Figuring out why cells don’t burst

April 10, 2014 § Leave a comment

Researchers have found one critical component of the protein machinery that stops cells from bursting. Image by Jorge Colombo.

Researchers have found one critical component of the protein machinery that stops cells from bursting. Image by Jorge Colombo.

Why don’t cells swell up with water and burst? For 30 years, scientists have known that there is an ion channel dedicated to regulating the volume of a cell, but its molecular identity has been a mystery. In a paper just out in the journal Cell, researchers identified an essential component of this channel.

Water comes and goes through cell membranes fairly easily. It flows in a way to balance out the concentration of solutes both inside and outside the cell. If solutes increase inside the cell, water flows into the cell. But what keeps the water from gushing in so much that the cell bursts like a balloon?

Experiments 30 years ago showed that there was an unidentified ion channel in the cell membrane called the volume-regulated anion channel (or VRAC for short). When a cell swells, VRAC opens to release chloride ions and some other negatively charged molecules. Water molecules follow these molecules out of the cell, and the cell doesn’t swell.

But technical challenges dogged the molecular identification of VRAC. Zhaozhu Qiu at The Scripps Research Institute and Genomics Institute of the Novartis Research Foundation, the first author of the new study, explains the lack of a specific high-affinity ligand prevented researchers from being able to directly pull out the protein from a cell. Expression cloning in cells, a technique that has worked well for other ion channel identifications, failed for VRAC because the large quantity of native VRACs in a cell drowned out the ones being artificially expressed.

So Qiu and the rest of the team, spearheaded by Ardem Patapoutian at the Howard Hughes Medical Institute and TSRI, decided to screen for the genes needed to produce VRAC. The screen used RNA molecules to selectively turn off genes. Qiu explains, “We chose one of the most commonly used cell lines, HEK293T, because it has native VRAC currents and, more importantly, it takes up small interfering RNA readily,” allowing the researchers to easily turn off genes that possibly were a part of VRAC.

Qiu adds, “Like many other genomewide screens, we got hundreds of potential hits. To prioritize, we reasoned that the candidate VRAC gene has to encode a membrane protein, a feature shared by all ion channels, and that it is most likely a relatively novel gene.”

With those criteria in mind, the investigators evaluated 51 gene candidates in a second screen. They homed in on one gene because VRAC activity diminished when the gene was turned off. Keeping in mind that there may be more than one component of VRAC, the investigators called this particular one “SWELL1.”

Besides being the water patrol for cells, SWELL1 may have other important roles. “Interestingly, the gene for SWELL1 was first noted by scientists because its mutant form is associated with agammaglobulinemia, a lack of antibody-producing B cells, in a patient,” says Patapoutian. “This suggests that SWELL1 might be somehow required for normal B cell development. There also have been suggestions from prior studies that the VRAC channel is involved in stroke, because of the brain-tissue swelling associated with stroke, and that it may be involved as well in the secretion of insulin by pancreatic beta cells. So there are lots of hints out there about its relevance to disease. We just have to go and figure it all out now.”

A proteomic probe for GTP-binding proteins

March 18, 2013 § 1 Comment

This affinity probe captures GTP-binding proteins for proteomics analysis. Image by Beth Cisar.

This affinity probe captures GTP-binding proteins for proteomics analysis. Image by Beth Cisar.

GTP-binding proteins make up  huge and ubiquitous portions of proteins that have essential functions in cell signaling, trafficking, cytoskeletal structure, nucelotide metabolism and translation. In a recent paper in the Journal of American Chemical Society, Hugh Rosen and colleagues at The Scripps Research Institute described a GTP affinity probe for proteomics. The probe, say the investigators, should help researchers identify a variety of GTP-binding proteins in a single shot by mass spectrometry.

The probe was designed to solve a problem in the Rosen laboratory, explains Beth Cisar, the first author on the paper. The laboratory is interested in the sphingosine-1-phosphate (S1P) receptors, which are five G-protein coupled receptors (GPCRs) that are needed for the proper functioning of many systems, including the cardiovascular and lymphatic systems. GPCRs work in concert with a variety of GTPases. But the investigators didn’t have a tool that would let them comprehensively pull out, in one shot, all the GTP-binding proteins involved in S1P receptor signaling pathways.

