Abstract Activation induced cytidine deaminase (AID) is an important enzyme that creates mutations in DNA via deamination of a cytosine base into a uracil. AID, also referred to as activation induced deaminase (AICDA), plays a crucial part in the human immune response as it is essential for isotype switching and cellular differentiation. However, aberrant expressions in some pathways has been implicated in a plethora of diseases. There is a pressing need for research and comparison of current literature that informs related therapies. Previous studies have explored potential mechanisms by which AID works and subsequently ways to target gene therapies based on this information. Due to AID’s complexity, there have been many challenges along the path that led to our current understanding of the beneficial and harmful nature of AID. Furthermore, a better understanding of the way AID works can aid with the development of more efficacious therapies. Although further research on the topic and additional testing in humans and animal models are needed, it is clear that AID may play an important role in the development of therapeutic treatments in diseases like cancer, lupus, and type 1 diabetes. "DUJWBUJPO�*OEVDFE�%FBNJOBTF�BOE� 1PUFOUJBM�5IFSBQFVUJD�"WFOVFT �ïw�¦×ÝÚ×�°åàê×è× Berkeley Pharma Tech Journal of Medicine Correspondence:�� XPKUBSBN@VNJDI�FEV Keywords: "*$%" -VQVT (FOF�5IFSBQZ "VUPJNNVOF $BODFS 5IFSBQFVUJD�"EWBODFT Submitted�"QSJM 2�, 202�� Accepted�JuOF���, 20��� 1VCMJTIFE�+VMZ��� ����� Full Open Access Creative Commons Attribution�� License 4.0 Activation Induced Cytidine Deaminase Image by Emw - Own work, CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?curid=8764396 Introduction Previous studies and research have established that AID regulates secondary antibody diversi�cation. There are many di�erent immunoglobulin (Ig) diversi�cation processes, such as somatic hypermutation (SHM), class switch recombination (CSR), and gene conversion (GC)4. SHM allows for B cells to diversify in order to respond to threats to the immune system,5 while CSR allows for the generation of di�erent classes of antibodies5. GC is a process in which mutations can occur in the antibody genes5. AID is central to CSR/SHM and plasma cell di�erentiation and is encoded by AICDA and B lymphocyte maturation protein 1 (Blimp-1) which is a transcription factor encoded by Prdm16. AID and its transcription factors underpin Ab and autoantibody responses7 . The deamination results in a change from a cytosine base to a uracil base in Ig genes, and this can result in either CSR or SHM, depending on the deoxyribonucleic acid (DNA) repair pathway. AID expression is upregulated by in�ammatory cytokines like interferon-γ and tumor necrosis factor (TNF)-α which induces p53 mutations in in�ammatory or cancer cells. Although AID is typically associated with and expressed in B-cells, it can also be expressed, for example, in embryonic germ cells or pluripotent cells like oocytes. AID proteins have been shown to be expressed during early B-cell development in both human fetal liver and adult bone marrow 9. It is important to note that AID is a potent enzyme which instigates genomic diversity for both bene�cial10 and harmful outcomes in humans. This can best be depicted in Figure 1, which summarizes much of the following section. AID di�ers from other Apolipoprotein B mrRNA Editing Catalytic Polypeptides (APOBECs) speci�cally due to the size and orientation of its substrate speci�city loop12. It has a larger loop that extends away from the active site and thus can accommodate two purines next to a target C12. Despite some sequence similarity to APOBEC cytidine deaminases, AID’s critical function in Ab diversi�cation in CSR cannot be substituted by other APOBEC proteins10. While aberrant deaminase activity can certainly threaten the genome, recent biotechnological e�orts have focused on harnessing and targeting deaminase activity in base editors that are related to AID11. Berkeley Pharma Tech Journal of Medicine | 108 Figure 1: A diagram summarizing some of the beneficial and harmful outcomes of AID AID, a potent DNA mutator, must be tightly regulated to prevent any o �-target e�ects which can result in a plethora of problems including mutations in non-Ig genes, genomic instability, interchromosomal translocations, and cellular neoplastic transformation13. AID has previously been implicated in the tumorigenic process in B cell tumors potentially through the induction of chromosomal translocations and mutations in tumor suppressor genes and oncogenes14. AID expression has also been implicated in the pathogenesis of human B cell