The Ocean holds secrets about genome editing

It’s being told that the ocean holds many secrets, of lovers and murders, of treasures and ships untouched, that it makes me wonder what other secrets could it possibly hold. We have to thank the ocean for giving us biochemists such special gifts as our beloved GFP (Green Fluorescent Protein), but what other cool proteins can come to us from the ocean?

Everyone has a favorite protein, what’s yours?

I was once asked by a biochemistry teacher: “everybody has a favorite protein, what’s yours?” I think I was way too young to have an answer back then. It’s been over a decade since I graduated and right now I don’t really think I have one favorite protein, but I have a group of favorites and among them are the TALEs. That’s why I think they deserve a post dedicated just to them.

Is it possible to edit the genome?

According to the great Wikipedia, editing is the process of selecting and preparing written, visual and audible media used to convey information. But editing can be applied in other aspects of life as well, even at the gene level. Genome editing is a type of genetic engineering in which the DNA is inserted, replaced or removed from a genome using artificial nucleases or “molecular scissors” or in other words is about re-writing the DNA code.

Genetic engineering emerged in the lab of Paul Berg in 1972 in the form of a recombinant DNA technology, when scientists combined the E. coli genome with the genes of a bacteriophage and the SV40 virus. Since then, this science has achieved tremendous success; many methods to manipulate DNA have been developed as well as vector systems and methods for their delivery to the cell.

From 1990 to 2003, the DNA sequence of human, E. coli, mouse and others was deciphered, retrieving information about nucleotide sequence only. In 2003, the U.S. National Human Genome Research Institute launched a new international project, ENCODE (Encyclopedia Of DNA Elements), which aim was to obtain a complete list of the functional elements of the human genome. Unfortunately, the methods used back then are not only expensive but also quite labor-intensive and they do not allow one to introduce precise changes into a strictly defined genome locus. Currently, researchers have several tools that allow them to solve the problems of precise plant’s, animal’s, and human’s genome editing.

A tale about TALEs

Bacteria of the genus Xanthomonas are pathogens of crop plants, such as rice, pepper and tomato that cause significant economic damage to agriculture. Cool thing about these bacteria is that they were found to inject proteins into the host plants where they mimic eukaryotic transcription factors thus hijacking the host’s transcriptional machinery to control gene expression. These proteins are known as TALEs for Transcription Activator-Like Effectors. But why would TALEs do that? Well, to increase the plant susceptibility to the pathogen, and they do it pretty much like a virus.

One of the most interesting things about TALEs is what I call their “outfit”. It turns out that TALEs bind to DNA in a specific manner and this specificity is given by its DNA-binding domain “outfit” which consists of tandem repeat arrays of amino acids, with each repeat binding a single DNA base. Moreover, The residues located at positions 12 and 13 in each repeat are highly variable and are called the BSR (base specifying residues), which are responsible for the recognition of a specific nucleotide. This TALE code is degenerate in the way that some BSRs can bind to several nucleotides with different efficiencies.

After TALEs were described, two other plant disease associated bacteria were found to encode for TALE-like proteins as well. These proteins are RipTALs from Ralstonia solanacaerum and Bats from endofungal bacterium Burkholderia rhizoxinica. TALE-likes seems to be united only by possession of DNA binding repeats with a conserved code, in fact Bats lack the domains necessary to function as eukaryotic transcription factors.

What can TALEs be used for?

So lets imagine: we have a cool protein, wearing a nice tandem repeat outfit, which holds a special code that allows to bind to DNA in a specific manner. So, what if we knew the code? Well, then one could predict the DNA binding element for any given TALE and to design them to match any DNA sequence of interest. Now THAT is interesting. But it gets even better! Genetic engineering in not called that just for nothing, so the idea is to make these proteins even cooler. Now, what could be done? Specific DNA-binding in one thing, but the function is another thing. So a great idea is to couple TALEs to a functional domain of choice and those chimeras are invaluable tools for precision manipulation of genome.

But we can go beyond: non-BSR polymorphisms might also be useful to tune DNA-binding properties and further expand the diversity of TALE-DNA interactions. One could then create libraries of designed TALEs with a range of binding strengths for the same DNA element, useful for the regulation of synthetic genetic circuits.

