Methylene-bridge tryptophan fatty acylation regulates PI3K-AKT signaling and glucose uptake.

Protein fatty acylation regulates numerous cell signaling pathways. Polyunsaturated fatty acids (PUFAs) exert a plethora of physiological effects, including cell signaling regulation, with underlying mechanisms to be fully understood. Herein, we report that docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) regulate PI3K-AKT signaling by modifying PDK1 and AKT2. DHA-administered mice exhibit altered phosphorylation of proteins in signaling pathways. Methylene bridge-containing DHA/EPA acylate δ1 carbon of tryptophan 448/543 in PDK1 and tryptophan 414 in AKT2 via free radical pathway, recruit both the proteins to the cytoplasmic membrane, and activate PI3K signaling and glucose uptake in a tryptophan acylation-dependent but insulin-independent manner in cultured cells and in mice. DHA/EPA deplete cytosolic PDK1 and AKT2 and induce insulin resistance. Akt2 knockout in mice abrogates DHA/EPA-induced PI3K-AKT signaling. Our results identify PUFA's methylene bridge tryptophan acylation, a protein fatty acylation that regulates cell signaling and may underlie multifaceted effects of methylene-bridge-containing PUFAs.

Nuclear dihydroxyacetone phosphate signals nutrient sufficiency and cell cycle phase to global histone acetylation.

Global histone acetylation varies with changes in the nutrient and cell cycle phases; however, the mechanisms connecting these variations are not fully understood. Herein, we report that nutrient-related and cell-cycle-regulated nuclear acetate regulates global histone acetylation. Histone deacetylation-generated acetate accumulates in the nucleus and induces histone hyperacetylation. The nuclear acetate levels were controlled by glycolytic enzyme triosephosphate isomerase 1 (TPI1). Cyclin-dependent kinase 2 (CDK2), which is phosphorylated and activated by nutrient-activated mTORC1, phosphorylates TPI1 Ser 117 and promotes nuclear translocation of TPI1, decreases nuclear dihydroxyacetone phosphate (DHAP) and induces nuclear acetate accumulation because DHAP scavenges acetate via the formation of 1-acetyl-DHAP. CDK2 accumulates in the cytosol during the late G1/S phases. Inactivation or blockade of nuclear translocation of TPI1 abrogates nutrient-dependent and cell-cycle-dependent global histone acetylation, chromatin condensation, gene transcription and DNA replication. These results identify the mechanism of maintaining global histone acetylation by nutrient and cell cycle signals.

Sensing and Transmitting Intracellular Amino Acid Signals through Reversible Lysine Aminoacylations.

Amino acids are known regulators of cellular signaling and physiology, but how they are sensed intracellularly is not fully understood. Herein, we report that each aminoacyl-tRNA synthetase (ARS) senses its cognate amino acid sufficiency through catalyzing the formation of lysine aminoacylation (K-AA) on its specific substrate proteins. At physiologic levels, amino acids promote ARSs bound to their substrates and form K-AAs on the ɛ-amine of lysines in their substrates by producing reactive aminoacyl adenylates. The K-AA marks can be removed by deacetylases, such as SIRT1 and SIRT3, employing the same mechanism as that involved in deacetylation. These dynamically regulated K-AAs transduce signals of their respective amino acids. Reversible leucylation on ras-related GTP-binding protein A/B regulates activity of the mammalian target of rapamycin complex 1. Glutaminylation on apoptosis signal-regulating kinase 1 suppresses apoptosis. We discovered non-canonical functions of ARSs and revealed systematic and functional amino acid sensing and signal transduction networks.

pGlyco 2.0 enables precision N-glycoproteomics with comprehensive quality control and one-step mass spectrometry for intact glycopeptide identification.

The precise and large-scale identification of intact glycopeptides is a critical step in glycoproteomics. Owing to the complexity of glycosylation, the current overall throughput, data quality and accessibility of intact glycopeptide identification lack behind those in routine proteomic analyses. Here, we propose a workflow for the precise high-throughput identification of intact N-glycopeptides at the proteome scale using stepped-energy fragmentation and a dedicated search engine. pGlyco 2.0 conducts comprehensive quality control including false discovery rate evaluation at all three levels of matches to glycans, peptides and glycopeptides, improving the current level of accuracy of intact glycopeptide identification. The N-glycoproteome of samples metabolically labeled with 15N/13C were analyzed quantitatively and utilized to validate the glycopeptide identification, which could be used as a novel benchmark pipeline to compare different search engines. Finally, we report a large-scale glycoproteome dataset consisting of 10,009 distinct site-specific N-glycans on 1988 glycosylation sites from 955 glycoproteins in five mouse tissues.Protein glycosylation is a heterogeneous post-translational modification that generates greater proteomic diversity that is difficult to analyze. Here the authors describe pGlyco 2.0, a workflow for the precise one step identification of intact N-glycopeptides at the proteome scale.

