Biology subjects

Cook, M. E.

Publications and source records attributed to Cook, M. E..

6 recordsLinked to original sources

bioRxiv2026

APOBEC3A is the predominant global editor of cytosines in human mRNAs and in single-strand RNA viruses

APOBEC cytidine deaminases can convert cytosines to uracils in DNA as well as in RNA. The knowledge of DNA deamination motifs preferred by individual APOBECs revealed APOBEC3A as a major source of hypermutation in cancer. However, the extent and relative contribution of specific APOBECs into RNA editing remains unclear as their preferred RNA-editing motifs have not been defined. Here, using a parallel DNA and RNA sequencing strategy, coupled with motif-centered statistical analyses, we sought to identify mRNA edits and diagnostic editing motifs in yeast and human cells overexpressing individual APOBEC enzymes. This approach revealed a prevailing global enrichment for the uCg trinucleotide motif with even greater preference to the motifs cytosines located in 3 base of a loop within a hairpin-loop secondary structure when APOBEC3A, but not any other tested APOBEC, was overexpressed. Further analysis revealed the APOBEC3A-like diagnostic motif enrichment in editing calls from human cancers and blood cells. The APOBEC3A-like editing motif also prevailed in the RNA genomes of SARS-CoV-2 pandemic isolates, as well as in infectious persistent rubella viruses, and in polioviruses emerging from live-attenuated vaccine strains. Together, our results indicate that APOBEC3A is the predominant global APOBEC RNA editor with a potential to impact cell physiology and viral evolution.

genetics↗

bioRxiv2025

Epitranscriptomic control of host epithelial responses to candidiasis via N6-Methyladenosine (m6A) methylation

Fungal infections represent a major global threat, yet to date there is a paucity of effective antifungal drugs and no licensed vaccines to any pathogenic fungi. The commensal pathobiont Candida albicans can cause severe oropharyngeal and mucocutaneous infections in immunocompromised individuals. The initial point of interaction of C. albicans in the oral mucosa is with superficial oral epithelial cells (OECs). Upon fungal encounter, OECs upregulate a program of anti-fungal defense genes, a process that has been extensively studied at the level of proximal signaling pathways, transcriptional induction, and epigenetic control. However, inflammatory genes are also subject to extensive regulation at the mRNA level, yet little is known about mechanisms that control C. albicans-induced genes post-transcriptionally. The importance of epitranscriptomic gene regulation by mRNA methylation is becoming increasingly appreciated, yet its contribution to host responses during fungal infection remains poorly understood. N6-methyladenosine (m6A) is the most abundant RNA modification and influences numerous transcript fates including mRNA stabilization. Here, we demonstrate that C. albicans-induced genes in human and mouse OECs are widely subject to m6A modification following infection. Silencing of a specific subclass of m6A binding proteins, the YTHDF1/2/3 readers, reprogrammed the OEC response to C. albicans by up- and down-regulating antifungal transcripts. The net effect of blocking the m6A pathway using a first-in-class METTL3 inhibitor was to amplify mRNA expression of antimicrobial peptides that reduce fungal burden. These studies establish a role for the m6A pathway as a modulator of immunity to mucosal candidiasis and suggest a new therapeutic avenue to treating this condition.

microbiology↗

bioRxiv2025

An estrogen-independent and IL-1-dependent pathway controls vulvovaginal candidiasis through combined IL-17/IL-22 signaling

Vulvovaginal candidiasis (VVC) affects >75% of women, with considerable morbidity and high medical cost burden. While Type 17 cytokines (IL-17, IL-22) are critical for oral and dermal immunity to C. albicans, their role in VVC has been less clear. Th17 gene signatures are potently upregulated in VVC, yet impairment of individual Th17 components (IL-17A, IL-17R subunits, IL-22) does not worsen disease. Rather, estrogen activity is tightly linked to VVC, leading to a paradigm that hormonal pathways rather than immune defense, dominate susceptibility. Here, we reveal a previously unappreciated role for IL-1/Type 17 in VVC that operates independently of estrogenic hormones. In contrast to mice lacking IL-17A, IL-17RA, IL-22, or IL-22R individually, mice lacking IL-17RA and IL-22RA1 together (Il17raIl22ra1-/-) exhibited high fungal loads and exacerbated tissue damage and inflammation. In human vulvar epithelial cells, IL-17 and IL-22 drive synergistic signaling. IL-1R signaling but surprisingly not IL-23 wa upstream of this response. Il17raIl22ra1-/- mice expressed high IL-1{beta} yet did not control disease, indicating that IL-1 is upstream but not downstream of Type 17 responses. Unexpectedly, Type 17-dependent control occurred in the absence of exogenous estrogen administration and persisted even when estrus was prevented by progesterone treatment. Collectively, these data indicate that susceptibility to VVC is driven not only by estrogen sensitization but through combinatorial loss of IL-17 and IL-22. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=168 SRC="FIGDIR/small/671995v2_ufig1.gif" ALT="Figure 1000"> View larger version (52K): org.highwire.dtl.DTLVardef@19946e9org.highwire.dtl.DTLVardef@1feac71org.highwire.dtl.DTLVardef@eb433aorg.highwire.dtl.DTLVardef@18ab622_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2025

Thymine DNA Glycosylase Binds to R-Loops and Excises 5-Formyl and 5-Carboxyl Cytosine from DNA/RNA Hybrids

