The protein MAEBL is critical for completing the life cycle of malaria parasites in mosquitoes, allowing the insects to transmit the potentially deadly infection to humans, a University of South Florida study has shown. The research may ultimately help provide a way to better control malaria by blocking development of the malaria parasite in the mosquito.

John Adams, PhD, and his team study the complex life cycle of the malaria parasite (on computer screen) to try to find ways to block transmission of the deadly infection.

Researchers with the USF Global Health Infectious Diseases Research team found that the transmembrane protein MAEBL is required for the infective stage of the malaria parasite Plasmodium falciparum to invade the mosquito’s salivary glands. Their findings were published May 28 in the online journal PLoS ONE.

“The mosquito is the messenger of death,” said the study’s principal investigator John Adams, PhD, professor of global health at the USF College of Public Health. “If we could eliminate the parasite from the mosquito, people wouldn’t become infected.”

Plasmodium falciparum causes three-quarters of all malaria cases in Africa, and 95 percent of malaria deaths worldwide. It is transmitted to humans by the bite of an infected mosquito, which injects the worm-like, one-celled malaria parasites from its salivary glands into the person’s bloodstream.

Dr. Adams, center, with his team including, l to r, Steven Maher, Fabian Saenz, PhD, lead author of the PLoS ONE paper, and Sandra Kennedy.

The study was done by genetically modifying the malaria parasites and feeding them in a blood meal to uninfected mosquitoes. Parasites in which MAEBL was deleted were not harbored in the salivary glands of mosquitoes, even though an earlier form of these parasites was observed in the gut of the mosquitoes. The researchers concluded that the transmembrane form of MAEBL is essential for the parasite to enter the mosquito’s salivary glands.

While more studies are needed, lead author Fabian Saenz, PhD, said the finding suggests that silencing the receptor for MAEBL in the mosquito salivary gland might block passage of the parasite through the mosquito, thereby preventing human infection through mosquito bites.

“Our study shows that MAEBL is a weak link in the parasite’s biology,” Dr. Adams said. “This could provide a potential way to block transmission in the mosquito, before the parasite ever has a chance to infect a new person. It is better to prevent the malaria infection from occurring in the first place than having to kill the parasite already inside humans with vaccines or drugs.”

The study was supported by a grant from the National Institute of Allergy and Infectious Diseases. Other study authors were Dr. Bharath Balu, Jonah Smith and Sarita Mendonca.

Microscopic view of an Anopeheles mosquito infected with malaria parasites.

Amyloid deposits in tissues and organs are linked to a number of diseases, including Alzheimer’s, Parkinson’s, type II diabetes, and prion diseases such as BSE. However, amyloids are not just pathological substances; they have potential as a nanomaterials. “The potential applications of these supramolecular assemblies exceed those of synthetic polymers,” state Ehud Gazit and co-author Izhack Cherny in the journal Angewandte Chemie, “since the building blocks may introduce biological function in addition to mechanical properties.”

© Wiley-VCH

Even in nature, amyloids are not merely abnormal, incorrectly folded proteins; they are physiological components of organisms. For example, they are an important protective material in the egg envelopes of insects and fish. They are also involved in the formation of the biofilms of many bacteria, a coating on the surface of the bacterial cells that protects them from antimicrobial substances and facilitates their attachment to surfaces.

Amyloid fibrils are bundles of highly ordered protein filaments made of ladder-like strands and can be several micrometers long. In cross-section, amyloids appear as hollow cylinders or ribbons. Although amyloid fibrils are proteins, they more closely resemble synthetic polymers (plastics) than the usual globular proteins. Amyloids can display amazing mechanical properties similar to spider silk. Spider silk is, by weight, significantly stronger than steel and can be stretched to many times its original length without tearing— properties that have not been reproducible with synthetic fibers.

“The self-assembly properties of amyloids, together with their observed plasticity, makes them attractive natural building blocks for the design of new nanostructures and nanomaterials,” according to the authors from the University of Tel Aviv (Israel). “These building blocks can be broadly varied by means of simple molecular biological techniques.” Surfaces could be given tailored and biocompatible coatings, for example, in analytical flow devices for medical technology or bioanalysis. Other ideas include amyloid hydrogels for the encapsulation and controlled release of drugs and for scaffolds for three-dimensional cell cultures and tissue engineering. Functional proteins such as enzymes could be bound to amyloid-forming sequences to mimic biological processes.

