Mommy, Where Does Blood Come From?

With the recent rise of the vampire star in popular culture, this scientist thinks we’ve all been seeing a little more blood in everything from movies, to television, to magazine ads. While it might not be immediately apparent from these corn-syrup-and-food-colouring imitations, our blood is pretty complex stuff. Like so much of our bodies, what seems like a simple (if slightly goopy) liquid is really a mixture of different types of cells and substances. Also like much of the rest of us, the makeup of this serological soup needs to be closely regulated to keep us alive. When it comes to controlling the composition of our bodies, enzymes have the gene-wrangling power that places them squarely in the drivers seat.

            If CSI: Miami has taught me anything, it’s that sunglasses are required to investigate crimes, and each person has unique DNA. In fact, each cell in your body carries the exact same DNA sequence, the same genetic code. So how do some cells end up as heart muscle, and some as fingernail, and some as bone marrow? If all of the DNA is the same, how can it hold information for making so many different kinds of cells? What makes different kinds of cells different isn’t their DNA, but the proteins that exist in that cell. Proteins are made by decoding specific parts of the DNA, so the body controls what cells are what by regulating what pieces of the DNA get decoded to make protein. It’s kind of like having a whole recipe book with many recipes that could make up many meals. If you want a pancake and poached egg breakfast, you use those specific recipes to come up with the desired end product.

            To control what proteins get made in each cell, we control which recipes are available to the chef. Our DNA isn’t just crammed willy-nilly into our cells, but has a defined structure in which it is wound up very tightly. There are proteins called histones that the DNA folds around to help condense it into a smaller space. The DNA itself, as well as the histones, can be modified by attaching specific chemical groups. In this way, the DNA can be effectively labeled with respect to which proteins should and shouldn’t be made in that cell. This labeling doesn’t happen on it’s own, however. There are specific enzymes that attach and detach these groups from our genetic material, thus controlling which genes get made into protein (expressed) or are wound up too tightly to be accessed (repressed).

            One of the chemical groups enzymes attach to DNA is the methyl group: one carbon atom attached to three hydrogen atoms. A team of researchers in Texas recentlyinvestigated what would happen if an enzyme responsible for methyl-labeling was removed from stem cells in the bone marrow. Normally, these cells are responsible for morphing into all the different types of cells that make up our blood and bone marrow, in addition to making more stem cells. When this DNA-labeling enzyme was absent, the cell made the wrong proteins at the wrong times and could not make functional blood cells. Their experiment showed that DNA labeling by this enzyme is a key part of how the body produces the blood cells we need to live.

None of us could survive if we only made one type of cell. It’s our bodies’ ability to pick and choose the right recipes for the right occasion that results in our different body parts and organs. With enzymes playing such a key role, maybe the vampires are just missing an enzyme they need to make their own blood. Human blood, a vampire’s complete breakfast.

Playing Evolution: Making Enzymes from Scratch

Photo credit www.jacehallshow.com

In researching almost any topic in biology I am consistently amazed at how natural selection produces things so well suited for their specific roles. From an animal perfectly filling an ecological niche, to an organ efficiently performing a biological task, to a molecule precisely providing the ideal chemistry for its function. It is truly astounding how a process that moves along through random mutation events can result in perfectly coordinated systems at every level. Although natural evolution results in sublimely well-tuned components for every biological system, it also takes a stupendously long time to go about its business. Enter the twenty-first century scientist. While scientists in nanorobotics and materials science are making game-changing leaps and bounds in our ability to manipulate ourselves and our environment, enzyme engineers are starting to give them a run for their money. Have a tough chemical job to do? One that has never been done before? Why not engineer a brand-spanking-new custom enzyme to catalyze your worries away?

            Many of the enzymes that exist naturally are made of protein. The protein itself is a long chain made of different amino acids linked together. You can think of it kind of like a long charm bracelet: the links of the chain form the structure of the chain, while different charms on each of the links make each part of it unique. With proteins, there are 20 types of amino acids or “charms” to choose from that can be strung together in different combinations.