Acetate functions as an epigenetic metabolite to promote lipid synthesis under hypoxia.

Besides the conventional carbon sources, acetyl-CoA has recently been shown to be generated from acetate in various types of cancers, where it promotes lipid synthesis and tumour growth. The underlying mechanism, however, remains largely unknown. We find that acetate induces a hyperacetylated state of histone H3 in hypoxic cells. Acetate predominately activates lipogenic genes ACACA and FASN expression by increasing H3K9, H3K27 and H3K56 acetylation levels at their promoter regions, thus enhancing de novo lipid synthesis, which combines with its function as the metabolic precursor for fatty acid synthesis. Acetyl-CoA synthetases (ACSS1, ACSS2) are involved in this acetate-mediated epigenetic regulation. More importantly, human hepatocellular carcinoma with high ACSS1/2 expression exhibit increased histone H3 acetylation and FASN expression. Taken together, this study demonstrates that acetate, in addition to its ability to induce fatty acid synthesis as an immediate metabolic precursor, also functions as an epigenetic metabolite to promote cancer cell survival under hypoxic stress.

In-depth mapping of the mouse brain N-glycoproteome reveals widespread N-glycosylation of diverse brain proteins.

N-glycosylation is one of the most prominent and abundant posttranslational modifications of proteins. It is estimated that over 50% of mammalian proteins undergo glycosylation. However, the analysis of N-glycoproteins has been limited by the available analytical technology. In this study, we comprehensively mapped the N-glycosylation sites in the mouse brain proteome by combining complementary methods, which included seven protease treatments, four enrichment techniques and two fractionation strategies. Altogether, 13492 N-glycopeptides containing 8386 N-glycosylation sites on 3982 proteins were identified. After evaluating the performance of the above methods, we proposed a simple and efficient workflow for large-scale N-glycosylation site mapping. The optimized workflow yielded 80% of the initially identified N-glycosylation sites with considerably less effort. Analysis of the identified N-glycoproteins revealed that many of the mouse brain proteins are N-glycosylated, including those proteins in critical pathways for nervous system development and neurological disease. Additionally, several important biomarkers of various diseases were found to be N-glycosylated. These data confirm that N-glycosylation is important in both physiological and pathological processes in the brain, and provide useful details about numerous N-glycosylation sites in brain proteins.

ITMSQ: A software tool for N- and C-terminal fragment ion pairs based isobaric tandem mass spectrometry quantification.

Tandem MS (MS2) quantification using the series of N- and C-terminal fragment ion pairs generated from isobaric-labelled peptides was recently considered an accurate strategy in quantitative proteomics. However, the presence of multiplexed terminal fragment ion in MS2 spectra may reduce the efficiency of peptide identification, resulting in lower identification scores or even incorrect assignments. To address this issue, we developed a quantitative software tool, denoted isobaric tandem MS quantification (ITMSQ), to improve N- and C-terminal fragment ion pairs based isobaric MS2 quantification. A spectrum splitting module was designed to separate the MS2 spectra from different samples, increasing the accuracy of both identification and quantification. ITMSQ offers a convenient interface through which parameters can be changed along with the labelling method, and the result files and all of the intermediate files can be exported. We performed an analysis of in vivo terminal amino acid labelling labelled HeLa samples and found that the numbers of quantified proteins and peptides increased by 13.64 and 27.52% after spectrum splitting, respectively. In conclusion, ITMSQ provides an accurate and reliable quantitative solution for N- and C-terminal fragment ion pairs based isobaric MS2 quantitative methods.

Up-regulation of fatty acid oxidation in the ligament as a contributing factor of ankylosing spondylitis: A comparative proteomic study.

