#EB2013: Trashing the cystic fibrosis protein

Last year, I turned 30. It’s one of those numbers that often causes humans to pause and think about what we want to accomplish in the next decade of our lives – run a(nother) marathon, travel the world, land an awesome job… But how many of give much consideration to the question of if we’ll be around to celebrate that next decade? For patients with cystic fibrosis, an inherited disease that affects the lungs and other organs, that if is a big one – the median survival age for cystic fibrosis patients is 38.

Cystic fibrosis is an incredible example of a truly molecular disease. It’s caused by mutation of one gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), a protein that helps maintain salt homeostasis in epithelial cells. Epithelial cells line surfaces and cavities of our bodies, things like the airway, sweat glands, and ducts in the liver and pancreas; these cells stick close together to create barriers between different environments and maintain a careful balance of fluid and electrolyte shuttling between these environments. CFTR transports chloride and other anions into mucus lining the epithelium, and sodium and then water follow to maintain normal, fairly fluid mucus. If CFTR is dysfunctional, the balance breaks down; thick mucus builds up in passages of the airway and digestive system, which become blocked. The obstructions alone cause serious respiratory and gastrointestinal problems, but the thick mucus in the airway also creates a cozy home for bacteria, setting the stage for respiratory infections. The mucus and associated complications significantly impact quality of life, but it’s the infections that typically kill.

Schematic of CFTR structure from CFTR.Info

CFTR is a massive and complex protein, that must be properly folded, embedded in a membrane, and shuttled to the cell surface. The fully functional, or mature, form weighs in at almost 190,000 Daltons (g/mol for the chemists out there ), which is more than 3 times the size of a typical protein. It’s initially expressed as a smaller precursor protein (~135,000 Daltons), but as CFTR passes quality control checkpoints, it’s decorated with sugar chains that tell cellular machinery that this CFTR protein is OK and ready for the next step of the process. With normal CFTR, only about 30% of CFTR that’s expressed actually transits the checkpoints successfully. The remaining protein is trashed without ever having done the job it was made to do. Just as sugars flag proteins that have passed quality control, ubiquitin commonly flags proteins that need to be degraded. Ubiquitin is a small regulatory protein that can be attached to a specific group (lysine) on other proteins. Ubiquitin can be conjugated to itself to create poly-ubiquitin chains, which target the proteins to the proteasome, a large multi-protein cylinder that cuts proteins into smaller pieces, or to the lysosome, an acidic intracellular sack filled with degradative proteins; in both cases, the end result is destruction of ubiquitinated proteins. At  Experimental Biology 2013, Dr. Seakwoo Lee, a research fellow in Pam  Zeitlin’s lab at Johns Hopkins Children’s Center, presented work on how ubiquitin modifies and regulates CFTR stability.

Two ways to ubiquitinated protein degradation (source)

The Zeitlin lab worked with Michelle McClure in Eric Sorscher’s lab at the University of Alabama at Birmingham to use mass spectrometry (“mass spec” to its friends) to identify residues of CFTR that had been modified by ubiquitin used. CFTR was isolated from cells and chopped into smaller fragments; this process removes ubiquitin but leaves behind a trace of it (glycine-glycine) where the ubiquitin tag once resided. Mass spec defined the amino acid sequences of fragments and the locations of glycine-glycine modifications and thereby ubiquitin. Lee mutated each glycine-glycine tagged site to arginine, a substitution that maintains the charge of the protein but that cannot be modified by ubiquitin. For each mutant, he looked at the protein expression levels of total and mature CFTR. He determined whether ubiquitination of each lysine targeted CFTR to the proteasome or the lysosome by using small molecule inhibitors. Because CFTR only functions at the cell surface, Lee  checked surface expression of the mutant by confocal microscopy and Western blotting. Finally he checked the ability of mutants to modulate expression of the inflammatory chemokine interleukin-8 in an epithelial cell line.

