Author: Michele Arduengo, PhD

Keeping Cool When It Hurts: An Engineered Alternative to Pharmacological Pain Management

Posted on by Michele Arduengo, PhD

Pain management, particularly long-term pain management, is a difficult problem. Opioids are effective at reducing pain, but they are addictive and carry with them the significant costs of addiction and overdose that affect not only the individual but society as well. 

Saltatory conduction in neurons. Localized cooling of neurons may provide a non-addictive method for pain management.

Recently, work presented in Science by Reeder and colleagues proposes an engineering solution to pain management: miniature, implantable devices that can deliver localized and reversible neural blocking to reduce pain. While such devices could deliver any kind of stimulus ranging from electrical to pharmacological, the researchers developed a device that temporarily blocks neural signals by cooling.

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Designing Better Therapeutic mAbs: An Assay for Rapid, Parallel Screening of Fc/ FcษฃR Interactions

Posted on by Michele Arduengo, PhD

The first monoclonal antibody (mAb) was produced in a lab 1975, and the first therapeutic mAb was introduced in the United States to prevent kidney transplant rejection in 1986. The first mAb used in cancer treatment the anti-CD20 mAb, rituximab, was used to treat non-Hodgkinโ€™s lymphoma and chronic lymphocytic leukemia. Today therapeutic mAbs have become a mainstay of cancer, autoimmune disease, and metabolic disease therapies and include HERCEPTINยฎ used to treat certain forms of breast cancer, Prolia used to treat bone loss in post-menopausal women, and Stelara used to treat autoimmune diseases like psoriatic arthritis and severe Crohn disease, among many others. Therapeutic mAbs bind targets with high specificity and affinity and they can recruit effector cells to drive target elimination through mechanisms such as antibody-dependent cellular cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP), making them highly specific, effective therapies.

3D rendering of a Lumit Assay which can be used for plate-based screening assay to measure the affinities of Fc interactions of therapeutic mAbs.
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Use of ProAlanase Digestion Increases Number of Identified Methylglyoxal (MGO)-Modified Proteins in Whole-Cell Lysates

Posted on by Michele Arduengo, PhD
space filling structural model methylglyoxal (MGO)
Methylglyoxal is responsible for post translational protein modifications, that result in advanced glycation endproducts (AGEs), which are associated with aging and disease.

Post-translational modifications (PTM) of proteins are essential for the function of many proteins, but aberrant modification of protein residues also can interfere with protein function. PTMs occur in two ways. Proteins may be modified through the activity of enzymes such as kinases, phosphorylases, glycosylases and others that add or remove specific chemical moieties to amino acid residues. PTMs can also result from non-enzymatic reaction between electrophilic compounds and nucleophilic arginine and lysine residues within a protein. Metabolites and metabolic by products produced during glycolysis, especially glyoxal and methylglyoxal (MGO), are examples of such electrophilic compounds. These compounds can react with arginine and lysine to produce advanced glycation end products (AGEs), which are biomarkers associated with aging and degenerative diseases such as Alzheimer disease, diabetes and others. MGO is also elevated in tumors that have switched from oxidative phosphorylation to glycolysis as their main energy production pathway.

Only limited information is available about site-specific MGO PTMs in mammal cells, and most studies have focused on measuring the amount of MGO modifications in a treatment scenario compared to a control. Donnellan and colleagues recently published work to identify specific MGO protein modifications.  They used a โ€œbottom-upโ€ proteomic analysis of WIL2-NS B lymphoblastoid whole-cell lysates to identify specific MGO-modified proteins. In particular, the group was looking for modifications in proteins that might explain how MGO activity contributes to aneuploidy.

For the study, 100ยตg of cellular protein extract was reduced with dithiothreitol and then alkylated with chloroacetamide. The sample was diluted to reduce urea concentration. Trypsin Gold was added and samples were digested for 8 hours at 37ยฐC. Digestion was terminated by adding formic acid. For ProAlanase digestion, 20ยตg of protein was reduced, alkylated and diluted to reduce urea concentration before adding digesting with ProAlanase for 4 hours at 37ยฐC.

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The authors identified 519 MGO-modified proteins.  Most of the modifications were identified in the trypsin digestion reactions; however, ProAlanase digestion increased the number of MGO modifications identified by approximately 25% (with less than 4% of the modification sites being detected in both the ProAlanase and trypsin digestion reactions. The authors suggest that ProAlanase increased sequence coverage to reveal sites not detected in the trypsin digestions. Therefore, they conclude that ProAlanase can be used along with trypsin digestion to increase the identification of MGO modifications.

