Dataset Information

Long-lived metabolic enzymes in the crystalline lens identified by pulse-labeling of mice and mass spectrometry.

ABSTRACT: The lenticular fiber cells are comprised of extremely long-lived proteins while still maintaining an active biochemical state. Dysregulation of these activities has been implicated in diseases such as age-related cataracts. However, the lenticular protein dynamics underlying health and disease is unclear. We sought to measure the global protein turnover rates in the eye using nitrogen-15 labeling of mice and mass spectrometry. We measured the 14N/15N-peptide ratios of 248 lens proteins, including Crystallin, Aquaporin, Collagen and enzymes that catalyze glycolysis and oxidation/reduction reactions. Direct comparison of lens cortex versus nucleus revealed little or no 15N-protein contents in most nuclear proteins, while there were a broad range of 14N/15N ratios in cortex proteins. Unexpectedly, like Crystallins, many enzymes with relatively high abundance in nucleus were also exceedingly long-lived. The slow replacement of these enzymes in spite of young age of mice suggests their potential roles in age-related metabolic changes in the lens.

SUBMITTER: Liu P

PROVIDER: S-EPMC6914337 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Long-lived metabolic enzymes in the crystalline lens identified by pulse-labeling of mice and mass spectrometry.

Liu Pan P Edassery Seby Louis SL Ali Laith L Thomson Benjamin R BR Savas Jeffrey N JN Jin Jing J

eLife 20191210

The lenticular fiber cells are comprised of extremely long-lived proteins while still maintaining an active biochemical state. Dysregulation of these activities has been implicated in diseases such as age-related cataracts. However, the lenticular protein dynamics underlying health and disease is unclear. We sought to measure the global protein turnover rates in the eye using nitrogen-15 labeling of mice and mass spectrometry. We measured the <sup>14</sup>N/<sup>15</sup>N-peptide ratios of 248 l ...[more]

PMID: 31820737

Similar Datasets

Project description:Numerous metabolic pathways have been implicated in diabetes-induced renal injury, yet few studies have utilized unbiased systems biology approaches for mapping the interconnectivity of diabetes-dysregulated proteins that are involved. We utilized a global, quantitative, differential proteomic approach to identify a novel retinoic acid hub in renal cortical protein networks dysregulated by type 2 diabetes.Total proteins were extracted from renal cortex of control and db/db mice at 20 weeks of age (after 12 weeks of hyperglycemia in the diabetic mice). Following trypsinization, (18)O- and (16)O-labeled control and diabetic peptides, respectively, were pooled and separated by two dimensional liquid chromatography (strong cation exchange creating 60 fractions further separated by nano-HPLC), followed by peptide identification and quantification using mass spectrometry. Proteomic analysis identified 53 proteins with fold change >or=1.5 and p<or=0.05 after Benjamini-Hochberg adjustment (out of 1,806 proteins identified), including alcohol dehydrogenase (ADH) and retinaldehyde dehydrogenase (RALDH1/ALDH1A1). Ingenuity Pathway Analysis identified altered retinoic acid as a key signaling hub that was altered in the diabetic renal cortical proteome. Western blotting and real-time PCR confirmed diabetes-induced upregulation of RALDH1, which was localized by immunofluorescence predominantly to the proximal tubule in the diabetic renal cortex, while PCR confirmed the downregulation of ADH identified with mass spectrometry. Despite increased renal cortical tissue levels of retinol and RALDH1 in db/db versus control mice, all-trans-retinoic acid was significantly decreased in association with a significant decrease in PPARbeta/delta mRNA.Our results indicate that retinoic acid metabolism is significantly dysregulated in diabetic kidneys, and suggest that a shift in all-trans-retinoic acid metabolism is a novel feature in type 2 diabetic renal disease. Our observations provide novel insights into potential links between altered lipid metabolism and other gene networks controlled by retinoic acid in the diabetic kidney, and demonstrate the utility of using systems biology to gain new insights into diabetic nephropathy.