They defined seven specific sites of modification, scattered throughout multiple domains of the protein. In all but one instance, lysine->arginine mutations increased the amount of total CFTR protein and, importantly, the amount of fully matured protein. Addition of ubiquitin to different CFTR domains targeted the protein to different pathways for destruction. Modification of the N-terminal and nucleotide-binding domains targeted CFTR to the lysosome, whereas modification of the regulatory domain targeted CFTR to the proteasome. Now you might think that more CFTR and more mature CFTR would also mean more CFTR on the cell surface where it’s needed to function. Yet mutants that prevented proteasomal degradation were actually expressed at lower levels than wild-type CFTR  on the cell surface, even though these mutations produced more mature CFTR  inside the cell. 

So Lee saw that CFTR expression was stabilized by introducing mutations that prevented ubiquitination, but he wanted to find out if it functioned properly. Previous studies had shown that CFTR surface expression and activity suppresses basal inflammatory signaling. In cultured epithelial cells, dysfunctional CFTR activates the transcription factor NFΚB, which goes to the nucleus and turns on genes associated with inflammation. Co-expression with functional CFTR counteracts this program. Lee used one of those inflammatory program genes (interleukin-8 or IL-8) to look at the functional outcomes of his CFTR mutations when co-expressed with a completely non-functional CFTR. Mutants expressed on the cell surface at levels approaching wild-type were non-inflammatory. With mutants that failed to localize to the surface, cells still produced

CFTR trafficking & degradation (source)

elevated IL-8. However, lysosomal inhibition increased surface expression of these mutants and decreased IL-8 synthesis. How did that happen? Lysosomes are tightly linked to endocytosis – that is, internalization of things bound to or embedded in the surface membrane. Like many surface receptors, CFTR is rapidly internalized and shuttled back to the surface (recycling) or shipped to the lysosome for degradation. Lysosomal inhibition ultimately piles up and inhibits endocytosis. Lee’s result might suggest that stabilizing CFTR expression alone is insufficient for boosting CFTR activity. Instead drug discovery and development might also need to CFTR internalization and recycling.

The FDA recently approved the first drug that actually treats the molecular cause of cystic fibrosis. However, this drug addresses only one aspect of CFTR dysfunction, its direct activity. Over a thousand mutations have been identified in cystic fibrosis patients, and mutations that alter CFTR stability and localization present more of a challenge for therapeutic targeting and will likely require a combination approaches. One day, researchers might be able to leverage the sites and roles of CFTR ubiquitination to develop a drug to enhance CFTR surface expression. However, Lee’s data are preliminary, and further studies are needed to confirm the mechanisms of ubiquitin regulation of CFTR. The Zeitlin lab also needs to identify small molecule modulators of the process and define the therapeutic benefit and limits of this approach. In the past few decades, the survival age of cystic fibrosis patients has increased dramatically, but there’s still a long way to go.

 

#EB2013: The Scientist-Advocate

 

Policy needs more science. Science needs more funding. Funding needs limited politically motivated restrictions. These are common refrains among the scientific community, but what do we need to do to get there?

On Saturday, the American Physiological Society hosted a session entitled “How to be a science advocate in your own backyard”, and the American Society of Biochemistry and Molecular Biology held a policy session, “How scientists can save the world”. Both societies fielded excellent panels of professionals engaged in science, science outreach, and science policy: for APS, Gina Schatteman (professor emeritus at UIowa, former AAAS policy fellow, and director of iExplore STEM), Tim Leshan (vice president for government relations at Northeastern University), Michele Sukup Jackson (PhD candidate and member of MIT Science Policy Initiative), and William Talman (professor at UIowa and former president of FASEB); for ASBMB, Tania Baker (professor and head of Department of Biology at MIT), Darlene Cavalier (founder of Science Cheerleader and SciStarter), and Craig Mello (professor at UMass Med and Nobel laureate).

I strongly believe that basic science holds the keys to the universe, and we have a responsibility to do important research and explain it to the world.

- Tania Baker

Advocacy can take many forms, from talking to your neighbor to coordinating with the White House Office of Science & Technology Policy. Schatteman divided advocacy into 2 categories: direct – working through official channels to affect policy, talking to your Congress representative; and indirect or “stealth”, being an advocate just by being a scientist and talking to people you know. Talking to your family and neighbors about what you do is a great way to get started. It’s a (mostly) non-threatening venue to practice talking about science to a non-science audience. Plus your family is kind of stuck listening to you but will also be honest about how you’re doing (Baker commented that your family will smack you if you’re doing a bad job ). Next try a science festival; the presentations are short, and you have multiple chances to give it and modify it as necessary. Or maybe teach a science course for non-majors. Baker mentioned that her department is integrating communications into all parts of the science curriculum. Cavalier is leveraging Science Cheerleader and SciStarter to increase interest and investment of the public in science. In other words, opportunities to engage with non-scientists and promote science are all around us.

