Protein linked to sex differences in age-related dopamine neuron loss

Protein linked to sex differences in age-related dopamine neuron loss

It is not every day that scientists come across a phenomenon so fundamental that it is observed across fruit flies, rodents and humans.

In a paper published today in Aging Cell, neuroscientists from the University of Pittsburgh Schools of the Health Sciences discovered that a single protein—a glutamate transporter on the membrane of vesicles that carry dopamine in neurons—is key to regulating sex differences in the brain’s vulnerability to age-related neuron loss.

The protein—named VGLUT—was more abundant in dopamine neurons of female fruit flies, rodents and human beings than in males, correlating with females’ greater resilience to age-related neuron loss and mobility deficiencies, the researchers found. Excitingly, genetically reducing VGLUT levels in female flies diminished their protection from neurodegeneration associated with aging, suggesting that VGLUT could be a new target for prolonging dopamine neuron resilience and delaying the onset of symptoms of aging in the brain.

“From flies to rodents to human beings, we found that VGLUT levels distinguish males from females during healthy aging,” said senior author Zachary Freyberg, M.D., Ph.D., assistant professor of psychiatry and cell biology at Pitt. “The fact that this marker of dopamine neuron survival is conserved across the animal kingdom suggests that we are looking at a fundamental piece of biology. Understanding how this mechanism works can help prolong dopamine neuron resilience and delay aging.”

Neurodegenerative disorders such as Parkinson’s disease are more likely to develop as we age. Parkinson’s disease—a slow but relentless loss of dopamine neurons in the brain that impairs one’s ability to move or talk—is known to predominantly affect men. But while biological sex differences, which arise from a combination of hormonal, genetic and environmental influences, seem to explain why females are protected from early stages of Parkinson’s, the driver and regulator of these protections was, until now, unknown.

Using a combination of biochemical and genetic techniques, as well as behavioral studies where flies’ locomotion was monitored for a 24-hour period, researchers found that age-related benefits afforded to females disappeared when the levels of VGLUT gene expression were significantly reduced in dopamine neurons.

“We found that VGLUT expression increases with age, and that flies become more vulnerable to dopamine neuron degeneration when we knock down VGLUT,” said lead author Silas Buck, a Ph.D. candidate at the Pitt Center for Neuroscience. “We also found that VGLUT expression is higher in females than males, suggesting that VGLUT may play a role in regulating sex differences in vulnerability to neurodegeneration in Parkinson’s and other neurological disorders where females are more resilient than males.”

As the rates of Parkinson’s disease are rapidly rising—the number of people affected by the illness worldwide is projected to reach 20 million by 2040—Pitt scientists hope to further probe the role of VGLUT in neuroprotection in humans.

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Researchers identify protein produced after stroke that triggers neurodegeneration

Researchers identify protein produced after stroke that triggers neurodegeneration

Researchers with the Peter O’Donnell Jr. Brain Institute at UT Southwestern have identified a new protein implicated in cell death that provides a potential therapeutic target that could prevent or delay the progress of neurodegenerative diseases following a stroke.

Scientists from the departments of pathology, neurology, biochemistry, and pharmacology at UTSW have identified and named AIF3, an alternate form of the apoptosis-inducing factor (AIF), a protein that is critical for maintaining normal mitochondrial function. Once released from mitochondria, AIF triggers processes that induce a type of programmed cell death.

In a study published in the journal Molecular Neurodegeneration, the UT Southwestern team collaborated with researchers at The Johns Hopkins University School of Medicine and found that, following a stroke, the brain switches from producing AIF to producing AIF3. They also reported that stroke triggers a process known as alternative splicing, in which a portion of the instructions encoding AIF is removed, resulting in the production of AIF3. Defective splicing can cause disease, but modifying the splicing process may offer potential for new therapies.

In both human brain tissue and mouse models developed by researchers, AIF3 levels were elevated after a stroke. In mice, the stroke-induced production of AIF3 led to severe progressive neurodegeneration, hinting at a potential mechanism for a severe side effect of stroke observed in some patients. Stroke has been recognized as the second most common cause of dementia, and it is estimated that 10 percent of stroke patients develop post-stroke neurodegeneration within one year.

The molecular mechanism underlying AIF3 splicing-induced neurodegeneration involves the combined effect of losing the original form of AIF in addition to gaining the altered AIF3, leading to both mitochondrial dysfunction and cell death.

“AIF3 splicing causes mitochondrial dysfunction and neurodegeneration,” says senior author Yingfei Wang, Ph.D., assistant professor of pathology and neurology and a member of the O’Donnell Brain Institute. “Our study provides a valuable tool to understand the role of AIF3 splicing in the brain and a potential therapeutic target to prevent or delay the progress of neurodegenerative diseases.”

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Inhibition of Meprin Β enzyme can trigger the development of Alzheimer's disease, cancer

Researchers at Johannes Gutenberg University Mainz (JGU) in Germany and the Institute of Molecular Biology of Barcelona in Spain have discovered how the blood plasma protein fetuin-B binds to the enzyme meprin β and used a computer model to visualize their findings.

These results could lead to the development of new drugs to treat serious diseases such as Alzheimer's and cancer. Meprin β releases proteins from cell membranes, thus controlling important physiological functions in the human body.

