The RAG1 Ubiquitin Ligase Domain Stimulates Recombination of TCRß and TCRα Genes and Influences Development of αß T Cell Lineages.

RAG1/RAG2 (RAG) endonuclease-mediated assembly of diverse lymphocyte Ag receptor genes by V(D)J recombination is critical for the development and immune function of T and B cells. The RAG1 protein contains a ubiquitin ligase domain that stabilizes RAG1 and stimulates RAG endonuclease activity in vitro. We report in this study that mice with a mutation that inactivates the Rag1 ubiquitin ligase in vitro exhibit decreased rearrangements and altered repertoires of TCRß and TCRα genes in thymocytes and impaired thymocyte developmental transitions that require the assembly and selection of functional TCRß and/or TCRα genes. These Rag1 mutant mice present diminished positive selection and superantigen-mediated negative selection of conventional αß T cells, decreased genesis of invariant NK T lineage αß T cells, and mature CD4+ αß T cells with elevated autoimmune potential. Our findings reveal that the Rag1 ubiquitin ligase domain functions in vivo to stimulate TCRß and TCRα gene recombination and influence differentiation of αß T lineage cells, thereby establishing replete diversity of αß TCRs and populations of αß T cells while restraining generation of potentially autoreactive conventional αß T cells.

Evaluation of therapeutic potential of VB-001, a leave-on formulation, for the treatment of moderate adherent dandruff.

BACKGROUND: Dandruff is a common scalp condition characterized by excessive scaling and itch. Aberrant colonization of the scalp by commensal Malassezia spp. is a major contributor in the multifactorial etiology of dandruff. Literature based understanding of Malassezia linked pathophysiology of dandruff allowed us to comprehend a strategy to potentiate the efficacy of a known antifungal agent used in dandruff therapy. The aim of this study was to determine the efficacy and skin safety of VB-001 antidandruff leave-on formulation in comparison with marketed antidandruff ZPTO shampoo in patients with moderate adherent dandruff of the scalp. METHODS: Healthy males or females aged ≥ 15 years and ≤ 65 with a clinical diagnosis of moderate adherent dandruff of the scalp were recruited for the study to monitor the effects of topical VB-001 versus those of marketed antidandruff ZPTO shampoo. RESULTS: 168 subjects were randomized to the treatment (VB-001, n = 84) and control (ZPTO shampoo, n = 84) groups. The efficacy of each product was evaluated by comparing proportion of subjects who have shown reduction in flaking by ASFS (adherent scalp flaking score) and pruritus by IGA (investigator global assessment) score. VB-001 imparted consistently better reduction in ASFS and enabled early reduction of pruritus in comparison to marketed ZPTO shampoo. CONCLUSION: VB-001, a leave-on formulation with ingredients chosen to selectively disturb the Malassezia niche on dandruff scalp by denying extra nutritional benefits to the microbe, provides unique advantages over existing best in class ZPTO shampoo therapy. It has the potential to emerge as an attractive novel treatment for moderate adherent dandruff. TRIAL REGISTRATION: CTRI Registration number: CTRI/2013/01/003283 . Registered on: 02/01/2013.

Antibiotic-resistant acne: getting under the skin.

Propionibacterium acnes is a key pathogenic factor in the development of acne. Antibiotics are the first choice of treatment for mild-to-moderate, mixed, papular/pustular, and moderate nodular acne, and an alternative choice in severe, nodular/conglobate acne. The emergence of resistance to the currently available antibiotics poses a serious set-back to this algorithm, and the reduced arsenal can diminish efficacy of treatment. This emerging situation should catalyze innovations in dermatology; for example, newer drugs and technologies such as next-generation antibiotics with excellent potency and low propensity to develop resistance, rapid diagnostic platforms to select responders and nonresponders, and delivery technologies that target the bacteria. Such innovations can dramatically expand the arsenal for dermatologists in the management of acne.

Treatment recalcitrant cases of tinea corporis/cruris caused by T. mentagrophytes - interdigitale complex with mutations in ERG11 ERG 3, ERG4, MDR1 MFS genes & SQLE and their potential implications.

BACKGROUND: Recalcitrant dermatophyte infections are being reported from various parts of the world due to varied causes including strain variation, steroid misuse, SQLE mutations, and variable quality of itraconazole pellet formulations. The oral drug preferred in endemic areas is itraconazole, to which MIC levels remain low, and clinical failures to itraconazole reported defy a sound scientific explanation. OBJECTIVES: The objective of the study was to conduct a proteomic and genomic analysis on isolates from therapeutically recalcitrant case with isolation of gene mutations and enzymatic abnormalities to explain azole failures. METHODS: Trichophyton mentagrophyte interdigitale complex strains were isolated from seven clinically non-responding tinea corporis/cruris patients, who had failed a sequential course of 6 weeks of terbinafine 250 mg QD and itraconazole 100 mg BID. After AFST 1 strain, KA01 with high MIC to most drugs was characterized using whole genome sequencing, comparative proteomic profiling, and total sterol quantification. RESULTS: Sterol quantification showed that the standard strain of Trichophyton mentagrophytes (MTCC-7687) had half the ergosterol content than the resistant KA01 strain. Genomic analysis revealed mutations in SQLE, ERG4, ERG11, MDR1, MFS genes, and a novel ERG3 mutation. Proteomic analysis established the aberrant expression of acetyl Co-A transferase in the resistant strain and upregulation of thioredoxin reductase and peroxiredoxin. CONCLUSION: Our findings demonstrate possible reasons for multidrug resistance in the prevalent strain with mutations in genes that predict terbinafine (SQLE) and azole actions (ERG4, ERG11, ERG3) apart from efflux pumps (MDR1, MFS) that can explain multidrug clinical failures.

