RxPG News : Proteins

Scientists develop a general control switch for protein activity

Wed, 20 Jun 2007 19:59:37 PST

( from http://www.rxpgnews.com ) Our bodies could not maintain their existence without thousands of proteins performing myriad vital tasks within cells. Since malfunctioning proteins can cause disease, the study of protein structure and function can lead to the development of drugs and treatments for numerous disorders. For example, the discovery of insulin's role in diabetes paved the way for the development of a treatment based on insulin injections. Yet, despite enormous research efforts led by scientists worldwide, the cellular function of numerous proteins is still unknown. To reveal this function, scientists perform various genetic manipulations to increase or, conversely, decrease the production of a certain protein, but existing manipulations of this sort are complicated and do not fully meet the researchers' needs.

Prof. Mordechai "Moti" Liscovitch and graduate student Oran Erster of the Weizmann Institute's Biological Regulation Department, together with Dr. Miri Eisenstein of Chemical Research Support, have recently developed a unique 'switch' that can control the activity of any protein, raising it several-fold or stopping it almost completely. The method provides researchers with a simple and effective tool for exploring the function of unknown proteins, and in the future the new technique may find many additional uses.

The switch has a genetic component and a chemical component: Using genetic engineering, the scientists insert a short segment of amino acids into the amino acid sequence making up the protein. This segment is capable of binding strongly and selectively to a particular chemical drug, which affects the activity level of the engineered protein by increasing or reducing it. When the drug is no longer applied, or when it is removed from the system, the protein returns to its natural activity level.

As reported recently in the journal Nature Methods, the first stage of the method consists of preparing a set of genetically engineered proteins (called a 'library' in scientific language) with the amino acid segment inserted in different places. In the second stage, the engineered proteins are screened to identify the ones that respond to the drug in a desired manner. The researchers have discovered that in some of the engineered proteins the drug increased the activity level, while in others this activity was reduced. Says Prof. Liscovitch: "We were surprised by the effectiveness of the method - it turns out that a small set of engineered proteins is needed to find the ones that respond to the drug. With their greater resources, biotechnology companies will be able to create much larger sets of engineered proteins in order to find one that best meets their needs."

The method developed by the Weizmann Institute scientists is ready for immediate use, both in basic biomedical research and in the pharmaceutical industry, in the search for proteins that can serve as targets for new drugs. Beyond offering a potent tool that can be applied to any protein, the method has an important advantage compared with other techniques: It allows the total and precise control over the activity of an engineered protein. Such activity can be brought to a desired level or returned to its natural level, at specific locations in the body and at specific times - all this by giving exact and well-timed doses of the same simple drug.

In addition, the method could be used one day in gene therapy. It may be possible to replace damaged proteins that cause severe diseases with genetically engineered proteins, and to control these proteins' activity levels in a precise manner by giving appropriate doses of the drug. Another potential future application is in agricultural genetic engineering. The method might make it possible, for example, to create genetically engineered plants in which the precise timing of fruit ripening would be controlled using a substance that increases the activity of proteins responsible for ripening. Moreover, numerous proteins are used in industrial processes, as biological sensors and in other applications. The possibility of controlling these applications - strengthening or slowing the rate of protein activity in an immediate and reversible manner - can be of great value.

MITOMI Device Enables Huge Leaps in Mapping Protein Function

Thu, 11 Jan 2007 13:06:01 PST

( from http://www.rxpgnews.com ) Howard Hughes Medical Institute researchers have designed a laboratory about the size of a quarter that is capable of conducting thousands of experiments simultaneously to measure how specialized proteins bind their DNA targets. This tool provides a new way of measuring the activity and function of proteins. Having those measurements may help scientists predict the behavior of individual proteins in biological systems without making any direct measurements on model organisms.

Howard Hughes Medical Institute investigator Stephen Quake, whose lab is at Stanford University, and co-author Sebastian Maerkl published a research article describing their new approach in the January 12, 2007, issue of the journal Science.

“The goal of systems biology is to understand how biological networks act in concert to enable an organism to function,” said Quake. “Very powerful high-throughput methods have been developed to map the network topologies, revealing what interacts with what. But if you point to a particular node on the network and ask how it is functioning, frequently all you can say is that there is some interaction going on. You rarely have the biochemical depth of knowledge to properly understand network function from the bottom up.”

“We ought to be able to make biophysical characterizations of all these protein machines, and if we understand how these machines work, then, in principle, the only other information we need to predict how the organism works is a blueprint from the genome,” he said. The microlaboratory that Quake and Maerkl have developed could help bring researchers closer to this goal.

