Method To Produce Proteins In Laboratory Has Now Been Discovered

Main Category: Biology / Biochemistry
Article Date: 13 Dec 2011 - 1:00 PST

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The most abundant and important molecules in all living organisms are proteins; after all they manage to participate in every single one of life's essential reactions. So it is easy to see why scientists have been making such a fuss trying to learn how to synthesise them in laboratory as this would provide them with a tool of extraordinary potential. Unfortunately, this has not proved easy. But an article just out in the journal Science by Bruno Correia, Mihai Azoitei, William Schief and colleagues from the Biochemistry Department of Washington University and the Institute Gulbenkian of Science in Portugal might have found a solution.

The study describes a new method, which the researchers show to work by synthesising a totally new protein that they think can help in the development of a highly effective vaccine against HIV. This result proves the potential of a protocol that, like Correia points out "can now be used to design any protein whether to treat disease, create food, new sources of energy and even for totally new, not yet imagined, functions".

Synthesis of Proteins in Laboratory - one of the "holy grails" of molecular biology, much talked, much chased but not really achieved. The problem is that the function of a protein is determined by its three-dimensional (3D) structure, which in turn is the result on the physicochemical interactions of its different aminoacids (the "bricks" that form the protein). And to predict the combination of amino acids that will give origin to the 3D structure that have the function we want has proved, so far, too difficult.

The new approach by Correia and Azoitei tries to overcome the problem by using a combination of two well known methods - the so called evolutional protocol, in which proteins with a function similar to the one we want are mutated in an attempt to "evolve" into the desired protein and computational design where computers, departing from the different amino acids' characteristics, attempt to predict their right combination to generate the 3D structure with the function we want. In fact,alone, each of these methods has proven to have impossible limitations for example, in the first case, the number of mutated proteins to test is just too large as even a (small) protein of just 100 amino acids will have more than 20 to the power of 100 different mutations (and there is no guarantee that at least one will be functional). In the second method, the problem resides in our still limited lack of computational power that make accurate predictions except for a few very simple proteins impossible . "Our strategy, on the other hand, works explains Correia because we take the best of each of the 2 methods, using the capacity of the computation programs to explore an enormous space of sequences, and the efficiency of the evolutional method to select those that perform the desired function. "

To test the effectiveness of the new strategy the researchers next tried to create a protein that could serve as basis for a more effective vaccine against HIV then the ones developed so far.

So vaccines work by injecting a dead, attenuated or partial pathogen into the organism we want to protect against the pathogen. This triggers an immune response (without disease) that leaves an "immune memory", that if the organism is one day confronted with the "real thing" , rapidly sets off a fast and powerful immune defence. Anti-viral vaccines have a problem though and this is the reason why there are so few and also why we need a new flu vaccine every year and that is that viruses mutate/change rapidly making "immune memories" too often quickly irrelevant. To overcome the problem Correia and Azoitei used the new method to create a totally new protein, one that contained essential "bits" of HIV , essential in the sense that they are known to never mutate. In this case were used parts of the gp120 protein, the molecule used by the virus to get inside the cells to infect.

So how exactly does the new method works?

To start computational methods are used to find the best protein to transplant the gp120 "bits" into (so a protein with a shape that would not disrupt their 3D structure) and then the resulting molecule is mutated (to make it "evolve"). The proteins obtained (each with a different mutation) are then analysed, again by computers, to find the one with a 3D structure closer to HIV. This assures that the antibodies against the vaccine would be highly effective against the live virus.

"And although we are at early stages concludes Correia we hope that our protein can be used to develop a more effective vaccine against HIV as is one that is not expected to lose "power" (memory) over time. And since the method seems to work the possibilities now are endless, after all from diabetes to haemophilia, to mad cow disease and even cancer or Alzheimer's, the problem is always the same: a defective or absent protein..."

