Proteomic changes in the photoreceptor outer segment upon intense light exposure.

Acute light-induced photoreceptor degeneration has been studied in experimental animals as a model for photoreceptor cell loss in human retinal degenerative diseases. Light absorption by rhodopsin in rod photoreceptor outer segments (OS) induces oxidative stress and initiates apoptotic cell death. However, the molecular events that induce oxidative stress and initiate the apoptotic cascade remain poorly understood. To better understand the molecular mechanisms of light-induced photoreceptor cell death, we studied the proteomic changes in OS upon intense light exposure by using a proteolytic (18)O labeling method. Of 171 proteins identified, the relative abundance of 98 proteins in light-exposed and unexposed OS was determined. The quantities of 11 proteins were found to differ by more than 2-fold between light-exposed OS and those remaining in darkness. Among the 11 proteins, 8 were phototransduction proteins and 7 of these were altered such that the efficiency of phototransduction would be reduced or quenched during light exposure. In contrast, the amount of OS rhodopsin kinase was reduced by 2-fold after light exposure, suggesting attenuation in the mechanism of quenching phototransduction. Liquid chromatography multiple reaction monitoring (LC-MRM) was performed to confirm this reduction in the quantity of rhodopsin kinase. As revealed by immunofluorescence microscopy, this reduction of rhodopsin kinase is not a result of protein translocation from the outer to the inner segment. Collectively, our findings suggest that the absolute quantity of rhodopsin kinase in rod photoreceptors is reduced upon light stimulation and that this reduction may be a contributing factor to light-induced photoreceptor cell death. This report provides new insights into the proteomic changes in the OS upon intense light exposure and creates a foundation for understanding the mechanisms of light-induced photoreceptor cell death.

Proteolytic 18O-labeling strategies for quantitative proteomics.

A number of proteomic techniques have been developed to quantify proteins in biological systems. This review focuses on the quantitative proteomic technique known as "proteolytic 18O-labeling." This technique utilizes a protease and H(2)18O to produce labeled peptides, with subsequent chromatographic and mass spectrometric analysis to identify and quantify (relative) the proteins from which the peptides originated. The technique determines the ratio of individual protein's expression level between two samples relative to each other, and can be used to quantitatively examine protein expression (comparative proteomics) and post-translational modifications, and to study protein-protein interactions. The present review discusses various aspects of the 18O-labeling technique, including: its history, the advantages and disadvantages of the proteolytic 18O-labeling technique compared to other techniques, enzymatic considerations, the problem of variable incorporation of 18O atoms into peptides with a discussion on recent advancements of the technique to overcome it, computational tools to interpret the data, and a review of the biological applications.

pH dependency of the carboxyl oxygen exchange reaction catalyzed by lysyl endopeptidase and trypsin.

The pH dependency of the carboxyl oxygen exchange reaction catalyzed by lysyl endopeptidase (Lys-C) and trypsin has been studied. The reaction was quantitatively monitored by measuring the incorporation of 18O atom into the alpha-carboxyl group of N(alpha)-acetyl-L-lysine from H2(18)O solvent. The optimum pHs of the carboxyl oxygen exchange reaction catalyzed by Lys-C and trypsin were found to be pH 5.0 and 6.0, respectively, which were significantly shifted toward acidic pHs compared to the most favorable pHs of their amidase activities for N(alpha)-acetyl-L-lysine amide in the pHs examined. Steady-state kinetics parameters were also determined for both enzymes at two different pHs, one at the pH optimum for their carboxyl oxygen exchange activity (pH 5-6) and the other at the favorable pH for their amidase activity (pH 8-9). Significantly lower Km (2-fold lower for Lys-C, 3-fold lower for trypsin), and higher kcat values (1.5-fold higher for Lys-C, 5-fold higher for trypsin) were obtained at the acidic pHs compared to the alkaline pHs, suggesting that Lys-C and trypsin have higher substrate binding affinities and higher catalytic rates at the acidic pHs than at the alkaline pHs. The higher carboxyl oxygen exchange activities at the acidic pHs were also confirmed with peptide substrates derived from apomyoglobin. These findings are significant toward the goal of improving the efficiency of the Lys-C and trypsin catalyzed 18O labeling reactions and are thus pertinent to improving the accuracy and reliability of quantitative proteomic experiments utilizing 18O labeling.

Peptidyl-Lys metalloendopeptidase-catalyzed 18O labeling for comparative proteomics: application to cytokine/lipolysaccharide-treated human retinal pigment epithelium cell line.

We recently proposed a comparative proteomic method utilizing proteolytic 18O labeling of peptides catalyzed by peptidyl-Lys metalloendopeptidase (Lys-N) (Rao, K. C. S., Carruth, R. T., and Miyagi, M. (2005) Proteolytic 18O labeling by peptidyl-Lys metalloendopeptidase for comparative proteomics. J. Proteome Res. 4, 507-514). Unlike trypsin, which generates a mixture of isotopic isoforms resulting from the incorporation of one or two 18O atoms into each peptide species, Lys-N incorporates only a single 18O atom into the carboxyl terminus of each proteolytically generated peptide in H2(18)O solvent. This study reports the first biological application of the Lys-N-based proteolytic 18O labeling method, characterizing the proteome changes of cytokine/lipopolysaccharide-treated verses untreated human retinal pigment epithelium (ARPE-19) cells. The study resulted not only in the identification of 584 proteins but also the determination of the relative abundances of 562 proteins in the two proteomes. The results demonstrate the usefulness of the Lys-N-based proteolytic 18O labeling method in comparative proteomic studies. The results also provide the most comprehensive description of the retinal pigment epithelium proteome to date.

Proteolytic 18O labeling by peptidyl-Lys metalloendopeptidase for comparative proteomics.

The potential capabilities of a new proteolytic 18O labeling method employing peptidyl-Lys metalloendopeptidase (Lys-N) have been demonstrated for use in comparative proteomics. Conditions (pH>or=9.5) have been found such that Lys-N incorporates only a single 18O atom into the carboxyl terminus of each proteolytically generated peptide. This 18O labeling method has a major advantage over current protelytic 18O labeling methods that generate a mixture of isotopic isoforms resulting from the incorporation of one or two 18O atoms into each peptide species by the proteases (trypsin, Lys-C, or Glu-C) used. We demonstrate that the single 18O atom incorporation property of Lys-N overcomes the major problem of the current proteolytic 18O labeling methods and provides accurate quantification results for isotopically labeled peptides.

Nigerloxin, a novel inhibitor of aldose reductase and lipoxygenase with Free radical scavenging activity from Aspergillus niger CFR-W-105.

An enzyme inhibitor, nigerloxin, with inhibition against soy bean lipoxygenase-I (LOX-1), rat lens aldose reductase (RLAR) as well as free radical scavenging activity was isolated from the fermented wheat bran using Aspergillus niger CFR-W-105. Its chemical structure was identified as 2-amido-3-hydroxy-6-methoxy-5-methyl-4-(prop-1'-enyl) benzoic acid by NMR and GCEIMS data. The IC50 values against LOX-1 and RLAR were found to be 79 microM and 69 microM and ED50 against 1,1-diphenyl-2-picrylhydrazyl (DPPH) was 66 microM.