The Effect of Long Term Calorie Restriction on in Vivo Hepatic Proteostatis: A Novel Combination of Dynamic and Quantitative Proteomics

Abstract

Calorie restriction (CR) promotes longevity. A prevalent mechanistic hypothesis explaining this effect suggests that protein degradation, including mitochondrial autophagy, is increased with CR, removing damaged proteins and improving cellular fitness. At steady state, increased catabolism must be balanced by increasing mitochondrial biogenesis and protein synthesis, resulting in faster protein replacement rates. To test this hypothesis, we measured replacement kinetics and relative concentrations of hundreds of proteins in vivo in long-term CR and ad libitum-fed mice using metabolic 2H2O-labeling combined with the Stable Isotope Labeling in Mammals protocol and LC-MS/MS analysis of mass isotopomer abundances in tryptic peptides. CR reduced absolute synthesis and breakdown rates of almost all measured hepatic proteins and prolonged the half-lives of most (∼80%), particularly mitochondrial proteins (but not ribosomal subunits). Proteins with related functions exhibited coordinated changes in relative concentration and replacement rates. In silico expression pathway interrogation allowed the testing of potential regulators of altered network dynamics (e.g. peroxisome proliferator-activated receptor gamma coactivator 1-alpha). In summary, our combination of dynamic and quantitative proteomics suggests that long-term CR reduces mitochondrial biogenesis and mitophagy. Our findings contradict the theory that CR increases mitochondrial protein turnover and provide compelling evidence that cellular fitness is accompanied by reduced global protein synthetic burden.

Protein kinetic signatures of the remodeling heart following isoproterenol stimulation

“…Also expectedly, immunoblots failed to detect evidence for increased expression of the tested glycolytic proteins. Taken together, these data suggest that changes in protein turnover and protein abundance in a system are largely independent parameters, an observation that is consistent with a recent study in 2 H 2 O and SILAC doubly labeled mice (41).…”

Reduced in vivo hepatic proteome replacement rates but not cell proliferation rates predict maximum lifespan extension in mice

“…To capture changes in both the RR constants ( k , fraction new protein per day) and RPS of individual proteins, we combined heavy water labeling with internal S table I sotope La beling in M ammals (SILAM) standards and measured both by LC‐MS/MS. The rate of incorporation of deuterium from heavy water into proteins was used to calculate RRs, as previously described (Price et al ., 2012b). To calculate the RPS of proteins, liver homogenates were spiked with an exogenous heavy‐labeled SILAM standard (derived from the liver of a mouse fully labeled with 15 N).…”

“…aRelative to control counterparts.bHomozygous for loss‐of‐function recessive mutation in Pit1 gene.cDuration of intervention taken to be the age of the animals at the time of euthanasia.d25% calorie‐restricted relative to AL.eAdapted from (Price et al ., 2012b). …”

Circulating protein synthesis rates reveal skeletal muscle proteome dynamics

“…We (25,(29)(30)(31) and others (32,33) have shown that the second problem can be addressed by combining stable isotope label incorporation with tandem mass spectrometric-based proteomics techniques. In particular, isotope ratio measurements using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) with quadrupole-time-of-flight (Q-ToF) instruments can be performed in the scan mode on trypsinderived peptides with sufficient analytic accuracy to quantify synthesis rates of hundreds of proteins concurrently after relatively low-level in vivo 2 H 2 O labeling (25,(29)(30)(31). Here, to address the third problem noted above, we report the development and validation of blood test approach for measuring the integrated rate of muscle protein synthesis over days to weeks.…”

“…This minimally invasive method can be seen as a virtual biopsy for monitoring skeletal muscle protein synthesis rates. We asked whether the labeling kinetics of proteins in plasma that were synthesized in skeletal muscle, such as creatine kinase M-type (CK-M) and carbonic anhydrase 3 (CA-3) the dynamics of the skeletal muscle proteome, using 2 H 2 O labeling combined with LC-MS/MS analysis (25,(29)(30)(31)(32)(33). In addition, the synthesis and turnover rates of multiple proteins across the proteome (i.e., proteome dynamics) had not previously been characterized in human skeletal muscle.…”