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Biosynthesis and turnover of DOPA-containing proteins by human cells
Free Radical Biology & Medicine, Vol. 37, No. 11, pp. 1756–1764, 2004 Copyright D 2004 Elsevier Inc. Printed in the USA. All rights reserved 0891-5849/$-see front matter
doi:10.1016/j.freeradbiomed.2004.08.009
Original Contribution BIOSYNTHESIS AND TURNOVER OF DOPA-CONTAINING PROTEINS BY HUMAN CELLS KENNETH J. RODGERS,* PETER M. HUME,* RACHAEL A. DUNLOP,* and ROGER T. DEANy * Cell Biology Unit, The Heart Research Institute, Camperdown, Sydney, NSW 2050, Australia; and y The University of Canberra, Canberra, ACT 2601, Australia (Received 31 March 2004; Revised 6 July 2004; Accepted 12 August 2004) Available online 11 September 2004
Abstract — Protein-bound 3,4-dihydroxyphenylalanine (PB-DOPA) is a major product of hydroxyl radical attack on tyrosine residues of proteins. Levels of PB-DOPA in cells and tissues have been shown to be greatly elevated in agerelated diseases. We demonstrate for the first time that l-DOPA (levodopa) can be biosynthetically incorporated into cell proteins by human cells (THP-1 monocytes and monocyte-derived macrophages). The DOPA-containing proteins generated were selectively visualized on PVDF membranes using a redox-cycling staining method. Many cell proteins contained DOPA and seemed to be synthesized as their full-length forms. The cellular removal of DOPA-containing proteins by THP-1 cells was by proteolysis involving both the proteasomal and the lysosomal systems. The rate of cellular proteolysis of DOPA-containing proteins increased at lower levels of DOPA incorporation but decreased at higher levels of DOPA incorporation. The decreased rate of degradation was accompanied by an increase in the activity of cathepsins B and L but the activity of cathepsin S increased only at lower levels of DOPA incorporation. These data raise the possibility that PB-DOPA could be generated in vivo from l-DOPA, which is the most widely used treatment for Parkinson disease. D 2004 Elsevier Inc. All rights reserved. Keywords—DOPA, Proteasome, Lysosome, Cathepsin, Protein oxidation, Free radicals
shown that oxidizing and reducing species which are capable of initiating secondary reactions and transferring damage to other biomolecules can also be generated [8]. The major reducing species is protein-bound (PB) DOPA (3,4-dihydroxyphenylalanine) [9], a product of hydroxyl radical attack on tyrosine residues and one of the major oxidized species elevated in pathological tissues [10,11]. Previous studies have described the potentially toxic effects of PB-DOPA, such as promoting further radicalgenerating reactions by replenishing levels of reduced metals (for review see [6]); in addition DOPA can react with a number of amino acids, generating products such as 5-S-cysteinyl-DOPA, and so may initiate protein aggregation through intermolecular cross-linking [12]. We have previously shown that DOPA-containing proteins can be experimentally generated in mouse macrophages from the biosynthetic incorporation of DOPA [13]. The aims of the present studies were to determine if DOPA is biosynthetically incorporated into proteins by human cells in culture and to investigate the cellular proteolysis of these modified proteins.
INTRODUCTION
Proteins in living tissues are constantly exposed to free radical-derived reactive oxygen species (ROS) originating from both endogenous and exogenous sources (reviewed in [1,2]). Despite extensive cellular antioxidant defenses oxidized proteins are continually generated in living systems and can accumulate during age-related pathologies [3] and in the aging process itself [1,4]. The primary mechanism for the removal of oxidized proteins in biological systems is thought to be complete enzymatic hydrolysis [5,6] but this defense is sometimes inadequate as evidenced by their accumulation. ROS attack on proteins can result in modification of amino acid side chains, protein fragmentation, or crosslinking [2,7]. Side-chain modification commonly generates relatively inert chemical species but we have
Address correspondence to: Ken Rodgers, The Heart Research Institute, 145 Missenden Road, Camperdown, Sydney, NSW 2050, Australia; Fax: +61 2 9550 3302; E-mail: [email protected]. 1756
Biosynthetic incorporation of DOPA MATERIALS AND METHODS
Materials Lactacystin, p-nitrocatechol sulfate, l-DOPA, nitro blue tetrazolium, amido black, pepstatin A, N-Cbz-GlyGly-Arg-7-aminomethylcoumarin (AMC), N-Suc-LeuLeu-Val-Tyr-AMC, N-t-Boc-Leu-Ser-Thr-Arg-AMC, and E64 were purchased from the Sigma–Aldrich Chemical Co. (St. Louis, MO, USA). THP-1 cells were from American Type Culture Collection (ATCC). All radiochemicals were obtained from Amersham Life Science (Buckinghamshire, England). Mercaptoacetic acid was from Merck (Kilsyth, VIC, Australia). Culture medium was from JRH Biosciences (Lenexa, KS, USA). Z-Val-Val-Arg-AMC, Z-Arg-Arg-AMC, Z-Phe-ArgAMC, and Cbz-Leu-Leu-Glu-AMC were purchased from Calbiochem–Behring (La Jolla, CA, USA). Epoxomicin was from Calbiochem (Darmstadt, Germany). Water was from a Milli-Q four-stage system (Millipore-Waters, Lane Cove, NSW, Australia). All electrophoresis materials and equipment were from Bio-Rad Laboratories (Hercules, CA, USA). The electrophoresis molecular weight markers used were Bio-Rad Kaleidoscope. Other chemicals, solvents, and chromatographic materials were AR or HPLC grade. Isolation of human peripheral blood monocytes Monocytes were isolated from the peripheral blood of healthy volunteers by counterflow centrifugal elutriation as described previously [14]. Briefly, a white cell concentrate was prepared using Lymphoprep (Nycomed) and monocytes were isolated by counterflow centrifugal elutriation using a Beckman Coulter Avanti J-20 XPI centrifuge and a 4 ml elutriation chamber. Collected fractions were examined using a Cytospin system (Shandon) and cells stained using WrightTs stain (DiffQuick, Lab-Aids). Monocyte purity was greater than 95%. Monocytes were plated at 1 106 cells per well in 12 well plates in RPMI medium (serum free) for 60 min and then cultured in RPMI containing 10% heatinactivated human serum. Culture medium was changed every 48 h.
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centrifugation (THP-1 cells). For degradation studies using THP-1 cells the medium was replaced with DMEM containing 10% FCS and 5 mM tyrosine—bdegradation mediumQ for the degradation period (0 to 48 h). The cells were then washed in PBS and pelleted by centrifugation. In the inhibition studies, sterile stock solutions of lactacystin (10 mM in DMSO), E64 (1 mM in water), and NH4Cl (1 M in water) were freshly prepared and added to the culture medium immediately before use. The final concentrations used were lactacystin 10 AM, E64 10 AM, and ammonium chloride 50 mM. Isolated human peripheral blood monocytes were used after 5 d in culture by which time they displayed characteristic macrophage morphology. Cell culture for degradation studies using [14C]DOPA Degradation medium was added to THP-1 cells in which [14C]DOPA-containing proteins had been generated for 24 h. The cultures were terminated (0 to 24 h), the cells were lysed with 0.1% Triton X-100, and the radioactivity in the culture medium and cell lysate was measured by liquid scintillation counting. TCA (50%) was added to give a final concentration of 5%, and free amino acid (TCA soluble) and protein-incorporated (TCA precipitable) radiolabel in cell lysate and culture medium was quantified by liquid scintillation counting as we have previously described [13]. Cell pellets were washed three times with 5% TCA and the pellet was dissolved in formic acid. Examination of cell lysates by electrophoresis Cells were washed three times with PBS and pelleted. The pellet was lysed in Triton X-100 (0.1%), the lysate centrifuged at 13,000 g for 30 min, and the supernatant collected. The protein content of the supernatant was measured using the BCA assay with BSA as a standard. Cell proteins were mixed with standard SDS–PAGE sample buffer and heated to 958C for 5 min. Electrophoresis was carried out using 10% acrylamide 7 cm 1.5 mm gels using a standard Tris–glycine buffer system (Bio-Rad). Transfer to PVDF membranes was carried out overnight (100 mA) using the Towbin buffer system.
THP-1 cell culture
Redox and protein staining of membranes
THP-1 cells (a human macrophage cell line) were maintained in 750 cm2 flasks in DMEM containing 10% FCS.
