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Q&A: Mass Spec Study Says Nicotine May Affect Cell Interactions More Broadly Than Thought

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Canon0005.jpgName: Edward Hawrot
Position: Associate dean for the program in biology, Brown University, 2007 to present
Background: Professor in pharmacology, Brown University, 2001 to present; chairman department of molecular pharmacology, physiology and biotechnology, Brown, 1996 to 2007; PhD, biochemistry, Harvard University, 1976

The alpha-7 nicotinic acetylcholine receptor is an established high-affinity alpha bungarotoxin-binding protein in mammalian brain associated with learning and memory. Some studies using immunolabeling and co-immunoprecipitation, yeast two-hybrids, and Western blotting have identified potential binding partners of the receptor.

Using a mass spectrometry-based approach, however, researchers at Brown University have performed what they said is the "first large-scale proteomics-based analysis of the alpha-7 nicotinic acetylcholine receptor interactome."

They identified 55 proteins in wild-type mice that were not present in knockout mice, many of which are novel proteomics candidates for interaction partners of the receptor. In addition, many are associated with multiple signaling pathways that may be implicated in alpha-7 function in the central nervous system.

The results suggest nicotine may affect cellular interactions in more ways than previously thought.

The work is described in a study in the current issue of the Journal of Proteome Research.

ProteoMonitor spoke with the corresponding author on the study, Edward Hawrot, this week about the research. Below is an edited version of the conversation.

Describe the work that you did.

The thrust of the work is to understand what nicotine does in the brain and what it interacts with in the brain.

There is a reasonably good basis of prior work on that in terms of identification of what's called a nicotinic receptor, but there's been very little done as to what other cellular proteins are interacting with that receptor.

Two things drew me to this work. One is the observation that nicotine has multiple effects in cognition and behavior. One of the reasons nicotine is addictive or people smoke is to sharpen their focus or enhance their ability to focus and increase some memory function.

One of the questions that is out there is: Can we somehow capture the beneficial effects of nicotine without involving the considerable negative effects?

In terms of central nervous system function and behavior and being a biochemist, my initial focus would be on what are the other proteins and signaling pathways in the cell that might be intersecting with the nicotinic acetylcholine receptor.

We know from the structure of the receptor, or at least from the homologs of this receptor, that there's a large loop of peptide sequence, this so-called M3, M4 loop — M3 and M4 refer to the two transmembrane helices. Between M3 and M4 there's a relatively long stretch of amino acids [and] based on the topology, the receptor must be on the inside of the cell interacting with the cytoplasm.

And in previous work, people mostly focused on sites within that sequence that could be phosphorylated through protein kinase A, protein kinase C, a number of enzymes that phosphorylate membrane proteins, the thinking being these phosphorylations oftentimes are regulatory in nature — they can dampen the function or alter the function.

That's where the most focus has been in the past. I wanted to use the proteomic approach to, rather than just study a particular kinase or enzyme, to open the door to examine as much as possible, all the proteins, or get a listing or read-out of all the proteins in the cell that might be interacting with this cytoplasmic loop of the nicotinic receptor.

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And this particular nicotinic receptor is called the alpha-7 nicotinic receptor and there are related gene products, but alpha-7 is the one that's considered most primitive, going furthest back in evolution. And unlike the newer genes that produce receptors that interact with nicotine, this one has a similar overall structure, but the receptor needs only one subunit that is assembled into a pentamer, so there are five of these alpha-7 subunits, and that makes a functional receptor.

And that's primitive. … The newer forms of the receptor require more than one subunit. There's been some evolution presumably to put in some specialization.

But the other reason I was drawn to [this alpha-7 receptor] is that in … brain tissue, this is the only protein that is recognized by a snake toxin called alpha-bungarotoxin.

Alpha-bungarotoxin was made famous, if you will, because it comes from the venom of a cobra-like snake in Taiwan and mainland China, and that snake, Bungarus multicinctus, has this venom that contains large amounts of this protein … which has high specificity for the neuromuscular form of the nicotinic receptor.

Is there a parallel between the way it acts and the way that nicotine acts?

It unfortunately gets a little complicated, but nicotine in most parts of the nervous system and in the muscles will initially act very much like acetylcholine, so it will activate the receptor and cause muscle contraction.

Nicotine used to be found in high amounts in insecticides. About 10 percent of the insecticides that are used in the world [still] contain nicotine.

You don't see as many cases now, but it was more common to see nicotine poisoning in children who accidentally ingested a cigarette, actually ate a cigarette. For a small child, two cigarettes approaches the lethal dose of nicotine, and it acts like acetylcholine and it causes the muscle to contract, but at high sustained concentration, it will just sort of sit there and immobilize the receptor and then you have paralysis.

With nicotine poisoning, eventually you can't breathe because your respiratory muscles are blocked.

So nicotine does act very similarly to acetylcholine and at high concentrations, it will sort of act like a toxin but at a molecular level, [there's a] different mechanism. It will initially stimulate, but then at sustained levels it will cause the receptor to stop functioning.

The toxin gave us an additional biochemical tool to help enrich for the receptor, so we use a toxin that is immobilized to beads to then fish out the receptor from a general mixture of all the proteins in a cell in the brain.

So we grind up the mouse brain tissue, then incubate this mixture of all the proteins with beads to which alpha-bungarotoxin has been immobilized from the surface so that the receptors can bind to the beads, and any proteins that are associated with the receptors would similarly be tagging along.

Then we can let the beads settle, or we centrifuge down the beads, wash them … and use this procedure to enrich greatly for the receptor proteins out of the entire mixture.

It's sort of like fishing … using the alpha-bungarotoxin as a bait and a hook to pull out the proteins of interest.

