Shadowing Good Cholesterol While It Walks the Dark Side

Good cholesterol, or high-density lipoprotein (HDL), doesn’t always live up to its reputation. Instead, it sometimes runs with the wrong crowd, turns awry, and loses its cardioprotective properties. Good cholesterol can even become harmful, promoting inflammation and atherosclerosis.

The bad influence on good cholesterol is myeloperoxidase (MPO). As indicated by recent studies, MPO can oxidize good cholesterol’s major structural protein, apolipoprotein A1 (apoA1). Specifically, the studies show that in vitro oxidation of either apoA1 or HDL particles by MPO impairs their cholesterol acceptor function.

Determined to uncover additional details, a group of researchers at the Cleveland Clinic decided to investigate the structural details of dysfunctional apoA1. Not only did they learn which part of apoA1 goes awry, they gathered clues about apoA1’s subsequent disease-promoting ways.

The researchers were led by Stanley Hazen, M.D., Ph.D., vice chair of translational research for the Lerner Research Institute and section head of Preventive Cardiology & Rehabilitation in the Miller Family Heart and Vascular Institute at Cleveland Clinic. “Identifying the structure of dysfunctional apoA1 and the process by which it becomes disease-promoting instead of disease-preventing is the first step in creating new tests and treatments for cardiovascular disease,” said Dr. Hazen.

Dr. Hazen and his colleagues used phage display affinity maturation to develop a method for identifying dysfunctional apoA1/HDL. (In particular, they generated a high-affinity monoclonal antibody that specifically recognizes both apoA1 and HDL that have been modified by the MPO-H₂O₂-Cl⁻ system.) In addition, they discovered the process by which it is oxidized and turned dysfunctional in the artery wall. Finally, they then tested the blood of 627 Cleveland Clinic cardiology patients for the dysfunctional HDL and found that higher levels raised the patient’s risk for cardiovascular disease.

Dr. Hazen’s team published its results January 26 in Nature Medicine, in an article entitled “An abundant dysfunctional apolipoprotein A1 in human atheroma.” In this article, the authors wrote, “An oxindolyl alanine (2-OH-Trp) moiety at Trp72 of apoA1 is the immunogenic epitope. Mutagenesis studies confirmed a critical role for apoA1 Trp72 in MPO-mediated inhibition of the ATP-binding cassette transporter A1 (ABCA1)-dependent cholesterol acceptor activity of apoA1 in vitro and in vivo.”

In other words, apoA1 loses its ability to transfer cholesterol out of the artery wall and deliver it to the liver, from which cholesterol is excreted. Instead, a large proportion of apo1 in the artery wall becomes oxidized during atherosclerosis.

According to the researchers, “ApoA1 containing a 2-OH-Trp72 group (oxTrp72-apoA1) is in low abundance within the circulation but accounts for 20% of the apoA1 in atherosclerosis-laden arteries. OxTrp72-apoA1 recovered from human atheroma or plasma is lipid poor [and] virtually devoid of cholesterol acceptor activity. Moreover, the researchers found that the modified apoA1 demonstrated both a potent proinflammatory activity on endothelial cells and an impaired HDL biogenesis activity in vivo.”

Reflecting on the significance of these results, Dr. Hazen said, “Now that we know what this dysfunctional protein looks like, we are developing a clinical test to measure its levels in the bloodstream, which will be a valuable tool for both assessing cardiovascular disease risk in patients and for guiding development of HDL-targeted therapies to prevent disease.” The research also points toward new therapeutic targets for pharmaceuticals, such as those designed to prevent the formation of dysfunctional HDL and the development or progression of atherosclerosis.

WHO to launch multinational trial to jumpstart search for coronavirus drugs

The World Health Organization said Wednesday that it would launch a multiarm, multicountry clinical trial for potential coronavirus therapies, part of an aggressive effort to jumpstart the global search for drugs to treat Covid-19.

Four drugs or drug combinations already licensed and used for other illnesses will be tested, said WHO Director-General Tedros Adhanom Ghebreyesus. Ten countries have already indicated they will take part in the trial.

The mere fact the WHO is sponsoring the trial suggests that efforts in China to test these drugs may not have come up with enough data to indicate whether any were of use to prevent patients from developing severe disease or save those with severe disease from death.

The study, which Tedros said he hopes other countries will join, has been named the SOLIDARITY trial. Countries that have already signed on are: Argentina, Bahrain, Canada, France, Iran, Norway, South Africa, Spain, Switzerland, and Thailand.

“Multiple small trials with different methodologies may not give us the clear strong evidence we need about which treatments help to save lives,” he said during a briefing in Geneva

Ana Maria Henao-Restrepo, unit head for the WHO’s research and development “blueprint” group, said the trial design was deliberately kept simple “to enable even hospitals that have been overloaded to participate.”

“This trial focuses on the key priority questions for the public. Do any of these drugs reduce mortality? Do any of these drugs reduce the time a patient is in hospital and whether or not the patients receiving any of the drugs needed ventilation or intensive care units,” Henao-Restrepo said.

