No pregnant woman gets excited about invasive procedures. But, for the diagnosis of some complications, pregnant women have to undergo procedures such as amniocentesis. Researchers have been aiming to come up with less invasive tests. In a paper in this month’s issue of Molecular & Cellular Proteomics, scientists describe how a class of proteins can be reliably tracked in blood samples taken from pregnant women and found hints that changes in expression patterns of this class of proteins may indicate if a pregnant woman is at risk for premature delivery.

The class of proteins that Danielle Ippolito and colleagues at the Madigan Army Medical Center studied were apolipoproteins. “Apolipoproteins are lipid-transport proteins in plasma” which, along with lipids and cholesterols, increase exponentially in pregnancy to support fetal development, explains Ippolito. Apolipoproteins exist stably in the blood plasma, suggesting they can be reliable indicators of different biological processes.

Ippolito says she, her colleagues and other researchers had found earlier that maternal plasma had different concentrations of apolipoprotein subtypes, depending on whether or not the women developed preemclampsia. Based on that finding, the investigators reasoned that, if they could track these various subtypes, they would be able to find proteins that signaled early on whether or not a pregnancy was going to be complicated.

To see if their hypothesis bore out, the investigators analyzed by mass spectrometry the plasma collected from women at different time points of their pregnancies. They found that modified subtypes of an apolipoprotein called Apo A-II were significantly higher in plasma from mothers who delivered preterm babies than those who didn’t.

Ippolito says that, as the next step, her team intends “to analyze plasma from patients with different obstetric outcomes to compare apolipoprotein profile in patients who deliver without complications versus women who develop gestational diabetes, severe preeclampsia and preterm premature rupture of the membranes leading to premature birth.”

The journal Molecular & Cellular Proteomics is establishing guidelines for reporting glycomics data. Before the guidelines become official, the editors would like scientists to give their feedback.

“MCP has been a leader in the -omics fields and establishes guidelines by which publications that use mass spectrometry can be standardized,” says MCP Co-editor Ralph Bradshaw at the University of California, San Francisco. Proteomics was one field for which standards were introduced by MCP so that researchers could both reproduce and rely on the experiments described in the papers. The field of glycomics, the comprehensive analysis of all glyproteins and other glycoconjugates in a biological unit, has now reached that same point.

“This is the first time in history that the technology has existed to actually do glycomics” at a detailed level, says MCP Associate Editor Gerald Hart of Johns Hopkins University. “New instrumentation, like electron transfer dissociation and MALDI techniques, didn’t even exist five years ago for sugars. Now we’re on the cutting edge of where this instrumentation could have a huge impact on the field of glycobiology.”

The new instrumentation and methods will unleash a deluge of data and new insights. “Carbohydrates are quite different from proteins. If you identify a protein by mass spectrometry, you’ve identified the polypeptide,” explains Hart. “If you identify carbohydrates, they have extreme structural diversity that depends on the biology of the system and the level of the analysis.”

Al Burlingame, the other MCP co-editor at the University of California, San Francisco, says that the field of glycomics, because of its potential as a clinical tool for disease diagnostics and therapeutics, has attracted a diversity of scientists, not all of whom are trained in complex sugar chemistry and mass spectrometry. For this reason, he, Bradshaw, Hart and MCP editorial board member Lance Wells at the University of Georgia, Athens, say it’s important that rigor be introduced in the way the data are reported in publications. The proposed MCP guidelines, which come along with a checklist, are meant to instill some order into the burgeoning field so that all researchers can understand the diverse types of sugar analyses.

The draft guidelines and checklist for glycobiology are similar to the guidelines and checklists MCP already has in place for clinical data and mass spectrometric analyses of peptides. The new checklist will help authors of manuscripts ensure that they are clearly communicating how they did their analyses so that reviewers and readers can understand their experiments.

Burlingame, Bradshaw, Hart and Wells all emphasize that the checklist is just that, a checklist. It’s not a quality check of the data. It, and the guidelines on which it is based, just make sure that all the bits and pieces of information are there so that everyone can understand how the experiments were carried out.

Bradshaw explains that the goal is to set standards so that only technically strong reports make it into the literature. He adds that “getting universal buy-in is an important aspect of” setting standards. For this reason, MCP is making its proposed guidelines and checklist available for public comment for a 30-day period.