However, a dysregulation of this process can trigger the development of Alzheimer's and cancer. Meprin β is regulated by fetuin-B binding to the enzyme when required, thereby preventing the release of other proteins. Presenting their findings in the journal Proceedings of the National Academy of Sciences, the researchers are now the first to describe this binding in detail.

The team at Mainz University produced both meprin β and fetuin-B in insect cells and then allowed them to react with one other in a test tube. By means of measurement of enzyme kinetics and biophysical analyses, the researchers determined that this reaction resulted in an exceptionally stable, high-molecular-mass complex.

Their colleagues in Barcelona subsequently managed to crystallize the complex and determine its three-dimensional structure using X-ray crystallography. This involved X-rays being fired at the protein crystals, which allowed the atomic structure of the crystals to be calculated from the diffraction of the X-rays. A computer model of the structure was then generated.

"Thanks to the model, we can now see exactly how meprin β and fetuin-B bind together," said Professor Walter Stöcker, who conducted the research at JGU together with Dr. Hagen Körschgen and Nele von Wiegen. "This research represents an excellent starting point for gaining a better understanding of diseases such as Alzheimer's and for developing the drugs to combat them."

Meprin β is already known to be involved in the formation of so-called beta-amyloid plaques, which are a characteristic feature of the condition. Moreover, people with Alzheimer's disease have relatively little fetuin-B in their blood, which in turn may lead to a lack of regulation of meprin β.

If it is possible to develop a drug that binds to the enzyme and inhibits it in a similar way to fetuin-B, this could be a new way of treating Alzheimer's."

Walter Stöcker, Professor, Johannes Gutenberg University Mainz (JGU), Germany

Source:

Johannes Gutenberg University Mainz

Journal reference:

Eckhard, U., et al. (2021) The crystal structure of a 250-kDa heterotetrameric particle explains inhibition of sheddase meprin β by endogenous fetuin-B. Proceedings of the National Academy of Sciences. doi.org/10.1073/pnas.2023839118.

Posted in: Medical Research News | Medical Condition News

Tags: Alzheimer's Disease, Blood, Cancer, Cell, Crystallography, Diffraction, Drugs, Enzyme, Molecular Biology, Protein, Research, X-Ray

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Updated Moderna vaccines neutralize South African SARS-CoV-2 variant in mice

Researchers in the United States have conducted a pre-clinical study demonstrating the efficacy of two updated versions of the Moderna mRNA-1273 vaccine against variants of severe acute respiratory syndrome coronavirus 2 – the agent that causes coronavirus disease 2019 (COVID-19).

Study: Variant SARS-CoV-2 mRNA vaccines confer broad neutralization as primary or booster series in mice

The updated vaccine candidates include the monovalent mRNA-1273.351, which is designed to target the B.1.351 variant that emerged in South Africa, and the multivalent mRNA1273.211, which comprises a mixture of the original mRNA-1273 vaccine and mRNA-1273.351.

The vaccines were evaluated in mice as both a primary vaccination series and as a booster dose among animals that had previously been immunized with two doses of mRNA-1273.

The team reports that while the mRNA-1273.351 vaccine elicited high levels of neutralizing antibody titers against B.1.351, the multivalent mRNA-1273.211 was most effective at providing broad cross-variant neutralization.

In addition, a booster dose of mRNA-1273.351 dramatically increased neutralization titers against both wild-type SARS-CoV-2 and the B.1.351 variant.

“Both mRNA-1273.351 and mRNA-1273.211 are currently being evaluated in additional pre-clinical challenge models and in phase 1/2 clinical studies,” says the team from Moderna Inc and the National Institute of Allergy and Infectious Diseases.

Global surveillance for the emergence of further variants of concern (VOCs) is also ongoing, as are efforts to test the ability of mRNA-1273 to neutralize VOCs.

“If additional variants emerge that reduce the neutralization capacity of mRNA-1273 further, additional mRNA vaccine designs may be developed and evaluated,” writes Kai Wu and colleagues.

A pre-print version of the research paper is available on the bioRxiv* server, while the article undergoes peer review.

Model of S protein. mRNA-1273.351 encodes the B.1.351 lineage S variant. Surface representation of the trimeric S protein in the vertical view with the locations of surface-exposed mutated residues highlighted in red spheres and labeled on the grey monomer. The inset shows superimposition of ACE-2 receptor domain and the RBD. S protein structure, 6VSB. ACE2-RBD structure, 6M0J. ACE2, angiotensin converting enzyme 2; NTD, N-terminal domain; RBD, receptor-binding domain.

Variants increasingly pose a threat to vaccination

The emergence of SARS-CoV-2 variants has raised concerns that the virus may have evolved the ability to escape vaccine-induced immunity. Several variants have demonstrated resistance to neutralization by vaccinated sera, particularly the South African B.1.351 lineage that was first identified in December 2020.

The initial stage of the SARS-CoV-2 infection process is mediated by the viral spike protein, which attaches to the host cell receptor via its receptor-binding domain (RBD). This spike RBD is the primary target of the neutralizing antibodies that are generated following infection or vaccination. A neutralization “supersite” has also been identified in the N-terminal domain (NTD) of the spike protein.

Studies have shown that several recently emerged SARS-CoV-2 variants harbor mutations in the RBD and NTD of spike that may confer resistance to vaccine-induced neutralization activity.