           

            Much like how a ball of wool doesn’t do you a heck of a lot of good on a cold day, a enzyme doesn’t do much at all as one long strand. Mittens or a scarf, however, can be very functional, and so can an enzyme when it is tangled up into a very specific shape. In fact, it is the three dimensional shape that gives a folded enzyme its ability to perform chemistry. So how do enzymes stay folded in the right shape? Going back to the charm bracelet example, some charms (amino acids) can interact specifically with each other to reinforce specific shapes. The “chain” itself also has a tendency to form certain structures: telephone chord-like coils (alpha-helices) and flat sheets formed by the chain doubling back over itself repeatedly (beta-sheets). Additionally, over one third of all proteins contain atoms of iron, zinc, copper, or other metals. By interacting strongly with specific groups of amino acids, these metals can lock part of an enzyme structure in place, helping the rest of the enzyme to also hold its active shape.

            If the interactions that keep an enzyme folded properly and functional seem complex, that’s because they are. A single enzyme has hundreds of individual interactions between individual amino acids and metal groups, some of which can be removed or replaced, and some of which are vitally important either to keep the enzyme in the right shape, or to perform specific chemical functions. Scientists have been trying to tease this apart for years by deleting or swapping out specific amino acids in natural enzymes and observing the effects of these changes. Much like tinkering with an engine vs. building one from scratch, however, you can only learn so much about how something works without creating one of your own. This is exactly what a group of scientists from the University of Michigan reported recently in Nature Chemistry. These intrepid researchers designed an enzyme from scratch: designing a structural “scaffold” (complete with a structure-stabilizing metal) into which they incorporated a small area capable of performing similar chemistry to one of our body’s own enzymes. By testing which enzyme design concepts make the chemistry faster or slower, these researchers are testing and applying the basic principals we currently think govern how all enzymes work.

            Designing enzymes from scratch to perform tasks that natural enzymes already perform is almost the definition of reinventing the wheel. It is, however, incredibly valuable in at least two respects: it demonstrates that we understand how enzymes work well enough to design them from the bottom up, and it opens the door to engineering brand new enzymes that catalyze chemistry not found in nature. Imagine creating an enzyme that quickly breaks down Styrofoam or other long-lasting pollutants. Enzymes are the most powerful chemists in nature, and if we can design their abilities, rather than simply co-opting them, they can be our most powerful industrial and biotechnological tools as well.

Making Mutants, the Safe Way

The practice of medicine has come a long way from the days of the four humours. Instead of being filled with a delicate balance blood, snot, and various forms of “bile,” we now understand that our bodies are made of millions of cells, each with a specific job to do. We are now able to pinpoint the exact type of cell not doing its job in many diseases, and to figure out just what job it’s not doing. This knowledge can help us change or replace parts in our cells in order to treat some diseases. In the future, making our own engineered human mutants may help us accomplish medical feats not possible with traditional drugs.

            Each cell in our body is specialized to perform a specific function. Our nerve cells conduct electricity to pass messages around the body, our red blood cells carry oxygen, and cells on the inside of our stomachs secrete acids to help with digestion. In order to carry out these jobs, our different types of cells produce specific enzymes and other proteins. Without these specialized components, the cells don’t function like they should. For some diseases there may be only a single protein that is not being produced, and this deficiency results in a whole host of symptoms. For example, type I diabetes is an entire disease caused by the body being unable to make a single protein: insulin. All proteins are made by decoding DNA sequences, and researchers are looking at treating diseases like diabetes by adding back the specific DNA needed to make the proteins that are missing or damaged. By inserting these specific DNA sequences into the genetic material already in our cells, we hope to engineer helpful cellular mutants that can produce missing proteins and reverse disease.

