Protein turnover: Measurement of proteome dynamics by whole animal metabolic labelling with stable isotope labelled amino acids

Claydon, Amy J.; Thom, Michael D.; Hurst, Jane L.; Beynon, Robert J.

doi:10.1002/pmic.201100556

Cited by 77 publications

(104 citation statements)

References 38 publications

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“…Rules Governing Turnover Rate of Proteins-Other metabolic labeling studies suggest that protein turnover rates differ across mammalian organs (20,21). Our results demonstrate that such tissue-specific differences are preserved in the mitochondrial proteome (Fig.…”

Section: Rates Of Protein Turnover In Cardiac and Hepatic Mitochondrisupporting

confidence: 58%

Metabolic Labeling Reveals Proteome Dynamics of Mouse Mitochondria

Kim

Wang

Kim

et al. 2012

Molecular & Cellular Proteomics

161

187

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Mitochondrial dysfunction is associated with many human diseases. Mitochondrial damage is exacerbated by inadequate protein quality control and often further contributes to pathogenesis. The maintenance of mitochondrial functions requires a delicate balance of continuous protein synthesis and degradation, i.e. protein turnover. To understand mitochondrial protein dynamics in vivo, we designed a metabolic heavy water ( 2 H 2 O) labeling strategy customized to examine individual protein turnover in the mitochondria in a systematic fashion. Mice were fed with 2 H 2 O at a minimal level (<5% body water) without physiological impacts. Mitochondrial proteins were analyzed from 9 mice at each of the 13 time points between 0 and 90 days (d) of labeling. A novel multiparameter fitting approach computationally determined the normalized peak areas of peptide mass isotopomers at initial and steady-state time points and permitted the protein halflife to be determined without plateau-level 2 H incorporation. We characterized the turnover rates of 458 proteins in mouse cardiac and hepatic mitochondria and found median turnover rates of 0.0402 d ؊1 and 0.163 d ؊1 , respectively, corresponding to median half-lives of 17.2 d and 4.26 d. Mitochondria in the heart and those in the liver exhibited distinct turnover kinetics, with limited synchronization within functional clusters. We observed considerable interprotein differences in turnover rates in both organs, with half-lives spanning from hours to months (ϳ60 d). Our proteomics platform demonstrates the first large-scale analysis of mitochondrial protein turnover rates in vivo, with potential applications in translational research. Molecular & Cellular

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Section: Rates Of Protein Turnover In Cardiac and Hepatic Mitochondrisupporting

confidence: 58%

Metabolic Labeling Reveals Proteome Dynamics of Mouse Mitochondria

Kim

Wang

Kim

et al. 2012

Molecular & Cellular Proteomics

161

187

View full text Add to dashboard Cite

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“…The magnitude of this process should not be underestimated: a young mouse may replace all of its liver protein every 24 h, dropping in the adult to 50% per day (21). Turnover rates in skeletal muscle are lower, perhaps reflecting the relative mass of the two tissues, in addition to their different metabolic profiles and roles (11,22). Moreover, the age-dependent decline in protein turnover in skeletal muscle is more pronounced than in liver, which might reflect the decline in muscle function in older animals.…”

mentioning

confidence: 99%

“…The individually evolved susceptibility of each protein to the degradative process is thus modulated and tempered by the overall activity of the proteolytic machinery in each tissue; it could therefore be assumed that the baseline activity of the degradative machinery (probably the commitment step) is quantitatively different between two tissues, and the rate of entry of an individual protein into this process will thus place it in the same position in the overall degradation profile in each tissue. If this is true, then a prediction might be that for a set of proteins that are expressed in two tissues, the relationship between the first-order rate constants should be linear with a slope that is not unity; initial indications are that with some exceptions, this prediction will hold (22).…”

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confidence: 99%

“…Deviations from simple exponential labeling curves are to be anticipated, and will be revealed by higher granularity in time sampling (22). Factors such as metabolic reutilization of stable isotope atoms, precursor pool equilibration, and recycling of amino acids released from protein degradation back into the protein pool will all complicate analysis, once there are sufficient data to parameterize such complications (6).…”

mentioning

confidence: 99%

“…In more complex systems, such as animals, it is more challenging to design dynamic labeling experiments in which the precursor RIA is unity, particularly when the precursor is a specific amino acid. Diets with a high level of labeling are generated by compounding new dietary formulations derived from algal, bacterial, or yeast cultures that have been grown in the presence of a labeled precursor and subsequently incorporated into a feed (22,26,(42)(43)(44). The challenge in single amino acid labeling for animal studies has been met in two ways.…”

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confidence: 99%

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Proteome Dynamics: Revisiting Turnover with a Global Perspective

Claydon

Beynon

2012

Molecular & Cellular Proteomics

Self Cite

115

168

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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...

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Proteomics in Nutritional Systems Biology: Defining Health

Kussmann

Fay

2013

Foodomics

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Protein turnover: Measurement of proteome dynamics by whole animal metabolic labelling with stable isotope labelled amino acids

Cited by 77 publications

References 38 publications

Metabolic Labeling Reveals Proteome Dynamics of Mouse Mitochondria

Metabolic Labeling Reveals Proteome Dynamics of Mouse Mitochondria

Proteome Dynamics: Revisiting Turnover with a Global Perspective

Proteomics in Nutritional Systems Biology: Defining Health

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