The implications of APOBEC3-mediated C-to-U RNA editing for human disease.

Intra-organism biodiversity is thought to arise from epigenetic modification of constituent genes and post-translational modifications of translated proteins. Here, we show that post-transcriptional modifications, like RNA editing, may also contribute. RNA editing enzymes APOBEC3A and APOBEC3G catalyze the deamination of cytosine to uracil. RNAsee (RNA site editing evaluation) is a computational tool developed to predict the cytosines edited by these enzymes. We find that 4.5% of non-synonymous DNA single nucleotide polymorphisms that result in cytosine to uracil changes in RNA are probable sites for APOBEC3A/G RNA editing; the variant proteins created by such polymorphisms may also result from transient RNA editing. These polymorphisms are associated with over 20% of Medical Subject Headings across ten categories of disease, including nutritional and metabolic, neoplastic, cardiovascular, and nervous system diseases. Because RNA editing is transient and not organism-wide, future work is necessary to confirm the extent and effects of such editing in humans.

Enhanced anti-tumor efficacy with multi-transgene armed mesenchymal stem cells for treating peritoneal carcinomatosis.

BACKGROUND: Mesenchymal stem cells (MSCs) have garnered significant interest for their tumor-tropic property, making them potential therapeutic delivery vehicles for cancer treatment. We have previously shown the significant anti-tumour activity in mice preclinical models and companion animals with naturally occurring cancers using non-virally engineered MSCs with a therapeutic transgene encoding cytosine deaminase and uracil phosphoribosyl transferase (CDUPRT) and green fluorescent protein (GFP). Clinical studies have shown improved response rate with combinatorial treatment of 5-fluorouracil and Interferon-beta (IFNb) in peritoneal carcinomatosis (PC). However, high systemic toxicities have limited the clinical use of such a regime. METHODS: In this study, we evaluated the feasibility of intraperitoneal administration of non-virally engineered MSCs to co-deliver CDUPRT/5-Flucytosine prodrug system and IFNb to potentially enhance the cGAS-STING signalling axis. Here, MSCs were engineered to express CDUPRT or CDUPRT-IFNb. Expression of CDUPRT and IFNb was confirmed by flow cytometry and ELISA, respectively. The anti-cancer efficacy of the engineered MSCs was evaluated in both in vitro and in vivo model. ES2, HT-29 and Colo-205 were cocultured with engineered MSCs at various ratio. The cell viability with or without 5-flucytosine was measured with MTS assay. To further compare the anti-cancer efficacy of the engineered MSCs, peritoneal carcinomatosis mouse model was established by intraperitoneal injection of luciferase expressing ES2 stable cells. The tumour burden was measured through bioluminescence tracking. RESULTS: Firstly, there was no changes in phenotypes of MSCs despite high expression of the transgene encoding CDUPRT and IFNb (CDUPRT-IFNb). Transwell migration assays and in-vivo tracking suggested the co-expression of multiple transgenes did not impact migratory capability of the MSCs. The superiority of CDUPRT-IFNb over CDUPRT expressing MSCs was demonstrated in ES2, HT-29 and Colo-205 in-vitro. Similar observations were observed in an intraperitoneal ES2 ovarian cancer xenograft model. The growth of tumor mass was inhibited by ~ 90% and 46% in the mice treated with MSCs expressing CDUPRT-IFNb or CDUPRT, respectively. CONCLUSIONS: Taken together, these results established the effectiveness of MSCs co-expressing CDUPRT and IFNb in controlling and targeting PC growth. This study lay the foundation for the development of clinical trial using multigene-armed MSCs for PC.

Cytosine Deaminase-Overexpressing hTERT-Immortalized Human Adipose Stem Cells Enhance the Inhibitory Effects of Fluorocytosine on Tumor Growth in Castration Resistant Prostate Cancer.

