Btla signaling in conventional and regulatory lymphocytes coordinately tempers humoral immunity in the intestinal mucosa

The Btla inhibitory receptor limits innate and adaptive immune responses, both preventing the development of autoimmune disease and restraining anti-viral and anti-tumor responses. It remains unclear how the functions of Btla in diverse lymphocytes contribute to immunoregulation. Here, we show that Btla inhibits activation of genes regulating metabolism and cytokine signaling, including Il6 and Hif1a, indicating a regulatory role in humoral immunity. Within mucosal Peyer\u27s patches, we find T-cell-expressed Btla-regulated Tfh cells, while Btla in T or B cells regulates GC B cell numbers. Treg-expressed Btla is required for cell-intrinsic Treg homeostasis that subsequently controls GC B cells. Loss of Btla in lymphocytes results in increased IgA bound to intestinal bacteria, correlating with altered microbial homeostasis and elevations in commensal and pathogenic bacteria. Together our studies provide important insights into how Btla functions as a checkpoint in diverse conventional and regulatory lymphocyte subsets to influence systemic immune responses

Mode of action of DNA-competitive small molecule inhibitors of tyrosyl DNA phosphodiesterase 2

TDP2 is a 5’-tyrosyl DNA phosphodiesterase important for the repair of DNA adducts generated by non-productive (abortive) activity of topoisomerase II. TDP2 facilitates therapeutic resistance to topoisomerase poisons, which are widely used in the treatment of a range of cancer types. Consequently, TDP2 is an interesting target for the development of small molecule inhibitors that could restore sensitivity to topoisomerase-directed therapies. Previous studies identified a class of deazaflavin-based molecules that showed inhibitory activity against TDP2 at therapeutically useful concentrations, but their mode of action was uncertain. We have confirmed that the deazaflavin series inhibits TDP2 enzyme activity in a fluorescence-based assay, suitable for HTS-screening. We have gone on to determine crystal structures of these compounds bound to a ‘humanised’ form of murine TDP2. The structures reveal their novel mode of action as competitive ligands for the binding site of an incoming DNA substrate, and point the way to generating novel and potent inhibitors of TDP2

Engineered Picornavirus VPg-RNA Substrates: Analysis of a Tyrosyl-RNA Phosphodiesterase Activity

Using poliovirus, the prototypic member of Picornaviridae, we have further characterized a host cell enzymatic activity found in uninfected cells, termed “unlinkase,” that recognizes and cleaves the unique 5′ tyrosyl-RNA phosphodiester bond found at the 5′ end of picornavirus virion RNAs. This bond connects VPg, a viral-encoded protein primer essential for RNA replication, to the viral RNA; it is cleaved from virion RNA prior to its engaging in protein synthesis as mRNA. Due to VPg retention on nascent RNA strands and replication templates, but not on viral mRNA, we hypothesize that picornaviruses utilize unlinkase activity as a means of controlling the ratio of viral RNAs that are translated versus those that either serve as RNA replication templates or are encapsidated. To test our hypothesis and further characterize this enzyme, we have developed a novel assay to detect unlinkase activity. We demonstrate that unlinkase activity can be detected using this assay, that this unique activity remains unchanged over the course of a poliovirus infection in HeLa cells, and that unlinkase activity is unaffected by the presence of exogenous VPg or anti-VPg antibodies. Furthermore, we have determined that unlinkase recognizes and cleaves a human rhinovirus-poliovirus chimeric substrate with the same efficiency as the poliovirus substrate

Tandem mass tag-based quantitative proteomic profiling identifies candidate serum biomarkers of drug-induced liver injury in humans

Diagnosis of drug-induced liver injury (DILI) and its distinction from other liver diseases are significant challenges in drug development and clinical practice. We used Tandem Mass Tag-labeled quantitative proteomics detecting 2323 proteins in a cohort comprising patients with DILI [at onset (DO) and follow-up (DF)], acute non-DILI [at onset (NDO) and follow-up (NDF)], and healthy volunteers (HV) to identify novel serum biomarkers. Thirteen candidates selected based on differential expression, liver-specific expression, and mechanistic relevance to liver pathology, were assessed in confirmatory and replication cohorts of HV (n=94), DO (n=123), DF (n=110), NDO (n=58) and NDF (n=37) using a targeted label-free SureQuant assay. Area under the receiver operating characteristic curve (AUC) ranging between 0.94 and 0.99 across cohorts for five of these biomarkers, reflected differentiation between DO and HV with high sensitivity and specificity. In addition, fructose-1,6-bisphosphatase 1 distinguished NDO from DO (AUC: 0.75 and 0.65) on its own or in combination with glutathione S-transferase A1 and leukocyte cell derived chemotaxin 2 (AUC: 0.78 and 0.68). These can potentially differentiate DILI and acute liver injury from non-drug etiologies

Engineered Picornavirus VPg-RNA Substrates: Analysis of a Tyrosyl-RNA Phosphodiesterase Activity