Article adapted by Medical News Today from original press release. Source: Ciência Viva
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Cellular Processing Of Proteins Found In Congolese Child Birthing Tea

Main Category: Nursing / Midwifery
Also Included In: Women's Health / Gynecology
Article Date: 13 Dec 2011 - 1:00 PST

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Many plants produce compounds that serve as a defense against predators or pathogens. Some are also used by humans for a variety of beneficial purposes, such as in medicines. As recently as the early 1990s, a unique class of proteins previously unknown to science, the cyclotides, was discovered. First noted through African tribal use as a tea given to speed up delivery during childbirth, cyclotides have since been determined to serve as a powerful insecticidal and nematocidal defense in the plants that produce them, and they also have anti-HIV and antimicrobial properties, with obvious benefits for humans. However, scientists are still working on unlocking much of the basic science of these fascinating proteins, including how they work and where in the plant cell they are produced.

Among the scientists interested in cyclotides, as well as other immune proteins, is Marilyn Anderson of LaTrobe University, Australia.

"Cyclotides are small cyclic peptides of only 28-37 residues that most plant biologists may not have heard of, yet they form the largest family of cyclic proteins described to date in any organism," explains Anderson. "Cyclic cyclotides are widespread in members of the Rubiaceae, Violaceae, Cucurbitaceae and Fabaceae families, yet linear cyclotides are also produced by major monocots such as rice, corn and barley."

"The big question is what do they do?" she continues. "We have discovered that some are potent insecticidal and nematocidal molecules but it is likely that some have other functions as yet undescribed."

Indeed, cycolotides have a unique shape resulting from three disulfide bonds and a peptide backbone that twists in such as way as to produce a cystine knot. This cyclic configuration provides the protein with a very stable structure that is hard to break down - which is how it maintains its bioactivity despite, for example, the high temperatures used to brew the tea used to aid childbirth in the Congo.

"The tea, called kalata kalata, was prepared by boiling the leaves so the active constituent had to be stable to boiling, as well as passage through the human intestinal tract where sufficient amounts were absorbed into the bloodstream to stimulate the uterus," comments Anderson. "Some years after its use was noted, in 1995, the structure of kalata B1, the active constituent from the tea, was solved and its cyclic structure was discovered."

Since then, Anderson, in collaboration with other researchers, has discovered that cyclotides are gene encoded - and in fact are encoded by a single gene, which was at the time unique for a cyclic protein from a eukaryote - and continues to investigate how plants make these cyclic peptides. Her most recent discovery is published in the December issue of the American Journal of Botany. She and her colleagues successfully determined where in the plant cell the cyclotide kalata B1 is produced and how its precursor protein, Oak1, is directed to the appropriate processing location*.

Kalata B1 is found in the leaves of Oldenlandia affinis (Rubiaceae) and, as with all proteins, is made up of building blocks of amino acids put together in a genetically determined sequence. The precursor protein to kalata B1, called Oak1, is linear and is made up of a series of domains, the centerpiece of which contains a cyclotide domain sandwiched between the N- and C-terminal segments. Other cyclotide precursors may contain up to three cyclotide domains.

Anderson and co-authors used a novel approach to determine where in the plant cell the precursor protein was sent and which segment contained the signal sequence responsible for directing it there. They split the precursor protein Oak1 up into its component parts and tagged each segment with the green fluorescent protein (GFP). They then transferred the different constructs into Agrobacterium, injected the Agrobacterium into living leaves of Nicotiana benthamiana, and two days later injected a dye to visually highlight the plasma membrane and the intracellular membranes, such as the tonoplast, which surrounds the vacuole.

Their first finding was that the precursor protein Oak1 was sent to the vacuole to be processed. When viewed under a microscope, cells that were injected with the Oak1-GFP construct had vacuoles that were entirely filled with florescent green dye.

This was a very exciting finding because enzymes hypothesized to play a key role in the ring formation of cyclotides are naturally found in the vacuole. These two pieces of information led the authors to conclude that the vacuole must be the location where Oak1 is converted into kalata B1.

But how is the precursor protein directed to the vacuole?

When the authors looked at cells injected with the different constructs, they found that the only cells that had green florescent vacuoles were those containing constructs with segments of the Oak1 precursor protein that contained propeptides from the N-terminal region. Thus, while the C-terminal segment of the precursor protein is critical for the formation of the ring structure, the N-terminal segments get it to the appropriate cyclization processing location.