            Sticking helpful genes into human cells can be a great way to fill the gap of a missing protein, but there are many dangers inherent in introducing new DNA. If a new piece of DNA is inserted into one of your chromosomes, it can land in any number of places, including in the middle of another gene. This can potentially result in mutation of a different protein, in which case you might cure the first disease but could actually cause another. A more common and potentially more dangerous scenario is if the new piece of DNA disrupts an oncogene (see DNA Repair just doesn’t give me the same Buzz). In this case adding the extra DNA can cause cancer.

            While humans have only been sticking extra bits of DNA into cells for a few years, viruses have been accomplishing this task for millennia. For many viruses, part of their life cycle involves taking part of their DNA and inserting it into a chromosome of the cell they’re invading. In order to do this efficiently, viruses use a specific DNA-insertion tool: an enzyme called an integrase. This enzyme recognizes specific sequences on the DNA to be inserted and on the host cell DNA and stitches in the inserted piece at specific sites on the host chromosome.

            Nature doesn’t always do what we want it to (sometimes disease happens), but it also produces some pretty useful tools that we can borrow. In the case of gene-based therapies, the danger is not knowing where an introduced piece of DNA will insert into our own DNA, and whether or not that might cause even more problems. By borrowing the ability of viral integrases to insert DNA pieces into specific places in the genome, we drastically cut down this risk. Scientists are now working on giving patients the specific DNA segments needed to replace proteins missing in certain diseases and including viral integrase enzymes to make sure that DNA is inserted in safe spots in our genome.

            To most people messing with their DNA, the prospect of cancer, and viral enzymes are all pretty scary concepts. By understanding how each component works, however, we are able to use these concepts to treat diseases in innovative ways. Knowing more about almost anything can help it to be less worrisome and more useful.

Animals on Steroids – Steroids on Oxygen?

In today’s society, the word “steroid” conjures up images of hulking body-builders and sheepish forfeitures of Olympic medals. What most people don’t realize is that steroids actually play a role in all of our lives. In fact, right now there are a whole host of steroids performing the very important role of keeping you alive. Whether we’re pumping up or vegging out, we’re all chock full of steroids. What might be even more amazing is that in addition to keeping animals, plants, and fungi functioning like they should, steroids may also be able to tell us a bit about how and when such multi-celled beings came to be.

            As I alluded to above, steroids aren’t just for greased-up gym junkies. On the contrary, they are enormously prevalent substances produced by all kinds of plants, animals, and fungi. Steroids are molecules made of four rings of carbon fused together in a bent line. This basic structure contains 17 atoms of carbon, and modifying this welded-ring core with additional carbon or oxygen atoms makes hundreds of different steroids. The central steroid in human biology is cholesterol, and it is a vital component of every cell in your body. We also use it to make other steroids like the dreaded hormones that complicate adolescence and control much of our development and body maintenance. While fruit bats, magnolias, and button mushrooms don’t have the same type of awkwardness during puberty, they also produce a preponderance of different steroids nonetheless critical for their own lifestyles.

Cholesterol

            While cholesterol is indeed present in your hamburger, your body also makes it from the simpler molecules you get from digesting all kinds of food. There are three major atomic ingredients in the cholesterol recipe: carbon, hydrogen, and oxygen. The first two come from the food-begotten hydrocarbon building blocks. The necessary oxygen comes from the O₂ in the air we breathe. Like so many things our body makes, the real construction workers are our enzymes. There are many enzymes involved in making steroids, some of which are dedicated to incorporating oxygen from O₂ into the steroid being produced. These enzymes are aptly named “oxygenases”. The job of an oxygenase is not an easy one, and the reaction between O₂ and the hydrocarbons is incredibly slow if no oxygenase is around to speed it along. Many oxygenases use tools like metals and vitamins to manipulate the oxygen into reacting with the carbon in order to come up with the final steroid product.