OBJECTIVES: The present study first utilized a standardized shotgun proteomic analysis method to determine differences in protein expression of fibroblasts in the ligament between AS patients and healthy controls. METHODS: Proteins extracted from primarily cultured FLLs from 35 AS patients and 10 normal subjects were analyzed by automated 2D-Nano-LC-ESI-MS/MS. Differentially expressed proteins were screened by 2-sample t-test and fold change. Bioinformatics analysis of differentially expressed proteins was based on the IPA. Fatty acid ß-oxidation-related proteins and INSR pathway-related proteins in the ligament were confirmed by real-time PCR and Western blot. RESULTS: A total of 556 differential proteins were screened in AS. Of them, 322 proteins were up-regulated and the remaining 234 proteins were down-regulated. GO and pathway analyses showed that six fatty acid ß-oxidation-related proteins (HADHB, ECHS1, ACSL4, ACADM, ACSL1 and HADH) were up-regulated in FLL cells, which was consistent with the results obtained from real-time PCR, Western blot and MS, while INSR pathway-related proteins (INSR, IRS1, PI3K and PKC) was low in the ligament of AS as compared with that in healthy controls. CONCLUSION: The lower body fat level in AS maybe due to up-regulation of fatty acid ß-oxidation-related enzymes regulated by INSR/PI3K/PKC pathway. BIOLOGICAL SIGNIFICANCE: Ankylosing spondylitis (AS), a common spondyloarthropathy, is an inflammatory rheumatic disease with a predilection for the axial skeleton. Clinical hallmarks of AS include sacroiliitis, uveitis, enthesitis and persistent spinal inflammation. The pathogenic mechanism of disease causation and perpetuation remains poorly understood. In this study, we primarily cultured fibroblast cells from ligament biopsies, knowing that fibroblast cells are dominant cells in the diseased ligament. One of the characteristic pathologic changes in AS is inflammation of the attachment points, including the muscle, ligament and bone or joint capsule. Inflammation of the tendon attachment point is usually non-bacterial and can lead to pain and swelling of the tendon ligament. To obtain more information, we used Shotgun proteomic analysis based on multidimensional liquid chromatography tandem mass spectrometry (LC-MS/MS). we firstly mixed the lysates of FLL cells derived from the ligaments of 35 AS patients and 10 normal subjects, identified proteins by automated 2D-Nano-LC-ESI-MS/MS method, GO and pathway analyses showed that six fatty acid ß-oxidation-related proteins (HADHB, ECHS1, ACSL4, ACADM, ACSL1 and HADH) were up-regulated in the ligament, which was consistent with the results obtained from real-time PCR, Western blot and MS, while INSR pathway-related proteins (INSR, IRS1, PI3K and PKC) was low in the ligament of AS as compared with that in healthy controls. We also find that AS subjects had significantly lower body mass index (BMI) and BMI Z-scores compared with that in healthy controls. The results remind us that up-regulation of fatty acid ß-oxidation-related proteins lower the body fat content, which is a new discovery contributing to the progression of AS. This is the first report on fatty acid oxidation in AS. It was found that the body fat level was low in AS due to high fatty acid oxidation, suggesting that insulin signaling may play an important role in the metabolic switch from predominant to fatty acid metabolism that characterizes the ligament of AS. One mechanism for this transition is increased expression of genes that regulate the rate of fatty acid oxidation. This effect may be mediated by PI3K, a downstream mediator of many receptor tyrosine kinases, including the INSR. This is a newly discovered factor contributing to the progression of AS.

A novel quantitative proteomics workflow by isobaric terminal labeling.

Quantification by series of b, y fragment ion pairs generated from isobaric-labeled peptides in MS2 spectra has recently been considered an accurate strategy in quantitative proteomics. Here we developed a novel MS2 quantification approach named quantitation by isobaric terminal labeling (QITL) by coupling (18)O labeling with dimethylation. Trypsin-digested peptides were labeled with two (16)O or (18)O atoms at their C-termini in H(2)(16)O or H(2)(18)O. After blocking all ε-amino groups of lysines through guanidination, the N-termini of the peptides were accordingly labeled with formaldehyde-d(2) or formaldehyde. These indistinguishable, isobaric-labeled peptides in MS1 spectra produce b, y fragment ion pairs in the whole mass range of MS2 spectra that can be used for quantification. In this study, the feasibility of QITL was first demonstrated using standard proteins. An accurate and reproducible quantification over a wide dynamic range was achieved. Then, complex rat liver samples were used to verify the applicability of QITL for large-scale quantitative analysis. Finally, QITL was applied to profile the quantitative proteome of hepatocellular carcinoma (HCC) and adjacent non-tumor liver tissues. Given its simplicity, low-cost, and accuracy, QITL can be widely applied in biological samples (cell lines, tissues, and body fluids, etc.) for quantitative proteomic research.