“Importantly, mutations in the NTD domain, and specifically the neutralization supersite, are extensive in the B.1.351 lineage virus,” says Wu and colleagues.

Furthermore, “studies have demonstrated reduced neutralization titers against the full B.1.351 variant following mRNA-1273 vaccination,” they add.

What did the researchers do?

Now, Wu and colleagues have evaluated the efficacy of two updated vaccines.

The monovalent mRNA-1273.351 vaccine encodes the spike protein found in the B.1.351 lineage, while the multivalent mRNA-1273.211 comprises a 1:1 mix of mRNA-1273.351 and mRNA-1273.

The original mRNA-1273 vaccine targets the ancestral wild-type virus (Wuhan-Hu-1 variant) that contains a mutation called D614G.

The vaccines were administered in mice as a two-dose primary series and their immunogenicity against D614G and B.1.351 pseudoviruses was evaluated 2 weeks following the first and second immunizations.

The mRNA-1273.351 vaccine was also evaluated as a booster dose in animals that had previously received two doses of mRNA-1273.

What did the study find?

Vaccine mRNA-1273.351 elicited approximately 4-fold higher neutralization titers against B.1.351 than against D614G.

Vaccine mRNA-1273.211, on the other hand, elicited robust neutralization responses against both D614G and B.1.351, with no significant difference observed in neutralization titers.

“Thus, as a primary vaccination series, a multivalent approach appears most effective in broadening immune responses,” says the team.

Next, the researchers tested the ability of mRNA-1273.351 to serve as a booster shot for neutralization against both D614G and B.1.351.

Mice were injected with mRNA-1273 on days 1 and 22 and then evaluated for antibody responses over the course of 7 months before being vaccinated a third time with mRNA-1273.351.

Following this booster injection, neutralization titers against D614G increased 4.5-fold, while titers against B.1.351 increased 15-fold.

The mRNA-1273.351 vaccine is an “effective booster”

“mRNA-1273.351 is an effective third (booster) dose in animals previously vaccinated with a primary vaccination series of mRNA-1273,” says Wu and the team.

“Ongoing studies will evaluate the ability of mRNA-1273, mRNA-1273.351, and mRNA-1273.211 to effectively boost immunity driven by a primary vaccination series of mRNA-1273,” they add.

The team says the mRNA platform approach against SARS-CoV-2 VOCs has now been demonstrated in mice to effectively broaden neutralization across variants and boost antibody levels when applied as a third dose.

“The mRNA platform allows for rapid design of vaccine antigens that incorporate key mutations, allowing for rapid future development of alternative variant-matched vaccines should they be needed,” writes Wu and colleagues.

“Additional VOC designs can be rapidly developed and deployed in the future if needed to address the evolving SARS-CoV-2 virus,” they conclude.

*Important Notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Journal reference:
  • Wu K, et al. Variant SARS-CoV-2 mRNA vaccines confer broad neutralization as primary or booster series in mice. bioRxiv, 2021. doi: https://doi.org/10.1101/2021.04.13.439482, https://www.biorxiv.org/content/10.1101/2021.04.13.439482v1

Posted in: Medical Research News | Disease/Infection News

Tags: ACE2, Allergy, Angiotensin, Antibodies, Antibody, Cell, Coronavirus, Coronavirus Disease COVID-19, Efficacy, Enzyme, Infectious Diseases, Mutation, Protein, Receptor, Research, Respiratory, SARS, SARS-CoV-2, Severe Acute Respiratory, Severe Acute Respiratory Syndrome, Spike Protein, Syndrome, Vaccine, Virus

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Written by

Sally Robertson

Sally first developed an interest in medical communications when she took on the role of Journal Development Editor for BioMed Central (BMC), after having graduated with a degree in biomedical science from Greenwich University.

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Nanobody cocktails neutralize SARS-CoV-2 and variants

Researchers report a collection of nanobodies derived from llamas, some of which can work synergistically and work against newer virus variants.

Although several strategies have been implemented to combat the COVID-19 pandemic, it continues unabated. Despite several vaccines being approved and used, vaccination rates have been slow, and there have been challenges in the equitable distribution of vaccines. These, along with decreasing immunity in persons already infected and the emergence of new virus variants, make it difficult to contain the pandemic.

Several therapeutic strategies have used convalescent sera and human monoclonal antibodies. However, new variants have emerged, with mutations that can evade these therapeutics.

An alternative to monoclonal antibodies is nanobodies. These are smaller proteins derived from animals like llamas and alpacas. Their small size allows them to bind to regions that are generally not accessible to the larger monoclonal or polyclonal antibodies. They are also simpler to manufacture as they can be easily cloned and expressed in bacteria. They can also be delivered directly to the lungs via nebulization.

However, the nanobodies also recognize regions of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein receptor-binding domain (RBD), which can cause escape mutations, reducing their potency.

Pre-screening protocol to select llamas with naturally strong immune responses, as determined by activity against standard animal vaccines

Testing nanobody neutralization capability

In a study published in the bioRxiv* preprint server, researchers report a collection of a large number of nanobodies that could potentially be resistant to escape mutations.

To build their suite of nanobodies, the team refined and optimized their previous method. They chose llamas with a strong immune response to SARS-CoV-2 and selected 113 nanobodies for further testing. A large proportion of the antibodies bind to the RBD, while the other bind to the non-RBD portion of the S1 subunit of the spike protein and the rest to the S2 subunit.