After protein transfer, PVDF membranes were washed in water for 5 min and incubated in nitro blue tetrazolium (0.24 mM) in alkaline glycinate as described by Paz [15]. Color was allowed to develop for up to 30 min in the dark and then the membranes were scanned using a Umax Powerlook 1120 scanner. The color was then completely stripped with methanol (100%) and the membranes were stained with amido black (0.1% in 40% methanol and 10% acetic acid) and scanned as before. Densitometric analysis was carried out using a
Cell culture for DOPA incorporation For incorporation of DOPA, the culture medium was replaced with tyrosine-free DMEM (JRH Biosciences) containing 10% FCS and DOPA (or [14C]DOPA) (50 to 1000 AM). After 24 h the medium was removed and cells were washed three times with PBS and recovered by
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Umax Powerlook 1120 scanner and Bio-Rad Quantity One software. The parameters were identical in all of the scanning and densitometric data presented. Assays for cathepsins B and L Activity against fluorogenic peptide substrates was determined by continuously monitoring fluorescence of the released AMC using a Cytofluor II plate reader with an excitation wavelength of 360 nm and an emission wavelength of 460 nm. The rates of substrate cleavage were determined from the initial linear portions of the curves (10–30 min). Assays were carried out in triplicate in microtiter plates and the change in fluorescence was measured at 258C. Cathepsin B activity in cell lysates was determined against Z-Arg-Arg-AMC and cathepsin L against Z-Phe-Arg-AMC at a substrate concentration of 50 AM. Samples (5–20 Al) were activated for 5 min at 258C in 0.1 M phosphate buffer, pH 6 (cathepsin B) or pH 5.5 (cathepsin L), containing 0.005% Brij 35, EDTA (2.5 mM), and DTT (2.5 mM). Samples were then brought to 100 Al in the aforementioned buffer without DTT but containing the protease inhibitors to give the concentrations benzamidine 5 mM, EDTA 5 mM, and pepstatin A 1 AM and incubated for 15 min at room temperature before addition of substrate. DMSO concentration in the final assay buffer was always less than 0.5%. Determination of cathepsin S activity Cathepsin S activity was determined essentially as described for cathepsins B and L; however, an additional step was employed in which cell lysates were incubated in pH 7.5 buffer at 408C for 60 min to inactivate cathepsins other than cathepsin S [16]. Lysates were incubated in 50 mM Tris acetate, pH 5.5, containing 2 mM EDTA, 0.01% Triton X-100, and 2 mM dithiothreitol at 378C for 15 min; the pH was then adjusted to 7.5 by the addition of 0.2 M phosphate buffer containing 5 mM EDTA and 0.01% Triton X-100 and the samples were incubated at 408C for 60 min. Samples were then brought to 100 Al with 50 mM Tris acetate, pH 5.5, containing proteinase inhibitors to give the final concentrations benzamidine 5 mM, EDTA 5 mM, and pepstatin A 1 AM. Samples were incubated for a further 15 min at room temperature and the substrate (Z-Val-Val-ArgAMC) was added (50 AM). The change in fluorescence was measured at 258C under conditions under which a linear relation between time and product generation was obtained as described for cathepsins B and L. Determination of proteasome activity Proteasome activity against fluorogenic peptide substrates was determined by continuously monitoring the fluorescence of the released AMC using a Cytofluor II plate reader. The assays were performed in 50 mM Tris,
20 mM KCl, 0.5 mM MgOAc, 2 mM ATP, 1 mM dithiothreitol, pH 7.8 [17], in a final reaction volume of 60–100 Al. The peptide substrates were used at 100 AM and the change in fluorescence was measured with an excitation wavelength of 360 nm and an emission wavelength of 460 nm. Chymotryptic activity was measured using N-Suc-Leu-Leu-Val-Tyr-AMC, peptidylglutamyl peptide hydrolyzing (PGPH) activity using Cbz-Leu-Leu-Glu-AMC, and tryptic activity using BocLeu-Arg-Arg-AMC. The rates of substrate cleavage were determined from the initial linear portions of the curves (5–20 min). Activity was determined in the presence and absence of the proteasome inhibitor epoxomycin (50 AM). Proteasome activity was calculated from the differences in the activity in samples with and without epoxomycin. Assays were carried out in 96 well microtiter plates and the change in fluorescence was measured after incubation at 258C. Determination of arylsulfatase activity Arylsulfatase activity was determined by measuring the enzymatic hydrolysis of the ester sulfate bond of p-nitrocatechol sulfate using methods based on those of Orange and Moore [18]. Briefly, 10 Al aliquots of cell lysates were incubated for up to 60 min with 90 Al of 10 mM sodium acetate buffer (pH 5.0) containing 7.5 mM p-nitrocatechol sulfate and 0.01% (v/v) Triton X-100. The reaction was stopped with the addition of 100 Al of freshly prepared alkaline quinol solution (1 ml of 0.1 M HCl containing 4% (w/v) hydroquinone (UNILAB) mixed with 20 ml of 2.5 M NaOH containing 5% (w/v) Na2SO3 d 7H2O). Absorbance was measured at 540 nm to determine the free nitrocatechol released by arylsulfatase enzyme activity (Sunrise Remote Control Plate Reader, Tecan, Austria). Protein hydrolysis and HPLC analysis Cell lysates were precipitated with TCA (5%), washed, and delipidated by resuspending the pellet twice in TCA (5%) containing sodium deoxycholate (0.02%) and sodium borohydride (25 Ag/ml) and then washed twice in ice-cold acetone and once in diethyl ether. The delipidated protein samples were then freezedried and hydrolyzed under anaerobic conditions using a standard gas-phase acid-catalyzed method (HCl containing mercaptoacetic acid) [9]. HPLC analysis of DOPA was performed on an LC-10A HPLC system (Shimadzu) equipped with a column oven (Waters; 308C) with methods developed in our laboratory as described in detail previously [11]. System operation was automated by Class LC-10 software. Chromatography was on a Zorbax ODS column (250 4.6 mm) with a Pelliguard guard column (LC-18). The mobile phase was a gradient of solvent A (10 mM sodium phosphate buffer (pH 2.5)
Biosynthetic incorporation of DOPA
with 100 mM sodium perchlorate) and solvent B (80% v/v methanol) at a flow rate of 1 ml/min. The following gradient was used: isocratic elution with 100% A for 12 min, then to 80% A over 8 min, elution at 80% A for 3 min before changing to 50% A in 3 min, isocratic elution at 50% A for a further 3 min, and then reequilibration with 100% A for 10 min. The eluate was monitored by a UV detector (Shimadzu) and a fluorescence detector (Hitachi F-1080) in series. The fluorescence detector was set at an excitation wavelength of 280 nm and an emission wavelength of 320 nm. The unmodified p-tyrosine was quantified by UV measurement when there was an off-scale response by fluorescence detection. The elution positions of the amino acids and oxidized amino acids were defined on the basis of standards using both UV and fluorescence spectra. Statistical analysis Results are presented as means plus or minus standard errors of the mean or the standard deviations (when indicated). Differences between samples were evaluated using a one-way ANOVA and a Newman–Keuls multiple comparison t test. RESULTS
Incorporation of DOPA into THP-1 cell proteins by protein synthesis l-DOPA (or DOPA) was supplied to THP-1 cells (a human monocyte cell line) in tyrosine-free culture medium for 24 h. The cell proteins were isolated, washed, and hydrolyzed and the levels of tyrosine and DOPA in the hydrolysate quantified by HPLC. The level of DOPA in the cell proteins increased linearly with increasing the concentration of DOPA supplied to the cells (Fig. 1). To determine if DOPA incorporation was by protein synthesis, THP-1 cells were incubated in medium containing 500 AM DOPA (of which 0.02 AM was present as [14C]DOPA) and cycloheximide (0 and 0.5 Ag/ml) for 24 h. The incorporation of [14C]DOPA into cell proteins was reduced by 95% (standard deviation 1.2%) when cycloheximide was present in the medium, demonstrating that protein synthesis was required to generate PB-DOPA. To visualize DOPA-containing proteins, THP-1 cells were cultured for 24 h in medium containing DOPA (0, 150, and 500 AM) with and without cycloheximide (0.5 Ag/ ml). The cell proteins were isolated, separated by SDS– PAGE, transferred to PVDF membranes, and visualized on the membranes using a redox-cycling staining method previously used to detect naturally occurring quinoproteins [15]. Quinone-containing proteins were present in lysates from DOPA-treated cells but were not present in lysates from DOPA-free cultures or when cyclo-
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Fig. 1. THP-1 cells were incubated in medium containing DOPA (0 to 1000 AM) for 24 h. The 5% TCA-precipitated cell proteins were washed and hydrolyzed and the DOPA and tyrosine contents determined after separation by HPLC. DOPA content of the proteins is expressed as a ratio to the parent amino acid tyrosine. The correlation coefficient is 0.962.
