June 30, 2016 § Leave a comment
Seven milliliters of a king cobra’s venom can kill 20 people. But what exactly is in the snake’s venom? Researchers have pursued that question for decades.
Now, in a paper published in the journal Molecular & Cellular Proteomics, a team of researchers reveals a detailed account of the proteins in the venom of king cobras. “I believe this study to be one of the most complete and precise catalogues of proteins in a venom yet obtained,” states Neil Kelleher at Northwestern University, one of the study’s senior investigators.
Snake venoms always have intrigued scientists, because they “have a rich diversity of biological activities,” says Kelleher’s collaborator Gilberto Domont at Universidade Federal do Rio de Janeiro in Brazil. Among other things, venoms contain various proteases, lipases, nerve growth factors and enzyme inhibitors. Besides understanding how venoms function, researchers want to develop better antidotes to snake venom and identify molecules from venom that can be exploited as drugs, such as painkillers, anticlotting medications and blood pressure treatments. Domont points to captopril, a drug now commonly used to treat high blood pressure and heart failure. It was derived from a molecule found in the venom of a poisonous Brazilian viper.
Although the venom of the king cobra, the largest venomous snake in the world, which can stretch up to 13 feet, has been analyzed previously, questions persist about the venom. How do the sequences of the toxins evolutionarily vary? How do some post-translational modifications on proteins make the venom lethal? But to answer these questions, researchers need a proper count of the proteins in king cobra venom.
The advent of proteomics has allowed scientists to survey the rich diversity of proteins in a given sample. There are different approaches that rely on mass spectrometry to carry out proteomic analyses. One approach is called top-down proteomics. It allows researchers to look at proteins as whole, intact entities. In the more conventional approach, called bottom-up proteomics, proteins are cut into bite-sized fragments for analysis.
In bottom-up proteomics, researchers have to use computer algorithms to stitch back together protein fragments identified by mass spectrometry. Top-down proteomics avoids this problem. Its biggest advantage is that it can capture variations within the proteins as well as post-translational modifications.
Kelleher’s group is one of the leaders in developing top-down proteomics, so that’s what the investigators decided to use to analyze king cobra venom. Domont, Kelleher, Domont’s graduate student Rafael Melani and colleagues obtained venom from two Malaysian king cobras held at the Kentucky Reptile Zoo. They analyzed the venom by top-down proteomics in two modes, denatured and native. In the denatured mode, the protein complexes were taken apart; in the native mode, the venom was kept as is so the protein complexes remained intact.
The investigators identified 113 proteins in king cobra venom as well as their post-translational modifications. To date, only 17 proteins had been known in king cobra venom.
November 21, 2014 § Leave a comment
Pharmaceutical compounds in waterways have become a growing environmental concern. Researchers want to know how the human and veterinary drugs affect fish and other aquatic animals as well as how these drug-bearing animals affect the food chain on which we rely.
In a paper just out in the journal Molecular & Cellular Proteomics, investigators analyzed how three common human drugs affect Atlantic salmon. They chose to study Atlantic salmon because wild stocks of the fish are dwindling. Water quality is thought to be one cause of the decline.
The investigators studied the pain-killer acetaminophen, the hypertension drug atenolol, and epilepsy and antidepression drug carbamazepine. These drugs represent different classes of pharmaceuticals.
By carrying out proteomics based on mass spectrometry, the investigators found that exposure to environmentally relevant concentrations of the three drugs changed the protein-expression profile in the livers of the salmon.
In particular, the investigators observed that levels of enzymes involved in energy metabolism, such as mitochondrial ATP synthase, acetyl-CoA acetyltransferase, and glyceraldehydes-3-phosphate dehydrogenase, changed. The first author on the paper, Miriam Hampel at the Andalusian Centre for Marine Science and Technology in Spain, says that it was surprising to see changes in protein-expression levels in the fish even in the presence of low concentrations of the drugs.
Hampel and colleagues now want to see how molecular mechanisms in which these enzymes are involved are affected. As Hampel explains, the most important step will be “to link these and similar findings with physiological effects in exposed organisms that could indicate long-term ecological effects.”
The ecological effects are not just limited to the Atlantic salmon, notes Hampel. “Humans are also more and more exposed to these compounds through drinking water,” she says.