The four drugs or combinations will be compared to what is called standard of care — the regular support hospitals treating these patients use now, such as supplementary oxygen when needed.

The drugs to be tested are the antiviral drug remdesivir; a combination of two HIV drugs, lopinavir and ritonavir; lopinavir and ritonavir plus interferon beta; and the antimalarial drug chloroquine. All show some evidence of effectiveness against the SARS-CoV 2 virus, which causes Covid-19, either in vitro and/or animal studies.

Remdesivir is made by Gilead. Lopinavir and ritonavir are combined and sold as Kaletra or Aluvia by AbbVie.

Later in the day, after close of business in Geneva, the New England Journal of Medicine published a study from China that reported finding that the lopinavir-ritonavir combination did not improve survival or speed recovery, though the authors noted that the very high death rates among patients who received the drugs and those who received only standard care suggest they had enrolled “a severely ill population.”

Of the 199 patients in the trial, 22% died, which was “substantially higher than the 11% to 14.5% mortality reported in initial descriptive studies of hospitalized patients with Covid-19,” they said. The trial was also not blinded — meaning the doctors knew which patients were receiving the drugs — which they acknowledge could have influenced their clinical decision making.

“These early data should inform future studies to assess this and other medication in the treatment of infection with SARS-CoV-2,” wrote the authors. “Whether combining lopinavir–ritonavir with other antiviral agents, as has been done in SARS and is being studied in MERS-CoV, might enhance antiviral effects and improve clinical outcomes remains to be determined.”

Henao-Restrepo said chloroquine — which is cheap and used regularly around the world — will be tested two ways. Some countries will test chloroquine against the standard of care while others will test hydroxychloroquine, a related drug.

“The good thing about the trial is … that the randomization could be adjusted to the drugs available in each individual hospital over time,” Henao-Restrepo said. “The other good thing … is that we can include additional arms or drop arms as our global data safety and monitoring committee advises we should do.”

Enrolling patients across a number of countries should speed the world to an answer about which drugs, if any could be effective in reducing the toll of Covid-19. The WHO launched a similar trial in the Democratic Republic of the Congo in November 2018 to test four therapies against Ebola.

At the time of that launch, it was thought that the trial might need to draw data from several Ebola outbreaks before it could reach an answer. But the North Kivu outbreak, which could be declared over next month, was so large results were announced in August 2019. Given the high number of cases globally of Covid-19 and the number of countries participating, results should come faster with this trial.

This story had been corrected to remove an error about where hydroxychloroquine can be used. It has also been updated.

Coronavirus Treatment Could Lie in Existing Drugs

As the global number of COVID-19 cases passes 81,000, collaborating European scientists have identified 31 existing broad-spectrum antiviral agents (BSAAs) that they say may represent candidates for repurposing against the infection. The researchers suggest that repositioning existing approved and investigational drugs may represent the key to future fights against viral infections — including the SARS-CoV-2 virus, and other emerging viruses — and they have compiled a database that summarizes the activity and development status of more than 100 safe-in-man BSAAs.

“Drug repurposing is a strategy for generating additional value from an existing drug by targeting diseases other than that for which it was originally intended,” said Denis Kainov, PhD, senior author on the paper and an associate professor at the Norwegian University of Science and Technology (NTNU). “For example, teicoplanin, oritavancin, dalbavancin, and monensin are approved antibiotics that have been shown to inhibit corona- and other viruses in the laboratory.” Kainov and his co-authors say that these and other already tested safe-in-man, broad-spectrum antiviral agents (BSAAs) are good starting candidates.

Drug virus info
Safe-in-man broad-spectrum antiviral agents and coronaviruses they inhibit, from drugvirus.info website. Different shadings indicate different development status of BSAAs. Gray shading indicates that the antiviral activity has not been either studied or reported.
The researchers are making their database freely available, and report on their findings in the International Journal of Infectious Diseases, in a paper titled “Discovery and development of safe-in-man broad-spectrum antiviral agents.” Their report coincides with the start of U.S. trials with Gilead Sciences’ antiviral drug, remdesivir, which was previously tested as a treatment for Ebola virus.

BSAAs are small-molecules that may inhibit different types of human viruses that exploit similar pathways and host factors to replicate inside cells, the authors explained. The advantage of repurposing a drug is that all of the details surrounding the drug development are already known, from the chemical synthesis steps and manufacturing processes to information regarding the different phases of clinical testing.