So the investigators designed the GTP-BP-yne probe. The molecule covalently binds to GTP-binding proteins through a photocrosslinking reaction and has an alkyne handle that lets reporter tags, such as avidin and rhodamine, latch onto
it. This allows the investigators to analyze GTP-binding proteins either by mass spectrometry or in-gel fluorescence.

With their probe, the investigators pulled down many GTP-binding proteins found in human embryonic kidney cells, which were their test case, by mass spectrometry. Rosen and colleagues got 33 proteins, including members of several known classes of GTP-binding proteins.

But, much to their surprise, they also found ATP-binding proteins, including three related kinases called Src, Lyn and Yes. Src had been shown previously to bind GTP, but Lyn and Yes’ ability to do so was unknown until this study. This finding highlights “the idea that purine nucleotide selectivity is often not as strict as it is thought to be,” notes Cisar.

There are fluorescent or radioactive GTP analogs to study GTPases and other
GTP-binding proteins. There is a commercial GTP probe for proteomics, but Cisar says that their GTP-BP-yne probe labels targets via a different mechanism that allows identification of not only GTP-binding proteins but also proteins that bind GTP-binding proteins.

With their probe now in hand, Cisar says, the investigators are going back to their original problem, which was the study of S1P receptor signaling pathways. “We are particularly interested in determining whether the probe can distinguish between targets’ active and inactive states,” she says.

Biomarker for river blindness

February 25, 2013 § Leave a comment

Life cycle of the filarial worm Onchocerca volvulus. Image from WHO:

Life cycle of the filarial worm Onchocerca volvulus. Image from WHO:

Onchocerciasis, also known as “river blindness,” is a parasitic infection that strikes millions of people in Africa, Latin America and other tropical regions. There are treatment options but monitoring the disease’s progression during treatments and conducting surveillance of the disease in a population are challenging. In a recent paper in the Proceedings of the National Academy of Sciences, researchers described the identification of a unique biomarker in urine that could lay the foundation for a cheap and easy diagnostic tool to track the disease during treatment and eradication campaigns.

Onchocerciasis is caused by the filarial worm Onchocerca volvulus. The worm is transmitted to humans as larvae through the bites of infected blackflies. In humans, the larvae mature to adult worms. After mating, the female adult worm can release up to 1,000 microfilariae a day. These early-stage worms move through the body; when they die, they cause blindness, skin rashes, lesions, intense itching and skin depigmentation.

The combination of an antimicrofiliarial drug called ivermectin and the antibiotic doxycycline can kill the worms. The World Health Organization’s African Programme for Onchocerciasis Control has set a target date of 2025 for the eradication of the disease in that region. Indiscriminate treatment with the drugs could lead to drug resistance and render the treatnent ineffective so it’s important to have a way to track the disease and ensure eliminiation programs are on course.

To track the disease, researchers need a signature molecule that reflects the status of the infection. Kim Janda at The Scripps Research Institute explains that he and his colleagues initially focused on finding biomarkers for O. volvulus infection in blood. They found 14  but what Janda says he really wanted was a single biomarker so that a simple diagnostic tool could be based on this biomarker. “In my opinion, a single biomarker is needed that tracks the progression of onchocerciasis and monitor whether years of mass treatment of people infected by O. volvulus is working toward the goal of its eliminination,” he says.

So the investigators decided to look in urine, with the rationale that the urine-production pathway would generate a different profile of metabolites that was different from those in blood. They analyzed samples from infected and uninfected people by liquid chromatography-mass spectrometry.

The investigators identified a single biomarker, N-acetyltyramine-O,β-glucuronide (NATOG), a metabolite derived from an O. volvulus neurotransmitter. They showed that NATOG is upregulated at the time of infection. If an infected patient gets treated with doxycycline, the level of NATOG goes down. Janda explains that this finding gives “confidence that the biomarker tracks with the worm’s metabolic life cycle.”

The researchers are now working on developing a simple dipstick test based on NATOG.

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