To understand complex biological systems and predict their behavior under particular circumstances, it is essential to characterize molecular interactions in a quantitative way, Quake said. Binding energy-the energy with which one protein bind to another or to DNA-is one important quantitative measurement researchers would like to know. But these interactions are highly transient and often involve extremely low binding affinities, so they are difficult to measure on a large scale. To overcome this hurdle, Quake and Maerkl set out to develop a microlaboratory that could trap a type of protein known as a transcription factor. Once the transcription factor was trapped, the scientists hoped to measure the binding energy of the transcription factor bound to specific DNA sequences.

But simply measuring the binding energy between a transcription factor and a single DNA sequence is not enough, Quake said. He said it would be more meaningful to know the energy involved in a transcription factor binding to many different DNA sequences. This would give researchers a more complete picture of the “DNA binding energy landscape” of each transcription factor.

To determine the binding energy landscape, Quake and Maerkl's microlaboratory needed to conduct thousands of binding-energy experiments at once. The apparatus they created, which they called mechanically induced trapping of molecular interactions (MITOMI), consists of 2,400 individual reaction chambers, each controlled by two valves and including a button membrane. Each of the chambers is less than a nanoliter in volume. That's one-billionth of a liter—enough to hold a snippet of human hair only as long as the hair's diameter. The MITOMI apparatus fits over a 2,400-unit DNA microarray, or gene chip, onto which the researchers can dab minute amounts of DNA sequences. Each spot of DNA is then enclosed in its own reaction chamber.

The researchers constructed the MITOMI apparatus by first producing silicon molds using the same photolithography process used to make microelectronic circuits. They then cast the elements of the MITOMI apparatus in rubber, before bonding them together to create the finished device.

The MITOMI process begins by pumping a transcription factor into the chambers containing the thousands of slightly varied DNA sequences anchored to the microarray chip. There, the transcription factor interacts with the DNA. The scientists then lower the button membrane in each chamber onto the microarray surface, physically trapping the transcription factors that have bound to the DNA. The researchers can then conduct measurements that reveal the binding energy of the trapped transcription factor. Quake emphasized that MITOMI can be used to measure the interactions between any two proteins, as well as between a protein and DNA.

To demonstrate the technique, Quake and Maerkl characterized the binding energy landscape of two versions of a human transcription factor called MAX, as well as two yeast transcription factors, called Pho4p and Cbf1p. They chose the transcription factors because they represent a large family of proteins that have a characteristic structure called a basic helix-loop-helix. This family regulates genes involved in an array of cellular processes ranging from cell proliferation and development to metabolism. The MAX transcription factors also play a role in some cancers.

Quake said the MITOMI technique enabled them to predict the biological function of these transcription factors purely from the physical measurements of the binding energies. “We discovered a wealth of interesting things—to me the most important being that using the binding energy data for different sequences, we could predict which genes the yeast transcription factors would regulate,” he said.

Quake and Maerkl also used MITOMI to test the widely held assumption that each nucleotide unit of a DNA sequence functions independently in contributing to the binding of a transcription factor. Their measurements of how the transcription factors bound to a multitude of slightly different DNA sequences revealed a flaw in that assumption: They showed that the nucleotides act cooperatively in establishing the binding energy of the transcription factor.

As a rapid, high-throughput technique for measuring binding between proteins, MITOMI is a powerful new approach to mapping biological networks, said Quake. “We would like to use this technique to map all the protein-protein binding energies in an organism. For even a small bacterium, this means millions of interactions, but with MITOMI, we can hope to accomplish such measurements,” he said. The next effort in his laboratory will be to use measurements of the binding energy of unknown transcription factors as clues to identify the genes they regulate.

According to Quake, MITOMI brings scientists closer to an important goal. “To test theories of systems biology, we should now be able to predict biology without making any measurements on the organism itself,” he said.

First major study of mammalian 'disorderly' proteins

Tue, 10 Oct 2006 12:58:37 PST

( from http://www.rxpgnews.com ) Investigators at St. Jude Children's Research Hospital turned up the heat on "disorderly" proteins and confirmed that most of these unruly molecules perform critical functions in the cell. The St. Jude team completed the first large-scale collection, investigation and classification of these so-called intrinsically unstructured proteins (IUPs), a large group of molecules that play vital roles in the daily activities of cells.

The new technique for collecting and identifying IUPs is important because although scientists have been aware of the existence of flexible proteins for many years, they have only recently realized that these molecules play major biological roles in the cell, according to Richard Kriwacki, Ph.D., an associate member of the St. Jude Department of Structural Biology. Moreover, he said, previous work by other researchers suggested that a large proportion of IUPs in mammalian cells play key roles in transmitting signals and coordinating biochemical and genetic activities that keep the cell alive and functioning. Kriwacki is senior author of a report on this work that appears in the prepublication online issue of Journal of Proteome Research.

