APOBEC3 enzymes form part of the innate immune system by deaminating cytosine to uracil in single-stranded DNA (ssDNA) and thereby preventing the spread of pathogenic genetic information. However, APOBEC mutagenesis is also exploited by viruses and cancer cells to increase rates of evolution, escape adaptive immune responses, and resist drugs. This raises the possibility of APOBEC3 inhibition as a strategy for augmenting existing antiviral and anticancer therapies. Here we show that, upon incorporation into short ssDNAs, the cytidine nucleoside analogue 2′-deoxyzebularine (dZ) becomes capable of inhibiting the catalytic activity of selected APOBEC variants derived from APOBEC3A, APOBEC3B, and APOBEC3G, supporting a mechanism in which ssDNA delivers dZ to the active site. Multiple experimental approaches, including isothermal titration calorimetry, fluorescence polarization, protein thermal shift, and nuclear magnetic resonance spectroscopy assays, demonstrate nanomolar dissociation constants and low micromolar inhibition constants. These dZ-containing ssDNAs constitute the first substrate-like APOBEC3 inhibitors and, together, comprise a platform for developing nucleic acid-based inhibitors with cellular activity.
APOBEC3G has an important role in human defense against retroviral pathogens, including HIV-1. Its single-stranded DNA cytosine deaminase activity, located in its C-terminal domain (A3Gctd), can mutate viral cDNA and restrict infectivity. We used time-resolved nuclear magnetic resonance (NMR) spectroscopy to determine kinetic parameters of A3Gctd's deamination reactions within a 5=-CCC hot spot sequence. A3Gctd exhibited a 45-fold preference for 5=-CCC substrate over 5=-CCU substrate, which explains why A3G displays almost no processivity within a 5=-CCC motif. In addition, A3Gctd's shortest substrate sequence was found to be a pentanucleotide containing 5=-CCC flanked on both sides by a single nucleotide. A3Gctd as well as fulllength A3G showed peak deamination velocities at pH 5.5. We found that H216 is responsible for this pH dependence, suggesting that protonation of H216 could play a key role in substrate binding. Protonation of H216 appeared important for HIV-1 restriction activity as well, since substitutions of H216 resulted in lower restriction in vivo. Human APOBEC3G (A3G) is a member of a family of Zn 2ϩ -dependent polynucleotide cytosine deaminases. This family was named after APOBEC1 (apolipoprotein B mRNA-editing enzyme catalytic polypeptide 1) and also includes the antibody gene diversification enzyme AID (activation-induced cytidine deaminase) (reviewed in references 1-5). A3G can restrict HIV-1 replication by packaging into assembling viral particles for delivery to target cells, where it deaminates cytosine to uracil in newly transcribed viral DNA. These cDNA uracils base pair with adenine during plus-strand synthesis and result in G-to-A hypermutation and, in turn, inactivation of the viral genome. A3G has two Zn 2ϩ binding domains that span residues 1 to 196 and 197 to 384, but only the C-terminal domain is catalytically active (6-8). The Nterminal domain interacts with HIV-1 Vif, RNA, and singlestranded DNA (ssDNA) (e.g., see references 7 and 9-11). A3G predominantly deaminates the 3= cytosine (underlined) in a 5=-CCC sequence, although the middle cytosine can also be deaminated in subsequent reactions following deamination of the 3= cytosine (12-16). In longer ssDNA substrates with multiple 5=-CCC sites, A3G deamination exhibits a 3=¡5= spatial preference in vitro (9,17,18). In the present study, we use the catalytic domain of A3G (A3Gctd) to determine kinetic parameters. Our results provide kinetic constants for two independent deaminations within a 5=-CCC sequence, which explain A3G's catalytic site preference for the 3= cytosine. We identify a strong pH dependence of the reaction speed, which implies that a histidine residue is involved in substrate binding. In addition, we identify the shortestlength ssDNA substrate for A3Gctd to be a pentanucleotide. MATERIALS AND METHODSPurification of A3Gctd. The APOBEC3G C-terminal domain (A3Gctd), comprising amino acids 191 to 384, was expressed and purified as previously described (19). Briefly, the glutathione S-transferase (GST)-fused A3Gctd was...
The novel Ras effector mNore1, capable of inducing apoptosis, is a multidomain protein. It comprises a C1 domain homologous to PKC and an RA domain similar to the Ras effectors AF-6 and RalGDS. Here, we determine the affinity of these two domains to the active forms of Ras and Rap1 using isothermal calorimetric titration. The interaction of Ras/Rap1-GTP with the RA domain of mNore1 is weakened significantly by direct binding of the C1 domain to the RA domain. In order to analyze this observation in atomic detail, we solved the C1 solution structure by NMR. By determining chemical shifts and relaxation rates, we can show an intramolecular complex of C1-RA. GTP-Ras titration and binding to RA disrupts this complex and displaces the C1 domain. Once the C1 domain tumbles freely in solution, a lipid binding interface becomes accessible. Furthermore, we provide evidence of phosphatidylinositol 3-phosphate binding of the free C1 domain.
The APOBEC3 (APOBEC3A‐H) enzyme family is part of the human innate immune system that restricts pathogens by scrambling pathogenic single‐stranded (ss) DNA by deamination of cytosines to produce uracil residues. However, APOBEC3‐mediated mutagenesis of viral and cancer DNA promotes its evolution, thus enabling disease progression and the development of drug resistance. Therefore, APOBEC3 inhibition offers a new strategy to complement existing antiviral and anticancer therapies by making such therapies effective for longer periods of time, thereby preventing the emergence of drug resistance. Here, we have synthesised 2′‐deoxynucleoside forms of several known inhibitors of cytidine deaminase (CDA), incorporated them into oligodeoxynucleotides (oligos) in place of 2′‐deoxycytidine in the preferred substrates of APOBEC3A, APOBEC3B, and APOBEC3G, and evaluated their inhibitory potential against these enzymes. An oligo containing a 5‐fluoro‐2′‐deoxyzebularine (5FdZ) motif exhibited an inhibition constant against APOBEC3B 3.5 times better than that of the comparable 2′‐deoxyzebularine‐containing (dZ‐containing) oligo. A similar inhibition trend was observed for wild‐type APOBEC3A. In contrast, use of the 5FdZ motif in an oligo designed for APOBEC3G inhibition resulted in an inhibitor that was less potent than the dZ‐containing oligo both in the case of APOBEC3GCTD and in that of full‐length wild‐type APOBEC3G.
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