A promising de novo approach for the treatment of Castration-resistant prostate cancer (CRPC) exploits cell-mediated enzyme prodrug therapy comprising cytosine deaminase (CD) and fluorouracil (5-FC). The aim of this study was to determine the potential of bacterial CD-overexpressing hTERT-immortalized human adipose stem cells (hTERT-ADSC.CD) to suppress CRPC. A lentiviral vector encoding a bacterial CD gene was used to transfect and to generate the hTERT-ADSC.CD line. The ability of the cells to migrate selectively towards malignant cells was investigated in vitro. PC3 and hTERT-ADSC.CD cells were co-cultured. hTERT-ADSC.CD and 1 × 106 PC3 cells were administered to nude mice via intracardiac and subcutaneous injections, respectively, and 5-FC was given for 14 days. hTERT-ADSC.CD were successfully engineered. Enhanced in vitro hTERT-ADSC.CD cytotoxicity and suicide effect were evident following administration of 5 µM 5-FC. hTERT-ADSC.CD, together with 5-FC, augmented the numbers of PC3 cells undergoing apoptosis. In comparison to controls administered hTERT-ADSC.CD monotherapy, hTERT-ADSC.CD in combination with 5-FC demonstrated a greater suppressive effect on tumor. In CPRC-bearing mice, tumor suppression was enhanced by the combination of CD-overexpressing ADSC and the prodrug 5-FC. Stem cells exhibiting CD gene expression are a potential novel approach to treatment for CRPC.

Multiplexed in-situ mutagenesis driven by a dCas12a-based dual-function base editor.

Mutagenesis driving genetic diversity is vital for understanding and engineering biological systems. However, the lack of effective methods to generate in-situ mutagenesis in multiple genomic loci combinatorially limits the study of complex biological functions. Here, we design and construct MultiduBE, a dCas12a-based multiplexed dual-function base editor, in an all-in-one plasmid for performing combinatorial in-situ mutagenesis. Two synthetic effectors, duBE-1a and duBE-2b, are created by amalgamating the functionalities of cytosine deaminase (from hAPOBEC3A or hAID*Δ ), adenine deaminase (from TadA9), and crRNA array processing (from dCas12a). Furthermore, introducing the synthetic separator Sp4 minimizes interference in the crRNA array, thereby facilitating multiplexed in-situ mutagenesis in both Escherichia coli and Bacillus subtilis. Guided by the corresponding crRNA arrays, MultiduBE is successfully employed for cell physiology reprogramming and metabolic regulation. A novel mutation conferring streptomycin resistance has been identified in B. subtilis and incorporated into the mutant strains with multiple antibiotic resistance. Moreover, surfactin and riboflavin titers of the combinatorially mutant strains improved by 42% and 15-fold, respectively, compared with the control strains with single gene mutation. Overall, MultiduBE provides a convenient and efficient way to perform multiplexed in-situ mutagenesis.

Exploring Product Release from Yeast Cytosine Deaminase with Metadynamics.

The yeast cytosine deaminase (yCD) enzyme/5-fluorocytosine prodrug system is a promising candidate for targeted chemotherapeutics. After conversion of the prodrug into the toxic chemotherapeutic drug, 5-fluorouracil (5-FU), the slow product release from the enzyme limits the overall catalytic efficiency of the enzyme/prodrug system. Here, we present a computational study of the product release of the anticancer drug, 5-FU, from yCD using metadynamics. We present a comparison of the 5-FU drug to the natural enzyme product, uracil. We use volume-based metadynamics to compute the free energy landscape for product release and show a modest binding affinity for the product to the enzyme, consistent with experiments. Next, we use infrequent metadynamics to estimate the unbiased release rate from Kramers time-dependent rate theory and find a favorable comparison to experiment with a slower rate of product release for the 5-FU system. Our work demonstrates how adaptive sampling methods can be used to study the protein-ligand unbinding process for engineering enzyme/prodrug systems and gives insights into the molecular mechanism of product release for the yCD/5-FU system.

Protein Interaction Map of APOBEC3 Enzyme Family Reveals Deamination-Independent Role in Cellular Function.