If we’re not passionate about keeping science going, we probably shouldn’t be doing it anyway.

- Bill Talman

However, direct advocacy – engaging with legislators – remains critical. Leshan put it nicely: “If you don’t advocate for what you care about, a million other people will be advocating for what they care about, and you will not be heard.” Talman encouraged scientists to not be “the typical NPR listener – all listen and no pay”. In science, we should pay our dues for the opportunity to do science by advocating for science. But approaching a Congressional representative might be an intimidating prospect for many scientists. Starting with local, “stealth” advocacy allows us to become comfortable discussing our science, but we must take a different approach to legislative advocacy. A few key points emerged:

• Do your homework. Find out the representative’s record and stance on issues.
• Hone your message. Have one or two key points.
• Have a clear pitch. Your goal should be to inform legislators of your research and its importance, but there needs to be a clear ‘ask’.
• Make evident how the issue affects the representative’s constituency and how you can help him or her with that issue. Talman recommended writing your areas of expertise on the back of your business card, so that staffers know with which issues you can help.
• Learn how to present your message in a way that others will listen to it – especially when dealing with controversial topics. Schatteman provided herself, a stem cell biologist, as an example.  If she’s talking to someone she knows is a proponent for stem cell research, her approach is straightforward, easy; she can say “stem cell biologist” and know that she hasn’t shut down the discussion immediately. If someone has a record of voting against embryonic stem cell work, she emphasizes “adult stem cells”. Some people may immediate shut down at the mention of “stem cells”, so she starts with a target disease and implications.

If we based [science] policy on science, the world would be a different place. We base policy on emotions and politics.

- Gina Schatteman

• Be flexible. Leshan shared an experience of a meeting in which the representative requested that the scientist end the meeting with a prayer. The scientists obliged and has maintained a strong connection with the representative.
• Follow up and use multimedia to your advantage. For example, make sure that you have a webpage that includes a summary of your research and public engagement in terms that a non-scientist can understand.
• Work with allies such as your institution’s government relations office or your professional society’s policy committee to coordinate efforts.
• If you’re interested and willing, learn the ropes from the inside. Schatteman started off with her university conflict of interest committee; she progressed to first to the university’s and then to a professional society’s legislative affairs committee before spending a year as a AAAS policy fellow – this after she had established here independent research career.
• Legislative advocacy cycles back to the public. The public – our our families, friends, and neighbors – help decide science policy by voting! Mello made the point that as long as the public doesn’t understand or even really care about science, “Congressional representation [of science] will be deficient”. We have to make the benefits of science clear.

If science is to continue to thrive, someone needs to advocate for it – and scientists must advocate for it. We don’t all need to be sitting in meetings on Capitol Hill. We don’t need to do all the advocacy. But we can each make a contribution to keep science and science policy moving forward

Further thoughts

Storify from APS Session

Storify from ASBMB Session

Decompression

It should have been a beautiful day.

Sunny, a little cool, a bit breezy, but on the whole, not bad for spring in Boston. A fantastic day for a run.

I worked from home a bit before heading to the lab. A couple of hours after lunch, as is my typical routine, I headed to the gym. After about 45 minutes of strength training, I took off out the door, following one of my short loops to tack a couple of miles onto my workout. I passed people with their yellow bags and blue jackets, reminding me that it was Marathon Monday.

For my first four years in the city, I lived only a couple of blocks from a section of the route, and Paramed and I would walk up, watch with inspiration as the wheelchair division zipped by and with awe as the elite runner zoomed past. We’d meander down the last few miles of the course, cheering on runners and feeding on the energy and excitement of the crowd. Last summer, we moved into one of the more distant suburbs of Boston. I had briefly considered trekking downtown to spectate, but I had things to do, and there simply hadn’t seemed to be enough time of late.