heximide was included in the culture medium to block the synthesis of new proteins (Fig. 2). Direct incubation of THP-1 cell lysates with DOPA (0, 150, and 500 AM) for 24 h at 378C did not generate any redox-cycling positive proteins (data not shown). THP-1 cells were then incubated in medium containing 500 AM DOPA (0.02 AM as [14C]DOPA) to allow a comparison between detection of DOPA-containing proteins by redox-cycling staining and detection of radiolabeled DOPA by phosphorimage analysis. Cell proteins were isolated, electrophoretically separated, and transferred to a PVDF membrane. The membrane was stained for redox-cycling activity, the stain was then removed using methanol, and the membrane was restained with amido black (for total protein). The same membrane was then analyzed using a phosphorimager (28 d) to detect 14C-labeled proteins (Fig. 3). Visualization of DOPA-containing proteins by the redoxcycling method and that by phosphorimage analysis of [14C]DOPA-labeled proteins were indistinguishable; the newly synthesized DOPA-containing proteins visualized by these methods covered a molecular weight range similar to that obtained from staining for total cell proteins. A number of protein bands were detectable by protein staining that were absent when DOPA-containing proteins were selectively visualized; the relative band intensities also varied between protein staining and detection of DOPA-containing proteins (Fig. 3). Redoxcycling staining was faster and more sensitive than phosphorimage analysis of [14C]DOPA and avoided the use and expense of radioisotopes, so it was used by preference in the following studies. The correlation between protein loading and staining intensity was then assessed for the redox-cycling staining
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protein loading with a correlation coefficient of 0.964 for redox staining and 0.953 for amido black protein staining (plot not shown). Cellular proteolysis of DOPA-containing proteins—visualization by redox-cycling staining
Fig. 2. THP-1 cells were incubated in medium containing DOPA, 0 AM (lanes 1 and 2), 150 AM (lanes 3 and 4), and 500 AM (lanes 5 and 6), for 24 h. Cycloheximide was present in the culture medium of the cell cultures examined in lanes 2, 4, and 6. Cells were lysed and proteins were precipitated, separated by SDS–PAGE, and blotted onto PVDF. Visualization of DOPA-containing proteins was by redox-cycling staining. Lane 7 contains prestained molecular weight markers with molecular weights as indicated. 50 Ag of protein was loaded onto each lane.
method. DOPA-containing proteins were generated by incubating THP-1 cells with DOPA (500 AM) for 24 h. The cell proteins were isolated and the protein concentration in the lysate was quantified using the BCA assay. Increasing amounts of protein were electrophoresed in each lane (10, 20, 30, 40, and 50 Ag) and the proteins transferred to a PVDF membrane. After staining, five proteins were chosen, covering the full molecular weight range of proteins, and the staining intensity of the band was measured in each lane by densitometry. The band intensity was then plotted against protein loading. With both redox staining and amido black staining, a linear correlation was demonstrated between band intensity and
Fig. 3. THP-1 cells were incubated in medium containing 500 AM DOPA and 0.02 AM [14C]DOPA for 24 h. Cell proteins were isolated, electrophoresed, and analyzed for redox-cycling activity (lane 2). The stain was removed and the membrane stained with amido black for total protein (lane 1); the membrane-bound radioactivity was then measured by phosphorimage analysis (28 d) (lane 3). Lane 4 contains prestained molecular weight markers with molecular weights as indicated. 100 Ag of protein was loaded onto each lane.
To examine the cellular removal of DOPA-containing proteins, DOPA (500 AM) was incorporated into THP-1 cell proteins for 24 h and the turnover of redox-positive (DOPA-containing) proteins monitored over the following 48 h. After 48 h most of the detectable redoxpositive proteins had been removed (Fig. 4A). The membrane was then washed in methanol and restained with amido black to confirm equal protein loading in each lane (Fig. 4B). Inhibition of turnover of DOPA-containing proteins To determine the effects of proteinase inhibitors on the cellular removal of DOPA-containing proteins THP-1 cells were incubated for 24 h with 250 AM DOPA (of which 0.02 AM was present as [14C]DOPA). Cells were then washed in PBS and incubated in degradation medium. After 2, 4, and 7 h cells were harvested and the degradation of the [14C]DOPA-containing proteins was quantified using the methods we have described previously [13]. The inclusion of an inhibitor of the proteasome (lactacystin 10 AM) inhibited degradation of DOPA-containing proteins by an average of 68% (standard deviation 3.5%) and inhibitors of the lysosomal proteinases (ammonium chloride 50 mM and E64 10 AM) inhibited the degradation of DOPA-containing proteins by an average of 32% (standard deviation 3.2%) (Fig. 5).
Fig. 4. THP-1 cells were incubated in medium containing 500 AM DOPA for 24 h. The medium was then replaced with degradation medium. The cultures were terminated and the cell lysates examined after 24, 12, 8, 4, and 0 h (lanes 2 to 6, respectively). Lane 1 contains prestained molecular weight markers with molecular weights as indicated. (A) DOPA-containing proteins were visualized by redoxcycling staining. (B) The membrane was then scanned and the stain removed by washing in methanol. The membrane was restained for total protein using amido black. 50 Ag of protein was loaded onto each lane.
Biosynthetic incorporation of DOPA
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some activity without a separation step to remove other proteases [19,20], but because insufficient sample was available for this procedure, activity was measured in the presence or absence of the specific proteasomal inhibitor epoxomycin. No change in the proteasome activity could be detected with increasing DOPA incorporation into cell proteins (Fig. 6B—only chymotryptic shown). The rates of degradation of DOPA-containing proteins by THP-1 cells were then assessed at different levels of supply (and incorporation). [14C]DOPA was included to allow the degradation of [14C]DOPA-containing proteins to be selectively assessed. Degradation was assessed after 8 h by measuring the release into the culture medium of free radiolabel [13]. An initial increase in the rates of [14C]DOPA-containing protein degradation was demonstrated at lower levels of DOPA supply; however, at higher levels of DOPA the turnover rates of these modified proteins started to decrease (Fig. 7). To Fig. 5. THP-1 cells were incubated in medium containing 250 AM DOPA for 24 h (0.02 AM was present as [14C]DOPA). The medium was then replaced with degradation medium and the cells were allowed to degrade the labeled proteins. Parallel cultures contained the proteasome inhibitor lactacystin (10 AM, gray bars) or the inhibitors of lysosomal proteinases E64 and ammonium chloride (10 AM and 50 mM, respectively, black bars). Cultures were terminated at 2, 4, and 7 h and the degradation of the [14C]DOPA-containing proteins was quantified. The degradation in the inhibitor-containing cultures was expressed as a percentage of that in the inhibitor-free cultures. Error bars show the standard deviation obtained from triplicate cell cultures.
Effect of DOPA-containing proteins on the activity of proteasomes and lysosomes In order to assess if the biosynthetically generated DOPA-containing proteins altered the activity of the major cellular degradation machinery, THP-1 cells were incubated in a range of DOPA concentrations (0 to 750 AM) for 24 h, the DOPA-containing medium was removed, and the cells were allowed to degrade the native and modified proteins for 8 h. Cells were then harvested and the activity of the proteasomes and some lysosomal enzymes was measured (Fig. 6). The activity of the lysosomal enzyme arylsulfatase was unchanged at all levels of DOPA supply (Fig. 6B), a small but significant decrease in cathepsin B and L activities was found at the lower levels of DOPA supply (up to 250 AM supply or 12 mmol DOPA/mol Tyr incorporation), but the activity of these proteinases significantly increased at higher levels of DOPA supply (Fig. 6A) (500 AM supply or 18 mmol/mol Tyr incorporation). The activity of cathepsin S, however, showed a significant increase at the lower DOPA levels, decreasing to the levels in the DOPA-free cultures at higher levels (Fig. 6B). Proteasomal activity was assessed by measuring tryptic, chymotryptic, and PGPH activities. As has been shown previously, it is difficult to accurately measure protea-
Fig. 6. THP-1 cells were incubated in medium containing DOPA (0, 125, 250, 500, and 750 AM) for 24 h. The medium was then replaced with degradation medium and the cells were allowed to degrade proteins for 8 h. (A) The cells were then lysed and the activity of cathepsins B (.) and L (o) and the chymotryptic activity of the proteasome (D) were measured. (B) The activity of the lysosomal proteinase cathepsin S (5) and the lysosomal sulfatase arylsulfatase (n) was also measured. Activity was normalized to cell protein and expressed as a percentage of the control (DOPA-free cultures).
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Fig. 7. THP-1 cells were incubated for 24 h in medium containing DOPA (0, 125, 250, 500, and 750 AM) of which 0.02 AM was present as [14C]DOPA. The medium was then replaced with degradation medium and the cells allowed to degrade proteins for 8 h. Cells were then lysed and the degradation was measured from the release of free [14C]DOPA into the culture medium over the degradation period. Degradation was expressed as a percentage of the total amount of labeled protein present at the start of the degradation period.
