Activating the Inflammasome: A New Tool Brings New Understanding

Innate immunity, the first line of immune defense, uses a system of host pattern recognition receptors (PRRs) to recognize signals of “danger” including invariant pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). These signals in turn recruit and assemble protein complexes called inflammasomes, resulting in the activation of caspase-1, the processing and release of the pro-inflammatory cytokines IL-1ß and IL-18, and the induction of programmed, lytic cell death known as pyroptosis.

Innate immunity and the activity of the inflammasome are critical for successful immunity against a myriad of environmental pathogens. However dysregulation of inflammasome activity is associated with many inflammatory diseases including type 2 diabetes, obesity-induced asthma, and insulin resistance. Recently, aberrant NLRP3 inflammasome activity also has been associated with age-related macular degeneration and Alzheimer disease. Understanding the players and regulators involved in inflammasome activity and regulation may provide additional therapeutic targets for these diseases.

Currently inflammasome activation is monitored using antibody-based techniques such as Western blotting or ELISA’s to detect processed caspase-1 or processed IL-1ß. These techniques are tedious and are only indirect measures of caspase activity. Further, gaining information about kinetics—relating inflammasome assembly, caspase-1 activation and pyroptosis in time—is very difficult using these methods. O’Brien et al. describe a one-step, high-throughput method that enables the direct measurement of caspase-1 activity. The assay can be multiplexed with a fluorescent viability assay, providing information about the timing of cell death and caspase-1 activity from the same sample. Continue reading “Activating the Inflammasome: A New Tool Brings New Understanding”

Obesity: Can Simple Approaches Reduce Complex Risks?

Obesity Prevalance 2017
Prevalence of Self-Reported Obesity Among U.S. Adults by State and Territory, Behavioral Risk Factor Surveillance System (BRFSS) 2017. Prevalence estimates reflect BRFSS methodological changes started in 2011. These estimates should not be compared to prevalence estimates before 2011. Source: BRFSS, Centers for Disease Control and Prevention

The Obesity Epidemic

For over a decade, obesity has been called an “epidemic”, both in the popular and scientific literature. Traditionally, the term “epidemic” is associated with a highly contagious disease that carries with it a significant risk of mortality. A comprehensive review of observational studies (1) suggested that obesity did not fit this definition, despite the use of the term in a widely disseminated report by the World Health Organization in 2002.

Regardless of the etymological fine points, the worldwide prevalence of obesity and its associated health risks are clear. These risks include type 2 diabetes, hypertension, several cancers, gall bladder disease, coronary artery disease and stroke (2). Yet, the debate over obesity and options for reducing its risks has become increasingly polarized. As a result, some health researchers are advocating a “health at every size” (HAES) approach to address the social, cultural and lifestyle implications of obesity (2).

Continue reading “Obesity: Can Simple Approaches Reduce Complex Risks?”

PROTACs, PHOTACs and LYTACs: How to Target a Protein for Degradation

PROTACs for Targeted Protein Degradation
An illustration of PROTAC structure and the proteins it binds.

Targeting a single protein and making it disappear from the cell is quite the magic trick, and there are various molecular tools available for this task. You can use RNA interference, which prevents a protein from being made, inhibitors that bind the protein, rendering it unavailable for use or even gene editing tools like CRISPR that can remove it from the genome. But did you know that you can target an existing protein for destruction, using the cell’s own garbage disposal system to degrade the protein? All you need is a molecule that can connect your protein to one with a role in cellular protein degradation and your protein can be destroyed. Continue reading “PROTACs, PHOTACs and LYTACs: How to Target a Protein for Degradation”

When Proteins Get Together: Shedding (Blue) Light on Cellular LOV

NanoBRETNo protein is an island. Within a cell, protein-protein interactions (PPIs) are involved in highly regulated and specific pathways that control gene expression and cell signaling. The disruption of PPIs can lead to a variety of disease states, including cancer.

Two general approaches are commonly used to study PPIs. Real-time assays measure PPI activity in live cells using fluorescent or luminescent tags. A second approach includes methods that measure a specific PPI “after the fact”; popular examples include a reporter system, such as the classic yeast two-hybrid system.

