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...

A new quantitative strategy has generated a comprehensive rate control map for protein synthesis in exponentially growing yeast cells. This analysis reveals the modularity of the system as well as highly non-stoichiometric relationships between components.

OBJECTIVEThe induction of hepatic glucose 6-phosphatase (G6pc) by glucose presents a paradox of glucose-induced glucose intolerance. We tested whether glucose regulation of liver gene expression is geared toward intracellular homeostasis.RESEARCH DESIGN AND METHODSThe effect of glucose-induced accumulation of phosphorylated intermediates on expression of glucokinase (Gck) and its regulator Gckr was determined in hepatocytes. Cell ATP and uric acid production were measured as indices of cell phosphate homeostasis.RESULTSAccumulation of phosphorylated intermediates in hepatocytes incubated at elevated glucose induced rapid and inverse changes in Gck (repression) and Gckr (induction) mRNA concomitantly with induction of G6pc, but had slower effects on the Gckr-to-Gck protein ratio. Dynamic metabolic labeling in mice and liver proteome analysis confirmed that Gckr and Gck are low-turnover proteins. Involvement of Max-like protein X in glucose-mediated Gck-repression was confirmed by chromatin immunoprecipitation analysis. Elevation of the Gck-to-Gckr ratio in hepatocytes was associated with glucose-dependent ATP depletion and elevated urate production confirming compromised phosphate homeostasis.CONCLUSIONSThe lowering by glucose of the Gck-to-Gckr ratio provides a potential explanation for the impaired hepatic glucose uptake in diabetes. Elevated uric acid production at an elevated Gck-to-Gckr ratio supports a role for glucose regulation of gene expression in hepatic phosphate homeostasis.

The measurement of protein turnover in tissues of intact animals is obtained by whole animal dynamic labelling studies, requiring dietary administration of precursor label. It is difficult to obtain full labelling of precursor amino acids in the diet and if partial labelling is used, calculation of the rate of turnover of each protein requires knowledge of the precursor relative isotope abundance (RIA). We describe an approach to dynamic labelling of proteins in the mouse with a commercial diet supplemented with a pure, deuterated essential amino acid. The pattern of isotopomer labelling can be used to recover the precursor RIA, and sampling of urinary secreted proteins can monitor the development of liver precursor RIA non-invasively. Time-series analysis of the labelling trajectories for individual proteins allows accurate determination of the first order rate constant for degradation. The acquisition of this parameter over multiple proteins permits turnover profiling of cellular proteins and comparisons of different tissues. The median rate of degradation of muscle protein is considerably lower than liver or kidney, with heart occupying an intermediate position.

BackgroundEjaculates contain a diverse mixture of sperm and seminal fluid proteins, the combination of which is crucial to male reproductive success under competitive conditions. Males should therefore tailor the production of different ejaculate components according to their social environment, with particular sensitivity to cues of sperm competition risk (i.e. how likely it is that females will mate promiscuously). Here we test this hypothesis using an established vertebrate model system, the house mouse (Mus musculus domesticus), combining experimental data with a quantitative proteomics analysis of seminal fluid composition. Our study tests for the first time how both sperm and seminal fluid components of the ejaculate are tailored to the social environment.ResultsOur quantitative proteomics analysis reveals that the relative production of different proteins found in seminal fluid – i.e. seminal fluid proteome composition – differs significantly according to cues of sperm competition risk. Using a conservative analytical approach to identify differential expression of individual seminal fluid components, at least seven of 31 secreted seminal fluid proteins examined showed consistent differences in relative abundance under high versus low sperm competition conditions. Notably three important proteins with potential roles in sperm competition – SVS 6, SVS 5 and CEACAM 10 – were more abundant in the high competition treatment groups. Total investment in both sperm and seminal fluid production also increased with cues of heightened sperm competition risk in the social environment. By contrast, relative investment in different ejaculate components was unaffected by cues of mating opportunities.ConclusionsOur study reveals significant plasticity in different ejaculate components, with the production of both sperm and non-sperm fractions of the ejaculate strongly influenced by the social environment. Sperm competition risk is thus shown to be a key factor in male ejaculate production decisions, including driving plasticity in seminal fluid composition.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-015-0197-2) contains supplementary material, which is available to authorized users.