ProAlanase can be used along with trypsin digestion to increase the identification of MGO modifications.

MGO-modified proteins from the WIL2-NS whole cell lysates included proteins involved in glycolysis, translation initiation, protein folding, mRNA splicing, cell-to-cell adhesion, heat response, nucleosome assembly, protein SUMOylation and the G2/M cell cycle transition. More work to further characterize the sites of these modifications and their potential effects on the function of the modified proteins is ongoing.

Read more about ProAlanase, a new site-specific endoprotease from Promega.

Literature Cited

Donnelian, L et al. (2022) Proteomic Analysis of Methylglyoxal Modifications Reveals Susceptibility of Glycolytic Enzymes to Dicarbonyl Stress Int. J. Mol. Sci. 23(7), 3689 doi.org/10.3390/ijms23073689

PROTAC Virus Vaccines: A New Approach to Vaccine Development

Posted on by Michele Arduengo, PhD

Vaccine research and development is a major area of focus for life scientists across the globe. Clinical trials have shown that vaccines that target tumors show promise for cancer treatment. Additionally, the emergence of new zoonotic diseases has revealed a need to develop vaccines quickly as the world becomes more global and human populations interact more often with each other and wild habitats. Importantly, these vaccines need to be suitable for distribution in a variety of settings, including those that do not have easy access to refrigeration.

Influenza Virus. Si et al used influenza as a model to engineer and test PROTAC Virus vaccines

There are many ways to classify the different types of vaccines that are currently available. The National Institute of Allergy and Infectious Diseases in the United States, categorizes vaccines as: whole pathogen vaccines, subunit vaccines, and nucleic acid vaccinesโ€”based on how the antigen that stimulates the immune response is delivered to the host.

Whole-pathogen vaccines, which include many of vaccines used in clinical settings, use the entire pathogen (organism that causes the disease) that has been either weakened or killed to elicit a protective immune response. Killed vaccines are what their name implies: the pathogen has been killed so that it cannot cause disease, but enough of its structure remains to generate antibody response. Often, the immune response generated with killed vaccines is not as robust as that generated with other kinds of vaccines. 

Weakened or attenuated vaccines use whole pathogens that have been weakened in the laboratory through long-term culture or other means. Our modern MMR (measles, mumps and rubella) vaccine is an example of an attenuated vaccine. These vaccines tend to generate strong, long-lasting immune responses, but have increased risk for immunocompromised individuals.

Engineering an Influenza A PROTAC Virus Vaccine

A recent paper by Si et al published in Nature Biotechnology describes a new type of live-attenuated whole pathogen vaccine: the PROTAC virus. PROTAC viruses are prevented from replicating by targeting critical viral proteins for degradation using the host cell protein degradation pathway. The vaccine is live-attenuated by the host cells that degrade critical proteins.

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Detecting Disulfide Bond Shuffling in Biologics Using Trypsin Platinum

Posted on by Michele Arduengo, PhD
polypeptide chain showing protease cleavage sites
Learn about the new Trypsin Platinum Protease in this blog.

Biologic therapeutics such as monoclonal antibodies and biosimilars are complex proteins that are susceptible to post-translational modifications (PTMs). These chemical modifications can affect the performance and activity of the biologic, potentially resulting in decreased potency and increased immunogenicity. Such modifications include glycosylation, deamidation, oxidation and disulfide bond shuffling. These PTMs can be signs of protein degradation, manufacturing issues or improper storage. Several of these modifications are well characterized, and methods exist for detecting them during biologic manufacture. However, disulfide shuffling is not particularly well characterized for biologics, and no methods exist to easily detect and quantify disulfide bond shuffling in biologics.

Disulfide bond shuffling occurs when the S-S linkage is not between a Cys and its normal partner
Disulfide bonds are important for protein conformation and function

Normally the cysteines in a protein will pair with a predictable or โ€œnormalโ€ partner residue either within a polypeptide chain or between two polypeptide chains when they form disulfide bonds. These normal disulfide bonds are important for final protein conformation and stability. Indeed, disulfide bonds are considered an important quality indicator for biologics.

In a recently published study, Coghlan and colleagues designed a semi-automated method for characterizing disulfide bond shuffling on two IgG1 biologics: rituximab (originator drug Rituxanยฎ and biosimilar Acellbiaยฎ) and bevacizumab (originator Avastinยฎ and biosimilar Avegraยฎ).