To send your thoughts and opinions, click here. The journal would like to formally adopt the guidelines and checklist by March 1.

Bacterial vaginosis in pregnant women could lead to preterm labor and birth. Image from http://en.wikipedia.org/wiki/File:Premature_infant_with_ventilator.jpg

Bacterial vaginosis is an imbalance of vaginal bacteria and linked to a variety of serious health problems in women. In a recent paper in the Journal of Biological Chemistry, researchers described how a class of enzymes involved in bacterial vaginosis attacked glycoproteins in vaginal mucus.

Bacterial vaginosis can be an asymptomatic condition, meaning that women can walk around without knowing that the bacterial composition in their vaginal mucus is out of whack. But, explains Amanda Lewis at Washington University in St. Louis, who was the senior author on the paper, the condition in pregnant women is known to be associated with an elevated risk of preterm birth. “Despite being the most common reason for a woman to seek treatment from her (obstetrician/gynecologist), bacterial vaginosis is not often discussed,” she says.

Researchers know that in bacterial vaginosis the vaginal mucus shifts away from its normal state. The shift is caused by the action of hydrolytic enzymes, which researchers think come from bacteria, that chew down glycoproteins in the vaginal mucus. These glycoproteins have a protective function.

However, “despite the clinical significance of bacterial vaginosis, experimental systems for studying the condition are lacking, the bacteria involved are difficult to grow in the lab, and other tools used in studies of infectious diseases are difficult to apply since bacterial vaginsos is a polymicrobial condition,” says Lewis.

So she and her colleagues decided to use vaginal swabs to observe the breakdown of protective vaginal molecules. They observed that sialidases attacked the mucus glycoproteins and removed their sialic acid residues. “Glycobiologists have known for a long time that once sialic acid residues are removed other glycosidase enzymes can better act on carbohydrates underneath, leading to more extensive deglycosylation of proteins,” says first author and Lewis’ husband, Warren Lewis. This means that the protection offered by these vaginal glycoproteins is destroyed.

Lewis says her group will be working to identify which bacteria produce the sialidases. “We are also interested in developing a better understanding of which combinations of enzymes and bacteria produce the greatest degree of mucus destruction,” she says, adding they would like to understand which combinations are correlated with symptomatic bacterial vaginosis or adverse outcomes, such as preterm birth.

Researchers in Taiwan are testing potential cancer vaccines based on carbohydrates. Image from http://en.wikipedia.org/wiki/File:Syringe2.jpg

In a paper just out in Proceedings of the National Academy of Sciences, researchers describe the development of carbohydrate-based vaccines against breast cancer. One of the vaccines is in clinical trials for terminal-stage breast cancer patients.

Chi-Huey Wong at the Academia Sinica in Taiwan says he and his colleagues have been searching for unique cancer biomarkers as targets for vaccine development. Unfortunately, most cancer-related proteins are either upregulated or downregulated instead of being uniquely expressed. These proteins are not good candidates for vaccine design “as the antibodies generated from immunization may also attack normal cells and thus cause autoimmune disease,” explains Wong.

Instead, the investigators decided to look at sugars attached to either proteins or lipids that were expressed on the surface of cancer cells. By using enzymatic methods and mass spectrometry, they were able to identify a number of unusual sugar structures on the cell surfaces of several types of cancers, including breast cancer and breast cancer stem cells.

The investigators decided to design vaccines that would induce antibodies, in particular IgG, to target three unique sugar structures called SSEA4, GloboH and Gb5. But they had to proceed with caution. “We know carbohydrates are not good immunogens and often induce IgM antibodies, which are less desirable,” says Wong.

But he and his colleagues found that they could develop carbohydrate-based vaccines that targeted these markers. When they carefully chose the vaccine carrier proteins, adjuvant and other molecules, they were able to coax the immune system to generate more IgG than IgM antibodies.

One of the vaccines Wong and colleagues developed, called GloboH-KLH/QS21, is now undergoing phase II and III clinical trials in terminal-stage breast cancer patients. The investigators also are focusing on another vaccine, SSEA4-DT/C34, which is more selective for  SSEA4, GloboH and Gb5 and generates more IgGs than the GloboH-KLH/QS21 vaccine. The SSEA4-DT/C34 vaccine is targeted against breast cancer but also may have applications in pancreatic, ovarian and lung cancers, says Wong.

