Measurement of protein turnover rates by heavy water labeling of nonessential amino acids
Introduction
Protein synthesis and turnover are essential for life, and much effort has gone into quantifying the rates of these processes in various biological settings by use of isotopic labels. Each of the commonly used labeling approaches, however, has drawbacks, especially for applications in vivo. Radioisotopes are too hazardous for routine use in humans, so non-radioactive, stable isotope labels are preferable. Most commonly, deuterium- (2H-) or 13C-labeled essential amino acids (AAs) are used. Their incorporation into proteins of interest, via protein synthesis, is tracked following bolus or continuous administration, using mass spectrometric (MS) analysis of protein hydrolyzates or peptide fragments [1].
A central challenge in any biosynthetic labeling experiment is to determine or infer the amount of label in the precursor pool [2], i.e., in the relevant AA-tRNAs when protein synthesis is measured. The intracellular pools of AAs and AA-tRNAs used for protein synthesis often are not in isotopic equilibrium with plasma AA, and a complex subcellular organization exists that is usually inaccessible to direct measurement [1], [2], [3], [4], [5], [6], [7]. One way to overcome this problem of the true precursor pool enrichment is to administer the labeled AA precursor continuously over several half-lives (t1/2) of the protein of interest. Eventually, label enrichment in the protein-bound AA approaches a plateau that equals the enrichment in the precursor pool [2]. This obviates the need to measure intracellular AA label content directly. In the case of protein biosynthetic labeling, the plateau isotopic enrichment, at 100% replacement of the protein by newly synthesized molecules, reflects labeling of the tRNA-AA pool during the labeling period [2], [6].
MS analysis of stable isotope incorporation into proteins also permits another approach for determining precursor enrichment. The label distributes into different mass variants (mass isotopomers), which differ in mass, due to different isotope content, but not in chemical structure. The pattern of mass isotopomers after a labeling experiment contains information about the precursor pool enrichment [2], [8], [9], [10], [11]. This information can be extracted by comparing the results of biosynthetic labeling experiments to predictions of computational modeling by mass isotopomer distribution analysis (MIDA) [2], [8], [9], [10], [11]. MIDA has been used successfully in measurements of protein synthesis [1], [5], [7], [9]. The combinatorial probability algorithms used for MIDA calculations have been reviewed in detail previously [9].
Particular difficulties in establishing the precursor/product relationship arise when isotope-tagged AAs are used to label slow-turnover proteins. In this setting, short-term labeling often results in insufficient isotope incorporation for MIDA. Continuous labeling to plateau can sometimes be achieved with stable isotope-labeled essential AAs [1], but is usually too expensive or impractical. We therefore looked for a novel biosynthetic labeling approach for applying the rise-to-plateau principle to slow-turnover proteins.
Labeling with heavy water (2H2O) has been used safely for decades as a tracer for physiologic studies of body water turnover [12], [13], [14] and the synthesis and turnover of lipids [8], [11], [14], [15], [16]. More recently, synthesis rates of nucleic acids have been measured with 2H2O [11], [17], [18], [19], [20], [21], [22]. No toxicities are observed below about 20 mol% 2H in body water [13], [14]. At lower doses (≈1–2% 2H in body water), no adverse effects have been observed in humans labeled continuously for several months [12], [18], [21]. Rodents have been kept at 3–20% 2H in body water for months or years without adverse effects to the labeled animals or their progeny [11], [17], [21].
The dynamics of 2H label in body water makes oral 2H2O a particularly useful biosynthetic label. In humans given oral 2H2O, the 2H label equilibrates in water throughout all tissues within ≈1 h, and when 2H2O administration is discontinued, the label decays with the half-life of body water (≈1 week) [23], [24], [25]. Constant 2H levels in body water (precursor pool enrichment) can therefore be achieved for extended periods through simple oral administration protocols. From body water, 2H label rapidly enters free nonessential AAs (NEAAs) through intermediary metabolic pathways (Fig. 1A); moreover, all free AAs are labeled at their α carbon (C2) positions via transamination. The 2H-labeled AAs then are incorporated into newly synthesized proteins via AA-tRNAs (Fig. 1B). Importantly, the enzymes of intermediary metabolism through which 2H-label enters free AAs do not act on AA residues after incorporation into the protein backbone. Unlike labile N–H or O–H bonds, C–H bonds do not exchange hydrogen spontaneously with body water. Thus, 2H from 2H2O does not enter into C–H bonds of AA posttranslationally. These features of NEAA metabolism make 2H2O a promising label for measuring protein synthesis and turnover.
Here, we address the general applicability of 2H2O labeling for measuring protein synthesis in vivo by the rise-to-plateau approach.
Section snippets
Animal studies
Sprague–Dawley rats (female, 7–8 weeks old, 200–250 g, Simonsen Inc., Gilroy, CA) and C57Bl/6J male mice, 4–6 weeks old, 10–15 g, Jackson Laboratories, Bar Harbor, ME) were housed individually (rats) or in groups of 5 (mice) in a specific pathogen-free facility with a 12 h light/12 h dark cycle, controlled temperature and humidity. Feeding (with Purina® rodent chow) and physical activity were ad libitum. All studies received prior approval from Animal Care and Use Committees at UC Berkeley and
Experimental approach
We wished to test whether continuous biosynthetic labeling with oral 2H2O, measured by GC/MS analysis of 2H incorporation into NEAA residues of proteins and by application of the rise-to-plateau kinetic approach, allows reliable measurement of turnover rates of long-lived proteins in vivo. The general experimental strategy consisted of the following steps: (1) Oral administration of 2H2O to animals or humans, to achieve stable 2H enrichments in body water; (2) isolation of proteins of interest
Discussion
The attractiveness of 2H2O as a metabolic label for long-term biosynthetic studies and its proven utility for measuring lipid and DNA turnover led us to examine its general utility for measuring protein turnover. Recently, we [39] used 2H2O labeling of Ala to measure dynamic exchange between tubulin α/β dimers and polymerized microtubules. Previs et al. tracked the time course of 2H incorporation from body water into Ala residues of human albumin [40], [41]. However, 2H2O labeling potentially
Acknowledgments
These studies were supported in part by NIH grants AI44767 and AI41401 (to M.K.H.) and by an unrestricted gift from KineMed, Inc. We thank Glen Lindwall, Abraham J. Bautista, Tim Riiff, and Kristen L. LaPrade for assistance with protein purification, Ablatt Mahsut for assistance with mass spectrometry, and Glen Lindwall for critically reading the manuscript. Marc K. Hellerstein is Chief of the Scientific Advisory Board and owns stock in KineMed, Inc.
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Contributed equally to this work.