Biomedical subjects

Deborah A Ferrington

Publications and source records attributed to Deborah A Ferrington.

At least 19 recordsLinked to original sources

PubMed2006

Myosin and actin expression and oxidation in aging muscle.

While the age-related loss in muscle mass partially explains the decline in strength, other yet undefined mechanisms contribute. This study investigates whether changes in myosin-actin stoichiometry and oxidative modification could help explain the decrement in muscle strength with aging. Protein expression and oxidation were evaluated in myosin and actin isolated from the soleus and semimembranosus muscles from young adult, old, and very old Fischer 344 rats. In the soleus muscle, actin and myosin content did not change with aging. In the semimembranosus, actin content was stable, but myosin exhibited decreased content in muscles from very old rats, resulting in a decrease in the myosin-to-actin ratio. 3-Nitrotyrosine and 4-hydroxy-2-nonenal were used as markers of protein oxidative damage. Although myosin and actin are modified with 3-nitrotyrosine and 4-hydroxy-2-nonenal, the extent of chemical modification does not increase with age. The results suggest that the decline in force production with age is not due to the accumulation of these two specific markers of protein oxidation on the myofibrillar proteins. Additionally, age-dependent changes in myofibrillar stoichiometry do not contribute to the decline in force production in the soleus, but may play a role in the semimembranosus with advanced age.

Actins↗

PubMed2006

Different death stimuli evoke apoptosis via multiple pathways in retinal pigment epithelial cells.

Loss of retinal pigment epithelial (RPE) cells via apoptosis plays a prominent role in several retinal degenerative diseases, such as age-related macular degeneration, and with light damage. Strategies for preservation of vision that would interrupt the apoptotic cascade require understanding the molecular events associated with apoptosis. This study investigated the susceptibility of RPE to caspase-dependent and -independent apoptotic pathways when challenged with different stimuli, including oxidants, anti-Fas antibody, and activated cytotoxic T lymphocytes (CTLs). These experiments used novel RPE cell lines developed from wildtype and heterozygous mice with reduced levels of either Mn superoxide dismutatse (SOD) or CuZnSOD. Peroxide and 4-hydroxynonenal induced apoptosis through both caspase-independent and -dependent pathways, respectively. With both oxidants, translocation of apoptosis inducing factor into the nucleus was observed. Cells containing reduced levels of CuZnSOD were the most susceptible to oxidant-induced cell death. Targeted killing by CTLs and activation of the Fas death receptor induced caspase-dependent apoptosis. These results show stimulus-specific activation of either the caspase-dependent or -independent pathway. Since cultured RPE express the protein components required for different apoptotic pathways, they provide a good model system for studying molecular events associated with multiple signals that lead to cell death.

Animals↗

PubMed2006

Retinal proteins modified by 4-hydroxynonenal: identification of molecular targets.

The reactive aldehyde, 4-hydroxynonenal (HNE), is a product of lipid peroxidation that can covalently modify and inactivate proteins. Previously, we reported increased HNE modification of select retinal proteins resolved by one-dimensional gel electrophoresis in aged Fisher 344 x Brown Norway rats (Louie, J.L., Kapphahn, R.J., Ferrington, D.A., 2002. Proteasome function and protein oxidation in the aged retina. Exp. Eye Res. 75, 271-284). In the current study, quantitative assessment of HNE molar content using slot blot immunoassays showed HNE content is increased 30% in aged rat retina. In contrast, there was no age-related difference in HNE content in individual spots resolved by 2D gel electrophoresis suggesting the increased modification is likely on membrane proteins that are missing on 2D gels. The HNE-immunoreactive proteins resolved by 2D gel electrophoresis were identified by MALDI-TOF mass spectrometry. These proteins are involved in metabolism, chaperone function, and fatty acid transport. Proteins that were frequently modified and had the highest molar content of HNE included triosephosphate isomerase, alpha enolase, heat shock cognate 70 and betaB2 crystallin. Immunochemical detection of HNE adducts on retinal sections showed greater immune reaction in ganglion cells, photoreceptor inner segment, and the inner plexiform layer. Identification of HNE modified proteins in two alternative model systems, human retinal pigment epithelial cells in culture (ARPE19) and human donor eyes, indicated that triosephosphate isomerase and alpha enolase are generally modified. These results identify a common subset of proteins that contain HNE adducts and suggest that select retinal proteins are molecular targets for HNE modification.

Aging↗

PubMed2006

Protein nitration with aging in the rat semimembranosus and soleus muscles.

