@article{LiangRiosMiguelJaricketal.2021, author = {Liang, Chunguang and Rios-Miguel, Ana B. and Jarick, Marcel and Neurgaonkar, Priya and Girard, Myriam and Fran{\c{c}}ois, Patrice and Schrenzel, Jacques and Ibrahim, Eslam S. and Ohlsen, Knut and Dandekar, Thomas}, title = {Staphylococcus aureus transcriptome data and metabolic modelling investigate the interplay of Ser/Thr kinase PknB, its phosphatase Stp, the glmR/yvcK regulon and the cdaA operon for metabolic adaptation}, series = {Microorganisms}, volume = {9}, journal = {Microorganisms}, number = {10}, issn = {2076-2607}, doi = {10.3390/microorganisms9102148}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-248459}, year = {2021}, abstract = {Serine/threonine kinase PknB and its corresponding phosphatase Stp are important regulators of many cell functions in the pathogen S. aureus. Genome-scale gene expression data of S. aureus strain NewHG (sigB\(^+\)) elucidated their effect on physiological functions. Moreover, metabolic modelling from these data inferred metabolic adaptations. We compared wild-type to deletion strains lacking pknB, stp or both. Ser/Thr phosphorylation of target proteins by PknB switched amino acid catabolism off and gluconeogenesis on to provide the cell with sufficient components. We revealed a significant impact of PknB and Stp on peptidoglycan, nucleotide and aromatic amino acid synthesis, as well as catabolism involving aspartate transaminase. Moreover, pyrimidine synthesis was dramatically impaired by stp deletion but only slightly by functional loss of PknB. In double knockouts, higher activity concerned genes involved in peptidoglycan, purine and aromatic amino acid synthesis from glucose but lower activity of pyrimidine synthesis from glucose compared to the wild type. A second transcriptome dataset from S. aureus NCTC 8325 (sigB\(^-\)) validated the predictions. For this metabolic adaptation, PknB was found to interact with CdaA and the yvcK/glmR regulon. The involved GlmR structure and the GlmS riboswitch were modelled. Furthermore, PknB phosphorylation lowered the expression of many virulence factors, and the study shed light on S. aureus infection processes.}, language = {en} } @article{FischerHelfrichFoersterPeschel2016, author = {Fischer, Robin and Helfrich-F{\"o}rster, Charlotte and Peschel, Nicolai}, title = {GSK-3 Beta Does Not Stabilize Cryptochrome in the Circadian Clock of Drosophila}, series = {PLoS ONE}, volume = {11}, journal = {PLoS ONE}, number = {1}, doi = {10.1371/journal.pone.0146571}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-180370}, year = {2016}, abstract = {Cryptochrome (CRY) is the primary photoreceptor of Drosophila's circadian clock. It resets the circadian clock by promoting light-induced degradation of the clock protein Timeless (TIM) in the proteasome. Under constant light, the clock stops because TIM is absent, and the flies become arrhythmic. In addition to TIM degradation, light also induces CRY degradation. This depends on the interaction of CRY with several proteins such as the E3 ubiquitin ligases Jetlag (JET) and Ramshackle (BRWD3). However, CRY can seemingly also be stabilized by interaction with the kinase Shaggy (SGG), the GSK-3 beta fly orthologue. Consequently, flies with SGG overexpression in certain dorsal clock neurons are reported to remain rhythmic under constant light. We were interested in the interaction between CRY, Ramshackle and SGG and started to perform protein interaction studies in S2 cells. To our surprise, we were not able to replicate the results, that SGG overexpression does stabilize CRY, neither in S2 cells nor in the relevant clock neurons. SGG rather does the contrary. Furthermore, flies with SGG overexpression in the dorsal clock neurons became arrhythmic as did wild-type flies. Nevertheless, we could reproduce the published interaction of SGG with TIM, since flies with SGG overexpression in the lateral clock neurons shortened their free-running period. We conclude that SGG does not directly interact with CRY but rather with TIM. Furthermore we could demonstrate, that an unspecific antibody explains the observed stabilization effects on CRY.