July 28, 2014 § Leave a comment
How do you get into a mutually beneficial relationship? That is the question researchers asked in a recent paper in the journal Molecular & Cellular Proteomics, albeit for a squid and its bacterial partner. The researchers showed that in order for the Hawaiian bobtail squid to form a symbiotic relationship with the bioluminescent Vibrio fischeri, proteomic changes have to occur in a set of cells of the squid.
The Hawaiian bobtail squid, formally known as Euprymna scolopes, has a light organ that is exclusively colonized by V. fischeri. The squid feeds the bacterium a solution of sugar and amino acids and, in return for the steady food supply, the bacterium gives off the light that masks the squid’s silhouette while it goes hunting for various species of shrimp for its own meals.
Scientists study the squid and its bacterial partner as a model to understand how beneficial bacteria form associations with multicellular organisms and help animals develop. “Our lab is interested in understanding the role of the host’s innate immune system in establishing specificity,” says Tyler Schleicher at the University of Connecticut, the first author on the MCP paper. “Each generation of squid is colonized by V. fischeri from the environment, and they must distinguish between the symbiont and a huge background of nonsymbiotic bacteria that are found in seawater.” The question is how the squid achieves this feat.
To answer the question, the investigators used two quantitative proteomic techniques to compare a set of special cells taken from squid colonized with bacteria and those that were uncolonized. “This is the first time that two independent, high-throughput proteomic techniques have been applied to the squid-Vibrio association,” says Spencer Nyholm, also at the University of Connecticut, and the senior author on the paper.
The cells the investigators chose to look at are hemocytes, which are blood cells in the squid’s light organ; these cells have properties of the immune system’s macrophages and interact with the symbiotic bacteria present at the light organ.
From the investigators’ analyses of the differences in protein expression in hemocytes taken from colonized and uncolonized squid, they saw that the presence of V. fischeri in the light organ induced changes in the hemocyte proteome to promote the cell’s tolerance of the bacteria and favor symbiosis. The changes involved the cytoskeleton, lysosome function, proteases and receptors. Because scientists still don’t understand the precise mechanisms that contribute to host-symbiont specificity, the investigators are now focusing on studying several proteins identified in this study that appear to influence the bacterium’s adhesion to the squid’s light organ.
“A growing body of evidence from a variety of animal model systems suggests that beneficial microbes influence a host’s innate immune system to foster these associations,” says Nyholm. Because macrophagelike cells similar to hemocytes are found in almost all animals, he adds, “our study may provide insight into other host-microbe associations.”
June 11, 2014 § 3 Comments
Honeybees give us our honey, royal jelly, pollen, propolis and beeswax and support the ecological structure of the environment by transferring pollen between plants. Despite their ecological and economic importance, very little is known about how honeybee workers develop as embryos into the adult stage at molecular level. In a paper just out in Molecular & Cellular Proteomics, researchers tackled a proteomic analysis of honeybee worker embryos.
They found that while there is a central set of proteins involved in common biological pathways to drive development, “embryos at different developmental stages have their own specific proteome and pathway signatures,” says Jianke Li at the Chinese Academy of Agricultural Science in Beijing. “These findings provide a vital resource as a starting point for further functional analysis and genetic manipulation for both the honeybee embryos” and other eusocial insects, such as wasps, ants and termites.
The investigators studied the worker bees, which are responsible for building the honeycomb, cleaning it, defending the colony, foraging nectar and feeding the larvae. The worker bees, all sterile females, rise out of fertilized eggs in four stages. The first stage is the egg during which the body plan of the insect is established. Li and colleagues used liquid chromatography combined with mass spectrometry as well as bioinformatics to see what proteins were present and how expression changed in embryos during their 72-hour lifecycle.
The investigators found that the core proteome of all stages of embryonic development consisted of proteins involved in protein synthesis, metabolic energy generation and consumption, development, and molecular transporters. But each embryonic stage had specific sets of proteins turned up on top of the core proteome.
Embryos younger than 24 hours had more proteins involved in nutrient storage and nucleic acid metabolism which could correlate with the cell proliferation that happens at the early stage. Embryos during the 24-to-48-hour span expressed proteins responsible for cell cycle control, transporters, antioxidant activity and the cytoskeleton. These proteins may be present to support early formations of organs. The late-stage embryos, during the 48-to-72-hour time frame, produced proteins implicated in fatty acid metabolism and morphogenesis. These proteins could be responsible for the final formation of organs.