R-loops are three-stranded nucleic acid structures consisting of a DNA/RNA hybrid and a displaced single-stranded DNA. Once considered rare byproducts of transcription, R-loops are now recognized as important regulators of various nuclear processes. In particular, evidence indicates a role for R-loops in regulating DNA methylation dynamics. R-loops have been shown to promote active DNA demethylation--the enzymatic reversal of 5-methylcytosine (5mC) back into cytosine--by recruiting associated proteins, providing an attractive targeting mechanism. Nevertheless, many important aspects of this process, including whether the associated proteins bind to and function on R-loops, remain to be substantiated. In this study, we demonstrate for the first time that thymine DNA glycosylase (TDG), a key enzyme in the active DNA demethylation pathway, binds tightly to R-loops in vitro and can excise DNA demethylation intermediates 5-formylcytosine (5fC) and 5-carboxycytosine (5caC) from DNA in DNA/RNA hybrid duplexes. We also show that R-loops guide the strand-specific activity of TDG at CpG sites, potentially explaining the asymmetric distribution of 5fC/5caC at gene promoters. Furthermore, we provide important mechanistic insights into base excision on DNA/RNA hybrid duplexes using 19F NMR. Finally, our findings suggest that TDG-R-loop interactions may be widespread in human cells. Collectively, our results provide strong evidence that R-loops play a critical role in DNA demethylation and support a mechanism in which 5fC/5caC are directly removed from DNA/RNA hybrids in cells. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC="FIGDIR/small/668694v1_ufig1.gif" ALT="Figure 1"> View larger version (13K): org.highwire.dtl.DTLVardef@1ac2095org.highwire.dtl.DTLVardef@18f4952org.highwire.dtl.DTLVardef@16c403org.highwire.dtl.DTLVardef@9ed96c_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2019

Structure of the cell-binding component of the Clostridium difficile binary toxin reveals a novel macromolecular assembly

Targeting Clostridium difficile infection (CDI) is challenging because treatment options are limited, and high recurrence rates are common. One reason for this is that hypervirulent CDI often has a binary toxin termed the C. difficile toxin (CDT), in addition to the enterotoxins TsdA and TsdB. CDT has an enzymatic component, termed CDTa, and a pore-forming or delivery subunit termed CDTb. CDTb was characterized here using a combination of single particle cryoEM, X-ray crystallography, NMR, and other biophysical methods. In the absence of CDTa, two novel di-heptamer structures for activated CDTb (aCDTb; 1.0 MDa) were solved at atomic resolution including a symmetric (SymCDTb; 3.14 [A]) and an asymmetric form (AsymCDTb; 2.84 [A]). Roles played by two receptor-binding domains of aCDTb were of particular interest since RBD1 lacks sequence homology to any other known toxin, and the RBD2 domain is completely absent in other well-studied heptameric toxins (i.e. anthrax). For AsymCDTb, a novel Ca2+ binding site was discovered in RBD1 that is important for its stability, and RBD2 was found to be critical for host cell toxicity and the novel di-heptamer fold for both forms of aCDTb. Together, these studies represent a starting point for structure-based drug-discovery strategies to targeting CDT in the most severe strains of CDI. SIGNIFICANCE STATEMENTThere is a high burden from C. difficile infection (CDI) throughout the world, and the Center for Disease Control (CDC) reports more than 500,000 cases annually in the United States, resulting in an estimated 15,000 deaths. In addition to the large clostridial toxins, TcdA/TcdB, a third C. difficile binary toxin (CDT) is associated with the most serious outbreaks of drug resistant CDI in the 21st century. Here, structural biology and biophysical approaches were used to characterize the cell binding component of CDT, termed CDTb, at atomic resolution. Surprisingly, two novel structures were solved from a single sample that help to explain the molecular underpinnings of C. difficile toxicity. These structures will also be important for targeting this human pathogen via structure-based therapeutic design methods.

biophysics↗

bioRxiv2019

Deficiency in Bhlhe40 impairs resistance to H. polygyrus bakeri and reveals novel Csf2rb-dependent regulation of anti-helminth immunity

The cytokines GM-CSF and IL-5 are thought to possess largely divergent functions despite a shared dependence on the common beta ({beta}C) chain to initiate signaling. Although IL-5 is part of the core type 2 cytokine signature and is required for protection against some helminths, it is dispensable for immunity to others, such as Heligmosomoides polygyrus bakeri (H. polygyrus). Whether this is due to compensatory mechanisms is unclear. The transcription factor Bhlhe40 has been shown to control GM-CSF production and is proposed to be a novel regulator of T helper type 2 cells. We have found that Bhlhe40 is required in T cells for a protective memory response to secondary H. polygyrus infection. H. polygyrus rechallenge elicited dramatic Bhlhe40-dependent changes in gene and cytokine expression by lamina propria CD4+ T cells and in vitro-polarized TH2 cells, including induction of GM-CSF and maximal production of type 2 cytokines including IL-5. {beta}C chain-deficient, but not GM-CSF-deficient, mice rechallenged with H. polygyrus had severely impaired protective immunity. Our results demonstrate that Bhlhe40 is an essential regulator of TH2 cell immunity during helminth infection and reveal unexpected redundancy of {beta}C chain-dependent cytokines.

immunology↗