The Molecular Floodgates of Stress-Induced Senescence Reveal Translation, Signalling and Protein Activity Central to the Post-Mortem Proteome

Abstract

The transitioning of cells during the systemic demise of an organism is poorly understood. Here, we present evidence that organismal death is accompanied by a common and sequential molecular flood of stress-induced events that propagate the senescence phenotype, and this phenotype is preserved in the proteome after death. We demonstrate activation of “death” pathways involvement in diseases of ageing, with biochemical mechanisms mapping onto neurological damage, embryonic development, the inflammatory response, cardiac disease and ultimately cancer with increased significance. There is sufficient bioavailability of the building blocks required to support the continued translation, energy, and functional catalytic activity of proteins. Significant abundance changes occur in 1258 proteins across 1 to 720 h post-mortem of the 12-week-old mouse mandible. Protein abundance increases concord with enzyme activity, while mitochondrial dysfunction is evident with metabolic reprogramming. This study reveals differences in protein abundances which are akin to states of stress-induced premature senescence (SIPS). The control of these pathways is significant for a large number of biological scenarios. Understanding how these pathways function during the process of cellular death holds promise in generating novel solutions capable of overcoming disease complications, maintaining organ transplant viability and could influence the findings of proteomics through “deep-time” of individuals with no historically recorded cause of death.

Serum proteome of dogs with chronic enteropathy

“…Total protein concentration was assessed using the 2D Quant kit (Cytiva, Massachusetts, USA) as per manufacturer instructions. Preparation of samples was as described 28 . Briefly, samples of 100 μg total protein were mixed in 50 μL AMBIC buffer (50 mM Ammoniumbicarbonate, 10 mM DTT, 2 M urea at pH 8) and digested with trypsin at 25°C for 16 hours in a 1:100 enzyme‐to‐protein ratio based on the calculated serum protein concentration.…”

“…Digested peptides were reconstituted in 10 μL 0.1% formic acid and separated by nano‐LC using an Ultimate 3000 HPLC and autosampler (Dionex, Amsterdam, Netherlands) and followed methods similar to those described previously 28 . Briefly, the sample, 1.7 μL from 10 μL, was loaded onto a micro C18 pre‐column (300 μm × 5 mm, Dionex) with H 2 O:CH 3 CN (98:2, 0.1% TFA) at 10 μL min −1 .…”

Tryptophan Metabolism ‘Hub’ Gene Expression Associates with Increased Inflammation and Severe Disease Outcomes in COVID-19 Infection and Inflammatory Bowel Disease

“…RNA Seq analysis was used to compare expression values across 5 control and 4 paired I and NI ileal CD biopsy samples [ 67 ]. Global and Parallel Reaction Monitoring (PRM) proteomics was carried out on 100 µg protein extracted from frozen bowel tissue using a cell shearing method as previously described [ 71 ]. Serums were sampled from CD patients enrolled in the Confocal Endoscopy study measuring fluorescein leak.…”

“…Study two analysed proteome and metabolome of nasal epithelium (NE) swabs. NE swabs were resuspended in 400 µL of 80% methanol with 6mg of 1.0 mm zirconium beads and used a cell shearing method [ 71 ]. Proteins were pelleted and 50 µg enzymatically treated with trypsin, while supernatant containing metabolites were stored at −80 °C until required.…”

“…Mass spectrometry was carried out using a QExactive (Thermo Electron, Bremen, Germany) mass spectrometer run in DDA mode using 1.5 μg (2.0 μL from 10 μL) as previously described [ 71 ]. PRM was used to analyse all product ions resulting from specific precursor ions.…”