Vaccination of at least a quarter of the school population resulted in decreased influenza rates and improved school attendance. Herd immunity for unvaccinated children may occur in schools with vaccination coverage approaching 50%.

f Anaplasma phagocytophilum is a tick-borne rickettsial pathogen that provokes an acute inflammatory response during mammalian infection. The illness caused by A. phagocytophilum, human granulocytic anaplasmosis, occurs irrespective of pathogen load and results instead from host-derived immunopathology. Thus, characterizing A. phagocytophilum genes that affect the inflammatory process is critical for understanding disease etiology. By using an A. phagocytophilum Himar1 transposon mutant library, we showed that a single transposon insertion into the A. phagocytophilum dihydrolipoamide dehydrogenase 1 gene (lpda1 [APH_0065]) affects inflammation during infection. A. phagocytophilum lacking lpda1 revealed enlargement of the spleen, increased splenic extramedullary hematopoiesis, and altered clinicopathological abnormalities during mammalian colonization. Furthermore, LPDA1-derived immunopathology was independent of neutrophil infection and correlated with enhanced reactive oxygen species from NADPH oxidase and nuclear factor (NF)-B signaling in macrophages. Taken together, these findings suggest the presence of different signaling pathways in neutrophils and macrophages during A. phagocytophilum invasion and highlight the importance of LPDA1 as an immunopathological molecule.

SUMOylation plays important roles in many key physiological and pathological processes. The SUMOylation cascade involves a heterodimer of activating enzyme, E1 (Aos1/Uba2); a conjugating enzyme, E2 (Ubc9); and many ligase enzymes, E3. Focusing on the activation step of the SUMOylation process, we examined the interaction of E1 with its substrates. Previous studies reported the Km of E1 enzymes in ubiquitin and other ubiquitin-like pathways, but the Km of the SUMO paralogs (SUMO2 and SUMO3) is unknown. Here, by using quantitative FRET to measure the SUMO E1 enzyme kinetics of SUMO1, 2, and 3 and ATP under steady state conditions, we found that the enzyme kinetics from the quantitative FRET method are comparable to those from conventional radioactive assays. Additionally, the kinetic constants, Km , of SUMO2 (3.418 ± 0.9131 μM) and SUMO3 (2.764 ± 0.75 μM) [FW1] are approximately four to five times higher than that of SUMO1 Km (0.7458 ± 0.1105 μM). These results demonstrate the advantages of FRET technology for determining Km , including the ability to monitor reaction progress in real-time with high-throughput and high-sensitivity in an environmentally friendly manner. The processes discussed here extend the utility of quantitative FRET in characterizing protein-protein interactions and enzyme kinetics.

The ubiquitin—proteasome system and ubiquitin-like protein pathways, such as SUMOylation, are critical in protein homeostasis and activities in vivo and are emerging as a new strategy to treat many acute and chronic human diseases, such as cancers. Although various kinase inhibitors have been developed as target-based therapy, solid tumors are still challenges in clinical therapy because various resistant are developed after kinase inhibitor treatments, and therapeutic agents with novel mechanisms are urgently needed. SUMO has been shown to modify various critical proteins, such as p53, MDM2, Estrogen receptor and androgen receptors. More recently, a genome-wide siRNA screening shown that inhibition of SUMO E1 ligases can lead to synergistically lethality of c-Myc overexpressed breast cancer cells. However, so far, specific inhibitor of SUMOylation is still not available for the scientific or pharmaceutical communities. We developed a novel quantitative Förster resonance energy transfer (FRET) technology platform for both basic kinetics parameter determinations and high-through screening (HTS) assays for SUMOylation cascade. The novel theoretical and experimental procedures for protein interactions affinity (Kd) determinations in the SUMOylation cascade, including the interaction between SUMO1 and its E2 ligase, Ubc9, E1 heterodimers(Aos1 and Uba2), E1 and E2 interactions(Uba2 and Ubc9), and E2 and substrate interactions(Ubc9 and RanGap1c) and protease kinetics, Kcat/KM of SENP1 endopeptidase activity have been developed in a systems biology manner. The data are in good agreement with traditional methods. Multiple FRET-based HTS assays have also been developed and HTS campaigns have led to very promising hit that can preferentially kill Non-small cell lung cancer cells. The novel SUMOylation inhibitor can be used for cancer treatments by synergistically lethality strategy. Note: This abstract was not presented at the conference. Citation Format: Jiayu Liao, Hilda Wiryawan, Yang Song, Yan Liu, Ling Jiang, Harbani Kaur Malik, Amanda N. Saaredra. Quantitative FRET technology for SUMOylation cascade and high-throughput screening assay for SUMOylation inhibitor in cancer drug discovery. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr A39.

The ubiquitin–proteasome system and ubiquitin-like protein pathways, such as SUMOylation, are critical in protein homeostasis and activities in vivo and are emerging as a new strategy to treat many acute and chronic human diseases, such as cancers. Although various kinase inhibitors have been developed as target-based therapy, solid tumors are still challenges in clinical therapy because various resistant are developed after kinase inhibitor treatments, and therapeutic agents with novel mechanisms are urgently needed. SUMO has been shown to modify various critical proteins, such as p53, MDM2, Estrogen receptor and androgen receptors. More recently, a genome-wide siRNA screening shown that inhibition of SUMO E1 ligases can lead to synergistically lethality of c-Myc overexpressed breast cancer cells. However, so far, specific inhibitor of SUMOylation is still not available for the community. Fig. 1. SUMOylation in human diseases and quantitative systems biology approach for basic and translational research of SUMOylation. We developed a novel quantitative Förster resonance energy transfer (FRET) technology platform for both basic kinetics parameter determinations and high-through screening(HTS) assays for SUMOylation cascade. The novel theoretical and experimental procedures for protein interactions affinity(Kd) determinations in the SUMOylation cascade, including the interaction between SUMO1 and its E2 ligase, Ubc9, E1 heterodimers(Aos1 and Uba2), E1 and E2 interactions(Uba2 and Ubc9), and E2 and substrate interactions(Ubc9 and RanGap1c) and protease kinetics, Kcat/KM of SENP1 endopeptidase activity have been developed in a systems biology manner. The data are in good agreement with traditional methods. Multiple FRET-based HTS assays have also been developed and HTS campaigns have led to very promising hit that can preferentially kill Non-small cell lung cancer cells. The novel SUMOylation inhibitor can be used for cancer treatments by synergistically lethality strategy. Citation Format: Jiayu Liao, Hilda Wiryawan, Yang Li, Yang Song, Yan Liu, Jiacong You, Ling Jiang, Harbani Kaur Malik, Amanda N. Saavedra, Sophie Qu. Quantitative FRET technology for SUMOylation cascade and high-throughput screening assay for SUMOylation inhibitor in cancer drug discovery. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Susceptibility and Cancer Susceptibility Syndromes; Jan 29-Feb 1, 2014; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(23 Suppl):Abstract nr 21. doi:10.1158/1538-7445.CANSUSC14-21