As you can see, the study gives researchers an idea of the processes happening at the different stages of embryonic development. Scientists now can use the data to see if particular processes lend themselves well to creating genetically modified honeybees, an active area of research.
February 19, 2014 § Leave a comment
Learning is complicated business, but typical research studies into the molecular basis of learning and memory measure only one or a few proteins. In a study just reported in the journal Molecular & Cellular Proteomics, researchers cast a wider net and looked at 80 proteins in the brain of mice. By looking at more proteins, the study leader’s Katheleen Gardiner at the University of Colorado says researchers can get a better appreciation of “the greater complexity of molecular events underlying learning and memory, how components of a single pathway change in concert and how many pathways and processes respond.”
Gardiner’s research focus is on Down syndrome, two characteristics of which are that patients suffer from some level of intellectual disability and eventually develop Alzheimer’s disease. Gardiner’s group aims to find drugs that can lessen the learning disability. But, in order to do that, researchers need to better understand the molecular events associated with learning, memory, and neurodegeneration.
To get a grasp of the proteins involved in a particular learning process, the investigators studied context fear conditioning in mice. In this type of experiment, mice are put in a new cage and given a small electrical shock. Researchers can tell when a mouse has learned to be fearful of the same cage when the mouse freezes when put back in the cage. This approach “has the advantage that it requires only a single trial, lasting less than five minutes, for mice to learn,” explains Gardiner. “This means that we have a clear window in time where we know molecular events associated with successful learning occur.”
Context fear conditioning demands that the hippocampus, a region of the brain important for memory formation, be functional. The hippocampus is also a part of the brain that degenerates in Alzheimer’s disease.
The investigators gave the mice a drug called memantine, which is used to treat moderate to severe cases of Alzheimer’s disease. The drug has been shown to correct for learning impairment in a mouse model of Down syndrome.
Gardiner’s group used proteins arrays to see how protein expression changed in the brains of mice that underwent context fear conditioning and were given memantine compared with control mice. They found levels of 37 proteins changed in the nuclear fraction of hippocampus. Abnormalities in 13 proteins had been reported in brains of Alzheimer’s patients. “One surprise was that many proteins that increased in level with normal learning also increased, although not as much, with treatment with memantine alone,” says Gardiner. “Memantine induces responses in a substantial number of proteins that we measured, and it does this without impairing or enhancing learning. This indicates that there is considerable flexibility in the timing and extent of protein responses that still result in successful learning.”
In particular, Gardiner’s group identified the MAPK and MTOR pathways to be affected in their experiments, as well as subunits of glutamate receptors and the NOTCH pathway modulator called NUMB. NUMB is known to be essential for some aspects of brain development.
Gardiner says her group is now looking at data from a similar experiment done with a mouse model of Down syndrome. Those mice were unsuccessful with context fear conditioning, but they did as well as wild-type mice when they were treated with memantine.
October 4, 2013 § Leave a comment
Tilapia that grace our dinner tables are interesting critters: The fish can change the workings of their gills based on the saltiness of the water they are in. In a recent Molecular & Cellular Proteomics paper, researchers looked into the molecular details of how tilapia change protein expression in their gills to accommodate for different concentrations of salt.
There are four species of tilapia, which belong to the large family of cichlids, and they all easily mate with one another. The hybrids are grown in fish farms around the world. “These fish have a large economic value as a source of protein and other nutrients,” explains Dietmar Kültz at the University of California, Davis, who was the first author on the MCP paper.
Kültz says tilapia’s ability to easily adapt to their environment has made them an invasive species. They’ve left their native Africa and are swarming into places in North America, such as Florida and Hawaii.
He also points out that projected effects of climate change include rises in sea levels and more frequent droughts. “Knowing the molecular basis of tilapia’s high environmental stress tolerance will offer insight into potential strategies for managing their aquaculture performance and invasiveness,” says Kültz. “In addition, such research reveals the mechanisms that equip fish with an extreme capacity for tolerating salinity stress. Those mechanisms will likely be under great selection pressure in many species of fish exposed to future climate changes.”
