Blood for all: Making universal blood through enzymes

April 23, 2015 § 3 Comments

Making universal blood. Image provided by Stephen Withers and David Kwan.

Making universal blood. Image provided by Stephen Withers and David Kwan.

Universal blood is an appealing notion because it could be transfused into anyone regardless of blood type. Researchers have been kicking around the idea of using enzymes to create universal blood since the early 1980s, “but a major limitation has always been the efficiency of the enzymes,” says Stephen Withers at the University of British Columbia. “Impractically large amounts of enzyme were needed.”

Now in a paper just out in the Journal of the American Chemical Society, Withers, David Kwan, Jay Kizhakkedathu at the Centre for Blood Research and others describe the development of an improved enzyme that takes us a step closer to having universal and others describe the development of an improved enzyme that takes us a step closer to having universal blood.

Blood comes in four major types: A, B, AB and O. The difference between them lies in the sugar structures that festoon the surface of red blood cells. Both blood type A and B have the same core sugar structure as blood type O, but differ in an additional sugar at the tip of the sugar structure. Type A has an N-acetylgalactosamine residue. Type B has a galactose residue. Type AB has a mix of both residues. The moiety carrying the additional residue can be tacked onto the core structure in various ways, giving rise to subtypes of A and B blood.

The additional residue presents trouble during blood transfusions: It can trigger life-threatening immune responses. Type A people can’t take type B blood; type B people can’t take in type A blood. Type AB can take A or B. Only type O blood can be freely shared without the fear of immune responses.

The idea from the 1980s has been to use enzymes to remove the moieties with the terminal N-acetylgalactosamine or the galactose residues to leave the core sugar structure on red blood cells, just like in type O blood. But to date, sugar hydrolases have not been sufficiently efficient to make the idea practical.

So Withers’ group, which had some success in engineering different classes of sugar enzymes, tackled the creation of more efficient sugar hydrolases by directed evolution. In directed evolution, researchers carry out iterative rounds of mutations on a gene to ultimately produce a protein that performs better than the original gene product.

Kwan, Withers and the rest of the team carried out directed evolution on the family 98 glycoside hydrolase from a strain of Streptococcus pneumoniae. Kwan explains that the structure of the enzyme is known, which helped the investigators design their variants.

Kwan adds that the enzyme also is good at cleaving most A and B subtypes with the exception of a few A subtypes. The investigators decided to engineer the enzyme so it had better activity against those A subtypes. By directed evolution, the investigators got an engineered enzyme with a 170-fold greater efficiency than the original enzyme.

However, the engineered enzyme still doesn’t remove every single moiety with the N-acetylgalactosamine. Withers says that the immune system is sensitive enough to small amounts of the moiety to start an immune response. He says, “Before our enzyme can be used clinically, further improvements by directed evolution will be necessary to effect complete removal” of all moieties with the terminal N-acetylgalactosamine residues.

The investigators are now looking to tackle the remaining subtypes of type A blood that the S. pneumoniae hydrolase struggles to cut. Withers says, “Given our success so far, we are optimistic that this will work.”

How a plant molecule is a “superb” sunscreen

October 30, 2014 § Leave a comment

 

 

Plants make their own sunblock to protect against UV damage. Image from http://en.wikipedia.org/wiki/Plant#mediaviewer/File:Leaf_1_web.jpg

Like us, plants worry about sunburn. Research now shows that a molecule called sinapoyl malate found in plants is one hardworking sunblock. It captures some of the most damaging radiation from the sun.

Ultraviolet radiation is split into nine groups. One of the groups is UV-B radiation in the 280 to315 nm range. Overexposure to UV-B radiation in humans causes sunburn and some forms of skin cancer. In plants, the radiation can cause DNA damage and interfere with photosynthesis.

Timothy Zwier at Purdue University, who led the recent study published in the Journal of the American Chemical Society, says he got intrigued by sinapoyl malate when a Purdue colleague, Clint Chapple, discovered that the molecule was responsible for acting as a sunscreen in plants. While sinapoyl malate generally was known to absorb UV-B radiation in aqueous solution, the inherent spectral properties of the bare molecule were unknown.

Zwier’s lab’s specialty is studying the UV spectroscopy of isolated molecules in the gas phase. But the problem is that sinapoyl malate, like most other biological molecules, can’t be heated into a gas because it breaks down.

So Zwier and colleagues zapped the molecule off a thin film with a laser to get it into the gas phase. Once the molecule was in the gas phase, the investigators cooled it down to a temperature within a few degrees of absolute.