"Until now there was no way to separate IUPs in large numbers from the more structured proteins and confirm their roles in the cell," Kriwacki said. "Our new technique selectively concentrates the IUPs that are involved in regulating functions in the cell and transmitting signals within them."

Unlike the classic description of proteins described in science textbooks, IUPs are not completely locked into rigid, 3-D shapes that determine their function in the cell. Instead, IUPs have varying amounts of flexibility within their sometimes spaghetti-like structures that is critical for function. For example, one protein named p27 initially looks like a SlinkyTM toy. However, when p27 goes to work, it puts a vise-like grip on an enzyme that otherwise would promote uncontrolled cell division.

The St. Jude team developed a technique that uses heat to isolate IUPs in large, purified quantities from extracts of a standard type of cultured mouse cells called NIH3T3 fibroblasts. The IUPs were resistant to the heat, unlike more structured proteins, which fell apart. Based on these studies, the investigators were able to classify all proteins into one of three categories: IUPs; intrinsically folded proteins (IFPs, i.e., fully folded into specific shapes); or mixed ordered or disordered proteins (MPs), which have both structured and unstructured parts.

"This work further illustrates that the disorderliness of IUPs isn't just a curiosity," said Charles Galea, Ph.D., a postdoctoral fellow in Kriwacki's lab. "This characteristic is a fundamental part of how these proteins work. So determining their exact nature, including the parts that are disordered, is an important part of understanding how they work. This is especially important in the case of IUPs linked to cancer and other diseases." The paper's first author, Galea, did much of the work on this project.

Exploring mechanics of chromatid cohesion

Wed, 05 Jul 2006 14:52:37 PST

( from http://www.rxpgnews.com ) Over the long course of life's history, the appearance of a new function in an organism may be accompanied by a new protein. But, more often, the work is done by an old one that adds a new role to its repertoire. Such proteins are likely to be found in a wide variety of organisms, reflecting their ancient lineage and continuing relevance. Proteins never act in isolation, of course; instead, they bind to one or more others to carry out their tasks. And so, if one member of a protein pair has taken on a new function, it's a good bet the other may have done so as well. In a new study, Vlad Seitan, Tom Strachan, and colleagues show that two proteins, whose interactions in yeast help chromosomes divide, have counterparts in a full range of other organisms, including humans. And true to prediction, the proteins don't just continue to play their old rolesin animals, they also appear to help guide multicellular development.

The focus of the study is a pair of yeast proteins, Scc2 and Scc4. Bound together, they load the protein complex cohesin onto chromosomes to link together sister chromatids, ensuring proper separation in mitosis. Scc2 has orthologsproteins with similar structure sharing a common ancestorin both fruit flies and humans, known respectively as Nipped-B and delangin. But, until very recently, orthologs of Scc4 have not been found outside of a few fungal species.

The authors set out to find binding partners for Nipped-B and delangin. Using Nipped-B as the bait, they snagged the protein product of the fly gene CG4203. The human counterpart of this protein, called KIAA0892, bound to delangin. Because both CG4203 and KIAA0892 are related to a nematode protein called MAU-2, the authors dubbed them fly and human MAU-2. Each of these was about the same size as Scc4 and, using specialized bioinformatics approaches, they confirmed that the sequences of all three were related. Thus, Scc2 is to Scc4 as Nipped-B is to fly MAU-2, and delangin is to human MAU-2.

Up to this point, the only demonstrated functional similarity between Scc4 and the MAU-2s was their ability to bind their respective partners. To test whether human MAU-2 had a similar role in linking sister chromatids, the authors used RNA interference to diminish MAU-2 expression. When the level of MAU-2 was low, less cohesin was loaded onto the chromosomes, and sister chromatids prematurely separated, just as in yeast.

The nematode version of MAU-2 was originally identified as having a role in guiding cell movements and growth of axons during development. Did it also play a part in chromatid cohesion in the worm? Once again, RNA interference showed it did. Finally, if MAU-2 has a developmental role in the worm, what about in other organisms? When the authors used antisense to reduce MAU-2 in the frog, early development was delayed and the embryo displayed multiple defects. Reduction of frog delangin caused similar defects, indicating the two likely pair in this organism as well.

These findings shed light on the mechanics of chromatid cohesion, and will be useful for further elucidating the complex means by which chromatids remain together and then separate during mitosis. They also indicate that both subunits take part in shaping development. How they do so is not yet clear, but the role of the pair in controlling chromosome structure suggests they may help modify chromatin outside of the events of mitosis. Further study of this activity will likely help illuminate the pathologic mechanism of a rare human developmental disorder, Cornelia de Lange syndrome, which can be caused by a mutation in the delangin gene and which is characterized by low birth weight, slow growth, and multiple physical abnormalities.