To identify nanobodies that will be resistant to virus mutations, the team selected a large number of high RBD binding nanobodies and tested them against the B.1.1.7 variant. Of the seven nanobodies they tested, they found six showed strong binding to the variants. In addition, they also chose nanobodies that bind to the non-RBD regions of the spike protein.

The authors then categorized the antibodies depending on what parts of the RBD they bind to. Their analysis revealed the RBD-binding nanobodies could be classified into three distinct groups. Within each group, the nanobodies could be grouped into bins so that some bind to distinct epitopes and partially overlap, binding to other separate epitopes.

This suggests two or more nanobodies can bind to the RBD at the same time. Further analysis revealed at least three different nanobodies could bind to the RBD simultaneously, which will be important in designing nanobody cocktails for therapies.

The nanobodies were potent in neutralizing SARS-CoV-2 pseudoviruses, with 16 nanobodies neutralizing the pseudovirus at less than 20 nM concentration. Nanobodies that target the non-RBD regions and the S2 subunit also neutralized the virus but at higher concentrations. The authors write this is the first evidence of nanobody neutralization targeting regions outside the RBD.

Nanobody cocktails more potent against escape variants

The team also tested the best nanobodies against pseudoviruses carrying the spike protein of the B.1.351 variant. Some nanobodies showed no neutralization activity against this variant, while one showed similar neutralization as the wild-type virus. Two nanobodies, however, showed increased neutralization activity against the variant. The nanobodies that neutralized the pseudovirus also neutralized the real SARS-CoV-2 virus and human airway epithelial cells. The team also found some nanobody combinations can work synergistically and increase potency dramatically.

Using nanobody cocktails mixed with the SARS-CoV-2 virus and allowing multiple replications, the authors also identified mutations that resist neutralization. Some potent nanobodies caused mutations at the same location as generated by antibodies in human convalescent sera, such as E484K, indicating the ACE2 binding site is a point of vulnerability for neutralization.

However, nanobody cocktails also increased the genetic barrier for escape. Mixing two nanobodies required the virus to undergo two different amino acid substitutions, making it more difficult for it to escape neutralization. Carefully choosing mixtures with more nanobodies may further decrease escape mutations.

Thus, the escape experiments show that currently used or newer therapeutics may yet lose their potency with newer virus variants emerging, which is very likely as some of the mutations seen in the experiments have not been seen in human isolates so far. However, using a combination of nanobodies, which can work synergistically to improve neutralization potency, the large collection of nanobodies generated by the team may help develop more potent treatments.

*Important Notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Journal reference:
  • Mast, F. D. et al. (2021) Nanobody Repertoires for Exposing Vulnerabilities of SARS-CoV-2. bioRxiv, https://doi.org/10.1101/2021.04.08.438911, ​https://www.biorxiv.org/content/10.1101/2021.04.08.438911v1

Posted in: Medical Science News | Medical Research News | Disease/Infection News

Tags: ACE2, Amino Acid, Antibodies, Bacteria, Coronavirus, Coronavirus Disease COVID-19, Genetic, Immune Response, Lungs, Nanobodies, Pandemic, Protein, Pseudovirus, Receptor, Respiratory, SARS, SARS-CoV-2, Severe Acute Respiratory, Severe Acute Respiratory Syndrome, Spike Protein, Syndrome, Therapeutics, Virus

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Lakshmi Supriya

Lakshmi Supriya got her BSc in Industrial Chemistry from IIT Kharagpur (India) and a Ph.D. in Polymer Science and Engineering from Virginia Tech (USA).

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Researchers discover a new monoclonal antibody that is effective against SARS-CoV-2 variants

A new monoclonal antibody targets a particular region of the receptor-binding domain (RBD) on the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This region is usually not accessible to immune cells,  which may be why it has broad neutralizing capabilities.

With the coronavirus disease 2019 (COVID-19) pandemic continuing around the globe, new mutants of SARS-CoV-2 are emerging. These new variants are likely more infectious and can better evade our immune response.

The SARS-CoV-2 spike protein, in particular the RBD, is key in binding to host receptors, mainly the angiotensin-converting enzyme 2 (ACE2) in humans. One highly conserved region of the RBD, called antigenic site II, can elicit neutralizing antibodies. However, this region is generally inaccessible because of the RBD conformation, and there is a low fraction of antibodies targeting this site in infected individuals.

Study: Structural basis for broad sarbecovirus neutralization by a human monoclonal antibody. Image Credit: Design_Cells / Shutterstock

In a new study published in the bioRxiv* preprint server, researchers report a new monoclonal antibody that is targeted toward site II and has a broad neutralizing capability.

Testing potency of monoclonal antibody

The authors sorted spike protein-specific memory B cells from a convalescent individual 75 days after symptom onset. They found one monoclonal antibody, called S2X259, which reacted with 29 of 30 spike proteins of sarbecoviruses, including SARS-CoV-2 and its new variants. The antibody also reacted with bat sarbecoviruses, suggesting its broad neutralizing capability.

The antibody also bound strongly to 10 RBDs from different sarbecoviruses. The binding of this antibody was not affected by the different single-point RBD mutations seen in the new variants of SARS-CoV-2, including the United Kingdom, South African, Brazilian, and the B.1.427/B.1.429 variants.