Fig. 8. Human monocyte-derived macrophages were incubated in medium containing DOPA, 0 AM (lanes 2 and 5), 500 AM (lanes 3 and 6), and 500 AM with cycloheximide (lanes 4 and 7), for 24 h. Cells were lysed and proteins were precipitated, separated by SDS–PAGE, and blotted onto PVDF. Visualization of DOPA-containing proteins was by redox-cycling staining (lanes 2, 3, and 4). The membrane was stripped and restained for protein using amido black (lanes 5, 6, and 7). 30 Ag of protein was loaded onto each lane.
determine if free DOPA (rather than PB-DOPA) had a direct inhibitory effect on the activity of the cathepsins, cathepsin B and L activity was compared in the lysates from cells incubated with DOPA for 24 h (150 and 500 AM) both with and without cycloheximide. No significant difference in cathepsin activity was found in the cycloheximide-treated cultures; however, cathepsin B and L activities were both significantly inhibited in the cycloheximide-free cultures (Table 1).
containing 500 AM DOPA (of which 0.02 AM was present as [14C]DOPA), or leucine-free medium containing 0.02 AM [14C]leucine, and cycloheximide (0 and 0.5 Ag/ml) was added to the cells. After 24 h cell proteins were isolated and the incorporation of [14C]DOPA or [14C]leucine was assessed by liquid scintillation counting. The incorporation of [14C]DOPA into cell proteins was reduced by 79% (standard deviation 6.8%) when cycloheximide was present and the incorporation of [14C]leucine was reduced by 73% (standard deviation 2.6%), demonstrating that DOPA-containing proteins were also generated in these primary human cells by protein synthesis.
Incorporation of DOPA into cell proteins by isolated human monocytes
Visualization of DOPA-containing proteins in human monocyte-derived macrophages
In order to examine DOPA incorporation into cell proteins by a primary human cell, monocytes were isolated from peripheral human blood and maintained in culture. After 5 d in culture, tyrosine-free medium
Cell proteins were generated in human monocytederived macrophages as described above and the cell proteins isolated and examined by redox staining. No redox-staining-positive bands were detected in cell
Table 1. DOPA-containing Proteins Were Generated by Incubating THP-1 Cells with DOPA (0, 150, and 500 AM); Some Cultures Contained Cycloheximide to Prevent Protein Synthesis Cathepsin L activity (% of control) DOPA (AM) 0 150 500
Cathepsin B activity (% of control)
No cycloheximide
With cycloheximide
No cycloheximide
With cycloheximide
100 (5.5) 80.3* 85.7* (0.6)
100 (1.0) 100.7 (3.5) 101.0 (4.0)
100 (3.5) 84.7* (2.5) 92.3* (4.6)
100 (2.0) 97 (1.7) 106 (5.3)
The cells were washed and lysed and the activities of cathepsins L and B in the lysates measured using peptide substrates; these were normalized to the protein concentration. The specific activity in the DOPA-treated cultures is expressed relative to that of the DOPA-free cultures (set at 100%). Data were from triplicate cultures. * Statistically significant change in activity ( p b .05).
Biosynthetic incorporation of DOPA
proteins isolated from human monocytes; a number of redox-staining-positive bands were present in cell proteins from cells which had been incubated for 24 h in medium containing DOPA (500 AM); these were not present when cycloheximide was included in the culture medium to block the synthesis of new proteins (Fig. 8). Stripping and restaining of the membrane with amido black confirmed equal protein loading in each lane (Fig. 8, lanes 5, 6, and 7). DISCUSSION
We have previously demonstrated that DOPA and other oxidized amino acids can be biosynthetically incorporated into proteins by murine cells in culture [13]. Here we demonstrate that a human cell line (THP1 monocytes) and primary human cells (monocytederived macrophages) can also generate protein-bound DOPA by protein synthesis. Using a redox-cycling staining method which was first introduced to detect native quino-proteins [15] we were able to visualize for the first time the range of biosynthetically generated DOPA-containing cell proteins. The staining method utilizes the capacity of the catechol group of DOPA to redox cycle (catechol to quinone) at an alkaline pH in the presence of glycine, reducing nitro blue tetrazolium to formazan, producing a purple color. No quinonecontaining proteins were detectable in cell proteins when no DOPA was present in the culture medium or when DOPA was supplied together with an inhibitor of protein synthesis, providing further evidence that incorporation of DOPA is by protein synthesis. Incubation of DOPA with cell lysates did not result in any positive quinone staining, eliminating the possibility that there were any SDS–PAGE stable chemical interactions between DOPA and cell proteins. The staining patterns obtained from THP-1 cell proteins by measuring PB-[14C]DOPA by phosphorimage analysis and PB-DOPA by redox staining were very similar, supporting the validity of the redox staining as a sensitive and selective method to detect DOPA-containing proteins. In addition, a linear correlation was demonstrated between the intensity of the redox staining and the amount of sample examined. The levels of [14C]DOPA used in these studies required 28 d to detect DOPA-containing proteins, whereas redox staining could be performed in 30 min. The redox staining was therefore shown to be a fast and quantifiable method for the detection of DOPA-containing proteins. The range of the newly synthesized proteins which contained DOPA was shown to be similar to the total protein staining, suggesting that many proteins contained biosynthetically incorporated DOPA and that many of these proteins were synthesized as the fulllength forms.
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Most of the DOPA-containing proteins were removed by the THP-1 cells after 24 h; this process could be delayed by inhibitors of both the proteasomal and the lysosomal pathways, suggesting that both pathways are involved in their degradation. The proteasome seemed to be the primary pathway for the removal of DOPAcontaining proteins. Inhibitors of the lysosomal proteinases reduced the degradation of DOPA-containing proteins by around 30%, consistent with our previous studies using a mouse macrophage cell line. These data support a role for the endosomal–lysosomal system in the turnover of these endogenously generated DOPA-containing proteins but indicate that the proteasome is the primary pathway responsible for their turnover. At the lower levels of DOPA incorporation into proteins their turnover was greatly accelerated without any detectable increases in the activity of proteasomes or the lysosomal enzymes, suggesting that the increased turnover rate of the modified proteins did not require upregulation in either of the major proteolytic pathways. A small but significant decrease in cathepsin B and L activity was found at lower levels of DOPA incorporation. The ability of DOPA to transfer damage to other proteins has been described and this process may be amplified in the acid environment of the lysosome, which may also contain redox-active iron [21,22]. DOPA could also directly inactivate lysosomal cysteine proteinases by cross-linking with the active-site cysteine residues to form cysteinyl-DOPA, a reaction which is known to occur in vivo [23]. No decrease in cathepsin activity was found when protein synthesis was blocked; this would suggest that inhibition was from protein-bound DOPA rather than free DOPA. As we reported previously, at higher levels of DOPA incorporation into proteins their turnover rate begins to decrease; this may reflect the situation in pathological tissues in which these proteins begin to accumulate. The activity of the proteasome in THP-1 cells was not altered when DOPA-containing proteins were present at higher levels of incorporation, suggesting that there was no direct inhibition of proteasomes by DOPA-containing proteins; this is consistent with our previous studies, which showed that the turnover of DOPA-containing proteins did not alter the degradation rates of native proteins, which are thought to be degraded mainly by the proteasomal pathway [13]. The decrease in turnover of DOPA-containing proteins was accompanied by a substantial increase in the activity of the lysosomal proteinases cathepsins B and L. A similar increase in cathepsin activity (or immunereactive cathepsin) has been reported in response to aggregated protein accumulation in both experimental systems [24] and pathological tissues as a response to protein aggregation [25]. Here we demonstrate that an
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increase in lysosomal proteinase activity may also be a compensatory response to the presence of oxidized amino-acid-containing proteins, which are less readily degraded by the cells. Cathepsin S activity was significantly increased at lower levels of DOPA incorporation but returned to basal levels at higher levels of DOPA incorporation, which may suggest a more specialized role for cathepsin S in the cell, such as in the generation of MHC class II epitopes. DOPA-containing proteins were also generated in human monocyte-derived macrophages on incubation of the cells with DOPA. The inclusion of an inhibitor of protein synthesis blocked the incorporation of DOPA, demonstrating that incorporation was by biosynthesis. As was observed for the THP-1 cells, a range of DOPAcontaining proteins was generated by the monocytederived macrophages and could be visualized by redoxcycling staining. These data raise the possibility that DOPA could be biosynthetically incorporated into human proteins in vivo. In summary, we demonstrate that DOPA can be biosynthetically incorporated into a broad range of proteins by a human monocyte cell line (THP-1) and primary human cells (monocyte-derived macrophages). The cellular removal of DOPA-containing proteins by THP-1 cells is by proteolysis, involving both the proteasomal and the lysosomal systems. At higher levels of incorporation the activity of certain lysosomal cathepsins is selectively increased but the rate of removal of the proteins is reduced. These studies raise the possibility that DOPA-containing proteins can be generated in vivo from l-DOPA (levodopa), which is the primary treatment for Parkinson disease. Acknowledgments — This study was supported by the National Health and Medical Research Council of Australia (K.R., P.H., R.T.D.) and by an Australian Postgraduate Award (R.A.D.). REFERENCES [1] Ames, B. N.; Shigenaga, M. K.; Hagen, T. M. Oxidants, antioxidants, and the degenerative diseases of aging. Proc. Natl. Acad. Sci. USA 90:7915 – 7922; 1993. [2] Dean, R. T.; Fu, S.; Stocker, R.; Davies, M. J. The biochemistry and pathology of radical-mediated protein oxidation. Biochem. J. 324:1 – 18; 1997. [3] Dean, R. T.; Dunlop, R.; Hume, P.; Rodgers, K. J. Proteolytic ddefencesT and the accumulation of oxidized polypeptides in cataractogenesis and atherogenesis. Biochem. Soc. Symp. 70: 135 – 146; 2003. [4] Linton, S.; Davies, M. J.; Dean, R. T. Protein oxidation and ageing. Exp. Gerontol. 36:1503 – 1518; 2001. [5] Dunlop, R. A.; Rodgers, K. J.; Dean, R. T. Recent developments in the intracellular degradation of oxidized proteins. Free Radic. Biol. Med. 33:894 – 906; 2002. [6] Rodgers, K. J.; Dean, R. T. The metabolism of protein-bound DOPA in mammals. Int. J. Biochem. Cell Biol. 32:945 – 955; 2000. [7] Berlett, B. S.; Stadtman, E. R. Protein oxidation in aging, disease, and oxidative stress. J. Biol. Chem. 272:20313 – 20316; 1997.