“This gave us a chance to study the inherent spectroscopic signatures of this UV-B-sunscreen molecule as an isolated molecule, free from the effects of solvent and cooled to low temperatures, where it can best reveal its secrets,” says Zwier.

However, sinapoyl malate had a surprise. Even in the stripped-down experimental conditions, the molecule’s spectrum was inherently broad, absorbing continuously over all wavelengths encompassed in the UV-B radiation range. Zwier notes that in its natural environment in the plant cell’s aqueous condition, sinapoyl malate probably works in conjunction with other factors to cover even more of the UV spectrum.

“Nevertheless, at the most fundamental quantum mechanical level, sinapoyl malate has what it takes to be a superb UV-B sunscreen — large absorption cross-section in the right wavelength range and complete spectral coverage,” says Zwier. “Nature has chosen a molecule to serve as its UV-B sunscreen that is extraordinarily well-suited for the task.”

On the hook: A new pipette to capture single cells

July 23, 2014 § Leave a comment

The handheld single-cell pipette. Image provided by Lidong Qin.

The handheld single-cell pipette. Image provided by Lidong Qin.

Seeking out individuals from a crowd has become increasingly important for biologists interested in single-cell analyses. Studies on individual cells allow scientists to understand processes that happen within this basic unit of life that often get masked by approaches that analyze groups of cells.

But there is “one very basic problem — how can one precisely pick up an individual cell?” asks Lidong Qin at the Houston Methodist Research Institute. Current methods of isolating single cells can be inefficient, expensive, time-consuming, biased or labor-intensive. Now, in a paper just out in the Journal of the American Chemical Society, Qin and colleagues describe a new kind of pipette specially designed to pick out single cells. “The pipette platform is almost a universal tool that is easy, cheap and reproducible,” says Qin.

The instrument, called the handheld single-cell pipette or hSCP, has dimensions much like a conventional handheld air-displacement pipette. It has two channels and one tip.

A researcher uses the hSCP to suck up cells from a Petri dish. Inside the pipette tip is a hook small enough to catch a single cell at random from the cells that get sucked up from the dish. Those cells that are not on the hook get removed by a flow of liquid, while the single cell on the hook can be deposited into standard 96-well or similar types of plates, Petri dishes or vials for single-cell biochemical analyses, such as cloning or the polymerase chain reaction.

Qin says the next priority is to develop the prototype into a device that can be mass-produced and commercialized.

How a polymerase bypasses the rules of Watson-Crick base-pairing

March 20, 2014 § 3 Comments

The half-sized ASFV DNA polymerase X does not follow the dogma of binding DNA first followed by MgdNTP. It binds synthetic dGTP prior to DNA in order to catalyze dG:dGTP incorporation. Image by Wen-Jin Wu.

The half-sized ASFV DNA polymerase X does not follow the dogma of binding DNA first followed by MgdNTP. It binds synthetic dGTP prior to DNA in order to catalyze dG:dGTP incorporation. Image by Wen-Jin Wu.

A pairs with T; G pairs with C. This is the golden rule in DNA base pairing, known as Watson-Crick base pairing. But some polymerases thumb their noses at this golden rule and can get the bases to pair with different partners. In a paper recently published in the Journal of the American Chemical Society, researchers demonstrate how a polymerase found in the African swine fever virus bypasses the golden base-pairing rule by tackling the process of making DNA in a different way.

DNA polymerases are the enzymes involved in fixing and making DNA. Ming-Daw Tsai’s laboratory at Academia Sinica in Taiwan has a longstanding interest in DNA polymerase X. This polymerase, known as Pol X, is found in the African swine fever virus. The polymerase is unusual because it can get G to pair with itself; it also can carry out canonical base pairing. So, how does the polymerase accomplish the noncanonical base pairing?

“Kinetic studies suggested that Pol X does not follow the established mechanistic paradigm that DNA polymerases bind DNA” before binding to a nucleotide complexed with a magnesium ion, explains Tsai. “However, structural studies took us a long time since Pol X does not crystallize.”

The investigators were left with studying the enzyme in solution by nuclear magnetic resonance spectroscopy. “Even though the structure of the free enzyme in 2001 was solved by NMR, using NMR to solve the structures of complexes with MgdGTP, in the absence and presence of DNA, was a lot harder,” he says.

After spending 10 years on the problem, Tsai’s group now has an answer. DNA polymerases that strictly stick to Watson-Crick base pairing, known as hgh-fidelity polymerases, first bind to the DNA and then to the necessary nucleotide complexed with a magnesium ion. The investigators found that Pol X binds to a nucleotide-magnesium ion complex first and then to the DNA. Whatever nucleotide this low-fidelity polymerase is carrying could get inserted into the DNA strand.

