Papers of the Month
By Kelley Christensen and Janelle Weaver
DNTP study shows that a BPA derivative induces uterine fibrosis
A ubiquitous environmental contaminant called tetrabromobisphenol A (TBBPA) causes the induction of fibrosis and cell proliferation of human uterine fibroid cells, according to researchers from the Division of the National Toxicology Program (DNTP).
TBBPA is a bisphenol A (BPA) derivative that is widely used as a flame retardant across the globe. The prevalent and persistent chemical readily accumulates in plants, humans, and wildlife. TBBPA exposure can disrupt hormones in animals, raising concerns over its potential toxicity. Animal studies have shown that neonatal BPA exposure can cause uterine leiomyomas — hormone-responsive benign smooth muscle tumors called fibroids that are commonly diagnosed in women. Whether TBBPA has a similar effect is not clear.
Researchers developed a 3-D culture system consisting of human uterine-fibroid cells to closely mimic tumors in humans. Short-term TBBPA treatment increased cell proliferation and promoted fibrosis of uterine fibroid cells by activating fibrosis genes and the transforming growth factor-beta signaling pathway.
According to the authors, the results demonstrate that the new in vitro model is a successful, much-needed strategy for studying the effects of environmental exposures on human uterine fibroids. Moreover, the findings shed light on the molecular mechanisms by which TBBPA exposure contributes to fibroid development, and thereby potentially poses a health risk to women. (JW)
Citation: Liu J, Yu L, Castro L, Yan Y, Clayton NP, Bushel P, Flagler ND, Scappini E, Dixon D. 2022. Short-term tetrabromobisphenol A exposure promotes fibrosis of human uterine fibroid cells in a 3D culture system through TGF-beta signaling. FASEB J 36(2):e22101.
Why birch allergens trigger strong immune responses
Birch allergens are more stable and more abundant than other birch pollen proteins, according to NIEHS researchers and their collaborators. The study supports the immunological role of abundance and stability, consistent with prevailing models of allergic sensitization.
Only a small fraction of environmental proteins encountered by the human immune system consistently induce allergic sensitization. This suggests that the presence of specific biophysical properties may distinguish allergens from non-allergens. Consistent with this idea, past research has shown that allergens from cockroaches, dust mites, ragweed, and grass are more stable and more abundant than non-allergens.
The researchers tested whether this trend extends to birch pollen proteins. They assessed protein stability by testing resistance to proteolytic cleavage — the breakdown of proteins into smaller polypeptides or amino acids. The results showed that allergens such as Bet v1 are more stable and abundant than their non-allergenic counterparts.
Taken together with previous research, the new findings suggest that the slow degradation of allergens could explain why they generate robust immune reactions. According to the authors, the study extends this stability hypothesis to the proteome level. But they noted that genetics, exposure, cross-reactivity, and environmental factors can also influence allergic sensitization. (JW)
Citation: Cabrera A, Foo ACY, Fitzgerald MC, Mueller GA. 2022. Bet v 1 and other birch allergens are more resistant to proteolysis and more abundant than other birch pollen proteins. Allergy; doi: 10.1111/all.15209 [Online 7 January 2022].
Purifying SARS-CoV-2 proteins may produce targeted therapies
NIEHS researchers have developed a simple protocol to produce pure and high quantities of two proteins required for the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
To date, the U.S. Food and Drug Administration has approved only one drug, remdesivir, to treat coronavirus disease 2019 (COVID-19), which is caused by SARS-CoV-2. Because remdesivir is a broad-spectrum antiviral drug, more targeted therapeutics are urgently needed. Understanding the core replication complex of SARS-CoV-2 is essential for developing novel coronavirus-specific drugs.
Two proteins required for the faithful replication of the SARS-CoV-2 genome are nonstructural protein 14 (NSP14) and NSP10. Lack of straightforward purification protocols for NSP10 and NSP14 has hampered biochemical and structural studies of this important SARS-CoV-2 complex.
