https://hdl.handle.net/10481/14660 2026-04-11T22:32:51Z https://hdl.handle.net/10481/109761 Identification of polyketide inhibitors targeting 3-dehydroquinate dehydratase in the shikimate pathway of Enterococcus faecalis Cheung, Vivian W.N.; Xue, Bo; Hernandez-Valladares, Maria; Go, Maybelle K.; Tung, Alvin; Aguda, Adeleke H.; Robinson, Robert C.; Yew, Wen S. Due to the emergence of resistance toward current antibiotics, there is a pressing need to develop the next generation of antibiotics as therapeutics against infectious and opportunistic diseases of microbial origins. The shikimate pathway is exclusive to microbes, plants and fungi, and hence is an attractive and logical target for development of antimicrobial therapeutics. The Gram-positive commensal microbe, Enterococcus faecalis, is a major human pathogen associated with nosocomial infections and resistance to vancomycin, the "drug of last resort". Here, we report the identification of several polyketide-based inhibitors against the E. faecalis shikimate pathway enzyme, 3-dehydroquinate dehydratase (DHQase). In particular, marein, a flavonoid polyketide, both inhibited DHQase and retarded the growth of Enterococcus faecalis. The purification, crystallization and structural resolution of recombinant DHQase from E. faecalis (at 2.2 Å resolution) are also reported. This study provides a route in the development of polyketide-based antimicrobial inhibitors targeting the shikimate pathway of the human pathogen E. faecalis. This research was supported by grants from the Ministry of Education, the National Medical Research Council and the National Research Foundation of Singapore to W.S.Y, and grants from the Biomedical Research Council of A*STAR to R.C.R. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. https://hdl.handle.net/10481/109760 The role of palmitoylation in regulating Ras localization and function Eisenberg, Sharon; Laude, Alex J.; Beckett, Alison J.; Mageean, Craig J.; Aran, Verónica; Hernández Valladares, María del Carmen; Henis, Yoav I.; Prior, Ian A. Ras GTPases are important regulators of pathways controlling proliferation, differentiation and transformation. Three ubiquitously expressed almost identical Ras genes are not functionally redundant; this has been attributed to their distinctive trafficking and localization profiles. A palmitoylation cycle controls the correct compartmentalization of H-Ras and N-Ras. We review recent data that reveal how this cycle can be regulated by membrane organization to influence the spatiotemporal signalling of Ras. We gratefully acknowledge funding from the Royal Society, North West Cancer Research Fund, the Wellcome Trust (to I.A.P.) and from the DGF-DIP [grant numbers KL 1948/1-1 and GA 309/10-1] and the Ministry of Science & Technology, Israel (to Y.I.H.). Y.I.H. is an incumbent of the Zalman Weinberg Chair in Cell Biology. https://hdl.handle.net/10481/109759 Actin Polymerization Dynamics - Insights from In vitro TIRF Microscopy Kannan, Balakrishnan; Larsson, Marten; Lee, Wei Lin; Hernandez-Valladares, Maria; Robinson, Robert C Actin elongation is a bi-molecular reaction between monomeric actin (G-actin) and filamentous actin (F-actin), in the first approximation. It can be controlled by changing the ability of either G-actin or F-actin to participate in the reaction. Either of the two mechanisms alone is not sufficient to maintain a large pool of G-actin ready to polymerize in a signal-controlled fashion [1]. Mammalian cells have hundreds of actin-binding proteins (ABP) which bind either or both the forms of actin. Profilin sequesters G-actin and makes them pre-disposed towards F-actin barbed-end addition, cofilin severs F-actin and deploymerizes it into G-actin. On the other hand, capping protein (CP) caps the barbed-end and stops further elongation of F-actin [2]. Gelsolin-family of proteins [3] sever F-actin as well as cap filaments. In vitro TIRF microscopy [4] has been used to monitor real-time actin dynamics in the presence of ABPs [5], [6]. Representative results on some ABPs which alter actin assembly will be presented. https://hdl.handle.net/10481/109758 Quantitative proteomic analysis of compartmentalized signaling networks Hernández-Valladares, María; Aran, Verónica; Prior, Ian A. Ras proteins operate predominantly from the plasma membrane; however, they have also been localized to most intracellular compartments. Various functions and signaling outputs have been ascribed to endomembranous Ras although systematic comparison and measurement of potential outputs have not yet been carried out. We describe the methodology for isolating and measuring compartment-specific signaling networks using quantitative proteomics. This approach reveals the potential of a subcellular platform for supporting specific outputs and will inform subsequent studies of endogenous isoform-specific Ras signaling. The authors thank J. R. Wis´niewski, G. Palmisano, and T. Geiger for sharing further details of their phosphoproteomic protocols. Our research is funded by North West Cancer Research, the BBSRC (BB/G018162), and the Wellcome Trust (WT085201). https://hdl.handle.net/10481/109757 Host susceptibility to malaria in human and mice: compatible approaches to identify potential resistant genes Hernández-Valladares, María; Rihet, Pascal; Iraqi, Fuad A. There is growing evidence for human genetic factors controlling the outcome of malaria infection, while molecular basis of this genetic control is still poorly understood. Case-control and family-based studies have been carried out to identify genes underlying host susceptibility to malarial infection. Parasitemia and mild malaria have been genetically linked to human chromosomes 5q31-q33 and 6p21.3, and several immune genes located within those regions have been associated with malaria-related phenotypes. Association and linkage studies of resistance to malaria are not easy to carry out in human populations, because of the difficulty in surveying a significant number of families. Murine models have proven to be an excellent genetic tool for studying host response to malaria; their use allowed mapping 14 resistance loci, eight of them controlling parasitic levels and six controlling cerebral malaria. Once quantitative trait loci or genes have been identified, the human ortholog may then be identified. Comparative mapping studies showed that a couple of human and mouse might share similar genetically controlled mechanisms of resistance. In this way, char8, which controls parasitemia, was mapped on chromosome 11; char8 corresponds to human chromosome 5q31-q33 and contains immune genes, such as Il3, Il4, Il5, Il12b, Il13, Irf1, and Csf2. Nevertheless, part of the genetic factors controlling malaria traits might differ in both hosts because of specific host-pathogen interactions. Finally, novel genetic tools including animal models were recently developed and will offer new opportunities for identifying genetic factors underlying host phenotypic response to malaria, which will help in better therapeutic strategies including vaccine and drug development.