On the basis of the accelerated age-related effects in type II muscle, we hypothesized that with aging the semimembranosus (type II) muscle would accumulate a greater amount of oxidized proteins compared to proteins in the soleus (type I) muscle. In this study, 3-nitrotyrosine (3-NT) was used as a stable marker of protein oxidative damage. The presence of 3-NT was evaluated in muscles from young adult, old, and very old Fischer 344 rats to provide an indication of the time course of muscle protein oxidative damage. A significant age-associated increase in nitrotyrosine-modified proteins was observed. The modified proteins identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry include the sarcoplasmic reticulum Ca(+2)-ATPase, aconitase, beta-enolase, triosephosphate isomerase, and carbonic anhydrase III. These proteins, involved in metabolism and calcium homeostasis, exhibited an age-dependent increase in 3-NT content in both muscles. However, significant levels of 3-NT modification were present at an earlier age in the semimembranosus muscle.

Aging↗

PubMed2006

Proteomics of the retinal pigment epithelium reveals altered protein expression at progressive stages of age-related macular degeneration.

PURPOSE: Age-related macular degeneration (AMD) is characterized clinically by changes in the retinal pigment epithelium (RPE), formation of drusen between the RPE and the underlying vasculature, geographic atrophy, and choroidal neovascularization. Later clinical stages are accompanied by impaired central vision. A limited understanding of the molecular events responsible for AMD has constrained the development of effective treatments. A proteomics approach was used to investigate the underlying mechanisms of AMD and to identify proteins exhibiting significant changes in expression with disease onset and progression. METHODS: Human donor eyes were categorized into one of four progressive stages of AMD. Proteins from the RPE were resolved and quantified by two-dimensional (2-D) gel electrophoresis. Proteins exhibiting significant expression changes at different disease stages were identified by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. 2-D and semiquantitative one dimensional (1-D) Western blot analyses were used to determine whether changes identified by the proteomic analysis were specific for a protein subpopulation or representative of the entire protein population. RESULTS: Proteins were identified from several critical pathways that changed at early and late disease stages, indicating potential causal mechanisms and secondary consequences of AMD, respectively. Proteins involved in protecting from stress-induced protein unfolding and aggregation, mitochondrial trafficking and refolding, and regulating apoptosis changed early in the disease process. Late-stage changes occurred in proteins that regulate retinoic acid and regeneration of the rhodopsin chromophore. CONCLUSIONS: These results provide the first direct evidence of AMD stage-specific changes in human RPE protein expression and provide a basis for functional investigation of AMD that may ultimately suggest new therapeutic strategies.

Adult↗

PubMed2006

The proteome of central and peripheral retina with progression of age-related macular degeneration.

PURPOSE: A growing understanding of the molecular events in age-related macular degeneration (AMD) has lead to targeted therapies for a select group of patients with advanced AMD. Development of therapies for the earlier stages requires further elucidation of disease mechanisms. In this study, a proteomics approach was used to identify proteins that had altered content in human donor eyes with progression of AMD. METHODS: The early molecular events associated with AMD were identified by comparing the proteome of the macular and peripheral neurosensory retina during four progressive stages of AMD. Proteins were resolved and quantified by two-dimensional gel electrophoresis. Twenty-six proteins exhibited changes in content and were identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Two-dimensional (2-D) and semiquantitative one-dimensional (1-D) Western blot analyses were used to determine whether changes identified by proteomic analysis were specific for a protein subpopulation or representative of the entire protein population. RESULTS: Twenty-six proteins were identified that exhibited changes at disease onset or with progression (indicating potential causal mechanisms) and at end-stage disease (indicating potential secondary consequences). These proteins are involved in key functional pathways, such as microtubule regulation and protection from stress-induced protein unfolding. Approximately 60% of the proteins exhibited changes specific to either the macula or periphery, with the remaining 40% changing in both regions. These results imply that both the macula and periphery are affected by AMD. CONCLUSIONS: This study provides the first direct evidence of AMD stage- and region-specific changes in retinal protein levels and highlights potential novel, disease-related proteins and biochemical pathways for future studies of AMD.

Adult↗

PubMed2006

RPE cells resist bystander killing by CTLs, but are highly susceptible to antigen-dependent CTL killing.