Amyloid fibrils are also suitable as matrices for nanostructures. For example, it has been possible to produce a conducting nanoscale coaxial cable by filling amyloid nanotubes with sliver and externally coating them with gold.

A team of researchers at the National Institute of Standards and Technology (NIST) have demonstrated a new device that creates nanodroplet “test tubes” for studying individual proteins under conditions that mimic the crowded confines of a living cell. “By confining individual proteins in nanodroplets of water, researchers can directly observe the dynamics and structural changes of these biomolecules,” says physicist Lori Goldner, a coauthor of the paper* published in Langmuir.

With the flip of a switch: Nanodrop “test tubes” are created by an electronic switch that causes a micropipette to jerk back and leave behind a droplet less that 1 micron in diameter for study.
Credit: NIST

Researchers recently have turned their attention to the role that crowding plays in the behavior of proteins and other biomolecules—there is not much extra space in a cell. NIST’s nanodroplets can mimic the crowded environment in cells where the proteins live while providing advantages over other techniques to confine or immobilize proteins for study that may interfere with or damage the protein. This more realistic setting can help researchers study the molecular basis of disease and supply information for developing new pharmaceuticals. For example, misfolded proteins play a role in many illnesses including Type 2 diabetes, Alzheimer’s and Parkinson’s diseases. By seeing how proteins fold in these nanodroplets, researchers may gain new insight into these ailments and may find new therapies.

The NIST nanodroplet delivery system uses tiny glass micropipettes to create tiny water droplets suspended in an oily fluid for study under a microscope. An applied pressure forces the water solution containing protein test subjects to the tip of the micropipette as it sits immersed in a small drop of oil on the microscope stage. Then, like a magician whipping a tablecloth off a table while leaving the dinnerware behind, an electronic switch causes the pipette to jerk back, leaving behind a small droplet typically less than a micrometer in diameter.

The droplet is held in place with a laser “optical tweezer,” and another laser is used to excite fluorescence from the molecule or molecules in the droplet. In one set of fluorescence experiments, explains Goldner, “The molecules seem unperturbed by their confinement—they do not stick to the walls or leave the container—important facts to know for doing nanochemistry or single-molecule biophysics.” Similar to a previous work (see “‘Micro-boxes’ of Water Used to Study Single Molecules”, Tech Beat July 20, 2006), researchers also demonstrated that single fluorescent protein molecules could be detected inside the droplets.

Fluorescence can reveal the number of molecules within the nanodroplet and can show the motion or structural changes of the confined molecule or molecules, allowing researchers to study how two or more proteins interact. By using only a few molecules and tiny amounts of reagents, the technique also minimizes the need for expensive or toxic chemicals.

Reference:

* J. Tang, A.M. Jofre, G.M. Lowman, R.B. Kishore, J.E. Reiner, K. Helmerson, L.S. Goldner and M.E. Greene. Green fluorescent protein in inertially injected aqueous nanodroplets. Published in Langmuir, ASAP Article, Web release date: March 27, 2008.

A new research study discovered that scrapie can be transmitted to lambs through milk. The study was published in the online open access journal BMC Veterinary Research. The study provides important information on the transmission of this prion-associated disease and the control of scrapie in affected flocks. Scrapie is a fatal neurodegenerative disease of sheep and goats. Clinical signs include itchiness, head tremor, wool loss and skin lesions as well as changes in behaviour and gait.Timm Konold and colleagues from the Veterinary Laboratories Agency in Weybridge, UK, investigated the transmission of scrapie by feeding milk from scrapie-affected ewes to lambs that are genetically susceptible to contracting scrapie. The researchers were looking for the presence of the prion protein, PrPd, which is associated with the disease.

Eighteen lambs were fed milk from scrapie-affected ewes. Three of these lambs were culled and two were found to have PrPd in intestinal tissues. The prion protein was also detected in lymphoid tissue of the gut of the surviving lambs and in some control lambs mixed with the scrapie milk recipients after weaning. This suggested that scrapie milk recipients were able to shed the infectious agent and infect other lambs. There was no sign of PrPd in tissue samples from a control group of 10 lambs(one culled and the rest alive), which were housed in the same building but fed milk from healthy ewes. The research will continue, to see whether the lambs with PrPd develop the disease as they get older.

