Two biologists at the University of California, San Diego have discovered the first of a new class of cellular motor proteins that “rewind” sections of the double-stranded DNA molecule that become unwound, like the tangled ribbons from a cassette tape, in “bubbles” that prevent critical genes from being expressed.

“When your DNA gets stuck in the unwound position, your cells are in big trouble, and in humans, that ultimately leads to death” said Jim Kadonaga, a professor of biology at UCSD who headed the study.  “What we discovered is the enzyme that fixes this problem.”

The discovery represents the first time scientists have identified a motor protein specifically designed to prevent the accumulation of bubbles of unwound DNA, which occurs when DNA strands become improperly unwound in certain locations along the molecule.

The UCSD researchers’ findings, detailed in the October 31 issue of Science, are also important because they provide biomedical scientists with a greater understanding of the molecular mechanisms that lead to a rare genetic disorder called Schimke immuno-osseous dysplasia.  The discovery will eventually allow medical researchers to design future treatments for this devastating genetic disorder, which causes strokes, congestive heart failure, kidney failure and death in young children.

“We knew this particular protein caused this disease before we started the study,” said Kadonaga.  “That’s why we investigated it.  We just didn’t know what it did.”

What this protein, called HARP for HepA-related protein, did astounded Kadonaga and Timur Yusufzai, a postdoctoral fellow working in his laboratory.  The two molecular biologists initially discovered that this motor protein burns energy in the same way as enzymes called helicases and, like helicases, attached to the dividing sections of DNA.  But while helicases use their energy to separate two annealed nucleic acid strands—such as two strands of DNA, two strands of RNA or the strands of a RNA-DNA hybrid— the scientists found to their surprise that this protein did the opposite; that is, it rewinds sections of defective DNA and thus seals the two strands together again.

As a consequence, the UCSD biologists termed their new enzyme activity an “annealing helicase.”

“We didn’t even consider the idea of annealing helicases before this study started,” said Kadonaga.  “It didn’t occur to us that such enzymes even existed.  In fact, we never knew until now what happened to DNA when it got stuck in the unwound position.”

Now scientists who study the action of helicases on DNA and RNA have an entirely new class of proteins to investigate.

“This will open up a whole new area of study,” said Kadonaga.  “There are very few enzymes known that alter DNA structure.  And we’ve discovered an entirely new one.  This was not expected to happen in the year 2008.  We should have found them all by now.”

“I believe it’s going to go beyond DNA.  Just as there are DNA-DNA helicases, there are RNA-DNA helicases and RNA-RNA helicases.  So it doesn’t take a lot of imagination to foresee that there are probably going to be RNA-DNA annealing helicases and RNA-RNA annealing helicases.  The field potentially can be fairly large.  And as more and more people discover additional annealing helicases, this field will expand.”

Kadonaga and Yusufzai are already searching for more annealing helicases, but they also plan to continue their studies of HARP.

“First, what we want to do is find more of these proteins, so we’re looking for more right now,” said Kadonaga.  “We also want to see what other specific processes are affected by this particular protein, HARP, in the cell.”

Amoebas glide toward their prey with the help of a protein switch that controls a molecular compass, biologists at the University of California, San Diego have discovered.

Their finding, detailed in this week’s issue of the journal Current Biology, is important because the same molecular switch is shared by humans and other vertebrates to help immune cells locate the sites of infections.

The amoeba Dictyostelium finds bacteria by scent and moves toward its meal by assembling a molecular motor on its leading edge. The active form of a protein called Ras sets off a cascade of signals to start up that motor, but what controlled Ras was unknown.

Richard Firtel, professor of biology along with graduate student Sheng Zhang and postdoctoral fellow Pascale Charest tested seven suspect proteins by disrupting their genes. One called NF1, which matches a human protein, proved critical to chemical navigation.

NF1 turns Ras off. Without this switch mutant amoebas extended false feet called pseudopodia in all directions and wandered aimlessly as Ras flickered on and off at random points on their surfaces. “You have to orient Ras in order to drive your cell in the right direction,” Firtel said.

