Abstract
The prognostic and therapeutic relevance of molecular subtypes for the most aggressive isocitrate dehydrogenase 1/2 (
IDH
) wild-type glioblastoma (GBM) is currently limited due to high ...molecular heterogeneity of the tumors that impedes patient stratification. Here, we describe a distinct binary classification of
IDH
wild-type GBM tumors derived from a quantitative proteomic analysis of 39
IDH
wild-type GBMs as well as
IDH
mutant and low-grade glioma controls. Specifically, GBM proteomic cluster 1 (GPC1) tumors exhibit Warburg-like features, neural stem-cell markers, immune checkpoint ligands, and a poor prognostic biomarker, FKBP prolyl isomerase 9 (
FKBP9
). Meanwhile, GPC2 tumors show elevated oxidative phosphorylation-related proteins, differentiated oligodendrocyte and astrocyte markers, and a favorable prognostic biomarker, phosphoglycerate dehydrogenase (
PHGDH
). Integrating these proteomic features with the pharmacological profiles of matched patient-derived cells (PDCs) reveals that the mTORC1/2 dual inhibitor AZD2014 is cytotoxic to the poor prognostic PDCs. Our analyses will guide GBM prognosis and precision treatment strategies.
In bacteria, nascent proteins bear the pretranslationally generated N-terminal (Nt) formyl-methionine (fMet) residue. Nt-fMet of bacterial proteins is a degradation signal, termed fMet/N-degron. By ...contrast, proteins synthesized by cytosolic ribosomes of eukaryotes were presumed to bear unformylated Nt-Met. Here we found that the yeast formyltransferase Fmt1, although imported into mitochondria, could also produce Nt-formylated proteins in the cytosol. Nt-formylated proteins were strongly up-regulated in stationary phase or upon starvation for specific amino acids. This up-regulation strictly required the Gcn2 kinase, which phosphorylates Fmt1 and mediates its retention in the cytosol. We also found that the Nt-fMet residues of Nt-formylated proteins act as fMet/N-degrons and identified the Psh1 ubiquitin ligase as the recognition component of the eukaryotic fMet/N-end rule pathway, which destroys Nt-formylated proteins.
In this single-center prospective study of 20 patients receiving maintenance hemodialysis (HD), we compared the therapeutic effects of medium cut-off (MCO) and high flux (HF) dialyzers using ...metabolomics and proteomics. A consecutive dialyzer membrane was used for 15-week study periods: 1st HF dialyzer, MCO dialyzer, 2nd HF dialyzer, for 5 weeks respectively.
H-nuclear magnetic resonance was used to identify the metabolites and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was used to identify proteins. To compare the effects of the HF and MCO dialyzers, orthogonal projection to latent structure discriminant analysis (OPLS-DA) was performed. OPLS-DA showed that metabolite characteristics could be significantly classified by 1st HF and MCO dialyzers. The Pre-HD metabolites with variable importance in projection scores ≥ 1.0 in both 1st HF versus MCO and MCO versus 2nd HF were succinate, glutamate, and histidine. The pre-HD levels of succinate and histidine were significantly lower, while those of glutamate were significantly higher in MCO period than in the HF period. OPLS-DA of the proteome also substantially separated 1st HF and MCO periods. Plasma pre-HD levels of fibronectin 1 were significantly higher, and those of complement component 4B and retinol-binding protein 4 were significantly lower in MCO than in the 1st HF period. Interestingly, as per Ingenuity Pathway Analysis, an increase in epithelial cell proliferation and a decrease in endothelial cell apoptosis occurred during the MCO period. Overall, our results suggest that the use of MCO dialyzers results in characteristic metabolomics and proteomics profiles during HD compared with HF dialyzers, which might be related to oxidative stress, insulin resistance, complement-coagulation axis, inflammation, and nutrition.
Various forms of protein (proteoforms) are generated by genetic variations, alternative splicing, alternative translation initiation, co- or post-translational modification and proteolysis. Different ...proteoforms are in part discovered by characterizing their N-terminal sequences. Here, we introduce an N-terminal-peptide-enrichment method, Nrich. Filter-aided negative selection formed the basis for the use of two N-blocking reagents and two endoproteases in this method. We identified 6,525 acetylated (or partially acetylated) and 6,570 free protein N-termini arising from 5,727 proteins in HEK293T human cells. The protein N-termini included translation initiation sites annotated in the UniProtKB database, putative alternative translational initiation sites, and N-terminal sites exposed after signal/transit/pro-peptide removal or unknown processing, revealing various proteoforms in cells. In addition, 46 novel protein N-termini were identified in 5' untranslated region (UTR) sequence with pseudo start codons. Our data showing the observation of N-terminal sequences of mature proteins constitutes a useful resource that may provide information for a better understanding of various proteoforms in cells.
