Density dependent regulation of inflammatory responses in macrophages

Macrophage distribution density is tightly regulated within the body, yet the importance of macrophage crowding during in vitro culture is largely unstudied. Using a human induced pluripotent stem cell (iPSC)-derived macrophage model of tissue resident macrophages, we characterize how increasing macrophage culture density changes their morphology and phenotype before and after inflammatory stimulation. In particular, density drives changes in macrophage inflammatory cytokine and chemokine secretion in both resting and activated states. This density regulated inflammatory state is also evident in blood monocyte derived-macrophages, the human monocytic THP-1 immortalized cell line, and iPSC-derived microglia. Density-dependent changes appear to be driven by a transferable soluble factor, yet the precise mechanism remains unknown. Our findings highlight cell plating density as an important but frequently overlooked consideration of in vitro macrophage research relevant to a variety of fields ranging from basic macrophage cell biology to disease studies

Radical-SAM dependent nucleotide dehydratase (SAND), rectification of the names of an ancient iron-sulfur enzyme using NC-IUBMB recommendations

In 1789, the influential French chemist Antoine-Laurent Lavoisier described his view of science and its langague in his book Traité élémentaire de chimie. According to the Robert Kerr’s translation it states (Lavoisier, 1790): “As ideas are preserved and communicated by means of words, it necessarily follows that we cannot improve the language of any science without at the same time improving the science itself; neither can we, on the other hand, improve a science without improving the language or nomenclature which belongs to it.” This view reminds us of Confucius’s earlier doctrine, the rectification of names (Steinkraus, 1980; Lau, 2000). Confucius believed that rectification of names is imperative. He explained (Steinkraus, 1980; Lau, 2000): “If language is incorrect, then what is said does not concord with what was meant, what is to be done cannot be affected. If what is to be done cannot be affected, then rites and music will not flourish. If rites and music do not flourish, then mutilations and lesser punishments will go astray. And if mutilations and lesser punishments go astray, then the people have nowhere to put hand or foot. Therefore the gentleman uses only such language as is proper for speech, and only speaks of what it would be proper to carry into effect. The gentleman in what he says leaves nothing to mere chance.” Inspired by these views, we make the analogy that the progress of science and the language used to describe it are two entangled electrons. This entanglement highlights the importance of introducing systemic names for enzymes using EC classification and the ever-growing problem of protein names (McDonald and Tipton, 2021). Here, we tackle one specific case of iron-sulfur ([FeS]) enzymes. We show that the language used to describe a conserved [FeS] enzyme of the innate immune system, i.e., viperin or RSAD2, is now inadequate and disentangled from its science. We discuss that the enzyme has cellular functions beyond its antiviral activity and that eukaryotic and prokaryotic enzymes catalyse the same chemical reactions. To prevent bias towards antiviral activity while studying various biochemical activities of the enzyme and using scientifically incorrect terms like “prokaryotic viperins,” we rectify the language describing the enzyme. Based on NC-IUBMB recommendations, we introduce the nomenclature S-adenosylmethionine (SAM) dependent Nucleotide Dehydratase (SAND)

Mechanism of Diol Dehydration by a Promiscuous Radical‐SAM Enzyme Homologue of the Antiviral Enzyme Viperin (RSAD2)

3´‐deoxy nucleotides are an important class of drugs because they interfere with metabolism of nucleotides and their incorporation into DNA or RNA terminates cell division and viral replication. These compounds have largely been produced via multistep chemical synthesis and an enzyme with the ability to catalyse removal of 3´‐deoxy group from different nucleotides has yet to be described. Here, using a combination of HPLC, high‐resolution mass spectrometry, and NMR spectroscopy we demonstrate that a thermostable fungal radical S‐adenosylmethionine (SAM) enzyme with similarity to the vertebrate antiviral enzyme viperin (RSAD2) can catalyze transformation of CTP, UTP, and 5‐bromo‐UTP to their 3ʹ‐deoxy‐3′,4ʹ‐didehydro analogues. We show that unlike the fungal enzyme human viperin can only catalyse transformation of CTP. Using electron paramagnetic resonance (EPR) spectroscopy and molecular docking and dynamics simulations in combination with mutagenesis studies we provide insight into the origin of the unprecedented substrate promiscuity of the enzyme and the mechanism of dehydration of a nucleotide. Our findings highlight the evolution of substrate specificity in a member of the radical‐SAM enzymes. We predict that our work will help in utilizing a new class of radical‐SAM enzymes for biocatalytic synthesis of 3ʹ‐deoxy nucleotide/nucleoside analogues