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Heart failure, brain ischemia, and strokes have one thing in common: an intracellular overload of calcium. In a recent Paper of the Week in the Journal of Biological Chemistry, researchers described how this calcium overload comes about and leads to cell death.

The calcium overload is caused by a hyperactivation of acid-sensing ion channels. One class of these channels are the degenerin/epithelial amiloride-sensitive sodium channels (DEG/ENaC) . The hyperactivation of DEG/ENaC channels has been shown to lead to apoptosis in mammalian cells. But the mechanism of how these channels contribute to cell death remains unclear.

A team led by Wei-Xing Zong at Stony Brook University in New York demonstrated that the hyperactivation of DEG/ENaC channels and the calcium overload activated a mechanism of apoptosis mediated by the ubiquitin-binding protein p62 and the autophagy-related protein LC3. These two proteins ultimately led to cell death via the proapoptotic protein caspase-8.

The authors concluded in their paper that the mechanism involving p62 and LC3 was a novel mechanism for caspase-8-mediated apoptosis.

To learn more about the details of this work, listen to this podcast where the public outreach coordinator for the American Society for Biochemistry and Molecular Biology, Geoff Hunt, chats with Zong.

A mutated and hyperactive DEG/ENaC channel promotes interactions between LC3, caspase-8, and p62.

The enzyme disulfide isomerase’s two pairs of vicinal thiols are about to interact with an unfolded protein has arsenical maleimide moeities attached to it. Image provided by Colin Thorpe.

Over the past two decades, arsenic-based compounds have been under investigation in clinical trials as possible therapeutics for leukemias and solid tumors. However, the current cohort of compounds is rather limited. In a recent Journal of American Chemical Society paper, researchers demonstrated a new type of arsenic-based compound that inhibits a wide range of proteins involved in disease processes.

Arsenic-based chemistry for disease treatment goes back to the early 1900s but fell by the wayside with the rise of antibiotics and other medicines in the 1940s. But there has been renewed interested in arsenic-based compounds for cancer therapies; indeed, the U.S. Food and Drug Adminstration approves the use of arsenic trioxide to treat acute promyelocytic leukemia. 

But rather than synthesizing arsenic-based compounds for specific diseases, which isn’t an efficient use of time and labor, Colin Thorpe and Aparna Sapra at the University of Delaware wondered if they could make new tools for producing arsenic-based compounds. “We wanted a versatile approach that could be easily implemented with one simple arsenical that could be quickly coupled to a wide range of peptide or protein scaffolds,” says Thorpe.

So they developed arsenical-maleimide, a simple compound that appears to quickly and efficiently attack a range of proteins and peptides that have exposed thiol (-SH) groups on them. The maleimide component of the compound latches onto one set of -SH groups. Then the arsenic, which is a good chelator of exposed thiol groups, binds to any other pairs of thiol groups that may be in the vicinity.

The investigators showed by using model peptides that they could get anywhere from one to eight molecules of arsenical-maleimide onto a chain, depending on the number of exposed thiol groups it contains. Because proteins interact with each other, proteins that had arsenical-maleimide attached to them carried the compound to their binding partners and specifically inhibited them through the arsenic moiety. ”We hope that the renewed interest in arsenicals by a number of research groups might contribute to a renaissance in the use of arsenic-based therapies,” says Thorpe.

Mice fed 6F-engineered tomato powder in a high-cholesterol diet had less plaque buildup (right) than mice who weren’t given the genetically engineered tomato powder. Image courtesy of Srinivasa Reddy.

Special tomatoes to stave off heart attacks and strokes? In a recent paper in the Journal of Lipid Research, a group of researchers describe a genetically engineered tomato that contains a protein that helps stave off atherosclerosis, the buildup of plaque in arteries that leads to heart attacks and strokes.

ApoA-I mimetic therapy is currently one way to treat atherosclerosis. Apolipoprotein (apo)A-I, which has 243 amino acids, is the main component in high-density lipoprotein, also known as good cholesterol. In animal models and humans, infusions of apoA-I have been associated with improvements in atherosclerosis. But given its length, it’s an expensive protein to produce and has to be given intravenously.