The birth of a new pair of genetic scissors: TALENs

After deciphering the code of DNA recognition by TALE proteins, which attracted the attention of researchers across the world due to its simplicity (one monomer – one nucleotide), the first chimeras of TALEs and nucleases were created: the sequence encoding the DNA-binding domain of TALE was inserted into a plasmid vector previously used for creating Zinc Finger Nucleases. This resulted in the generation of genetic constructs expressing artificial chimeric nucleases (TALENs) and this was so amazing that in 2011 Nature Methods named the methods of precise genome editing, including the TALEN system, method of the year.

How to ‘pimp’ your TALE

One approach to TALE repeat engineering is random mutagenesis and screening. Alternatively, mutations could be introduced in a more targeted fashion, but this requires information on the impact of different types of polymorphisms at different positions in the TALE repeat. So, where do we get information on what sites should be good targets?....in mother nature. Natural variation would provide useful information on what residues can or cannot be tolerated at which positions and with what effect. Unfortunately, TALEs sequence diversity is very low and residues clustered around the BSR are largely invariant across all currently known TALEs, RipTALs and Bats. Good news is RipTALs and Bats are only near 40% identical to TALEs, making them useful for repeat engineering.

TALEs from the Ocean

The ocean is quite an interesting place to do research. We often look for answers or work on the model of terrestrial organisms, but the ocean holds many secrets.

On the Gulf of Mexico/Yucatan Channel, during the Global Ocean Sampling (GOS) expedition, biological samples were taken and used for DNA sequencing. Among them, a particular sample was analyzed, likely from bacterial origin based on size filtering of the biological material that was used. During sequence analysis two predicted proteins came into the light: MOrTL1 and MOrTL2 (Marine Organism TALE-likes, names that reflect the limited information scientist had regarding their provenance).

These sequences have been previously suggested to encode modular DNA binding repeats, but no functional analysis had been reported. And guess what, both proteins are tandem repeat arrays … does it ring a bell?

This MOrTLs repeats differ at more than 60% of positions from each other and from all other TALE-likes. Although MOrTL sequences are incomplete and likely to be fragments of larger, incompletely sequenced genes, they were synthesized and MOrTL1 was further expressed and purified from E. coli. As analyzed by electrophoretic mobility shift assay (EMSA), MOrTL1 showed a weak DNA binding, inconsistent with TALE-likes. This would likely reflect the incomplete sequence of MOrTL1, yielding a incomplete functional protein.

Then, how to study a predicted protein, based on an incomplete sequence, if even when expressed it’s not fully functional? Well….  CHIMERAS!

MOrTL repeats were embedded within the repeat domain of a Bat named Bat1. Now there were ready to be studied.

Can Bat1-MOrTL chimeras bind to DNA?

Yes they can! And this was confirmed by mixing Bat1-MOrTL chimeras and their cognate TALE-code predicted DNA binding element and analyzed by two approaches: a qualitative classical EMSA and the quantitative MicroScale Thermophoresis (MST) which can quantify the affinity of the binding and calculate binding constants (K_D) of any intermolecular interactions with high precision. In fact, Bat1-MOrTL chimeras bind with similar strength to the wild type Bat1 protein.

Is this binding specific?

When you prove that a TALE-like protein binds to its predicted on-target sequence, does it prove adherence to the TALE code? NOT NECESSARILY!

How to prove it then? Well, specificity needs to be tested and by this I mean proving the binding to the worst predicted match DNA sequence based on the TALE code. How can this experiment be done? By competition experiments. So when analyzing probe-protein interactions (DNA-binding elements-MOrTLs interactions) with on and off-target competitors it was confirmed that both MOrTL1 and MOrTL2 TALE-code consistence base preference. Additionally, when quantifying the protein-DNA interactions with the off-target probe by MST a very interesting result came into light: the Bat1-MOrTL1 chimera had an affinity 19 times lower than the on-target interaction, while Bat1-MOrTL2 was only two times lower, thus they differ in the discriminating power. The higher discriminatory power of MOrTL1 repeats thus make them better for integration into TALE-like repeat arrays for biotechnological applications. A very nice piece of information.

Could protein stability account for the differences observed?

Functional differences among proteins could be due to stability issues. Protein stability can be analyzed by inducing thermal denaturation and then monitoring the fluorescence of SYPRO Orange, which binds non-specifically to hydrophobic surfaces. So, when the protein unfolds, the exposed hydrophobic surfaces bind the dye, resulting in an increase in fluorescence. This method is called DSF (Differential Scanning Fluorimetry), also known as Thermofluor. But as the music band ‘Outkast’ asks “what’s cooler than being cool?”, well the answer is not “Ice Cold”, it is "nanoDSF". This technology allows to do the same as DSF but taking advantage of the Tryptophan’s intrinsic fluorescence and by using this pretty cool technology, it was shown that the functional difference between MOrTL1 and MOrTL2 chimeras are not due to differences in protein stability.