Continue reading “When Proteins Get Together: Shedding (Blue) Light on Cellular LOV”

Evaluating CAR NK Immunotherapy in Patient-Derived Colorectal Organoids

In recent years, great advances have been made in the field of immunotherapy to treat cancer. One of the most promising treatments involves engineering immune cells to express chimeric antigen receptors (CAR). These receptors are carefully designed to recognize antigens expressed on the surface of tumor cells. Once the target is recognized, the CAR-engineered immune cells can attack and kill the tumor cells. CAR T cells have been successfully used to treat certain blood cancers—three CAR T therapies for lymphoma and leukemia have gained US FDA approval. In these cases, T cells were taken from individual patients, grown and genetically-altered in the lab, then reintroduced into the same patient. Continue reading “Evaluating CAR NK Immunotherapy in Patient-Derived Colorectal Organoids”

Studying Autophagy in Flies Using CRISPR

 

Transcribed RNA can be used to study RNA structure and how it relates to function or how proteins and RNA interact. It can also be used for gene silencing using RNAi (studied more often as a possible therapeutic option) or simply serve as a molecular standard in Real-time RT-PCR. Transcribed RNA is also used in Class 2 Clustered Regularly Interspaced Short Palindromic Repeat systems, or CRISPR.

The CRISPR system, which is naturally occurring in bacteria, has been manipulated to perform gene editing in a laboratory environment. To perform CRISPR in the laboratory environment, you need two main reagents:

  1. The Brains: Guide RNA (gRNA or sgRNA) – Small piece of RNA containing a nucleotide sequence that is capable of binding the chosen Cas Protein, and contains a portion of the sequence that can bind the DNA the researcher intends to modify – the target DNA.
  2. The Brawn: CRISPR-associated endonuclease (Cas Protein) – The protein that cleaves the target DNA; the most popular Cas protein is called Cas9. The Cas protein is guided by the (gRNA).

Recently, Guo et al. used Promega’s RiboMAX™ Large-Scale RNA Production System to produce gRNA to be used in CRISPR for their study to determine the effects of the loss of, or mutations in, a specific gene in fruit flies (1).  Atg101 is a gene that plays an important role in autophagy, an intracellular pathway for removing toxins or damaged parts of cells. Continue reading “Studying Autophagy in Flies Using CRISPR”

How Do You Solve a Problem Like Malaria?

malaria_researcher
Photo courtesy of NIH/NIAID

Malaria affects nearly half of the world’s population, with almost 80% of cases in sub-Saharan Africa and India. While there have been many strides in education and prevention campaigns over the last 30 years, there were over 200 million cases documented in 2017 with over 400,000 deaths, and the majority were young children. Despite being preventable and treatable, malaria continues to thrive in areas that are high risk for transmission. Recently, clinicians started rolling out use of the first approved vaccine, though clinical trials showed it is only about 30% effective. Meanwhile, researchers must continue to focus on innovative efforts to improve diagnostics, treatment and prevention to reduce the burden in these areas.

Continue reading “How Do You Solve a Problem Like Malaria?”

Characterizing Compound Binding in Cell-Free Systems

Dioxins (e.g., 2,3,7,8-Tetrachlorodibenzo-p-dioxin, TCDD) and related compounds (DRCs) are persistent environmental pollutants that gradually accumulate through the food chain, mainly in the fatty tissues of animals. Dioxins are highly toxic and can cause reproductive and developmental problems, damage the immune system, interfere with hormones and also cause cancer. This broad range of toxic and biological effects of DRCs is mostly mediated by the aryl hydrocarbon receptor (AHR).

In animal cells, DRCs bind to AHR in the cytoplasm and then translocate into the nucleus, where they affect the transcription of multiple target genes, including xenobiotic-metabolizing enzymes, such as CYP1A isozymes. AHR is also involved in immune system maintenance, protein degradation and cell proliferation.