“This result is contrary to the thought that maybe low-fidelity polymerases are just poor polymerases that cannot achieve high fidelity,” explains Tsai. In fact, Pol X “is highly specific and seems to know what it is doing.”

He says the data show that there are two competing factors in a DNA polymerase’s function: The DNA may impose the rules of Watson-Crick base pairing on the polymerase, but the enzyme may control the DNA by prebinding its preferred nucleotide-magnesium ion complex.

Tsai emphasizes that the work by his group specifically applies to Pol X. “There has been an explosion in the study of the structure and mechanism of low-fidelity and translesion synthesis DNA polymerases in the past decade. Many beautiful structural studies have been published, and it appears that each polymerase has some of its own interesting features,” he says.

Indeed, his group will test other low-fidelity polymerases to see how they work. Also, he notes, “there could be some clinical implication as well, though it is speculative at this point.” For making new drugs against the African swine fever virus, Tsai says it’s possible “we can target Pol X alone instead of Pol X-DNA complex. The same concept can be applied to some disease-relevant DNA polymerase mutants in human, which may have developed the ability to pre-bind dNTP.”

Splitting up kinases

February 26, 2014 § Leave a comment

A ligand-inducible split-kinase is activated by a small molecule. Image by Karla Camacho-Soto.

A ligand-inducible split-kinase is activated by a small molecule. Image by Karla Camacho-Soto.

Researchers have figured out a way to bend kinases at their will. In a paper recently published in the Journal of the American Chemical Society, Indraneel Ghosh and colleagues at the University of Arizona describe engineering these enzymes, which are critical for signaling pathways, so that they can be controlled by researchers. These engineered kinases should “allow for a more precise understanding of signaling cascades that are currently unavailable,” says Ghosh.

Scientists have been interested in manipulating enzymes with exquisite precision because the manipulation helps them to understand how these molecular machines work. Kinases also represent a lucrative class for drug targets because many of them have been identified to be involved in disease conditions, such as cancer. Knowing how kinases work in detail helps to develop highly targeted drugs.

Ghosh and colleagues demonstrated that one way to turn kinases on and off at will was to split the enzymes into two. They then connected the two parts of the enzyme with a special linker. When given a specific small molecule, such as the immunosuppressant rapamycin, two parts of the split enzyme came together and became activated like their normal counterparts. Because these enzymes could be activated with a small molecule, Ghosh and colleagues called them “ligand inducible split-kinases.”

The researchers showed that the approach could be used on four different kinases, suggesting that the method is broadly applicable. Ghosh says the long-term goal of the project is to have multiple ligand inducible split-kinases within a cell so that researchers can understand how these enzymes work relative to one another in the complex network of signaling pathways.

No more secondary antibodies: Universal adaptor for protein detection

September 5, 2013 § 1 Comment

ELISAs would be easier without having to deal with secondary antibodies. Image from http://en.wikipedia.org/wiki/File:ELISA.jpg

ELISAs would be easier without having to deal with secondary antibodies. Image from http://en.wikipedia.org/wiki/File:ELISA.jpg

Imagine being able to do your Western blots, ELISAs and immunohistochemical reactions without fiddling around with finicky secondary antibodies. How cool would that be? In a paper recently out in the Journal of the American Chemical Society, researchers have come up with a new molecule that can detect proteins without relying on secondary antibodies. “This will greatly move the protein detection forward, impacting a variety of fields, such as biosensing and diagnostics, anything related to protein detections,” says Dan Luo of Cornell University who spearheaded the work.

The molecule, which Luo and colleagues call the universal adaptor, has two parts to it: an antibody-binding component and a DNA tag. The antibody component is derived from an engineered variant of protein A, which binds to most types of IgG primary antibodies. The DNA tag can hybridize to DNA-modified reporter molecules that give off signals, such as DNA nanobarcodes, quantum dots and enzymes.

When the universal adaptor is used with an IgG primary antibody in an experiment, the antibody component of the adaptor latches onto the primary antibody. The DNA tag attaches to a chosen reporter molecule, such as horseradish peroxidase, and labels the primary antibody.