In contrast, normal amoebas with working versions of NF1 elongate in a single direction and head straight for the most intense concentration of bacterial chemicals, the team reports.

The biochemical components of the system match those found in vertebrate immune cells called neutrophils that hunt down bacterial invaders, suggesting that the switch might be a key navigational control for many types of cells, Firtel said. “The pathway and responses are very similar and so are the molecules.”

In a paper published today in Nature, the team led by Professor Nigel Robinson have revealed a mechanism that ensures the right metal goes to the right protein. Proteins are essential and involved in just about every process in living cells.

Life, microbe, plant or human, is a painstaking assembly of trillions of atoms. The atoms include metals such as copper and manganese which act as catalysts in proteins. The proteins wrap around the metal atoms.

The research team has shown that to ensure a copper and a manganese protein wrap around the correct metal atoms they do this in different parts of the cell, in zones which contain different metals. Therefore, which protein attaches to which metal is determined by where the folding action takes place in the cell.

Previously, a common view was that the right metals were simply those which were most attracted to the protein, but in this work that is not the case.

Professor Nigel Robinson at Newcastle University who led the research says: “This has taken us one step closer to understanding why metals and proteins assemble in the ways they do.”

“One motive behind the work is pure curiosity, but as so many proteins need metals this type of work has many potential uses - for example, in synthetic biology which is striving to produce green power from bacteria by using energy from sunlight to produce hydrogen gas, a process which needs nickel and iron.

“It may also help in diseases such as Alzheimers where there are unexplained links to proteins binding metals such as copper. There’s also application in controlling infections by Staphylococcus aureus; a bacterium which our bodies defences succeed - or sometimes fail - in killing by removing manganese and zinc from abscesses.”

The researchers have shown that the way the metals attach is identical for a protein that binds manganese to one that binds copper. In both cases the metals bind inside protein barrels with the same type of metal-attractions.

Carrying out the work in a blue-green algae, a cyanobacterium, the team has been able to show that a protein requiring copper transports to the periplasm, the outer area of the cell, where it then folds around the available metal, which is copper.

Conversely, manganese but not copper atoms are found in the cytosol, in the middle of the cell. The team has demonstrated that a protein requiring manganese folds in the cytosol. The manganese protein is then transported to the periplasm having first trapped its manganese.

The cyanobacterium organism was chosen because it has a high demand for these two metals which are required for proteins involved in photosynthesis. These metals were chosen because they lie towards opposite ends of a chemical series called the Irving-Williams series, such that selecting these metals for proteins should be especially demanding.

In the work funded by the BBSRC, the Newcastle University team first developed a new approach to discover metal-binding proteins. This is now being swiftly applied to lots of other types of living cells and other essential metals (zinc, nickel, cobalt, iron). Unexpectedly, x-ray crystal structures showed that the identified proteins, MncA for manganese and CucA for copper, were both cupins (Latin for barrels) with identical sets of atoms for binding to the metals. Consistent with the chemical series, a ten-thousand times excess of manganese over copper was needed to fill the MncA barrel with manganese when folding is done in the laboratory.

Once folded, the manganese site is buried, the metal trapped inside the protein, and so the manganese protein can subsequently co-exist with the copper protein because its’ metal becomes impervious to replacement by metals further up the Irving-Williams series.

The work exemplifies a cell overcoming the metal binding preferences of proteins.

The new discipline of synthetic biology aims to engineer cells to carry out useful tasks, for example to generate valuable compounds. Because metals are the catalysts for so much of biology, knowing how to engineer a supply of the right metals to the right proteins will be important to the success of these ventures.

Using computer models and live cell experiments, biomedical engineers at the Johns Hopkins University School of Medicine have discovered more than 100 human protein fragments that can slow or stop the growth of cells that make up new blood vessels.

Reporting online last week in the Proceedings of the National Academy of Sciences, the researchers say the findings could lead to developing treatments to fight diseases that depend on the growth of new blood vessels, including cancer, macular degeneration and rheumatoid arthritis.