Project description:IntroductionThe aim of our study was to identify new early rheumatoid arthritis (RA) autoantibodies.MethodsSera obtained from 110 early untreated RA patients (<6 months) were analyzed by western blot using HL-60 cell extract, separated on one-dimensional and two-dimensional gel electrophoresis (1-DE, 2-DE). Sera from 50 healthy blood donors and 20 patients with non-RA rheumatisms were used as controls for 1-DE and 2-DE, respectively. The immunoreactive proteins were identified by MALDI-TOF mass spectrometric analysis and the presence of potential sites of citrullination in each of these proteins was evaluated. FT-ICR mass spectrometry was used to verify experimentally the effect of citrullination upon the mass profile observed by MALDI-TOF analysis.ResultsThe 110 1-DE patterns allowed detection of 10 recurrent immunoreactive bands of 33, 39, 43, 46, 51, 54, 58, 62, 67 and 70 kDa, which were further characterized by 2-DE and proteomic analysis. Six proteins were already described RA antigens: heterogeneous nuclear ribonucleoprotein A2/B1, aldolase, alpha-enolase, calreticulin, 60 kDa heat shock protein (HSP60) and BiP. Phosphoglycerate kinase 1 (PGK1), stress-induced phosphoprotein 1 and the far upstream element-binding proteins (FUSE-BP) 1 and 2 were identified as new antigens. Post-translational protein modifications were analyzed and potentially deiminated peptides were found on aldolase, alpha-enolase, PGK1, calreticulin, HSP60 and the FUSE-BPs. We compared the reactivity of RA sera with citrullinated and noncitrullinated alpha-enolase and FUSE-BP linear peptides, and showed that antigenicity of the FUSE-BP peptide was highly dependent on citrullination. Interestingly, the anti-cyclic citrullinated peptide antibody (anti-CCP2) status in RA serum at inclusion was not correlated to the reactivity directed against FUSE-BP citrullinated peptide.ConclusionsTwo categories of antigens, enzymes of the glycolytic family and molecular chaperones are also targeted by the early untreated RA autoantibody response. For some of them, and notably the FUSE-BPs, citrullination is involved in the immunological tolerance breakdown observed earlier in RA patients. Autoantibodies recognizing a citrullinated peptide from FUSE-BP may enhance the sensibility for RA of the currently available anti-CCP2 test.

Project description:BackgroundNon-typeable H. influenzae (NTHi) is a nasopharyngeal commensal that can become an opportunistic pathogen causing infections such as otitis media, pneumonia, and bronchitis. NTHi is known to form biofilms. Resistance of bacterial biofilms to clearance by host defense mechanisms and antibiotic treatments is well-established. In the current study, we used stable isotope labeling by amino acids in cell culture (SILAC) to compare the proteomic profiles of NTHi biofilm and planktonic organisms. Duplicate continuous-flow growth chambers containing defined media with either "light" (L) isoleucine or "heavy" (H) (13)C6-labeled isoleucine were used to grow planktonic (L) and biofilm (H) samples, respectively. Bacteria were removed from the chambers, mixed based on weight, and protein extracts were generated. Liquid chromatography-mass spectrometry (LC-MS) was performed on the tryptic peptides and 814 unique proteins were identified with 99% confidence.ResultsComparisons of the NTHi biofilm to planktonic samples demonstrated that 127 proteins showed differential expression with p-values ≤0.05. Pathway analysis demonstrated that proteins involved in energy metabolism, protein synthesis, and purine, pyrimidine, nucleoside, and nucleotide processes showed a general trend of downregulation in the biofilm compared to planktonic organisms. Conversely, proteins involved in transcription, DNA metabolism, and fatty acid and phospholipid metabolism showed a general trend of upregulation under biofilm conditions. Selected reaction monitoring (SRM)-MS was used to validate a subset of these proteins; among these were aerobic respiration control protein ArcA, NAD nucleotidase and heme-binding protein A.ConclusionsThe present proteomic study indicates that the NTHi biofilm exists in a semi-dormant state with decreased energy metabolism and protein synthesis yet is still capable of managing oxidative stress and in acquiring necessary cofactors important for biofilm survival.

Dataset Information

Long-lived metabolic enzymes in the crystalline lens identified by pulse-labeling of mice and mass spectrometry.

Publications

Long-lived metabolic enzymes in the crystalline lens identified by pulse-labeling of mice and mass spectrometry.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets