Cedars-Sinai Parker Lab - ACE10 Bernstein Study

ACE over expression in myeloid cells increases oxidative metabolism and cellular ATP

  • Organism: Mus musculus
  • Instrument: QTRAP 6500+
  • SpikeIn: Yes
  • Keywords: ACE overexpression, Mouse Monocyte Metabolism, Mitoplex
  • Lab head: Sarah Parker
Abstract
Angiotensin converting enzyme (ACE) affects blood pressure. In addition, ACE over expression in myeloid cells increases their immune response. We identified marked changes of intermediate metabolites in ACE over expressing macrophages and neutrophils, with increased cellular ATP and Krebs cycle intermediates including citrate, isocitrate, succinate, and malate. Increased ATP is due to ACE activity; it is reversed by an ACE inhibitor but not by an angiotensin II AT1 receptor antagonist. ACE over expression does not change cell or mitochondrial size or number. However, expression levels of the electron transport chain proteins NDUFB8 (complex I), ATP5A, and ATP5β (complex V) are increased in macrophages and neutrophils, and MTCO1 and COX2 (complex IV) in macrophages, over expressing ACE. Macrophages over expressing ACE have increased mitochondrial ATP production rates and maximal respiratory rates. Increased cellular ATP underpins increased myeloid cell superoxide production and phagocytosis associated with increased ACE expression. Myeloid cells over expressing ACE indicate the existence of a novel pathway in which myeloid cell function can be enhanced, with a key feature being increased cellular ATP.
Experiment Description
Comparison of mitochondrial protein expression between ACE10-overexpressing and wild-type mouse monocytes
Sample Description
Monocyte pellets (N=4 per mouse genotype, ACE10 vs WT) were lysed in 8M Urea dissolved in 50mM TRIS-HCl buffer, pH 8.0. Lysis was facilitated by high pressure treatment on a Pressure Biosciences (location) barocycler (Model number), with 60 one-minute cycles consisting of 50 seconds of 45 kPSI followed by 10 seconds at Atmospheric Pressure. Following extraction, protein concentration was assayed using the BCA (Pierce, Company Location). Fifty micrograms of protein from each sample were aliquoted for digestion, and each sample was reduced using 10mM Dithiothreitol (10mM) and subsequently alkylated with iodoacetamide (100mM). Samples were diluted with 200mM Ammonium Bicarbonate buffer and supplemented with 10% Acetonitrile, to a final Urea concentration < 2M. Trypsin was added at a ratio of 1 ug to 50ug total protein and subjected to an additional series of 60 cycles (45kPSI for 50s, ATM for 10s) at 37 degrees C, after which they were left to incubate at ATM overnight at 37C. Digestion was quenched with 1% Trifluoroacetic acid and samples were desalted on Nest C18 tips (Nest Group, Location). Peptides were dried to completion and re-suspended in a solution of 0.1% Formic Acid (FA) in ddH20 into which a 1:250 dilution of stable isotope labeled reference peptides derived for targeted mitochondrial protein analysis as described in detail in Stotland et al., 2019 (https://www.biorxiv.org/content/10.1101/820167v1). A total of 8ug of digested peptides, injected twice as duplicate technical replicates, were separated on a Prominence UFLCXR HPLC system (Shimadzu, Japan) with a Waters Xbridge BEH30 C18 2.1mm x 100mm, 3.5µm column (Waters) flowing at 0.25 mL/min and 36 °C coupled to a QTRAP® 6500 (SCIEX, Framingham, MA). Mobile phase A consisted of 2% ACN, 98% water, and 0.1% formic acid and mobile phase B of 95% ACN, 5% water, and 0.1% formic acid. After loading, the column was equilibrated with 5% B for 5 minutes. Peptides were then eluted over 30 minutes with a linear 5% to 35% gradient of buffer B. The column was washed with 98% B for 10 minutes and then returned to 5% B for 5 minutes before loading the next sample. A scheduled, targeted acquisition method optimized for each peptide was used to monitor fragments within a 2-minute window of the optimized retention time.
Created on 11/14/19, 3:58 PM
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This data is available under the CC BY 4.0 license.