To address this need, the researchers developed a simple method for the bacterial expression and purification of NSP10 and NSP14 and for the in vitro assembly and purification of an NSP10/14 complex with high yields. These purification methods and biochemical analyses pave the way for future structural studies investigating the mechanism of SARS-CoV-2 viral synthesis. The researchers determined that the NSP14 exoribonuclease was stimulated several hundred-fold by binding NSP10. Ultimately, this research could lead to the design of targeted therapies that could be used alone or in combination with remdesivir to treat COVID-19 and potentially other coronaviruses. (JW)
Citation: Riccio AA, Sullivan ED, Copeland WC. 2022. Activation of the SARS-CoV-2 NSP14 3'-5' exoribonuclease by NSP10 and response to antiviral inhibitors. J Biol Chem 298(1):101518.
Studying regulatory genes may lead to targeted cancer treatments
NIEHS researchers investigated how dexamethasone (Dex), a synthetic anti-inflammatory hormone, regulates transcription of the gene DNA-damage-inducible transcript 4 (DDIT4) at four specific glucocorticoid receptor (GR) sites.
Using cultured human breast cancer cells and the gene editing technique known as CRISPR, the researchers individually deleted four GR binding sites (GBSs) within the DDIT4 super enhancer, which is a regulatory region of the genome that increases the transcription of target genes. The scientists sought to determine how the GBSs contribute to Dex induction of DDIT4 transcription and how they function as parts of the super enhancer. ChIP-seq showed that GR binding at the GBSs within the super enhancer was not uniform and highlighted the role chromatin plays in the transcriptional response. Each binding site is uniquely required to promote or suppress DDIT4 expression.
A variety of hormone-dependent cancer types — including breast, skin, lung, and colon cancers — have been found to occur when the transcription of DDIT4 is dysregulated. The higher the levels of DDIT4, the worse the prognosis. However, Dex treatment reorganizes the enhancer landscape in breast cancer cells. By targeting individual regulatory elements within the hormone responsive DDIT4 super enhancer, DDIT4 can be modulated. (KC)
Citation: Hoffman JA, Trotter KW, Day CR, Ward JM, Inoue K, Rodriguez J, Archer TK. 2022. Multimodal regulatory elements within a hormone-specific super enhancer control a heterogeneous transcriptional response. Mol Cell 82(4):803–815.e5. Story.
Polymerase mutations disrupt DNA replication, increase cancer risk
Using a Saccharomyces cerevisiae yeast model, a team of NIEHS researchers and their collaborators determined that two mutations in the polymerase epsilon exonuclease domain, a region responsible for DNA excision and repair known as proofreading, are strong mutators. These variants have similar, but slightly different, mutational specificity and were identified in human tumors. The work provides insight into how some tumors may form.
One variant, pol2-Y473F, reduces the ability of the enzyme to edit mistakes following the initial process of nucleotide selectivity by polymerase epsilon, resulting in a 20-fold increase in mutations. The other variant, pol2-N378K, additionally stimulates polymerase activity, increasing the chances of both incorporation and extension of a mismatch. The combined effect increases the mutation rate by 40-fold. Mutants for these experiments were generated via polymerase chain reaction and CRISPR and then analyzed using a combination of in vitro biochemistry and in vivo forward mutation assay and fluctuation analysis, respectively.
The scientists demonstrated that replication fidelity can be diminished by directly reducing proofreading via impairing exonuclease activity or indirectly by promoting mismatch incorporation and extension. They also determined that mutations in the exonuclease domain can affect DNA binding, polymerase activity, and the chemistry in active sites of both variants. (KC)
Citation: Dahl JM, Thomas N, Tracy MA, Hearn BL, Perera L, Kennedy SR, Herr AJ, Kunkel TA. 2022. Probing the mechanisms of two exonuclease domain mutators of DNA polymerase epsilon. Nucleic Acids Res 50(2):962–974.