I sort of buried my announcement at the end of my last post and, if you are like me, once you hit the word "references," you check out.
I'm getting married and will be away for two weeks! I'll be back to posting in the middle of May.
Happy Spring!
Saturday, April 30, 2011
Tuesday, April 26, 2011
Gene and Protein Names (Biochemistry)
I studied chemistry in college. Not biochemistry, not biology, not anatomy; chemistry. My first job out of college was as a research technician in a biological research lab. The techniques, the discussions, and the research topics were 100% foreign to me.
“Well, MAPK signaling isn’t turned on in that situation.”
“Did you get the knockout mice yet? Are they viable?”
“Run a northern and tell me about RNA levels.”
What?
I quickly figured out some of this stuff, but the most troublesome topic for me was the convention of naming proteins. In case you didn’t know, our cells are full of proteins, which number into the thousands. They all need names so scientists and can share information, but, much like naming children or pets, it can be your basic free-for-all.
In chemistry, most things have a scientific name: atom, proton, quark, neutron, etc. New molecules even have a universal convention for naming them. Granted, the names are huge and bulky, but they are not ambiguous! Often, they are shortened to something else, but there is always the universal name and, sometimes, a CAS number to fully identify the chemical.
In biology, everything is named with letters and numbers, sometimes arbitrarily picked (or not), that either stand for longer names (or not). For example, I worked heavily in graduate school on p53 (Cancerous Mutational Problems post). Why is it called that? Because when scientists were trying to isolate a protein known as Large T antigen (this is only one of its names, by the way), another protein kept hanging around. Scientists didn’t know anything about this other protein except that its size was 53 kilodaltons (don’t worry about what that means for this post) so…
P = protein
53 = weight
Protein name = p53
Genius.
I hated sitting through talks while I was a technician. The names of some proteins were ridiculous. I often wondered why they couldn’t name them all logical things. Some proteins really do have logical names!
Example 1: phosphofructokinase
Function: This protein is an enzyme that adds a phosphate group to phosphofructose*.
Phosphofructo = phosphofructose
Kinase = adds a phosphate group to
Name = phosphofructokinase
Now that makes sense!
Example 2: E6AP
Function: This is a protein known to bind to another protein called E6.
E6AP = E6 Associating Protein
Logical!
Other names are just completely batty. My personal favorite is when I started reading about RING proteins. You think it sounds nice, maybe the proteins form a ring-like structure, maybe they are involved in making rings of something else… maybe?? No. RING is an acronym for Really Interesting New Gene. I loved when people asked me in talks what RING meant.
One name stands out in my mind as one of the most ridiculous, somewhat contentious, but still high profile proteins in science: Sonic Hedgehog. Bob Riddle of the Harvard Medical School named this fly developmental gene in 1993. Luckily, Sega isn’t too threatened by it. However, Velcro and Pokemon don’t want genes or proteins named after them and both have threatened legal action unless their names were removed from recently characterized genes. Not all advertising is good advertising, apparently.
Guidelines do exist for unambiguously naming both genes and proteins. However, just as with chemicals, people tend to use the colloquial names because the correct nomenclature hasn’t caught on yet or it’s simply easier. It is always fun to read “protein A, more commonly known as protein ABC” in literature. I once spent several days looking for two papers that I was told existed: structural information about the CH1 and CH3 domains of a protein known as p300 (or CBP, take your pick). I could not find them and was convinced everyone was delusional. It was later that someone mentioned they were published under the names TAZ1 and TAZ2.
Really? Science.
CAS Number – Numbers assigned by the Chemical Abstracts Service to every chemical reported in scientific literature. Universal names can be long and difficult so some names are shortened. Different companies will list chemicals under different names, but CAS numbers will always be listed with the chemical so you can be 100% certain of its identity.
Kilodalton – Unit of protein weight. Proteins can be described by how much they weigh, which is useful information to give scientists an idea how large or small a particular protein is. The more amino acids a protein has, the heavier it is so the more kilodaltons it has.
1 Dalton (Da) = 1.66 X 10-24 g
1 kDa = 1.66 x 10-21 g
One protein weighing 53 kDa = 8.8 X 10-20 g (Really small!)