Mimics of apoA-I have been produced with only 18 to 26 amino acids. They don’t have sequence similarities with apoA-I but they bind lipids in the same way. Srinivasa Reddy and Alan Fogelman at the David Geffen School of Medicine at the University of California Los Angeles, along with their colleagues, have been studying an apoA-I mimetic peptide called 4F.

4F has been demonstrated in animal models to reduce inflammation, atherosclerosis and other disease processes associated with inflammation. The animal studies spawned two clinical trials. Data from the trials led the investigators to conclude that 4F was most effective when taken orally and processed in the small intestine.

But the problem was that the necessary oral dose was high. “The 4F peptide can only be made by chemical synthesis,” explains Reddy. “The cost of producing enough 4F peptide by chemical synthesis to achieve efficacy prevented this from being pursued as a therapy in humans.”

So the investigators began a search for a peptide that didn’t require extensive chemical synthesis and could be produced by genetic engineering. They came up with another peptide called 6F and decided to see if they could produce it in tomatoes. “We wanted to produce the peptide in a plant that could be eaten without cooking because we felt that cooking the peptide might denature it,” says Fogelman. “The tomato was a convenient and tasty choice.”

The investigators genetically engineered tomatoes to produce the 6F peptide, freeze-dried the fruit, ground them into a powder and added the powder to a high-fat, high-cholesterol Western diet for mice. “We found that, some hours after feeding the peptide, it was still intact in the small intestine,” says Reddy. “Markers of inflammation in the blood were significantly reduced, HDL-cholesterol and HDL function were significantly improved, and atherosclerosis of the aorta was significantly reduced.”

Put together, the investigators say, the work demonstrates that tomatoes engineered to produce an apoA-I mimic could potentially be used as-is to reduce inflammation and atherosclerosis without having to extract and purify the peptide from the plant.

Life cycle of the filarial worm Onchocerca volvulus. Image from WHO: http://www.who.int/tdr/diseases-topics/onchocerciasis/en/

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.

Structure of a Streptomyces albus beta-lactamase (http://en.wikipedia.org/wiki/File:PDB_1bsg_EBI.jpg)

Resistance against antibiotics, such as penicillin and cephalosporins, are haunting many hospitals and clinical practices. In a recent paper  in the Journal of the American Chemical Society, researchers reported that they made laboratory versions of the ancient ancestors of the enzymes that lead to antibiotic resistance. By studying the ancient forefathers of these enzymes, researchers hope to understand how modern antibiotic resistance evolved and figure out new ways to deal with it. “Antibiotic-resistant organisms cause thousands of human deaths every year. Anything new we can learn about antibiotic resistance may be potentially useful in coping with this problem,” says Jose Sanchez-Ruiz at the University of Granada in Spain, one of the study’s coauthors.

Antibiotic resistance isn’t a modern phenomenon that only arose in the face of clinical antibiotic use in the past 60 years. Bacteria have been toting around enzymes to disarm antibiotics for millenia. Indeed, genes for antibiotic resistance have been found in 30,000-year-old permafrost sediment and in places largely untouched by human activity, such as remote Alaskan soil and the bottom of the Pacific Ocean.

The class of enzymes that render drugs like penicillin useless are called beta-lactamases. The question is whether current pathogenic bacteria are simply recruiting, or improving upon, enzymes like beta-lactamases or other molecules that confer resistance in natural environments.

The permafrost sediment study dated antibiotic resistance as 30,000 years old. “But we knew that it is actually much older than just 30,000 years, because the resistance enzyme beta-lactamase is widely distributed throughout the bacterial domain of life. Phylogenetic analysis places its origin at about 2 to 3 billion years ago,” says Valeria Risso, also at the University of Granada.

To figure out how these enzymes functioned way back when, Risso and Sanchez-Ruiz, along with Eric Gaucher at the Georgia Institute of Technology, used bioinformatics tools to reconstruct ancestral lactamase sequences corresponding to the Precambrian nodes in the bacterial evolutionary trees. They then used molecular biology approaches to make the corresponding proteins in the laboratory and studied their structure, stability and function.

From their analyses, the investigators found that the Precambrian beta-lactamases were highly stable and promiscuous, with the ability to degrate a variety of antibiotics. This finding has biotechnological applications because highly stable and promiscuous enzymes are “in the top of the wish list of any protein engineer,” says Sanchez-Ruiz.