“Before, there were only 40 known antiangiogenesis peptides,” says Aleksander Popel, Ph.D., a professor of biomedical engineering at Hopkins.  “Now, using a whole-genome, computer-based approach, we have identified more than 100 new ones, all of which can be further researched for their ability to fight the more than 30 known diseases affected by excessive blood vessel growth.”

To identify short protein fragments — peptides — that can block blood vessel growth, the team started by looking at 40 known peptides that have been studied and characterized by other experts in the field to stop blood vessel growth in animal models of disease.  Working under the assumption that the antivessel activity of these peptides can be attributed to similar features that are shared by a number of proteins, like the sequence of the peptide building blocks, the team first categorized the 40 known peptides by where they are located and what they look like.

Having defined nine families, the researchers then used computer programs and compared the peptide families to all of the proteins encoded by the genome.  They found more than 120 peptides contained in 82 different proteins, many of which were not previously known to have any activity on blood vessel development.

“Computational methods only identify potential candidates,” says Popel.  “We next had to do the experiments on live cells to see if they had any real activity.  Of the 82 proteins we identified, most were not previously known to have any antiangiogenic activity.”

To test the activity of these candidate peptides, the researchers applied them to blood vessel cells growing in the lab and examined whether they had any effect on the growth, survival and movement of these cells.  To test growth and survival, they added different amounts of peptide to dishes containing roughly 2,000 cells and after three days, counted how many cells were still alive.

To test cell movement, they placed cells in double-chambered dishes and treated the cells with a growth factor known to encourage cells to move.  To some of the dishes they added the test peptides.  After 20 hours, they measured the number of cells that had crawled from one chamber to the other.  They then identified the protein receptors that the peptides bind to and were able to show in some cases that combinations of more than one peptide were better able to stop the cells than using single peptides.

“Basic, computational studies like this are critical to understanding normal blood vessel growth,” says Popel.  “A better understanding of normal growth gives us a better idea of what happens in disease.”

The next step, Popel says, is to test these peptides in animal models of human disease and to identify the diseases most appropriately treated by these newly identified peptide inhibitors.

Researchers at the University of Toronto have shown that the EBNA1 protein of Epstein-Barr virus (EBV) disrupts structures in the nucleus of nasopharyngeal carcinoma (NPC) cells, thereby interfering with cellular processes that normally prevent cancer development.  The study, published October 3rd in the open-access journal PloS Pathogens, describes a novel mechanism by which viral proteins contribute to carcinogenesis.

EBV is a common herpesvirus whose latent infection is strongly associated with several types of cancer including NPC, a tumor that is endemic in several parts of the world.  With NPC only a few EBV proteins are expressed, including EBNA1.  EBNA1 is required for the persistence of the EBV genomes, however, whether or not EBNA1 directly contributes to the development of tumors has not been clear, until now.

In this study Frappier and her team examined PML nuclear bodies and proteins in EBV-positive and EBV-negative NPC cells.  Manipulation of EBNA1 levels in each cell type clearly showed that EBNA1 expression induces the loss of PML proteins and PML nuclear bodies through an association of EBNA1 with the PML bodies.  PML nuclear bodies are known to have tumor-suppressive effects due to their roles in regulating DNA repair and programmed cell death, and accordingly, EBNA1 was shown to interfere with these processes.

The researchers conclude that there is “an important role for EBNA1 in the development of NPC, in which EBNA1-mediated disruption of PML nuclear bodies promotes the survival of cells with DNA damage.”  Since EBNA1 is expressed in all EBV-associated tumors, including B-cell lymphomas and gastric carcinoma, these findings raise the possibility that EBNA1 could play a similar role in the development of these cancers.  The cellular effects of EBNA1 in other EBV-induced cancers will require further investigation.

