Evolutionary trajectories of DNA/RNA-editing enzymes involved in adaptive and innate immunity

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Vertebrate immune cells purposefully induce mutations in their genomes, or in the genomes of invading viruses, through specific DNA/RNA-editing enzymes. The Activation-induced cytidine deaminase/Apolipoprotein B editing catalytic like polypeptide (AID/APOBEC) family of cytidine deaminase enzymes are central players in immunity. AID acts on the endogenous immunoglobulin genes and initiates antibody affinity maturation, while APOBECs mutate the genomes of viruses to incapacitate their coding ability. The evolutionary pressure imposed on AID/APOBECs due to their functions in immunity has made these highly diverse among species, making them a fascinating subject for enzyme structure:function evolution. However, as with other molecules involved in immunity, while the human and mouse versions have been intensely studied, there is a glaring gap in our understanding of AID/APOBEC (or similar) enzymes in distantly evolved species. We pioneered the study of divergent AID/APOBECs. Focusing on species representing key evolutionary junctures, we contributed several advances: 1. discovered and characterized new enzymes 2. co-discovered the phenomenon of enzyme diversification within the same species 3. discovered the first instance of natural AID inactivation correlating with a deficiency in antibody response, and a novel mode of compensatory co-evolution with expanded innate immunity 4. Carried out the first ancestral reconstruction (ASR) of an enzyme involved in immunity to decipher its evolutionary trajectory Here we propose to: Objective 1. Study the function of DNA/RNA-editing enzymes from extant and ancestral evolutionary distant species, and examine their co-evolution with immunity We will focus on understanding the evolution and co-evolution with immunity, of AID/APOBEC enzymes from key evolutionary junctures of invertebrates, jawless and jawed vertebrates. Extant and predicted ancestral enzymes of key evolutionary nodes will be studied in enzyme assays and cell-based assays of function. Enzyme properties will then be considered in the context of the adaptive and innate immune components of each species. Objective 2. Combined application of enzymology, structural biology and artificial intelligence to decipher structure:function relationships in DNA/RNA-editing enzymes We will apply machine learning to decipher the structure:function features that underlie evolutionary adaptations of enzyme properties. To this end, we will produce the largest dataset of any enzyme in terms of breadth (number of orthologs) and depth (biochemical parameters). High throughput biochemical analysis will be complemented by computational and experimental insights into 3D structures. Impact. We will discover new enzymes and structure:function relationships, and gain insights into unknown evolutionary distant immune systems. Our work will also represent a novel application of machine learning in enzymology.