Using pseudotyped virus systems, the team found that the antibody neutralized SARS-CoV-2 and did not lose its potency against the different variants or the N439K or Y453F mutation. The antibody not only neutralized a variety of sarbecoviruses that use the ACE2 receptor but also cross-reacts with sarbecoviruses that do not use ACE2 for infection.

To understand how this antibody has high neutralizing potency, the team imaged the complex formed between the spike protein and the antibody using cryo-electron microscopy. They found that the antibody recognizes a glycan-free site, which requires two RBDs to be in the open conformation. It forms contacts with residues 369-386, 404-411, and 499-508 in the RBD.

The epitope the antibody binds to is conserved in all the circulating SARS-CoV-2 variants. In addition, it does not target the 417 or 484 residues (mutations here are found in B.1.351 and P.1), and this could be why it is potent against the different variants.

The action of this antibody does not affect the neutralization effect of class 1 and class 3 antibodies. The majority of approved antibodies for clinical use belong to these classes. Hence, the new antibody can be used in combination with other antibodies to increase neutralization breadth.

The S2X259 broadly neutralizing sarbecovirus mAb recognizes RBD antigenic site II. a-b, CryoEM structure of the prefusion SARS-CoV-2 S ectodomain trimer with three S2X259 Fab fragments bound to three open RBDs viewed along two orthogonal orientations. c. The S2X259 binding pose involving contacts with multiple RBD regions. Residues corresponding to prevalent RBD mutations are shown as red spheres. d-e, Close-up views showing selected interactions formed between S2X259 and the SARS-CoV-2 RBD. In panels a-e, each SARS-CoV-2 S protomer is coloured distinctly (cyan, pink and gold) whereas the S2X259 light and heavy chain variable domains are coloured magenta and purple, respectively. N-linked glycans are rendered as blue spheres in panels a-c.

Potential use against a broad range of sarbecoviruses

Using computational analysis, the team determined what RBD mutations could escape the antibody from binding. They found only a few RBD mutations disrupt the binding of this antibody. The substitution of the residue at position 504 gave the most significant disruption in binding.

When they replicated a pseudotyped SARS-CoV-2 virus in the presence of the S2X259 antibody, the only mutation they found caused by selective pressure was G504D. This mutation has rarely been seen in human isolates so far.

The selection of a single escape mutation suggests the region targeted by the antibody might not tolerate amino acid substitutions without changing viral fitness. Hence it is conserved across different sarbecoviruses. Thus, there is a high barrier for the emergence of mutations against this antibody, suggesting it could become key in combating the pandemic.

When Syrian hamsters were challenged with SARS-CoV-2, with the antibody administered 48 hours before virus infection, the authors found more than two orders of magnitude decrease in virus in the lungs compared to hamsters that did not receive any treatment. In addition, the antibody also protected hamsters infected with the B.1.351 strain.

The detection of a large variety of sarbecoviruses in bats and other mammals, along with the increased human-animal interactions, makes it likely that more cross-species transmission of viruses can occur. With increasing evidence that antibodies targeting the RBD form a major proportion of neutralizing activity, RBD-based vaccines could elicit high levels of antibodies like S2X259 with high potency. Such strategies can help overcome the current COVID-19 pandemic and help prepare for future sarbecovirus infections.

*Important Notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Journal reference:
  • Tortorici, M. A. et al. (2021) Structural basis for broad sarbecovirus neutralization by a human monoclonal antibody. bioRxiv. https://doi.org/10.1101/2021.04.07.438818, https://www.biorxiv.org/content/10.1101/2021.04.07.438818v1

Posted in: Medical Science News | Medical Research News | Miscellaneous News | Disease/Infection News | Healthcare News

Tags: ACE2, Amino Acid, Angiotensin, Angiotensin-Converting Enzyme 2, Antibodies, Antibody, Coronavirus, Coronavirus Disease COVID-19, Electron, Electron Microscopy, Enzyme, Glycan, Glycans, Immune Response, Lungs, Microscopy, Monoclonal Antibody, Mutation, Pandemic, Protein, Receptor, Respiratory, SARS, SARS-CoV-2, Severe Acute Respiratory, Severe Acute Respiratory Syndrome, Spike Protein, Syndrome, Virus

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Written by

Lakshmi Supriya

Lakshmi Supriya got her BSc in Industrial Chemistry from IIT Kharagpur (India) and a Ph.D. in Polymer Science and Engineering from Virginia Tech (USA).

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Study reveals molecular mechanisms of drug resistance in Mycobacterium tuberculosis

A consortium of researchers from Russia, Belarus, Japan, Germany and France led by a Skoltech scientist have uncovered the way in which Mycobacterium tuberculosis survives in iron-deficient conditions by utilizing rubredoxin B, a protein from a rubredoxin family that play an important role in adaptation to changing environmental conditions.

The new study is part of an effort to study the role of M. tuberculosis enzymes in developing resistance to the human immune system and medication. The paper was published in the journal Bioorganic Chemistry.

According to the World Health Organization, every year 10 million people fall ill with tuberculosis and about 1.5 million die from it, making it the world's top infectious killer. The bacterium that causes TB, Mycobacterium tuberculosis, is notorious for its ability to survive within macrophages, cells of the immune system that destroy harmful bacteria.