T-cell acute lymphoblastic leukemia (T-ALL)

The white blood cells in our body combat foreign intruders, such as viruses and bacteria.  However, in leukemia, the formation of white blood cells is disturbed: the cells that should develop into white blood cells multiply out of control without fully maturing.  This process disrupts the production of normal blood cells, making patients more susceptible to infections.  T-ALL, a particular form of leukemia, is the most prevalent cancer in children under 14 years of age and occurs predominantly between the ages of two and three.  At the moment, with an optimal treatment using chemotherapy, over half of the children are cured.  But scientists hope to be able to develop targeted therapies that are less toxic than chemotherapy, based on knowledge of the biological processes behind T-ALL.

Importance of the location

Oncogenes are often at the root of cancer.  So, scientists around the world are concentrating on identifying oncogenes and their related proteins.  Recent research by Kim De Keersmaecker and colleagues in Jan Cools’ research group (VIB-K.U.Leuven) indicates that the location in the cell where these proteins are found plays an important role in the entire carcinogenic mechanism.  In collaboration with Maarten Fornerod (Nederlands Kanker Instituut, Amsterdam) and Gary Gilliland (Harvard Medical School, Boston), the VIB researchers have demonstrated that NUP214-ABL1, a fusion of two proteins, is carcinogenic only when it is in a protein complex near the nucleus of the cell.  Located at another place in the cell, NUP214-ABL1 does not lead to cancer.  This finding sheds new light on the study of carcinogenic processes.

A new therapeutic approach?

Many forms of cancer are caused by genetic defects in which a certain kinase becomes too active and this is the case with NUP214-ABL1.  The most obvious solution is to make the carcinogenic kinase inactive, and so kinase inhibitors are usually used to combat these kinds of cancers.  However, the carcinogenic kinase often becomes resistant to these inhibitors which is certainly true for T-ALL.  So, scientists are actively seeking alternative approaches.

De Keersmaecker’s recent research results now offer a possibility.  Indeed, the scientists have shown in cells that NUP214-ABL1 is no longer carcinogenic when it cannot bind with the protein complex in the vicinity of the cell nucleus.  On the basis of these results, the researchers want to further investigate the therapeutic possibilities of compounds that render binding between the complex and NUP214-ABL1 impossible.  This study also indicates that the location of proteins can play an important role in other forms of cancer/leukemia as well.

Wei Tong and colleagues, at Children’s Hospital of Philadelphia, have provided new insight into the molecular control of hematopoietic stem cells (HSCs), the cells that give rise to all types of blood cell, by explaining why mice lacking an inhibitory protein known as Lnk have more HSCs than normal mice.

In the study, it was observed that a greater proportion of HSCs in mice lacking Lnk were not undergoing cell division and were said to be quiescent (i.e., in a state of inactivity or in silent mode).  Lnk was found to regulate HSC quiescence by binding a signaling protein known as JAK2 after it was activated following binding of the soluble factor TPO to its receptor Mpl.  The authors therefore hypothesize that in the absence of the inhibitory molecule Lnk, TPO-initiated signaling from Mpl to JAK2 goes unchecked and the number of HSCs produced is increased to a level at which they do not need to undergo cell division as often to maintain their population.

Xiao-Jing Wang and colleagues, at Oregon Health & Science University, Portland, have provided new insight into the role of the signaling molecule Smad2 in skin cancer by analyzing human skin cancer tissue and a mouse model of skin cancer.

In the study, human squamous cell skin cancer samples were found to frequently lose expression of Smad2.  In particular, Smad2 expression was lost in all samples characterized as “poorly differentiated” (which means they had progressed to become aggressive tumors).  Consistent with this, mice lacking Smad2 in cells of the skin known as keratinocytes developed chemically induced skin cancer more rapidly than normal mice, and the cancers were all characterized as “poorly differentiated”.  The mouse cancers also underwent a process known as epithelial-mesenchymal transition (EMT) and this was found to contribute to the accelerated progression of the skin cancer to an aggressive form.  These data identify Smad2 as a suppressor of skin cancer development and progression to an aggressive form, and future studies will investigate in more detail the mechanisms underlying the role of Smad2 loss in human skin cancer progression.