The investigators also say that when a microorganism develops resistance toward a drug,  it is repeating an adaptation process that likely took place in natural environments for several billion years. “The availability of resurrected Precambrian lactamases opens up new possibilities to understand such evolutionary adaptations and may therefore provide useful information to cope with modern antiobiotic-resistance problem,” says Risso.

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The chaperone protein Hsp90 is a potential cancer drug target but to date, the clinical efficacy of Hsp90 inhibitors has been less than satisfactory. In a recent Paper of the Week in the Journal of Biological Chemistry, a team led by Ahmed Chadli at the Georgia Regents University demonstrated that the natural plant product, gedunin, may be able to stall the Hsp90 machinery.

Gedunin comes from the neem tree extracts. The plant extracts have been used to treat malaria and other infectious diseases in traditional Indian medicine.

Chadli and colleagues demonstrated that gedunin could potentially also be used for cancer therapies. In their work, the investigators showed that the molecule binds p23, a co-chaperone to Hsp90. The gedunin binding prevents p23 from partnering with Hsp90 and stops them from inducing the overexpression of antiapoptotic proteins, Hsp70 and Hsp27. By using molecular docking along with mutational and functional analysis, the investigators provided evidence that gedunin inhibits p23 chaperoning activity, blocks its interaction with Hsp90 and interferes with p23-mediated gene regulation.

To hear more about the work, check out this podcast I recently did with Chadli.

Mounted Neanderthal skeleton at the American Museum of Natural History. Image by Claire Houck http://en.wikipedia.org/wiki/File:Neanderthalensis.jpg

It very well may be possible that compromised leukotriene signaling is one of the reasons Neanderthals are not among us today. In a recent paper in the Journal of Lipid Research, investigators compared the genome sequences of Homo neanderthalensis and Homo sapiens to see if the two hominid subspecies shared genes for the biosynthesis of leukotrienes and other inflammatory mediators. The investigators, led by Hartmut Kuhn at the University Medicine Berlin-Charité, found that the Neanderthal genome contained six genes encoding for six different lipid-peroxidizing isoenzymes called lipoxygenases (LOXs). Previous work has shown that we too have six LOX genes. However, the cDNA for two of the enzymes contained premature stop codons in the Neanderthal sequence, suggesting that expression of these enzymes was compromized.

Neanderthals are our closest evolutionary relatives. The youngest Neanderthal fossils have been dated to some 30,000 years ago, but there is evidence that Neanderthals may have survived in southwestern Europe until about 25,000 years ago.

A draft sequence of the Neanderthal genome was recently published. Global comparison of the genomic sequences of H. sapiens and H. neanderthalensis hinted that a number of genomic regions were different between the two subspecies of hominids. “Among them was the gene encoding the cysteinyl leukotriene receptor 2, which was mutated in the Neanderthal genome,” says Kuhn. “Although no direct functional studies have been carried out, the sequence data suggest that Neanderthals might have suffered from compromised leukotriene signaling.”

Leukotriene signaling, in which cysteinyl leukotriene receptor 2 is involved during processes of inflammation, requires the biosynthesis of leukotrienes. These molecules are made by LOXs. LOXs are lipid peroxidizing enzymes that have been implicated in cell differentiation and in the pathogenesis of inflammatory, hyperproliferative and neurodegenerative diseases. “Except from a large number of genomic LOX sequences that have been deposited in the publically available databases, virtually nothing is known is know about the evolution of this enzyme family,” says Kuhn.

Kuhn and colleagues carried out a series of bioinformatic experiments and protein biochemical assays to compare and contrast the LOX genes of Neanderthals and modern humans. They established that the genomes of H. sapiens and H. neanderthalensis contained six LOX genes – nALOX15, nALOX12, nALOX5, nALOX15B, nALOX12B and nALOXE3 – and one functionless pseudogene. “Since this pseudogene is functional in mice, it appears to have been corrupted later on in mammal evolution,” says Kuhn.

The sequences of the LOX genes confirmed that the two subspecies were related closely in evolutionary terms. But nALOX12 and nALOXE3 had two premature stop codons, hinting that the expression of these two LOX isoforms in Neanderthals might have been compromised. But Kuhn cautions that this conclusion should be interpreted carefully because there may be sequencing artifacts and problems with sample collection.