This work raises the possibility that other prion diseases could be transmitted in sheep via milk although it should have no direct implications for human health. Scrapie has been found in sheep and has not been shown to be transmissible to humans. BSE has not been found naturally in sheep and occurrence in sheep in the UK is considered to be unlikely. This research adds to our understanding of the transmission of prion diseases in sheep and would help to inform measures needed to protect human health if BSE were ever to be found in sheep.

References on Prion Disease from Milk1. Evidence of scrapie transmission via milk
Timm Konold, S. Jo Moore, Susan J. Bellworthy, and Hugh A. Simmons
BMC Veterinary Research (in press)

Many scientists have tried for decades to understand the mechanism that allows these channels to open. Using cryo-electron microscopy, in which samples frozen at extremely low temperatures are examined under an electron microscope, some researchers obtained images of the closed ion channel. More recently, others used X-ray crystallography to image the closed-channel conformation. This technique involves crystallizing the protein, creating a lattice that reveals many details of its three-dimensional structure.

But until the Illinois team developed a new method for probing the interior of the open channel, no studies had been able to infer the structure of the open channel conformation in a living cell. The Illinois team was able to do this by exploiting electrical properties of these membrane proteins.

Much like the flow of electrons through an electrical wire, the flow of ions through a cell membrane is a current. In the 1970s, two German researchers developed a technique for measuring the current through a single ion channel, an innovation (known as the patch-clamp technique) that won them a Nobel Prize in 1991. Claudio Grosman, a professor of molecular and integrative physiology at Illinois, and Gisela D. Cymes, a postdoctoral associate in his lab, adopted this technique, and predicted that they could use it as a tool for what they call “in vivo, time-resolved structural biology.”

In a study published in 2005, the Grosman lab showed that ionizable amino acids (that is, those that may alternately be charged or neutral) can be engineered into the inner lining of the channel pore. These changes to the amino acid sequence alter the current, revealing the structure of the open-channel conformation in unprecedented detail.

The neurotransmitter acetylcholine is an essential chemical communicator, carrying impulses from neurons to skeletal muscle cells and many parts of the nervous system. Now researchers at the University of Illinois have painstakingly mapped the interior of a key component of the relay system that allows acetylcholine to get its message across. Their findings, which appear in the current issue of Nature Structure & Molecular Biology, reveal how the muscle nicotinic acetylcholine receptor responds to a burst of acetylcholine on the surface of a cell.

The muscle nicotinic receptor is a neurotransmitter-gated ion channel. This “gate” regulates the flow of information, in the form of charged particles, or ions, across the cell membrane. Although normally closed, when the ion channel encounters acetylcholine – or nicotine – on the surface of the cell the interaction causes the gate to open, allowing positively charged ions (called cations) to flow into the cell.

“As the ionizable amino acids bind and release protons from the watery environment, the pore gains or loses a positive charge that interferes with the normal flow of cations through the channel,” Grosman said.
After analyzing the data, Grosman’s team demonstrated that the discrete changes in current reflect the position of each mutated amino acid in the channel and the extent to which water molecules penetrate the membrane protein.

This approach allowed Grosman’s team to map the relative position of every amino acid that formed the ion channel.

The new study extends this work to more distant portions of the protein.

After comparing these findings to direct studies of the structure of the closed channel, Grosman concluded that the conformational changes that allow the channel to open are quite subtle. The five subunits that make up the protein channel do not rotate or pivot dramatically when opening the gate.

“There are many good reasons why I think a subtle conformational change is advantageous from an evolutionary point of view,” Grosman said.

Muscle nicotinic receptors must respond to acetylcholine with staggering speed, opening within microseconds of their exposure to the neurotransmitter.

“These ion channels are meant to be quick,” he said. “If they are too slow, we have disease.”

Grosman said that the approach developed in his lab is the first to allow scientists to infer the structure of an ion channel in its open conformation as it functions in a living cell.

“I know when the protein is open, because in single-molecule experiments the distinction between open and closed conformations is simple; the channel either passes a current or not,” he said.

