Protein behavior might hold the key to synthetic silk

A trans-Atlantic collaboration of scientists has revealed the structure of a key protein of silk and discovered a previously unknown behavior of this protein: to self-organize into tiny fibrils a single molecule in diameter. This discovery sets the stage for the eventual creation of synthetic silk—not just the luxury fabric that’s a product of silkworms, but also the manufacture of ultra-tough spider silk familiar to fans of the Marvel superhero Spider-Man.

Hannes Schniepp cautions that the world’s textile mills aren’t likely to start producing “spidey silk” in the near future, but says his work describing the structure of the silk protein and its self-organizing behavior is an important step in that direction. Schniepp is an assistant professor in the Department of Applied Science at the College of William & Mary. Along with graduate student Minzhen Cai and a set of collaborators at the University of Oxford in the United Kingdom, he has published a paper describing silk at the molecular level.

“Silk is a polymer,” Schniepp explained. “It’s not a synthetically-made polymer, but it’s a polymer made out of proteins.”

Synthetic plastics are polymers, but these macromolecules are common in the natural world, too. Schniepp pointed out that much of the human body—including DNA—is constructed of various polymers. 

“What’s so fascinating about silk is that in terms of its mechanical properties, silk is better than any polymer that we can make synthetically,” he said. “Particularly, certain spider silks are even tougher than Kevlar, the best high-performance polymer we have.”

You can’t farm spiders

For millennia, people have been using the cocoons of silkworms to weave silk cloth. Humans have used spider silk to a much lesser degree, but spiders have proven to be impossible to cultivate: “They start eating each other,” Schniepp says.

Figuring out a process to make synthetic silk has been a sort of Holy Grail of materials science for nearly as long as people have been making silk. After years of scientific study, the exact natures of both the biochemistry and the mechanics of silk creation by silkworms and spiders remain elusive.

“The big question really is how does the spider do it? How does the silkworm do it?,” Schniepp says. “The problem is it’s a tiny animal and it happens really in very small dimensions inside the animal, and it’s really almost impossible to watch what’s going on there.”

He said that most of the scientific study on structure of silk has focused on examination of the product through microscopy and other analytical tools. The study has yielded a fair amount of understanding about the structural nature of silk, but scientists had no idea of what shape an individual silk protein had.

Schniepp and his team at William & Mary took a different approach than most materials scientists, sampling “silk dope,” the gel-like material inside the silkworm that the worm exudes to spin its cocoon.

“A lot of these biomolecules, they’re very sensitive to changes. So the closer you can be to the native state, the more valuable this information is that you get,” he said.

Working with silk dope

Schniepp and his research group examined the silk dope in their McGlothlin-Street Hall laboratory, using an atomic force microscope (AFM), an instrument capable of looking at materials at the nanoscale. Before placing them in the AFM, they prepared their silk dope, diluting the samples with a bit of water, then spun the sample on a plate, so that the silk spread out on the surface.

“When you spin liquid on a plate like this, you shear it. And that does something to these proteins that’s similar to the way that the animals do,” he explained. “They have a gland that produces this material and at the end is something like a nozzle. So they squeeze this material out through the nozzle. To create a similar effect, you shear the solution. By spinning it very quickly, the liquid is forced away, and it is similar to what happens when the animal pushes the silk out.”

A number of curious things happen when the material is sheared. For one thing, the water-soluble silk dope has been transformed into something waterproof. More importantly, the shearing somehow induces individual proteins to “find each other,” as Schniepp describes, and to self-organize into fibrils. One molecule thick, the fibrils are the thinnest possible threads of silk and are precursors to silk fibers.

Seen through AFM magnification, each fibril shows where the individual proteins have conglomerated. The magnification resembles a string of pearls. It’s the first time that the structure of the native silkworm protein has been imaged at such high resolution.

The work on silk is supported by the Jeffress Memorial Trust. Schniepp published his findings in a paper, “Shear-Induced Self-Assembly of Native Silk Proteins into Fibrils Studied by Atomic Force Microscopy” in the journal Biomacromolecules. Fritz Vollrath of Oxford University is a co-author, as is Cai. They are continuing their work on the structure of the material.

“We don’t know what other secrets silk has hidden for us,” Schniepp says.

Provided by The College of William & Mary

'Genetic biopsy' of human eggs might help pick the best for IVF

Given the stakes of in vitro fertilization, prospective parents and their doctors need the best information they can get about the eggs they will extract, attempt to fertilize, and implant. New research at Brown University and Women & Infants Hospital of Rhode Island has found a way to see which genes each egg cell is expressing without harming it. As researchers learn more about how those genes affect embryo development, the new technique could ultimately give parents and doctors a preview of which eggs are likely to make the most viable embryos.

In the research, now in press in the Journal of Biological Chemistry, the team of physicians and biologists were able to sequence the transcribed genetic material, or mRNA, in egg cells and, in a scientific first, in smaller structures pinched off from them called "polar bodies." By comparing the gene expression sequences in polar bodies and their host eggs, the researchers were able to determine that the polar bodies offer a faithful reflection of the eggs' genetic activity.

