The RAG1 and RAG2 proteins are required to assemble mature Ag receptor genes in developing lymphocytes. Hypomorphic mutations in the gene encoding RAG1 are associated with Omenn syndrome, a primary immunodeficiency. We explored the biochemical defects resulting from a mutation identified in an Omenn syndrome patient which generates an amino acid substitution in the RAG1 RING finger/ubiquitin ligase domain (C325Y in murine RAG1) as well as an adjacent substitution (P326G). RAG1 C325Y demonstrated a 50-fold reduction in recombination activity in cultured pro-B cells despite the fact that its expression and localization to the nucleus were similar to the wild-type protein. The C325Y substitution severely abrogated ubiquitin ligase activity of the purified RAG1 RING finger domain, and the tertiary structure of the domain was altered. The P326G substitution also abrogated ubiquitin ligase activity but had a less severe effect on protein folding. RAG1 P326G also demonstrated a recombination impairment that was most pronounced when RAG1 levels were limiting. Thus, a correctly folded RAG1 RING finger domain is required for normal V(D)J recombination, and RAG1 ubiquitin ligase activity can contribute when the protein is present at relatively low levels.

Histone variant H3.3 is associated with transcriptionally active chromatin and accumulates at loci undergoing preparation for V(D)J recombination, a DNA rearrangement required for the assembly of antigen receptors and development of B and T lymphocytes. Here we demonstrate that the RAG1 V(D)J recombinase protein promotes ubiquitylation of H3.3 that has been heavily acetylated and phosphorylated on serine 31 (acetyl-H3.3 S31p). A fragment of RAG1 promoted formation of a mono-ubiquitylated H3 product that was identified using mass spectrometry as ubiquitylated acetyl-H3.3 S31p. H3 was ubiquitylated at multiple lysine residues, and correspondingly, di-, tri- and higher-order ubiquitylated products were detected at low levels. Ubiquitylation was dependent on an intact RAG1 RING finger/ubiquitin ligase domain and required additional regions of the RAG1 amino terminus that are likely to interact with H3. Acetylated residues within the H3 amino terminal tail were also required. Purified, recombinant H3.1 and H3.3 were not good substrates, suggesting that post-translational modifications enhance recognition by RAG1. A complex including damage-DNA binding protein has also been shown to ubiquitylate H3 in response to UV treatment, suggesting the H3 ubiquitylation may be a common step in multiple DNA repair pathways.

Summary The really interesting new gene (RING) finger ubiquitin ligase domain of the recombinase activating gene 1 (RAG1) V(D)J recombinase protein adopts a standard cross‐brace architecture but co‐ordinates three zinc ions as opposed to the canonical two. We demonstrated previously that disruption of the conserved zinc co‐ordination sites resulted in loss of structural integrity and ubiquitin ligase (E3) activity and interfered with the ability of full‐length RAG1 to support recombination. Here we present evidence that amino acids surrounding the third, non‐canonical site also contribute to functional interaction with the ubiquitin conjugating (E2) enzyme CDC34, while certain residues on the RING domain’s surface important for interaction between other E2–E3 pairs are less critical to the functional RAG1–CDC34 interaction in this assay. Partial reduction of ubiquitin ligase activity was significantly correlated with reduction in the ability of RAG1 to support recombination of extra‐chromosomal substrates (r = 0·805, P = 0·009). While poly‐ubiquitin chains could be generated, RAG1 did not promote rapid chain extension following mono‐ubiquitylation of substrate, regardless of the E2 enzyme used. No single ubiquitin lysine mutant disrupted the ability of CDC34 to form ubiquitin chains on RAG1, and mass spectrometric analysis of the poly‐ubiquitylated products indicated ubiquitin chain linkages through lysines 48 and 11. These data suggest that RAG1 promotes a mono‐ubiquitylation reaction that is required for optimal levels of V(D)J recombination.

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The prevalence of drug-resistant pathogenic fungi is a major global health challenge. There is an urgent need for novel drugs that can exert a potent antifungal activity and overcome resistance. Newly discovered anti-fungal properties of existing compounds can potentially offer a rapid solution to address this persistent threat. We rationalized that structures which disrupt the fungal cell membrane could address the above unmet need. As fatty acids underpin the formation and stability of cell membranes, we used computational simulations to evaluate the interactions between selected short chain fatty acids and a model cell membrane. Here, we report that caprylic acid could penetrate and perturb the membrane in silico. Based on the in silico findings, we identified a derivative of this fatty acid that disrupts fungal membranes as detected using steady-state fluorescence anisotropy. We show that this fatty acid derivative is potent against a variety of fungal pathogens like Candida and Trichophyton. We further demonstrated the ability of this fatty acid derivative to potentiate some azoles in vitro and enhance the efficacy of antifungal formulations in vivo. Our data suggests the emergence of a novel therapy for effective disease management and overcoming anti-fungal drug resistance.