TY - JOUR AB - Infectious diseases remain a major burden to global health. Despite implementation of successful vaccination campaigns and efficient drugs, the increasing emergence of pathogenic vaccine or treatment resistance demands novel therapeutic strategies. The development of traditional therapies using small molecule drugs is based on modulating protein function and activity through occupation of active sites, such as enzyme inhibition or ligand-receptor binding. These pre-requisites result in the majority of host and pathogenic disease-relevant, non-enzymatic and structural proteins being labelled 'undruggable'. Targeted protein degradation (TPD) emerged as a powerful strategy to eliminate proteins of interest, including those of the 'undruggable' variety. Proteolysis-targeting chimeras (PROTACs) are rationally designed hetero-bifunctional small molecules that exploit the cellular ubiquitin-proteasome system to specifically mediate the highly selective and effective degradation of target proteins. PROTACs have shown remarkable results in the degradation of various cancer-associated proteins and several candidates are already in clinical development. Significantly, PROTAC mediated TPD holds great potential for targeting and modulating pathogenic proteins, especially in the face of increasing drug-resistance to the best-in-class treatments. In this review, we discuss advances in development of TPD in the context of targeting the host-pathogen interface and speculate on their potential use to combat viral, bacterial and parasitic infection. AU - Grohmann, C.* AU - Marapana, D.S.* AU - Ebert, G. C1 - 63632 C2 - 51636 CY - 111 River St, Hoboken 07030-5774, Nj Usa TI - Targeted protein degradation at the host-pathogen interface. JO - Mol. Microbiol. PB - Wiley PY - 2022 SN - 0950-382x ER - TY - JOUR AB - A prerequisite for any rational drug design strategy is understanding the mode of protein-ligand interaction. This motivated us to explore protein-substrate interaction in Type-II NADH:quinone oxidoreductase (NDH-2) from Staphylococcus aureus, a worldwide problem in clinical medicine due to its multiple drug resistant forms. NDHs-2 are involved in respiratory chains and recognized as suitable targets for novel antimicrobial therapies, as these are the only enzymes with NADH:quinone oxidoreductase activity expressed in many pathogenic organisms. We obtained crystal and solution structures of NDH-2 from S. aureus, showing that it is a dimer in solution. We report fast kinetic analyses of the protein and detected a charge-transfer complex formed between NAD(+) and the reduced flavin, which is dissociated by the quinone. We observed that the quinone reduction is the rate limiting step and also the only half-reaction affected by the presence of HQNO, an inhibitor. We analyzed protein-substrate interactions by fluorescence and STD-NMR spectroscopies, which indicate that NADH and the quinone bind to different sites. In summary, our combined results show the presence of distinct binding sites for the two substrates, identified quinone reduction as the rate limiting step and indicate the establishment of a NAD(+) -protein complex, which is released by the quinone. AU - Sena, F.V.* AU - Batista, A.P.* AU - Catarino, T.* AU - Brito, J.A.* AU - Archer, M.* AU - Viertler, M. AU - Madl, T. AU - Cabrita, E.J.* AU - Pereira, M.M.* C1 - 47194 C2 - 39179 SP - 272-288 TI - Type-II NADH:quinone oxidoreductase from Staphylococcus aureus has two distinct binding sites and is rate limited by quinone reduction. JO - Mol. Microbiol. VL - 98 IS - 2 PY - 2015 SN - 0950-382x ER - TY - JOUR AB - The enzymatic dearomatization of aromatic ring systems by reduction represents a highly challenging redox reaction in biology and plays a key role in the degradation of aromatic compounds under anoxic conditions. In anaerobic bacteria, most monocyclic aromatic growth substrates are converted to benzoyl-coenzyme A (CoA), which is then dearomatized to a conjugated dienoyl-CoA by ATP-dependent or -independent benzoyl-CoA reductases. It was unresolved whether or not related enzymes are involved in the anaerobic degradation of environmentally relevant polycyclic aromatic hydrocarbons (PAHs). In this work, a previously unknown dearomatizing 2-naphthoyl-CoA reductase was purified from extracts of the naphthalene-degrading, sulphidogenic enrichment culture N47. The oxygen-tolerant enzyme dearomatized the non-activated ring of 2-naphthoyl-CoA by a four-electron reduction to 5,6,7,8-tetrahydro-2-naphthoyl-CoA. The dimeric 150kDa enzyme complex was composed of a 72kDa subunit showing sequence similarity to members of the flavin-containing old yellow enzyme' family. NCR contained FAD, FMN, and an iron-sulphur cluster as cofactors. Extracts of Escherichia coli expressing the encoding gene catalysed 2-naphthoyl-CoA reduction. The identified NCR is a prototypical enzyme of a previously unknown class of dearomatizing arylcarboxyl-CoA reductases that are involved in anaerobic PAH degradation; it fundamentally differs from known benzoyl-CoA reductases. AU - Eberlein, C.* AU - Estelmann, S.* AU - Seifert, J. AU - von Bergen, M. AU - Müller, M.* AU - Meckenstock, R.U. AU - Boll, M.