The 2H2O method allowed the detection of early in vivo impairments in HDL metabolism and function that were related to hyperglycemia-induced glycation of ApoAI in T2DM.

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.

Type 2 diabetes mellitus (T2DM) is associated with oxidative stress and perturbed iron metabolism. Serotransferrin (Trf) and ceruloplasmin (Cp) are two key proteins involved in iron metabolism and anti-oxidant defense. Non-enzymatic glycation and oxidative modification of plasma proteins are known to occur under hyperglycemia and oxidative stress. In this study, shotgun proteomics and 2H2O-based metabolic labeling were used to characterize post-translational modifications and assess the kinetics of Trf and Cp in T2DM patients and matched controls in vivo. Six early lysine (Amadori) and one advanced arginine glycation were detected in Trf. No glycation, but five asparagine deamidations, were found in Cp. T2DM patients had increased fractional catabolic rates of both Trf and Cp that correlated with HbA1c (p < 0.05). The glycated Trf population was subject to an even faster degradation compared to the total Trf pool, suggesting that hyperglycemia contributed to an increased Trf degradation in T2DM patients. Enhanced production of Trf and Cp kept their levels stable. The changes in Trf and Cp turnover were associated with increased systemic oxidative stress without any alteration in iron status in T2DM. These findings can help better understand the potential role of altered Trf and Cp metabolism in the pathogenesis of T2DM and other diseases.

Background: Endoproteolytic ␣-cleavage of cellular prion protein (PrP C ) regulates PrP C toxicity and functions; the responsible protease(s) is uncertain. Results: ADAM8 performs ␣-cleavage of PrP C and PrP C overexpression up-regulates ADAM8 in muscle. Conclusion: ADAM8 is the primary protease for the ␣-cleavage of PrP C that appears self-regulated through ADAM8 in muscle. Significance: This advances our understandings on physiological processing and functions of PrP C .

Nonalcoholic fatty liver disease (NAFLD) is associated with hepatic mitochondrial dysfunction characterized by reduced ATP synthesis. We applied the HO-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 5.15 ± 0.49 day, < ). In particular, the WD reduced stability of oxidative phosphorylation subunits, including cytochrome b- complex subunit 1 (5.9 ± 0.1 3.4 ± 0.8 day), ATP synthase subunit α (6.3 ± 0.4 5.5 ± 0.4 day) and ATP synthase F(0) complex subunit B1 of complex V (8.5 ± 0.6 6.5 ± 0.2 day) ( < ). 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, theHO-metabolic labeling approach shows that increased degradation of hepatic oxidative phosphorylation subunits contributed to mitochondrial impairment in NAFLD mice.