malignancies15. Indeed, accumulating evidence suggests AID is pro-oncogenic and induces cancer- promoting mutations or chromosomalrearrangements16. Another detrimental impact of AID is the generation of autoimmunity, which can occur after on-target point mutations in variable genes produce antibodies with high a�nity for self-proteins11. These detrimental e�ects are important to consider when choosing to target AID in potential research projects. Other studies have proposed more novel functions for AID. For instance, one has suggested that AID functions as an adaptor protein that represses viral transcription, which would have implications for the development of anti-HIV therapeutics and other therapies 17. Moreover, AID can exert non canonical functions when aberrantly expressed in epithelial cells and was Berkeley Pharma Tech Journal of Medicine | 109 long known to lack speci�c inhibitors which prevented therapeutic applications to modulate AID functions18. Mechanism Historically, the discovery of AID and its essential role in antibody diversi�cation kindled a debate over AID’s nucleic acid substrate, which inspired the DNA deamination model and identi�cation of downstream players in the CSR pathway such as the DNA glycosylase UNG19. Subsequently, this discovery led to a race to uncover ssDNA as the target of AID’s enzymatic activity and its dependence on transcription for deamination19. It is important to note that AID deaminates C→U only on ssDNA and does not function on dsDNA or RNA12. Figure 2 depicts this type of deamination reaction. Nearly two decades prior to this research, there was an observation that noncoding RNA transcripts originated from within the IgH locus and could be playing a role in CSR19. Figure 2: Cytosine to Uracil deamination reaction wherein an amine group is removed SHM generates point mutations in the Ig variable regions while CSR exchanges the Ig heavy chain constant region, and ultimately this gives rise to antibodies with enhanced a�nity and new e�ector functions20. AID initiates both these processes through deamination of cytosine to uracil in Ig variable and switch gene regions, and the resultingU:G mismatches are Berkeley Pharma Tech Journal of Medicine | 110 subsequently processed by uracil-DNA glycosylase (UNG2) and a pathway that requires The mechanism by which AID works has not been entirely discerned. Namely, the mechanism of AID targeting has especially been a long-standing mystery10. Currently, there are many di�erent �ndings that are piecing together the puzzle of how exactly AID works. In terms of frequency, the number of molecules containing deamination in both DNA strands at the acceptor switch region corresponds to its class switch e�ciency. It has been proposed that the minimal requirement for a DNA double-strand break (DSB) formation is as low as only one AID deamination event on both DNA strands4. There are also several proposed mechanisms for AID function. AID may target template and non-template strands at similar frequencies and predominantly after R-loops are processed by cellular enzymes that expose DNA on both DNA strands4. Additionally, AID footprints may be distributed evenly across the entire length of the S region, unlike SHM, which is not evenly distributed over a distance; thus AID deaminates S and V regions with distinct mechanisms. It has also been suggested that AID-mediated DNA demethylation occurs due to the deamination of methylated cytidine residues in single-stranded DNA, followed by DNA repair7. A long-standing hypothesis on AID targeting, known as the hotspot hypothesis, has recently been under re-evaluation. This hypothesis considered a short sequence motif (AGCT) conserved in all S regions as functionally important for CSR, proposing that it exerts its function via its overlapping AID hotspot structure23. However, an initial weakness of this theory was that these sequences are very common in the genome23. Another study determined one of the �rst crystal structures of maltose-binding protein (MBP)-fused AID and its complex with cytidine (C), deoxycytidine (dC), and deoxycytidine monophosphate (dCMP). These structures can help explain the discrimination between DNA and RNA in AID catalysis and reveal that AID has a bifurcated substrate-binding surface10. This supports the theory that one AID recognizes two adjacent ssDNA overhangs from one structured substrate to achieve high a�nity10. G4 structured substrates induce AID cooperative Berkeley Pharma Tech Journal of Medicine | 111 oligomerization, which could promote clustered mutations in the Ig S regions10. Overall, the bifurcated substrate binding surface and oligomerization interface are both an