“Although the concept of BSAAs has been around for almost 50 years, the field received a new impetus with recent outbreaks of Ebola, Zika, Dengue, influenza and other viral infections, the discovery of novel host-directed agents as well as development of drug repositioning methodology,” the investigators noted. “Therefore, repositioning of launched or even failed drugs to viral diseases provides unique translational opportunities, including a substantially higher probability of success to market as compared with developing new virus-specific drugs and vaccines, and a significantly reduced cost and timeline to clinical availability.”
SBAAs could thus hold promise for treating infection with the SARS-CoV-2 virus. “No vaccines and drugs are available for prevention and treatment of coronavirus infections in humans,” they stated. “However, safe-in-man BSAAs could be effective against 2019-nCoV [SARS-CoV-2 virus] and other coronaviruses.” For example, they pointed out, chloroquine and remdesivir effectively inhibited infection by the SARS-CoV-2 virus in vitro. “Moreover, teicoplanin, oritavancin, dalbavancin, monensin, and emetine could be repurposed for treatment of 2019-nCoV infections,” the team noted. “Oritavancin, dalbavancin, and monensin are approved antibiotics, whereas emetine is an anti-protozoal drug. These drugs have been shown to inhibit several corona- as well as some other viral infections.”

The researchers reviewed information on the discovery and development of BSAAs, and summarized what they found for 120 approved, investigational and experimental drugs that had already been shown to be safe for humans use, and which inhibit 86 human viruses in 25 viral families. “The BSAAs inhibit viral or host factors and block viral replication, reduce the viral burden to a level at which host immune responses can deal with it or facilitate apoptosis of infected cells,” the authors noted. They suggested that 31 of the SBAAs could represent possible candidates for prophylaxis and treatment of infection by SARS-CoV-2.

The team has compiled their findings into a BSAA database, which is freely available at Drugvirus.info. “The database allows interactive exploration of virus-BSAA interactions,” they stated. “It also includes information on BSA targets.”

The investigators also highlight the potential to combine drugs against viruses. “By contrast to individual drugs, combinations of 2-3 BSAAs could be used to target even broader range of viruses,” they noted. “Such combinations could serve as front line therapeutics against poorly characterized emerging viruses or re-emerging drug-resistant viral strains.” Kainov and colleagues further suggest that making the results of relevant clinical trials publicly available and standardizing data collection will aid prioritization and translation of emerging and existing BSAAs into clinical practice. “This would allow BSAAs to play a pivotal role in the battle against emerging and re-emerging viral diseases.”

Triple-Negative Breast Cancer in Mice Inhibited by Migraine Drug

A drug previously approved for treating migraine and epilepsy has been found to slow the growth of triple negative breast cancer in mouse models. Researchers in the U.S., Taiwan, and China carried out screening studies to identify known pharmacologically active compounds that trigger degradation of N-Ras, a key protein that drives the development of aggressive basal-like breast cancer (BLBC). The assays highlighted flunarizine (FLN) as a promising candidate, and subsequent in vitro studies confirmed that the drug selectively blocked the growth of BLBC cells, but not other subtypes of breast cancer cells, and also inhibited tumor growth in an in vivo BLBC xenograft model.

“We focused on finding ways to disrupt the effects of a class of protein called Ras, which are powerful drivers of a wide range of cancers,” comments Eric C. Chang, Ph.D., associate professor of molecular and cellular biology at the Lester and Sue Smith Breast Center in the Dan L. Duncan Comprehensive Cancer Center at Baylor College of Medicine. “In this proof-of-concept study, we have established a strategy to target N-Ras for therapy.”

The Baylor College of Medicine team, headed by Ze-Yi Zheng, Ph.D., together with colleagues at the Houston Methodist Research Institute, the Hospital (TCM) Affiliated to Southwest Medical University (China), and the National Taiwan University Hospital and National Taiwan University College of Medicine (Taiwan), report on their work in a paper published today in Scientific Reports, which is titled, “Induction of N-Ras degradation by flunarizine-mediated autophagy.”

Humans have three RAS genes, H-, N-, and K-Ras, the authors explain. More than 30% of all human tumors contain oncogenic RAS mutations, and while K-Ras is the most frequently mutated RAS gene in cancers generally, N-Ras mutations also occur in certain cancer types. Yet despite the importance of Ras in cancer, there are no drugs that specifically target the Ras proteins. Current approaches are designed to reduce membrane affinity for Ras proteins, block Ras-effector interaction, or inhibit the activity of effector protein kinases such as B-Raf, the authors continue. However, tumors commonly develop resistance through mechanisms that thwart the continued effectiveness of these therapeutic approaches. “Therefore it seems highly desirable to target Ras proteins themselves in order to shut down all of their oncogenic potential at the root,” the team writes.

One potential therapeutic avenue is to control Ras post-transcriptionally by proteolysis. The researchers devised a screening strategy that aimed to identify and repurpose already FDA-approved drugs that can also induce Ras degradation. “Our assay allows us to visually determine which drugs promote N-Ras degradation,” Dr. Chang explains. “We tagged N-Ras proteins with a green fluorescent tag. If the fluorescent N-Ras proteins were destroyed, the fluorescence would be lost.”