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World Art Day: Experiences of Fine Art That Disrupt and Drive Innovation

Posted on by Michele Arduengo, PhD
This metal sculpture adorns the parking garage that serves Promega Kornberg and Feynman buildings on the Madison, WI, USA campus.
This metal sculpture adorns the parking garage that serves Promega Kornberg and Feynman buildings on the Madison, WI, USA campus.

World Art Day embraces art as a means for nurturing creativity and innovation, gaining greater understanding of cultural experiences that are different from our own, and showcasing the contributions of art and artists to sustainable development. It is no accident that World Art Day is celebrated on the birthday of Leonardo da Vinci, the celebrated Italian polymath who used art to express emotions, articulate technological concepts well before their time, and understand the workings of human anatomy.

Takaski Mitachi, a moderator of the 2019 global conference panel on Innovation through Art: Leveraging Disruption for a Sustainable Ecosystem, said that โ€œArt can add value so that we could drive innovation beyond logic and data.โ€ In a sense, the fine arts are a way of expressing our observations about the world around us, similar to how an original scientific hypothesis attempts to explain phenomena we observe and want to test. While scientific investigation builds on data and logic to test a hypothesis, art gives us a different way of knowing our world that extends beyond just data and logic. When we explore the arts, we broaden our understanding and interpretation of the world and bring these new perspectives to our scientific and technological explorations. Art is a disruptor of staid ways of thinking about and approaching problems, and like many other disruptors in our world, it can drive innovation.

In an article in the MIT Technology Review, author Sarah Lewis discusses the relationship between the arts and scientific breakthroughs. She notes one study that found a disproportionately high number of Nobel Prize-winning scientists also pursue art, writing or music as serious avocations. When asked why achievement in art and science seem to go together, she replied: โ€œWhat the arts allow us to do is develop the muscle required for discernment and also strengthen our sense of agency to determine for ourselves how weโ€™re going to tackle a given problemโ€ฆUltimately itโ€™s up to the person creating the work to determine what that path is, and that kind of agency is whatโ€™s required for innovation.โ€

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Using Structural Computation Models to Predict Productive PROTAC Ternary Complexes

Posted on by Michele Arduengo, PhD

With use and time things wear out. Tires get worn on a car, and you have the old tires removed, recycled, and replaced with new ones. Sometimes a part or piece of something isnโ€™t made properly. For instance, if you are assembling a piece of furniture and you find a screw with no threads, you throw it out and get a screw that was made properly. The same thing holds true for cells. Components wear out (like tires) or get improperly made (a screw with no threads), or they simply have a limited lifetime so that they are available in the cell only when needed. These used and worn components need to be removed from the cell. One system that allows cells to recycle components and remove old or improperly functioning proteins is the Ubiquitin-Proteasome System (UPS). ย The UPS system relies on a series of small peptide tags, ubiquitin, to mark a protein for degradation. Researchers are now harnessing the UPS to target aberrant proteins in diseased cells through PROteolysis TArgeting Chimeras or PROTACs. PROTACs hold promise as highly efficacious therapeutics that can be directed to eliminate only a single protein. To take full advantage of the power of PROTACs, researchers need to understand the molecular underpinnings that are responsible for successful protein degradation. Here we review a paper that seeks to develop a computer model for predicting whether PROTAC ternary complex formation leads to ubiquitination and successful degradation of a target protein.

Diagram of ubiquitination of a protein. ThePROTAC ternary complex is formed the E2/E3 complex, PROTAC and target protein are bound simultaneously
Proteins are targeted for degradation by the proteasome. A small chain of ubiquitin peptides (Ub) is added to available lysine residues of the target protein through the actions of three enzymes: E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; and E3 ubiquitin ligase. After the addition of the Ub chain, the proteasome is recruited and the protein degraded.

Addressing the Intractable Target

Research to understand diseases including cancers, neurodegeneration, and auto-immune conditions has revealed that in many disease states, affected cells produce growth factors or enzymes that are constitutively active (โ€œalways onโ€). These proteins are targets for small molecule inhibitors that bind specific sites preventing the constitutive activity or signaling. More recently, biologics, or protein-based therapeutics, including monoclonal antibodies (mAb), have been developed that can bind and block inappropriate signaling pathways, especially those that allow cancer cells to escape immune system surveillance.