[8] Simpson, J. A.; Narita, S.; Gieseg, S.; Gebicki, S.; Gebicki, J. M.; Dean, R. T. Long-lived reactive species on free-radical-damaged proteins. Biochem. J. 282:621 – 624; 1992. [9] Gieseg, S. P.; Simpson, J. A.; Charlton, T. S.; Duncan, M. W.; Dean, R. T. Protein-bound 3,4-dihydroxyphenylalanine is a major reductant formed during hydroxyl radical damage to proteins. Biochemistry 32:4780 – 4786; 1993. [10] Fu, S.; Davies, M. J.; Stocker, R.; Dean, R. T. Evidence for roles of radicals in protein oxidation in advanced human atherosclerotic plaque. Biochem. J. 333:519 – 525; 1998. [11] Fu, S.; Dean, R.; Southan, M.; Truscott, R. The hydroxyl radical in lens nuclear cataractogenesis. J. Biol. Chem. 273:28603 – 28609; 1998. [12] Ito, S.; Kato, T.; Shinpo, K.; Fujita, K. Oxidation of tyrosine residues by tyrosinase—formation of protein-bound 3,4-dihydroxyphenylalanine and 5-S-cysteinyl-3,4-dihydroxyphenylalanine. Biochem. J. 222:407 – 411; 1984. [13] Rodgers, K. J.; Wang, H.; Fu, S.; Dean, R. T. Biosynthetic incorporation of oxidized amino acids into proteins and their cellular proteolysis. Free Radic. Biol. Med. 32:766 – 775; 2002. [14] Garner, B.; Dean, R. T.; Jessup, W. Human macrophage-mediated oxidation of low-density lipoprotein is delayed and independent of superoxide production. Biochem. J. 301:421 – 428; 1994. [15] Paz, M. A.; Fluckiger, R.; Boak, A.; Kagan, H. M.; Gallop, P. M. Specific detection of quinoproteins by redox-cycling staining. J. Biol. Chem. 266:689 – 692; 1991. [16] Kirschke, H.; Wiederanders, B.; Bromme, D.; Rinne, A. Cathepsin S from bovine spleen: purification, distribution, intracellular localization and action on proteins. Biochem. J. 264:467 – 473; 1989. [17] Sitte, N.; Huber, M.; Grune, T.; Ladhoff, A.; Doecke, W. D.; Von Zglinicki, T.; Davies, K. J. Proteasome inhibition by lipofuscin/ ceroid during postmitotic aging of fibroblasts. FASEB J. 14:1490 – 1498; 2000. [18] Orange, R. P.; Moore, E. G. Functional characterization of rat mast cell arylsulfatase activity. J. Immunol. 117:2191 – 2196; 1976. [19] Rodgers, K. J.; Dean, R. T. Assessment of proteasome activity in cell lysates and tissue homogenates using peptide substrates. Int. J. Biochem. Cell Biol. 35:716 – 727; 2003. [20] Rivett, A. J.; Bose, S.; Pemberton, A. J.; Brooks, P.; Onion, D.; Shirley, D.; Stratford, F. L.; Forti, K. Assays of proteasome activity in relation to aging. Exp. Gerontol. 37:1217 – 1222; 2002. [21] Marzabadi, M. R.; Sohal, R. S.; Brunk, U. T. Effect of ferric iron and desferrioxamine on lipofuscin accumulation in cultured rat heart myocytes. Mech. Ageing Dev. 46:145 – 157; 1988. [22] Persson, H. L.; Yu, Z.; Tirosh, O.; Eaton, J. W.; Brunk, U. T. Prevention of oxidant-induced cell death by lysosomotropic iron chelators. Free Radic. Biol. Med. 34:1295 – 1305; 2003. [23] Ito, S.; Kato, T.; Fujita, K. Covalent binding of catechols to protein through the sulphydryl group. Biochem. Pharmacol. 37:1707 – 1710; 1988. [24] Bendiske, J.; Bahr, B. A. Lysosomal activation is a compensatory response against protein accumulation and associated synaptopathogenesis—an approach for slowing Alzheimer disease? J. Neuropathol. Exp. Neurol. 62:451 – 463; 2003. [25] Ii, K.; Ito, H.; Kominami, E.; Hirano, A. Abnormal distribution of cathepsin proteinases and endogenous inhibitors (cystatins) in the hippocampus of patients with AlzheimerTs disease, parkinsonismdementia complex on Guam, and senile dementia and in the aged. Virchows Arch. A Pathol. Anat. Histopathol. 423:185 – 194; 1993. ABBREVIATIONS
DMEM — DulbeccoTs minimal essential medium DOPA — 3,4-dihydroxyphenylalanine E64 — l-transepoxysuccinylleucylamide-(4-guanido) butane FCS — fetal calf serum PGPH — peptidylglutamyl peptide hydrolyzing TCA — trichloroacetic acid
doi:10.1016/j.freeradbiomed.2004.08.009
Original Contribution BIOSYNTHESIS AND TURNOVER OF DOPA-CONTAINING PROTEINS BY HUMAN CELLS KENNETH J. RODGERS,* PETER M. HUME,* RACHAEL A. DUNLOP,* and ROGER T. DEANy * Cell Biology Unit, The Heart Research Institute, Camperdown, Sydney, NSW 2050, Australia; and y The University of Canberra, Canberra, ACT 2601, Australia (Received 31 March 2004; Revised 6 July 2004; Accepted 12 August 2004) Available online 11 September 2004
Abstract — Protein-bound 3,4-dihydroxyphenylalanine (PB-DOPA) is a major product of hydroxyl radical attack on tyrosine residues of proteins. Levels of PB-DOPA in cells and tissues have been shown to be greatly elevated in agerelated diseases. We demonstrate for the first time that l-DOPA (levodopa) can be biosynthetically incorporated into cell proteins by human cells (THP-1 monocytes and monocyte-derived macrophages). The DOPA-containing proteins generated were selectively visualized on PVDF membranes using a redox-cycling staining method. Many cell proteins contained DOPA and seemed to be synthesized as their full-length forms. The cellular removal of DOPA-containing proteins by THP-1 cells was by proteolysis involving both the proteasomal and the lysosomal systems. The rate of cellular proteolysis of DOPA-containing proteins increased at lower levels of DOPA incorporation but decreased at higher levels of DOPA incorporation. The decreased rate of degradation was accompanied by an increase in the activity of cathepsins B and L but the activity of cathepsin S increased only at lower levels of DOPA incorporation. These data raise the possibility that PB-DOPA could be generated in vivo from l-DOPA, which is the most widely used treatment for Parkinson disease. D 2004 Elsevier Inc. All rights reserved. Keywords—DOPA, Proteasome, Lysosome, Cathepsin, Protein oxidation, Free radicals
shown that oxidizing and reducing species which are capable of initiating secondary reactions and transferring damage to other biomolecules can also be generated [8]. The major reducing species is protein-bound (PB) DOPA (3,4-dihydroxyphenylalanine) [9], a product of hydroxyl radical attack on tyrosine residues and one of the major oxidized species elevated in pathological tissues [10,11]. Previous studies have described the potentially toxic effects of PB-DOPA, such as promoting further radicalgenerating reactions by replenishing levels of reduced metals (for review see [6]); in addition DOPA can react with a number of amino acids, generating products such as 5-S-cysteinyl-DOPA, and so may initiate protein aggregation through intermolecular cross-linking [12]. We have previously shown that DOPA-containing proteins can be experimentally generated in mouse macrophages from the biosynthetic incorporation of DOPA [13]. The aims of the present studies were to determine if DOPA is biosynthetically incorporated into proteins by human cells in culture and to investigate the cellular proteolysis of these modified proteins.