essential component of CSR and help elucidate recognition of structured substrates as an important AID-targeting mechanism, speci�cally in the Ig S regions10. It has therefore been suggested that G4 substrates mimicking Ig S regions are preferred AID targets in vitro. This recent �nding is a departure from our previous understanding of AID targeting. This data also posits that AID preference for these substrates is likely due to their bundled ssDNA overhangs structure rather than the primary sequence motif, which was long believed to bewhy AID preferred these substrates10. It is important to recognize that a de�nitive complex structure with fully characterized substrate conformation is still lacking and must be developed10. Many proposed therapies suggest that selective inhibition of AID may ameliorate the conditions. Ultimately,further experimentation and analysis with more sensitive techniques that may eventually be developed is needed to more fully understand the mechanism of AID inhibition. Given that the crystal structure of AID has recently been resolved, future e�orts would certainly bene�t from structural modeling approaches10. A more de�nitive structure could serve as a template for potential therapeutic intervention against AID10. Progress on AID structure is very timely alongside the growing knowledge about Ig class switch region nucleic acid structures, which are supported by functional studies24. Already, we are seeing promising results from initiatives focusing on AID. Platforms like GENEVESTIGATOR consolidate publicly available studies from microarrays, mRNA sequencing, and more under healthy conditions versus diseased states25. Using these comparisons is one potential strategy for a comprehensive analysis of the role of AID in the pathobiology of immune- or in�ammatory-based diseases and cancer25. It has also been suggested that we may eventually be able to analyze AID gene signatures to get decisive determinants of patient-speci�c or patient-group-speci�c antiviral response, which could allow us to understand how viruses can impact di�erent individuals25. Berkeley Pharma Tech Journal of Medicine | 112 Estrogen and AID Estrogen has been found to reverse the repression of AID, resulting in a subsequent boost in AID expression. This is proposed to occur through the upregulation of HoxC4, which, together with NF-κB, critically mediates AID promoter activation6. There may, however, be additional epigenetic mechanisms at play that serve to regulate AID expression. Estrogen reverses HDI-mediated inhibition of AID and CSR in Ab and autoantibody responses through the downregulation of B cell miR-26a, which targets AID mRNA’s 3’UTR6. As epigenetic modi�ers, SCFA HDIs, like miR-26a and miR-125a, inhibit AID expression and CSR through the upregulation of select B cell miRNAs, which silence AID26. This is interesting as it may provide an explanation for the female bias in autoantibody-mediated autoimmune diseases like lupus2. Yet, an experimental and fully functioning in vivo model of the human immune system is needed in order to understand the epigenetic mechanisms relating to the human Ab and autoantibody response6. Autoimmune Diseases Cellular reprogramming, broadly, is a mechanism that must be further explored. Currently, there are three approaches to induce reprogramming: cell fusion, nuclear transfer, and iPSC14. Cell fusion is a great way to understand nuclear plasticity and is a main element of many cancer processes14. Nuclear transfer, more commonly referred to as cloning, has potential therapeutic applications, although ethical concerns exist14. iPSC technology is anexcellent option given that it has potential therapeutic applications for clinical use without ethical concerns and can be used to model human diseases and screen potential new treatments27. DNA methylation is a major barrier to induced pluripotent stem (iPS) cell reprogramming, and putative DNA demethylase protein AID can erase DNA methylation at pluripotency gene promoters, which will subsequently allow cellular reprogramming14. Autoimmune diseases are detrimental to the health and wellbeing of individuals globally. Berkeley Pharma Tech Journal of Medicine | 113 One example of such a disease is common variable immunode�ciency (CVID), which is a primary immunode�ciency characterized by hypogammaglobulinemia and di�erent degrees of B cell compartment alteration28. We found reduced Bcl-2 protein levels in memory B cells from CVID. Hypertension is another medical condition where the study of AID can be useful. In the USA, nearly 50% of the adult population has hypertension, and prevalence increases to ~80% at advanced age29. B cell Ig