There are two main benefits to the universal adaptor, says Luo. First, researchers are no longer limited to the relatively few commercially available primary and secondary antibody pairs. They can also use several primary antibodies in their experiments, throw in the universal adaptor, and use a variety of reporter molecules to get multiplexed detection without having to worry about secondary antibodies inadvertently crossreacting with the wrong primary antibody. The second benefit is that the DNA portion of the hybrid molecule lends itself to amplification, which means signals from experiments can be turned up for easier detection.

Under pressure: mechanism of DNA injection by herpes viruses

July 26, 2013 § Leave a comment

High pressure inside herpes viruses allows them to shove their DNA into human cells. Credit: National Institute of Allergy and Infectious Diseases

High pressure inside herpes viruses allows them to shove their DNA into human cells.
Credit: National Institute of Allergy and Infectious Diseases

According to research just out in the Journal of the American Chemical Society, the herpes viruses that cause shingles, chickenpox, Hodgkin’s lymphoma and other serious illnesses push their DNA into human cells with great force. In fact, the investigators involved in the research say that the herpes simplex virus 1, which causes cold sores, has an internal pressure eight times greater than that of a car tire. The virus uses this pressure to shove its DNA into cells.

“The mechanism of DNA release from herpes viruses, as well as the majority of other viruses, is poorly understood,” says Alex Evilevitch at the Carnegie Mellon University, who spearheaded the work. “This work provides important evidence of how this happens for herpes viruses.”

The investigators demonstrated that HSV-1 goes into a cell, docks with portals that are on the cell’s nucleus and pushes its DNA into the organelle with high pressure. The high pressure is caused by tight packing of the capsid, the shell that contains the viral genome.

Bacteriophages, which are viruses that infect bacteria, are known to use the same high-pressure mechanism to shoot their DNA into nuclei. This suggests that evolution has found this method to be very effective for viral infection and has repurposed it for other viruses.

The mechanism also gives researchers a new handle on developing antiviral therapies. “As the DNA inside herpes viruses has a unique condensed state, which is not present anywhere else in the cell, decreasing this pressure will make the virus noninfectious,” explains Evilevitch. “This provides a new drug target that is resistant to mutations since it will target a universal physical mechanism, rather than specific viral proteins.”

Tracking how multiple proteins interact with each other

July 10, 2013 § Leave a comment

The role of SNARE proteins in a cellular process known as exocytosis. Image from http://en.wikipedia.org/wiki/File:Exocytosis-machinery.jpg

The role of SNARE proteins in a cellular process known as exocytosis. Image from http://en.wikipedia.org/wiki/File:Exocytosis-machinery.jpg

Proteins are great believers in hands-on teamwork. They come together in groups to directly interact with each other and set off reactions. But because these interactions happen over extremely tiny distances, watching them has been almost impossible. In a paper recently out in the Journal of the American Chemical Society, researchers described a method that allows them to track multiple proteins that closely interact with each other below the optical diffraction limit at the single-molecule level.

The optical diffraction limit has been the long-standing problem of optical microscopy because it stops researchers from seeing events that happen at distances less than 200 nm, the limit of light diffraction. In the past decade, optical techniques have emerged that allow researchers to bypass this limit. The new techniques, such as single-molecule fluorescence resonance energy transfer (FRET), have allowed researchers to view molecules on the scale of a few nanometers, distances that often take place in biological systems.

“But the single-molecule FRET technique has been able to follow only one protein complex,” explains Tae-Young Yoon at KAIST in South Korea, and multiple protein complexes within one diffraction-limited spot cannot be distinguished from one another.

So Yoon and colleagues developed a way to use single-molecule FRET on multiple proteins below the diffraction limit. As their proof of concept, they used SNARE proteins, which are involved in the fusion of lipid membranes. “It is now believed that, with only few exceptions, membrane fusion processes in eukaryotic cells are all mediated by the SNARE proteins, which underscores the importance of the SNARE family and their complex formation,” says Yoon. “Moreover, it has been long anticipated that SNARE proteins are in a clustered state and that multiple SNARE complexes are formed in a concurrent manner to maximize their impact to membrane fusion. The SNARE complex is an ideal and important model system for protein complexes working as a team.”

The investigators labeled all the proteins in a SNARE complex with the appropriate dyes. During conventional optical imaging, all the photons coming out from the dyes are detected at once, which makes it impossible to tell one dye from another. Yoon and colleagues defined quantized FRET states, where each state corresponded to a specific number of protein complexes activated at a given moment. They knew the value of the maximum FRET efficiency when all the protein complexes were activated at the same time. Then the researchers measured the total FRET efficiency of the protein complexes and compared this measurement with one of the quantized states. From the maximum value and their measurements, the investigators came up with a ratio that gave information about the number of activated protein complexes out of the total number of possible SNARE complexes.