Shape of a Common Protein Module Munc-13 Suggests Role as Molecular Switch

Sat, 10 Jun 2006 13:06:37 PST

( from http://www.rxpgnews.com ) A vital aspect of a neuron's job is deciding when to pass their cache of chemicals on to neighboring cells. To do this in a way that ensures effective communication, neurons must keep tight reins on their neurotransmittersthe chemical messengers they release to influence neighboring cells. Neurons quickly collect and then jettison these neurotransmitters, cycling through this process many times per second.

Now a new study reveals the structure of a fragment of Munc-13a key protein in this processshowing how it could act as a switch that neurons use to toggle quickly between storing and releasing their neurotransmitters. For this switching role, the key part of the protein is one of its C2 domains (the C2A domain). This domain is widespread, suggesting that other proteins may function by a similar mechanism.

Neurons store their neurotransmitters in vesicles, tiny sacs that amass at the synapse between cells. There, they dock at the inside of the cell membrane and undergo one or more reactions that prime the vesicles to be ready to quickly unload their neurotransmitters outside the cell when the neuron fires. A cascade of protein interactions mediates these different steps.

These protein cascades not only allow neurons to do their usual duty, but they also help neurons process information. If neurons react quickly or sluggishly, reliably or not, to a neuron firing, this affects the signal that gets passed on to neighboring neurons. And, if their reactions change with time, this plasticity can help the animal remember, learn, and adapt.

To understand how the proteins interact, a key step is figuring out the shapes the proteins assume. Once they have the shapes, researchers can see how proteins, like pieces of a jigsaw puzzle, fit together. For example, this helps researchers make sense of chemical and biological data that suggest how proteins bind together and influence each other.

Josep Rizo and colleagues targeted a protein called Munc13-1 since its family of proteins plays important roles in preparing vesicles to release neurotransmitters, and Munc13-1 itself is known to have a role in the plasticity of synapses. Rizo and colleagues homed in on one of the key zones of Munc13-1, called the C2A domain. C2 domains are well known for being able to bind to phospholipids, the fatty acids that make up cell and vesicle membranes. But the researchers found an unexpected role for the Munc13-1 C2A domain: it can bind to other proteins as well.

To figure out the shape of Munc13-1's C2A domain, down to the atomic level, the researchers combined two methods: X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy. X-ray crystallography is the classic method for solving the mystery of a protein's shape. But not all proteins crystallize well, and so are not amenable to the method. Or if they do crystallize, their shape may be distorted from the form it normally takes when floating in a cell's watery interior.

Rizo and colleagues combined the two methods to create a detailed picture of the protein's shape. They used NMR spectroscopy to check how well the protein behaves in solution and to optimize the crystallization, aiding the X-ray studies. Also, data from the NMR spectroscopy on the shape of the protein helped them interpret the X-ray results. In the end, they were able to figure out the natural shape of the protein's C2A domain to atomic resolution.

These structural data also helped Rizo and colleagues make sense of their experiments designed to determine Munc13-1's binding partners. The researchers found that, in a pure solution, Munc13-1 proteins bound to each other in pairs. But when mixed with RIM2α (another protein that plays a key role in neurotransmitter release and neuron plasticity), then Munc13-1 formed pairs with RIM2α instead.

Combining the structural and binding data, the researchers were able to piece together a picture of how Munc13-1 could work as a switch. They found that the C2 domain of Munc13-1 has two adjacent and partially overlapping regions that can bind to other proteins. So two C2 domains could bind each other using one part of that domain, and another part of the domain could bind to RIM2α. But the C2 domain can't bind both at the same time. The researchers could see, once they had the shapes of the proteins, that they simply can't all fit together at once. Thus it seems that there is competition for Munc13-1 to bind either to itself or to other proteins. This mechanism could prepare the vesicles to be ready to unload the neurotransmitters when the neuron fires.

Since the C2 domain is widespread among proteinsand is highly similar among distantly related speciesthe findings could hold clues to understanding more roles for this common sequence of amino acids.

New process to inhibit zinc finger protein, HIV NCp7

Wed, 31 May 2006 17:09:37 PST

( from http://www.rxpgnews.com ) Using small molecules containing platinum, Virginia Commonwealth University Massey Cancer Center researchers have created a process to inhibit a class of proteins important in HIV and cancer.

The findings may help researchers develop new drugs to fight HIV or cancer by selectively targeting proteins known as zinc fingers.

In the May 30 issue of the journal Chemistry & Biology, researchers reported that a zinc finger protein, known as HIV NCp7, can be inhibited when it is exposed to a platinum complex. They observed that when the HIV NCp7 protein interacts with platinum, the zinc portion of the molecule is ejected from the protein chain. This causes the protein to lose its tertiary structure or overall shape. For these molecules, shape is an important property that enables the protein to carry out certain biological functions.

