Dataset Information

Multiple experiments: Analysis of the HTPS FT; Kinetics of Thrombin and Chymotrypsin; Effect of temperature on Thrombin and Chymotrypsin

ABSTRACT: 1) Analysis of HTPS FT (analysis of the background, without protease) 2) Analysis of specificity and activity of Thrombin and Chymotrypsin at multiple time point (0,5,15,30,60,120,240 min) 3) Analysis of specificity and activity of Thrombin and Chymotrpysin with 2 hr incubation at 20C, 25C and 37C

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Federico Uliana

LAB HEAD: Ruedi Aebersold

PROVIDER: PXD022972 | Pride | 2021-02-04

REPOSITORIES: Pride

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Publications

Mapping specificity, cleavage entropy, allosteric changes and substrates of blood proteases in a high-throughput screen.

Uliana Federico F Vizovišek Matej M Acquasaliente Laura L Ciuffa Rodolfo R Fossati Andrea A Frommelt Fabian F Goetze Sandra S Wollscheid Bernd B Gstaiger Matthias M De Filippis Vincenzo V Auf dem Keller Ulrich U Aebersold Ruedi R

Nature communications 20210316 1

Proteases are among the largest protein families and critical regulators of biochemical processes like apoptosis and blood coagulation. Knowledge of proteases has been expanded by the development of proteomic approaches, however, technology for multiplexed screening of proteases within native environments is currently lacking behind. Here we introduce a simple method to profile protease activity based on isolation of protease products from native lysates using a 96FASP filter, their analysis in ...[more]

PMID: 33727531

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Project description:Maintenance of the correct level and organization of nucleosomes is crucial for genome function. Here we uncover a role for a conserved bromodomain AAA-ATPase, Abo1, in maintenance of nucleosome architecture in fission yeast. Cells lacking abo1+ experience both a reduction and mis-positioning of nucleosomes at transcribed sequences in addition to increased intragenic transcription, phenotypes that are hallmarks of defective chromatin re-establishment behind RNA polymerase II. Abo1 is recruited to gene sequences and associates with histone H3 and the histone chaperone FACT. Furthermore, the distribution of Abo1 on chromatin is disturbed by impaired FACT function. The role of Abo1 extends to some promoters and also to silent heterochromatin. Abo1 is recruited to pericentromeric heterochromatin independently of the HP1 ortholog, Swi6, where it enforces proper nucleosome occupancy. Consequently, loss of Abo1 alleviates silencing and causes elevated chromosome mis-segregation. We suggest that Abo1 provides a histone chaperone function that maintains nucleosome architecture genome-wide. A chromatin-seq/MNase-seq approach called Chromatin Particle Spectrum Analysis (Kent et al., (2011) Nucleic Acids Res. 39:e26) was used to map and compare nucleosome position in wild-type (strain 972) and isogenic abo1 knock-out (strain HM463) fission yeast cells. For each strain, three independent in vivo MNase digest bio-reps were performed and the purified DNA pooled. CPSA was performed using paired-end mode Illumina technology with pooled samples multiplexed over two HiSeq2000 lanes. Each CPSA paired read describes a microcococcal nuclease (MNase) resistant DNA species from chromatin, with the insert-size equivalent to the size of DNA protection. For each strain type, two analysed data sets are provided here: one listing the genomic distribution of MNase-protected DNAs of 150bp (â150bp CPSA size classâ) and corresponding to mono-nucleosomes; the other listing the genomic distribution of MNase-protected DNAs of 300bp (â300bp CPSA size classâ) and corresponding to di-nucleosomes.

Dataset Information

Multiple experiments: Analysis of the HTPS FT; Kinetics of Thrombin and Chymotrypsin; Effect of temperature on Thrombin and Chymotrypsin

Publications

Mapping specificity, cleavage entropy, allosteric changes and substrates of blood proteases in a high-throughput screen.

Similar Datasets

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