PURPOSE: Retinal pigmented epithelial (RPE) cells maintain the blood-retinal barrier, sustain retinal photoreceptor cell health and function, and may play a role in ocular immune privilege. If RPE immunomodulatory activities were antigen specific, their expression would require antigen presentation. In a study of antigen processing and major histocompatibility complex (MHC) class I-restricted presentation by RPE cells, the cells' sensitivity to the activity of cytotoxic T lymphocytes (CTLs) was determined. METHODS: RPE was cultured, with and without proinflammatory cytokines and antigen, followed by the addition of beta-galactosidase (beta-gal)-specific CTLs. Cytotoxic activity was measured by the CTL-dependent activation of caspase-3 in the RPE. Sensitivity to the CTLs was used to evaluate the activity of pathways of antigen processing and presentation using an antigen (beta-gal) that was either applied to or expressed in RPE. RESULTS: RPE cells were sensitive targets for activated CTL-mediated killing in vitro only if prepulsed with cognate peptide, or if beta-gal-expressing RPE was pretreated to induce upregulation of immunoproteasome. Activated CTLs induced apoptosis in RPE within 3 hours of coculture with antigen-positive RPE monolayers. Application of CTLs in a resting state to antigen-positive RPE led to their activation in the absence of exogenous antigen-presenting cells (APCs). This antigen-dependent activation and killing required 24 hours of co-incubation of RPE with resting CD8 T cells specific for beta-gal. Although RPE cells are highly phagocytic, functional evidence for processing that allowed phagocytosed antigens to load into class I MHC was not detected. RPE was minimally sensitive to bystander killing by activated CTLs. CONCLUSIONS: Although there are many reports of T-cell inhibition by RPE, we found that CTLs efficiently killed RPE cells by induction of apoptosis in an antigen-dependent manner. The survival of RPE in the face of extensive CTL destruction of adjacent photoreceptor cells in vivo appears to be based on their insensitivity to injury via bystander mechanisms.

Animals↗

PubMed2005

Altered proteasome structure, function, and oxidation in aged muscle.

The proteasome is the main protease for degrading oxidized proteins. We asked whether altered proteasome function contributes to the accumulation of oxidized muscle proteins with aging. Proteasome structure, function, and oxidation state were compared in young and aged F344BN rat fast-twitch skeletal muscle. In proteasome-enriched homogenates from aged muscle, we observed a two- to threefold increase in content of the 20S proteasome that was due to a corresponding increase in immunoproteasome. Content of the regulatory proteins, PA700 and PA28, relative to the 20S were reduced 75% with aging. Upon addition of exogenous PA700, there was a twofold increase in peptide hydrolysis in aged muscle, suggesting the endogenous content of PA700 is inadequate for complete activation of the 20S. Measures of catalytic activity showed a 50% reduction in specific activity for proteasome-enriched homogenates with aging. With purification of the 20S, proteasome specific activity was equivalent between ages, indicating that endogenous regulators inhibit proteasome in aged muscle. Significantly less degradation of oxidized calmodulin by the 20S from aged muscle was observed. Partial rescue of activity for aged 20S by DTT implies oxidation of functionally significant cysteines. These results demonstrate significant age-related changes in proteasome structure, function, and oxidation state that could inhibit removal of oxidized proteins.

Aging↗

PubMed2005

Age-related differences in the adaptive potential of type I skeletal muscle fibers.

This study was designed to investigate whether the relationship of fiber size and force is maintained with aging and inactivity. We hypothesized that fiber size and fiber force-generating capacity would decrease in parallel, thus resulting in no change in specific force with either age or inactivity. Thirty male Fischer 344/Brown Norway F1 hybrid rats, 6-23-month old (young adult), 24-33-month old (middle-aged) and 34-40 month old (old) were hindlimb unweighted for 14 days. Single permeabilized type I fibers from the soleus and gastrocnemius muscles were evaluated for size and contractile function. The diameter of the fibers from the soleus muscle declined with age and unweighting. In contrast, the fibers from the gastrocnemius showed no age-related atrophy. In both the soleus and gastrocnemius, there was a significant decrease in the force generation with age and unweighting. When comparing size and peak absolute force in the type I fibers from control young and middle-aged animals a positive relationship was observed whereas no significant relationship between size and peak absolute force was observed in the old animals. Following unweighting, fibers from young and middle aged rats showed a significant relationship between force and size. Fibers from old animals did not exhibit a relationship between size and force following unweighting. These results suggest aged skeletal muscle has an attenuated ability to adapt to inactivity by altering its size in response to inactivity.

Adaptation, Physiological↗

PubMed2005

Declines in arrestin and rhodopsin in the macula with progression of age-related macular degeneration.

PURPOSE: Biochemical analysis of age-related macular degeneration (AMD) at distinct stages of the disease will help further understanding of the molecular events associated with disease progression. This study was conducted to determine the ability of a new grading system for eye bank eyes, the Minnesota Grading System (MGS), to discern distinct stages of AMD so that retinal region-specific changes in rod photoreceptor protein expression from donors could be determined. METHODS: Donor eyes were assigned to a specific level of AMD by using the MGS. Expression of the rod photoreceptor proteins rhodopsin and arrestin was evaluated by Western immunoblot analysis in the macular and peripheral regions of the neurosensory retina from donors at different stages of AMD. RESULTS: A significant linear decline in both arrestin and rhodopsin content correlated with progressive MGS levels in the macula. In contrast, the peripheral region showed no significant correlation between MGS level and the content of either protein. CONCLUSIONS: The statistically significant relationship between decreasing macular rod photoreceptor proteins and progressive MGS levels of AMD demonstrates the utility of the clinically based MGS to correspond with specific protein changes found at known, progressive stages of degeneration. Future biochemical analysis of clinically characterized donor eyes will further understanding of the pathobiochemistry of AMD.

Adult↗

PubMed2004

Catalytic site-specific inhibition of the 20S proteasome by 4-hydroxynonenal.

The proteasome is responsible for most intracellular protein degradation and is essential for cell survival. Previous research has shown that the proteasome can be inhibited by a number of oxidants, including 4-hydroxynonenal (HNE). The present study demonstrates that HNE rapidly inhibits the chymotrypsin-like activity of the 20S proteasome purified from liver. Subunits containing HNE-adducts were identified following 2D gel electrophoresis, Western immunoblotting, and analysis by MALDI-TOF MS. At a time when only the chymotrypsin-like activity was inhibited, the alpha 6/C2 subunit was uniquely modified. These results provide important molecular details regarding the catalytic site-specific inhibition of proteasome by HNE.

Aldehydes↗

PubMed2004

Identification of a novel phosphorylation site in ataxin-1.

Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disease resulting from an expanded CAG repeat in the SCA1 gene that leads to an expanded polyglutamine tract in the gene product. Previous studies have demonstrated that serine at site 776 is phosphorylated [E.S. Emiamian, M.D. Kaytor, L.A. Duvick, T. Zu, S.K. Tousey, H.Y. Zoghbi, H.B. Clark, H.T. Orr, Serine 776 of ataxin-1 is critical for polyglutamine-induced disease in SCA1 transgenic mice, Neuron 38 (2003) 375-387.]. Studies of ataxin-1 S776 and serine mutated to an alanine, A776, have also shown differential protein-protein interactions and reduced neurodegeneration [H.K. Chen, P. Fernandez-Funez, S.F. Acevedo, Y.C. Lam, M.D. Kaytor, M.H. Fernandez, A. Aitken, E.M. Skoulakis, H.T. Orr, J. Botas, H.Y. Zoghbi, Interaction of Akt_phosphorylated ataxin-1 with 14-3-3 mediates neurodegeneration in spinocerebellar ataxia type 1.]. However, mutation of the site serine 776 to an alanine did not abolish all phosphorylation of the protein ataxin-1, suggesting the presence of additional phosphorylation sites [E.S. Emiamian, M.D. Kaytor, L.A. Duvick, T. Zu, S.K. Tousey, H.Y. Zoghbi, H.B. Clark, H.T. Orr, Serine 776 of ataxin-1 is critical for polyglutamine-induced disease in SCA1 transgenic mice, Neuron 38 (2003) 375-387.]. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and mutational analysis demonstrated a novel phosphorylation site at serine 239 of ataxin-1.

Amino Acid Sequence↗

PubMed2004

Altered proteasome function and subunit composition in aged muscle.

Myofibrillar protein degradation is mediated through the ubiquitin-proteasome pathway. To investigate if altered proteasome activity plays a role in age-related muscle atrophy, we examined muscle size and proteasome function in young and aged F344BN rats. Significant age-related muscle atrophy was confirmed by the 38% decrease in cross-sectional area of type 1 fibers in soleus muscle. Determination of proteasome function showed hydrolysis of fluorogenic peptides was equivalent between ages. However, when accounting for the 3-fold increase in content of the 20S catalytic core in aged muscle, the lower specific activity suggests a functional loss in individual proteins with aging. Comparing the composition of the catalytic beta-subunits showed an age-related 4-fold increase in the cytokine-inducible subunits, LMP2 and LMP7. Additionally, the content of the activating complexes, PA28 and PA700, relative to the 20S proteasome was reduced 50%. These results suggest significant alterations in the intrinsic activity, the percentage of immunoproteasome, and the regulation of the 20S proteasome by PA28 and PA700 in aged muscle.

