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1.
Sci Rep ; 11(1): 13084, 2021 06 22.
Article in English | MEDLINE | ID: mdl-34158536

ABSTRACT

The eukaryotic chaperonin TRiC/CCT is a large ATP-dependent complex essential for cellular protein folding. Its subunit arrangement into two stacked eight-membered hetero-oligomeric rings is conserved from yeast to man. A recent breakthrough enables production of functional human TRiC (hTRiC) from insect cells. Here, we apply a suite of mass spectrometry techniques to characterize recombinant hTRiC. We find all subunits CCT1-8 are N-terminally processed by combinations of methionine excision and acetylation observed in native human TRiC. Dissociation by organic solvents yields primarily monomeric subunits with a small population of CCT dimers. Notably, some dimers feature non-canonical inter-subunit contacts absent in the initial hTRiC. This indicates individual CCT monomers can promiscuously re-assemble into dimers, and lack the information to assume the specific interface pairings in the holocomplex. CCT5 is consistently the most stable subunit and engages in the greatest number of non-canonical dimer pairings. These findings confirm physiologically relevant post-translational processing and function of recombinant hTRiC and offer quantitative insight into the relative stabilities of TRiC subunits and interfaces, a key step toward reconstructing its assembly mechanism. Our results also highlight the importance of assigning contacts identified by native mass spectrometry after solution dissociation as canonical or non-canonical when investigating multimeric assemblies.


Subject(s)
Chaperonin Containing TCP-1/chemistry , Chaperonin Containing TCP-1/metabolism , Chaperonins/chemistry , Chaperonins/metabolism , Cryoelectron Microscopy/methods , Humans , Mass Spectrometry/methods , Protein Conformation , Protein Folding , Protein Subunits/metabolism
2.
PLoS Comput Biol ; 10(1): e1003449, 2014 Jan.
Article in English | MEDLINE | ID: mdl-24499934

ABSTRACT

Identifying enhancers regulating gene expression remains an important and challenging task. While recent sequencing-based methods provide epigenomic characteristics that correlate well with enhancer activity, it remains onerous to comprehensively identify all enhancers across development. Here we introduce a computational framework to identify tissue-specific enhancers evolving under purifying selection. First, we incorporate high-confidence binding site predictions with target gene functional enrichment analysis to identify transcription factors (TFs) likely functioning in a particular context. We then search the genome for clusters of binding sites for these TFs, overcoming previous constraints associated with biased manual curation of TFs or enhancers. Applying our method to the placenta, we find 33 known and implicate 17 novel TFs in placental function, and discover 2,216 putative placenta enhancers. Using luciferase reporter assays, 31/36 (86%) tested candidates drive activity in placental cells. Our predictions agree well with recent epigenomic data in human and mouse, yet over half our loci, including 7/8 (87%) tested regions, are novel. Finally, we establish that our method is generalizable by applying it to 5 additional tissues: heart, pancreas, blood vessel, bone marrow, and liver.


Subject(s)
Enhancer Elements, Genetic , Transcription Factors/metabolism , Algorithms , Amino Acid Motifs , Animals , Automation , Binding Sites , Cluster Analysis , Computational Biology , Computer Simulation , Epigenomics , Female , Gene Expression Profiling , Gene Expression Regulation , Humans , Mice , Placenta/physiology , Pregnancy , Trophoblasts/cytology
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