            Steroids, produced with the help of oxygenases, are one of the major things separating multi-cellular plants, animals, and fungi from life forms made of a single,simple cell, like bacteria. While all “higher” life forms produce steroids, as a rule, bacteria don’t. In fact, steroids are so important to the membranes of more complicated organisms that evolution of everything from venus flytraps to the mailman may not have been possible if our common ancestors couldn’t make steroids. When it comes to steroid production, we need a lot of oxygenases and a lot of oxygen. Way back in the history of life on earth, however, there wasn’t much oxygen floating around in the air like there is today. When there was no oxygen, there were also no steroid-producing oxygenases. As oxygen levels rose, these enzymes evolved, steroids were produced, and in a few hundred million years you were born, went through puberty, and learned all about it. Because of this necessary sequence of events, scientists are able to look at the emergence of steroid production and make connections with changes in the earth’s atmosphere and the evolution of the earliest common relative we share with the coconut.

Studying modern biology can not only help us understand how we are now, but give us insight into where we came from. As for me, the next time someone questions whether an athlete is on the ‘roids, I’ll turn to them and ask, “Isn’t everyone?”

Every Bug for Themselves! How Changing Enzymes Create Resistant Infections

Sooner or later pretty much everyone is going to have to take a course of antibiotics. Whether it’s a bacterial chest infection, strep throat, or a cut finger that gets out of hand, a bottle of “twice a day, with food, until finished” meds will eventually find its way onto our kitchen counters. Like most of the medicines we take, many of us don’t worry about how they let us reclaim our own bodies, as long as our symptoms get better and stay that way. Unlike many of the medications we take,  antibiotics target processes that aren’t controlled by our own bodies, but rather by the chemistry of another life form. Unfortunately for us, these little buggers multiply faster than we do, adapt faster than we do, and have big population numbers on their side. Lets take a look at how antibiotics effect bacterial enzymes, and what the bugs do to bite back.

In humans, enzymes are vital catalysts for much of the chemistry we need to sustain life. This includes breaking down our food, building up new body parts, and keeping everything repaired, supplied with nutrients, and responsive to our environment. The same is true for bacteria. Fortunately for modern medicine, many of the mechanisms and enzymes bacteria use are very different from those in humans. Additionally, there are many things that bacterial cells do, such as building cell walls, that human cells don’t. Many antibiotics work by targeting the enzymes that are different between the bacteria and people. As an example, bacteria need their cell walls to live, so a drug that shuts down an enzyme needed to make the cell wall will kill the bacterium but will not affect human cells that don’t have the same cellular machinery. By picking on enzymes bacteria need that humans don't have, antibiotic drugs are able to kill bacteria, and fight bacterial infections, without causing damage to the human patient.

If antibiotics kill bacteria by shutting down enzymes needed for important chemistry, how can misuse of antibiotics result in resistant strains of bacteria? To understand the answer to this question, we have to understand that bacteria reproduce much, much faster than people do. In fact, some bacteria produce more than one new generation per hour. To reproduce they have to copy their DNA, and sometimes mistakes are made resulting in changes to the DNA sequence. These changes can result in changes in the sequences of proteins within the cell, some of which will be in the enzymes targeted by the drugs. Some of the changes might result in enzymes that don't work and a new bacterium that will die, but the odd bacterium will coincidentally have an enzyme that still works, but is no longer affected by the drug. This can happen if the place the drug binds to the enzyme is disrupted, the enzyme interacts differently with something else in the cell, or even if the enzyme itself is not changed but something else interacts with the drug, stopping it from effecting its original target. Changes can also occur in other enzymes so that they pick up the catalytic slack and the original enzyme isn’t necessary anymore. 

Individual DNA mutation events are pretty infrequent and random, so a very very small percentage of the bacterial population will experience a mutation leading to a change in the target of a specific drug. These lucky few, however, will survive when the drug kills all of the bacteria without a lucky mutation. So in a typical infection with many drug-susceptible bacteria and a tiny percentage of drug-resistant bugs, the drug will kill the bacteria that it can, and those resistant to the drug will survive, eventually making up the whole bacterial population in that patient. This means that using antibiotics selectively encourages the growth of resistant bacteria, and every time a single antibiotic is used, resistant strains have an opportunity to become more common.