The process, active site displacement, involved design of a platinum drug with higher affinity for the protein peptide backbone, thus eliminating the zinc from its active site.

In the specific case discussed in the paper, the HIV NCp7 protein is responsible for the proliferation of the HIV virus. If researchers can inhibit the action of this zinc finger protein, they can stop the spread of the virus.

"When we target specific viruses with drugs, over time patients can become resistant to treatment and the drug becomes ineffective. Therefore, novel biological processes and proteins are attractive targets for antiviral drug development," said lead author Nicholas Farrell, Ph.D., professor and chair in the Department of Chemistry at VCU and a member scientist with the VCU Massey Cancer Center.

According to Farrell, these study findings may also one day be applied to the selective targeting of zinc fingers involved in the biological processes responsible for the spread of cancer. By applying the concept to development of anticancer drugs, the researchers hope to design more specific clinical agents with reduced side effects compared to the very useful, but toxic, cisplatin and congeners.

New methods of structural genomics have accelerated studies of individual proteins

Thu, 11 May 2006 17:32:37 PST

( from http://www.rxpgnews.com ) Biologists have long been thwarted in determining the three-dimensional structure of proteins that carry out their jobs only after intimately embracing other proteins. Although the structures of these complexes could reveal a bounty of new details about how proteins function, this information has been slow in coming because the work is difficult and time consuming.

Meanwhile, says Howard Hughes Medical Institute investigator David Eisenberg, the new methods of structural genomics have accelerated studies of individual proteins, but fail in many cases for proteins that are members of complexes.

Eisenberg and his colleagues at the University of California, Los Angeles, decided the time was right to develop a new approach to tackling the structure of protein complexes. In an article published during the week of May 8, 2006, in the Early Online Edition of the Proceedings of the National Academy of Sciences (PNAS), they describe the development of a genomic and structural-analytical approach that promises to rescue partner proteins from obscurity.

In the experiments presented in PNAS, Eisenberg, his graduate student Michael Strong, and others offer a proof-of-principle example of how this technique plays out in a real world application. They used their new approach to determine the structure of a protein complex unique to the tuberculosis bacterium. The fact that the protein complex, called PE/PPE, is only found in the bacterium Mycobacterium tuberculosis makes it a possible target for anti-tuberculosis drugs that would be more effective and specific than current antibiotics.

In the major NIH-sponsored structural genomics projects, a large fraction of proteins have been essentially lost from the research pipeline because either they are not expressed or they are expressed in an insoluble form, said Eisenberg. A protein's solubility is a critical factor for further structural analysis by widely used techniques such as nuclear magnetic resonance spectroscopy and x-ray crystallography.

Eisenberg and his colleagues sought to develop a technique that would enable expression of both partners of a protein complex. The technique involves first performing computational genomic analysis to infer whether two proteins might be functionally linked. For example, if the genes for the proteins lie close together in the genome, they might be more likely to exist as partners in the cell.

Second, the researchers engineered the genes for the proteins so that they would be expressed together in the bacterium E. coli. They could then use co-purification techniques to determine whether the proteins form a paired complex. Finally, the researchers used standard structural analytical techniques to determine the structure of the purified protein pair.

Our idea is that you could rescue from the trash bin of structural genomics many of the proteins that fall out of the pipeline, and then restore them in complexes, said Eisenberg. That approach has two advantages. First, you learn the structure of the protein that you started out to study. But probably more important, because it's in a complex with other proteins and you might know something about those other proteins you begin to learn about its molecular biology because it's through protein-protein interactions that the cell operates.

In their proof-of-principle demonstration, the researchers determined the structure of a pair of members of two large families of proteins called PE and PPE found uniquely in the tuberculosis bacterium. While the proteins make up a significant fraction of the tuberculosis bacterial genome, their structures and functions remain unknown, said Eisenberg. Despite extensive efforts in Eisenberg's laboratory, neither the PE nor PPE proteins could be isolated.

Also, we thought this family might be a good family to target because there are no human homologs for them; therefore, drugs against those proteins might interfere with TB but would be less likely to produce side effects, said Eisenberg

Genomic analysis by the paper's first author, Michael Strong, revealed that the genes for members of the two protein families were often found near one another. Such proximity implied that the proteins would be produced together and would function together, said Eisenberg.

The researchers chose two particular members of the PE and PPE families that were very closely associated on the genome and had size and structural characteristics that seemed likely to make them easier to analyze. The researchers found that they could, indeed, express the two proteins together in E. coli. And when they purified the proteins together, they found clear evidence that the proteins existed only as a complex in the cell. Using the complexed proteins to produce crystals, the researchers performed x-ray crystallographic analysis, developing the structure of the complex. This analysis revealed that the proteins' structures made them natural fits for one another.