Continuing spread of drug resistance of M. tuberculosis to widely used therapeutics over recent decades became a substantial clinical problem. In this regard, the identification of novel molecular drug targets and deciphering the molecular mechanisms of drug resistance are of pivotal importance.

Natallia Strushkevich, Assistant Professor at the Skoltech Center for Computational and Data-Intensive Science and Engineering (CDISE), and her colleagues studied the crystal structure and function of rubredoxin B (RubB), a metalloprotein that ensures the proper functioning of cytochrome P450 (CYP) proteins essential to bacterial survival and pathogenicity.

The team hypothesizes that M. tuberculosis switched over to more iron-efficient RubB to survive iron starvation when granulomas are formed (these are largely unsuccessful attempts at defense against TB by the immune system).

During the long-term co-evolution with mammals, M. tuberculosis developed a variety of strategies to subvert or evade the host innate immune response, from recognition of the bacterium and phagosomal defenses within infected macrophages, to adaptive immune responses by antigen presenting cells. Iron assimilation, storage and utilization is essential for M. tuberculosis pathogenesis and also involved in emergence of multi- and extensively-drug resistant strains. Heme is the preferable iron source for M. tuberculosis and serves as a cofactor for various metabolic enzymes."

Natallia Strushkevich, Assistant Professor, Skoltech Center for Computational and Data-Intensive Science and Engineering (CDISE)

Based on our finding, we linked rubredoxin B to heme monoooxygenases important for metabolism of host immune oxysterols and anti tubercular drugs. Our findings indicate that M. tuberculosis has its own xenobiotics transformation system resembling human drug metabolizing system," explains Natallia Strushkevich.

According to Natallia: New targets for drug design efforts are in great demand and the cytochrome P450 enzymes have emerged as novel targets for the development of tuberculosis therapeutic agents.

The classic approaches to block these enzymes are not straightforward. Finding the alternative redox partner, such as RubB, enables further understanding of their function in different host microenvironments. This knowledge could be exploited to identify new ways to block their function in M. tuberculosis.

Earlier research by the consortium showed that one of the CYPs enabled by RubB can act against SQ109, a promising drug candidate against multidrug-resistant tuberculosis. Another study focused on how Mycobacterium tuberculosis protects itself by intercepting human immune signaling molecules — a hurdle that limits drug discovery.

Source:

Skolkovo Institute of Science and Technology (Skoltech)

Journal reference:

Sushko, T., et al. (2021) A new twist of rubredoxin function in M. tuberculosis. Bioorganic Chemistry. doi.org/10.1016/j.bioorg.2021.104721.

Posted in: Molecular & Structural Biology | Microbiology | Biochemistry

Tags: Aging, Antigen, Artificial Intelligence, Bacteria, Biochemistry, Cytochrome P450, Drug Discovery, Drugs, Evolution, Immune Response, Immune System, Metabolism, Photonics, Protein, Research, Structural Biology, Therapeutics, Tuberculosis

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Team identifies protein that blocks body’s ability to clear bad cholesterol

Team identifies protein that blocks body's ability to clear bad cholesterol

A team of researchers at the University of Alberta has uncovered a long-sought link in the battle to control cholesterol and heart disease.

The protein that interferes with low-density lipoprotein (LDL) receptors that clear ‘bad’ cholesterol from the blood was identified in findings recently published in Nature Communications by Dawei Zhang, associate professor of pediatrics in the Faculty of Medicine & Dentistry. Excess LDL cholesterol can lead to atherosclerosis—a narrowing and hardening of arteries—and ultimately, heart attack.

“We have known for many years that these receptors could be cleaved, but nobody knew which protein was responsible,” said Zhang. “There had been several attempts around the world but nobody else was successful.”

Now that the culprit has been identified, Zhang’s lab is already at work to find a drug to target the protein, allowing the receptors to clear more LDL.

A cholesterol-reducing class of drugs called statins—Lipitor and Crestor are two well-known brand names—has been shown to reduce cardiac events by 20 to 40 percent, but they have side-effects that mean they can’t be given in high enough doses to work for everyone. The new drug would be used in combination with statins to boost their effect, Zhang said.

Zhang’s team stumbled upon the role of the protein—membrane type 1 matrix metalloproteinase—by accident while studying another protein involved in heart function. They then set out to repeat and confirm their findings in mouse, rat and human cells, working in collaboration with researchers in China and other faculty members at the U of A. Their study was funded by the Heart and Stroke Foundation of Canada and the Canadian Institutes of Health Research. Zhang is also a member of the Women and Children’s Health Research Institute.

The protein has other critical physiological functions, Zhang explained, so his lab will work to identify and focus on the specific region within the protein that acts on the LDL receptor. They are also working with a new technique to further target their potential drug so it will work only within the liver, further reducing the likelihood of unwanted side-effects. Their early results are encouraging, Zhang said.

Zhang noted the protein is also critical for cancer tumor invasion, so the team will collaborate with U of A oncology experts to learn more.

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Identification of specific anti-SARS-CoV-2 antibodies with cross-neutralization potency

A team of scientists from the USA and Canada recently characterized the cross-neutralizing potency of anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies isolated from coronavirus disease 2019 (COVID-19) patients. The findings reveal that some of the isolated antibodies targeting the spike receptor-binding domain (RBD) and the S2 subunit are capable of cross-neutralizing other members of the human beta-coronavirus family. The study is currently available on the bioRxiv* preprint server.

