A high-fat diet increases hepatic mitochondrial turnover through restricted acetylation in a NAFLD mouse model.

Lysine acetylation of proteins has emerged as a key posttranslational modification (PTM) that regulates mitochondrial metabolism. Acetylation may regulate energy metabolism by inhibiting and affecting the stability of metabolic enzymes and oxidative phosphorylation (OxPhos) subunits. Although protein turnover can be easily measured, due to the low abundance of modified proteins, it has been difficult to evaluate the effect of acetylation on the stability of proteins in vivo. We applied 2H2O-metabolic labeling coupled with immunoaffinity and high-resolution mass spectrometry method to measure the stability of acetylated proteins in mouse liver based on their turnover rates. As a proof-of-concept, we assessed the consequence of high-fat diet (HFD)-induced altered acetylation in protein turnover in LDL receptor-deficient (LDLR-/-) mice susceptible to diet-induced nonalcoholic fatty liver disease (NAFLD). HFD feeding for 12 wk led to steatosis, the early stage of NAFLD. A significant reduction in acetylation of hepatic proteins was observed in NAFLD mice, based on immunoblot analysis and label-free quantification with mass spectrometry. Compared with control mice on a normal diet, NAFLD mice had overall increased turnover rates of hepatic proteins, including mitochondrial metabolic enzymes (0.159 ± 0.079 vs. 0.132 ± 0.068 day-1), suggesting their reduced stability. Also, acetylated proteins had slower turnover rates (increased stability) than native proteins in both groups (0.096 ± 0.056 vs. 0.170 ± 0.059 day-1 in control, and 0.111 ± 0.050 vs. 0.208 ± 0.074 day-1 in NAFLD). Furthermore, association analysis revealed a relationship between the HFD-induced decrease in acetylation and increased turnover rates for hepatic proteins in NAFLD mice. These changes were associated with increased expressions of the hepatic mitochondrial transcriptional factor (TFAM) and complex II subunit without any changes to other OxPhos proteins, suggesting that enhanced mitochondrial biogenesis prevented restricted acetylation-mediated depletion of mitochondrial proteins. We conclude that decreased acetylation of mitochondrial proteins may contribute to adaptive improved hepatic mitochondrial function in the early stages of NAFLD.NEW & NOTEWORTHY This is the first method to quantify acetylome dynamics in vivo. This method revealed acetylation-mediated altered hepatic mitochondrial protein turnover in response to a high-fat diet in a mouse model of NAFLD.

Hepatic Mitochondrial Defects in a Nonalcoholic Fatty Liver Disease Mouse Model Are Associated with Increased Degradation of Oxidative Phosphorylation Subunits.

Nonalcoholic fatty liver disease (NAFLD) is associated with hepatic mitochondrial dysfunction characterized by reduced ATP synthesis. We applied the 2H2O-metabolic labeling approach to test the hypothesis that the reduced stability of oxidative phosphorylation proteins contributes to mitochondrial dysfunction in a diet-induced mouse model of NAFLD. A high fat diet containing cholesterol (a so-called Western diet (WD)) led to hepatic oxidative stress, steatosis, inflammation and mild fibrosis, all markers of NAFLD, in low density cholesterol (LDL) receptor deficient (LDLR-/-) mice. In addition, compared with controls (LDLR-/- mice on normal diet), livers from NAFLD mice had reduced citrate synthase activity and ATP content, suggesting mitochondrial impairment. Proteome dynamics study revealed that mitochondrial defects are associated with reduced average half-lives of mitochondrial proteins in NAFLD mice (5.41 ± 0.46 versus 5.15 ± 0.49 day, p < 0.05). In particular, the WD reduced stability of oxidative phosphorylation subunits, including cytochrome b-c1 complex subunit 1 (5.9 ± 0.1 versus 3.4 ± 0.8 day), ATP synthase subunit α (6.3 ± 0.4 versus 5.5 ± 0.4 day) and ATP synthase F(0) complex subunit B1 of complex V (8.5 ± 0.6 versus 6.5 ± 0.2 day) (p < 0.05). These changes were associated with impaired complex III and F0F1-ATP synthase activities. Markers of mitophagy were increased, but proteasomal degradation activity were reduced in NAFLD mice liver, suggesting that ATP deficiency because of reduced stability of oxidative phosphorylation complex subunits contributed to inhibition of ubiquitin-proteasome and activation of mitophagy. In conclusion, the 2H2O-metabolic labeling approach shows that increased degradation of hepatic oxidative phosphorylation subunits contributed to mitochondrial impairment in NAFLD mice.

d2ome, Software for in Vivo Protein Turnover Analysis Using Heavy Water Labeling and LC-MS, Reveals Alterations of Hepatic Proteome Dynamics in a Mouse Model of NAFLD.

Metabolic labeling with heavy water followed by LC-MS is a high throughput approach to study proteostasis in vivo. Advances in mass spectrometry and sample processing have allowed consistent detection of thousands of proteins at multiple time points. However, freely available automated bioinformatics tools to analyze and extract protein decay rate constants are lacking. Here, we describe d2ome-a robust, automated software solution for in vivo protein turnover analysis. d2ome is highly scalable, uses innovative approaches to nonlinear fitting, implements Grubbs' outlier detection and removal, uses weighted-averaging of replicates, applies a data dependent elution time windowing, and uses mass accuracy in peak detection. Here, we discuss the application of d2ome in a comparative study of protein turnover in the livers of normal vs Western diet-fed LDLR-/- mice (mouse model of nonalcoholic fatty liver disease), which contained 256 LC-MS experiments. The study revealed reduced stability of 40S ribosomal protein subunits in the Western diet-fed mice.

Cellular prion protein regulates its own α-cleavage through ADAM8 in skeletal muscle.

The ubiquitously expressed cellular prion protein (PrP(C)) is subjected to the physiological α-cleavage at a region critical for both PrP toxicity and the conversion of PrP(C) to its pathogenic prion form (PrP(Sc)), generating the C1 and N1 fragments. The C1 fragment can activate caspase 3 while the N1 fragment is neuroprotective. Recent articles indicate that ADAM10, ADAM17, and ADAM9 may not play a prominent role in the α-cleavage of PrP(C) as previously thought, raising questions on the identity of the responsible protease(s). Here we show that, ADAM8 can directly cleave PrP to generate C1 in vitro and PrP C1/full-length ratio is greatly decreased in the skeletal muscles of ADAM8 knock-out mice; in addition, the PrP C1/full-length ratio is linearly correlated with ADAM8 protein level in myoblast cell line C2C12 and in skeletal muscle tissues of transgenic mice. These results indicate that ADAM8 is the primary protease responsible for the α-cleavage of PrP(C) in muscle cells. Moreover, we found that overexpression of PrP(C) led to up-regulation of ADAM8, suggesting that PrP(C) may regulate its own α-cleavage through modulating ADAM8 activity.