Renal dysfunction, a major complication of type 2 diabetes, can be predicted from estimated glomerular filtration rate (eGFR) and protein markers such as albumin concentration. Urinary protein ...biomarkers may be used to monitor or predict patient status. Urine samples were selected from patients enrolled in the retrospective diabetic kidney disease (DKD) study, including 35 with good and 19 with poor prognosis. After removal of albumin and immunoglobulin, the remaining proteins were reduced, alkylated, digested, and analyzed qualitatively and quantitatively with a nano LC-MS platform. Each protein was identified, and its concentration normalized to that of creatinine. A prognostic model of DKD was formulated based on the adjusted quantities of each protein in the two groups. Of 1296 proteins identified in the 54 urine samples, 66 were differentially abundant in the two groups (area under the curve (AUC):
-value < 0.05), but none showed significantly better performance than albumin. To improve the predictive power by multivariate analysis, five proteins (ACP2, CTSA, GM2A, MUC1, and SPARCL1) were selected as significant by an AUC-based random forest method. The application of two classifiers-support vector machine and random forest-showed that the multivariate model performed better than univariate analysis of mucin-1 (AUC: 0.935 vs. 0.791) and albumin (AUC: 1.0 vs. 0.722). The urinary proteome can reflect kidney function directly and can predict the prognosis of patients with chronic kidney dysfunction. Classification based on five urinary proteins may better predict the prognosis of DKD patients than urinary albumin concentration or eGFR.
The 5-year survival rate in the early and late stages of ovarian cancer differs by 63%. In addition, a liquid biopsy is necessary because there are no symptoms in the early stage and tissue ...collection is difficult without using invasive methods. Therefore, there is a need for biomarkers to achieve this goal. In this study, we found blood-based metabolite or protein biomarker candidates for the diagnosis of ovarian cancer in the 20 clinical samples (10 ovarian cancer patients and 10 healthy control subjects). Plasma metabolites and proteins were measured and quantified using mass spectrometry in ovarian cancer patients and control groups. We identified the differential abundant biomolecules (34 metabolites and 197 proteins) and statistically integrated molecules of different dimensions to better understand ovarian cancer signal transduction and to identify novel biological mechanisms. In addition, the biomarker reliability was verified through comparison with existing research results. Integrated analysis of metabolome and proteome identified emerging properties difficult to grasp with the single omics approach, more reliably interpreted the cancer signaling pathway, and explored new drug targets. Especially, through this analysis, proteins (PPCS, PMP2, and TUBB) and metabolites (L-carnitine and PC-O (30:0)) related to the carnitine system involved in cancer plasticity were identified.
The redox-dependent inhibition of thioredoxin (TRX) by thioredoxin-interacting protein (TXNIP) plays a pivotal role in various cancers and metabolic syndromes. However, the molecular mechanism of ...this regulation is largely unknown. Here, we present the crystal structure of the TRX-TXNIP complex and demonstrate that the inhibition of TRX by TXNIP is mediated by an intermolecular disulphide interaction resulting from a novel disulphide bond-switching mechanism. Upon binding to TRX, TXNIP undergoes a structural rearrangement that involves switching of a head-to-tail interprotomer Cys63-Cys247 disulphide between TXNIP molecules to an interdomain Cys63-Cys190 disulphide, and the formation of a de novo intermolecular TXNIP Cys247-TRX Cys32 disulphide. This disulphide-switching event unexpectedly results in a domain arrangement of TXNIP that is entirely different from those of other arrestin family proteins. We further show that the intermolecular disulphide bond between TRX and TXNIP dissociates in the presence of high concentrations of reactive oxygen species. This study provides insight into TRX and TXNIP-dependent cellular regulation.