*Name was shortened from fructose-6-phosphate so people could more easily understand the name
References
Evans et al. “Mutations in the active site of Escherichia coli phosphofructokinase.” Nature (1987) 327, pgs 437 – 439.
Huibregtse et al. “Cloning and expression of the cDNA for E6-AP, a protein that mediates the interaction of the human papillomavirus E6 oncoprotein with p53” Mol. Cell Biol. (1993) 13(2) pgs 775 – 784.
Simonite, T. “Pokemon blocks gene name” Nature (2005) 438 pg 897
Maclean, K. “Humor of gene names lost in translation to patients.” Nature (2006) 439 pg 266
HUGO Gene Nomenclature Committee: www.genenames.org
De Guzman et al. “Solution structure of the TAZ2 (CH3) Domain of the Transcriptional Adaptor Protein p300” J Mol Biol. (2000) 303 pgs 243 – 253
De Guzman et al. “CBP/p300 TAZ1 Domain Forms a Structured Scaffold for Ligand Binding” Biochemistry (2005) 44 pgs 490 – 497
NOTE: Amedeo needs to take a short break because she is getting married. I'll return in the middle of May!
Monday, April 18, 2011
Crushing Defeat, aka The Peer Review Process (Lab Life)
I would imagine that this is how a successful actor feels the morning following his big movie’s opening night. He is sitting in his home enjoying some coffee in the predawn light and thinking back over the previous evening’s festivities. Everyone had come to see his performance, which he knew he gave his all. He had, after all, spent countless hours preparing for the part, studying the character’s personality, and really trying to embed himself in the role. He also knows his performance would not have been possible without the insight and input from his gracious director. The film, of which they were truly proud, was a labor of love between the two of them. The actor smiles to himself, quietly remembering to buy the director a gift and thank him graciously for the opportunity.
It is at this moment and with this joy that his picks up the morning paper. Splattered across each and every page are scathing reviews of his performance. Reviewers are exclaiming his inability to act, their disbelief that he was even cast, and ruminations of the director’s sanity.
Yeah. It’s kind of like that.
For the past five years, I dedicated a tremendous amount of time, effort, and brain power to a project that most people felt would never work. (Forgive me; I love a challenge.) I had other projects in lab, most of them fruitful, but this five year whopper was my baby. It was the project on which I cut my scientific teeth. It was the first project I touched on Day One and what I was still working on come Day Seventhousandeighthundredandeightytwo, when I was packing up my stuff to move on to my post doc lab. In fact, I worked it on for several months at night after starting in my new lab.
A few weeks ago, we finally pulled all the data together, wrapped up the story in a nice article, and sent it off to a journal to be considered for publication. The process is rather straightforward. The paper is first read by an editor who decides if the work is decent and lies within the scope of the journal. If so, typically three or four reviewers are chosen to read the paper. Reviewers are scientists who work in a similar field as the paper’s main topic or with the paper’s primary techniques. After several weeks, the authors of the paper and the journal’s editors receive the reviewers’ comments. Several things can happen after this: 1. The paper is published with minor editing, 2. The paper can be resubmitted following major editing, or 3. The paper is just flat out rejected. Through the editing process, the authors have the ability to respond to reviewers comments directly and incorporate their thoughts and criticisms into the work.
As an author, you have no idea who the reviewers are to your paper. However, the reviewers know exactly who the authors are because the names are listed with the paper. This leads to some …unfairness… at times, but I’m going to let that go for now. The peer review process in its most idealized form allows for outside reviewers to consider the work and highlight the flaws in protocols, conclusions, and theories posited by the authors. It is a way for the field of science to self-police and minimize the amount of errors published in the name of scientific advancement.
Today, my paper, which has several authors on it aside from me (but I am first author), was sent back with reviewers comments. My baby is bruised and bleeding. I don’t want to take the comments so personally or feel like I’m going to throw up when I see my hard work demolished, but it is hard to take. This isn’t even my 1st first author paper I’ve published (not my first rodeo!), but it is work that I fought to make publishable and to which I dedicated a tremendous amount of myself. I feel very emotionally invested in it.
I think a stiff drink is in order this evening before I face the long task of responding to each comment and rewriting the paper.