By demonstrating that MOrTL repeats mediate DNA binding behavior analogous to that of other TALE-likes repeats, scientists have gain insights into the nature of the whole TALE-like family… hopefully this amazing discovered secrets from the ocean will enable further research into the distribution and functions of these fascinating DNA binding proteins.

1.    

11. DNA-binding proteins from marine bacteria expand the known sequence diversity of TALE-like repeats. Nucl. Acids Res. first published online October 19, 2015 doi:10.1093/nar/gkv1053

The holly venom: How can a bee sting save your life

Have you ever been stung by a bee? I have. I was in one of those movie-like moments, enjoying a beautiful sunny day in a wonderful park, surrounded by nature, filling my lungs with fresh air and of course without shoes so that I could deeply feel the softest, moist and green morning grass of all… slipping through my toes in a wonderful massage given by mother nature, when BANG!!!!!!!! A terrible pain as if I had stepped on a box of nails started flowing from my feet up to my brain….. what the hell just happened!!!!!!!!! OUCHHHH!!! I can tell you IT DOES HURT!.

I grew up listening that in case that happened, you should quickly remove the bee sting and suck all the venom you can, it never occurred to me that letting the poison flow through my bloodstream could have any benefit. When I grew older, I recall watching on the TV this emerging therapy called “apitherapy” in which some guy was picking up bees from a flask with tweezers and putting them in some other person’s body to sting …. On purpose!!!! How crazy can people be!!!!!!

But what is it behind this bee venom. As a scientist I just had to know and now I want you to read probably the most amazing bee story of your life. It goes like this.

Who do you think would win a battle between bees and bacteria?

In the spring of 1996, at the age of 27, Ellie Lobel an active woman and mother of three kids, was bitten by a tick. Ellie didn’t know to look for the characteristic bull’s-eye rash when she was bitten- she thought it was just a weird spider bite.

Then came three months with flu-like symptoms and horrible pains that moved around the body. She was incapacitated. Her first doctor told her it was just a virus and it would run its course. So did the next. Ellie went to doctor after doctor, each giving her a different diagnosis. Multiple slecrosis, lupus, rheumatoid arthritis, fibromyalgia. None of them realized she was infected with Borrelia, a bacteria transmitted by ticks, until more than a year after she contacted the disease-and by then, it was far too late. Lyme bacteria, as it is called, are exceptionally good at adapting and capable of dodging both the immune system and the arsenal of antibiotics currently available. Although she kept doing treatment after treatment, she got worse. She describes being stuck in bed or a wheelchair and not being able to think clearly, but she kept fighting, with every antibiotic, every pharmaceutical, every holistic treatment she could find¹.     

After fifteen years, she gave up. So she packed up everything and moved to California to die. And she almost did. Less than a week after moving, Ellie was attacked by a swarm of bees. Ellie was in California for three days before her attack. At this point, Ellie was struggling to stand on her own. She had a caregiver on hand to help her.

She was just standing near a broken wall and a tree when the first bee appeared, she remembers, “just hitting me in the head. All of a sudden-boom!-bees everywhere.” Her caregiver ran. But Ellie couldn’t run. She couldn’t even walk. “They were in my hair, in my head, all I heard was this crazy buzzing in my ears. I thought, THIS IS IT. I’m just going to die right here”.

Ellie, like 1-7% of the world’s population, is severely allergic to bees. When she was two, a sting put her into anaphylaxis, a severe reaction of the body’s immune system that can include sweeling, nausea and narrowing of the airways. She nearly died. She stopped breathing and had to be revived by defibrillation. Her mother drilled a fear to bees into her to ensure she never ended up in the same dire situation again. So when the bees descended, Ellie was sure that this was the end.

Bees are armed with a potent sting that many of us are all too aware of. Their venom is a mixture of many compounds. Perhaps the most important is a tiny 26-amino-acid peptide called Melittin, which constitutes more than half of the venom of honey bees. This little compound is responsible for the burning pain associated with bee stings. It tricks our bodies into thinking that they are quite literally on fire.

 “I just went limp. I put my hands up and covered my face because I didn’t want them stinging me into the eyes… The next thing I know, the bees are gone”, says Ellie.

