Comparative Proteomics Analysis of the Postmitochondrial Supernatant Fraction of Human Lens-Free Whole Eye and Liver

Abstract

The increasing incidence of ocular diseases has accelerated research into therapeutic interventions needed for the eye. Ocular enzymes play important roles in the metabolism of drugs and endobiotics. Various ocular drugs are designed as prodrugs that are activated by ocular enzymes. Moreover, ocular enzymes have been implicated in the bioactivation of drugs to their toxic metabolites.The key purpose of this study was to compare global proteomes of the pooled samples of the eye (n 5 11) and the liver (n 5 50) with a detailed analysis of the abundance of enzymes involved in the metabolism of xenobiotics and endobiotics. We used the postmitochondrial supernatant fraction (S9 fraction) of the lens-free whole eye homogenate as a model to allow accurate comparison with the liver S9 fraction. A total of 269 proteins (including 23 metabolic enzymes) were detected exclusively in the pooled eye S9 against 648 proteins in the liver S9 (including 174 metabolic enzymes), whereas 424 proteins (including 94 metabolic enzymes) were detected in both the organs. The major hepatic cytochrome P450 and UDP-glucuronosyltransferases enzymes were not detected, but aldehyde dehydrogenases and glutathione transferases were the predominant proteins in the eye. The comparative qualitative and quantitative proteomics data in the eye versus liver is expected to help in explaining differential metabolic and physiologic activities in the eye. SIGNIFICANCE STATEMENTInformation on the enzymes involved in xenobiotic and endobiotic metabolism in the human eye in relation to the liver is scarcely available. The study employed global proteomic analysis to compare the proteomes of the lens-free whole eye and the liver with a detailed analysis of the enzymes involved in xenobiotic and endobiotic metabolism. These data will help in better understanding of the ocular metabolism and activation of drugs and endobiotics.

Exploring the Interactome of Cytochrome P450 2E1 in Human Liver Microsomes with Chemical Crosslinking Mass Spectrometry

“…To probe the effect of CYP2E1 modification by benzophenone derivatives on the functional properties of the enzyme, we incorporated CYP2E1-BPM and CYP2E1-BPS into insect cell microsomes (Supersomes TM ) containing cDNA-expressed human NADPH-cytochrome P450 reductase (CPR) along with human cytochrome b 5 (SS(CPR+b5)) and studied their activity in deethylation of 7-Ethoxy-4-trifluoromethylcoumarin (7-EFC). Although this fluorogenic substrate is commonly used as a CYP2B6-specific probe, it can also be metabolized by CYP2E1 with a reasonable turnover rate [ 25 , 33 , 34 ]. To obtain the appropriate reference points, we also studied 7-EFC metabolism by SS(CPR+b5) with incorporated unmodified CYP2E1 (SS(CPR+b5)2E1) and commercial preparation of Supersomes containing cDNA-expressed full-length CYP2E1 (SS(2E1)).…”

Activity and Expression of Carboxylesterases and Arylacetamide Deacetylase in Human Ocular Tissues

“…Our quantitative data confirms this ocular profile for CES1 and AADAC, but in contrast, clearly indicates the presence of CES2. Another qualitative study indicated the presence of CES4/CES1P1 while no other CES isoform was identified in the S9 fraction from pooled human whole eyes (Balhara et al, 2021). This sampling approach minimized interindividual variation but diluted the individual tissue contents.…”

“…RPE/choroid (Skeie and Mahajan, 2014). In contrast, a recent study identified only CES4/CES1P1 among CES isoforms in S9 fractions of pooled human eye homogenates (Balhara et al, 2021). Such qualitative data cannot fully estimate the metabolic profile of human eye tissues.…”

Exploring the Interactome of Cytochrome P450 2E1 in Human Liver Microsomes with Chemical Crosslinking Mass Spectrometry

“…After washing and drying, the gel fragments were subjected to trypsin digestion using a previously described procedure [22]. MS analysis was carried out using a nanoLC coupled to a Q Exactive HF hybrid quadrupole-orbitrap™ mass spectrometer (Thermo Fisher Scientific, Rockwell, IL, USA) [30]. The obtained raw data were processed using the MaxQuant software (https://maxquant.org) with the built-in search engine Andromeda.…”

“…Following subsequent treatment with 55 mM iodoacetamide in 0.1 M ammonium bicarbonate in the dark at room temperature for 20 min, the gel fragments were dried with pure acetonitrile and digested with trypsin as described in [29]. Further LC-MS/MS analysis was carried out using nanoLC coupled to a Q Exactive HF hybrid quadrupole-orbitrap™ mass spectrometer (Thermo Fisher Scientific, Rockwell, IL, USA) [30].…”