The assay identified the calcium ion channel blocker flunarazine, which is commonly used to treat conditions including epilepsy.  “Flunarizine has been used in medical practice for decades to treat dizziness and vertigo and to prevent migraines,” Dr. Chang comments. Interestingly, while flunarazine hasn’t previously been used to treat cancer, prior research has shown that it can boost the sensitivity of cancer cells to some chemotherapy agents.

The team’s subsequent studies indicated that flunarazine induced N-Ras degradation by autophagy, and in vitro tests showed that the drug inhibited the growth of BLBC cells specifically. This growth inhibition could be further enhanced by combining flunarazine with drugs that target other components of the N-Ras pathway.

In a final round of experiments the researchers demonstrated that flunarazine therapy reduced the growth of human BLBC tumors in mice. “Our data showed that when FLN was added at levels comparable to those used in humans, tumor growth was efficiently inhibited, mimicking the effect of doxycycline (DOX)-inducible N-Ras silencing,” they comment.

The authors acknowledge that further studies will be needed to define why and how flunarazine therapy leads to N-Ras degradation in BLBC cells. Nevertheless, they write, “ … this proof-of-principle study presents evidence that the autophagy pathway can be coerced by small molecule inhibitors, such as FLN, to degrade Ras as a strategy to treat cancer. FLN has low toxicity and should be further investigated to enrich the toolbox of cancer therapeutics.”

They also note that the studies validate the use of their screening approach to identify and repurpose existing drug compounds against cancer. “Screening existing relatively safe drugs for new functions is a valuable strategy for identifying drugs that can potentially be used to treat diseases for which currently there are no available treatments,” Dr. Chang states. “Reprogramming pathways that degrade cellular materials may be an effective strategy to remove a cancer driver that is otherwise hard to target.”

New Vaccine Protects Cattle from RSV, Provides Roadmap for Human Therapy

• The respiratory syncytial virus (RSV) is responsible for a common childhood illness. Researchers are hopeful that results from this new study will provide a model for developing an effective human RSV vaccine. [NIH]

  A team of investigators led by scientists at the National Institute of Allergy and Infectious Diseases (NIAID), the Pirbright Institute, U.K., and the Institute for Research in Biomedicine in Switzerland has developed a novel vaccine that can protect cattle from respiratory syncytial virus (RSV) infection. The RSV strain that the researchers used for this study naturally infects cattle and is closely related to human RSV. The results suggest that a similar human RSV vaccine construct may provide protection to humans.

  The findings from this study were published recently in NPJ Vaccines through an article entitled “Protection of Calves by a Prefusion-Stabilized Bovine RSV F Vaccine.”

  RSV is a primary cause of respiratory disease in cattle, resulting in significant economic costs to the industry. In humans, RSV can cause serious bronchiolitis and pneumonia in young children and the elderly, as well as adults with compromised immune systems. RSV infections are estimated to cause more than 250,000 human deaths annually around the world. There is no licensed vaccine to prevent RSV infection in humans, and vaccines currently in use for cattle have noted safety and effectiveness problems.

  For the current study, the research team created an investigational vaccine containing a single structurally engineered RSV protein that elicited high levels of neutralizing antibodies in mice. The protein is a stabilized version of the RSV fusion (F) glycoprotein in its initial conformation, called pre-F. Other vaccines have used the same protein in its final conformation (called post-F), but investigators found the immune response to that vaccine was much lower.

  We used “a combination of structure-based design, antigenic characterization, and X-ray crystallography to translate human RSV F stabilization into the bovine context,” the authors wrote. “A ‘DS2’ version of bovine respiratory syncytial virus F with subunits covalently fused, fusion peptide removed, and pre-fusion conformation stabilized by cavity-filling mutations and intra- and inter-protomer disulfides was recognized by pre-fusion-specific antibodies, AM14, D25, and MPE8, and elicited bovine respiratory syncytial virus-neutralizing titers in calves >100-fold higher than those elicited by post-fusion F.”

  The investigators immunized five 3- to 6-week-old calves with the pre-F protein via two injections 4 weeks apart. They vaccinated another five calves with a post-F protein, while the third group of five calves received two placebo injections of saline. Four weeks after the second immunization, investigators infected all three groups with RSV. The calves vaccinated with the pre-F protein had high levels of neutralizing antibodies (more than 100-fold higher than those that received the post-F protein), and four of five were protected from RSV viral replication in the upper and lower respiratory tracts. In contrast, RSV was detected in all calves immunized with either the post-F protein or placebo.

  According to authors“Our results demonstrate proof-of-concept that DS2-stabilized RSV F immunogens can induce highly protective immunity from RSV in a native host with implications for the efficacy of prefusion-stabilized F vaccines in humans and the prevention of bovine respiratory syncytial virus in calves,”

Revolutionizing Biotechnology with Artificial Restriction Enzymes

• Restriction enzymes are essential tools for recombinant DNA technology that have revolutionized modern biological research; however, they have limited sequence specificity and availability. The Pyrococcus furiosus Argonaute (PfAgo)-based platform for generating artificial restriction enzymes (AREs) is capable of recognizing and cleaving DNA sequences at virtually any arbitrary site and generating defined sticky ends of varying length. [Behnam Enghiad and Huimin Zhao/University of Illinois at Urbana-Champaign]

  Scientists at the University of Illinois say they have developed a new technique of genetic engineering for basic and applied biological research and medicine. Their work (“Programmable DNA-Guided Artificial Restriction Enzymes”), reported in ACS Synthetic Biology, could open new doors in genomic research by improving the precision and adherence of sliced DNA, according to the investigators.

