Project description:This is supplemental raw data for Abe et al., 2022. Proteome analysis and phospho-proteome analysis using mouse liver prepared at 6 circadian time points (CT2, 6, 10, 14, 18, 22). In the delta-RRE mutant, two RRE sequences are removed from 5'UTR of mouse Bmal1 gene.
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:The PIK3CA gene is frequently mutated in human cancers. To study the signaling mechanisms responsible for cell growth and invasion phenotypes induced by mutant PIK3CA molecules, we carried out a SILAC-based quantitative phosphoproteomic analysis of MCF10A, a spontaneously immortalized normal mammary epithelial cell line, and two MCF10A knockin cell lines containing different activating mutations of the PIK3CA gene. MCF10A and PIK3CA mutation knock in cells were propagated in DMEM/F12 SILAC media deficient in both L-lysine and L-arginine and supplemented with light lysine (K) and arginine (R) for light, 2H4-K and 13C6-R for medium state and 13C615N2-K and 13C615N4-R for heavy state labeling. Cell lysates were prepared in urea lysis buffer containing 20 mM HEPES pH 8.0, 9 M urea, 1 mM sodium orthovanadate, 2.5 mM sodium pyrophosphate, 1 mM ß-glycerophosphate and 5mM sodium fluoride. The lysates were reduced, alkylated and digested by trypsin. Tryptic peptides were desalted by C18 reverse phase column and followed by strong cation exchange (SCX) fractionation. Fractionated peptides were subjected to TiO2-based phosphopeptide enrichment. LC-MS/MS analysis of enriched phosphopeptides was carried out using a reverse-phase liquid chromatography system interfaced with an LTQ-Orbitrap Velos mass spectrometer. Proteome Discoverer (v 1.3) suite was used for quantitation and database searches. The tandem mass spectrometry data were searched using Mascot (2.2.0) and SEQUEST search algorithms against a Human RefSeq database supplemented with frequently observed contaminants.
Project description:The intrinsically disordered protein, α-synuclein, implicated in synaptic vesicle homeostasis and neurotransmitter release, is also associated with several neurodegenerative diseases. The different roles of α-synuclein are characterized by distinct structural states (membrane-bound, dimer, tetramer, oligomer, and fibril), which are originated from its various monomeric conformations. The pathological states, determined by the ensemble of α-synuclein monomer conformations and dynamic pathways of interconversion between dominant states, remain elusive due to their transient nature. Here, we use inter-dye distance distributions from bulk time-resolved Förster resonance energy transfer as restraints in discrete molecular dynamics simulations to map the conformational space of the α-synuclein monomer. We further confirm the generated conformational ensemble in orthogonal experiments utilizing far-UV circular dichroism and cross-linking mass spectrometry. Single-molecule protein-induced fluorescence enhancement measurements show that within this conformational ensemble, some of the conformations of αsynuclein are surprisingly stable, exhibiting conformational transitions slower than milliseconds. Our comprehensive analysis of the conformational ensemble reveals essential structural properties and potential conformations that promote its various functions in membrane interaction or oligomer and fibril formation.
Project description:Formaldehyde is a widely used fixative in biology and medicine. The current chemical model for formaldehyde cross-linking of proteins is the formation of a methylene bridge that incorporates one carbon atom into the link. Here, we present mass spectrometry data that largely refute this model. Instead, our data show that cross-linking of structured proteins mainly involves a reaction that incorporates two carbon atoms into the link. Under MS/MS fragmentation, the link cleaves symmetrically to yield unusual fragments with a modification of one carbon atom. We apply this new understanding of the underlying cross-linking chemistry to the structural approach of cross-linking coupled to mass spectrometry. First, we cross-linked a mixture of purified proteins with formaldehyde. Our new analysis readily identified tens of cross-links from these proteins, which fit well with their atomic structures. We then perform in-situ cross-linking of human cells in culture. We identified 469 intra-protein and 90 inter-protein cross-links, which also fit well with available atomic structures. Interestingly, many of these cross-links could not be mapped onto a known structure and thus provide new structural insights. We highlight an example in which formaldehyde cross-links localize the binding site of βNAC on the ribosome. We also find several interactions of actin with auxiliary proteins. Our findings not only expand our understanding of formaldehyde reactivity and toxicity, but also clearly demonstrate how to use this potent reagent for structural studies.