In an effort to laugh it all off (or at least not cry myself to sleep tonight), I gathered the following from Ph.D. Comics (www.phdcomics.com). This website is fantastic for anyone in graduate school and I highly recommend checking out their comic archives.
References
From www.phdcomics.com:
Wednesday, April 13, 2011
The Atom (Chemistry, Biochemistry)
I suppose this would be a good time to admit it: I read and re-read my posts after I put them up. It’s not unusual for me to read a post (for the fourth or fifth time) and edit it. My brain works faster than my fingers and I’ve been known to forget words, miss a letter, or sometimes I get angry that the color scheme I have going on is not exactly the way I want it. (If you haven’t noticed yet, all figures are colored green, all post names are colored purple, defined-at-the-end words are blue, and all parenthetical thoughts are gray). I also read them for content. I spend a tremendous amount of time pretending to know nothing about chemistry and reading my posts as an outsider. I try to decide if everything makes sense. Let me know how I’m doing, dear readers!
This brings me to today’s topic. I don’t think I did a good job explaining the difference between the left and right panels of this figure (Figure 7.4, Protein Folding). To more thoroughly explain, I’m going to first talk about an atom. Information in this post will be helpful towards Protein Folding, Spanish Influenza, Part 3, and Fun with Radioactivity.
Around 400 B.C., some Greeks were supporting the idea that matter was made up of small, indivisible particles, which Democritus called atomos. Other Greeks felt that matter was continuous. Unfortunately, this controversy was as far as they got because scientists then busied themselves with alchemy for about 2000 years. But, the Greeks did leave us with our present name for particles: atoms.
So what does an atom look like?
It’s made up of three smaller particles: protons, neutrons, and electrons. Yeah, yeah – you’re thinking that you knew that. Okay, but I’m going to tell you more, anyway.
Protons and neutrons hang out in the nucleus. They are of similar size and mass, however protons have a charge of +1 and a neutron, which is aptly named, is neutral. Nuclei are pretty fascinating. Think about what happens when you have two magnets. Each has a positive pole and a negative pole. A positive pole attracts a negative pole. But, if you try to put two positive poles together, you feel a very strong repulsion. How does an atomic nucleus handle all those positive charges together without completely exploding? Ah, a topic for another day.
Electrons, which have a charge of -1, balance out the positive charge in the nucleus to make a neutral atom. The number of protons in the nucleus is called the atomic number and defines the atom. 6 protons in your nucleus? You are carbon. I don’t care how many electrons or neutrons you have; 6 protons always always always means you are a carbon atom.
The number of neutrons can vary, giving rise to isotopes. For example, some carbon atoms have 6 proton and 6 neutrons (called 12C or carbon twelve); others have 6 protons and 8 neutrons (called 14C). Carbon twelve and carbon fourteen are isotopes of carbon. 32P is a particular isotope of phosphorous, which also happens to be radioactive (aha!). Figure 13.1 shows you how chemists thoroughly describe different atoms so others know exactly what they are talking about.
The electrons are “floating” around the outside of the nucleus. Some people think that “floating” means they are just statically there, like stars. Others think that electrons orbit the nucleus like planets around the sun. Neither option is correct. Electrons are zipping around the nucleus – yes – but we don’t know exactly where. All scientists really know is probably where the electrons are. Isn’t that interesting? In reality, an electron could be anywhere*. It could be 75 feet away from the nucleus. Of course that is not probable, but according to the math, it is possible. Weird. (I’ll explain the math another day. It’s really complicated and involves a crazy theory called “particle in a box.” Really, just say “particle in a box” to introductory chemistry students and watch them dissolve into tears. It’s a kinda funny.)
The majority of atomic size is due to its electrons. If we are thinking in terms of a stadium, the nucleus takes up about as much room as a dime placed at midfield and the remaining space is due to electrons flying about.
When pictures like those in Figure 7.4 are shown, they depict an atom like a sphere. If we only know probably where an electron is, how can we definitely say how big an atom is?