Dimethylation of the histone H3 protein at lysine residue 9 (H3K9) is mediated by euchromatin histone methyltransferase II (EHMT2) and results in transcriptional repression of target genes. Recently, ...chemical inhibition of EHMT2 was shown to induce various physiological outcomes, including endoplasmic reticulum stress-associated genes transcription in cancer cells. To identify genes that are transcriptionally repressed by EHMT2 during apoptosis, and cell stress responses, we screened genes that are upregulated by BIX-01294, a chemical inhibitor of EHMT2. RNA sequencing analyses revealed 77 genes that were upregulated by BIX-01294 in all four hepatic cell carcinoma (HCC) cell lines. These included genes that have been impli-cated in apoptosis, the unfolded protein response (UPR), and others. Among these genes, the one encoding the stress-response protein Ras-related GTPase C (RRAGC) was upregulated in all BIX-01294-treated HCC cell lines. We confirmed the regulatory roles of EHMT2 in RRAGC expression in HCC cell lines using proteomic analyses, chromatin immune precipitation (ChIP) assay, and small guide RNA-mediated loss-of-function experiments. Upregulation of RRAGC was limited by the reactive oxygen species (ROS) scavenger N-acetyl cysteine (NAC), suggesting that ROS are involved in EHMT2-mediated transcriptional regulation of stress-response genes in HCC cells. Finally, combined treatment of cells with BIX-01294 and 5-Aza-cytidine induced greater upregulation of RRAGC protein expression. These findings suggest that EHMT2 suppresses expression of the RRAGC gene in a ROS-dependent manner and imply that EHMT2 is a key regulator of stress-responsive gene expression in liver cancer cells.
Dimethylation of the histone H3 protein at lysine residue 9 (H3K9) is mediated by euchromatin histone methyltransferase II (EHMT2) and results in transcriptional repression of target genes. Recently, ...chemical inhibition of EHMT2 was shown to induce various physiological outcomes, including endoplasmic reticulum stress-associated genes transcription in cancer cells. To identify genes that are transcriptionally repressed by EHMT2 during apoptosis, and cell stress responses, we screened genes that are upregulated by BIX-01294, a chemical inhibitor of EHMT2. RNA sequencing analyses revealed 77 genes that were upregulated by BIX-01294 in all four hepatic cell carcinoma (HCC) cell lines. These included genes that have been implicated in apoptosis, the unfolded protein response (UPR), and others. Among these genes, the one encoding the stress-response protein Ras-related GTPase C (RRAGC) was upregulated in all BIX-01294-treated HCC cell lines. We confirmed the regulatory roles of EHMT2 in RRAGC expression in HCC cell lines using proteomic analyses, chromatin immune precipitation (ChIP) assay, and small guide RNA-mediated loss-of-function experiments. Upregulation of RRAGC was limited by the reactive oxygen species (ROS) scavenger N-acetyl cysteine (NAC), suggesting that ROS are involved in EHMT2-mediated transcriptional regulation of stress-response genes in HCC cells. Finally, combined treatment of cells with BIX-01294 and 5- Aza-cytidine induced greater upregulation of RRAGC protein expression. These findings suggest that EHMT2 suppresses expression of the RRAGC gene in a ROS-dependent manner and imply that EHMT2 is a key regulator of stress-responsive gene expression in liver cancer cells. BMB Reports 2020; 53(11): 576-581
Objective
The need for a biomarker with robust sensitivity and specificity in diagnosing systemic lupus erythematosus (SLE) remains unmet. Compared with blood samples, urine samples are more easily ...collected; thus, we aimed to identify such a biomarker based on urinary proteomics which could distinguish patients with SLE from healthy controls (HCs).
Methods
Urine samples were collected from 76 SLE patients who visited rheumatology clinic in 2019 at Asan medical center and from 25 HCs. Urine proteins were analyzed using sequential windowed acquisition of all theoretical fragment ion spectra-mass spectrometry, and the candidate marker was confirmed by enzyme-linked immunosorbent assay (ELISA). Receiver operating characteristic curve analysis was used to determine the diagnostic value of the candidate biomarker.
Results
Of 1157 proteins quantified, 153 were differentially expressed in urine samples from HCs. Among them were previously known markers including α-1-acid glycoprotein 1, α-2-HS-glycoprotein, ceruloplasmin, and prostaglandin-H2 D-isomerase. Moreover, the amount of β-2 glycoprotein (APOH) was increased in the urine of patients with SLE. The ELISA results also showed the level of urine APOH was higher in patients with SLE than in HCs and patients with rheumatoid arthritis. Moreover, the level was not different between SLE patients with and without nephritis. The urine APOH had an area under the curve value of 0.946 at a cut-off value of 228.53 ng/mg (sensitivity 91.5%, specificity 92.0%) for the diagnosis of SLE.
Conclusion
The results indicate that the urine APOH level can be an appropriate screening tool in a clinical setting when SLE is suspected.
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