INTRODUCTION
Proteins in living tissues are constantly exposed to free radical-derived reactive oxygen species (ROS) originating from both endogenous and exogenous sources (reviewed in [1,2]). Despite extensive cellular antioxidant defenses oxidized proteins are continually generated in living systems and can accumulate during age-related pathologies [3] and in the aging process itself [1,4]. The primary mechanism for the removal of oxidized proteins in biological systems is thought to be complete enzymatic hydrolysis [5,6] but this defense is sometimes inadequate as evidenced by their accumulation. ROS attack on proteins can result in modification of amino acid side chains, protein fragmentation, or crosslinking [2,7]. Side-chain modification commonly generates relatively inert chemical species but we have
Address correspondence to: Ken Rodgers, The Heart Research Institute, 145 Missenden Road, Camperdown, Sydney, NSW 2050, Australia; Fax: +61 2 9550 3302; E-mail: [email protected]. 1756
Biosynthetic incorporation of DOPA MATERIALS AND METHODS
Materials Lactacystin, p-nitrocatechol sulfate, l-DOPA, nitro blue tetrazolium, amido black, pepstatin A, N-Cbz-GlyGly-Arg-7-aminomethylcoumarin (AMC), N-Suc-LeuLeu-Val-Tyr-AMC, N-t-Boc-Leu-Ser-Thr-Arg-AMC, and E64 were purchased from the Sigma–Aldrich Chemical Co. (St. Louis, MO, USA). THP-1 cells were from American Type Culture Collection (ATCC). All radiochemicals were obtained from Amersham Life Science (Buckinghamshire, England). Mercaptoacetic acid was from Merck (Kilsyth, VIC, Australia). Culture medium was from JRH Biosciences (Lenexa, KS, USA). Z-Val-Val-Arg-AMC, Z-Arg-Arg-AMC, Z-Phe-ArgAMC, and Cbz-Leu-Leu-Glu-AMC were purchased from Calbiochem–Behring (La Jolla, CA, USA). Epoxomicin was from Calbiochem (Darmstadt, Germany). Water was from a Milli-Q four-stage system (Millipore-Waters, Lane Cove, NSW, Australia). All electrophoresis materials and equipment were from Bio-Rad Laboratories (Hercules, CA, USA). The electrophoresis molecular weight markers used were Bio-Rad Kaleidoscope. Other chemicals, solvents, and chromatographic materials were AR or HPLC grade. Isolation of human peripheral blood monocytes Monocytes were isolated from the peripheral blood of healthy volunteers by counterflow centrifugal elutriation as described previously [14]. Briefly, a white cell concentrate was prepared using Lymphoprep (Nycomed) and monocytes were isolated by counterflow centrifugal elutriation using a Beckman Coulter Avanti J-20 XPI centrifuge and a 4 ml elutriation chamber. Collected fractions were examined using a Cytospin system (Shandon) and cells stained using WrightTs stain (DiffQuick, Lab-Aids). Monocyte purity was greater than 95%. Monocytes were plated at 1 106 cells per well in 12 well plates in RPMI medium (serum free) for 60 min and then cultured in RPMI containing 10% heatinactivated human serum. Culture medium was changed every 48 h.
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centrifugation (THP-1 cells). For degradation studies using THP-1 cells the medium was replaced with DMEM containing 10% FCS and 5 mM tyrosine—bdegradation mediumQ for the degradation period (0 to 48 h). The cells were then washed in PBS and pelleted by centrifugation. In the inhibition studies, sterile stock solutions of lactacystin (10 mM in DMSO), E64 (1 mM in water), and NH4Cl (1 M in water) were freshly prepared and added to the culture medium immediately before use. The final concentrations used were lactacystin 10 AM, E64 10 AM, and ammonium chloride 50 mM. Isolated human peripheral blood monocytes were used after 5 d in culture by which time they displayed characteristic macrophage morphology. Cell culture for degradation studies using [14C]DOPA Degradation medium was added to THP-1 cells in which [14C]DOPA-containing proteins had been generated for 24 h. The cultures were terminated (0 to 24 h), the cells were lysed with 0.1% Triton X-100, and the radioactivity in the culture medium and cell lysate was measured by liquid scintillation counting. TCA (50%) was added to give a final concentration of 5%, and free amino acid (TCA soluble) and protein-incorporated (TCA precipitable) radiolabel in cell lysate and culture medium was quantified by liquid scintillation counting as we have previously described [13]. Cell pellets were washed three times with 5% TCA and the pellet was dissolved in formic acid. Examination of cell lysates by electrophoresis Cells were washed three times with PBS and pelleted. The pellet was lysed in Triton X-100 (0.1%), the lysate centrifuged at 13,000 g for 30 min, and the supernatant collected. The protein content of the supernatant was measured using the BCA assay with BSA as a standard. Cell proteins were mixed with standard SDS–PAGE sample buffer and heated to 958C for 5 min. Electrophoresis was carried out using 10% acrylamide 7 cm 1.5 mm gels using a standard Tris–glycine buffer system (Bio-Rad). Transfer to PVDF membranes was carried out overnight (100 mA) using the Towbin buffer system.
THP-1 cell culture
Redox and protein staining of membranes
THP-1 cells (a human macrophage cell line) were maintained in 750 cm2 flasks in DMEM containing 10% FCS.
After protein transfer, PVDF membranes were washed in water for 5 min and incubated in nitro blue tetrazolium (0.24 mM) in alkaline glycinate as described by Paz [15]. Color was allowed to develop for up to 30 min in the dark and then the membranes were scanned using a Umax Powerlook 1120 scanner. The color was then completely stripped with methanol (100%) and the membranes were stained with amido black (0.1% in 40% methanol and 10% acetic acid) and scanned as before. Densitometric analysis was carried out using a
Cell culture for DOPA incorporation For incorporation of DOPA, the culture medium was replaced with tyrosine-free DMEM (JRH Biosciences) containing 10% FCS and DOPA (or [14C]DOPA) (50 to 1000 AM). After 24 h the medium was removed and cells were washed three times with PBS and recovered by
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Umax Powerlook 1120 scanner and Bio-Rad Quantity One software. The parameters were identical in all of the scanning and densitometric data presented. Assays for cathepsins B and L Activity against fluorogenic peptide substrates was determined by continuously monitoring fluorescence of the released AMC using a Cytofluor II plate reader with an excitation wavelength of 360 nm and an emission wavelength of 460 nm. The rates of substrate cleavage were determined from the initial linear portions of the curves (10–30 min). Assays were carried out in triplicate in microtiter plates and the change in fluorescence was measured at 258C. Cathepsin B activity in cell lysates was determined against Z-Arg-Arg-AMC and cathepsin L against Z-Phe-Arg-AMC at a substrate concentration of 50 AM. Samples (5–20 Al) were activated for 5 min at 258C in 0.1 M phosphate buffer, pH 6 (cathepsin B) or pH 5.5 (cathepsin L), containing 0.005% Brij 35, EDTA (2.5 mM), and DTT (2.5 mM). Samples were then brought to 100 Al in the aforementioned buffer without DTT but containing the protease inhibitors to give the concentrations benzamidine 5 mM, EDTA 5 mM, and pepstatin A 1 AM and incubated for 15 min at room temperature before addition of substrate. DMSO concentration in the final assay buffer was always less than 0.5%. Determination of cathepsin S activity Cathepsin S activity was determined essentially as described for cathepsins B and L; however, an additional step was employed in which cell lysates were incubated in pH 7.5 buffer at 408C for 60 min to inactivate cathepsins other than cathepsin S [16]. Lysates were incubated in 50 mM Tris acetate, pH 5.5, containing 2 mM EDTA, 0.01% Triton X-100, and 2 mM dithiothreitol at 378C for 15 min; the pH was then adjusted to 7.5 by the addition of 0.2 M phosphate buffer containing 5 mM EDTA and 0.01% Triton X-100 and the samples were incubated at 408C for 60 min. Samples were then brought to 100 Al with 50 mM Tris acetate, pH 5.5, containing proteinase inhibitors to give the final concentrations benzamidine 5 mM, EDTA 5 mM, and pepstatin A 1 AM. Samples were incubated for a further 15 min at room temperature and the substrate (Z-Val-Val-ArgAMC) was added (50 AM). The change in fluorescence was measured at 258C under conditions under which a linear relation between time and product generation was obtained as described for cathepsins B and L. Determination of proteasome activity Proteasome activity against fluorogenic peptide substrates was determined by continuously monitoring the fluorescence of the released AMC using a Cytofluor II plate reader. The assays were performed in 50 mM Tris,
