Mitochondrial proteome Wt and MitoPPX cells under control conditions and heat shock
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Thermo Fisher Scientific
ORGANISM(S): Homo Sapiens (ncbitaxon:9606)
SUBMITTER: 
Maria E Solesio
PROVIDER: MSV000095168 | MassIVE | Thu Jun 27 12:13:00 BST 2024
REPOSITORIES: MassIVE
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Project description:HeLa cells were treated or untreated with heat shock at 42°C for 5, 30, and 60 min, and were then fixed in medium containing 1% formaldehyde at 37°C for 30 min. Chromatin fraction was sonicated with the Branson Sonifier 450 (BRANSON Ultrasonics) into fragmented DNA around 200-300 bp. HSF1 bound to genomic DNA was immunoprecipitated with 2 ul of rabbit immune serum for mHSF1 (anti-mHSF1j or Millipore ABE1044) or preimmune serum conjugated to Dynabeads Protein A at 4°C for 3 h (ChIP). Immunoprecipitated sample was eluted by incubation at 90°C for 30 min in 1× Laemmli sample buffer (2% SDS, 60 mM Tris-HCl (pH 6.8), 100 mM DTT, 10% glycerol, 0.001% bromophenol blue). Protein samples were loaded on 12% SDS-PAGE for preparation of gel slices for mass spectrometry (MS). Gel bands were cut-out and subjected to in-gel digestion with trypsin. Resulting peptides were dissolved in a solution containing 0.1% trifluoroacetic acid and 2% acetonitrile and analyzed by an LTQ Orbitrap Velos Pro mass spectrometer coupled with a nanoLC instrument and HTC-PAL autosampler.
2022-06-19 | PXD031821 | JPOST Repository
Project description:Environmental stress, such as oxidative or heat stress, induces the activation of the heat shock response
(HSR) and leads to an increase in the heat shock proteins (HSPs) level. These HSPs act as molecular
chaperones to maintain cellular proteostasis. Controlled by highly intricate regulatory mechanisms,
having stress-induced activation and feedback regulations with multiple partners, the HSR is still
incompletely understood. In this context, we propose a minimal molecular model for the gene
regulatory network of the HSR that reproduces quantitatively different heat shock experiments both
on heat shock factor 1 (HSF1) and HSPs activities. This model, which is based on chemical kinetics
laws, is kept with a low dimensionality without altering the biological interpretation of the model
dynamics. This simplistic model highlights the titration of HSF1 by chaperones as the guiding line of
the network. Moreover, by a steady states analysis of the network, three different temperature stress
regimes appear: normal, acute, and chronic, where normal stress corresponds to pseudo thermal
adaption. The protein triage that governs the fate of damaged proteins or the different stress regimes
are consequences of the titration mechanism. The simplicity of the present model is of interest in
order to study detailed modelling of cross regulation between the HSR and other major genetic
networks like the cell cycle or the circadian clock.
Sivéry, A., Courtade, E., Thommen, Q. (2016). A minimal titration model of the mammalian dynamical heat shock response. Physical biology, 13(6), 066008.
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Project description:How heat shock induces the heat shock response (HSR) - a gene expression program encoding chaperones and other protein homeostasis (proteostasis) factors - remains an unresolved question in eukaryotic cell biology. Here we show that subcellular localization of the conserved J-protein Sis1 is a key regulator of the HSR in yeast. Under nonstress conditions, nucleoplasmic Sis1 promotes interaction between the chaperone Hsp70 and the transcription factor Hsf1 to repress the HSR. Heat shock triggers Sis1 to localize to the periphery of the nucleolus and to condense on the ER surface. Sis1 recruits the proteasome to this spatial network along with disaggregases and the ribosome quality control complex. Through localization dynamics, Sis1 relays the condition of the proteome to Hsf1. We conclude that the activation state of the HSR is determined by the spatial organization of the proteostasis network.
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Project description:The outer membrane proteins (OMPs) of Gram-negative bacterial cells, as well as the mitochondrion and chloroplast organelles, possess unique and highly stable ?-barrel structures. Biogenesis of OMPs in Escherichia coli involves such periplasmic chaperones as SurA and Skp. In this study, we found that the ?surA ?skp double-deletion strain of E. coli, although lethal and defective in the biogenesis of OMPs at the normal growth temperature, is viable and effective at the heat shock temperature. We identified FkpA as the multicopy suppressor for the lethal phenotype of the ?surA ?skp strain. We also demonstrated that the deletion of fkpA from the ?surA cells resulted in only a mild decrease in the levels of folded OMPs at the normal temperature but a severe decrease as well as lethality at the heat shock temperature, whereas the deletion of fkpA from the ?skp cells had no detectable effect on OMP biogenesis at either temperature. These results strongly suggest a functional redundancy between FkpA and SurA for OMP biogenesis under heat shock stress conditions. Mechanistically, we found that FkpA becomes a more efficient chaperone for OMPs under the heat shock condition, with increases in both binding rate and affinity. In light of these observations and earlier reports, we propose a temperature-responsive OMP biogenesis mechanism in which the degrees of functional importance of the three chaperones are such that SurA > Skp > FkpA at the normal temperature but FkpA ? SurA > Skp at the heat shock temperature.
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Project description:Chaperones are fundamental to regulating the heat shock response, mediating protein recovery from thermal-induced misfolding and aggregation. Using the QconCAT strategy and selected reaction monitoring (SRM) for absolute protein quantification, we have determined copy per cell values for 49 key chaperones in Saccharomyces cerevisiae under conditions of normal growth and heat shock. This work extends a previous chemostat quantification study by including up to five Q-peptides per protein to improve confidence in protein quantification. In contrast to the global proteome profile of S. cerevisiae in response to heat shock, which remains largely unchanged as determined by label-free quantification, many of the chaperones are upregulated with an average two-fold increase in protein abundance. Interestingly, eight of the significantly upregulated chaperones are direct gene targets of heat shock transcription factor-1. By performing absolute quantification of chaperones under heat stress for the first time, we were able to evaluate the individual protein-level response. Furthermore, this SRM data was used to calibrate label-free quantification values for the proteome in absolute terms, thus improving relative quantification between the two conditions. This study significantly enhances the largely transcriptomic data available in the field and illustrates a more nuanced response at the protein level.
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Project description:Post-embryonic plant development must be coordinated in response to and with environmental feedback. Development of above-ground organs is orchestrated from stem cells in the center of the shoot apical meristem (SAM). Heat can pose significant abiotic stress to plants and induce a rapid heat shock response, developmental alterations, chromatin decondensation, and activation of transposable elements (TEs). However, most plant heat-stress studies are conducted with seedlings, and we know very little about cell-type-specific responses. Here we use fluorescent-activated nuclear sorting to isolate and characterize stem cells of wild type and mutants defective in TE defense and chromatin compaction after heat shock and after a long recovery. Our results indicate that stem cells can suppress heat shock response pathways to maintain developmental programs. Furthermore, mutants defective in DNA methylation fail to recover efficiently from heat stress and persistently activate heat shock factors and heat-inducible TEs. Heat stress also induces DNA methylation epimutations, especially in the CHG context, and we find hundreds of DNA methylation changes three weeks after stress. Our results underline the importance of disentangling cell type-specific environmental responses for understanding plant development.
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