Human APOBEC3 enzymes are a family of single-stranded (ss)DNA and RNA cytidine deaminases that act as part of the intrinsic immunity against viruses and retroelements. These enzymes deaminate cytosine to form uracil which can functionally inactivate or cause degradation of viral or retroelement genomes. In addition, APOBEC3s have deamination-independent antiviral activity through protein and nucleic acid interactions. If expression levels are misregulated, some APOBEC3 enzymes can access the human genome leading to deamination and mutagenesis, contributing to cancer initiation and evolution. While APOBEC3 enzymes are known to interact with large ribonucleoprotein complexes, the function and RNA dependence are not entirely understood. To further understand their cellular roles, we determined by affinity purification mass spectrometry (AP-MS) the protein interaction network for the human APOBEC3 enzymes and mapped a diverse set of protein-protein and protein-RNA mediated interactions. Our analysis identified novel RNA-mediated interactions between APOBEC3C, APOBEC3H Haplotype I and II, and APOBEC3G with spliceosome proteins, and APOBEC3G and APOBEC3H Haplotype I with proteins involved in tRNA methylation and ncRNA export from the nucleus. In addition, we identified RNA-independent protein-protein interactions with APOBEC3B, APOBEC3D, and APOBEC3F and the prefoldin family of protein-folding chaperones. Interaction between prefoldin 5 (PFD5) and APOBEC3B disrupted the ability of PFD5 to induce degradation of the oncogene cMyc, implicating the APOBEC3B protein interaction network in cancer. Altogether, the results uncover novel functions and interactions of the APOBEC3 family and suggest they may have fundamental roles in cellular RNA biology, their protein-protein interactions are not redundant, and there are protein-protein interactions with tumor suppressors, suggesting a role in cancer biology. Data are available via ProteomeXchange with the identifier PXD044275.

Direct inhibition of human APOBEC3 deaminases by HIV-1 Vif independent of the proteolysis pathway.

HIV-1 Vif is known to counteract the antiviral activity of human apolipoprotein B mRNA-editing catalytic polypeptide-like (A3), a cytidine deaminase, in various ways. However, the precise mechanism behind this interaction has remained elusive. Within infected cells, Vif forms a complex called VßBCC, comprising CBFß and the components of E3 ubiquitin ligase, Elongin B, Elongin C, and Cullin5. Together with the ubiquitin-conjugating enzyme, VßBCC induces ubiquitination-mediated proteasomal degradation of A3. However, Vif exhibits additional counteractive effects. In this study, we elucidate that VßBCC inhibits deamination by A3G, A3F, and A3B independently of proteasomal degradation. Surprisingly, we discovered that this inhibition for A3G is directly attributed to the interaction between VßBCC and the C-terminal domain of A3G. Previously, it was believed that Vif did not interact with the C-terminal domain. Our findings suggest that inhibiting the interaction between VßBCC and the C-terminal domain, as well as the N-terminal domain known to be targeted for ubiquitination, of A3G may be needed to prevent counteraction by Vif.

Enzymatic DNA repairing amplification-powered construction of an Au nanoparticle-based nanosensor for single-molecule monitoring of cytosine deaminase activity in cancer cells.

APOBEC3A (A3A) is a cytidine deaminase with critical roles in molecular diagnostics. Herein, we demonstrate the enzymatic DNA repairing amplification-powered construction of an Au nanoparticle-based nanosensor for single-molecule monitoring of A3A activity in cancer cells. Target A3A can convert cytosine (C) in substrate probe to uracil (U), and then the template binds with substrate probe to form a dsDNA containing U/A base pairs. Uracil DNA glycosylase (UDG) excises the U base to produce an apurinic/apyrimidinic (AP) site that can be cleaved by apurinic/apyrimidic endonuclease 1 (APE1) to obtain the substrate fragment with 3'-OH end. Subsequently, the substrate fragment initiates cyclic enzymatic repairing amplification (ERA), releasing trigger-1 and trigger-2. The resultant trigger-1 can act as the primer to induce multiple cycles of cyclic ERA, producing numerous trigger-1 and trigger-2. The hybridization of trigger-2 with signal probe forms the dsDNA duplexes with an AP site, inducing the cyclic cleavage of signal probes by APE1 to release abundant Cy5 molecules from the AuNPs. Released Cy5 molecules can be easily quantified by single-molecule imaging. This nanosensor allows for specific and sensitive detection of A3A activity with a detection limit of 0.855 aM, and it can further measure kinetic parameters, screen inhibitors, and quantify endogenous A3A activity at the single-cell level, with prospect application in disease diagnostics and therapy.