Among the first cells of the immune system to respond to microorganisms that invade our body are neutrophils.  Although neutrophils are considered the “good guys” in such circumstances, they also contribute to the noninfectious chronic inflammation that underlies various diseases, including autoimmune diseases such as rheumatoid arthritis.  One mechanism by which neutrophils protect us is to internalize microorganisms and destroy them using proteins known as neutrophil serine proteases (NSPs), but whether NSPs have a role in noninfectious chronic inflammation has not been clearly determined.  However, using mice lacking two very similar NSPs, PR3 and NE, a team of researchers at the Max-Planck-Institute of Neurobiology, Germany, have now shown that these two NSPs have a crucial role in one form of noninfectious chronic inflammation.  Detailed analysis revealed that PR3 and NE destroy an anti-inflammatory molecule known as PGRN and in this way help to promote inflammation in the absence of invading microorganisms.  The authors therefore suggest that these data provide rationale for considering inhibitors of NSPs as anti-inflammatory drugs.

A small molecule that locks an essential enzyme in an inactive form could one day form the basis of a new class of unbeatable, species-specific antibiotics, according to researchers at Fox Chase Cancer Center.

Their findings, highlighted on the cover of the June 23 issue of the journal Chemistry & Biology, take advantage of an emerging body of science regarding “morpheeins” – proteins made from individual components that are capable of spontaneously reconfiguring themselves into different shapes within living cells.

The researchers discovered a small molecule, which they have named morphlock-1, binds the inactive form of a protein known as porphobilinogen synthase (PBGS), an enzyme used by nearly all forms of cellular life.  The functioning form of PBGS is built from eight identical component parts – in what is called an octamer configuration – and is essential among nearly all forms of life in the processes that enable cells to use energy.  The other configuration is made of six parts – or a hexamer configuration – and serves as a “standby” mode for the protein.

“As the name suggests, morphlock-1 essentially locks the hexamer configuration into place, preventing its protein subunits from reconfiguring into the active assembly,” says lead investigator Eileen Jaffe, Ph.D, a Senior Member of Fox Chase.  “Targeting morpheeins in their inactive assemblies provides an entirely new approach to drug discovery.”

While their study was performed using a pea plant-version of PBGS, the researchers have reason to believe the principle could apply to bacterial versions of PBGS as well.  “Using morphlock-1 as a base, we are seeking to fine tune the molecule so that it blocks just the bacterial version of the PBGS enzyme, ” Jaffe says.

“Because PBGS is so crucial for life, the part of the enzyme where chemistry happens is highly conserved through evolution,” Jaffe says, meaning that an all-around PBGS-inhibiting drug would harm bacteria, peas and people alike.  The area where the potential drug binds to the hexamer form of the protein, however, has been found to differ among species, depending how far the organisms have evolved from each other.

When PBGS is in its inactive hexamer form, there is a small cavity on the surface of the assembled complex.  Using computer docking techniques, Jaffe and her Fox Chase colleagues identified a suite of small molecules predicted to bind to this cavity.

The researchers then bought and tested a selection of these molecules in the lab to see if any of them stabilized the pea PBGS in its hexamer assembly.  One inhibitor in particular, given the name morphlock-1, potently drove the formation of the hexamer in pea PBGS, but not in that of humans, fruit flies, or the infectious bacteria Pseudomonas aeruginosa, or Vibrio cholerae, the latter of which causes cholera.  Morphlock-1 is a potent inhibitor of pea PBGS, but not of the PBGS from these other organisms.

Jaffe coined the term “morpheein” in 2005 after a study of the structure of PBGS revealed its shape-shifting tendencies.  While initially met with skepticism because the existence of morpheeins contradicts some classic concepts about protein structure and function, subsequent studies have reinforced that PBGS (and perhaps other proteins) exhibits this behavior.  According to Jaffe, this study is the first to make use of alternate morpheein shapes as a potential strategy for drug discovery, in general, particularly for antibiotics.

“Multi-drug resistance drives the need for developing new antibiotics,” Jaffe says.  “Since drugs that stabilize the inactive PBGS hexamer need not be chemically similar to each other, it will be difficult for the bacterium to develop complete resistance to a cocktail of such compounds.”

