Parasitic helminths and helminth-derived molecules have demonstrated to possess powerful anti-inflammatory properties and confirmed therapeutic effects on inflammatory diseases. The helminth Fasciola hepatica has been reported to suppress specific Th1 specific immune responses induced by concurrent bacterial infections, thus demonstrating its anti-inflammatory ability in vivo. In this study, we demonstrate that native F. hepatica glutathione S-transferase (nFhGST), a major parasite excretory-secretory antigen, majorly comprised of Mu-class GST isoforms, significantly suppresses the LPS-induced TNFα and IL1β of mouse bone-marrow derived macrophages in vitro and the pro-inflammatory cytokine/chemokine storm within C57BL/6 mice exposed to lethal doses of LPS increasing their survival rate by more than 85%. Using THP1-Blue CD14 cells, a human monocyte cell line, we also demonstrate that nFhGST suppresses NF-κB activation in response to multiple TLR-ligands, including whole bacteria clinical isolates and this suppression was found to be dose-dependent and independent of the timing of exposure. Moreover, the suppressive effect of nFhGST on NF-κB activation was shown to be independent of enzyme activity or secondary structure of protein. As part of its anti-inflammatory effect nFhGST target multiple proteins of the canonic and non-canonic NF-κB signaling pathway as well as also JAK/STAT pathway. Overall, our results demonstrate the potent anti-inflammatory properties of nFhGST and its therapeutic potential as an anti-inflammatory agent.

High-throughput thermal screening (HTTS) has generally been used to analyze the stability of libraries of mutant proteins, as well as stability changes for 388a

Protein expression functions as a biomarker for precision drug therapy to more effectively treat cancer or as a measurement of drug resistance. Expression levels are commonly determined in bulk populations of cells at single time points. However, cells often undergo dynamic expression during therapeutic response that may influence drug resistance. Therefore, how changing expression levels dynamically impact cellular response remains largely unknown, limiting determination of bona fide protein biomarkers while leaving mechanisms of therapeutic resistance underappreciated. Drugs inhibiting the DNA repair protein poly(ADP-ribose) polymerase (PARP) have been increasingly used to treat various cancers. PARP inhibitors (PARPi) are especially potent in cancers exhibiting deficient homologous recombination, or BRCAness. Yet, acquired PARPi resistance is common and remains a major area of concern, underscoring the importance of discovering how to overcome resistance mechanisms. To determine the impact of dynamic protein expression on PARPi resistance, we have developed a platform approach that dynamically tracks specific protein expression during drug treatment in single cells. We used CAS9-directed plasmid insertion to precisely couple fluorescent protein expression with translation of PARP1 and, combined with longitudinal (days) single cell tracking, correlated PARP1 expression levels with cellular fate during drug exposure. Remarkably, we found that dynamic expression of PARP1 controls cellular fate - division, death, or senescence. PARPi were effective in breast cancer cells when PARP1 expression remained steady. However, cells with similar initial PARP1 expression that significantly increased expression proved resistant. Furthermore, while all PARPi enzymatically inhibit the target protein, the 5 approved drugs display a differential ability to trap PARP1 onto damaged DNA, producing increased potency. We found dynamic PARP1 expression impacted PARPi resistance differently based on drug trapping ability, suggesting a mechanism to select the most effective PARPi for each patient. Guided by a CRISPR knockout screen, we then determined dynamic expression of other important proteins in PARPi response, finding dynamic expression influence in cellular resistance. These results highlight the role of dynamic DNA damage repair in response to PARPi and present potential combination therapy targets. Complementary to dynamic transcriptional profiling of cell lines under drug treatment, our approach displays significant resistance dependence on dynamic protein expression, but provides increased sensitivity through tracking single cells. Overall, our novel approach to measure dynamic protein expression levels highlights previously unknown avenues of cellular resistance. Citation Format: Bianca Fernandez, J Matthew Dubach. Dynamic protein expression governs cellular PARP inhibitor response and represents a pathway to single cell drug resistance [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 619.

