-venom is not a cure. It is a severely outdated and archaic backyard remedy that is long past its prime. Anti-venom is created by injecting an animal (like a horse) with the venom you wish to create an anti-venom for. After a day or two, the animal's now antibody-rich blood is drawn and used to create a serum that helps fight the toxins (13). While this may have been a clever idea back in 1895 when it was first invented, we now have the technological ability to come up with something better, and we need to. Here's why.

First, anti-venom is severely expensive. This is because in order to make it, you need copious amounts of real venom produced by the animal in question. This makes anti-venom hard to obtain, especially in third world countries, where death by invenomation is most common. We need something cheaper than anti-venom.

Second, anti-venom is dangerous. Anti-venom serum contains dozens of proteins from the body of a horse/goat/sheep/rabbit. Severe allergic reactions upon administration can and have killed patients. Delayed allergic reactions, known as serum sickness, are also quite common. We need something less risky than anti-venom.

Third, anti-venom doesn't always work. Its efficacy is dependent on how quickly it is administered and it is completely unable to reverse the damage already done by the venom when it is given.

In short, anti-venom is an expensive, dangerous treatment that doesn't always work. It might even kill you. So why don't we try anything else? Well because, as previously mentioned most kinds of venom consists of dozens of toxic components and making a treatment that counteracts all of them is extremely difficult. Before we can create a new treatment for these complex kinds of venom, we need to have a proof of concept by creating a new treatment for a much simpler venom.

In this way, the Sydney Funnel-Web Spider is the ideal candidate because its venom contains one component: δ-ACTX. In fact, some researchers down under have begun doing just such a thing and were able to create a vaccine that protected a pair of monkey's from a lethal dose of δ-ACTX. See my Assignment #2 post for more details.

 

3. We can learn from it

δ-ACTX could provide a useful tool for understanding voltage-gated sodium ion channels, especially how they are regulated. This understanding could lead to the creation of more effective drugs for all conditions that involve voltage-gated sodium ion channels (which is a lot).

 

4. Crocodile Hunter would vote for it

As I'm sure everybody knows, the late Steve Irwin was super awesome. I mean, who didn't love the Crocodile Hunter as a kid? Steve Irwin loved animals, and the Sydney Funnel-Web was so exception (he actually picked one up once) . If he was still alive I'm positive he would vote for δ-ACTX to be POTY, and that's kind of like a celebrity endorsement. What other reason could you possibly need?

5. Steve Irwin

Bibliography

1. “Australian Funnel-Web Spider.” Wikipedia, the free encyclopedia 11 Jan. 2015. Wikipedia. Web. 6 Mar. 2015.

2. Comis, Alfio et al. “Immunization with a Synthetic Robustoxin Derivative Lacking Disulphide Bridges Protects against a Potentially Lethal Challenge with Funnel-Web Spider (Atrax Robustus) Venom.” Journal of Biosciences 34.1 (2009): 35–44. Print.

3. Craik, D. J., N. L. Daly, and C. Waine. “The Cystine Knot Motif in Toxins and Implications for Drug Design.” Toxicon: Official Journal of the International Society on Toxinology 39.1 (2001): 43–60. Print.

4. “Delta Atracotoxin.” Wikipedia, the free encyclopedia 22 Jan. 2015. Wikipedia. Web. 6 Mar. 2015.

5. Fletcher, Jamie I et al. “The Structure of Versutoxin (δ-Atracotoxin-Hv1) Provides Insights into the Binding of Site 3 Neurotoxins to the Voltage-Gated Sodium Channel.” Structure 5.11 (1997): 1525–1535. ScienceDirect. Web. 6 Mar. 2015.

6. Gilles, Nicolas et al. “Variations in Receptor Site-3 on Rat Brain and Insect Sodium Channels Highlighted by Binding of a Funnel-Web Spider Δ-Atracotoxin.” European Journal of Biochemistry 269.5 (2002): 1500–1510. Wiley Online Library. Web. 6 Mar. 2015.

7. Harris, J.B., S. Sutherland, and M.A. Zar. “Actions of the Crude Venom of the Sydney Funnel-Web Spider, Atrax Robustus on Autonomic Neuromuscular Transmission.” British Journal of Pharmacology 72.2 (1981): 335–340. Print.

8. “Inhibitor Cystine Knot.” Wikipedia, the free encyclopedia 6 Oct. 2014. Wikipedia. Web. 22 Mar. 2015.

9. Little, Michelle J et al. “Δ-Atracotoxins from Australian Funnel-Web Spiders Compete with Scorpion Α-Toxin Binding on Both Rat Brain and Insect Sodium Channels.” FEBS Letters 439.3 (1998): 246–252. ScienceDirect. Web. 6 Mar. 2015.

10. Pallaghy, Paul K et al. “Solution Structure of Robustoxin, the Lethal Neurotoxin from the Funnel-Web Spider Atrax Robustus.” FEBS Letters 419.2–3 (1997): 191–196. ScienceDirect. Web. 6 Mar. 2015.

11. “Sodium Channel.” Wikipedia, the free encyclopedia 5 Mar. 2015. Wikipedia. Web. 22 Mar. 2015.