  “Using our technology, we can create highly active artificial restriction enzymes with virtually any sequence specificity and defined sticky ends of varying length,” said Huimin Zhao, Ph.,D., professor of chemical and biomolecular engineering, who leads a synthetic biology research group at the Carl R. Woese Institute for Genomic Biology at Illinois. “This is a rare example in biotechnology where a desired biological function or reagent can be readily and precisely designed in a rational manner.”

  Restriction enzymes cut DNA at a specific site and create a space wherein foreign DNA can be introduced for gene-editing purposes. This process is not achieved only by naturally occurring restriction enzymes; artificial restriction enzymes, or AREs, have risen to prominence in recent years. CRISPR/Cas9, a bacterial immune system used for “cut-and-paste” gene editing, and TALENs, or transcription activator-like effector nucleases, which are modified restriction enzymes, are two popular examples of such techniques.

  Though useful in genetic engineering, no AREs generate defined “sticky ends”—an uneven break in the DNA ladder structure that leaves complementary overhangs, improving adhesion when introducing new DNA. “If you can cleave two different DNA samples with the same restriction enzyme, the sticky ends that are generated are complementary,” explained graduate student Behnam Enghiad. “They will hybridize with each other, and if you use a ligase, you can stick them together.”

  However, restriction enzymes themselves have a critical drawback: the recognition sequence that prompts them to cut is very short, usually only four to eight base pairs. Because the enzymes will cut anywhere that sequence appears, researchers rely on finding a restriction enzyme whose cut site appears only once in the genome of their organism or plasmid, an often difficult proposition when the DNA at hand might be thousands of base pairs long.

  This problem has been partially solved simply by the sheer number of restriction enzymes discovered: more than 3600 have been characterized, and over 250 are commercially available. “Just in our freezer, for our other research, we have probably over 100 different restriction enzymes,” said Enghiad. “We look through them all whenever we want to assemble something. The chance of finding the unique restriction site is so low.”

  “Our new technology unifies all of those restriction enzymes into a single system consisting of one protein and two DNA guides. Not only have you replaced them, but you can now target sites that no available restriction enzymes can.”

  The new method creates AREs through the use of an Argonaute protein (PfAgo) taken from Pyrococcus furiosus, an archeal species. Led by a DNA guide, PfAgo is able to recognize much longer sequences when finding its cut site, increasing specificity and removing much of the obstacles posed by restriction enzymes. Furthermore, PfAgo can create longer sticky ends than even restriction enzymes, a substantial benefit as compared to other AREs.

  “When we started, I was inspired by a paper about a related protein—TtAgo. It could use a DNA guide to cleave DNA, but only up to 70 degrees,” continued Enghiad. “DNA strands start to separate over 75 degrees, which could allow a protein to create sticky ends. If there were a protein that was active at higher temperatures, I reasoned, that protein could be used as an artificial restriction enzyme. So I started looking for that, and what I found was PfAgo.”

  In addition to replacing restriction enzymes in genetic engineering processes, Enghiad and Dr. Zhao believe their technology will have broad applications in the biological research. By creating arbitrary sticky ends, PfAgo could make assembly of large DNA molecules easier and would enable cloning of large DNA molecules, such as biochemical pathways and large genes.

  The application of these techniques is broad-reaching, they say,  ranging from discovery of new small-molecule drugs to engineering of microbial cell factories for synthesis of fuels and chemicals to molecular diagnostics of genetic diseases and pathogens, which are the areas Dr. Zhao and Enghiad are currently exploring.

  “Due to its unprecedented simplicity and programmability (a single protein plus DNA guides for targeting), as well as accessibility…we expect PfAgo-based AREs will become a powerful and indispensable tool in all restriction enzyme or nuclease-enabled biotechnological applications and fundamental biological research,” predicts Dr. Zhao. “It is to molecular biology as the CRISPR technology is to cell biology.”

Team Shows How Zika Crosses Placental Barrier to Cause Birth Defects

• Investigators from the Florida campus of The Scripps Research Institute (TSRI) say they have uncovered the details behind the virus’s unique ability to cross the placental barrier and expose the fetus to a range of birth defects that often go beyond microcephaly to include eye and joint injury, and even other types of brain damage. The new study (“AXL-Dependent Infection of Human Fetal Endothelial Cells Distinguishes Zika Virus from Other Pathogenic Flaviviruses”), led by TSRI associate professor Hyeryun Choe, Ph.D., was published online ahead of print this week in the journal Proceedings of the National Academy of Sciences.