}, language = {en} } @article{FrankDengjelWilflingetal.2015, author = {Frank, Daniel O. and Dengjel, J{\"o}rn and Wilfling, Florian and Kozjak-Pavlovic, Vera and H{\"a}cker, Georg and Weber, Arnim}, title = {The Pro-Apoptotic BH3-Only Protein Bim Interacts with Components of the Translocase of the Outer Mitochondrial Membrane (TOM)}, series = {PLoS ONE}, volume = {10}, journal = {PLoS ONE}, number = {4}, doi = {10.1371/journal.pone.0123341}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-143301}, pages = {e0123341}, year = {2015}, abstract = {The pro-apoptotic Bcl-2-family protein Bim belongs to the BH3-only proteins known as initiators of apoptosis. Recent data show that Bim is constitutively inserted in the outer mitochondrial membrane via a C-terminal transmembrane anchor from where it can activate the effector of cytochrome c-release, Bax. To identify regulators of Bim-activity, we conducted a search for proteins interacting with Bim at mitochondria. We found an interaction of Bim with Tom70, Tom20 and more weakly with Tom40, all components of the Translocase of the Outer Membrane (TOM). In vitro import assays performed on tryptically digested yeast mitochondria showed reduced Bim insertion into the outer mitochondrial membrane (OMM) indicating that protein receptors may be involved in the import process. However, RNAi against components of TOM (Tom40, Tom70, Tom22 or Tom20) by siRNA, individually or in combination, did not consistently change the amount of Bim on HeLa mitochondria, either at steady state or upon de novo-induction. In support of this, the individual or combined knockdowns of TOM receptors also failed to alter the susceptibility of HeLa cells to Bim-induced apoptosis. In isolated yeast mitochondria, lack of Tom70 or the TOM-components Tom20 or Tom22 alone did not affect the import of Bim into the outer mitochondrial membrane. In yeast, expression of Bim can sensitize the cells to Bax-dependent killing. This sensitization was unaffected by the absence of Tom70 or by an experimental reduction in Tom40. Although thus the physiological role of the Bim-TOM-interaction remains unclear, TOM complex components do not seem to be essential for Bim insertion into the OMM. Nevertheless, this association should be noted and considered when the regulation of Bim in other cells and situations is investigated.}, language = {en} } @article{DusikSenthilanMentzeletal.2014, author = {Dusik, Verena and Senthilan, Pingkalai R. and Mentzel, Benjamin and Hartlieb, Heiko and W{\"u}lbeck, Corina and Yoshii, Taishi and Raabe, Thomas and Helfrich-F{\"o}rster, Charlotte}, title = {The MAP Kinase p38 Is Part of Drosophila melanogaster's Circadian Clock}, series = {PLoS Genetics}, volume = {10}, journal = {PLoS Genetics}, number = {8}, issn = {1553-7404}, doi = {10.1371/journal.pgen.1004565}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-119433}, pages = {e1004565}, year = {2014}, abstract = {All organisms have to adapt to acute as well as to regularly occurring changes in the environment. To deal with these major challenges organisms evolved two fundamental mechanisms: the p38 mitogen-activated protein kinase (MAPK) pathway, a major stress pathway for signaling stressful events, and circadian clocks to prepare for the daily environmental changes. Both systems respond sensitively to light. Recent studies in vertebrates and fungi indicate that p38 is involved in light-signaling to the circadian clock providing an interesting link between stress-induced and regularly rhythmic adaptations of animals to the environment, but the molecular and cellular mechanisms remained largely unknown. Here, we demonstrate by immunocytochemical means that p38 is expressed in Drosophila melanogaster's clock neurons and that it is activated in a clock-dependent manner. Surprisingly, we found that p38 is most active under darkness and, besides its circadian activation, additionally gets inactivated by light. Moreover, locomotor activity recordings revealed that p38 is essential for a wild-type timing of evening activity and for maintaining ∼ 24 h behavioral rhythms under constant darkness: flies with reduced p38 activity in clock neurons, delayed evening activity and lengthened the period of their free-running rhythms. Furthermore, nuclear translocation of the clock protein Period was significantly delayed on the expression of a dominant-negative form of p38b in Drosophila's most important clock neurons. Western Blots revealed that p38 affects the phosphorylation degree of Period, what is likely the reason for its effects on nuclear entry of Period. In vitro kinase assays confirmed our Western Blot results and point to p38 as a potential "clock kinase" phosphorylating Period. Taken together, our findings indicate that the p38 MAP Kinase is an integral component of the core circadian clock of Drosophila in addition to playing a role in stress-input pathways.