Improved proteomic analysis pipeline for LC-ETD-MS/MS using charge enhancing methods.

Electron transfer dissociation (ETD) is a useful and complementary activation method for peptide fragmentation in mass spectrometry. However, ETD spectra typically receive a relatively low score in the identifications of 2+ ions. To overcome this challenge, we, for the first time, systematically interrogated the benefits of combining ion charge enhancing methods (dimethylation, guanidination, m-nitrobenzyl alcohol (m-NBA) or Lys-C digestion) and differential search algorithms (Mascot, Sequest, OMSSA, pFind and X!Tandem). A simple sample (BSA) and a complex sample (AMJ2 cell lysate) were selected in benchmark tests. Clearly distinct outcomes were observed through different experimental protocol. In the analysis of AMJ2 cell lines, X!Tandem and pFind revealed 92.65% of identified spectra; m-NBA adduction led to a 5-10% increase in average charge state and the most significant increase in the number of successful identifications, and Lys-C treatment generated peptides carrying mostly triple charges. Based on the complementary identification results, we suggest that a combination of m-NBA and Lys-C strategies accompanied by X!Tandem and pFind can greatly improve ETD identification.

In vivo termini amino acid labeling for quantitative proteomics.

Quantitative proteomics is one of the research hotspots in the proteomics field and presently maturing rapidly into an important branch. The two most typical quantitative methods, stable isotope labeling with amino acids in cell culture (SILAC) and isobaric tags for relative and absolute quantification (iTRAQ), have been widely and effectively applied in solving various biological and medical problems. Here, we describe a novel quantitative strategy, termed "IVTAL", for in vivo termini amino acid labeling, which combines some advantages of the two methods above. The core of this strategy is a set of heavy amino acid (13)C(6)-arginine and (13)C(6)-lysine and specific endoproteinase Lys-N and Arg-C that yield some labeled isobaric peptides by cell culture and enzymatic digestion, which are indistinguishable in the MS scan but exhibit multiple MS/MS reporter b, y ion pairs in a full mass range that support quantitation. Relative quantification of cell states can be achieved by calculating the intensity ratio of the corresponding reporter b, y ions in the MS/MS scan. The experimental analysis for various proportions of mixed HeLa cell samples indicated that the novel strategy showed an abundance of reliable quantitative information, a high sensitivity, and a good dynamic range of nearly 2 orders of magnitude. IVTAL, as a highly accurate and reliable quantitative proteomic approach, is expected to be compatible with any cell culture system and to be especially effective for the analysis of multiple post-translational modificational sites in one peptide.

iTRAQ-2DLC-ESI-MS/MS based identification of a new set of immunohistochemical biomarkers for classification of dysplastic nodules and small hepatocellular carcinoma.

The study aims to develop novel clinical immunohistochemical biomarkers for distinguishing small hepatocellular carcinoma (sHCC) from dysplastic nodules (DN). iTRAQ-2DLC-ESI-MS/MS technique was used to screen immunohistochemical biomarkers between precancerous lesions (liver cirrhosis and DN) and sHCC. A total of 1951 proteins were quantified, including 52 proteins upregulated in sHCC and 95 proteins downregulated in sHCC by at least 1.25- or 0.8-fold at p < 0.05. The selected biomarker candidates were further verified using Western blotting and immunohistochemistry. Furthermore, receiver operation characteristics (ROC) curves and logistic regression model were carried out to evaluate the diagnostic values of the biomarkers. Finally, aminoacylase-1 (ACY1) and sequestosome-1 (SQSTM1) were chosen as novel candidate biomarkers for distinction of sHCC from DN. A constructed logistic regression model included ACY1, SQSTM1, and CD34. The sensitivity and specificity of this model for distinguishing sHCC from DN was 96.1% and 96.7%. In conclusion, ACY1 and SQSTM1 were identified as novel immunohistochemical biomarkers distinguishing sHCC from DN. In conclusion, expression levels of CD34, ACY1, and SQSTM1 can be used to establish an accurate diagnostic model for distinction of sHCC from DN.