  How Zika virus crosses the placental barrier, while other closely related viruses in the flavivirus family including dengue and West Nile viruses do not, has puzzled researchers since the crisis began some 2 years ago in Brazil. Obstacles to reaching the fetal brain are substantial—a virus must move from the mother’s blood into fetal circulation, which is separated by placental barrier cells designed to prevent that very occurrence.

  The researchers found that human umbilical endothelial cells, derived from four donors in the study, proved far more susceptible to Zika infection than to other viruses, with viral counts as much as 100 or 1000 times higher than West Nile or dengue virus. The new research also suggests that the Zika virus learned to exploit something of a secret passage, a cell-surface molecule known as AXL, while West Nile and dengue viruses did not.

  “Zika uses AXL to efficiently slip past one of the major barrier cell types in the placenta—fetal endothelial cells, which are the gateway to access fetal circulation,” said Dr. Choe.

  What may help make the Zika virus particularly infectious in cells that other flaviviruses can’t infect, said TSRI research associate Audrey Richard, Ph.D., first author of the study, is that it profits from the built-in function of AXL. “The physiological function of AXL is to quench activated immune reactions, including the antiviral interferon response,” said Dr. Richard. “By using AXL, the Zika virus catches two birds with one stone; it enters cells and also gains favorable environment for its replication inside the cells.”

  Zika is able to take advantage of AXL by binding to an intermediate molecule known as Gas6, which is present in blood and other bodily fluids. Gas6 acts as an active bridge between the virus and AXL by binding AXL on one end and the virus membrane on the other, helping the virus utilize AXL and gain entry to host cells.

  These differences may help explain why, among related viruses, only Zika can efficiently access and infect the fetal bloodstream.

  “We don’t yet understand why the Zika virus uses AXL and the others don’t,” Dr. Choe  added. “The common belief is that all flaviviruses have similar structures, but our findings suggest that the Zika virus may have a different average population structure than others. This has significant scientific and clinical implications.”

  “Structural studies show that most of the infectious virion membrane is completely covered with viral proteins, which makes it difficult for Gas6 to bind to the Zika virus membrane underneath the protein shell,” said TSRI research associate Byoung-Shik Shim, Ph.D., the study’s other first author. “However, flavivirus particles assume many asymmetric shapes and are in continuous dynamic motion, which likely exposes patches of the virion membrane. Our study suggests that Zika virus exposes enough membrane for Gas6 binding, whereas West Nile and dengue viruses do not.”

  The researchers also speculated on the Zika virus’ pathology. AXL is also present in the blood–brain barrier, the eye–blood barrier, and the testes, where it maintains integrity of the blood vessels and the functions of the testes. It may be used by the Zika virus to infect those cells and may explain the Zika virus’ ability to infect the fetal brain and eye and to transmit sexually.

Periodic Table of Protein Complexes Unveiled

• A new periodic table presents a systematic, ordered view of protein assembly, providing a visual tool for understanding biological function. [EMBL-EBI / Spencer Phillips]

  Move over Mendeleev, there’s a new periodic table in science. Unlike the original periodic table, which organized the chemical elements, the new periodic table organizes protein complexes, or more precisely, quaternary structure topologies. Though there are other differences between the old and new periodic tables, they share at least one important feature—predictive power.

  When Mendeleev introduced his periodic table, he predicted that when new chemical elements were discovered, they would fill his table’s blank spots. Analogous predictions are being ventured by the scientific team that assembled the new periodic table. This team, consisting of scientists from the Wellcome Genome Campus and the University of Cambridge, asserts that its periodic table reveals the regions of quaternary structure space that remain to be populated.

  The periodic table of protein complexes not only offers a new way of looking at the enormous variety of structures that proteins can build in nature, it also indicates which structures might be discovered next. Moreover, it could point protein engineers toward entirely novel structures that never occurred in nature, but could be engineered.

  The new table appeared December 11 in the journal Science, in an article entitled, “Principles of assembly reveal a periodic table of protein complexes.” The “principles of assembly” referenced in this title amount to three basic assembly types: dimerization, cyclization, and heteromeric subunit addition. In dimerization, one protein complex subunit doubles, and becomes two; in cyclization, protein complex subunits from a ring of three or more; and in heteromeric subunit addition, two different proteins bind to each other.

  These steps, repeated in different combinations, gives rise to enormous number of proteins of different kinds. “Evolution has given rise to a huge variety of protein complexes, and it can seem a bit chaotic,” explained Joe Marsh, Ph.D., formerly of the Wellcome Genome Campus and now of the MRC Human Genetics Unit at the University of Edinburgh. “But if you break down the steps proteins take to become complexes, there are some basic rules that can explain almost all of the assemblies people have observed so far.”