* C1 - 25505 C2 - 31875 SP - 1032-1039 TI - Identification and characterization of 2-naphthoyl-coenzyme A reductase, the prototype of a novel class of dearomatizing reductases. JO - Mol. Microbiol. VL - 88 IS - 5 PB - Wiley-Blackwell PY - 2013 SN - 0950-382x ER - TY - JOUR AB - P>Nitrate is a dominant form of inorganic nitrogen (N) in soils and can be efficiently assimilated by bacteria, fungi and plants. We studied here the transcriptome of the short-term nitrate response using assimilating and non-assimilating strains of the model ascomycete Aspergillus nidulans. Among the 72 genes positively responding to nitrate, only 18 genes carry binding sites for the pathway-specific activator NirA. Forty-five genes were repressed by nitrate metabolism. Because nirA- strains are N-starved at nitrate induction conditions, we also compared the nitrate transcriptome with N-deprived conditions and found a partial overlap of differentially regulated genes between these conditions. Nitric oxide (NO)-metabolizing flavohaemoglobins were found to be co-regulated with nitrate assimilatory genes. Subsequent molecular characterization revealed that the strongly inducible FhbA is required for full activity of nitrate and nitrite reductase enzymes. The co-regulation of NO-detoxifying and nitrate/nitrite assimilating systems may represent a conserved mechanism, which serves to neutralize nitrosative stress imposed by an external NO source in saprophytic and pathogenic fungi. Our analysis using membrane-permeable NO donors suggests that signalling for NirA activation only indirectly depends on the nitrate transporters NrtA (CrnA) and NrtB (CrnB). AU - Schinko, T.* AU - Berger, H.* AU - Lee, W. AU - Gallmetzer, A.* AU - Pirker, K.* AU - Pachlinger, R.* AU - Buchner, I.* AU - Reichenauer, T.* AU - Güldener, U. AU - Strauss, J. C1 - 6005 C2 - 28011 CY - Malden SP - 720-738 TI - Transcriptome analysis of nitrate assimilation in Aspergillus nidulans reveals connections to nitric oxide metabolism. JO - Mol. Microbiol. VL - 78 IS - 3 PB - Wiley-Blackwell Publishing PY - 2010 SN - 0950-382x ER - TY - JOUR AB - Repair under non-growth conditions of DNA double-stranded breaks (DSBs) and S1 nuclease-sensitive sites (SSSs; e.g. DNA damage which is processed by in vitro treatment with S1 nuclease to DSBs) induced by [60Co]-gamma-rays (200 Gy; anoxic conditions) was monitored in a diploid repair-competent strain of Saccharomyces cerevisiae. We used pulsed-field gel electrophoresis (PFGE), which allows the separation of chromosome-sized yeast DNA molecules, to determine the number of DSBs and SSSs in individual chromosome species of yeast. Our results indicate that SSSs which have been regarded as clusters of base damage in opposite DNA strands are repaired efficiently in a repair-proficient diploid strain of yeast. The time course of SSS repair is comparable to the one of DSB repair, indicating similarities in the molecular mechanism. Both types of repair kinetics are different for different chromosome species. AU - Geigl, E.-M. AU - Eckardt-Schupp, F. C1 - 19068 C2 - 12123 SP - 1615-1620 TI - The repair of double-strand breaks and S1 nuclease-sensitive sites can be monitored chromosome-specifically in Saccharomyces cerevisiae using pulsed-field gel electrophoresis. JO - Mol. Microbiol. VL - 5 IS - 7 PY - 1991 SN - 0950-382x ER - TY - JOUR AU - Geigl, E.-M. AU - Eckardt-Schupp, F. C1 - 17515 C2 - 10445 SP - 801-810 TI - Chromosome-specific Identification and Quantification of S1 Nuclease-Sensitive Sites in Yeast Chromatin by Pulsed-Field Gel Electrophoresis. JO - Mol. Microbiol. VL - 4 PY - 1990 SN - 0950-382x ER - TY - JOUR AB - Sites that are sensitive to the single-strand-specific endonuclease S1 ('S1-sensitive sites', SSS) occur in native chromatin and, like DNA double-stranded breaks (DSB), they are induced by DNA-damaging agents, such as ionizing radiation. We have developed a method to quantify SSS and DSB in yeast chromatin by using pulsed-field gel electrophoresis (PFGE) to separate the intact chromosomal-length DNA molecules from the lower molecular-weight broken ones. Direct evaluation of the photonegatives of the ethidium bromide-stained gels by laser densitometry enabled us to calculate the numbers of DSB and SSS per DNA molecule. These numbers were determined from the bulk of the non-separated genomic DNA of yeast, corresponding to a single band in the PFGE (pulse time 10 seconds), and in each of the eight largest yeast chromosomes, corresponding to distinct bands in the PFGE gels (pulse time 50 seconds), which were not superimposed by the smear of the broken, low molecular-weight DNA. Furthermore, the induction of DSB and SSS in a specific chromosome (circular chromosome III) was determined by Southern hybridization of the PFGE gels with a suitable centromere probe, followed by densitometry of the autoradiographs. Our method allows the chromosome-specific monitoring of DSB and all those DNA structures that are processed either in vivo or in vitro into DSB and which may not be distributed randomly within the genome. AU - Geigl, E.M. AU - Eckardt-Schupp, F. C1 - 42044 C2 - 40137 SP - 801-810 TI - Chromosome-specific identification and quantification of S1 nuclease-sensitive sites in yeast chromatin by pulsed-field gel electrophoresis. JO - Mol. Microbiol. VL - 4 IS - 5 PY - 1990 SN - 0950-382x ER -