Kültz and colleagues used proteomic methods to analyze many of the proteins in the gills of the fish, the organs that take up water to extract oxygen from it. First, the researchers looked at proteins known to be involved in handling salinity. They found that the expression of mitochondrial proteins, molecular chaperones and ion transport proteins was increased as salt concentrations increased.
Next, the investigators looked for novel proteins involved in salinity processing. They discovered a protein called NDRG1 whose expression decreased with increasing salinity. This protein never has been implicated in gill reconstruction, although it was known to be involved in cell proliferation and differentiation. The investigators suspect that NDRG1 stalls cell growth: When salinity increases and the fish need more cells in their gills to handle all the salt ions, they turn down levels of NDRG1.
Kültz explains the investigators are now interested in the mechanisms by which the protein expression levels are altered by salinity and how other organs in the fish cooperate with the gills to increase the fish’s tolerance to rising amounts of salt.
September 9, 2013 § Leave a comment
While much of the recent research focus has been on understanding what makes up a microbiome in various parts of our bodies and how it impacts health and disease, there is less of a focus on how we respond to these communities of bacteria. But in order to do that, researchers need to know which proteins in the huge mix of proteins are actually ours. Teasing out the host proteins has been a technical challenge.
In a paper just out in Molecular & Cellular Proteomics, researchers tackled this issue by developing a method that can pick out the proteome of the host gut and see how it changes to shape the gut environment and nurture its teeming community of microbes. “While it is important to enumerate all the microbes that inhabit our bodies, it may be even more important to measure the ways in which we, as hosts, have evolved ways to shape the microbes’ environment, and how changes in that environment can, in turn, shape our own health,” says senior author Josh Elias of Stanford University.
The investigators studied feces by mass spectrometry to understand how a host proteome responds to a microbiome. The lead author on the paper, Josh Lichtman, had suggested the idea to Elias while he was interviewing for admission into the Stanford University chemical and systems biology graduate program. “Immediately, I agreed that studying gut microbes with mass spectrometry would be a powerful tool for studying this hugely complex system, but that it could get ‘messy,’” notes Elias.
So when Lichtman joined the group, the investigators started on the project. “One of the biggest difficulties in understanding the gut environment is that there are potentially hundreds of thousands to millions of proteins from hundreds of distinct organisms that could be identified,” explains Elias. “The gut environment, with its active proteases, add a further layer of complexity” because the proteases generate peptides that complicate the signals obtained from the mass spectrometric analysis, which involves protease digestion steps. Because the number of bacterial species outnumbers the kinds of animal cells present in the gut, the proteomes of all the bacteria overwhelm that of the host.
To solely focus on proteins from the host, Lichtman devised an enrichment strategy during the step of protein extraction from the fecal samples that made sure that the proteins came from the animal’s intestines, not from bacteria. The method allowed the investigators to track changes in expression of more than 3000 host proteins.
The investigators, in collaboration with Justin Sonnenburg also at Stanford, collected feces from mice that were either germ-free or limited to up to five bacterial species. They also used mice that had the human version of the gut microbiome. These mice, with their increasingly complicated gut microbiomes, allowed the investigators to figure out how well their technique worked in distinguishing between bacterial and host proteomes as well as see how the proteomes changes with more complicated microbiomes. The investigators also applied their method to human samples.
The investigators established that as the gut microbiome got more complex, the host proteome responded in kind by becoming more complex in the types of proteins expressed. “Our work demonstrates that host responses can be readily measured, that these responses are correlated with the underlying microbiota, and we can measure them fairly simply with modern proteomics methods,” states Elias. “There are tremendous amounts of resources currently being spent on evaluating the microbes, in isolation or in aggregate, but often without coincident studies on their host’s biology. We believe our study provides a route towards reconciling the two.”
July 8, 2013 § Leave a comment
We now have the sperm proteome of a primate. In a paper just out in Molecular & Cellular Proteomics, researchers describe the sperm proteome of the rhesus macaque, the first primate to have its sperm proteome analyzed.
Sperm proteomes from non-primate species, such as rat, mouse and fruit fly, already have been determined. “For comparative evolutionary and functional genomics studies, a primate sperm proteome was highly desirable to include in this growing list of sperm proteomes,” explains Tim Karr at Arizona State University.