When the bees finally dissipated, her caregiver tried to take her to the hospital, but Ellie refused to go. “This is God’s way of putting me out of my misery even sooner”, she told him. “I’m just going to accept this. I locked myself in my room and told him to come collect the body tomorrow”.

But Ellie didn’t die. Not that day, and not the three to four months later. “I just can’t believe that was three years ago and I just can’t believe where I am now. I had all my blood work done. Everything. We tested everything. I’m so healthy”.

After the attack, Ellie watched the clock, waiting for anaphylaxis to set in, but it didn’t. Instead, three hours later, her body was racked with pains. Ellie thinks that these weren’t a part of an allergic response, but instead indicated a Jarisch-Herxheimer reaction-her body was being flooded with toxins from dying bacteria. A theory is that certain bacterial species go down swinging, releasing nasty compounds that cause fever, rash and other symptoms. For days she was in pain. Then, she wasn’t.

With a now-clear head, Ellie started wondering what had happened. So she did what anyone else would do: Google it. Disappointingly, her searches turned up very little. But she did find one 1997 study, where it was found that melittin killed Borrelia². Exposing cell cultures to purified melittin, they reported that the compound completely inhibited Borrelia growth. Soon after melittin was added, the bacteria were effectively paralysed, unable to move as their outer membranes were under attack and then they were killed.

My “friend-beans-Chagas disease-bees” connection

I know it may sound like tangled up spaghetti in your head. But thing is a couple days ago, my friend advice me to keep my daughter away from the beans that are given to her in her school lunch. Of course I asked why, since beans are the basis of any food here in Brazil. She told me that it is because of a Chagas disease outbreak and that it seems that people can get infected through parasites growing in beans. Chagas disease is a tropical parasitic disease caused by the protozoan Trypanosoma cruzi and is spread mostly by insects known as kissing bugs. The symptoms change over the course of the infection, going from fever, headaches, swollen lymph nodes, to enlargement of the ventricles of the heart leading to heart failure, among other symptoms.

Anyways, after she texted me sending pictures of this nasty parasites in beans, I immediately googled it. And guess what, what I found was an interesting 2013 paper entitled “Melittin peptide kills Trypanosoma cruzi parasites by inducing different cell death pathways”³. So I texted her back saying, ”ok, so if you or anyone you know get infected with Chagas disease, I highly recommend you to go play Winnie the Pooh chasing beehives or to take a walk in the park without shoes… or just try apitherapy”.

Melittin therefore, holds promises in the cure of several parasite-transmitted diseases, but also for viral diseases, since it has been shown that it inhibits replication of murine retroviruses, tobacco mosaic virus and herpes simplex virus. This anti-viral activity of Melittin has been attributed to direct lysis of viral membranes. However, Melittin also displays anti-viral activity at much lower non-virolytic concentrations, as shown for T cells chronically infected with human immunodeficiency virus 1 (HIV-1), where it as been shown that it suppress production of HIV-1 by acutely infected cells.

Venoms: a juicy target for pharmaceutical companies

Modern science has slowly begun to take apart venoms, piece by piece, to understand how they do the things they do. Most of them are complex cocktails of compounds, with dozens to hundreds of different proteins, peptides and other molecules. Each compound has a different task that allows the venom to work with maximum efficacy- any parts moving together to immobilize, induce pain or do whatever it is that the animal needs its venom for.

Venoms started to be screened against specific therapeutic targets in the 2000s. Of the seven venom-derived pharmaceuticals on the international market, the most successful, captopril, was derived from a peptide found in the venom of the Brazilian viper (Bothrops jararaca). 

This venom has been known for centuries for its potent blood-thinning ability (one tribe are said to have coated their arrow tips in it to inflict maximum damage) and the drug has made its parent company more than a billion dollars and become a common treatment for hypertension. It’s being said that captopril is not only one of the top twenty drugs of all time, but it has been one of the most persistent, outside of maybe aspirin.

There is a lot more research to do on these venoms…. But in the meantime, don’t be scared of bees… who knows what they could do to you.

1. http://mosaicscience.com/story/how-bee-sting-saved-my-life-poison-medicine
2. Lubke LL, Garon CF (July 1997). "The antimicrobial agent melittin exhibits powerful in vitro inhibitory effects on the Lyme disease spirochete". Clin. Infect. Dis. 25 (Suppl 1): S48–51.
3. Adade, C., Oliveira, I., Pais, J. and Souto-Padrón, T. 2013. Melittin peptide kills Trypanosoma cruzi parasites by inducing different cell death pathways. Toxicon 69: 227-239.