}, language = {en} } @phdthesis{Sibilski2014, author = {Sibilski, Claudia}, title = {Identification and characterization of the novel mKSR1 phosphorylation site Tyr728 and its role in MAPK signaling}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-114672}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2014}, abstract = {In mammals, KSR1 functions as an essential scaffold that coordinates the assembly of RAF/MEK/ERK complexes and regulates intracellular signal transduction upon extracellular stimulation. Aberrant activation of the equivalent MAPK signaling pathway has been implicated in multiple human cancers and some developmental disorders. The mechanism of KSR1 regulation is highly complex and involves several phosphorylation/dephosphorylation steps. In the present study, a number of novel in vivo phosphorylation sites were detected in mKSR1 by use of mass spectrometry analysis. Among others, Tyr728 was identified as a unique regulatory residue phosphorylated by LCK, a Src kinase family member. To understand how phosphorylation of Tyr728 may regulate the function of KSR1 in signal transduction and cellular processes, structural modeling and biochemical studies were integrated in this work. Computational modeling of the mKSR1(KD) protein structure revealed strong hydrogen bonding between phospho-Tyr728 and the residues surrounding Arg649. Remarkably, this pattern was altered when Tyr728 was non-phosphorylated or substituted. As confirmed by biochemical analysis, Arg649 may serve as a major anchor point for phospho-Tyr728 in order to stabilize internal structures of KSR1. In line with the protein modeling results, mutational studies revealed that substitution of Tyr728 by phenylalanine leads to a less compact interaction between KSR1 and MEK, a facilitated KSR1/B-RAF binding and an increased phosphorylation of MEK in complex with KSR1. From these findings it can be concluded that phospho-Tyr728 is involved in tightening the KSR1/MEK interaction interface and in regulating the phosphorylation of KSR1-bound MEK by either RAF or KSR1 kinases. Beside the Tyr728, Ser722 was identified as a novel regulatory phosphorylation site. Amino acid exchanges at the relevant position demonstrated that Ser722 regulates KSR1-bound MEK phosphorylation without affecting KSR1/MEK binding per se. Due to its localization, Ser722 might consequently control the catalytic activity of KSR1 by interfering with the access of substrate (possibly MEK) to the active site of KSR1 kinase. Together with Ser722, phosphorylated Tyr728 may further positively affect the kinase activity of KSR1 as a consequence of its vicinity to the activation and catalytic loop in the KSR1(KD). As revealed by structural modeling, phospho-Tyr728 builds a hydrogen bond with the highly conserved Lys685. Consequently, phospho-Tyr728 has a stabilizing effect on internal structures involved in the catalytic reaction and possibly enhances the phosphate transfer within the catalytic cleft in KSR1. Considering these facts, it seems very likely that the LCK-dependent phosphorylation of Tyr728 plays a crucial role in the regulation of KSR1 catalytic activity. Results of fractionation and morphology analyses revealed that KSR1 recruits LCK to cytoskeleton for its phosphorylation at Tyr728 suggesting that this residue may regulate cytoskeleton dynamics and, consequently, cell motility. Beside that, phosphorylation of Tyr728 is involved in the regulation of cell proliferation, as shown by a significantly reduced population doubling time of KSR1-Y728F cells compared to cells expressing wild type KSR1. Taken together, tyrosine phosphorylation in KSR1 uncovers a new link between Src family kinases and MAPK signaling. Tyr728, the novel regulatory phosphorylation site in murine KSR1, may coordinate the transition between the scaffolding and the catalytic function of KSR1 serving as a control point used to fine-tune cellular responses.}, subject = {MAP-Kinase}, language = {en} } @phdthesis{Wenzel2014, author = {Wenzel, Jens}, title = {Regulation of TLR-induced macrophage responses by cytoskeleton-associated phosphoproteins}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-98843}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2014}, abstract = {Toll-like receptors (TLR) are pattern recognition receptors (PRR) by which macrophages (M{\O}) sense pathogen-associated molecular patterns (PAMPs). The recognition of lipopolysaccharide (LPS), the PAMP of gram negative bacteria, by TLR4 triggers signaling cascades and leads to the pro-inflammatory activation of the cells. A recent quantitative and kinetic analysis of the phosphoproteome of LPS-activated primary macrophages highlighted the cytoskeleton as a cell compartment with an enriched protein phosphorylation. In total 44 cytoskeleton-associated proteins were regulated by this post-translational modification and thus might be involved in the control and regulation of key macrophage functions like spreading, motility and phagocytosis. To investigate the control of cytoskeleton-associated cell functions by TLR4 activation, we first developed a method to quantitatively measure the spreading response of bone marrow M{\O} after stimulation with LPS. Fluorescence microscopy was used for cell imaging and visualisation of the M{\O} contact area. In collaboration with the Fraunhofer Institute Erlangen, we developed and validated a software tool for