In a living cell the protein responds, in measurable ways, to changes in its structure and environment, he said. “It’s not frozen at super low temperatures. It’s not in a crystalline lattice. The cells are alive at the beginning of the experiment and when we finish the experiment, the cells keep living.”

Shown is an image of bacteriophage Epsilon15 studied by Wen Jiang, an assistant professor of biological sciences at Purdue. The bacteriophage is shown at a resolution of 4.5 angstrom — the highest resolution achieved for a living organism of this size. Credit Graphic/Wen Jiang lab

WEST LAFAYETTE, Ind. - A team led by a Purdue University researcher has achieved images of a virus in detail two times greater than had previously been achieved.

Wen Jiang, an assistant professor of biological sciences at Purdue, led a research team that used the emerging technique of single-particle electron cryomicroscopy to capture a three-dimensional image of a virus at a resolution of 4.5 angstroms. Approximately 1 million angstroms would equal the diameter of a human hair.

“This is one of the first projects to refine the technique to the point of near atomic-level resolution,” said Jiang, who also is a member of Purdue’s structural biology group. “This breaks a threshold and allows us to now see a whole new level of detail in the structure. This is the highest resolution ever achieved for a living organism of this size.”

Details of the structure of a virus provide valuable information for development of disease treatments, he said.

“If we understand the system - how the virus particles assemble and how they infect a host cell - it will greatly improve our ability to design a treatment,” Jiang said. “Structural biologists perform the basic science and provide information to help those working on the clinical aspects.”

A paper detailing the work was published in the Feb. 28 issue of Nature.

Roger Hendrix, a professor of biological sciences at the University of Pittsburgh, said what is learned about viruses can be applied to many other biological systems.

“Understanding the proteins that create the structure of a virus gives us insight into the tiny biological machines found throughout our bodies,” he said. “Getting to 4.5 angstrom using this technique is a watershed of sorts because it is the first time we can actually trace the polypeptide chain - the backbone of proteins. Now we can see the tiny gears and levers that allow the proteins to move and interact as they carry out their intricate biological roles.”

The imaging technique, called cryo-EM, has the added benefit of maintaining the sample being studied in a state very similar to its natural environment. Other imaging techniques used regularly, such as X-ray crystallography, require the sample be manipulated.

“This method offers a new approach for modeling the structure of proteins in other macromolecular assemblies, such as DNA, at near-native states,” Jiang said. “The sample is purified in a solution that is very similar to the environment that would be found in a host cell. It is as if the virus is frozen in glass and it is alive and infectious while we examine it.”

In addition to Jiang, Matthew L. Baker, Joanita Jakana and Wah Chiu from Baylor College of Medicine, and Peter R. Weigele and Jonathan King from Massachusetts Institute of Technology worked on the project, which was funded by the National Institutes of Health and the National Science Foundation.

The team obtained a three-dimensional map of the capsid, or protein shell, of the epsilon15 bacteriophage, a virus that infects bacteria and is a member of a family of viruses that are the most abundant life forms on Earth, Jiang said.

Other methods of determining the structure could not be used for this family of virus. None had been successfully crystallized, and the complexity of members of this family had prevented evaluation through the genome sequence alone.

“This demonstration shows that cryo-EM is doable and is a major step in reaching the full potential of this technique,” he said. “The goal is to have it reach a 3 to 4 angstrom resolution, which would allow us to clearly see the amino acids that make up a protein.”

In electron microscopy, a beam of electrons takes the place of the light beam used in a conventional microscope. The use of electrons instead of light allows the microscope to “see” in much greater detail.

Cryo-EM cools specimens to temperatures well below the freezing point of water. This decreases damage from the electron beam and allows the specimens to be examined for a longer period of time. Longer exposure time allows for sharper, more detailed images.

Researchers using cryo-EM had obtained images at a resolution of 6-9 angstroms but could not differentiate between smaller elements of the structure spaced only 4.5 angstroms apart.

“There are different elements that make up the protein building blocks of the virus,” Jiang said. “It is like examining a striped blanket. From a distance, the stripes blur together and the blanket appears to be one solid color. As you get closer you can see the different stripes, and if you use a magnifying glass you can see the strands of string that make up the material. The resolution needs to be smaller than the distance between the strands of thread in order to see two separate strands.

“By being able to zoom in, researchers were able to see components that blurred together at the earlier achieved resolution.”

