Measurement of protein turnover rates by heavy water labeling of nonessential amino acids

Abstract

In vivo measurements of protein synthesis using isotope-labeled amino acids (AAs) are hampered by the heterogeneity of AA pools and, for slow turnover proteins, the difficulty and expense of long-term labeling. Continuous oral heavy water (²H₂O) labeling can safely maintain stable body water ²H enrichments for weeks or months. ²H is metabolically incorporated into C–H bonds of nonessential AAs (NEAAs) and hence into proteins. No posttranslational label exchange occurs, so ²H incorporation into protein NEAAs, in principle, reports on protein synthesis. Here, we show by mass isotopomer distribution analysis (MIDA) of ²H₂O-labeled rodent tissue proteins that metabolic ²H flux into C–H bonds of Ala, Gly, or Gln used for protein synthesis is nearly complete. By ²H₂O labeling of rodents, turnover of bone and muscle mixed proteins was quantified and stimulation of liver collagen synthesis by CCl₄ was detected. Kinetics of several human serum proteins were also measured, reproducing published t_1/2 estimates. Plateau enrichments in Ala varied among different proteins. Moderate amounts of protein, isolated chromatographically or electrophoretically, sufficed for kinetic analyses. In conclusion, ²H₂O labeling permits sensitive, quantitative, operationally simple measurements of protein turnover in vivo by the rise-to-plateau approach, especially for proteins with slow constitutive turnover.

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 (t_1/2) 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.