Study: Isolation and Characterization of Cross-Neutralizing Coronavirus Antibodies from COVID-19+ Subjects. Image Credit: Corona Borealis Studio / Shutterstock

Background

SARS-CoV-2, the causative pathogen of COVID-19, is an enveloped, positive-sense, single-stranded RNA virus of the Coronaviridae family. The viruses belonging to the Coronaviridae family are capable of zoonotic transmission as observed in the current COVID-19 pandemic, as well as in previous outbreaks of SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV).

Given the 54% sequence similarity between different coronavirus strains, many studies have been conducted to investigate the cross-reactive potency of anti-SARS-CoV-2 antibodies. In this context, it has been observed that only a small fraction of antibodies isolated from SARS-CoV-2 infected patients are capable of cross-neutralizing SARS-CoV, MERS-CoV, or other beta (OC43 and HKU1) and alpha (229E and NL63) coronaviruses.

In the current study, the scientists have isolated and characterized a total of 198 monoclonal antibodies targeting SARS-CoV-2 spike protein. Specifically, they have explored the cross-neutralizing potency of these antibodies against different strains of coronaviruses.  

For antibody isolation, serum samples and peripheral blood mononuclear cells have been collected from four COVID-19 positive patients 3 to 7 weeks after the symptom onset.

Important observations

The analysis of serum samples using Enzyme-linked Immunosorbent assay (ELISA) revealed that all enrolled patients developed significant levels of anti-RBD bindings and neutralizing antibodies in response to SARS-CoV-2 infection. The highest virus neutralization potency was observed in samples collected at later timepoints.

By conducting a series of experiments using patient-obtained spike+ and RBD+ B cells (IgG+), the scientists finally isolated 198 anti-SARS-CoV-2 monoclonal antibodies. Moreover, they isolated 59 monoclonal antibodies from healthy individuals as experimental controls. By comparing relative frequencies of each heavy and light chain variable region sequence of patients and healthy controls, they observed that naïve B cell clones preferentially recognized the viral spike protein at the initial stages of infection. In contrast, the anti-spike B cell response predominated 3 – 7 weeks after the symptom onset. Moreover, they noticed that the sequences of heavy and light chain variable regions derived from later-timepoint samples harbored higher amino acid mutations than early-timepoint samples. This indicates that the evolution of B cells occurs continuously during SARS-CoV-2 infection.

Cross-reactive potency of anti-SARS-CoV-2 antibodies

Using various recombinant proteins, including spike S1 and S2 subunits, RBD, and N-terminal domain (NTD), the scientists observed that only a small fraction of isolated monoclonal antibodies recognized and bound the RBD. While exploring the cross-reactivity against SARS-CoV, MERS-CoV, OC43 and HKU1 beta-coronaviruses, and NL63 and 229E alpha-coronaviruses, they noticed that 81 out of 198 monoclonal antibodies bound various subdomains of SARS-CoV spike protein, with RBD being the highly recognized region. In contrast, a significantly lower cross-reactivity was observed against other coronaviruses.

Cross-neutralizing potency of anti-SARS-CoV-2 antibodies

Of 198 monoclonal antibodies, only 14 showed SARS-CoV-2 neutralization potency; of which, one targeted the NTD, one targeted the S2 subunit, and 12 targeted the RBD. Regarding cross-neutralization, only 4 out of 14 antibodies were found to effectively neutralize SARS-CoV. Of all cross-neutralizing antibodies, three were specific to the RBD, and one was specific to the S2 subunit. Importantly, all cross-neutralizing antibodies were found to effectively neutralize the South African variant of SARS-CoV-2 (lineage: B.1.351).  

The mechanistic analysis conducted in the study revealed that the most potent anti-RBD antibodies neutralized SARS-CoV-2 by blocking the angiotensin-converting enzyme 2 (ACE2)-RBD interaction. Moreover, there was an association between the degree of ACE2-RBD binding inhibition and the robustness of neutralization. A similar mechanism was observed for SARS-CoV neutralization.

Interestingly, the analysis revealed that the two most potent anti-SARS-CoV-2 neutralizing antibodies failed to neutralize SARS-CoV. This could be because these two antibodies interacted with the receptor-binding motif in the RBD, which is structurally not similar to that of SARS-CoV RBD.

To explore the infection preventing abilities of neutralizing antibodies, the scientists initially immunized the mice with a panel of neutralizing antibodies with different epitope specificities, followed by experimental infection with SARS-CoV-2. By analyzing the viral RNA in lung samples two days post-infection, they observed that only antibodies with high neutralization efficiency could provide protection against infection.

Study significance

The study reveals that the expansion of B cell populations expressing particular pairs of variable domains is not a prerequisite for generating SARS-CoV-2 and SARS-CoV cross-neutralizing antibodies. The study also identifies one epitope in the spike S2 subunit that is specific to at least four human beta-coronaviruses. Monoclonal antibodies targeting this S2 epitope have cross-neutralization potency.  