the semi-automated segmentation and quantitation of M{\O} fluorescence microscopy data, which allowed fast, robust and objective image analysis. Using this method, we observed that LPS caused time-dependent spreading, which was detectable after 1-2 h and maximal after 24 h. Next, the impact of genetic or pharmacological inhibition of known TLR signaling components was investigated. Deficiency in the adapter protein MYD88 strongly reduced spreading activity at the late time points, but had no impact early after LPS-stimulation. A similar effect was observed upon pharmacological inhibition of ERK1/2 signaling, indicating that ERK1/2 mediates MYD88-dependent M{\O} spreading. In contrast, M{\O} lacking the MAPK p38 were impaired in the initial spreading response but responded normally 8-24 h after stimulation. The genetic deletion of the MAPK phosphatases DUSP1 and DUSP16 resulted in impaired late spreading, corroborating the essential role for functional MAPK signaling in TLR4-driven M{\O} spreading. To identify the contribution of other cytoskeletal phosphoproteins to M{\O} spreading, siRNA knockdown of selected candidate genes in primary murine M{\O} was employed and combined with automated quantitative image analysis. These experiments revealed a functional role for the Myosins MYO1e and MYO1f in M{\O} spreading. These motor proteins are strongly phosphorylated in LPS-activated M{\O}. Because of their ability to simultaneously bind to actin filaments and cell membrane or other proteins, we investigated their role in phagocytosis, cytokine production and antigen presentation. Phagocytosis and killing of bacteria were not affected in Myo1e-/- macrophages. However, MYO1e plays a role in chemokine secretion and antigen presentation processes. MCP1 (CCL2) release was selectively increased in Myo1e-deficient M{\O} and dendritic cells (DC), while cytokine secretion was unaffected. Furthermore, macrophages and DCs lacking MYO1e showed lower levels of MHC-II on the cell surface. However, mRNA levels of CCL2 and of MHC-II were unaltered. These data suggest a role for MYO1e in the transport of selected chemokines and of MHC-II molecules to the cell surface. MHC-II-restricted antigen presentation assays revealed an impaired capacity of macrophages and DC lacking MYO1e to stimulate antigen-specific T cells, suggesting that the reduced MHC-II expression is functionally relevant. Taken together, in this study first a quantitative image analysis method was developed which allows the unbiased, robust and efficient investigation of the macrophage spreading response. Combination of this method with siRNA knockdown of selected cytoskeleton-associated phosphoproteins led to the identification of MYO1e and MYO1f as regulators of macrophage spreading. Furthermore, we identified MYO1e in M{\O} and DC to be essential for the intracellular transport of CCL2 and MHC-II to the cell surface and for optimal stimulation of antigen-specific CD4 T cells.}, subject = {Toll-like-Rezeptoren}, language = {en} } @article{RudelFaulstichBoettcheretal.2013, author = {Rudel, Thomas and Faulstich, Michaela and B{\"o}ttcher, Jan-Peter and Meyer, Thomas F. and Fraunholz, Martin}, title = {Pilus Phase Variation Switches Gonococcal Adherence to Invasion by Caveolin-1-Dependent Host Cell Signaling}, series = {PLoS Pathogens}, journal = {PLoS Pathogens}, doi = {10.1371/journal.ppat.1003373}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-96679}, year = {2013}, abstract = {Many pathogenic bacteria cause local infections but occasionally invade into the blood stream, often with fatal outcome. Very little is known about the mechanism underlying the switch from local to invasive infection. In the case of Neisseria gonorrhoeae, phase variable type 4 pili (T4P) stabilize local infection by mediating microcolony formation and inducing anti-invasive signals. Outer membrane porin PorBIA, in contrast, is associated with disseminated infection and facilitates the efficient invasion of gonococci into host cells. Here we demonstrate that loss of pili by natural pilus phase variation is a prerequisite for the transition from local to invasive infection. Unexpectedly, both T4P-mediated inhibition of invasion and PorBIA-triggered invasion utilize membrane rafts and signaling pathways that depend on caveolin-1-Y14 phosphorylation (Cav1-pY14). We identified p85 regulatory subunit of PI3 kinase (PI3K) and phospholipase Cγ1 as new, exclusive and essential interaction partners for Cav1-pY14 in the course of PorBIA-induced invasion. Active PI3K induces the uptake of gonococci via a new invasion pathway involving protein kinase D1. Our data describe a novel route of bacterial entry into epithelial cells and offer the first mechanistic insight into the switch from local to invasive gonococcal infection.}, language = {en} }