Unfortunately, up to 85% of targets have proven intractable to small molecule inhibitors, or they are not suitable for a biologics approach. Oftentimes, the target protein doesnโ€™t have a great place to bind a small molecule, so even though inhibitors might exist they cannot bind well enough to be effective. Or, as in the case of many cancers, the diseased cell manages to overcome the effect of the inhibitor by overexpressing the target. Still other aberrant proteins associated with diseases havenโ€™t gained function to cause a disease; they have instead, lost function, so designing an inhibitor of the protein is not a workable strategy. ย Enter the PROTAC.

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Run to Remember: A Mouse-Model Study Investigating the Mechanism of Exercise-Induced Neuroprotection

Posted on by Michele Arduengo, PhD

Research in animal models shows physical exercise can induce changes in the brain. In humans, studies also revealed changes in brain physiology and function resulting from physical exercise, including increased hippocampal and cognitive performance (1). Several studies in mice and rats also demonstrated that exercise can improve learning and memory and decrease neuroinflammation in models of Alzheimer’s disease and other neurodegenerative pathologies (2); these benefits are tied to increased plasticity and decreased inflammation in the hippocampus in mice (2). If regular time pounding the pavement does improve brain function, what is the underlying molecular biology of exercise-induced neuroprotection? Can we identify the cellular pathways and components involved? Can we detect important components in blood plasma? And, is the benefit of these components transferrable between organisms? De Miguel and colleagues set out to answer these questions and describe their results in a recent study published in Nature.

A recent study investigates the underlying molecular mechanisms of exercise-induced neuroprotection in a mouse model.
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How to Commit to Global Responsibility with Local Accountability

Posted on by Michele Arduengo, PhD

This summer, Dr. Anette Leue, Director of Digital Marketing and PR Promega GmbH, represented Promega Corporation in Sustainability Day activities sponsored by Smart Lab Connects. Dr. Leue presented Promega Corporationโ€™s corporate responsibility activities and joined a panel discussion about global responsibility with representatives from Eppendorf, Max Planck Sustainability Network, and NIUB Sustainability Consultants.

Dr. Anette Leue, pictured, talked about global responsibility as part of Sustainability Day.

As the Sustainability Day activities progressed, what became apparent is that calls for sustainable business growth are coming from all directions. Customers of life sciences companies are asking, โ€œwhat are you doing to be a responsible companyโ€? And, employees also are asking the same question of their employers. This interest sustainability and global responsibility by customers, employees and local communities is bringing into sharp focus the activities of companies to be good corporate citizens. Sustainability and global responsibility programs are no longer nice extras for life science companies, but rather are requirements for doing business.

โ€œSustainability is not a “nice to have”, but something that should be intrinsically implemented in the companies.โ€

Dr. Anette Leue
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The Stories in the Bones: DNA Forensic Analysis 20 Years after 9/11

Posted on by Michele Arduengo, PhD

September 11, 2001 is the day that will live in infamy for my generation. On that beautiful late summer day, I was at my desk working on the Fall issue of Neural Notes magazine when a colleague learned of the first plane hitting the World Trade Center. As the morning wore on, we learned quickly that it wasnโ€™t just one plane, and it wasnโ€™t just the World Trade Center.

Two beams of light recognized the site of the World Trade Center attack. Today DNA forensic analysis applies new technologies to bring closure to families of victims.

Information was sparse. The world wide web was incredibly slow, and social media wasnโ€™t much of a thingโ€”nothing more than a few listservs for the life sciences. Someone managed to find a TV with a rabbit-eared, foil-covered antenna, and we gathered in the cafeteria of Promega headquartersโ€”our shock growing as more footage became available. At Promega, conversation immediately turned to how we could bring our DNA forensic analysis expertise to help and support the authorities with the identification of victims and cataloguing of reference samples.

Just as the internet and social media have evolved into faster and more powerful means of communicationโ€”no longer do we rely on TVs with antennas for breaking newsโ€”the technology that is used to identify victims of a tragedy from partial remains like bone fragments and teeth has also evolved to be faster and more powerful.

Teeth and Bones: Then and Now

โ€œBones tell me the story of a personโ€™s lifeโ€”how old they were, what their gender was, their ancestral background.โ€  Kathy Reichs

Many stories, both fact and fiction, start with a discovery of bones from a burial site or other scene. Bones can be recovered from harsh environments, having been exposed to extreme heat, time, acidic soils, swamps, chemicals, animal activities, water, or fires and explosions. These exposures degrade the sample and make recovering DNA from the cells deep within the bone matrix difficult.

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