A tiny but powerful engine that propels the bacterium Bacillus subtilis through liquids is disengaged from the corkscrew-like flagellum by a protein clutch, Indiana University Bloomington and Harvard University scientists have learned. Their report appears in this week’s Science.Scientists have long known what drives the flagellum to spin, but what causes the flagellum to stop spinning — temporarily or permanently — was unknown.

“We think it’s pretty cool that evolving bacteria and human engineers arrived at a similar solution to the same problem,” said IU Bloomington biologist Daniel Kearns, who led the project. “How do you temporarily stop a motor once it gets going?”

The action of the protein they discovered, EpsE, is very similar to that of a car clutch. In cars, the clutch controls whether a car’s engine is connected to the parts that spin its wheels. With the engine and gears disengaged from each other, the car may continue to move, but only because of its prior momentum; the wheels are no longer powered.

EpsE is thought to “sit down,” as Kearns describes it, on the flagellum’s rotor, a donut-shaped structure at the base of the flagellum. EpsE’s interaction with a rotor protein called FliG causes a shape change in the rotor that disengages it from the flagellum’s proton-powered engine.

The discovery of EpsE and its function was accidental. Kearns and colleagues were actually interested in learning more about the genes that cause individual cells of B. subtilis to cease wandering in solitude and take up residence in a massively communal, stationary assemblage called a biofilm. The stability of biofilms can be jeopardized by hyperactive bacterial cells whose flagella continue to spin.

“We were trying to get at how the bacterium’s ability to move and biofilm formation are balanced,” Kearns said. “We were looking for the genes that affected whether the cells are mobile or stationary. Although B. subtilis is harmless, biofilms are often associated with infections by pathogenic bacteria. Understanding biofilm formation may eventually prove useful in combating bacterial infections.”

Once the scientists learned EpsE was involved in repressing flagellar motion, they devised two possible explanations for how EpsE acts. The first was that EpsE acts like a brake by pushing a non-moving part against a moving part and locking up the works. The other possibility, they imagined, was that EpsE acts like a clutch, disengaging one moving part from another. In this latter scenario, the engine can no longer drive flagellar spinning because key moving parts are no longer in contact. In this case, the flagellum would still have freedom of motion, listless as it might be.

To determine which hypothesis was correct, the scientists decided it best to let the tail wag the dog. They attached the tail end of the flagellum to a glass slide and examined the movement of the entire cell in the presence and absence of EpsE. In the absence of EpsE, the entire cell rotated once every five seconds. In the presence of EpsE, the cells stopped but could rotate passively, pushed by disturbances in the environment (Brownian motion). If EpsE acted like a brake, the cells would not have rotated at all.

The researchers also learned that when the cell begins producing EpsE, it takes about 15 minutes before the flagellar machinery is disabled.

“This makes a lot of sense as far as the cell is concerned,” Kearns said. “The flagellum is a giant, very expensive structure. Often when a cell no longer needs something, it might destroy it and recycle the parts. But here, because the flagellum is so big and complex, doing that is not very cost effective. We think the clutch prevents the flagellum from rotating when constrained by the sticky matrix of the biofilm.”

The discovery may give nanotechnologists ideas about how to regulate tiny engines of their own creation. The flagellum is one of nature’s smallest and most powerful motors — ones like those produced by B. subtilis can rotate more than 200 times per second, driven by 1,400 piconewton-nanometers of torque. That’s quite a bit of (miniature) horsepower for a machine whose width stretches only a few dozen nanometers.

Against currently held dogma, scientists at the Universities of Cambridge and Bristol have revealed that the interactions within membrane complexes can be maintained intact in the vacuum of a mass spectrometer. Their research is published in this week’s edition of Science Express.

The researchers were surprised to discover that membrane complexes could remain associated as it has always been assumed that they would not survive once transferred to the alien conditions inside the mass spectrometer.