I returned to the lab from my own, much shorter run. I grabbed a yogurt from the fridge. My boss wanted to talk briefly about a collaboration. We chatted briefly about another thing, I giving him a good-natured ribbing. I returned to my desk.

As I sat down, my phone vibrated with a text message vibration. Immediately it buzzed again. And then again. Messages from 3 different people, asking if I was in Boston, wanting to know if I was OK.

My stomach knotted. “I don’t know what’s going on, but something’s happened,” I commented to my labmate. I fired off one reply and started logging into my computer to find out what was going on.

My phone started vibrating again, a call this time from Paramed. When I picked up, I didn’t bother with pleasantries. “What is going on?”

“Where are you at? Are you OK?” he asked urgently.

“I’m at work. I just got back to my desk and had 3 different people asking if I was OK. What happened?” I asked.

As I asked, my labmate – having just gotten a text from her husband – answered. “Oh my god. There’s been some sort of explosion at the marathon.”

I thought I’d been punched in the gut. “Oh my god.”

At the same time, Paramed replied. “I wanted to be sure you were OK. There were two explosions at the marathon near the finish. I’m in class. We just heard about it.”

We exchanged a few more words. “I’m glad you’re OK. Please be careful,” he said before we hung up.

Stunned, I quickly began replying to the direct messages queueing up on Twitter, while pulling up the local newsfeed and checking Twitter. I fired off a text to my dad, knowing that my family would be hearing about this any minute. I assured my friends on Twitter that I was far from danger.

Then I called our other labmate. She’s a runner, a crazy runner, the kind who loves pushing through 26.2 miles and beyond. She had qualified and registered for Boston. But she’s also a new mom, and the time away from her daughter to train for a marathon was more than she was willing to give up, at the moment. So she’d decided not to run. But we had no idea where she was, whether she and her husband and adorable 5-month old daughter were somewhere along the course to watch.

The phone rang a few times, and she answered. “Belle, how could…?” Her voice cracked. She knew.

“Are you alright?”

Her voice steadied. “Yes. We were watching at Coolidge Corner [24 mi marker]. We’re almost home now.”

“Good,” I replied. “We weren’t sure if you were watching or where.”

“I just…” She took a breath. “First, who the fuck would do something like this? But second… if I had run, that’s about the time I would have been finishing. That’s my pace – 4 hours, starting at 10 o’clock.”

“I know.” I did know, and I didn’t know what else to say.

“My sister’s best friend is running, and she would have been in the same group. I hope she’s OK,” she continued. [Later she learned that the friend was fine; she'd finished 10 minutes before the blasts.]

“I hope she’s OK,” I replied. “I’m going to get off, free up a ‘line’. I’m sure everyone’s trying to get through, but I wanted to make sure you were fine.”

“Thanks,” she said. “This just hits close to home. I’m hugging my daughter tight right now.”

Then I returned to the coverage. I watched and listened, my brain numb. By the time I tuned in, the anchors were still being cautious, indicating that there was no information as to whether it was intentional or not. But watching the video, I knew in my gut it was. Some part of my brain tried to come up with an alternative but was quickly shut down. Later they reported that, asked whether the blasts were bombs, a law enforcement official responded, “Hell yes. What else could they be?” As the coverage continued, it became evident no one thought this was accident. It was also clear, unsurprisingly, this story was personal. One anchor commented, “I’m infuriated. I hope they get this SOB in a hurry.” In the confused tumble of emotion, this stuck with me.

And yet through all this, the sun kept shining. It didn’t seem right. No, that wasn’t it. It didn’t seem real. It seemed like something out of a movie. Where was the dimming of the light to set the mood?

After the Boston PD press conference, I packed up to head home, wondering how long it would take me. The bus ride was strangely silent, no one daring to speak a word. Terrible things happen all the time – a bombing, a shooting, a fire – but somehow it seemed more acute, because it happened in our city. More than that on a day that we celebrate human commitment, motivation, discipline, achievement, and joy in what is normally a very solitary activity.

We all disembarked at the train station, murmuring farewells to the driver. At the entrance to the train station, I discovered a full security check – bag searches and metal detectors. And yet, despite the hundred or so people waiting to get to their trains, there wasn’t a word of dissent or complaint or protests of missing a train. As the train pulled out of the station, I watched a helicopter hover over the city. It should have been there for the finale of a marathon; knowing it was there covering the aftermath of an attack was chilling.