Let’s think of a hydrogen atom, which is the simplest case. Hydrogen has one proton in its nucleus and one electron spinning about it. According to the math, the probability of the electron lying close to the nucleus is high and gets progressively weaker as you move away (Figure 13.2). Imagine drawing a sphere around the nucleus and saying “There is a 90% chance that the electron falls within this sphere and a 10% chance that it does not.” The 90% sphere that you just drew becomes the boundary of the atom. This same 90% line was drawn around all atoms allowing us to define atomic radii for each and every one. Each element’s atom has a slightly different size and atoms will follow certain trends. But, as I keep saying: another post, another post.
So let’s get back to Figure 7.4. What the hell is going on here?
LEFT panel: Back in the Protein Folding post, I talked about how all amino acids looked exactly the same except for the R group. I also said that all amino acids hook together in the exact same way. When you have a tertiary protein structure, you can see exactly how all those amino acids fold up on each other. Imagine you could draw a line from the very first NH3 group, through the first carbon, to the next carbon, to the next nitrogen, and onwards. (Ignore the R groups for this panel.) This is called the protein backbone (Figure 13.3). In three dimensions, your line would twist and travel all over the molecule. This is exactly what is being shown in the left panel. PyMOL (a quite lovely program created by Warren DeLano, who died last year. Following his death, PyMOL went corporate. Boo.) will trace this line for you. When you reach alpha helices, the program will put in nice loopy-loops and when you hit beta strand, it will draw thick arrows. If it is just random coil, then the line will follow the N-C-C-N bonds all over in their random ways.
In essence, the left panel is just showing the outline of the protein. Where, in general, the protein backbone is going and highlighting the secondary structure so it is easily seen.
RIGHT panel: This is the exact same protein structure, exact same everything, except that each atom in the molecule (backbone atoms and R group atoms included!) is now represented as a 90% probability sphere (as defined above). It’s called space-filling because it is showing you how all the atoms sit together. It’s also showing you what the surface of a protein would look like. Structural biologists can now see filled space from empty holes in the protein. (Some proteins have tunnels in them!) I’m sure you can also appreciate that it is difficult to decipher secondary structure from the right panel. For these reasons, structural biologists flip back and forth between these two representations of the molecule depending on what they are trying to show.
In the Protein Folding post, I said to think of the left panel as a tree without its leaves (it is easy to see how the branches twist and turn) but the right panel is showing how the tree looks when covered with leaves (giving the tree a “surface” and giving the tree a “fullness”).
Atoms: smallest form of an element that still retains the chemical properties of that element
Proton: elementary particle, positively charged, found in the nucleus of atoms
Neutrons: elementary particle, neutral in charge, found in the nucleus of atoms
Electrons: elementary particle, much smaller than protons or neutrons, negatively charged, found circling atomic nuclei
Isotopes: Atoms of the same element, but that have differing numbers of neutrons in their nuclei
Protein Backbone: N-C-C-N-C-C-N-etc bonds within a protein molecule
* - According to the math, there are nodes of probability - places where there is no chance of an electron being. But since this isn't a post about Schrodinger, I'm going to skip right over that complexity for now.
References
Zumdahl, Steven S. “Chemical Principles, 4th Edition” (2002) Houghton Mifflin Company, Boston, MA.
Alberts et al. “Molecular Biology of the Cell, 4th Edition.” Garland Science, New York, New York. (2002).
Thursday, April 7, 2011
Fun with Radioactivity (Lab Life)
This past week in lab will be known as “The week I used a lot of radioactivity.” I didn’t enjoy it. I had to get dolled up in my lab coat (yup, found it) bearing my dosimeter, another dosimeter on my right index finger, two layers of gloves, and some sweet yellow safety glasses. Not part of the official outfit, but necessary nonetheless, is having long hair tied up, closed toe shoes, and long pants. During an unexpectedly warm day a few weeks ago, I had this attire on and our lab was a sweltering 90 degrees. I was an unhappy post-doc.
So, why in the world was I playing with radioactivity all week?
Scientists use it quite often, actually. Aside from the safety headaches, radioactivity is incredibly useful to understanding exactly what is going on with our microscopic little proteins. To explain how, I’m going to use a technique that I often use with my chemistry tutorees or younger labmates: You have to crawl inside the experiment.
Let’s go.
Experiment: You have two tubes. One tube contains water. The other has a protein floating around in it. Which tube is which?