Nonetheless, Kuhn and colleauges are pressing ahead with doing more sequence comparisons. The complete genomic sequence of another ancient human ancestor, the Denisovan, recently was released. The Denisovan hominids share a number of anatomical similarities with Neanderthals. “We are about to apply the combined research strategy of our Neanderthal study to these sequences to find out of whether or not the take-home messages we concluded from the Neanderthal genome may also be applicable for Denisovan individuals,” says Kuhn. He says the hope is the sequence comparison will help to confirm or reject that there are premature stop codons in nALOX12 and nALOXE3.

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.

A dividing stem cell partitions and aligns its chromosomes according to the position of a bead coated with a Wnt protein. [Video courtesy of Shukry Habib, Roel Nusse, Bi-Chang Chen and Eric Betzig (HHMI, Janelia Farm Research Campus)]

It appears that Wnt proteins are like real-estate agents and think “location, location, location.” In a paper just out in Science, researchers report that these important developmental signaling molecules work locally on embryonic stem cells to guide them toward asymmetric division and differentiation.

Stem cells are pluripotent, meaning that they have the potential to become almost any kind of cell type in the body. They divide asymmetrically, not evenly down the middle. Asymmetric cell division also happens in cancer cells. Despite the importance of asymmetric cell division, not much is known at the single-cell level about its mechanics.

A signaling pathway involving Wnt proteins is known to be involved in asymmetric cell division. “We understand a great deal about Wnt signaling at the level of their transcription targets,” explains Roel Nusse, a Howard Hughes Medical Institute investigator at Stanford University who is the senior author on the paper. “But much less is known about how the Wnt signal leads to oriented cell divisions.”

Based on worm studies, there is evidence that Wnt proteins act locally, says Nusse. But no one had ever studied single stem cells using single Wnt proteins to see if they elicited particular directional responsesduring cell division.

So Nusse, first-author Shukry Habib, also at Stanford, and colleagues developed a method based on time-lapse microscopy and beads coated with various Wnt molecules. They exposed each bead to a single mouse embryonic stem cell to see how the Wnt molecule affected cell division.  

They demonstrated that Wnt3a, which maintains a stem cell’s pluripotency, caused daughter cells to have different fates. Daughter cells closest to the beads maintained their pluripotency, but daughter cells farthest from the beads began to differentiate.

Nusse says the next step is to study other cell types, including human stem cells, and tease out the molecular events that get triggered by a localized Wnt signal that ultimately leads to  asymmetric cell divisions.

Jon Lorsch, a professor at Johns Hopkins University, will be the next director of the National Institute of General Medical Sciences. He’ll arrive at the institute in Bethesda, Md., this summer.

Lorsch, an active member of the American Society for Biochemistry and Molecular Biology’s mentoring committee, will oversee a $2.4 billion budget that supports primarily fundamental research and scientific workforce training.

“With his reputation of being a broad-minded and visionary thinker with strong management skills, I am confident that Jon will lead NIH’s basic science flagship to keep the U.S. at the forefront of biomedical research,” Francis S. Collins, director of the National Institutes of Health, said in a statement announcing the appointment on March 25.

Lorsch will take the NIGMS reins from Judith H. Greenberg. Greenberg has served as the acting director of the institute since July 2011, when Jeremy Berg stepped down, after holding the director position for eight years, to become the University of Pittsburgh’s associate senior vice-chancellor of science strategy and planning.

Berg, who now is also president of the ASBMB, said he was pleased with the appointment: “Jon is a great choice. He is an outstanding scientist with ideas spanning many disciplines and with great teaching and training experience. He also led the curriculum reform efforts at Johns Hopkins and balanced clinical and basic perspectives very well.”

Berg continued: “He is very personable and is a good listener but is not at all afraid of tough issues. Jon is one of a small group of people whom I frequently reached out to when I was NIGMS director for his perspectives and advice.  NIGMS will be in good hands.”

Lorsch holds a Ph.D. in biochemistry from Harvard University and completed a postdoctoral stint at Stanford University. His group at Hopkins developed a fully reconstituted yeast translation initiation system, which the group used to understand the molecular mechanisms of the process by which the genetic blueprint in cells gets turned into working protein machines.

According to a Hopkins bio, Lorsch is thought to be the black sheep in his family “because, out of five males, he is the only one without a degree from Harvard Business School.”

