Project description:RNF213 is novel transthiolating E3 ligase that is activated by ATP. To determine whether cellular RNF213 is activated by elevated ATP levels, we electroplated an E3 activity-based probe into HEK293 cells stably expressing RNF213 in a knockout background. DIA analysis was performed in cells that were coelectroporated with the poorly hydrolysable ATP analog, ATPgS, or buffer control.
Project description:Homeostatic control of intracellular ionic strength is essential for protein, organelle and genome function, yet mechanisms that sense and enable adaptation to ionic stress remain poorly understood in animals. We find that the transcription factor NFAT5 directly senses solution ionic strength using a C-terminal intrinsically disordered region. Both in intact cells and in a purified system, NFAT5 forms dynamic, reversible biomolecular condensates in response to increasing ionic strength. This self-associative property, conserved from insects to mammals, allows NFAT5 to accumulate in the nucleus and activate genes that restore cellular ion content. Mutations that reduce condensation or those that promote aggregation both reduce NFAT5 activity, highlighting the importance of optimally tuned associative interactions. To investigate the composition of NFAT5 condensates in response to hypertonic stress, proteins in close proximity of NFAT5 were identified using a variant of NFAT5 fused to TurboID as bait. Hypertonic stress increases NFAT5 proximity to protein complexes belonging to the GO gene sets of “transcription coactivator activity” and “positive regulation of DNA templated transcription initiation.” Closer inspection revealed that the association between NFAT5 and two transcriptional co-activators (the mediator complex and BRD4) and RNAPII itself increased in response to hypertonic stress.
Project description:Methods to affinity purify proteins are widely used in protein research. One important application is to identify interacting proteins of an affinity-purified protein of interest (POI) by mass spectrometry. Here, we developed an optogenetics-derived and light-controlled affinity purification method based on the light-regulated reversible protein interaction between phytochrome B (PhyB) and its phytochrome interacting factor 6 (PIF6). We engineered a truncated variant of PIF6 comprising only 22 amino acids that can be genetically fused to the POI as an affinity tag. Thereby the POI can be purified with PhyB-functionalized resin material using 660 nm light for binding and washing and 740 nm light for elution. As proof-of-concept, we expressed PIF-tagged variants of the tyrosine kinase ZAP70 in ZAP70-deficient Jurkat T cells, purified ZAP70 and associating proteins using our light-controlled system and identified the interaction partners by mass spectrometry
Project description:Differential proteome composition of wild-type yeast cells (S. cerevisiae BY4741) grown at 30 ºC or 37 ºC was studied using SILAC based quantitative proteomics.
Project description:Blue-native gel electrophoresis (BN) is a powerful method for protein separation. Combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) it enables large-scale identification of protein complexes and their subunits. Current BN-MS approaches, however, are limited in size resolution, comprehensiveness and quantification. Here, we present a new methodology combining defined sub-mm slicing of BN gels by a cryo-microtome with high-performance LC-MS/MS and label-free quantification of proteins. Application of this cryo-slicing BN-MS approach (csBN-MS) to mitochondria from brain demonstrated a high degree of comprehensiveness, quantification accuracy and size resolution. The technique provided abundance-mass profiles for 743 mitochondrial proteins including all canonical subunits of the oxidative respiratory chain assembled into 13 distinct (super)complexes. Moreover, the data revealed COX7R as a constitutive subunit of distinct supercomplexes and identified novel assemblies of porins and TOM proteins. Together, csBN-MS enables quantitative profiling of complexomes with resolution close to the limits of native gel electrophoresis.
Project description:Fluoroquinolones (FQs) are an important class of potent broad-spectrum antibiotics. However, their general use is more and more limited by adverse side effects. While general mechanisms for the Fluoroquinolone-associated disability (FQAD) have been identified, the underlying molecular origins of toxicity remain elusive. In this study, focusing on the most commonly prescribed FQs Ciprofloxacin and Levofloxacin, whole proteome analyses revealed prominent mitochondrial dysfunction in human cells, specifically of the complexes I and IV of the electron transport chain. Furthermore, global untargeted chemo-proteomic methodologies such as photo-affinity profiling with FQ-derived probes, as well as derivatization-free thermal proteome profiling, were applied to elucidate human protein off-targets of FQs in living cells. Accordingly, the interactions of FQs with mitochondrial AIFM1 and IDH2 have been identified and biochemically validated for their contribution to mitochondrial dysfunction and adverse downstream effects, including impaired redox-homeostasis, elevated matrix metalloprotease activity and epigenetic modifications. This off-target discovery study provides unique insights into FQ toxicity enabling to utilize the identified molecular principles for the design of a safer FQ generation.