More Useful Information: The protein is a particular kind of protein known as a kinase, which is an enzyme. Enzymes are very specialized and typically just do one type of job. Think of a factory where cupcakes are being made (is the analogy getting old yet?): one person makes the batter, one person fills the cups, one person bakes them, one person ices them, etc… Each person in that line is doing a very specific job. In our cells, enzymes are much like those cupcake assembly line workers. Each enzyme has its own small task in the great scheme of a larger process. There are thousands of jobs to do so there are a lot of enzymes. Kinases are a particular class of enzyme. The job of all kinases is to move a phosphate group from ATP to another protein (target protein) (Figure 12.1).
So… good. What do we do now?
Plan 1: Let’s add a little ATP and target protein to each tube. The tube with the kinase will then have target proteins covered with phosphate groups (Figure 12.2).
Problems with Plan 1: How can we tell if the target protein is covered with phosphate groups? It’s not like we see the target protein.
Solutions to Problems: While there are ways that scientists can “see” proteins, the easiest experiment to do here is to use a little radioactivity*. 32P to be exact.
What is 32P? Phosphourous is element 15 on the periodic table. A particular type of phosphorous has 17 neutrons and 15 protons (15+17 = 32) in its nucleus. This nucleus is unstable and will decay very predictably. We can purchase ATP with a 32P incorporated such that when the kinase moves the phosphate group, it will take the 32P (Figure 12.3).
I’d like to take a moment and stress here that the phosphorous in our bodies is not radioactive. Much like gloves in different colors, elements can be found in different forms. Some of the forms are radioactive, but most are not. Since ATP contains phosphorous anyway, scientists can switch out a nonradioactive phosphorous for a radioactive one and sell the crazy molecule to post-docs like me to do experiments.
Plan 2: Add a little radioactive ATP and target protein to each tube. In the tube with kinase, the target protein will become radioactive. The target protein in the water tube will not be radioactive.
Problems with Plan 2: How do we separate the target protein from everything else? How can we tell if it is radioactive?
Solutions to Problems: Special paper exists that will bind protein molecules but not ATP. Special machines will tell you if that paper is radioactive or not.
Excellent! So off we go to do our experiment and the results are in Figure 12.4.
Which tube had the kinase and which tube had water?
Think about the caption of Figure 12.4, as well. Is what is bound to the paper makes sense given everything you’ve read above? Have you successfully “crawled inside the experiment?”
Experiments like this are done very often by scientists. Obviously they are asking more interesting questions than which tube holds the kinase, but the basic experimental set up is what I outlined above. These are the types of experiments I was doing this week. Unfortunately, I think that next week will be called “The week I used even more radioactivity.”
Fun Facts that I couldn’t fit in the post:
1. I’m sure most people know that radioactivity is unhealthy. One reason why is because the energy radioactive decay emits can cause mutations in our DNA. Ooooh… think about the last post (Cancerous Mutational Problems).
2. All work with 32P, which is known as a beta emitter, must be done behind a plexi-glass shield. It can be super awkward wrapping your arms around the shields to do your experiments.
3. Radioactive ATP comes from Perkin-Elmer in little containers that block the radioactive decay. They are quite cute.
4. Radioactive ATP is colored lime green. (I assume so that you can see it if you spill some.)
5. Scientists wear dosimeters so the powers that be can know how much radioactivity we are being exposed to. Using proper shielding should result is very little exposure, but they check the dosimeters to be sure we are working safely. I had a friend who hung his coat (plus dosimeter) by the radioactive waste one day. When checked, his dosimeter was off the charts. Yeah, they were certain he had cancer until he explained.
6. Purchasing radioactivity is incredibly regulated. 32P represents only one of hundreds of different products and one of several different kinds of available radioactive elements for laboratory use. Some common other ones: 3H, 35S, and 125I.
7. Scientists follow very strict guidelines on radioactive waste disposal. Most is safely stored until a large proportion of it has decayed. We certainly do not pour it down the sink or drop it in the trashcan when we’re finished!