20 mM KCl, 0.5 mM MgOAc, 2 mM ATP, 1 mM dithiothreitol, pH 7.8 [17], in a final reaction volume of 60–100 Al. The peptide substrates were used at 100 AM and the change in fluorescence was measured with an excitation wavelength of 360 nm and an emission wavelength of 460 nm. Chymotryptic activity was measured using N-Suc-Leu-Leu-Val-Tyr-AMC, peptidylglutamyl peptide hydrolyzing (PGPH) activity using Cbz-Leu-Leu-Glu-AMC, and tryptic activity using BocLeu-Arg-Arg-AMC. The rates of substrate cleavage were determined from the initial linear portions of the curves (5–20 min). Activity was determined in the presence and absence of the proteasome inhibitor epoxomycin (50 AM). Proteasome activity was calculated from the differences in the activity in samples with and without epoxomycin. Assays were carried out in 96 well microtiter plates and the change in fluorescence was measured after incubation at 258C. Determination of arylsulfatase activity Arylsulfatase activity was determined by measuring the enzymatic hydrolysis of the ester sulfate bond of p-nitrocatechol sulfate using methods based on those of Orange and Moore [18]. Briefly, 10 Al aliquots of cell lysates were incubated for up to 60 min with 90 Al of 10 mM sodium acetate buffer (pH 5.0) containing 7.5 mM p-nitrocatechol sulfate and 0.01% (v/v) Triton X-100. The reaction was stopped with the addition of 100 Al of freshly prepared alkaline quinol solution (1 ml of 0.1 M HCl containing 4% (w/v) hydroquinone (UNILAB) mixed with 20 ml of 2.5 M NaOH containing 5% (w/v) Na2SO3 d 7H2O). Absorbance was measured at 540 nm to determine the free nitrocatechol released by arylsulfatase enzyme activity (Sunrise Remote Control Plate Reader, Tecan, Austria). Protein hydrolysis and HPLC analysis Cell lysates were precipitated with TCA (5%), washed, and delipidated by resuspending the pellet twice in TCA (5%) containing sodium deoxycholate (0.02%) and sodium borohydride (25 Ag/ml) and then washed twice in ice-cold acetone and once in diethyl ether. The delipidated protein samples were then freezedried and hydrolyzed under anaerobic conditions using a standard gas-phase acid-catalyzed method (HCl containing mercaptoacetic acid) [9]. HPLC analysis of DOPA was performed on an LC-10A HPLC system (Shimadzu) equipped with a column oven (Waters; 308C) with methods developed in our laboratory as described in detail previously [11]. System operation was automated by Class LC-10 software. Chromatography was on a Zorbax ODS column (250 4.6 mm) with a Pelliguard guard column (LC-18). The mobile phase was a gradient of solvent A (10 mM sodium phosphate buffer (pH 2.5)
Biosynthetic incorporation of DOPA
with 100 mM sodium perchlorate) and solvent B (80% v/v methanol) at a flow rate of 1 ml/min. The following gradient was used: isocratic elution with 100% A for 12 min, then to 80% A over 8 min, elution at 80% A for 3 min before changing to 50% A in 3 min, isocratic elution at 50% A for a further 3 min, and then reequilibration with 100% A for 10 min. The eluate was monitored by a UV detector (Shimadzu) and a fluorescence detector (Hitachi F-1080) in series. The fluorescence detector was set at an excitation wavelength of 280 nm and an emission wavelength of 320 nm. The unmodified p-tyrosine was quantified by UV measurement when there was an off-scale response by fluorescence detection. The elution positions of the amino acids and oxidized amino acids were defined on the basis of standards using both UV and fluorescence spectra. Statistical analysis Results are presented as means plus or minus standard errors of the mean or the standard deviations (when indicated). Differences between samples were evaluated using a one-way ANOVA and a Newman–Keuls multiple comparison t test. RESULTS
Incorporation of DOPA into THP-1 cell proteins by protein synthesis l-DOPA (or DOPA) was supplied to THP-1 cells (a human monocyte cell line) in tyrosine-free culture medium for 24 h. The cell proteins were isolated, washed, and hydrolyzed and the levels of tyrosine and DOPA in the hydrolysate quantified by HPLC. The level of DOPA in the cell proteins increased linearly with increasing the concentration of DOPA supplied to the cells (Fig. 1). To determine if DOPA incorporation was by protein synthesis, THP-1 cells were incubated in medium containing 500 AM DOPA (of which 0.02 AM was present as [14C]DOPA) and cycloheximide (0 and 0.5 Ag/ml) for 24 h. The incorporation of [14C]DOPA into cell proteins was reduced by 95% (standard deviation 1.2%) when cycloheximide was present in the medium, demonstrating that protein synthesis was required to generate PB-DOPA. To visualize DOPA-containing proteins, THP-1 cells were cultured for 24 h in medium containing DOPA (0, 150, and 500 AM) with and without cycloheximide (0.5 Ag/ ml). The cell proteins were isolated, separated by SDS– PAGE, transferred to PVDF membranes, and visualized on the membranes using a redox-cycling staining method previously used to detect naturally occurring quinoproteins [15]. Quinone-containing proteins were present in lysates from DOPA-treated cells but were not present in lysates from DOPA-free cultures or when cyclo-
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Fig. 1. THP-1 cells were incubated in medium containing DOPA (0 to 1000 AM) for 24 h. The 5% TCA-precipitated cell proteins were washed and hydrolyzed and the DOPA and tyrosine contents determined after separation by HPLC. DOPA content of the proteins is expressed as a ratio to the parent amino acid tyrosine. The correlation coefficient is 0.962.
heximide was included in the culture medium to block the synthesis of new proteins (Fig. 2). Direct incubation of THP-1 cell lysates with DOPA (0, 150, and 500 AM) for 24 h at 378C did not generate any redox-cycling positive proteins (data not shown). THP-1 cells were then incubated in medium containing 500 AM DOPA (0.02 AM as [14C]DOPA) to allow a comparison between detection of DOPA-containing proteins by redox-cycling staining and detection of radiolabeled DOPA by phosphorimage analysis. Cell proteins were isolated, electrophoretically separated, and transferred to a PVDF membrane. The membrane was stained for redox-cycling activity, the stain was then removed using methanol, and the membrane was restained with amido black (for total protein). The same membrane was then analyzed using a phosphorimager (28 d) to detect 14C-labeled proteins (Fig. 3). Visualization of DOPA-containing proteins by the redoxcycling method and that by phosphorimage analysis of [14C]DOPA-labeled proteins were indistinguishable; the newly synthesized DOPA-containing proteins visualized by these methods covered a molecular weight range similar to that obtained from staining for total cell proteins. A number of protein bands were detectable by protein staining that were absent when DOPA-containing proteins were selectively visualized; the relative band intensities also varied between protein staining and detection of DOPA-containing proteins (Fig. 3). Redoxcycling staining was faster and more sensitive than phosphorimage analysis of [14C]DOPA and avoided the use and expense of radioisotopes, so it was used by preference in the following studies. The correlation between protein loading and staining intensity was then assessed for the redox-cycling staining
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protein loading with a correlation coefficient of 0.964 for redox staining and 0.953 for amido black protein staining (plot not shown). Cellular proteolysis of DOPA-containing proteins—visualization by redox-cycling staining
Fig. 2. THP-1 cells were incubated in medium containing DOPA, 0 AM (lanes 1 and 2), 150 AM (lanes 3 and 4), and 500 AM (lanes 5 and 6), for 24 h. Cycloheximide was present in the culture medium of the cell cultures examined in lanes 2, 4, and 6. Cells were lysed and proteins were precipitated, separated by SDS–PAGE, and blotted onto PVDF. Visualization of DOPA-containing proteins was by redox-cycling staining. Lane 7 contains prestained molecular weight markers with molecular weights as indicated. 50 Ag of protein was loaded onto each lane.
method. DOPA-containing proteins were generated by incubating THP-1 cells with DOPA (500 AM) for 24 h. The cell proteins were isolated and the protein concentration in the lysate was quantified using the BCA assay. Increasing amounts of protein were electrophoresed in each lane (10, 20, 30, 40, and 50 Ag) and the proteins transferred to a PVDF membrane. After staining, five proteins were chosen, covering the full molecular weight range of proteins, and the staining intensity of the band was measured in each lane by densitometry. The band intensity was then plotted against protein loading. With both redox staining and amido black staining, a linear correlation was demonstrated between band intensity and
Fig. 3. THP-1 cells were incubated in medium containing 500 AM DOPA and 0.02 AM [14C]DOPA for 24 h. Cell proteins were isolated, electrophoresed, and analyzed for redox-cycling activity (lane 2). The stain was removed and the membrane stained with amido black for total protein (lane 1); the membrane-bound radioactivity was then measured by phosphorimage analysis (28 d) (lane 3). Lane 4 contains prestained molecular weight markers with molecular weights as indicated. 100 Ag of protein was loaded onto each lane.
To examine the cellular removal of DOPA-containing proteins, DOPA (500 AM) was incorporated into THP-1 cell proteins for 24 h and the turnover of redox-positive (DOPA-containing) proteins monitored over the following 48 h. After 48 h most of the detectable redoxpositive proteins had been removed (Fig. 4A). The membrane was then washed in methanol and restained with amido black to confirm equal protein loading in each lane (Fig. 4B). Inhibition of turnover of DOPA-containing proteins To determine the effects of proteinase inhibitors on the cellular removal of DOPA-containing proteins THP-1 cells were incubated for 24 h with 250 AM DOPA (of which 0.02 AM was present as [14C]DOPA). Cells were then washed in PBS and incubated in degradation medium. After 2, 4, and 7 h cells were harvested and the degradation of the [14C]DOPA-containing proteins was quantified using the methods we have described previously [13]. The inclusion of an inhibitor of the proteasome (lactacystin 10 AM) inhibited degradation of DOPA-containing proteins by an average of 68% (standard deviation 3.5%) and inhibitors of the lysosomal proteinases (ammonium chloride 50 mM and E64 10 AM) inhibited the degradation of DOPA-containing proteins by an average of 32% (standard deviation 3.2%) (Fig. 5).