The mascot for the American Society for Biochemisty and Molecular Biology needs a name for the upcoming Experimental Biology 2013 meeting. Who better to select one than you? Post your favorite name to ASBMB’s Facebook page.

Schematic to show biomarker identification in Down syndrome patients that link to Alzheimer’s disease. Image provided by Tiziana Cabras.

In a paper just out in Molecular & Cellular Proteomics, researchers report to have identified molecules in the saliva from people with Down syndrome that differ from those in healthy people. The tantalizing thing is that some of the identified molecules are thought to play roles in Alzheimer’s disease. As I briefly note in my latest cover story in ASBMB Today, Down syndrome patients can be particularly susceptible to Alzheimer’s disease.

Saliva contains biomarkers that act as early indicators for various conditions. The appealing thing about saliva is that it’s easy to collect — just spit into a tube and you’re done.

Because of saliva’s importance as a diagnostic fluid, a team led by Tiziana Cabras at the University of Cagliari in Italy wanted to see if it could find differences in biomarkers in those with Down syndrome. The investigators used a method called top-down proteomics to analyze all the salivary proteins in samples taken from 36 Down syndrome people. They then compared the salivary proteomes to those of sex- and age-matched control groups

Most intriguingly, Cabras and colleagues found that the levels of three proteins involved in immune responses, S100A7, S100A8 and S100A12, were increased significantly in the saliva of those with Down syndrome. The increase “may be of particular interest as biomarkers of the early onset Alzheimer’s disease, which is frequently associated with Down syndrome,” says Cabras. S100A7 has been previously shown to be a possible biomarker for Alzheimer’s disease.

Given this tantalizing, preliminary observation, Cabras says the researchers will recruit more patients to their study to see if the observation holds out. They also want to check to see if the same proteins also are increased in the saliva of Alzheimer’s disease patients and those with other neurological diseases. This would confirm that these proteins can act as salivary biomarkers to track the onset and progression of such diseases.

Printing of extended droplet network: Time-lapse movie of an extended droplet network being printed in bulk lipid-in-oil solution. The interval between frames is 20 s. Each frame of the video was cropped around the constructed-network for ease of viewing, as the original recording focused on the stationary capillary-tip, whilst the oil-well was moved in relation to the tip during printing. [Video courtesy of Alexander Graham]

In a paper just out in Science, researchers at the University of Oxford in the U.K. describe making materials in three dimensions that can transmit electrical signals and move in ways similar to how neurons and muscles do. The goal is to make biomaterials  compatible with humans for controlled drug release and repair of damaged organs.

Hagan Bayley says the work began as a basic science project. The investigators were using lipid-coated aqueous droplets in a miniaturized platform  to carry out single-channel recordings of membrane channels and pores. “We quickly realized that we could make interesting devices from collections of droplets,” says Bayley. “Our initial efforts were with just a few droplets, less than 10, and in two dimensions.”

To set up droplets in a network in three dimensions, Gabriel Villar, one of the investigators, made a special 3D printer from scratch. Then taking advantage of the well-established fact that oil and water don’t mix, Villar, Bayley and Alex Graham injected aqueous picoliter droplets into an oil bath. The investigators were able to set up tens of thousands of these droplets and remove most of the oil to form a 3D network of droplets connected by single lipid bilayers.

Each droplet can carry its own specific set of chemicals or biochemicals. When the investigators added a membrane protein called staphylococcal alpha-hemolysin, a pore that incorporates into lipid bilayers, a droplet could use the membrane protein to communicate with its neighbors. It did so by allowing an electric current to flow through the membrane proteins. In this manner, the investigators were able to send a rapid electrical signal along a specific path through the droplet network, much like a neuron.

A rectangular printed droplet network spontaneously folding into a circle.
[Image courtesy of Gabriel Villar, Alexander D. Graham and Hagan Bayley (University of Oxford)]

Bayley says that the investigators would like to move onto making larger networks. (The ones described in the paper were on the order of a few hundred micrometers.) They aim to make more intricate patterns of droplets and fill the droplets with more complex solutions.”The basic principles of how this might be achieved are now clear and further progress will just require time and patience,” says Bayley. “Our ultimate goal is to make materials that can replace or enhance living tissues, but which lack the problems associated with the use of living, replicating cells.”