*Important Notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Journal reference:
  • Jennewein M. 2021. Isolation and Characterization of Cross-Neutralizing Coronavirus Antibodies from COVID-19+ Subjects. BioRxiv. doi: https://doi.org/10.1101/2021.03.23.436684, https://www.biorxiv.org/content/10.1101/2021.03.23.436684v1

Posted in: Medical Science News | Medical Research News | Disease/Infection News | Healthcare News

Tags: ACE2, Amino Acid, Angiotensin, Angiotensin-Converting Enzyme 2, Antibodies, Antibody, Assay, Blood, Cell, Coronavirus, Coronavirus Disease COVID-19, Enzyme, Evolution, MERS-CoV, Pandemic, Pathogen, Protein, Receptor, Respiratory, RNA, SARS, SARS-CoV-2, Severe Acute Respiratory, Severe Acute Respiratory Syndrome, Spike Protein, Syndrome, Virus

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Dr. Sanchari Sinha Dutta

Dr. Sanchari Sinha Dutta is a science communicator who believes in spreading the power of science in every corner of the world. She has a Bachelor of Science (B.Sc.) degree and a Master's of Science (M.Sc.) in biology and human physiology. Following her Master's degree, Sanchari went on to study a Ph.D. in human physiology. She has authored more than 10 original research articles, all of which have been published in world renowned international journals.

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Researchers identify new drug candidates for treating patients with severe COVID-19

University of Maryland School of Medicine (UMSOM) researchers have identified the most toxic proteins made by SARS-COV-2–the virus that causes COVID-19 – and then used an FDA-approved cancer drug to blunt the viral protein's detrimental effects.

In their experiments in fruit flies and human cell lines, the team discovered the cell process that the virus hijacks, illuminating new potential candidate drugs that could be tested for treating severe COVID-19 disease patients. Their findings were published in two studies simultaneously on March XX in Cell & Bioscience, a Springer Nature journal.

Our work suggests there is a way to prevent SARS-COV-2 from injuring the body's tissues and doing extensive damage."

Zhe "Zion" Han, PhD, Study Senior Author and Associate Professor of Medicine, Director of Center for Precision Disease Modeling, University of Maryland School of Medicine

He notes that the most effective drug against Covid-19, remdesivir, only prevents the virus from making more copies of itself, but it does not protect already infected cells from damage caused by the viral proteins.

Prior to the pandemic, Dr. Han had been using fruit flies as a model to study other viruses, such as HIV and Zika. He says his research group shifted gears in February 2020 to study SARS-COV-2 when it was clear that the pandemic was going to significantly impact the U.S.

SARS-COV-2 infects cells and hijacks them into making proteins from each of its 27 genes. Dr. Han's team introduced each of these 27 SARS-CoV-2 genes in human cells and examined their toxicity. They also generated 12 fruit fly lines to express SARS-CoV-2 proteins likely to cause toxicity based on their structure and predicted function.

The researchers found that a viral protein, known as Orf6, was the most toxic killing about half of the human cells. Two other proteins (Nsp6 and Orf7a) also proved toxic, killing about 30-40 percent of the human cells. Fruit flies that made any one of these three toxic viral proteins in their bodies were less likely to survive to adulthood. Those fruit flies that did live had problems like fewer branches in their lungs or fewer energy-generating power factories in their muscle cells.

For the remaining experiments, the researchers focused on just the most toxic viral protein, so they could figure out what cell process the virus hijacks during infection. Dr. Han's team found that the virus' toxic Orf6 protein sticks to multiple human proteins that have the job of moving materials out of the cell's nucleus–the place in the cell that holds the genome, or the instructions for life.

They then discovered that one of these human moving proteins, targeted by the virus, gets blocked by the cancer drug selinexor. The researchers tested selinexor on human cells and fruit flies making the toxic viral protein to see if the drug could help reverse the damage.

Selinexor, like many cancer drugs is itself toxic. However, after accounting for its toxic effects, the drug improved human cell survival by about 12 percent. Selinexor prevented early death in about 15 percent of the flies making the toxic viral protein. The drug also restored branches in the lungs and the energy-generators in the muscle cells. Selinexor is FDA-approved to treat certain blood cancers.

"More than 1,000 FDA-approved drugs are in clinical trials to test as treatments for Covid-19, and luckily a trial testing selinexor, the drug used in our study, is being performed already," says Dr. Han. "If this trial proves to be successful, our data will have demonstrated the underlying mechanism for why the drug works."

Albert Reece, MD, PhD, MBA, Executive Vice President for Medical Affairs, University of Maryland Baltimore, and the John Z. and Akiko K. Bowers Distinguished Professor and Dean, University of Maryland School of Medicine, commented, "Although we now have vaccines, it may still be a while before we will have Covid-19 infections under control, especially with the new variants emerging. We will need to tap into every tool in the arsenal available to protect people from needless sickness, disability or even death, and this study guides us towards a new target for potential therapeutics."

Source:

University of Maryland School of Medicine

Journal reference:

Lee, J-G., et al. (2021) Characterization of SARS-CoV-2 proteins reveals Orf6 pathogenicity, subcellular localization, host interactions and attenuation by Selinexor. Cell & Bioscience. doi.org/10.1186/s13578-021-00568-7.

Posted in: Medical Research News | Disease/Infection News

Tags: Blood, Cancer, Cell, Disability, Disease Modeling, Drugs, Fruit, Genes, Genome, HIV, Lungs, Mass Spectrometry, Medical Research, Medical School, Medicine, Muscle, Pandemic, Pharmacy, Protein, Remdesivir, Research, SARS, SARS-CoV-2, Spectrometry, Therapeutics, Virus

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