* Any scientists reading this are probably laughing. I realize this is like using a hammer a push in a thumbtack, but I’m using a simple and straightforward experiment to illustrate a point. Go with it. If this was real life, I’d use Bradford Reagent and know in five seconds. But this post isn’t about Bradford Reagent. Maybe I’ll do that one another day!
Dosimeter: a small plastic device (~ 2 inch square) that records how much radiation one has been exposed to while working with radioactivity.
Enzyme: a protein that catalyzes specific reactions
Kinase: a specific class of enzymes that transfer phosphate groups from ATP to target proteins
Target protein: the protein which receives a phosphate group in a reaction catalyzed by a kinase
Bradford Reagent: a red dye that turns blue in the presence of protein
REFERENCES
Me, myself, and I.
Perkin-Elmer: www.perkinelmer.com
Sunday, April 3, 2011
Cancerous Mutational Problems (Biochemistry)
Willy Wonka had all kinds of treasured secrets in his factory. The list includes, but is not limited to: a chocolate waterfall, orange skinned/green haired midgets, everlasting gobstoppers, and geese that laid eggs full of the goodness that is both chocolate and gold. Harboring these kinds of objects also lent itself to a great deal of paranoia on Wonka’s part. Arthur Slugworth was lurking outside the walls, desperate to get inside and steal the secrets for his own candy factory. Wonka should have really invested in some sort of protection that wasn’t his own madness.
Luckily, our cells (which run something like factories or, you know, bakeries) do have a police force. Aside from viruses breaking in (see Spanish Influenza Parts 1 – 3 posts), cells are susceptible to all sorts of problems, such as DNA damage, misfolded proteins or improper protein function. Several proteins within our cells have the sole job to maintain order, trust, and well being to our cellular processes. If things go awry, they have orders to suspend operations (also referred to as cell cycle arrest) or simply destroy all evidence of the cell (known as apoptosis).
One of the key “policing” proteins is called p53. It hangs around in the cell with its ear to the phone, waiting for word of some problem to reach it. A base of DNA changes from an A to a T? p53 is called in. A cell is breaking through its checkpoints in growth? Someone get p53 on the phone. Hemoglobin’s latest shipment of oxygen did not come in? p53 will take care of it.
p53 is kind of a big deal.
After receiving information that problems are afoot, p53 goes into the nucleus and binds the DNA in very specific places. This binding of DNA leads to cell cycle arrest or apoptosis (for this post, it is not important how - just understand that it has to bind DNA properly to fulfill its function.)
These problems that arise in the cell are serious and need to be taken care of swiftly. Think back to the Central Dogma post. What would happen if an A was changed to a T in DNA? Well… if the A to T mutation was in an area that coded for a protein, the mutation would be passed on to the RNA and the wrong amino acid would be incorporated into the growing protein at the ribosome. It may not seem like a big deal that one amino acid is wrong in a string of 300 amino acids, but it can be a huge deal. Wrong amino acids in key places of proteins can lead to disease, the most recognizable one being cancer.
I’m now going to use p53 as an example to explain how a single amino acid change can have such deadly consequences.
Cells are constantly growing and dividing. A series of checkpoints exist to ensure that the cell is preparing itself appropriately to divide into two cells. This is called the cell cycle (Figure 11.1). It is comprised of four phases: G1 = growth, S = synthesis, G2 = growth, M = mitosis. In the growth phases, the cell is getting bigger so that when it splits in half, the two daughter cells are not incredibly small. The S phase is when the entire DNA molecule is faithfully copied so that two exact genomes exist in the cell. The M phase splits the cell down the middle and ensures that each new cell gets one complete copy of DNA. This entire process is highly regulated (isn’t everything in a cell?). If the cell does not go through all the steps correctly, it is p53’s job to stop that cell from dividing by either arresting its growth or simply killing it. Better to kill a poorly run cell than allow it to split into two poorly run cells.
Cancer is defined as unregulated cell growth. Cancerous cells (also called transformed cells) don’t give a crap about the cell cycle and all its checkpoints. They just want to grow, divide, keep moving and wreak havoc. Who cares if the DNA is copied well? Let’s just go. Oxygen hasn’t come along in awhile? Let’s just build a blood vessel here to carry oxygen to us! Who cares that this human’s body wouldn’t normally grow a blood vessel here? Go, go, go!