The researchers also used computational analysis to compare the PE/PPE protein structure with proteins of known function, to deduce the possible role they might play in the cell. That analysis indicated that the complex had characteristics of proteins that function in a biological signaling pathway, perhaps in the cell membrane, said Eisenberg.

Eisenberg said that the findings with PE/PPE illustrate the value of the new approach to analyzing the structure of complexed proteins. To me, these findings say that protein partnering is a very sensible way to go in these structural genomics projects, he said. That is, you can identify those proteins that fail to be expressed individually and use computational genomic analysis to infer possible protein partners. And then on that basis, if there is a strong indication of one or more protein partners, you could co-express the protein with the others and determine whether they are, indeed, partners. The technique will help narrow the chasm between structural genomics and structural biology.

Although the functions of PE/PPE are still largely unknown, the new structural information offers hints of possible applications for treating tuberculosis. From such structures, one could try to design a compound that would interfere with the protein complex and keep it from forming, Eisenberg said. Interfering in such a way could kill the bacterium. And other critical proteins in the family might form the same kinds of complexes, so they may also be vulnerable to such compounds.

Eisenberg and his colleagues are now planning to apply their technique to a broader range of protein complexes. They are also exploring whether one partner protein might be used as a hook to draw another insoluble protein, such as a membrane protein, into solution for structural analysis.

Wnt - One Signal, Multiple Pathways: Diversity Comes from the Receptor

Wed, 05 Apr 2006 18:47:37 PST

( from http://www.rxpgnews.com ) Type Wnt into Google Scholar, and you'll get nearly 72,000 hits, revealing the pivotal role this widely conserved family of signaling proteins plays in development and disease. Wnt proteins trigger complex signaling cascades that regulate cell growth, migration, differentiation, and many other aspects of development with the help of numerous interacting components. In the best-understood, canonical pathway, Wnt signaling molecules (called ligands) bind simultaneously to two coreceptors on the cell surface (Frizzled and LRP), allowing β-catenin proteins to stabilize (avoid destruction), enter the nucleus, associate with the transcription factor complex TCF/LEF, and activate genes involved in cell survival, proliferation, or differentiation. Inappropriate activation of β-catenin has been linked to several types of cancer.

Wnt ligands have also been implicated in several alternative, noncanonical pathways, challenging researchers to figure out how proteins that appear so similar at the sequence level can produce such different results. Studies in frogs and zebrafish embryos suggest this diversity derives from engaging multiple pathways, with Wnt5a, for example, triggering an intracellular calcium release that activates calcium-dependent signaling molecules. It's also possible that Wnt5a signals through other receptors (besides the canonical Frizzled receptor) with a Wnt-binding domain, such as the receptor tyrosine kinase-like orphan receptor 2 (Ror2). But because isolating Wnt ligands in a soluble form has proven difficult, scientists have been forced to resort to indirect methods of studying the mechanisms of Wnt studies, which often provided varying and conflicting results.

In a new study, Amanda Mikels and Roel Nusse have developed a technique to purify the Wnt5a protein and directly investigate its contribution to different pathways. They show that soluble Wnt5a proteins can both inhibit and activate the canonical pathway, depending on which combination of receptors is expressed on the cell surface. When Wnt5a interacts with Ror2, the canonical Wnt/β-catenin pathway is inhibited; when it engages Frizzled and LRP, the β-catenin pathway is activated.

The researchers modified a Wnt purification technique previously established in their lab to harvest Wnt5a proteins from cells engineered to overexpress the mouse Wnt5a gene, and confirmed the identity of the protein by examining a key part of its amino acid sequence. Having confirmed the identity of the protein, they compared Wnt5a's capacity to mediate signaling in cells expressing different combinations of the Ror2, Frizzled, and LRP surface receptors. They also examined Wnt5a's capacity to modulate signaling by Wnt3a, which is known to activate the canonical pathway.

First, the researchers tested the possibility that Wnt5a could also activate β-catenin signaling in a cultured cell line (called 293 cells) and found that it could not. But when they treated cells with both Wnt3a and Wnt5a, they discovered that Wnt5a could prevent activation of the β-catenin-dependent TCF transcription factor by Wnt3a. It was initially unclear how this happened. Wnt5a could compete with Wnt3a for the Frizzled receptor, or it might activate a gene that targets β-catenin for destruction. Either way, β-catenin levels should drop following treatment with Wnt5a. Yet β-catenin levels were unaffected; furthermore, Wnt5a didn't interfere with β-catenin's entry into the nucleus. These results indicate that Wnt5a did not block Wnt3a signaling through either of these routes. The researchers also show that Wnt5a doesn't rely on calcium-dependent signals, as had been suggested in previous work. Thus, Wnt5a must act through some other pathway to block β-catenin signaling by canonical Wnts such as Wnt3a.