A potent antibiotic-loaded bone-cement implant against staphylococcal bone infections.

New antibiotics should ideally exhibit activity against drug-resistant bacteria, delay the development of bacterial resistance to them and be suitable for local delivery at desired sites of infection. Here, we report the rational design, via molecular-docking simulations, of a library of 17 candidate antibiotics against bone infection by wild-type and mutated bacterial targets. We screened this library for activity against multidrug-resistant clinical isolates and identified an antibiotic that exhibits potent activity against resistant strains and the formation of biofilms, decreases the chances of bacterial resistance and is compatible with local delivery via a bone-cement matrix. The antibiotic-loaded bone cement exhibited greater efficacy than currently used antibiotic-loaded bone cements against staphylococcal bone infections in rats. Potent and locally delivered antibiotic-eluting polymers may help address antimicrobial resistance.

The RAG1 N-terminal region regulates the efficiency and pathways of synapsis for V(D)J recombination.

Immunoglobulin and T cell receptor gene assembly depends on V(D)J recombination initiated by the RAG1-RAG2 recombinase. The RAG1 N-terminal region (NTR; aa 1-383) has been implicated in regulatory functions whose influence on V(D)J recombination and lymphocyte development in vivo is poorly understood. We generated mice in which RAG1 lacks ubiquitin ligase activity (P326G), the major site of autoubiquitination (K233R), or its first 215 residues (Δ215). While few abnormalities were detected in R1.K233R mice, R1.P326G mice exhibit multiple features indicative of reduced recombination efficiency, including an increased Igκ+:Igλ+ B cell ratio and decreased recombination of Igh, Igκ, Igλ, and Tcrb loci. Previous studies indicate that synapsis of recombining partners during Igh recombination occurs through two pathways: long-range scanning and short-range collision. We find that R1Δ215 mice exhibit reduced short-range Igh and Tcrb D-to-J recombination. Our findings indicate that the RAG1 NTR regulates V(D)J recombination and lymphocyte development by multiple pathways, including control of the balance between short- and long-range recombination.

Mechanistic Insight Into the Antifungal Effects of a Fatty Acid Derivative Against Drug-Resistant Fungal Infections.

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.

Correlation between recombinase activating gene 1 ubiquitin ligase activity and V(D)J recombination.

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.

A Rationally Designed Multifunctional Antibiotic for the Treatment of Drug-Resistant Acne.

Acne is a multifactorial skin disease, underpinned by colonization of Propionibacterium acnes and inflammation. The emergence of resistant P. acnes strains has affected the current acne treatment algorithm. This setback served as an impetus for rationally designing a library of next-generation antibiotics that exhibit a bactericidal effect on resistant P. acnes and exert an immunomodulatory function to reduce inflammation. In silico screening showed that one of the molecules, VCD-004, exhibits improved mode of binding to bacterial DNA gyrase. VCD-004 shows high potency against clinical isolates of resistant P. acnes and excellent efficacy in vivo. Furthermore, VCD-004 exhibits a superior mutant prevention index, suggesting that it impedes the development of resistance better than clindamycin. Additionally, it shows optimal skin penetration and has a potent anti-inflammatory effect via reduction of proinflammatory cytokines (IL-6) independent of its antibacterial action. VCD-004 affects P. acnes-induced nuclear accumulation of NF-κB in THP-1 cells. The in vitro viability of human keratinocytes in the presence of VCD-004 indicates a desirable therapeutic window for topical use. Such rationally designed bactericidal and immunomodulatory dual pharmacophore-based lipophilic molecule(s) can emerge as the next-generation topical therapy for acne with underlying resistant P. acnes etiology.

The RAG1 V(D)J recombinase/ubiquitin ligase promotes ubiquitylation of acetylated, phosphorylated histone 3.3.

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.

Requirement for ubiquitin conjugation and 26S proteasome activity at an early stage in V(D)J recombination.

V(D)J recombination, the process that rearranges gene segments to assemble mature antigen receptor genes, relies on a recombinase comprising the RAG1 and RAG2 proteins. RAG1 is a multi-functional enzyme including DNA binding and cleavage as well as ubiquitin ligase activities, all of which appear to contribute to its role in recombination. Here we demonstrate that components of the ubiquitin conjugation machinery and the 26S proteasome are required for an early step in V(D)J recombination. Inhibitors of the 26S proteasome and ubiquitin activating enzyme (E1) blocked both chromosomal and extra-chromosomal recombination when added 1h following transfection/induction, but they had no effect when added 16 h later. There was no effect on expression of RAG1, and recombination did not require transit through the cell cycle, confirming that inhibition was not due to an indirect effect on cell cycle arrest or protein expression. Experiments in which RAG1 translation was blocked with cyclohexamide after 16 h of expression indicated that many active recombination complexes were formed within this window, although recombination products continued to accumulate for 48 h. These data suggest that ubiquitin-dependent degradation is an early step in complex assembly or activation, and are consistent with our previous hypothesis that degradation of a negative regulator is required to trigger recombination.

Biochemical and folding defects in a RAG1 variant associated with Omenn syndrome.

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.