  The authors of the Science article noted that many protein complexes assemble spontaneously via ordered pathways in vitro, and these pathways have a strong tendency to be evolutionarily conserved. “[There] are strong similarities,” the authors added, “between protein complex assembly and evolutionary pathways, with assembly pathways often being reflective of evolutionary histories, and vice versa. This suggests that it may be useful to consider the types of protein complexes that have evolved from the perspective of what assembly pathways are possible.”

  To explore this rationale, the authors examined the fundamental steps by which protein complexes can assemble, using electrospray mass spectrometry experiments, literature-curated assembly data, and a large-scale analysis of protein complex structures. Ultimately, they derived their approach to explaining the observed distribution of known protein complexes in quaternary structure space. This approach, they insist, provides a framework for understanding their evolution.

  “In addition, it can contribute considerably to the prediction and modeling of quaternary structures by specifying which topologies are most likely to be adopted by a complex with a given stoichiometry, potentially providing constraints for multi-subunit docking and hybrid methods,” the authors concluded. “Lastly, it could help in the bioengineering of protein complexes by identifying which topologies are most likely to be stable, and thus which types of essential interfaces need to be engineered.”

  The rows and columns of the periodic table of the elements, called periods and groups, were originally determined by each element’s atomic mass and chemical properties, later by atomic number and electron configuration. In contrast, the rows and columns of the periodic table of protein complexes correspond to the number of different subunit types and the number of times these subunits are repeated. The new table is not, it should be noted, periodic in the same sense as the periodic table of the elements. It is in principle open-ended.

  Although there are no theoretical limitations to quaternary structure topology space in either dimension, the abridged version of the table presented in the Science article can accommodate the vast majority of known structures. Moreover, when the table’s creators compared the large variety of countenanced topologies to observed structures, they found that about 92% of known protein complex structures were compatible with their model.

  “Despite its strong predictive power, the basic periodic table model does not account for about 8% of known protein complex structures,” the authors conceded. “More than half of these exceptions arise as a result of quaternary structure assignment errors.

  “A benefit of this approach is that it highlights likely quaternary structure misassignments, particularly by identifying nonbijective complexes with even subunit stoichiometry. However, this still leaves about 4% of known structures that are correct but are not compatible with the periodic table.” The authors added that the exceptions to their model are interesting in their own right, and are the subject of ongoing studies.

Suppressing Aging Genes Extends Lifespans

• Click Image To Enlarge +

  A recurring theme in art, the three ages, features in a new study of age-related changes in gene expression. The study identified a set of highly conserved genes that could, when suppressed, improve health and extend lifespans. [The Three Ages of Man, by Giorgione, is a work in the public domain that appears in the Web Gallery of Art and Wikipedia]

  Youth. Maturity. Old age. Each stage in physiological aging is characterized by different patterns of gene expression, which complicates the search for “aging genes.” To deal with this complication, scientists centered at ETH Zurich compared aging-related patterns in gene expression in young, mature, and old model organisms—nematodes, zebrafish, and mice. By studying gene expression patterns in three different organisms, the scientists hoped to highlight genes that have been conserved through evolution, and are thus likely to be found in humans, too.

  After measuring the production of messenger RNA (mRNA)—a proxy for gene activity—for each of 40,000 genes and for each of the aging stages, the scientists were able to identify genes that were regulated similarly in each of the model organisms. They found that the three organisms have only 30 genes in common that significantly influence the aging process.

  What’s more, the scientists conducted experiments in which the mRNAs of the corresponding genes were selectively blocked. These experiments allowed the scientists to determine the effect of these genes on the aging process in nematodes. By blocking a dozen of these genes, the scientists were able to extend the lifespan of the nematodes by at least 5%.

  One of these genes proved to be particularly influential: the bcat-1 gene. “When we blocked the effect of this gene, it significantly extended the mean lifespan of the nematode by up to 25%,” said Michael Ristow, M.D., professor of energy metabolism at ETH Zurich.

  Dr. Ristow is the coordinating author of a study that appeared December 1 in the journal Nature Communications. The study—“Branched-chain amino acid catabolism is a conserved regulator of physiological ageing”—not only explains what bcat-1 does, it explains how the gene works.

  The bcat-1 gene carries the code for the enzyme of the same name, which degrades so-called branched-chain amino acids (BCAAs). Naturally occurring in food protein building blocks, these include the amino acids L-leucine, L-isoleucine, and L-valine.

  “BCAAs reduce a LET-363/mTOR-dependent neuro-endocrine signal, which we identify as DAF-7/TGFβ, and that impacts lifespan depending on its related receptors, DAF-1 and DAF-4, as well as ultimately on DAF-16/FoxO and HSF-1 in a cell-non-autonomous manner,” wrote the authors of the Nature Communications article. “The transcription factor HLH-15 controls and epistatically synergizes with BCAT-1 to modulate physiological ageing.”