Antibiotics can help us to get rid of infections by killing off most of the bacteria and giving our immune system a chance to fight off the resistant few. Also, these drugs are often used in combination for tricky infections so that bugs resistant to one drug will probably be killed off by the other. The fact remains, however, that all of the antibiotics currently available are getting less and less effective as more and more resistant strains emerge. In the face of this problem, we all need to be careful about the way that these drugs are used, effectively conserving the resource of antibiotic effectiveness. So the next time you have the option to buy a product with an antibiotic added, consider the old-fashioned version. 


Sometimes all you need is soap.

Moonlighting Enzymes: Supporting Keg Parties Everywhere

Our lab is situated in close proximity to a high concentration of fraternity houses, and as the fall term commences the warm autumn atmosphere reeks of fresh loose-leaf, cheap beer, and hang-over bacon. Any truly savvy and overindulgent undergrad should be writing a little thank-you note to one of the crucial factors that enables this year-opening bash: the enzyme alcohol dehydrogenase (ADH). This little microbrew miracle has been helping humans indulge in handcrafted libations for around nine thousand years and is perfectly suited to this function. Or is it? A recent study questions the evolutionary history of ADH and suggests that it might have more important things to do than aid in our enjoyment of an evening martini.

            Given that enzymes are integral components of so much of our body chemistry, it can be hard to remember that they haven’t always been there. Just like everything else, they had to come, to evolve, from somewhere. ADH is no exception. These days, it’s studied as the first enzyme in the metabolic pathway helping us to break down ingested alcohol (ethanol). Without it, we couldn’t efficiently clear alcohol from our systems and it could accumulate to lethal levels over the course of a lively cocktail party. In fact, one form of the enzyme acts in our stomachs, starting to break down the intoxicating molecule before it ever reaches our bloodstream. But where and how did ADH evolve? Were the molecules in our prehistoric ancestors preparing for a time when our species would start breaking out the booze?

            The rules of enzyme evolution parallel those of organism evolution: an ecological (or functional) niche has to exist before the process of natural selection can result in something filling that niche. In the case of ADH this means the availability of ingestible alcohol. If there is no dietary alcohol to break down, there should be no enzyme evolution specifically to break it down. By far, the greatest source of alcohol available for animal consumption is fermentation of fruit sugars, so the appearance of fruit in the prehistoric world should predate that of ADH. This is assuming that ADH really did evolve to break down alcohol. It is this assumption that is being questioned by a group of Mexican scientists.

            In a sobering study examining the functional capacities and evolutionary histories of a range of ADH enzymes, the real purpose of human ADH was called into question. In actuality, humans have several forms of ADH from three very different classes of enzyme using very different means to achieve ethanol transformation. Only one class of ADH is considered to be important for human alcohol breakdown. As it turns out, this class of ADH evolved before the appearance of fruit-bearing plants. That is to say that the type of enzyme we use today to reduce both inhibitions and alcohol toxicity showed up before there was any alcohol to indulge in.

If ADH’s only job was to enable aperitifs, it would not have evolved to do so before the functional niche existed. Instead, it is more likely that ADH has a somewhat serendipitous ability to metabolize the alcohol we drink, and we exploit that ability. In fact, all of the human ADHs are better at catalyzing transformation of different (non-liquor-related) compounds in our bodies, such as bile acids and serotonin. It seems that the party-pleasing activity human ADH is best known for is a handy trick this enzyme would file under “other abilities.”

How'd he DO that? Real Chemical Magicians

Enzymes 101: Peaking into the black box

            The term “enzyme” has a pretty specific meaning to people who define themselves by such terms as “enzymologists” (go figure). To most people, however, it’s a less well-understood term. For many of the folks I know, the term “enzyme” can be virtually interchangeable with “protein,” or perhaps only recognized in the context of specific and dubious dietary supplements. For the purposes of this and further discussions, I’d like to talk about enzymes as proteins or nucleic acid that act as a catalyst to speed along chemical reactions. That’s what enzymes are and what they do, but how on earth do they do it? In this post we’ll use broad strokes to discuss some of the general strategies enzymes use to put eons of biochemical evolution to work.