the correct functioning and identity of organelles, being thus essential for cell signaling and survival. Here, we describe a eukaryotic mechanism of lipid saturation sensing. We show that Mga2, a transcription factor conserved among fungi, operates as a sensor for lipid packing in the ER membrane and can control the production of unsaturated fatty acids. We employ extensive in vivo mutagenesis, molecular dynamics simulations, and electron paramagnetic resonance spectroscopy to show that the membrane environment stabilizes different configurations of dimers of Mga2 transmembrane helices. Such configurations are defined by alternative relative rotations of the helices. In this way, altered membrane properties can trigger the proteolytic activation of Mga2. This work establishes a novel eukaryotic strategy of lipid saturation sensing that differs significantly from the analogous bacterial mechanism of sensing the hydrophobic thickness of the membrane. 2506-Pos Board B113. RND-type multidrug efflux exporter AcrAB-TolC tripartite complex is a major drug exporter in Gram-negative bacteria. The crystal structures of each component have been determined and recently their tripartite structures were also visualized by single-particle cryo-electron microscopy. In this way tripartite complex formation is gradually being unraveled from the structural point of view. However, their dynamic property on cell membrane is not yet been well-investigated. Here we analyzed diffusion of AcrB on the plasma membrane of living E. coli cells to assess the possibility of the substratedependent mode change from 'free diffusion for searching substrate' to 'restricted diffusion by forming tripartite for substrate efflux'. Diffusion was measured under the microscope by FDAP (fluorescence decay after photoconversion) analysis using PA-GFP (photoactivatable-GFP)-labeled AcrB. In order to facilitate observation of fluorescence diffusion in the intracellular space, cells were extended by treatment of ceftazidime. Diffusion of AcrB was slowed when it was expressed in the acrB-deficient cells than in the acrB/tolC-deficient cells. When different substrates were added to the acrBdeficient cells, diffusion of AcrB differed depending on the type of the substrates. In the case where small size substrates that are bound at the distal pocket in the crystal structures were added, the diffusion speed was similar to that in the absence of substrate. On the other hand, diffusion is decelerated when large size substrates that bound at the proximal pocket is added. Introduction of mutations into AcrB to disturb transport of proximal binding substrates eliminated this substrate depended deceleration. These results suggest that association and dissociation of AcrAB-TolC tripartite complex is in dynamic equilibrium even if there is no substrate and the complex formation is more stabilized when proximal binding larger substrates are added. Residence time of substrates in the proximal pocket might have correlation with life time of the AcrAB-TolC complex. uez, Puert...

Fasciola hepatica is a parasite well known as a master of immunomodulation because of its ability to suppress the Th1 immune responses and elicit a strong Th2/Treg response. This occurs when the parasite releases excretory‐secretory products (ESPs), which are comprised of a myriad of glycoproteins that are believed to play an important role in the immune response. F. hepatica Glutathione S‐Transferase (FhGST) is one of the ESP components recognized for its anti‐oxidant properties and its capacity to provide part of the parasite's defense by detoxifying the secondary products of lipid peroxidation that are produced due to the immune free‐radical attack on the host or the parasite membrane. Our preliminary studies demonstrate that FhGST exerts a suppressive effect on the activation of TLR4 in macrophages which is evidenced by significant suppression of NF‐kB activation and down‐regulation of TNFα and IL1‐β in response to LPS‐stimuli. We speculated that the integrity of the tertiary structure, as well as the glycans that decorate the protein moiety of FhGST, could be essential for its capacity to suppress the NF‐kB activation. To test our hypothesis, we first subjected FhGST to a thermal denaturing process by boiling it at 90°C for 10 min followed by a treatment with PNGase F. Circular dichroism analysis (CD) comparing the FhGST spectra before and after heat denaturing served to confirm that FhGST was full and irreversibly denatured. Next, denatured or native FhGST were added alone or in the presence of the whole extract of 1 x 108 cells Klebsiella pneumonia (Kp) to a culture of human monocytes (THP1‐CD14) that express most TLRs as well as an NF‐kb inducible secreted embryonic alkaline phosphatase reporter gene. Cells stimulated with an attenuated whole extract of 1 × 108 cells Klebsiella pneumonia (Kp) were used as activation control. Our results demonstrated that native FhGST suppresses NF‐kB activation by 83%, whereas the heat‐denatured protein suppresses NF‐kB activation by 84%. These suppressions suggest that in contrast to expected, the capacity of FhGST to suppress the NF‐κB activation in THP1‐CD14 cells is independent of the integrity of its secondary/tertiary structure and is not associated to the glycans. Docking analysis in which the FhGST‐protein moiety was docked with the CD14 and MD2 co‐receptor structures suggested that FhGST could target these co‐receptors as part of its mechanisms of action.Support or Funding InformationThis study was supported by MBRS‐RISE R25GM061838‐13, 5R25GM061151‐16 and NIH grants G12MD007600 and 2P40OD012217This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.