Post-translational Modifications and Their Biological Functions: Proteomic Analysis and Systematic Approaches Biological function;Post-translational modifications;Proteomics;Mass spectrometry;Systemic analysis; Recently produced information on post-translational modifications makes it possible to interpret their biological regulation with new insights. Various protein modifications finely tune the cellular functions of each protein. Understanding the relationship between post-translational modifications and functional changes (

Many transcription coactivators interact with nuclear receptors in a ligand- and C-terminal transactivation function (AF2)-dependent manner. These include activating signal cointegrator 2 (ASC-2), a recently isolated transcriptional coactivator molecule, which is amplified in human cancers and stimulates transactivation by nuclear receptors and numerous other transcription factors. In this report, we show that ASC-2 belongs to a steady-state complex of approximately 2 MDa (ASC-2 complex [ASCOM]) in HeLa nuclei. ASCOM contains retinoblastoma-binding protein RBQ-3, α/β-tubulins, and trithorax group proteins ALR-1, ALR-2, HALR, and ASH2. In particular, ALR-1/2 and HALR contain a highly conserved 130- to 140-amino-acid motif termed the SET domain, which was recently implicated in histone H3 lysine-specific methylation activities. Indeed, recombinant ALR-1, HALR, and immunopurified ASCOM exhibit very weak but specific H3-lysine 4 methylation activities in vitro, and transactivation by retinoic acid receptor appears to involve ligand-dependent recruitment of ASCOM and subsequent transient H3-lysine 4 methylation of the promoter region in vivo. Thus, ASCOM may represent a distinct coactivator complex of nuclear receptors. Further characterization of ASCOM will lead to a better understanding of how nuclear receptors and other transcription factors mediate transcriptional activation.

Aims: Increased oxidative stress and mitochondrial dysfunction in obese adipocytes contribute to adipokine dysregulation, inflammation, and insulin resistance. Results: Through an advanced proteomic analysis, we found that peroxiredoxin 3 (Prx3), a thioredoxin-dependent mitochondrial peroxidase, is highly expressed in 3T3-L1 adipocytes compared to preadipocytes. Interestingly, in obese db/db mice and human subjects, adipose Prx3 levels were significantly decreased, indicating its association with obesity. We therefore employed Prx3 knockout (KO) mice and transfected 3T3-L1 cells to examine the role of endogenous Prx3 in adipocyte metabolism. Prx3 KO mice had increased fat mass compared to wild-type due to adipocyte hypertrophy. Increased adipogenic transcription factors and lipogenic gene expression during differentiation of adipose tissue-derived stem cells from Prx3-deficient mice confirmed that these adipocytes are likely to accumulate fat. Mitochondrial protein carbonylation in Prx3 KO adipose tissue and mitochondrial superoxide level in Prx3 knockdown 3T3-L1 cells were increased showing aberrant regulation of oxidative stress. Proteomic analysis and gene expression analysis of Prx3 KO mice adipocytes also showed defect in mitochondria biogenesis along with enzymes involved in glucose/lipid metabolism and oxidative phosphorylation. In addition, expression level of adiponectin was downregulated and plasminogen activator inhibitor-1 was upregulated in Prx3 KO adipocytes. Impaired glucose tolerance and insulin resistance further implied metabolic dysregulation in Prx3 KO mice. Innovation and Conclusion: These data suggest that endogenous Prx3 may play an essential role in maintaining normal characteristics of adipocytes and that defect in Prx3 alters mitochondrial redox state and function, and adipokine expression in adipocytes leading to metabolic alteration. Antioxid. Redox Signal. 16, 229–243.

Knowledge of the cellular targets of ROS (reactive oxygen species) and their regulation is an essential prerequisite for understanding ROS-mediated signalling. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) is known as a major target protein in oxidative stresses and becomes thiolated in its active site. However, the molecular and functional changes of oxidized GAPDH, the inactive form, have not yet been characterized. To examine the modifications of GAPDH under oxidative stress, we separated the oxidation products by two-dimensional gel electrophoresis and identified them using nanoLC-ESI-q-TOF MS/MS (nano column liquid chromatography coupled to electrospray ionization quadrupole time-of-flight tandem MS). Intracellular GAPDH subjected to oxidative stress separated into multiple acidic spots on two-dimensional gel electrophoresis and were identified as cysteine disulfide and cysteic acids on Cys¹⁵² in the active site. We identified the interacting proteins of oxidized inactive GAPDH as p54nrb (54 kDa nuclear RNA-binding protein) and PSF (polypyrimidine tract-binding protein-associated splicing factor), both of which are known to exist as heterodimers and bind to RNA and DNA. Interaction between oxidized GAPDH and p54nrb was abolished upon expression of the GAPDH active site mutant C152S. The C-terminal of p54nrb binds to GAPDH in the cytosol in a manner dependent on the dose of hydrogen peroxide. The GAPDH–p54nrb complex enhances the intrinsic topoisomerase I activation by p54nrb–PSF binding. These results suggest that GAPDH exerts other functions beyond glycolysis, and that oxidatively modified GAPDH regulates its cellular functions by changing its interacting proteins, i.e. the RNA splicing by interacting with the p54nrb–PSF complex.