Cryo-EM requires high-end electron microscopes and powerful computing resources. The research team used the Baylor College of Medicine’s cryoelectron microscope. It is expected that Purdue will install a state-of-the-art cryoelectron microscope in 2009.

In 2006 Purdue received a $2 million grant from the National Institute of Health to purchase the microscope. It will be installed in Hockmeyer Hall of Structural Biology, expected to open in 2009.

Computer programs are used to extract the signal from the microscope and to combine thousands of two-dimensional images into an accurate three-dimensional image that maps the structure of the virus. This requires use of a large data set and could not have been done without the resources of Purdue’s Office of Information Technology, or ItaP, Jiang said.

Jiang used Purdue’s Condor program - which links computers including desktop machines and large, powerful research computers - to create the largest distributed computing network at a university.

“ITaP provided us with computational power at the supercomputer scale that was necessary for this work,” he said. “Purdue’s Condor program allowed us to take advantage of the power of 7,000 computers. This was a critical element to our success.”

Jiang plans to continue to refine every step of the process to improve the capabilities of the technique and to examine more medically relevant virus species.

Purdue’s structural biology group studies a diverse group of problems, including cellular signaling pathways, RNA catalysis, bioremediation, molecular evolution, viral entry, viral replication and viral pathogenesis. Researchers use a combination of X-ray crystallography, electron cryomicroscopy, NMR spectroscopy, and advanced computational and modeling tools to study these problems.

HOUSTON — A protein known as REST blocks the expression of a microRNA that prevents embryonic stem cells from reproducing themselves and causes them to differentiate into specific cell types, scientists at The University of Texas M. D. Anderson Cancer Center report in the journal Nature.Researchers show RE1-silencing transcription factor (REST) plays a dual role in embryonic stem cells, said senior author Sadhan Majumder, Ph.D., professor in M. D. Anderson’s Department of Cancer Genetics. “It maintains self-renewal, or the cell’s ability to make more and more cells of its own type, and it maintains pluripotency, meaning that the cells have the potential to become any type of cell in the body.”

The paper posted online March 23 in advance of publication grew from M. D. Anderson research on the protein’s role in medulloblastoma – an exceptionally aggressive pediatric brain cancer.

Embryonic stem cells are essentially blank slates. They have the unique ability to develop from identical, unspecialized cells and then differentiate into distinct types of cells with special functions. In the laboratory, scientists have been able to induce embryonic stem cells to develop into heart muscle cells or insulin-producing cells of the pancreas. The hope is that embryonic stem cells might one day be used to restore or replace failing cells in the human body and perhaps treat a wide range of diseases.

“Embryonic stem cells have a very high potential in medicine,” Majumder said. “The critical thing is to learn the mechanisms that could be used to generate a lot of self-renewing embryonic stem cells and be able to differentiate them into various cell types.” REST could play a key role in maintaining a steady supply of these cells and in preserving their differentiation capability.

Suppressing MicroRNA-21

In studies using mouse embryonic stem cells, the researchers found that REST disarms a specific microRNA called microRNA-21 or miR-21. MicroRNAs are tiny pieces of RNA that control gene expression by binding to the gene’s messenger RNA.

The team found that MiR-21 suppresses embryonic stem cell self-renewal and is associated with a corresponding loss of expression of critical self-renewal regulators, such as Oct4, Nanog, Sox2 and c-Myc. REST counters this by suppressing miR-21 to preserve the cells’ self-renewal and pluripotency.

The researchers discovered the roles of REST and miR-21 in a series of experiments using cultured mouse embryonic stem cells in either a self-renewal state or a differentiating state. They found that REST expression was significantly higher in the self-renewal state. Withdrawing REST reduced the stem cells’ ability to reproduce themselves and started differentiation — even when the cells were grown under conditions conducive to self-renewal. Adding REST to differentiating cells maintained their self-renewal.

These experiments also revealed that REST is bound to the gene chromatin of a set of microRNAs with the potential to target self-renewal genes. REST controls transcription of 11 microRNAs.

REST Implicated in Pediatric Brain Cancer

Previous laboratory research suggests that the qualities that make REST beneficial in stem cell production and pluripotency may contribute to the development of medulloblastoma, an aggressive type of children’s brain tumor. Medulloblastomas are believed to develop from undifferentiated neural stem cells in the external granule layer of the cerebellum.

