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Sander, S.

Publications and source records attributed to Sander, S..

6 recordsLinked to original sources

Sample preparation for mass spectrometry-based tissue (phospho)proteomics

This protocol describes the workflow for the preparation of tissue samples for proteome and phosphoproteome analyses using mass spectrometry. The tissue samples are cryogenically pulverized and homogenized in a sucrose-based buffer to ensure proper tissue disruption. For depletion of lipid contaminants, proteins are purified using chloroform-methanol precipitation, followed by a resuspension in a urea-based buffer for enzymatic digestion. Peptides are desalted and enriched for phosphopeptides prior LC-MS/MS analysis. The workflow was developed for skin biopsies but is compatible with a broad range of tissue types. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=51 SRC="FIGDIR/small/732915v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@1bfe743org.highwire.dtl.DTLVardef@1e812deorg.highwire.dtl.DTLVardef@12ed3c0org.highwire.dtl.DTLVardef@1501c4d_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO C_FIG

biochemistry↗

An FGF7-FGFR2-KLF4 feedback loop sustains anti-inflammatory signaling in epithelial cells

Chronic or excessive inflammation are hallmarks of many human diseases, but the endogenous factors that limit inflammatory responses are insufficiently characterized. We discovered a broad anti-inflammatory function of fibroblast growth factor 7 (FGF7), which is mediated via epithelial cels. FGF7 suppressed the expression of pro-inflammatory and immunomodulatory genes in cultured human keratinocytes in the absence or presence of pro-inflammatory stimuli and in a psoriasiform inflammation model in mice. Mechanistically, this involves an FGF7-FGF receptor 2 (FGFR2)-MAPK-Kruppel-like factor 4 (KLF4) signaling axis. FGF7 induced significant alterations in the KLF4 interactome in human keratinocytes and suppressed the transcriptional activity of KLF4 at its pro-inflammatory target genes. Concomitantly, expression of FGFR2 and downstream signaling components was promoted by KLF4, identifying a KLF4-dependent regulatory feedback loop that sustains anti-inflammatory FGF signaling. These results suggest activation of FGF7-FGFR2-KLF4 signaling as a strategy for the treatment of inflammatory diseases involving the skin or other epithelial tissues and highlight the role of epithelial cells in the control of inflammation.

cell biology↗

Synergistic effects of tRNA modification defects in Escherichia coli K12.

Transfer RNAs (tRNAs) are essential components of the translation machinery and carry numerous post-transcriptional modifications that contribute to decoding accuracy, efficiency and cellular fitness. In Escherichia coli K-12, all tRNA modification pathways have been identified, yet the functional interactions between these pathways remain largely unexplored. Here, we systematically analyze genetic interactions between 29 non-essential tRNA modification genes using a pairwise synthetic lethal screen based on P1 transduction. Most combinations of tRNA modification gene deletions are tolerated during growth in rich medium; however, we identify five synthetically lethal pairs and fifteen additional combinations that display negative genetic interactions. Deletions of truA, which encodes the pseudouridine synthase responsible for modifications at positions 38-40 of multiple tRNAs, show the highest frequency of negative epistasis. Synthetic lethality associated with truA can be complemented by expression of truA in trans and, in specific cases, partially suppressed by overexpression of its tRNA substrates, indicating substrate-specific functional dependencies. Analysis of tRNA abundance by northern blotting and AQRNA-seq demonstrates that loss of individual tRNA modification enzymes does not generally lead to widespread tRNA destabilization. Instead, further phenotypic characterization of viable double mutants reveals condition-dependent growth defects influenced by carbon source, temperature and metabolic stress, as well as toxicity associated with overexpression of specific tRNAs. Together, these results reveal a limited but distinct set of genetic interactions among bacterial tRNA modification pathways and highlight the importance of physiological context in uncovering their cellular roles.