Rhesus monkeys bear many genetic and physiological similarities to humans, so they are used regularly as a nonhuman primate model system in biomedical research, including human reproduction research. “Knowing the rhesus sperm proteome will greatly expand the possibility for targeted molecular studies of spermatogenesis and fertilization in a commonly used model species for human infertility,” explains Karr. (I wrote about sperm and male infertility earlier this year.)
In their study, Karr and colleagues collected epididymis tissues from male monkeys that contained sperm cells. (The epididymis is a long tubular lumen through which sperm travel after they leave the testis, an essential part of sperm maturation and fertility.) The investigators separated the sperm from the tissue and then proceeded to extract all the proteins from the sperm. The investigators next carried out gel electrophoresis, protein digestion and high-throughput mass spectrometry to identify all the proteins in the rhesus sperm.
From their analysis, Karr and colleagues identified, among other things, new ADAM proteins, ADAMs 3, 4, 6, in the rhesus macaque that have been lost or are nonfunctional in humans. This gives a glimpse of how the two species evolutionarily diverged.
The investigators also identified almost all components of the 20S proteasome core, including known activators of the proteasome. “This suggests there exists an active form of the proteasome in mature sperm,” says Karr.
Karr says he and his colleagues are now “very excited about our developmental work on sperm maturation in the mouse and macaque.” Based on what is known about the two animal sperm proteomes, the investigators now are analyzing the process of sperm maturation during epididymal transport.
April 9, 2013 § 1 Comment
Despite conventional wisdom that pythons are not venomous, python owners may be surprised to learn that their favorite snakes bear traces of venom in their mouths. In a recent paper in the journal Molecular & Cellular Proteomics, researchers described how python oral glands bear the remnants of the venom-producing machinery of their venomous snake and lizard cousins. The finding has important implications for doctors who use snake-venom detection kits to decide if a bitten person needs antivenom.
Bryan Grieg Fry at the University of Queensland in Australia and colleagues have been studying pythons and iguanas for a while because their venom production methods have not been well explored. the team has using a variety of techniques to understand the setup and mechanism of toxin production in these animals.
In previous work, Fry says, the investigators showed that powerful constricting snakes “are not primitive, as is conventionally believed, but actually highly (evolutionary) derived, with constriction as a novel form of prey capture. We had shown in other snakes, such as egg-eating sea snakes, that venom has a use-it-or-lose-it character due to the high energetic costs of making it.” For this reason, constrictors are important to study to understand how venom production has evolved in animals.
Besides being interesting from a fundamental biology standpoint, the work has practical applications. Fry explains that in Australia doctors rely on snake-venom detection kits to diagnose snake bites and figure out which antivenoms to use. “False-positives could lead to patients getting very expensive antivenom they don’t need, potentially triggering life-threatening allergies, but without the benefit of curing a snakebite, as well reducing supply for patients who actually need it,” he says.
A previous study showed that pythons cross-react in the venom-detection kits “but this curious result was dismissed as an anomaly and never thought of again,” says Fry.
In previous work, Fry says, he and his colleagues demonstrated that all snakes evolved from a common ancestor, a venomous lizard. “Snakes have various degrees of venomosity, with some being extremely advanced, such as cobras or taipans, while others have lost almost all their venom, like egg-eating sea snakes or pythons,” he explains.
Their current work in the MCP paper demonstrated that pythons pose a surprising potential source of false-positives when using sVDKs. Fry states, “We show in this paper that even though python oral glands overwhelmingly secrete mucus to swallow their large prey, there is still a trace of venom in there.”
The amount of venom is not enough to harm a human or to kill prey, says Fry, but it’s enough “to mess up an extremely sensitive diagnostic tool.”
Fry adds that he and his colleagues are excited by finding of a low level of evolutionary ancient venom still being secreted by pythons, as the venom could contain novel proteins. “These novel molecules therefore represent an untapped resource for biodiscovery,” notes Fry.
The investigators also delved into understanding the rictal glands, organs that are associated with venom glands. “These glands had only been investigated in a pair of studies almost 100 years ago. The secretions were shown to be highly toxic to the birds injected with it but then no more investigations were undertaken and the glands were forgotten in the sands of time,” says Fry.
In their MCP paper, the investigators showed that these glands are derived from the well-studied venom glands. This means that the venom glands aren’t the only organs involved in the secretion of venom, implicating that venom production in snakes is much more complex than previously thought.