Knowing precisely how the precursor protein is directed to its target location (the vacuole) where the cyclotide domain is excised and the ligation of the N- and C- termini occurs is a critical step in understanding the biology of these proteins.

"After conducting the research described in this paper," Anderson concludes, "we now know that the cyclization reactions occur in the vacuole and this provides more insight into the pH conditions required for cyclization and supports our hypothesis that the vacuolar enzyme asparaginyl endoproteinase is the crucial enzyme involved."

Article adapted by Medical News Today from original press release. Click 'references' tab above for source.
Visit our nursing / midwifery section for the latest news on this subject. Conlan, Brendon F., Amanda D. Gillon, Barbara L. Barbeta, and Marilyn A. Anderson. (2011). Subcellular targeting and biosynthesis of cyclotides in plant cells. American Journal of Botany 98(12): 2018-2026. DOI: 10.3732/ajb.1100154 The full article in the link mentioned is available for no charge for 30 days following the date of this summary at http://www.amjbot.org/content/98/12/2018.full.pdf+html. American Journal of Botany Please use one of the following formats to cite this article in your essay, paper or report:

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Proteins Do Not Predict Outcome Of Herceptin Treatment In HER2-Positive Breast Cancer

Main Category: Breast Cancer
Article Date: 09 Dec 2011 - 2:00 PST

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Precisely quantifying the amount of three different HER growth proteins, along with several other proteins believed linked to breast cancer, did not predict a patient's outcome after treatment for HER2-Positive Breast Cancer with Herceptin, say Mayo Clinic researchers. HER2-positive breast cancer gets its name from a protein called human epidermal growth factor receptor 2 that promotes cancer cell growth.

The finding, presented at the 2011 CTRC-AACR San Antonio Breast Cancer Symposium, represents a disappointment to oncologists who had hoped to find distinct biomarkers beyond standard HER2 testing that could help them gauge how well Herceptin will work for patients.

"This study debunks the hopeful notion, strongly felt in the breast cancer community, that measuring levels of a number of different proteins in the HER2 family could help oncologists better tailor their use of Herceptin," says the study's senior investigator, Edith Perez, M.D., director of Mayo Clinic's Breast Program in Florida.

"Improving our ability to predict the benefit of Herceptin treatment beyond testing for HER2 protein and genes remains an important goal, but we are not there yet," she says.

Currently, patients are considered eligible for Herceptin if a pathologist estimates that at least 10 percent of their tumor samples test positive for HER2 growth proteins. However, the test is relatively subjective, based on a HER2 stain on a slide of tumor tissue. While the test can predict the outcome of Herceptin treatment, which shuts down the HER2 growth receptor for some patients, it cannot do so for all patients, Dr. Perez says.

Researchers used a tool that precisely measures the amount of a protein expressed in a cancer sample. According to Dr. Perez, this study was the first to meticulously measure protein levels, including HER2, HER3, HER4, EGFR (epidermal growth factor receptor), ER (estrogen receptor), and PTEN (a tumor suppressor gene) in almost 1,400 tumor biopsies.

Many researchers thought that analysis of the HER3 protein might be a good predictive marker because HER2 and HER3 interact together to promote cancer growth, Dr. Perez says.

"A biopsy could have 80 percent HER3 protein, and it wouldn't be any different in terms of a patient's outcome from Herceptin use than a tumor that had 5 percent HER3 protein," she says.

The next step to finding predictive biomarkers showing a benefit to Herceptin use will be to look at multi-gene profiles, not single biomarkers, Dr. Perez says.

The study was led by Dr. Perez and her team at Mayo Clinic with the collaboration of David Rimm, M.D., Ph.D., at Yale School of Medicine, and investigators who enrolled patients in the N9831 trial throughout the United States. The work was funded by the National Institutes of Health and the Breast Cancer Research Foundation. The original N9831 patient study was partially supported by the National Cancer Institute and Genentech.

Article adapted by Medical News Today from original press release. Source: Elsevier
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