Proteomics Moves From Expression to Turnover

Update and Future Perspective

Mary K Doherty; Phillip D Whitfield

Disclosures

Expert Rev Proteomics. 2011;8(3):325-334. 

In This Article

Defining Protein Turnover

Protein turnover refers to the continual renewal or replacement of protein. It is defined by the balance between protein synthesis and protein degradation.[6] During periods of steady state, the overall rate of protein synthesis is equal to the rate of protein degradation. When an organism or cell is growing, the rate of protein synthesis exceeds that of protein degradation (anabolism), while the opposite is true during periods of catabolism. This balancing act allows the cell to regulate function and provides flexibility to rapidly respond to cellular events. Protein synthesis is driven by the transcriptome and, although the concentration of mRNA transcript influences the downstream concentration of cognate protein, there is not always a direct correlation.[7–9] The rate of protein synthesis is also influenced by initiation of transcription and ribosomal activity. The ability to accurately define the rate of protein synthesis has wide applications in our understanding of genotype–phenotype relationships and in the study of pathophysiological processes. Proteins can be degraded by a number of pathways in eukaryotes. The ubiquitin proteasome system and autophagy have been of primary interest in recent years. The ubiquitin proteasome system allows for the selective degradation of proteins that have been tagged by a polyubiquitin chain and directed to the 26S proteasome. Autophagy is believed to be a system of bulk protein, and indeed organelle, degradation that is triggered during times of stress, such as starvation. The ability for a protein to replenish its protein population is essential for maintaining health and viability. Dysfunction of protein turnover has been linked to a range of diseases, particularly those prominent in an aging population, including Parkinson's disease,[10,11] Alzheimer's disease[12,13] and cancer.[14,15]

Measuring protein turnover is complex. Even in simple unicellular organisms such as bacteria and yeast, there are many up- and down-stream processes that must be considered. The major premise of any approach to calculate protein synthesis or degradation rates is the measurement of incorporation or loss of a labeled precursor into the protein. In general, it is imperative that we are able to accurately determine the extent to which the precursor pool has been labeled. Although the true precursor pool for protein synthesis is the aminoacyl-tRNA pool,[16–18] these are in low abundance in cells and are difficult to quantify with precision.[19–21] Therefore, it is common to see amino acids used as a surrogate for the aminoacyl-tRNA pool.

When using cells in culture, it is relatively easy to control the specific (radio)activity (SA) or relative isotope abundance (RIA) of the precursor by supplementation of the external medium with a substantial excess of the radiolabeled or stable isotope-labeled compound. The SA/RIA quickly reaches a plateau to unity, and subsequent sampling of the labeled proteins then permits assessment of the rate of turnover of the proteins. However, in more complex multicellular species, interactions occur between the different cellular pools. This leads to an inability to precisely control the precursor RIA or SA to the same extent, and therefore requires the labeling of the precursor pool to be determined experimentally (Figure 1).

Figure 1.

The requirement for relative isotope abundance calculation in multicellular systems. Proteins are continually being synthesized and degraded by cells. In animal systems, unlabeled amino acids are provided from the recycling of proteins from other tissues. When using stable isotope labeling approaches, it is therefore critical that the RIA of the precursor is accurately defined. Stable isotope-labeled amino acids are introduced to the system at a defined RIA. In this example, dietary RIA = 0.5. Dilution from other tissues will lower the true precursor RIA for protein synthesis. The precursor RIA can be calculated from the relative intensity of the mass spectral ions generated from the proteins of interest.
RIA: Relative isotope abundance.

Prior to the development of proteomic technologies, the principal way of determining protein turnover in complex organisms, particularly in human subjects, was to introduce tracers in either the flooding dose method or by continuous infusion. The tracer, usually a radioisotope, is introduced to the subject either via a bolus injection or through an intravenous line. Proteins can then be separated by 1 or 2DE and the level of incorporation of the tracer is then measured directly from the tissues of interest, typically by autoradiography. The practical aspects, advantages and disadvantages of these methods have been described extensively elsewhere.[3,22] The primary limitation of these approaches is that quantitative information is only easily obtained for bulk protein turnover. The lack of protein-specific information arose from the historical difficulties of labeling proteins to a sufficient degree that individual protein turnover could be assessed. Moreover, global studies were hindered by the ability to establish the true specific activity of the precursor pool and a lack of methods to measure the kinetics of long-lived proteins. Synthesis and degradation rates of specific individual proteins have been previously determined, but the approaches used do not lend themselves to the analysis of large cohorts of proteins simultaneously. Further issues relate to the observation that amino acids may, in some cases, influence protein turnover in intact organisms. Buse and Reid reported the potential for leucine to regulate protein turnover in muscle,[23] and Pannemans et al. reported that certain tracers can affect protein turnover in elderly women.[24]

Comments

3090D553-9492-4563-8681-AD288FA52ACE
Comments on Medscape are moderated and should be professional in tone and on topic. You must declare any conflicts of interest related to your comments and responses. Please see our Commenting Guide for further information. We reserve the right to remove posts at our sole discretion.
Post as:

processing....