I now sit at home, sipping a beer and processing this day. The seemingly overwhelming problems of yesterday seem rather small. The worries I had have been temporarily displaced by shock and staggering sadness. There is no sense to be made of this. It was a horrific act. It’s the sort of thing we want to believe is inconceivable – and yet we know from recent history it is not. Innocent people have been killed, and many, many others injured. It took only a few moments on a Monday afternoon to completely change how we viewed the world. Suddenly every neglected bag, opaque water bottle, or abandoned box had become a threat. But even as objects were eyed with suspicion, there was also a great outpouring of aid and compassion for people.

This city and this event are irrevocably changed…

Changed… but not defeated. There’s far too much spunk in this city for that.

via Facebook

Coming soon to Beantown

If you’re in the Boston area, check out these upcoming events.

Monday, April 15, is the dreaded tax day. But on the bright side, it’s also the date for the next SciO Beantown gathering. The first was a casual & fun affair. This one promises to be equally so, but with a dash of discussion about open access publishing.

A week & a half away is Experimental Biology 2013! Tons of great science, career development, and outreach talks are on the docket. If you can’t make it to the meeting, follow #EB2013 on Twitter. I’ll also be posting from EB2013 as one of this year’s ASBMB meeting bloggers. Expect lots of protein stuff from me. Have a poster I absolutely must see? Let me know

When the odds beat you

You know the odds. You see and hear the statistics – over and over and over again. You cannot help but know there’s a very good chance that your vision of the future will fall apart, that you will fail.

But you do it anyway. You cling to the hope that your experience will be different. Maybe you tell yourself you have something special or that you’ll work harder or smarter. You’ll do the “right” things, or maybe you’ll just get lucky. You’ll find obstacles in your path, but you’ll find a way around or over or through them. Somehow you will make it work. You have to believe that you will succeed. If you didn’t believe that, would you try? Would you stand a chance?

So you do it. You go all in. There are good days that buoy your confidence. There are bad days that test your resolve, but you find a way through them. Most of the days fall somewhere in between, just doing the day-to-day work to keep it all afloat, often without actively thinking about long game.

Then it happens. That fragile dream falls, hits the ground, and breaks into a thousand million pieces. And you feel like you shattered along with it.

While you’re picking up the shards, trying to figure out what to do with what remains and how to move forward, you inevitably find yourself thinking about how you got here. What did you do wrong? What should you have done differently? Should you have put in more or taken a step back? Were you blinded by pride – or naivete? If you’d been a touch more cynical, maybe you’d have been better prepared. Possibly the crash was inevitable, but if you’d held something back, maybe it wouldn’t have hurt so damn much.

That might be the hardest part about broken dreams: They can make you question everything – who and what you are, all you do and how you do it. It can be especially hard when you try to be self-sufficient, when you compartmentalize your life in an effort to keep one part from bleeding into the other, when you’re accustomed to putting forward an air of calm and control even when you’re a wreck inside. The people around you think you’re OK. You’re the strong one; you’re always OK. You say little, so they say little. When they do say something, it’s what they think you need or want to hear – more visions for the future, assurances that you’ll find something new/different/lasting, promises of the wonderful things that will come in time.

What you really need to hear is that you’ll simply make it through the here and now. That you’ll survive picking up the pieces, even though each one cuts deeply as you grab hold. That you’re not alone. That no matter how broken you feel, you’re still relevant, you’re still worth something to someone. Some days you know all of that, without question. Some days take a little more convincing, and you have to tell yourself that you really can do this, that the pain and doubt won’t last forever.

But some days you need a little help. You need to hear someone else say all those things that you know deep down. The thing is, people – even those who are very close – don’t necessarily think to tell you those things because they should be obvious. So you have to reach out and ask for help, which can be harder than it sounds. It means letting go of the pride or logic or whatever it is that’s holding you back. To keep from losing yourself entirely, you have to let some of the cracks show. You have to bare your weakness. The very thing you fear will crush you is exactly the thing you must do. Finding the strength to do that helps you realize you have the strength and support you need not just to survive but to keep playing against the odds.