Highlights d Discovery of prognosis-associated proteins and pathways at early stage of LUAD d Proteomics revealed three subtypes related to clinical and molecular features d Identification of subtype-specific kinases and cancerassociated phosphoproteins d Identification of potential prognostic biomarkers and drug targets in LUAD

Ubiquitin-specific protease 14 (USP14) is one of the major proteasome-associated deubiquitinating enzymes critical for proteome homeostasis. However, substrates of USP14 remain largely unknown, hindering the understanding of its functional roles. Here we conduct a comprehensive proteome, ubiquitinome and interactome analysis for USP14 substrate screening. Bioinformatics analysis reveals broad new potential roles of USP14, especially in lipid and carbohydrate metabolism. Among the potential substrates identified, we show that fatty acid synthase (FASN), a key enzyme involved in hepatic lipogenesis, is a bona fide substrate of USP14. USP14 directly interacts with and increases FASN stability. As a result, overexpression of USP14 promotes liver triglyceride accumulation in C57BL/6 mice, whereas genetic ablation or pharmacological inhibition of USP14 ameliorates hepatosteatosis, hyperglycemia and insulin resistance in obese mice. In conclusion, our findings reveal for the first time an indispensable role of USP14 in hepatosteatosis through FASN stabilization.

Type 2 diabetes (T2D) is a major chronic healthcare concern worldwide. Emerging evidence suggests that a histone-modification-mediated epigenetic mechanism underlies T2D. Nevertheless, the dynamics of histone marks in T2D have not yet been carefully analyzed. Using a mass spectrometry-based label-free and chemical stable isotope labeling quantitative proteomic approach, we systematically profiled liver histone post-translational modifications (PTMs) in a prediabetic high-fat diet-induced obese (DIO) mouse model. We identified 170 histone marks, 30 of which were previously unknown. Interestingly, about 30% of the histone marks identified in DIO mouse liver belonged to a set of recently reported lysine acylation modifications, including propionylation, butyrylation, malonylation, and succinylation, suggesting possible roles of these newly identified histone acylations in diabetes and obesity. These histone marks were detected without prior affinity enrichment with an antibody, demonstrating that the histone acylation marks are present at reasonably high stoichiometry. Fifteen histone marks differed in abundance in DIO mouse liver compared with liver from chow-fed mice in label-free quantification, and six histone marks in stable isotope labeling quantification. Analysis of hepatic histone modifications from metformin-treated DIO mice revealed that metformin, a drug widely used for T2D, could reverse DIO-stimulated histone H3K36me2 in prediabetes, suggesting that this mark is likely associated with T2D development. Our study thus offers a comprehensive landscape of histone marks in a prediabetic mouse model, provides a resource for studying epigenetic functions of histone modifications in obesity and T2D, and suggest a new epigenetic mechanism for the physiological function of metformin.