In earlier research, Majumder’s group at M. D. Anderson discovered that about half of these tumors overexpress REST, which is not found in most neural cells. “We found that REST is a critical factor in this group of children’s brain tumors,” Majumder said, “and that its major function is to keep a group of specific brain stem cells, or progenitor cells, in a state of stemness.”

The researchers hypothesize that by maintaining the neural stem cells’ ‘stemness,’ REST prevents their differentiation into normal and distinct types of cells, leading instead to tumor formation. The M. D. Anderson scientists are now exploring whether microRNAs might also play a role in medulloblastomas.

Understanding REST function has applications in both medulloblastoma and embryonic stem cell biology. “Just as blocking REST function has therapeutic potential in medulloblastoma, blocking REST function to allow for differentiation of embryonic stem cells is a potentially critical step in regenerative medicine,” Majumder said.

UPTON, NY - Biologists at the U.S. Department of Energy’s Brookhaven National Laboratory, Stony Brook University, and the University of Wurzburg, Germany, have deciphered the structure of a large protein complex responsible for adding sugar molecules to newly formed proteins - a process essential to many proteins’ functions. The structure offers insight into the molecular “sugar-coating” mechanism, and may help scientists better understand a variety of diseases that result when the process goes awry. The research will appear in the March 12, 2008, issue of the journal Structure.”Proteins perform their functions by interacting at their surfaces with other molecules. So you can imagine that adding or removing sugar molecules will change the protein’s surface structure, and therefore its function,” said Huilin Li, a biologist at Brookhaven Lab who holds a joint appointment at Stony Brook and is co-corresponding author on the Structure paper. “Messing up this process can lead to the production of malformed proteins that are unable to do their jobs,” he added.

The results can be devastating. Failure of glycosylation, as the “sugar-coating” process is known, can lead to a variety of genetic disorders characterized by neurological problems including seizures and stroke-like episodes, feeding disorders, and possibly even some forms of muscular dystrophy.

“We studied one enzyme involved in glycosylation, the one that recognizes the protein sequence and adds the sugar chains to the protein as it is being synthesized by the cell,” said William J. Lennarz of Stony Brook University, a coauthor on the paper. “The challenge is that the enzyme, known as oligosaccharide transferase (OT), is large by protein standards, has eight intricately linked components, and sits embedded in a membrane within the cell’s protein-manufacturing machinery.”

“Membrane proteins, particularly large ones, are very difficult to study structurally,” added Li.

So the scientists turned to a technique called cryo-electron microscopy (cryo-EM), which shows great promise in deciphering large membrane protein structures.

“We imaged the purified OT complex by cryo-EM and obtained a first snapshot of the complex by computer reconstruction of the micrographs,” said Li, a cryo-EM expert.

In cryo-EM, he explained, samples are frozen in vitreous ice and maintained at cryogenic temperatures (-274° Fahrenheit) using liquid nitrogen while the samples are photographed in the high vacuum of an electron microscope. The sophisticated cryo-EM machine resides in Brookhaven Lab’s biology department. Li and his collaborators also measured the mass of the OT complex at Brookhaven’s Scanning Transmission Electron Microscope (STEM) facility.

The structure deciphered by the group helps to explain many biochemical phenomena observed about the enzyme complex over the past two decades. It also offers hints as to how the enzyme performs its various jobs, from recognizing the sugar molecules to be added to the protein, scanning the protein as it is formed to identify the sites where sugars should be attached, and transferring the sugar molecules to the protein at the right positions.

“OT physically associates with the protein translocation channel which moves a protein across a membrane and the cell’s protein synthesis machinery, forming an efficient three-machine assembly line for protein translation, translocation, and glycosylation,” Li said.

The researchers say further research is needed to illuminate the molecular mechanisms of disorders of glycosylation involving oligosaccharide transferase. For example, they would like to do structural studies of the enzyme at higher resolution in complex with substrates or in association with the cell’s protein translocation and protein synthesis machinery. A new facility Brookhaven Lab hopes to begin construction on next year, known as the National Synchrotron Light Source II, would greatly increase the precision of this work.

This research was supported by the National Institutes of Health and by Brookhaven National Laboratory’s Laboratory-Directed Research and Development funds.