Other folks who do similar studies often use antibodies, but … in the case of nicotinic receptors, the antibody approach doesn't work as well because most of the antibodies are not that amenable to this kind of approach. They cross-react with lots of other proteins, so that's perhaps one of the reasons this hasn't been done before.

Is this alpha-bungarotoxin workflow something that's translatable to other proteomics research?

The way that it can translate is that we know a lot about the sequence of amino acids that are the target for bungarotoxin binding, and so our lab and a number of other labs have been able to introduce that sequence into other proteins, so in that sense it can be translated into other systems.

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Some people call this alpha-bungarotoxin binding site BBS. We've sometimes referred to it as a pharmatope because we've been able to introduce it into regions of a receptor where not only the bungarotoxin bind but also causes a pharmacological effect or inhibits the protein, so it's not only binding.

It could be extended to other proteins. It would have to be done through a recombinant expression system or knock-in. It's a fair amount of work, but it can be done, and then you can use this as a tag or a hook to pull out those proteins.

Were there other approaches that would be useful for other researchers?

The other big boost to this work was the availability of the knockout mouse that had the alpha-7 receptor protein removed. … As far as I know, no one else has used [the mouse] in this particular way to compare and contrast wild-type mice and knockout mice to facilitate the purification of the wild-type alpha-7 receptor.

The availability of that mouse has made it much more straightforward because we could validate our workflow by saying 'If our beads are pulling out the bungarotoxin binding protein and associated proteins, then if we apply the same strategy to the tissue derived from a knockout mouse, we should see much less protein.'

And we did.

When you started this work, what was generally known about the effects of nicotine on proteins, and how did you want to build upon this knowledge?

The traditional thinking is that nicotine is binding to the receptor as if it were a so-called agonist, like an acetylcholine type of molecule, but it's also known that nicotine is one of these small molecules that can permeate inside cells much more so than acetylcholine.

There's always been some potential that the effects of nicotine are due to these non-receptor mediated effects going directly [and] permeating into the cells and perhaps binding to other proteins in the cell.

This particular receptor, the alpha-7, is quite a bit of a puzzle. It's quite primitive and it goes back far in evolution and it's been very difficult to determine the exact role of this receptor in the brain because in many cases it does not act like the muscle type receptor where you get a simple, 'Yeah, it's acetylcholine' and the receptor then undergoes conformational change and an actual pore within the protein itself opens up and allows ions to go in.

That's been extremely difficult to demonstrate with the alpha-7 receptor, and there's always been this kind of background thinking that maybe it's doing something else. … It's not as simple as opening up a channel and that's all there is to it.

Maybe the conformational change is important for interaction with some other protein inside the cell or in the membrane, so that's one of the reasons we were particularly interested in this alpha-7.

More recently, in the last year or so, there have been these genome-wide association studies that have indicated that in the human genome, there's an area of so-called microdeletion that's been identified in some patients with schizophrenia, and also with generalized epilepsy.

There are like seven genes missing in this deletion and the alpha-7 receptor gene is one of them. There's also been a longstanding observation that people with schizophrenia tend to smoke a lot, so there's been this view without a lot of deep scientific investigation that schizophrenics are maybe self-medicating by smoking.

Nicotine binds to a couple of other receptors in brain — alpha-7 is not the only one — but the properties of the alpha-7 receptor in terms of its binding characteristic have been suggested to co-mediate the morning craving that occurs for a cigarette.

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The thought is that when you're smoking, your receptors are becoming desensitized and this alpha-7 receptor has been suggested to desensitize very quickly, so overnight while you're sleeping, alpha-7 could recover and now it's available for binding nicotine and some of the craving for a cigarette first thing in the morning could be due to involvement of the alpha-7 receptor.

You categorized these 55 novel proteins that you identified into five groups. Was there any category that is particularly important?

The one that's most intriguing are the proteins that involve the G-protein family in signal transduction. In signal transduction, the interest is that this receptor could be affecting signaling mechanisms that are normally mediated by other proteins in other receptors.

Five out of the 55 were these so-called guanine nucleotide-binding proteins and they're typically associated with a functionally and structurally very different group of receptors called the G-protein coupled receptors, which as a group are very similar to the protein rhodopsin in your eye … and they're also transmembrane receptors, but they have a completely different mechanism. Upon activation they react with these guanine nucleotide-binding proteins.

That's really a signature for those particular proteins and receptors … so the fact that these G-proteins were pulled down in our workflow and identified as being associated with the alpha-7 receptor is quite novel and could suggest the alpha-7 system has some kind of cross-wiring interaction with G-protein coupled receptors.

The interest in the G-protein coupled receptor family is based on the fact that 40 percent of the drugs that are used today target G-protein coupled receptors and there are a whole bunch of receptors that drug companies are very interested in pursuing as potential new drug targets.

Your study found 26 proteins out of the 55 that overlap with about 700 proteins that were identified by an earlier study. What's the significance of this overlap?

The significance … is that these post-synaptic density proteins are thought to involve all the participants in synaptic transmission in the brain, so the fact that we had an overlap suggests that at least in some places in the brain, the alpha-7 receptor is likely to be involved in synaptic transmission or modifying synaptic transmission.

And this fits into the schizophrenia thinking. The thought is that with schizophrenia there are some disruptions in the circuitry in the brain, some of the synapses don't seem to be functioning appropriately.

So the main conclusion was that alpha-7 is involved in some synaptic function and then the second, less likely [conclusion] but still in the realm of possibility, is that the … overlap of the two sets [of proteins] might suggest that alpha-7 in some cases is found in the same synapse as the glutamate receptors, and the glutamate receptor is the main receptor that we use in the brain for long-term memory storage.

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