It doesn’t take a genius to realize that one of the first proteins knocked in cancer is p53. Remove the roadblock to growth and division quickly and early on. Imagine that one cell loses p53, then it divides into two cells. Those two new cells don’t have p53 and they each divide into two more cells without p53. On and on… it is exponential growth of cells without p53. Any cell without p53 can’t stop growing, can’t fix DNA problems, can’t do anything but g-r-o-w. These transformed cells are factories gone completely off the rails (bakeries selling dough or everlasting gobstoppers laced with poison!) and are difficult to stop. But, I’m sure most people appreciate the difficulty of stopping cancer growth.
So how do you knock out p53’s function? I told you earlier that in order for p53 to tell the cell to stop growth or just die, it needs to be able to bind DNA.
A landmark paper was published in 1994 by Nikola Pavletich’s laboratory. It was the tertiary structure of p53 bound to DNA. This work was able to show which amino acids in p53 were necessary for proper DNA binding. Just as fingers need to be placed correctly on a baseball to throw curves, fastballs, or sliders… (Go Phillies!), the correct amino acids of p53 must be in the correct places to grab DNA and hold on.
The paper went on to look at various amino acid mutations of p53 found in cancerous cells. Figure 11.2 is directly from their Science article. The p53 primary sequence lies along the X axis and the number of times a particular amino acid is mutated in cancer is represented by the vertical black bar. The larger the bar, the more often that particular amino acid is found mutated in cancer.
Before this paper, no one could exactly explain why the amino acid arginine was so essential at position 273. But like fingers gripping a baseball, this structure showed that arginine was necessary for p53 to grab the DNA properly. Another amino acid simply wouldn’t do. Imagine trying to throw a knuckleball without your index finger. It would be difficult or nearly impossible. Sure, you still have a hand, but if the hand’s job is to throw knuckleballs, it’s going to need that index finger in the correct place. A p53 without certain amino acids is crippled in its function in much the same way. It can’t grab hold of the DNA properly and thus it can’t tell the cell to stop growing. Without this protein working properly, a sick cell continues to grow, continues to divide, and can, in time, lead to a tumor.
How does a cell obtain a mutation in their p53 protein to begin with? It can happen at the level of DNA, RNA or protein. The ribosome could simply incorporate the wrong amino acid. It happens. Or maybe the RNA was not transcribed faithfully from the DNA. However, so many p53s are floating around the cell that one wrong one will quickly be compensated for by all the correct ones. Proteins are also degraded after a certain period of time and replaced with freshly made proteins. This turnover will also dilute out any small errors in transcription or translation.
However, DNA mutations are a bigger deal. In the previous two cases, the DNA was correct so going back to the beginning fixes the problem. But, what if the beginning is wrong to begin with? We pick up DNA mutations throughout our entire lives. A mutation in the p53 gene will be carried on to daughter cells, which will make incorrect p53. We also could have inherited a mutated copy of p53 from our parents. All our cells from the very beginning could have 50% of its p53 proteins wrong. It’s hard to fix mutated DNA or incorrect p53 molecules, but many scientists are working on that very type of research.
This topic will lead us down a fruitful road of learning about cancer, cancer therapies, new avenues of research, and so much more about p53. We’ll get there all in good time.
Cell cycle – the organized growth and division of cells
Cell cycle arrest – the pausing of the cell cycle while repairs are made inside the cell
Apoptosis – organized cell death; the process a cell undergoes when it needs to commit suicide
Transformed cells – term used to describe cells that have gained cancerous characteristics
REFERENCES
Dahl, Roald. “Charley and the Chocolate Factory.” (1964) Penguin Group, New York, New York.
Stuart, Mel. “Willy Wonka and the Chocolate Factory.” (1971)
Suryadinata, R. et al. “Control of cell cycle progression by phosphorylation of cyclin-dependent kinase (CDK) substrates.” (2010) Bioscience Reports 30, pgs 243 – 255.
Cho et al. “Crystal Structure of a p53 Tumor Suppressor-DNA Complex: Understanding Tumorigenic Mutations.” (1994) Science 265, pgs 346 – 355.
Subscribe to:
Posts (Atom)