Previous studies had suggested that Wnt5a might be able to bind another cell-surface receptor, Ror2, based on evidence that blocking expression of either Wnt5a or Ror2 produces the same effects in animals. And this line of investigation proved fruitful: Mikels and Nusse found that Ror2 is needed for Wnt5a-mediated repression of canonical β-catenin signaling. Additionally, by creating multiple Ror2 constructs lacking different combinations of their binding domains, they showed that Wnt5a binding triggers Ror2-mediated signaling inside the cell.

Interestingly, under very specific conditionswhen the coreceptors Frizzled 4 (Frz4) and LRP5 are presentWnt5a can actually trigger β-catenin accumulation and activate canonical β-catenin gene targets. Since 293 cells do not normally express Frz4, but do express Ror2, the predominant signal prompted by Wnt5a in these cells is inhibition of β-catenin signalingindicating that different combinations of cell-surface receptors drive different signaling outcomes for Wnt5a.

Whether Wnt5a inhibits β-catenin signalingperforming its job as a tumor suppressoror activates β-catenin's cell growth and proliferation targetssetting the stage for tumor formationdepends on which receptors are present on the surface of the cell in question. The next challenge will be to identify the mechanism through which Wnt5a blocks the β-catenin pathway. By showing that one Wnt ligand can function through two separate pathways, Mikels and Nusse have opened the floodgates for researching the possibility of dual functionality in the 19 mammalian homologs identified so far. This ability to stimulate different receptors with distinct results is unique for Wnt proteins, but it has been documented in other systems and may well represent an alternate strategy for effecting flexible responses under changing conditions.

Prostate cancer manipulates Wnts signaling proteins in bony metastasis

Tue, 06 Sep 2005 20:39:38 PST

( from http://www.rxpgnews.com ) New research by scientists at the University of Michigan's Comprehensive Cancer Center suggests that prostate cancer manipulates an important group of signaling proteins called Wnts (pronounced wints) to establish itself in bone. By changing the amount and activity of Wnt proteins, prostate cancer cells upset the normal balance between formation and destruction of bony tissue.

There is strong evidence that Wnt proteins play a central role in regulating normal skeletal development in an embryo, says Christopher L. Hall, Ph.D., a senior research fellow in urology at U-M. But this is the first time Wnts have been shown to be involved in abnormal bone production in adult animals with prostate cancer.

Hall is first author of a paper to be published in the Sept. 1 issue of Cancer Research, which presents results from U-M studies of Wnt proteins in human prostate cancer cell lines and in laboratory mice injected with prostate cancer cells.

Normal bone growth and remodeling depends on a controlled balance between production of new bone and resorption of existing bone, says Evan T. Keller, D.V.M., Ph.D., a professor of urology and pathology in the U-M Medical School, who directed the U-M study. When a tumor forms in bone, it upsets this balance.

Several types of cancer metastasize to bone, according to Keller, but most of them tip the balance toward destruction producing what scientists call osteolytic lesions, or holes in the bone. Prostate cancer is unique in its ability to trigger increased bone production, which creates what's called an osteoblastic lesion.

In metastatic prostate cancer, we think that both processes are going on, Keller says. Our hypothesis is that prostate cancer cells first induce more bone resorption to help the invading cells become established in bone. But then there's a switch to increased bone production. Although we don't know the exact mechanism responsible for the switch, we know that it's related to the activity of Wnt proteins in prostate cancer cells.

In the first phase of their research, U-M scientists measured the amount of Wnt protein in cells from normal human prostate tissue, localized prostate cancer and metastatic prostate cancer cells. Using the same cell lines, they also looked for the presence of a protein called DKK-1, which is known to inhibit Wnt activity. They discovered that the amounts of Wnt and DKK-1 protein present in human prostate cells varied inversely with the developmental stage of prostate cancer.

As the cancer progressed, DKK-1 levels went down, Hall says. Cells with osteoblastic activity had high levels of Wnt activity and low levels of DKK-1, while cells with osteolytic activity showed decreased Wnt activity and high levels of DKK-1.

Our results suggest that DKK-1 may act like a switch on prostate cancer cell activity, Keller says. When we altered the cells to increase the amount of active DKK-1, it blocked Wnt's signal, changing prostate cancer cells from an osteoblastic to a highly osteolytic cell line.