Aging↗

PubMed2004

Myofibrillar myosin ATPase activity in hindlimb muscles from young and aged rats.

We tested the hypothesis that Ca(2+)-activated myosin ATPase activity is lower in muscles of aged rats relative to muscles of young rats, independent of changes in myosin isoform expression. Myofibrils were prepared from permeabilized fibers of soleus, plantaris, and semimembranosus muscles of young (8-12 months) and aged (32-38 months) F344 x BN rats and assayed for resting myosin ATPase, Ca(2+)-activated myosin ATPase, and myosin heavy chain (MHC) and myosin light chain (MLC) isoform compositions. Resting myosin ATPases were not affected by age in any muscle (P > or = 0.42). Ca(2+)-activated myosin ATPases of soleus and plantaris myofibrils were not affected by age (P > or = 0.31) but were 16% lower in semimembranosus myofibrils from aged rats (0.448 +/- 0.019 micromol P(i)/min/mg) compared to young rats (0.533 +/- 0.031 micromol P(i)/min/mg; P = 0.03). Correspondingly, maximal unloaded shortening velocity of single semimembranosus fibers from aged rats was slow (4.6 +/- 0.2 fiber lengths/s) compared with fibers from young rats (5.8 +/- 0.3 fiber lengths/s; P < 0.01). No age-related changes in MHC or regulatory MLC isoforms were detected in any muscle (P > or = 0.08) but changes in the essential MLC occurred in plantaris and semimembranosus muscles. The data indicate that Ca(2+)-activated myosin ATPase activity is reduced with age in semimembranosus muscle, independent of age-related changes in MHC isoform expression, and is one mechanism contributing to age-related slowing of contraction in that muscle.

Aging↗

PubMed2004

Continuous remodeling of adult extraocular muscles as an explanation for selective craniofacial vulnerability in oculopharyngeal muscular dystrophy.

Oculopharyngeal muscular mystrophy (OPMD) is an inherited disorder caused by mutations of the polyadenylate binding protein nuclear 1 (PABPN1) gene. While a pathogenic hypothesis has been formulated that links the genetic and molecular abnormalities to cellular abnormalities, there is no proven explanation for the targeting of the craniofacial muscles. We propose a hypothesis that bridges this gap. It is based on the phenomenon of continuous remodeling of normal adult extraocular muscles (EOMs). Unlike the EOMs, the myonuclei of other skeletal muscles are postmitotic in the adult unless the muscles are injured. Continuous myofiber remodeling most likely requires upregulation of genes involved in cell cycling, and in protein degradation and synthesis. PABPN1 is a nuclear protein that performs the essential function of controlling polyadenylation of mRNA and the fidelity of protein synthesis. In OPMD, the ongoing production of mutant PABPN1 in muscles undergoing continuous remodeling could result in a failure of accurate production of mRNA required for the maintenance of the myocytes. Over many years, this would lead to cumulative myonuclear loss and finally to myofiber loss. This hypothesis offers an explanation for the selective involvement of extraocular muscles affected in OPMD and the onset of symptoms in adulthood.

Facial Muscles↗

PubMed2003

Modified alpha A crystallin in the retina: altered expression and truncation with aging.

Crystallins are small heat shock proteins with chaperone function that prevent heat- and oxidative stress-induced aggregation of proteins. This is the first report describing modifications of alphaA crystallin in the sensory retina, including altered content and truncation with aging. Proteins from adult, middle age, and old Fischer 344 Brown Norway rats were compared. Western immunoblotting was used to evaluate alphaA crystallin content and identify protein spots on two-dimensional gels containing alphaA crystallin. The type and site of multiple post-translational modifications were identified by mass spectrometry. We found the content of alphaA crystallin was significantly decreased in the oldest rats. On two-dimensional gels, retinal crystallins resolved into multiple spots with altered migration, indicative of changes in intrinsic charge and/or truncation. Post-translational modifications that were identified included oxidation, phosphorylation, deamidation, acetylation, and truncation. In samples from rats of all ages, a highly modified N-terminus containing these modifications was found. We also observed an age-dependent difference in the extent of N- and C-terminal truncation. These results suggest that protection against stress-induced protein aggregation is compromised in the aged retina.