12. “Sydney Funnel-Web Spider.” Wikipedia, the free encyclopedia 14 Jan. 2015. Wikipedia. Web. 23 Mar. 2015.
13. “Antivenom.” Wikipedia, the free encyclopedia 27 Jan. 2015. Wikipedia. Web. 16 Apr. 2015.
Photo Credits
1. http://en.wikipedia.org/wiki/Sydney_funnel-web_spider
2. Generated with PyMol
3. http://en.wikipedia.org/wiki/Inhibitor_cystine_knot
4. http://en.wikipedia.org/wiki/Sodium_channel
5. http://i.telegraph.co.uk/multimedia/archive/02847/Steve-Irwin-crocod_2847616b.jpg

Friday, March 6, 2015

Assignment #2

Articles on Delta Atracotoxin(s)


 Article #1: The structure of versutoxin (d-atracotoxin-Hv1) provides insights
into the binding of site 3 neurotoxins to the voltage-gated
sodium channel

This article describes the structural properties and mechanism of δ-ACTX-Hv1, which is the variant of delta atracotoxin found in the Blue Mountain Funnel Web Spider (H. versuta). The article begins with an introduction to the protein.

This figure compares the primary structure of δ-ACTX-Hv1 to δ-ACTX-Ar1, which is the variant of delta atracotoxin found in the Sydney Funnel Web Spider (A. robustus). The article notes the intriguing fact that the venom of both subspecies "produce severe neurotoxic symptoms in primates and baby mice, but not other vertebrates." This is one of the most interesting aspects of δ-ACTX: it is toxic only to insects, the spiders' natural prey, and primates. The article reaffirms earlier research that these 42-residue atracotoxins are responsible for the "primate-specific effects" of their respective venom.

This article then presents the result of their experiment: the solution to the 3-dimensional structure of δ-ACTX-Hv1, pictured below.


 
 The structure they derived was compared to other known proteins and found to have "significant structural homology" two other proteins. The first is w-agatoxin-IVB from A. aperta, a significantly less toxic spider found in North America. The second was with a protein known as gurmarin. The article describes as "sweet-taste suppressing polypeptide from the leaves of G. sylvestre."

The article also describes that although not structurally similar, δ-ACTX-Hv1 has a similar effect on voltage-gated sodium channels to that of toxins found in some scorpions and in sea anemones. The researchers investigated this further and found that although structurally different, the toxin of the sea anemone contains several sites very similar to that of δ-ACTX-Hv1. The article uses this and subsequent experiments to confirm that δ-ACTX-Hv1 acts by binding to site 3 of voltage-gated sodium ion channels.

The article concludes by suggesting that these be areas of further research, and makes some remarks as to the evolutionary implications of the similarities found.

Article #2: Solution structure of robustoxin, the lethal neurotoxin from the
funnel-web spider Atrax robus tus

This article similarly presents the structure of  δ-ACTX-Ar1. However, I instead want to focus on this articles introduction to the toxin, which lists its clinical effects.

http://upload.wikimedia.org/wikipedia/commons/9/9f/Atrax_robustus.jpg
photo credit: wikipedia.org


A bite from the Sydney funnel-web spider is, one of, if not the most deadly spider bites. It is interesting for two reasons. First, as previously mentioned, the venom appears to be toxic to only primates and insects. Second, the venom acts by targeting site 3 of voltage-gated sodium ion channels. Its mechanism prevents them from inactivating, which results in prolonged synapses being fired by different neurons in the body. Rather than killing via paralysis, the venom of the Sydney funnel-web kills by over-exciting neurons.


The clinical symptoms onset within an hour of being bitten. The first symptom is pain at the sight of the injury (duh). Later symptoms include salivation, lachrymation (tears), skeletal muscle fasciculation, anomalies in heart rate and blood pressure, and finalizing in severe hypotension and cardiopulmonary failure.

A successful antivenom was produced in 1980, and while this has successfully prevented any further fatalities, more efficient treatments might be possible (more on that next.) The researchers in this article point out that δ-ACTX-Ar1 is still of significant clinical interest because of its usefulness in studying ion channels in primates.

Article #3: Immunization with a synthetic robustoxin derivative lacking disulphide
bridges protects against a potentially lethal challenge with funnel-web
spider (Atrax robustus) venom

In this article, researches attempted to create a vaccine for the venom of the funnel web spider. This is an incredibly interesting concept. Current treatment for venomous injuries consists of administration of (expensive) anti-venom after the bite has occurred. The ability to vaccinate somebody against a venom prior to invenomation is a really promising idea.

This article describes experiments run on 4 monkeys, with three different vaccine attempts. In all attempts the primary component of the vaccine was an inactivated δ-ACTX-Ar1 protein. In the first attempt, it was co-polymerized with another component and the resulting vaccine protected two monkeys from a lethal dose of the toxin. Subsequent attempts and alterations of the original did not protect the other monkeys. The researchers discuss these results and the implications they have on vaccinating against protein based toxins.

Bibliography:

Comis, Alfio et al. “Immunization with a Synthetic Robustoxin Derivative Lacking Disulphide Bridges Protects against a Potentially Lethal Challenge with Funnel-Web Spider (Atrax Robustus) Venom.” Journal of Biosciences 34.1 (2009): 35–44. Print.

Fletcher, Jamie I et al. “The Structure of Versutoxin (δ-Atracotoxin-Hv1) Provides Insights into the Binding of Site 3 Neurotoxins to the Voltage-Gated Sodium Channel.” Structure 5.11 (1997): 1525–1535. ScienceDirect. Web. 6 Mar. 2015.

Pallaghy, Paul K et al. “Solution Structure of Robustoxin, the Lethal Neurotoxin from the Funnel-Web Spider Atrax Robustus.” FEBS Letters 419.2–3 (1997): 191–196. ScienceDirect. Web. 6 Mar. 2015.