There are some limitations with the approach. For one, the proteins in the complex have to stay paired long enough for their interaction to be measured. Another limitation is that the dyes have to be precisely and painstakingly attached to the proteins at proper locations to obtain the maximum number of photons out of each interaction.

Novel probes for an important developmental signaling cascade, the Sonic Hedgehog pathway

June 10, 2013 § Leave a comment

BRD50837 is a novel small-molecule probe that inhibits Hedgehog signaling. Image by Giannina Schaefer.

BRD50837 is a novel small-molecule probe that inhibits Hedgehog signaling. Image by Giannina Schaefer.

Researchers have found novel probes that could help to better understand a critical developmental signal-transduction pathway. These probes are small-molecule inhibitors of the Sonic Hedgehog signaling pathway which, besides being essential for body segmentation in insect and vertebrate embryos, has been implicated in some cancers and developmental diseases. The work appears in the Journal of the American Chemical Society. (Yes, the name “Sonic Hedgehog” does come from the Sega video game character.)

“Many facets of the signaling network are not yet well understood,” explains Giannina Schaefer at the Broad Institute, the first author of the paper. “Small-molecule probes, especially those with novel mechanisms-of-action, may help to elucidate the biology of the signaling network.”

Some small-molecule probes for the complicated hedgehog signaling pathway do exist. One of them, vismodegib, is a drug approved by the U.S. Food and Drug Administration as a treatment of basal cell carcinoma, a cancer in which the Hedgehog pathway goes awry.

To discover new probes that could inhibit the pathway from angles inaccessible to existing probes, Schaefer and colleagues, including Stuart Schreiber of the Broad Institute who led the team, used a high-throughput, cell-based screen. “This type of cell-based screen allowed us to find inhibitors with targets at different points in the signaling network, in contrast to biochemical screens that find modulators of one particular target,” says Schaefer.

The investigators found two potent inhibitors, BRD50837 and BRD9526, that seem to interact with the Hedgehog pathway differently than existing probes when tested on a variety of different cell lines. The investigators don’t know yet with which molecules these two inhibitors are interacting in the Hedgehog pathway. But their structure/activity relationships suggests that they are selective and specific.

“Further elucidation of the mechanisms of action [of the two inhibitors] may help us understand why they perform differently,” says Schaefer. “It will increase their value as probes and possibly teach us more about the pathway.”

New peptide mimics inhibit pathogenic bacteria involved in toxic shock syndrome

May 13, 2013 § Leave a comment

The investigators adapted a hemolysis assay with red blood cells to quantitate the ability of the peptide analogs to inhibit S. aureus' quorum sensing. Image provided by Helen Blackwell.

The investigators adapted a hemolysis assay with red blood cells to quantitate the ability of the peptide analogs to inhibit S. aureus’ quorum sensing. Image provided by Helen Blackwell.

The pathogenic bacterium Staphylococcus aureus believes in the power of numbers to bring on infections, such as toxic shock syndrome. In a paper just out in the Journal of the American Chemical Society, researchers describe potent inhibitors that stop the bacterium from forming its armies.

Bacteria use small molecules or peptides as signals to communicate with each other. This phenomenon is known as quorum sensing and it plays a critical role in a bacterium’s virulence. “If we can block quorum sensing, we can effectively tune the infectivity of this major pathogen,” explains Helen Blackwell.

Blackwell’s laboratory at the University of Wisconsin-Madison is focused on understanding how these small molecules and peptides function and is developing non-native mimetics of these compounds. These mimetics can be used as chemical probes to study the fundamental mechanisms of quorum sensing.

For this work, Blackwell and her group studied the structure and function of a S. aureus peptide called AIP-III. This peptide is critical for the bacterium’s quorum sensing system and plays a central role in initiating toxic shock syndrome in humans. The investigators made a series of AIP-III analogs that mimicked the native peptide’s structure.  Then they tweaked small parts in these analogs to see if that part was important for the peptide’s function: They identified a series of parts they could tweak to make the peptide unable to activate quorum sensing but instead strongly inhibit it.

These S. aureus quorum-sensing inhibitors are “the most active peptide-based quorum-sensing inhibitors to be reported in S. aureus,” she explains. “They can block virulence pathways in wild-type S. aureus at very low concentrations. Such lead compounds are a great place to start for the development of new agents for possible anti-infective therapies.”

The investigators are currently working on improving the inhibitor’s chemical stability in biological media so that they can remain active for longer and be more potent.

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