The jungle crow (Corvus macrorhynchos) has been considered a suitable indicator for monitoring environmental chemicals such as DRCs. While mammals only have one AHR form, avian species have multiple AHR isoforms such as AHR1 and AHR2. To unveil the functional diversity of multiple avian AHR isoforms in terms of their contribution to responses to DRCs a recent study by Kim et al. investigated the molecular and functional characteristics of jungle crow AHR isoforms, cAHR1 and jcAHR2 (1).

cAHR1 and jcAHR2 proteins were synthesized using AHR proteins were synthesized using the TnT Quick-Coupled Reticulocyte Lysate System  to examine whether these jcAHRs have the potential to bind to TCDD. TCDD-binding affinity of the in vitro-expressed jcAHR protein was analyzed using the velocity sedimentation assay with a sucrose gradient.

The results demonstrate that both jcAHR1and jcAHR2 are capable of binding to TCDD.

Reference
Kim, E-Y (2019) The aryl hydrocarbon receptor 2 potentially mediates cytochrome P450 1A induction in the jungle crow (Corvus macrorhynchos). Ecotoxicology and Environmental Safety 171. 99–111

Cancer is a Scourge Most Ancient

Reconstruction of Pappochelys rosinae or grandfather turtle. Attribution: Rainer Schoch [CC BY-SA 4.0 (https://creativecommons.org/licenses/by-sa/4.0)], via Wikimedia Commons
The first words that come to mind when people hear “Triassic” are likely dinosaur or maybe ginkgo or possibly even phytoplankton, but probably not cancer. For all that we have learned about how cancer develops based on the efforts of numerous research scientists, this disease is not solely a modern affliction. In fact, cancer has deep roots in the past. From several thousand year old human mummies to fossils millions of years old, cancer has left evidence of its presence in the historical record. Yes, even in the era of dinosaurs, cancer existed.

Cancer is usually a soft-tissue-based malady, but occasionally, it can also be found on bones, altering the bone’s surface and leaving unmistakable signs. Alterations like those observed on the femur of a 240-million-year-old shell-less stem-turtle found in modern-day Germany and described in JAMA Oncology. Continue reading “Cancer is a Scourge Most Ancient”

When Good Proteins Go Bad

Ribbon model of p53 protein bound to DNA molecule.
Ribbon model of p53 protein bound to DNA molecule.

Following what feels like an exceptionally long and brutal winter, I for one couldn’t be happier about the arrival of Spring and the way it makes everything seem brighter and brand-new. Soaking in the soul-warming sunshine. Reveling in the sweet melody of chirping birds. Watching the earth literally coming alive again with greenery. And for those of us who love and are enthralled by scientific discoveries like myself, the report of a recent shiny new discovery in the world of cancer research is equally as day-brightening and spirit-lifting.

To suppress tumors or to not suppress tumors: that is the question.

In the world of oncology, the protein known as p53 has long proven itself to be a primary target of interest. p53 operates as a tumor suppressor protein, often lauded as the “guardian of the human genome”, due to its dedication to governing controlled cell division and assessing damaged DNA. There are a number of cellular stressors that can wreak havoc on your DNA, including exposure to ultraviolet light or radiation, oxygen deficiency (hypoxia), and contact with hazardous chemicals.

Consider a normal-functioning p53 protein as the quality control person in a production factory. The p53 protein evaluates the products, DNA, coming down the line and determines an appropriate course of action for those that do not meet the quality standards.

Let’s say some less-than-quality DNA comes down the pipe. If the DNA is not too severely injured, p53 will alert and activate additional genes to repair the damage. However, if the products coming through are too marred to repair, p53 will shut down the whole factory, if you will, by signaling for the cell to self-destruct via apoptosis. In doing so, p53 effectively impedes tumor development by inhibiting the ability for flawed DNA to further divide.

So, it would seem like p53 has proven itself to be an undeniably upstanding citizen of the protein variety, right? The unfortunate truth of the matter is p53 balances delicately on a double-edged sword, establishing itself as the veritable Dr. Jekyll and Mr. Hyde of the cellular world: usually unquestionably good, but sometimes unspeakably evil. Continue reading “When Good Proteins Go Bad”