"We can now consider the polar body a natural cytoplasmic biopsy," said study co-author Sandra Carson, professor obstetrics and gynecology at the Warren Alpert Medical School of Brown University and director of the Center for Reproduction and Infertility at Women & Infants Hospital.

Polar bodies are where egg cells dispense with the second copies of chromosomes that, as sex cells, they don't need. But the polar bodies also capture a microcosm of the egg's mRNA, the genetic material produced when genes have been transcribed and a cell is set to make proteins based on those genetic instructions.

Pairs of genes

Last year the team became the first to find mRNA in human polar bodies. Now they have transcribed it in 22 pairs of human eggs and their polar bodies, and confirmed that what is in the polar bodies is a good proxy for what is in the eggs.

Given how little mRNA is present in polar bodies, the task was not easy, said Gary Wessel, professor of biology, but through a combination of clever amplification and analysis techniques by lead author and graduate student Adrian Reich and second author Peter Klatsky, the team got it done.

"There's no reason this should have worked, just because there was so little material," Wessel said. "Single-cell sequencing is very challenging."

To hedge their bets the team analyzed most of their samples in two pools of 10 cells each, for instance comparing the mRNA in 10 eggs with the mRNA in the 10 related polar bodies. But to their pleasant surprise, they were also able to sequence two individual eggs and their polar bodies directly.

What they found is that more than 14,000 genes can be expressed in the eggs. Of those, more than 90 percent of the genes detected in the polar bodies were also detected in the eggs and of the 700 most abundant genes found in the polar bodies, 460 were also among the most abundant in the eggs.

Toward clinical use

"It seems that the polar body does reflect what is in the egg," Carson said. "Because the egg is the major driver of the first three days of human embryo development, what we find in the polar body may give us a clue into what is happening during that time."

But Carson and Wessel acknowledged that more research will be required to create a clinically useful tool.

Finding which genes affect embryo viability is the next major step. With the new knowledge and techniques developed in their study, the researchers said, scientists could analyze the mRNA from polar bodies of eggs that are fertilized and track the progress of the resulting embryos. Once the key genes are known, they could create fast assays to look for those genes in polar bodies so that clinicians and patients could pick the best eggs. A sufficiently developed technology could also be used for choosing which eggs to bank for later use.

"We don't quite have the answer of what those messages are doing exactly or necessarily the purpose of them in the cell function, but that's to come," Carson said. "Now we have the words, but not the sentences."

Provided by Brown University (news : web)

Combining two peptide inhibitors might block tumor growth

A new study suggests that combining two experimental anticancer peptide agents might simultaneously block formation of new tumor blood vessels while also inhibiting the growth of tumor cells.

This early test of the two agents in a breast cancer model suggests that the double hit can stifle tumor progression, avoid drug resistance and cause few side effects, say researchers at the Ohio State University Comprehensive Cancer Center – Arthur G. James Cancer Hospital and Richard J. Solove Research Institute (OSUCCC – James) who developed the agents and evaluated their effectiveness in laboratory and animal tests.

The scientists designed one of the agents to prevent human epithelial growth factor from interacting with HER-2, a molecule that marks a particularly aggressive form of breast cancer. The other inhibitor blocks the action of vascular endothelial growth factor (VEGF), which stimulates the growth of new blood vessels that tumors need to grow beyond a certain size.

The findings are described in two papers published online in the Journal of Biological Chemistry. One presents the development of a novel VEGF inhibitor; the other describes the HER-2 inhibitor and the preclinical testing of the two agents together.

"When we combined our peptide HER-2 inhibitor with the VEGF peptide that inhibits angiogenesis, we observed significant additive benefits in reducing tumor burdens in preclinical studies," says principal investigator Pravin Kaumaya, professor of obstetrics and gynecology, of molecular and cellular biochemistry, and of microbiology, and director of the division of vaccine development at the OSUCCC – James.

The strategy of targeting both HER-2 and VEGF pathways should also discourage the development of drug resistance, Kaumaya says, because it simultaneously inhibits two pathways that are essential for tumor survival. "Combined peptide inhibitors might be appropriate in several types of cancer to overcome acquired resistance and provide clinical benefit," he adds.

Peptide inhibitors consist of short chains of amino acids (the VEGF inhibitor is 22 amino acids long) that conform in shape to the active site of the target receptor. In addition, Kaumaya engineered the VEGF peptide to be resistant to protease, an enzyme, thereby increasing its efficacy. The shape of the peptide HER-2 inhibitor engineered by Kaumaya and his colleagues, for example, is highly specific for the HER-2 receptor. It physically binds to the receptor, which prevents another substance, called epithelial growth factor, from contacting the receptor and stimulating the cancer cells to grow.

Other categories of targeted drugs in clinical use are humanized monoclonal antibodies and small-molecule TKI inhibitors. Both groups are associated with severe side effects and are very expensive, Kaumaya says. "We believe peptide inhibitors offer non-toxic, less-expensive alternatives to humanized monoclonal antibodies and small-molecule inhibitors for the treatment of solid tumors, with the potential for improved efficacy and better clinical outcomes," he says.

Provided by Ohio State University Medical Center