            It can be a bit hard to really appreciate, and I mean fully appreciate, what enzymes do for us, and how difficult this task is chemically. When I think of the power of enzyme chemistry it always brings me back to early organic chemistry classes. As a bright-eyed, eager young undergrad I dutifully learned the rules of traditional organic chemistry: what bonds can be broken by what chemicals under what impossibly harsh boiling acidic conditions. We learned that by the natural laws of energy, matter, and all that is chemically holy there are just certain reactions that wouldn’t happen at room temperature during the length of an average PhD. How then, I would ask, could these types of reactions be magically popping up in textbooks looking perfectly and innocently plausible? The professor’s answer? Enzymes.

            Upon receiving such a disyllabic and seemingly self-evident answer I’m sure that I nodded my head knowingly, pretending that I fully understood how the biochemical black box could perform chemical black magic. Since then I’ve made a few strides toward actually understanding the mechanisms behind these baffling enzymatic chemoacrobatics, and continue to be amazed by the underlying simplicity and elegance in complicated enzyme systems. As an introduction to the broad concepts of enzyme mechanisms I’ll talk about the very basic catalytic strategies they use, and leave more detailed mechanisms for a later date. So let’s talk about shape, strain, orientation sequestration, and transformation. Lets talk about enzymes.

            One of the ways that enzymes are able to shift chemical reaction rates into high gear is simply by bringing the necessary components, or substrates, together. Understandably, this seems trivial on the surface but practically it is both very important and very effective. Being macromolecules (proteins or nucleic acid), enzymes have lots of different chemical groups: hydroxyl groups, aromatic groups, amine groups, etc. These have been positioned during evolution of specific enzymes to be in just the right places to interact very specifically with their substrate molecules. The “lock and key” analogy is used a lot in explaining these interactions, and is as apt an analogy as any. Basically the enzyme is able to bind very specific compounds, while completely ignoring others. By doing this for more than one substrate, they are able to bring two or more reacting molecules into very close proximity and hold them there, allowing them to interact much more efficiently. Additionally, enzymes don’t bind their substrates in just any old way, but in a precise orientation, effectively lining up slot A with tab B. This very simple enzyme function is kind of like a good match maker, taking the chance out of two compatible things coming together and setting the mood just right for the chemistry to happen.

            Another power that enzymes have is a consequence of their size; enzymes are huge compared to the small molecules involved in many of the reactions they catalyze. This means that they can completely surround these small molecules, and in so doing can control the immediate environment in which the reaction is taking place. Practically this can mean separating the substrates from interfering water molecules or creating pockets of positive or negative charge in specific places. This gives the substrate molecule(s) a custom-built productivity space where all of the elements are designed to help the chemistry along and exclude interfering substances.

            The general mechanisms we’ve talked about so far involve concepts that I like to think of in terms of “tweaking the chemical circumstances,” but enzymes can play a much more “active” role in chemical reactions as well. One way that enzymes can shove a reaction in the right direction is an offshoot of the “lock and key” tight interaction concept mentioned above. Some enzymes bind their substrates in such a way that they actually bend, twist, or otherwise strain their shape. This changes the energy state of the molecule and helps to push the chemical reaction to its conclusion. Alternatively, the enzymes themselves can actually react chemically with their substrates, forming a covalent bond between enzyme and substrate. This bond is broken by the end of the reaction cycle to release the product and regenerate the enzyme. This latter mechanism is often referred to as providing an alternate path for the chemical reaction to go through, effectively using the substrate-enzyme reaction as a handy detour to avoid more difficult chemistry.

            The concepts of catalytic mechanism explored above are incredibly general, and the specifics that happen in each individual system can be complex enough to fill the contents of a research career (or several). With all of the complicated and truly challenging chemistry that’s going on in our bodies every day, however, I think it’s inspiring that such simple and intuitive concepts are at the root of such complex mechanisms. It’s amazing what we can understand about our own chemistry if we choose to start looking inside the black box.