“Even if interactions between proteins within the membrane could be maintained we would not have expected them to remain associated with proteins in the cell’s interior,” says Carol Robinson, Principal Investigator and Royal Society Research Professor at the University of Cambridge’s Department of Chemistry.

Cellular membranes surround cells and provide the ultimate in cellular security; nothing can get into a cell without the say so of membrane proteins – the worker molecules that reside in the membrane wall and provide tightly regulated entry points. This natural home of membrane proteins excludes water, yet methods available to study proteins at high resolution revolve round aqueous environments. The ability to “fly” intact membrane proteins in a mass spectrometer paves the way for weighing the proteins and identifying the molecular partners they work with in nature.

The new research, funded by the Biotechnology and Biological Sciences Research Council, will enable scientists to investigate membrane complexes with from a variety of sources and with a range of small molecules. Since about 60% of all drug targets are membrane proteins this is a significant discovery.

Ever since Professor Robinson first flew soluble protein complexes in a mass spectrometer in 1996, she has wanted to do the same with membrane complexes. Collaborating with a membrane biochemistry group in Bristol, led by Professor Paula Booth, she began to think of ways of studying these most challenging assemblies.

Dr Nelson Barrera a post-doctoral researcher in Chile, though experienced in membrane biochemistry, was a new recruit to mass spectrometry. He was largely unaware of the difficulties that had previously been encountered and approached the problem in a new way. Rather than trying to remove the detergent (used to keep the protein intact in solution once outside the natural membrane) he maintained the detergent in unusually high amounts. He then deliberately destroyed this protective detergent layer once in the gas phase. This allowed him to liberate the intact assembly. He was also able to remove units from the modular assembly in the gas phase, just as in solution.

Professor Robinson adds: “I am very excited by this finding given the importance of membrane complexes in guarding the entrance and exit to cells. The type of proteins we have been studying, for example, are involved in drug resistance in cancer cells and antibiotic resistance of bacteria.

“I look forward to exploiting this discovery to the full; not only in characterising the many membrane complexes for which controversy exists but also in discovering new assemblies and in investigating the potential of this approach in drug discovery.”

Professor Paula Booth, at the University of Bristol added: “This is a major advance that helps us understand how nature constructs cellular life. The membrane wall of cells is a precision-made, complex and highly regulated structure. We are now much better equipped to understand this incredible, natural self-assembly feat.”

Coronaviruses, a group including the well-known SARS virus, are the causative agents of many respiratory and enteric infections in humans and animals. As with all viruses, virtually every step of their infection cycle depends on host cellular factors. As the first, most crucial step after their penetration into cells, coronaviruses assemble huge RNA replication “factory” complexes in association with characteristic, newly induced double membrane vesicles. The cellular pathways hijacked by these plus-strand RNA viruses to create these “factories” have thus far not been elucidated.

The researchers, led by Cornelis A. M. de Haan, showed that RNA replication of mouse hepatitis coronavirus (MHV) was inhibited by a drug — brefeldin A — that disrupts the central station in the cell’s secretory pathway, the Golgi complex. Consistently, depletion of both the cellular target of brefeldin A, a factor called GBF1, and its downstream target, ARF1, was also shown to negatively affect coronavirus infection.

The researchers conclude that “an intimate association exists between the early secretory pathway and MHV replication.” They speculate that, while GBF1 and ARF1 are not involved in the formation of the viral replication structures, they probably play a key role in their maturation or functioning. As this work was limited to the mouse hepatitis coronavirus, an interesting next step would be to study the importance of GBF1 and ARF1 in the replication of other coronaviruses.

Uncontrolled blood vessel growth is a key feature of many pathological conditions, including the degenerative diabetic eye disease known as diabetic retinopathy.  Understanding the factors involved in the process is vital to developing treatments for the disease.  In a new study, a team of researchers at Keio University, Japan, has revealed a role for the protein LIF in blood vessel growth in mice.

