The primary investigator is an MD/PhD trained infectious diseases physician with an interest in understanding enzymes that generate diversity in host-pathogen interactions. In the proposed work, the PI aims to bring his prior experience in enzyme mechanisms and develop new training through virologic experiments and immunologic studies. A remarkable group of enzymes, the polynucleotide cytidine deaminases of the AID/APOBEC family, play both constructive and destructive roles in struggle against HIV. On one hand, deamination by the family member APOBEC3G interferes with the integrity of the pathogen genome. In turn, HIV has evolved the lentiviral protein Vif as an evasive means to counteract human APOBEC3G. Infection with HIV is also associated with immune activation, which can result in increased expression of a B-cell specific deaminase family member, AID. AID physiologically serves as the chief catalyst governing antibody diversity through the introduction of targeted uracil lesions in antibody variable genes or switch regions which ultimately result in higher affinity antibodies of altered isotype. Aberrant regulation and expression of AID has increasingly been associated with Non-Hodgkins lymphoma, the leading AIDS-defining malignancy in HIV infected patients. Despite the importance of these cytidine deaminases, little is know about the nature of their interaction with their nucleic acid targets. This proposal addresses the hypothesis that the molecular interactions that lead to catalysis and binding of nucleic acids are critical determinants of their proper physiologic function. The studies aim to decipher and perturb these molecular interactions. Initially, structure-based hypotheses will be used to localize the protein determinants of sequence preference and resolve the mode of binding to the nucleic acid backbone. By utilizing novel loop graft mutant enzymes with altered sequence preference, the impact of perturbed sequence specificity on retroviral restriction (APOBEC3G) or antibody diversity and chromosomal translocations (AID) will be explored. To understand catalysis by AID/APOBEC enzymes, nucleoside analogs will be introduced into oligonucleotides via chemical or chemoenzymatic methods and are used to characterize the kinetics of deamination and the inhibition of pro-oncogenic AID activity. Taken together, a full characterization of the AID/APOBEC-nucleic acid complex ? binding and catalysis ? will provide a molecular basis for the action of this important enzyme family in vitro and in vivo. Through mentored training, the PI will develop the broad based research skills necessary to examine biological and biochemical aspects of diversity generation in host-pathogen interactions upon an ultimate transition to independence.

Public Health Relevance

/RELEVANCE The immune system uses enzymes that catalyze the targeted deamination of cytosine to restrict HIV and make high affinity antibodies, though the same mechanisms can potentially be pro-oncogenic. This proposal aims to biochemically define and biologically perturb the molecular interactions that allow polynucleotide cytidine deaminase enzymes of the AID/APOBEC family to protect against infection.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Clinical Investigator Award (CIA) (K08)
Project #
5K08AI089242-06
Application #
8604126
Study Section
Acquired Immunodeficiency Syndrome Research Review Committee (AIDS)
Program Officer
Sharma, Opendra K
Project Start
2010-02-15
Project End
2015-01-31
Budget Start
2014-02-01
Budget End
2015-01-31
Support Year
6
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of Pennsylvania
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Nabel, Christopher S; DeNizio, Jamie E; Carroll, Martin et al. (2017) DNA Methyltransferases Demonstrate Reduced Activity against Arabinosylcytosine: Implications for Epigenetic Instability in Acute Myeloid Leukemia. Biochemistry 56:2166-2169
Liu, Monica Yun; Torabifard, Hedieh; Crawford, Daniel J et al. (2017) Mutations along a TET2 active site scaffold stall oxidation at 5-hydroxymethylcytosine. Nat Chem Biol 13:181-187
Liu, Monica Yun; DeNizio, Jamie E; Schutsky, Emily K et al. (2016) The expanding scope and impact of epigenetic cytosine modifications. Curr Opin Chem Biol 33:67-73
Liu, M Y; DeNizio, J E; Kohli, R M (2016) Quantification of Oxidized 5-Methylcytosine Bases and TET Enzyme Activity. Methods Enzymol 573:365-85
Mo, Charlie Y; Birdwell, L Dillon; Kohli, Rahul M (2014) Specificity determinants for autoproteolysis of LexA, a key regulator of bacterial SOS mutagenesis. Biochemistry 53:3158-68
Gajula, Kiran S; Huwe, Peter J; Mo, Charlie Y et al. (2014) High-throughput mutagenesis reveals functional determinants for DNA targeting by activation-induced deaminase. Nucleic Acids Res 42:9964-75
Kohli, Rahul M; Zhang, Yi (2013) TET enzymes, TDG and the dynamics of DNA demethylation. Nature 502:472-9
Nabel, Christopher S; Lee, Jae W; Wang, Laura C et al. (2013) Nucleic acid determinants for selective deamination of DNA over RNA by activation-induced deaminase. Proc Natl Acad Sci U S A 110:14225-30
MacMillan, Alyssa L; Kohli, Rahul M; Ross, Susan R (2013) APOBEC3 inhibition of mouse mammary tumor virus infection: the role of cytidine deamination versus inhibition of reverse transcription. J Virol 87:4808-17
Nabel, Christopher S; Jia, Huijue; Ye, Yu et al. (2012) AID/APOBEC deaminases disfavor modified cytosines implicated in DNA demethylation. Nat Chem Biol 8:751-8

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