Degradation Of Individual Proteins Research Articles

Degradation of ribosomal and chaperone proteins is attenuated during the differentiation of replicatively aged C2C12 myoblasts.

BackgroundCell assays are important for investigating the mechanisms of ageing, including losses in protein homeostasis and ‘proteostasis collapse’. We used novel isotopic labelling and proteomic methods to investigate protein turnover in replicatively aged (>140 population doublings) murine C2C12 myoblasts that exhibit impaired differentiation and serve as a model for age‐related declines in muscle homeostasis.MethodsThe Absolute Dynamic Profiling Technique for Proteomics (Proteo‐ADPT) was used to investigate proteostasis in young (passage 6–10) and replicatively aged (passage 48–50) C2C12 myoblast cultures supplemented with deuterium oxide (D2O) during early (0–24 h) or late (72–96 h) periods of differentiation. Peptide mass spectrometry was used to quantify the absolute rates of abundance change, synthesis and degradation of individual proteins.ResultsYoung cells exhibited a consistent ~25% rise in protein accretion over the 96‐h experimental period. In aged cells, protein accretion increased by 32% (P < 0.05) during early differentiation, but then fell back to baseline levels by 96‐h. Proteo‐ADPT encompassed 116 proteins and 74 proteins exhibited significantly (P < 0.05, FDR < 5% interaction between age × differentiation stage) different changes in abundance between young and aged cells at early and later periods of differentiation, including proteins associated with translation, glycolysis, cell–cell adhesion, ribosomal biogenesis, and the regulation of cell shape. During early differentiation, heat shock and ribosomal protein abundances increased in aged cells due to suppressed degradation rather than heightened synthesis. For instance, HS90A increased at a rate of 10.62 ± 1.60 ng/well/h in aged which was significantly greater than the rate of accretion (1.86 ± 0.49 ng/well/h) in young cells. HS90A synthesis was similar in young (21.23 ± 3.40 ng/well/h) and aged (23.69 ± 1.13 ng/well/h), but HS90A degradation was significantly (P = 0.05) greater in young (19.37 ± 2.93 ng/well/h) versus aged (13.06 ± 0.76 ng/well/h) cells. During later differentiation the HS90A degradation (8.94 ± 0.38 ng/well/h) and synthesis (7.89 ± 1.28 ng/well/h) declined and were significantly less than the positive net balance between synthesis and degradation (synthesis = 28.14 ± 3.70 ng/well/h vs. degradation = 21.49 ± 3.13 ng/well/h) in young cells.ConclusionsOur results suggest a loss of proteome quality as a precursor to the lack of fusion of aged myoblasts. The quality of key chaperone proteins, including HS90A, HS90B and HSP7C was reduced in aged cells and may account for the disruption to cell signalling required for the later stages of differentiation and fusion.

Adaptation of rat fast-twitch muscle to endurance activity is underpinned by changes to protein degradation as well as protein synthesis.

Muscle adaptations to exercise are underpinned by alterations to the abundance of individual proteins, which may occur through a change either to the synthesis or degradation of each protein. We used deuterium oxide (2 H2 O) labeling and chronic low-frequency stimulation (CLFS) in vivo to investigate the synthesis, abundance, and degradation of individual proteins during exercise-induced muscle adaptation. Independent groups of rats received CLFS (10Hz, 24h/d) and 2 H2 O for 0, 10, 20, or 30 days. The extensor digitorum longus (EDL) was isolated from stimulated (Stim) and contralateral non-stimulated (Ctrl) legs. Proteomic analysis encompassed 38 myofibrillar and 46 soluble proteins and the rates of change in abundance, synthesis, and degradation were reported in absolute (ng/d) units. Overall, synthesis and degradation made equal contributions to the adaptation of the proteome, including instances where a decrease in protein-specific degradation primarily accounted for the increase in abundance of the protein.

Analysis of individual protein turnover in live animals on a proteome-wide scale.

Classical quantitative proteomics studies focus on the relative or absolute concentration of proteins at a given time. In contrast, the investigation of protein turnover reveals the dynamics leading to these states. Analyzing the balance between synthesis and degradation of individual proteins provides insights into the regulation of protein concentration and helps understanding underlying biological processes. Comparing the half-lives of proteins allows detecting functional relationships and common regulation mechanisms. Moreover, comparing turnover of individual brain and plasma proteins between control- and treatment-groups indicates turnover changes induced by the treatment.Here, we describe a procedure for determining turnover information of individual proteins in mice on a proteome-wide scale based on partial (15)N metabolic labeling. We will outline the complete experimental workflow starting from (15)N labeling the animals over sample preparation and mass spectrometric measurement up to the analysis of the data.

A proteomics strategy for determining the synthesis and degradation rates of individual proteins in fish

In order to study the protein dynamics in the tissues of fish we have developed a proteomics-based strategy to determine the rates of synthesis and degradation of individual proteins. We have demonstrated the feasibility of this approach by measuring the turnover of multiple isoforms of parvalbumin (β1-7) in the skeletal muscle of common carp (Cyprinus carpio). A stable isotope-labelled amino acid ([(2)H(7)] l-leucine) was administered to the carp via the diet and its incorporation into the isoforms of parvalbumin in muscle over time was monitored by LC-MS analysis of signature peptides. The relative isotope abundance was calculated and used to deconvolute the data. The β7 parvalbumin isoform had a rate of synthesis that was greater than the rate of degradation. In contrast the rate of degradation of the β5 isoform exceeded its rate of synthesis, whilst the analysis revealed that the other parvalbumin β-isoforms (β1, β2, β3, β4 and β6) had a rate of synthesis that was equal to the rate of degradation. This work has addressed a number of technical challenges and represents the first study to use proteomic approaches to measure the turnover of individual proteins in fish.

The effects of sensory stimulation on the free amino acid pool of the developing brain.

Developing chicks were subjected to three different paradigmata of sensory stimulation, and the effects on the free amino acid concentrations in the blood and in various brain regions were monitored. The free amino acid pool of individual brain regions was found to be affected in a treatment- and age-specific manner. The increased neuronal activity resulting from sensory stimulation seems to affect intrinsic factors involved in the regulation of the free amino acid pool, most likely via modulations of the rate of metabolization of individual amino acids and/or of the rate of synthesis and degradation of individual proteins in certain brain regions.

Intracellular proteolysis: the elusive intermediates.

~~ protein substrates that are susceptible to that machinery to a greater or lesser extent. The study of bulk proteolysis, usually determined as the appearance of acid-soluble low-M, products derived from a prelabelled protein pool, yields information primarily on the cellular locations and machinery that is responsible for the late events in cellular proteolysis. By contrast, the measurement of the half-life of native individual proteins allows the derivation of the rate but yields little about the route of degradation. However, information on absolute, relative and changing rates of degradation of individual proteins may be correlated with structural properties, permitting some analysis of the features of the substrate that might dispose