Evolutionary Comparative Analyses of DNA-Editing Enzymes of the Immune System: From 5-Dimensional Description of Protein Structures to Immunological Insights and Applications to Protein Engineering

Abstract

The immune system is unique among all biological sub-systems in its usage of DNA-editing enzymes to introduce targeted gene mutations and double-strand DNA breaks to diversify antigen receptor genes and combat viral infections. These processes, initiated by specific DNA-editing enzymes, often result in mistargeted induction of genome lesions that initiate and drive cancers. Like other molecules involved in human health and disease, the DNA-editing enzymes of the immune system have been intensively studied in humans and mice, with little attention paid (< 1% of published studies) to the same enzymes in evolutionarily distant species. Here, we present a systematic review of the literature on the characterization of one such DNA-editing enzyme, activation-induced cytidine deaminase (AID), from an evolutionary comparative perspective. The central thesis of this review is that although the evolutionary comparative approach represents a minuscule fraction of published works on this and other DNA-editing enzymes, this approach has made significant impacts across the fields of structural biology, immunology, and cancer research. Using AID as an example, we highlight the value of the evolutionary comparative approach in discoveries already made, and in the context of emerging directions in immunology and protein engineering. We introduce the concept of 5-dimensional (5D) description of protein structures, a more nuanced view of a structure that is made possible by evolutionary comparative studies. In this higher dimensional view of a protein’s structure, the classical 3-dimensional (3D) structure is integrated in the context of real-time conformations and evolutionary time shifts (4th dimension) and the relevance of these dynamics to its biological function (5th dimension).

Phylogeny, Structure, Functions, and Role of AIRE in the Formation of T-Cell Subsets

“…To recognize conformational antigens, higher chordates have another mechanism of specificity enhancement called affinity maturation of BCRs after a low-specific interaction with the antigen ( Figure 2 ) [ 45 , 46 ]. Presently, this process or its analogs are not revealed in jawless vertebrates [ 29 , 47 , 48 , 49 , 50 ]. The emergence of the antigen-presenting system in higher chordates also solved the issue of the recognition of self/altered-self due to the mechanism of presentation of self-antigens in all the cells of an organism.…”

Ancestral reconstruction reveals catalytic inactivation of activation-induced cytidine deaminase concomitant with cold water adaption in the Gadiformes bony fish

“…These data were obtained with bacterially expressed and purified GST-AID. Since this system has been used to demonstrate catalytic activity of many bony fish AID orthologs [71][72][73]80], it is unlikely but formally possible that the extreme enzymatic lethargy of Gm-AID is due to mis-folding when expressed in bacteria. To examine this possibility, we expressed Gm-AID in a different expression system (293 T cells), along with Dr-AID as a positive control.…”

Cooperativity between Cas9 and hyperactive AID establishes broad and diversifying mutational footprints in base editors

“…Cas9 engagement impacts DNA deaminase strand capture. There are multiple hypotheses addressing how AID targets both Ig strands in B-cells, 19 including antisense transcription, DNA topology, and R-loop collapse by endogenous RNAse H digestion, all of which would expose both strands as ssDNA. However, how Cas9 binding could create the right environment for AID to access either the NTS or TS has not yet been studied.…”

“…AID naturally functions to drive somatic hypermutation (SHM) in B-cells where it generates clustered mutations on the immunoglobulin (Ig) locus by acting on either strand in a processive fashion (18, 19). In addition to being processive, AID activity can be tuned as its deamination activity has been highly constrained (20), as evidenced by the isolation of various hyperactivating mutations that shift the balance towards increased genomic instability in B-cells (21, 22). Similar to the opportunity offered by systematically altering AID, perturbing Cas9 cleavage activity can offer a means to alter downstream replication and repair, and analyzing BE off-targets can provide a means to assess the impact of Cas9 engagement.…”