Specifically, mice lacking LIF were observed to have increased blood vessel growth in many regions of the body, but as this study was focused on the eye, the authors homed in on the increased blood vessel growth in the retina of the eye.  Further analysis showed that mice lacking LIF developed more aberrant blood vessels in a model of retinopathy.  Mechanistically, LIF was found to inhibit the proliferation of brain cells known as astrocytes as well as inhibit their production of a factor known to promote blood vessel growth, VEGF.  It therefore seems that LIF is an important part of the communication between tissues and developing blood vessels, meaning that LIF and the signaling pathway it triggers might serve as a target for new treatment approaches for preventing diabetic retinopathy and other diseases that are associated with uncontrolled blood vessel growth, such as cancer.

Montana State University scientists in the Department of Chemistry and Biochemistry published new research this week that could one day affect the lives of millions around the world who suffer from blood iron disorders.
Martin Lawrence (left) and Anoop Sendamarai (MSU photo by Kelly Gorham)
The paper, which will appear in the Proceedings of the National Academy of Sciences, details the work of Associate Professor Martin Lawrence and doctoral candidate Anoop Sendamarai. The pair have spent the past two years studying Steap3, a protein involved in regulating the body’s absorption of iron.

The results of their studies - the first three-dimensional maps of the atoms that make up Steap3 - could allow pharmaceutical companies to someday design drugs to regulate iron levels in the blood.

“Iron is essential,” Lawrence said. “You can’t live without it, but it’s a double-edged sword. Too much of a good thing can kill you.”

Iron serves several important functions in the bloodstream. It carries oxygen, transports electrons within cells and plays an important role in enzyme systems.

Iron irregularities are some of the most common blood disorders in the world. According to the World Health Organization, iron deficiency, which can lead to anemia, affects more than a billion people around the world and can cause developmental and immune system problems.

Conversely, having too much iron, a condition called hemochromatosis, can also hurt the body by releasing destructive free radicals, Lawrence said. Hemochromatosis affects about one in every 300 people and is most common in people of northern European ancestry. Left untreated, it can lead to early death, often by age 50.

“We’re struck by how many people have too much or too little iron,” Lawrence said.

To understand Steap3’s role in transporting and maintaining balanced levels of iron, Lawrence and Sendamarai first had find and purify samples of the protein and then turn those samples into crystals.

Lawrence said the result of the crystallization process, if done correctly, is analogous to the rigid structure of a brick wall. If done incorrectly, it more closely resembles a pile of bricks.

“It’s kind of a black art really more than a science,” Lawrence said. “You can’t always predict the kind of witch’s brew that needs to be around to get it to crystallize.”

He said only a handful of labs in the country are crystallizing iron transport proteins like Steap3, a fact that places MSU on the same shelf as places like Harvard Medical School.

Once crystallized, the samples are shot with a powerful X-ray beam. Electrons in the sample diffract the X-rays, creating patterns on a digital sensor. The technique, called X-ray crystallography, has been used since the 1950s to de-termine the structure of different substances.

In their basement lab in the campus’s New Chemistry Building, Lawrence and Sendamarai then examined the diffraction patterns created by Steap3.

“It’s kind of like a contour map,” Sendamarai said. “Whenever we see the peaks, we know there are atoms.”

Working backward, they can mathematically determine the position of atoms in the protein and display them in three dimensions.

The computer-drawn result, a three-dimensional image that resembles tangled ribbons and strings, is an picture of what the atoms of Steap3 look like.

Sendamarai said having that picture, which depicts all the nooks and crannies on the protein’s surface, could allow drug companies to design drugs to fit those spots like puzzle pieces.

If a future drug fits those nooks just right, it could help treat hemochromatosis. From there, Sendamarai said it would be conceivable to work backward and possibly treat iron deficiencies or anemia.

Lawrence said that Steap3 is only one in a family of proteins that affect iron transport. This summer, in addition to continuing to study Steap3, Lawrence and Sendamarai hope to learn whether the lab will receive a grant from the National Institutes of Health to work on other iron transport proteins.

“It’s a critical step towards toward learning to modulate iron levels in patients with too much or too little iron,” Sendamarai said. “But, there are a lot of question marks left in iron transport. It’s a big field.”