Using poliovirus, the prototypic member of Picornaviridae, we have further characterized a host cell enzymatic activity found in uninfected cells, termed “unlinkase,” that recognizes and cleaves the unique 5′ tyrosyl-RNA phosphodiester bond found at the 5′ end of picornavirus virion RNAs. This bond connects VPg, a viral-encoded protein primer essential for RNA replication, to the viral RNA; it is cleaved from virion RNA prior to its engaging in protein synthesis as mRNA. Due to VPg retention on nascent RNA strands and replication templates, but not on viral mRNA, we hypothesize that picornaviruses utilize unlinkase activity as a means of controlling the ratio of viral RNAs that are translated versus those that either serve as RNA replication templates or are encapsidated. To test our hypothesis and further characterize this enzyme, we have developed a novel assay to detect unlinkase activity. We demonstrate that unlinkase activity can be detected using this assay, that this unique activity remains unchanged over the course of a poliovirus infection in HeLa cells, and that unlinkase activity is unaffected by the presence of exogenous VPg or anti-VPg antibodies. Furthermore, we have determined that unlinkase recognizes and cleaves a human rhinovirus-poliovirus chimeric substrate with the same efficiency as the poliovirus substrate

Unlinkase activity in different cytoplasmic extracts from human cell lines.

<p>Increasing amounts of total protein (0.1 µg/µl, lanes 1, 4, 7, 10, and 13; 0.2 µg/µl, lanes 2, 5, 8, 11, and 14; 0.4 µg/µl, lanes 3, 6, 9, 12, and 15) from cytoplasmic extracts from different cell lines were incubated with ³⁵S-methionine radiolabeled W1-VPg 31 virion RNA substrate. HeLa (lanes 1–3), SK-OV-3 (lanes 4–6), NGP (lanes 7–9), and K562 (lanes 13–15) are all human cell lines and have differing levels of unlinkase activity; RRL (rabbit reticulocyte lysate) (lanes 10–12), contrary to previous publications, did not exhibit unlinkase activity in this assay (lanes 10–12).</p

Unlinkase activity remains unchanged during poliovirus infection.

<p>HeLa cells were infected with wild type poliovirus and cytoplasmic extracts were collected at 0 (lanes 3 and 9), 2 (lanes 4 and 10), 4 (lanes 5 and 11), or 6 (lanes 6 and 12) hours post-infection. 1 µg (lanes 1–6) or 5 µg (lanes 7–12) of total protein from these extracts was incubated with full-length ³⁵S-methionine radiolabeled W1-VPg 31 virion RNA substrate. No difference in activity was observed between the mock lanes (lanes 1, 2, 7, and 8) and the different time points collected during the infection.</p

Unlinkase activity on a poliovirus versus a rhinovirus substrate.

<p>(A) VPg sequences of HRV14 and S1-VPgR1 (wild type poliovirus mutated to encode an HRV14 VPg that encodes two methionine residues). (B) Single-cycle growth assay of wild type poliovirus (closed circles) and S1-VPgR1 (closed squares). ³⁵S-methionine radiolabeled W1-VPg 31 or S1-VPgR1 substrate that was treated with RNase T1 (C) or untreated (D) was incubated with increasing amounts of total protein from a partially-purified fraction of unlinkase activity (Fraction CA) ((C) 0, 5, 10, and 20 µg and (D) 0, 0.5, 1, 2, 5 and 10 µg) to demonstrate that unlinkase activity recognizes each substrate equally.</p

Optimal assay conditions for detecting unlinkase activity using full-length poliovirus virion RNA ³⁵S-methionine labeled substrate.

<p>A protein chromatography fraction enriched for unlinkase activity was incubated with full-length ³⁵S-methionine radiolabeled W1-VPg 31 virion RNA substrate with either (A) increasing amounts of protein (0.01, 0.02, 0.04, 0.1, 0.2, 0.4, and 0.6 µg/µl) from an enriched source of unlinkase activity (Fraction SA) at 30°C for 30 minutes or (B) increasing incubation time (0, 1, 2, 5, 10, 15, 20, and 30 minutes) with 0.4 µg/µl of protein from a partially-purified fraction of unlinkase activity (Fraction SA) to determine optimal assay conditions for the full-length substrate. (C) ³⁵S-methionine radiolabeled W1-VPg 31 virion RNA substrate was mock-incubated (lane 1), incubated with 0.8 µg/µl RSW (lane 2), 0.4 µg/µl of protein from a partially-purified fraction of unlinkase activity (Fraction SA) (lane 3), or one unit of RNase A (lane 4) to differentiate between non-specific nuclease activity and authentic unlinkase activity.</p

VPg-RNA covalent linkage and VPg sequences of wild type and mutated W1-VPg31 polioviruses.

<p>Schematic of the VPg-RNA covalent linkage between the single tyrosine residue in poliovirus VPg and the first uridine at the 5′ terminus of the genomic RNA (A). Image was generated using ChemDraw software. The arrow indicates where unlinkase cleaves the bond. (B) VPg sequences of wild type poliovirus and W1-VPg31 (wild type poliovirus mutated to encode two methionines in the VPg coding region).</p