To test their hypothesis, U-M scientists injected human prostate cancer cells into the tibias, or long leg bones, of one group of immune-deficient mice. Twelve weeks later, U-M researchers removed and examined bone tumors from the mice. They found that these mice produced tumors with a dense overgrowth of bone. A second group of mice, injected with prostate cancer cells made to express the Wnt inhibitor, DKK-1, developed highly osteolytic tumor lesions, which destroyed most of the bone.

This demonstrated that Wnts promote the overproduction of bone by prostate cancer cells, Keller says.

In previous research, the U-M team found that preventing the osteolytic changes associated with bone resorption also prevented prostate cancer from establishing itself in bone. By learning how DKK-1 blocks Wnt's signal to prostate cancer cells, they hope to learn how to control physical changes in bone that encourage the development of metastatic tumors.

Our goal is to find ways to manipulate this Wnt pathway to slow the growth of tumors in bone or decrease the tumor-associated pain, Keller says. We won't be able to stop the primary tumor from releasing cells, but by preventing early bone resorption, we may be able to prevent metastatic cells from getting a foothold in bone.

In future research, U-M scientists will try to identify which of the nearly 20 known Wnt proteins is involved in bone changes associated with metastatic prostate cancer.

Ryk, Wnts and Frizzled3 receptors in neuronal regeneration - Study

Mon, 15 Aug 2005 20:48:38 PST

( from http://www.rxpgnews.com ) The same family of chemical signals that attracts developing sensory nerves up the spinal cord toward the brain serves to repel motor nerves, sending them in the opposite direction, down the cord and away from the brain, report researchers at the University of Chicago in the September 2005 issue of Nature Neuroscience (available online August 14). The finding may help physicians restore function to people with paralyzing spinal cord injuries.

Growing nerve cells send out axons, long narrow processes that search out and connect with other nerve cells. Axons are tipped with growth cones, bearing specific receptors, which detect chemical signals and then grow toward or away from the source.

In 2003, University of Chicago researchers reported that a gradient of biochemical signals known as the Wnt proteins acted as a guide for sensory nerves. These nerves have a receptor on the tips of their growth cones, known as Frizzled3, which responds to Wnts.

In this paper, the researchers show that the nerves growing in the opposite direction are driven down the cord, away from the brain, under the guidance of a receptor, known as Ryk, with very different tastes. Ryk sees Wnts as repulsive signals.

"This is remarkable example of the efficiency of nature," said Yimin Zou, Ph.D., assistant professor of neurobiology, pharmacology and physiology at the University of Chicago. "The nervous system is using a similar set of chemical signals to regulate axon traffic in both directions along the length of the spinal cord."

It may also prove a boon to clinicians, offering clues about how to grow new connections among neurons to repair or replace damaged nerves. Unlike many other body components, damaged axons in the adult spinal cord cannot adequately repair themselves. An estimated 250,000 people in the United States suffer from permanent spinal cord injuries, with about 11,000 new cases each year.

This study focused on corticospinal neurons, which control voluntary movements and fine-motor skills. These are some of the longest cells in the body. The corticospinal neurons connect to groups of neurons along the length of spinal cord, some of which reach out of the spinal cord. They pass out of the cord between each pair of vertebrae and extend to different parts of the body, for example the hand or foot.

Zou and colleagues studied the guidance system used to assemble this complex network in newborn mice, where corticospinal axon growth is still underway. Before birth, axons grow out from the cell body of a nerve cell in the motor cortex. The axons follow a path back through the brain to the spinal cord.

By the time of birth, the axons are just growing into the cord. During the first week after birth they grow down the cervical and thoracic spinal cord until they reach their proper position, usually after seven to ten days.

From previous studies, Zou and colleagues knew that a gradient of various Wnt proteins, including Wnt4, formed along the spinal cord around the time of birth. Here they show that two other proteins, Wnt1 and Wnt5a are produced at high concentrations at the top of the cord and at consecutively lower levels farther down.

They also found that motor nerves are guided by Wnts through a different receptor, called Ryk, that mediates repulsion by Wnts. Antibodies that blocked the Wnt-Ryk interaction blocked the downward growth of corticospinal axons when injected into the space between the dura and spinal cord in newborn mice.

This knowledge, coupled with emerging stem cell technologies, may provide the most promising current approach to nervous system regeneration. If Wnt proteins could be used to guide transplanted nerve cells -- or someday, embryonic stem cells -- to restore the connections between the body and the brain, "it could revolutionize treatment of patients with paralyzing injuries to these nerves," Zou suggests.

"Although half the battle is acquiring the right cells to repair the nervous system," he said, "the other half is guiding them to their targets where they can make the right connections."

"Understanding how the brain and the spinal cord are connected during embryonic development could give us clues about how to repair damaged connections in adults with traumatic injury or degenerative disorders," Zou added.