microbiology↗

Corepressor inactivation through tandem linear motif-induced polymerization

While the elucidation of regulatory mechanisms of folded proteins is facilitated due to their amenability to high-resolution structural characterization, investigation of these mechanisms in disordered proteins is more challenging due to their structural heterogeneity, which can be captured by a variety of biophysical approaches. Here, we used the transcriptional master corepressor CtBP, which binds the putative metastasis suppressor RAI2 through repetitive SLiMs, as a model system. Using cryo-electron microscopy embedded in an integrative structural biology approach, we show that RAI2 unexpectedly induces CtBP polymerization through filaments of stacked tetrameric CtBP layers. These filaments lead to RAI2-mediated CtBP nuclear foci and relieve its corepressor function in RAI2-expressing cancer cells. The impact of RAI2-mediated CtBP loss-of-function is illustrated by the analysis of a diverse cohort of prostate cancer patients, which reveals a substantial decrease in RAI2 in advanced treatment-resistant cancer subtypes. As RAI2-like SLiM motifs are found in a wide range of organisms, including pathogenic viruses, our findings serve as a paradigm for diverse functional effects through multivalent interaction-mediated polymerization by disordered proteins in healthy and diseased conditions. The specific properties of these repeated interactions open up new therapeutic opportunities.

molecular biology↗

The crystal structure of TRPM2 MHR1/2 domain reveals a conserved Zn2+-binding domain essential for ligand binding and activity

Transient receptor potential melastatin 2 (TRPM2) is a Ca2+-permeable, non-selective cation channel involved in diverse physiological processes such as immune response, apoptosis and body temperature sensing. TRPM2 is activated by ADP-ribose (ADPR) and 2'-deoxy-ADPR in a Ca2+-dependent manner. While two species-specific binding sites exist for ADPR, a binding site for 2'-deoxy-ADPR is not known yet. Here, we report the crystal structure of the MHR1/2 domain of TRPM2 from zebrafish (Danio rerio) and show binding of both ligands to this domain. We identified a so-far unrecognized Zn2+-binding domain that was not resolved in previous cryo-EM structures and that is conserved in most TRPM channels. In combination with patch clamp experiments, we comprehensively characterize the effect of the Zn2+-binding domain on TRPM2 activation. Our results provide insight into a conserved structural element essential for channel activity.

biochemistry↗

Virtual reality-based sensorimotor adaptation shapes subsequent spontaneous and naturalistic stimulus-driven brain activity

Our everyday life summons numerous novel sensorimotor experiences, to which our brain needs to adapt in order to function properly. However, tracking plasticity of naturalistic behaviour and associated brain modulations is challenging. Here we tackled this question implementing a prism adaptation training in virtual reality (VRPA) in combination with functional neuroimaging. Three groups of healthy participants (N=45) underwent VRPA (with a spatial shift either to the left/right side, or with no shift), and performed fMRI sessions before and after training. To capture modulations in free-flowing, task-free brain activity, the fMRI sessions included resting state and free viewing of naturalistic videos. We found significant decreases in spontaneous functional connectivity between large-scale cortical networks - namely attentional and default mode/fronto-parietal networks - only for adaptation groups. Additionally, VRPA was found to bias visual representations of naturalistic videos, as following rightward adaptation, we found upregulation of visual response in an area in the parieto-occipital sulcus (POS) in the right hemisphere. Notably, the extent of POS upregulation correlated with the size of the VRPA induced after-effect measured in behavioural tests. This study demonstrates that a brief VRPA exposure is able to change large-scale cortical connectivity and correspondingly bias the representation of naturalistic sensory inputs. Significance statementIn the current work, we tested how a brief sensorimotor experience changes subsequent brain activity and connectivity. Using virtual reality (VR) as a tool for sensorimotor training opens a window for creating otherwise impossible sensory experiences and sensorimotor interactions. Specifically, we studied how VR adaptation training in ecological conditions modulates spontaneous functional connectivity and brain representation of naturalistic real-life-like stimuli. Previous adaptation studies used artificial, lab-designed setups both during adaptation and while measuring subsequent aftereffects. Testing brain response while observing naturalistic stimuli and in resting state allowed us to stay as close as possible to naturalistic real-life-like conditions, not confounded by performance during a task. The current work demonstrates how rapid changes in free-flowing brain activity and connectivity occur following short-term VR visuomotor adaptation training in healthy individuals. Moreover, we found a link between sensory responses to naturalistic stimuli and adaptation-induced behavioural aftereffect, thus demonstrating a common source of training-induced spatial recalibration, which affects both behaviour and brain representations of naturalistic stimuli. These findings might have meaningful implications both for understanding the mechanisms underlying visuomotor plasticity in healthy individuals and for using VR adaptation training as a tool for rehabilitating brain-damaged patients suffering from deficits in spatial representation.

neuroscience↗