Aging↗

PubMed2002

Comparable levels of Ca-ATPase inhibition by phospholamban in slow-twitch skeletal and cardiac sarcoplasmic reticulum.

Alterations in expression levels of phospholamban (PLB) relative to the sarcoplasmic reticulum (SR) Ca-ATPase have been suggested to underlie defects of calcium regulation in the failing heart and other cardiac pathologies. To understand how variation in PLB expression relative to that of the Ca-ATPase can modulate calcium transport, we have investigated the inhibition of the Ca-ATPase by PLB in native SR membranes from slow-twitch skeletal and cardiac muscle and in reconstituted proteoliposomes. Quantitative immunoblotting in combination with affinity-purified protein standards was used to measure protein concentrations of PLB and of the Ca-ATPase. Functional inhibition of the Ca-ATPase was determined from both the calcium concentrations for half-maximal activation (Ca(1/2)) and the shift in the calcium concentrations following release of PLB inhibition (i.e., (Delta)Ca(1/2)) by incubation with monoclonal antibodies against PLB, which are equivalent to phosphorylation of PLB by cAMP-dependent protein kinase. We report that equivalent levels of PLB inhibition and antibody-induced activation ((Delta)Ca(1/2) = 0.25 +/- 0.02 microM) are observed in SR membranes from slow-twitch skeletal and cardiac muscle, where molar stoichiometries of PLB expressed per Ca-ATPase vary, respectively, from 0.9 +/- 0.1 to 4.1 +/- 0.8. Similar levels of inhibition to those observed in isolated SR vesicles were observed using reconstituted proteoliposomes following co-reconstitution of affinity-purified Ca-ATPase with PLB. These results indicate that total expression levels of one PLB per Ca-ATPase result in full inhibition of the Ca-ATPase and, based on the measured K(D) (140 +/- 30 microM), suggests one PLB complexed with two Ca-ATPase molecules is sufficient for full inhibition of activity. Therefore, the excess PLB expressed in the heart over that required for inhibition suggests a capability for graded responses of the Ca-ATPase activity to endogenous kinases and phosphatases that modulate the level of phosphorylation necessary to relieve inhibition of the Ca-ATPase by PLB.

Animals↗

PubMed2002

Covalent modification of epithelial fatty acid-binding protein by 4-hydroxynonenal in vitro and in vivo. Evidence for a role in antioxidant biology.

4-Hydroxynonenal (4-HNE) is a cytotoxic alpha,beta-unsaturated acyl aldehyde that is naturally produced from lipid peroxidation and cleavage in response to oxidative stress and aging. Such reactive lipids covalently modify cellular target proteins, thereby affecting biological structure and function. Herein we report the identification of the epithelial fatty acid-binding protein (E-FABP) as a molecular target for 4-HNE modification both in vitro and in vivo. 4-HNE covalently modified (t(12) < 60 s) E-FABP in vitro, as revealed by a combination of matrix-assisted laser desorption ionization-time of flight mass spectrometry and immunochemical reactivity using antibodies directed to 4-HNE-protein conjugates. Identification of Cys-120 as the major site of modification was determined through tandem mass spectral sequencing of tryptic peptides, as well as analysis of E-FABP mutants C120A, C127A, and C120A/C127A. The in vitro modification of Cys-120 by 4-HNE was relatively insensitive to pH (6.4-8.4), and temperature (4-37 degrees C) but was markedly potentiated by noncovalently bound fatty acids. 4-HNE-modified E-FABP was more stable than unmodified E-FABP to chemical denaturation by guanidine hydrochloride, as assessed by changes in intrinsic tryptophan fluorescence. Analysis of soluble protein extracts from rat retina with antibodies directed to 4-HNE-protein conjugates revealed immunoreactivity with a 15-kDa protein that was identified by electrospray ionization and matrix-assisted laser desorption ionization-time of flight mass spectrometry as E-FABP. Evaluation of retinal pigment epithelial cell extracts derived from E-FABP null mice by two-dimensional gel electrophoresis using anti-4-HNE antibodies revealed increased modification in the null cells relative to those from wild type cells. These results indicate that E-FABP is a molecular target for 4-HNE modification and the hypothesis that E-FABP functions as an antioxidant protein by scavenging reactive lipids through covalent modification of Cys-120.

Aldehydes↗