production is dependent on a subset of B cells called GC B cells, which are dependent on AID and may play a causal role in the pathophysiology of hypertension. The GC reaction is driven by IL-21 and T follicular helper (Tfh) cells, which are transcription factors associated with AID and have been demonstrated to play a role in hypertension and hypertensive end-organ damage30. It is possible that B cells and Ig contribute to hypertension in speci�c cases as in autoimmune diseases or preeclampsia31. However, future studies should investigate inducible genetic B cell deletion in adult animals to determine if B cells are viable therapeutic targets for hypertension31. Multiple Sclerosis (MS) is another debilitating chronic disease. B cell depleting therapies are a potential way to ameliorate symptoms in MS given that B cells play a critical role in the MS disease process32. There is a presence of B cells in active lesions and the cerebrospinal �uid of MS patients32. In a recent study, the community was able toglean more information on the role of secondary diversity of the BCR in experimental autoimmune encephalomyelitis (EAE) and identify IgG class-switched B cells as potential therapeutic targets for the treatment of MS32. AID was also found to presumably still exert some subtle e�ect on rMOG-induced (myelin oligodendrocyte glycoprotein) disease trajectory32. Arthritis is a debilitating disease that can result in a lot of pain A potential novel treatment for in�ammatory arthritis includes Fraxinellone33. The therapeutic e�ect of fraxinellone was associated with the inhibition of cellular di�erentiation and activation. It has been shown to attenuate the clinical and histologic features of in�ammatory arthritis in mice33. There Berkeley Pharma Tech Journal of Medicine | 114 was a lower expression of AID and Blimp-1 following treatment with Fraxinellone33. Remarkably, it also alleviated synovial in�ammation and osteoclastogenesis in mice33. Other drugs such as belimumab, a targeted therapy approved for systemic lupus erythematosus (SLE), serve as examples of how targeted therapies that disrupt the AID pathway can be bene�cial34. Further investigation is needed to see the side e�ects on normal cells. Cancer AID, as previously mentioned, has been largely suggested to induce cancer- promoting mutation. AID is expressed in more than 40% of primary human chronic lymphocytic leukemia (CLL) cases, but AID expression can be harnessed for antileukemic e�ect after inhibition of the RAD51 homologous recombination (HR) factor 4,4’-diisothiocyanatostilbene-2-2’-disulfonic acid (DIDS)16. This is a novel antineoplastic role of AID that can be triggered by inhibition of HR, which is a new paradigm to treat AID-expressing tumors and has had proof of principle studies conducted16. This treatment has also been suggested for use in type 1 diabetes6. Another avenue that has been considered is the chronic administration of HSP90 inhibitors, which decreases AID protein levels and has been shown to reduce disease severity in a mouse model of acute B cell lymphoblastic leukemia in which AID accelerates disease progression18. This is promising, as a proof-of-concept study has been published that showed HSP90 inhibitors directly target AID in vivo, and endogenous humanAID is sensitive to them 18. Yet another study has suggested that targeting AID is bene�cial in the immunotherapy ofAID positive tumors because siRNA silencing of AID in plasmacytoma dramatically increases its susceptibility to immunotherapy by cytotoxic T lymphocytes15. Overall, AID has shown to be a promising target in the aforementioned instances and more research may yield additional insights. Berkeley Pharma Tech Journal of Medicine | 115 Conclusion Although further research will help the scientific community to glean more clear insights, it is clear that elucidating how AID works will help with the development of novel therapeutic strategies for a multitude of diseases. Disrupting the AID pathway can have potential therapeutic e�ects. However, it is important to remain cognizant of the fact that AID is a complex component of the human immune system, which is in and of itself a complex system. With that in mind, therapeutic approaches targeting AID must undergo a variety of testing and considerations. 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Abrogated AID Function Prolongs Survival and Diminishes Renal Pathology in the BXSB Mouse Model of Systemic Lupus Erythematosus. J Immunol. 2020;204(5):1091-1100. doi:10.4049/jimmunol.1900501 Berkeley Pharma Tech Journal of Medicine | 119 Front_Page_Wojtara_Revised_FINAL Wojtara Body Template Wojtara Reference Template-2