  When the researchers inhibited the gene activity of bcat-1, BCAAs accumulated, triggering a molecular signaling cascade that increased longevity in the nematodes. Moreover, the timespan during which the worms remained healthy was extended. As a measure of vitality, the researchers measured the accumulation of aging pigments, the speed at which the creatures moved, and how often the nematodes successfully reproduced.

  The scientists also achieved a life-extending effect when they mixed the three BCAAs into the nematodes’ food. However, the effect was generally less pronounced because the bcat-1 gene was still active, which meant that the amino acids continued to be degraded and their life-extending effects could not develop as effectively.

  In the present study, the investigators purposefully opted not to study the impact on humans. But a follow-up study is already being planned. “We cannot measure the life expectancy of humans for obvious reasons,” explained Dr. Ristow. Instead, the researchers plan to incorporate various health parameters such as cholesterol or blood sugar levels in their study to obtain indicators on the health status of their subjects.

  Dr. Ristow added that the multiple BCAAs are already being used to treat liver damage and are also added to sport nutrition products. “However, the point is not for people to grow even older, but rather to stay healthy for longer,” he asserted.

Stress Study Points to Mind-Mitochondria-Body Connection

• Your response to stress—hormonally, metabolically, and behaviorally—may depend on your mitochondrial genes. And so your mitochondrial genes may determine your long-term susceptibility to stress-related diseases. They may even have a role in human psychology and the biology of psychiatric and neurological diseases.

  These possibilities were raised by a study that aligned mutations in mitochondrial genes with unique whole-body stress-response signatures. The study, which was conducted at the Children’s Hospital of Philadelphia, indicates that even relatively mild mutations in mitochondrial genes can cause systemic alterations in energetic metabolism, and that these alterations affect physiological networks essential for health, including physiological networks that affect the brain.

  “The brain, constituting only 2% of human body weight, consumes 20% of the body’s energy,” said Douglas C. Wallace, Ph.D., director of the Center for Mitochondrial and Epigenomic Medicine at the Children’s Hospital of Philadelphia. “Hence, mild variations in mitochondrial bioenergetics will have significant effects on the brain.”

  Dr. Wallace and colleagues settled on the hypothesis that abnormal mitochondrial functions in an organism would differentially modulate that organism’s multisystemic response to psychological stress. Then they tested this hypothesis. They subjected mice to a standardized psychological stress: placing them in restraint for a brief period. Next they measured the effects of this stressor on the animals’ neuroendocrine, inflammatory, metabolic, and gene transcription systems.

  Crucially, the mice used in this study sustained alterations in their mitochondrial genes, alterations that were designed to selectively impair mitochondrial respiratory chain function, energy exchange, and mitochondrial redox balance. Such alterations included mutations or deletions in the mitochondrial genes encoded in the mitochondrial DNA (NADH dehydrogenase 6 and cytochrome c oxidase subunit I) or nuclear DNA (adenine nucleotide translocator 1 and nicotinamide nucleotide transhydrogenase).

  The results of the study appeared in the December 1 issue of the Proceedings of the National Academy of Sciences, in an article entitled, “Mitochondrial functions modulate neuroendocrine, metabolic, inflammatory, and transcriptional responses to acute psychological stress.” The article described how the mitochondrial gene changes impacted the physiological reactivity and recovery from restrain stress.

  “When analyzed collectively, stress-induced neuroendocrine, inflammatory, metabolic, and transcriptional responses coalesced into unique signatures that distinguish groups based on their mitochondrial genotype,” wrote the article’s authors. In particular, mitochondrial dysfunctions altered the hypothalamic–pituitary–adrenal axis, sympathetic adrenal–medullary activation and catecholamine levels, the inflammatory cytokine IL-6, circulating metabolites, and hippocampal gene expression responses to stress.

  “These results demonstrate the role of mitochondrial energetics and redox balance as modulators of key pathophysiological perturbations previously linked to disease,” the authors continued. “This work establishes mitochondria as stress-response modulators, with implications for understanding the mechanisms of stress pathophysiology and mitochondrial diseases.”

  The study, said Dr. Wallace, could have profound implications for the hereditary basis of neuropsychiatric diseases and for the role of stress in human health. He added that identifying the altered mitochondrial states associated with neuropsychiatric diseases could suggest new therapies.

  If such therapies were be devised, physicians would be able to more effectively ameliorate the effects of environmental stressors on human health. This could make people more resilient in environmental changes, reduce the long-term burden of stress-related diseases, and produce more effective therapies for psychiatric disorders.

  “While human differences in behavior and its relation to predisposition to mental illness as well as to a wide varied of pediatric and adult neurological diseases has been the subject of intense investigations for over a century, we still have a rudimentary understanding of the physiological, genetic, and environmental factors that mediate mental health and illness,” concluded Dr. Wallace. “Our recent papers strongly suggest that by reorienting our investigations from the anatomy of the brain and brain-specific genes to the mitochondria and the bioenergetics genes, we may have a more productive conceptual framework to understand neuropsychiatric disease. If so, this will spawn a whole new generation of neuropsychiatric therapeutics.”