Proteome Dynamics: Revisiting Turnover with a Global Perspective

Abstract

Although bulk protein turnover has been measured with the use of stable isotope labeled tracers for over half a century, it is only recently that the same approach has become applicable to the level of the proteome, permitting analysis of the turnover of many proteins instead of single proteins or an aggregated protein pool. The optimal experimental design for turnover studies is dependent on the nature of the biological system under study, which dictates the choice of precursor label, protein pool sampling strategy, and treatment of data. In this review we discuss different approaches and, in particular, explore how complexity in experimental design and data processing increases as we shift from unicellular to multicellular systems, in particular animals. The use of stable isotopes to trace metabolic processes, pioneered by Schoenheimer starting in 1935, elicited a paradigm shift in the perception of proteins, such that they were no longer considered as unchanging structural components of a cell that are replaced only when damaged by general "wear and tear" (1). These seminal studies introduced the concept of continual breakdown and re-synthesis as an ongoing metabolic process that truly reflects "The Dynamic State of Body Constituents" (2). This original work, which predates the discovery of the ribosome or the elucidation of the genetic code, placed protein turnover firmly in the category of highly active metabolic processes. In the ensuing period, huge progress has been made in clarification of the mechanisms of protein turnover, although our understanding of the subtleties of protein synthesis still exceeds our understanding of the corresponding destructive processes by which a protein is converted to constituent amino acids. Even now, it is difficult to describe the complete mechanistic details of the breakdown of any specific intracellular protein; we know the beginning (the mature protein), we know the end point (amino acids), and we may know some details of the intermediate processes (whether the protein is ubiquitylated prior to proteasomal degradation, whether the proteasome is involved, and so forth), but for most proteins, it is still not possible to define the exact route from specific intact protein to its pool of constituent amino acids. Part of the problem is that protein degradation is associated with a loss of tangibility; thus, loss of a band on a western blot is easy to observe, but monitoring of transiently existing intermediates in the process of degradation is rather difficult. Higher level questions, such as those posed in a recent review (3), define some of the challenges in the development of our understanding of proteome dynamics and may well require the development of new experimental approaches.It is (at least conceptually) convenient to distinguish between two distinct processes in the degradation of any protein: a commitment step and a completion step. The commitment step is the rate-limiting step and need not be proteolytic. For example, polyubiquitin conjugation and lysosomal in...

Protein Degradation Rate in Arabidopsis thaliana Leaf Growth and Development

“…Protein synthesis is a zero order process and is thus described as a proportional increase of its starting pool (K S /A) per day (Claydon and Beynon, 2012;Li et al, 2012). "A" is the starting abundance for a specific protein at the beginning of the labeling period (T0).…”

Protein kinetic signatures of the remodeling heart following isoproterenol stimulation

“…Malfunctions of protein turnover are increasingly recognized in numerous human disorders, including cystic fibrosis, neurodegenerative diseases, and heart diseases (1, 2). In the normal heart, protein turnover helps maintain the protein pool in homeostasis through continual synthesis and degradation (2,3). The turnover cycle becomes perturbed during cardiac injury and heart failure by factors including hypertrophic signaling (4), calcium regulation (5), and proteolytic stress (6,7), whereas increased oxidative stress compounds the reduced protein degradation capacity of the failing heart (8).…”

“…Although disrupted protein homeostasis is a hallmark of the remodeling heart, the technologies to quantify its effects on protein turnover have been lacking until very recently. Quantitative evaluations of protein synthesis and degradation have been well demonstrated in cultured cells using techniques such as stable isotope-labeled amino acid (SILAC) or fluorescence timers (14,15), but the proteome dynamics of freely dividing cells in vitro does not necessarily recapitulate the physical constraints and physiological regulations that occur in animals in vivo (3,16). Measuring protein turnover in vivo entails additional technical challenges including label delivery and tolerance, determination of precursor enrichment, and data interpretation (3,12,16).…”

“…Quantitative evaluations of protein synthesis and degradation have been well demonstrated in cultured cells using techniques such as stable isotope-labeled amino acid (SILAC) or fluorescence timers (14,15), but the proteome dynamics of freely dividing cells in vitro does not necessarily recapitulate the physical constraints and physiological regulations that occur in animals in vivo (3,16). Measuring protein turnover in vivo entails additional technical challenges including label delivery and tolerance, determination of precursor enrichment, and data interpretation (3,12,16). Stable isotope labeling using deuterium oxide ( 2 H 2 O) tracers has shown great potential for tracing protein turnover in mammals (17)(18)(19)(20)(21).…”

“…Because the rate of either protein synthesis or degradation (i.e., protein turnover) must deviate from normal values in order for the protein pool size to adjust to a new level, any permutation of proteomic states leaves behind a kinetic signature in the form of a fraction of the newly synthesized proteins in the protein pool (3,12). This kinetic signature can be measured through the incorporation of isotopes into proteins over time and may be exploited to identify unexpected disease proteins and their pathological implications independently of abundance measurements.…”