Fig. 4. THP-1 cells were incubated in medium containing 500 AM DOPA for 24 h. The medium was then replaced with degradation medium. The cultures were terminated and the cell lysates examined after 24, 12, 8, 4, and 0 h (lanes 2 to 6, respectively). Lane 1 contains prestained molecular weight markers with molecular weights as indicated. (A) DOPA-containing proteins were visualized by redoxcycling staining. (B) The membrane was then scanned and the stain removed by washing in methanol. The membrane was restained for total protein using amido black. 50 Ag of protein was loaded onto each lane.
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some activity without a separation step to remove other proteases [19,20], but because insufficient sample was available for this procedure, activity was measured in the presence or absence of the specific proteasomal inhibitor epoxomycin. No change in the proteasome activity could be detected with increasing DOPA incorporation into cell proteins (Fig. 6B—only chymotryptic shown). The rates of degradation of DOPA-containing proteins by THP-1 cells were then assessed at different levels of supply (and incorporation). [14C]DOPA was included to allow the degradation of [14C]DOPA-containing proteins to be selectively assessed. Degradation was assessed after 8 h by measuring the release into the culture medium of free radiolabel [13]. An initial increase in the rates of [14C]DOPA-containing protein degradation was demonstrated at lower levels of DOPA supply; however, at higher levels of DOPA the turnover rates of these modified proteins started to decrease (Fig. 7). To Fig. 5. THP-1 cells were incubated in medium containing 250 AM DOPA for 24 h (0.02 AM was present as [14C]DOPA). The medium was then replaced with degradation medium and the cells were allowed to degrade the labeled proteins. Parallel cultures contained the proteasome inhibitor lactacystin (10 AM, gray bars) or the inhibitors of lysosomal proteinases E64 and ammonium chloride (10 AM and 50 mM, respectively, black bars). Cultures were terminated at 2, 4, and 7 h and the degradation of the [14C]DOPA-containing proteins was quantified. The degradation in the inhibitor-containing cultures was expressed as a percentage of that in the inhibitor-free cultures. Error bars show the standard deviation obtained from triplicate cell cultures.
Effect of DOPA-containing proteins on the activity of proteasomes and lysosomes In order to assess if the biosynthetically generated DOPA-containing proteins altered the activity of the major cellular degradation machinery, THP-1 cells were incubated in a range of DOPA concentrations (0 to 750 AM) for 24 h, the DOPA-containing medium was removed, and the cells were allowed to degrade the native and modified proteins for 8 h. Cells were then harvested and the activity of the proteasomes and some lysosomal enzymes was measured (Fig. 6). The activity of the lysosomal enzyme arylsulfatase was unchanged at all levels of DOPA supply (Fig. 6B), a small but significant decrease in cathepsin B and L activities was found at the lower levels of DOPA supply (up to 250 AM supply or 12 mmol DOPA/mol Tyr incorporation), but the activity of these proteinases significantly increased at higher levels of DOPA supply (Fig. 6A) (500 AM supply or 18 mmol/mol Tyr incorporation). The activity of cathepsin S, however, showed a significant increase at the lower DOPA levels, decreasing to the levels in the DOPA-free cultures at higher levels (Fig. 6B). Proteasomal activity was assessed by measuring tryptic, chymotryptic, and PGPH activities. As has been shown previously, it is difficult to accurately measure protea-
Fig. 6. THP-1 cells were incubated in medium containing DOPA (0, 125, 250, 500, and 750 AM) for 24 h. The medium was then replaced with degradation medium and the cells were allowed to degrade proteins for 8 h. (A) The cells were then lysed and the activity of cathepsins B (.) and L (o) and the chymotryptic activity of the proteasome (D) were measured. (B) The activity of the lysosomal proteinase cathepsin S (5) and the lysosomal sulfatase arylsulfatase (n) was also measured. Activity was normalized to cell protein and expressed as a percentage of the control (DOPA-free cultures).
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Fig. 7. THP-1 cells were incubated for 24 h in medium containing DOPA (0, 125, 250, 500, and 750 AM) of which 0.02 AM was present as [14C]DOPA. The medium was then replaced with degradation medium and the cells allowed to degrade proteins for 8 h. Cells were then lysed and the degradation was measured from the release of free [14C]DOPA into the culture medium over the degradation period. Degradation was expressed as a percentage of the total amount of labeled protein present at the start of the degradation period.
Fig. 8. Human monocyte-derived macrophages were incubated in medium containing DOPA, 0 AM (lanes 2 and 5), 500 AM (lanes 3 and 6), and 500 AM with cycloheximide (lanes 4 and 7), for 24 h. Cells were lysed and proteins were precipitated, separated by SDS–PAGE, and blotted onto PVDF. Visualization of DOPA-containing proteins was by redox-cycling staining (lanes 2, 3, and 4). The membrane was stripped and restained for protein using amido black (lanes 5, 6, and 7). 30 Ag of protein was loaded onto each lane.
determine if free DOPA (rather than PB-DOPA) had a direct inhibitory effect on the activity of the cathepsins, cathepsin B and L activity was compared in the lysates from cells incubated with DOPA for 24 h (150 and 500 AM) both with and without cycloheximide. No significant difference in cathepsin activity was found in the cycloheximide-treated cultures; however, cathepsin B and L activities were both significantly inhibited in the cycloheximide-free cultures (Table 1).
containing 500 AM DOPA (of which 0.02 AM was present as [14C]DOPA), or leucine-free medium containing 0.02 AM [14C]leucine, and cycloheximide (0 and 0.5 Ag/ml) was added to the cells. After 24 h cell proteins were isolated and the incorporation of [14C]DOPA or [14C]leucine was assessed by liquid scintillation counting. The incorporation of [14C]DOPA into cell proteins was reduced by 79% (standard deviation 6.8%) when cycloheximide was present and the incorporation of [14C]leucine was reduced by 73% (standard deviation 2.6%), demonstrating that DOPA-containing proteins were also generated in these primary human cells by protein synthesis.
Incorporation of DOPA into cell proteins by isolated human monocytes
Visualization of DOPA-containing proteins in human monocyte-derived macrophages
In order to examine DOPA incorporation into cell proteins by a primary human cell, monocytes were isolated from peripheral human blood and maintained in culture. After 5 d in culture, tyrosine-free medium
Cell proteins were generated in human monocytederived macrophages as described above and the cell proteins isolated and examined by redox staining. No redox-staining-positive bands were detected in cell
Table 1. DOPA-containing Proteins Were Generated by Incubating THP-1 Cells with DOPA (0, 150, and 500 AM); Some Cultures Contained Cycloheximide to Prevent Protein Synthesis Cathepsin L activity (% of control) DOPA (AM) 0 150 500
Cathepsin B activity (% of control)
No cycloheximide
With cycloheximide
No cycloheximide
With cycloheximide
100 (5.5) 80.3* 85.7* (0.6)
100 (1.0) 100.7 (3.5) 101.0 (4.0)
100 (3.5) 84.7* (2.5) 92.3* (4.6)
100 (2.0) 97 (1.7) 106 (5.3)
The cells were washed and lysed and the activities of cathepsins L and B in the lysates measured using peptide substrates; these were normalized to the protein concentration. The specific activity in the DOPA-treated cultures is expressed relative to that of the DOPA-free cultures (set at 100%). Data were from triplicate cultures. * Statistically significant change in activity ( p b .05).
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proteins isolated from human monocytes; a number of redox-staining-positive bands were present in cell proteins from cells which had been incubated for 24 h in medium containing DOPA (500 AM); these were not present when cycloheximide was included in the culture medium to block the synthesis of new proteins (Fig. 8). Stripping and restaining of the membrane with amido black confirmed equal protein loading in each lane (Fig. 8, lanes 5, 6, and 7). DISCUSSION
We have previously demonstrated that DOPA and other oxidized amino acids can be biosynthetically incorporated into proteins by murine cells in culture [13]. Here we demonstrate that a human cell line (THP1 monocytes) and primary human cells (monocytederived macrophages) can also generate protein-bound DOPA by protein synthesis. Using a redox-cycling staining method which was first introduced to detect native quino-proteins [15] we were able to visualize for the first time the range of biosynthetically generated DOPA-containing cell proteins. The staining method utilizes the capacity of the catechol group of DOPA to redox cycle (catechol to quinone) at an alkaline pH in the presence of glycine, reducing nitro blue tetrazolium to formazan, producing a purple color. No quinonecontaining proteins were detectable in cell proteins when no DOPA was present in the culture medium or when DOPA was supplied together with an inhibitor of protein synthesis, providing further evidence that incorporation of DOPA is by protein synthesis. Incubation of DOPA with cell lysates did not result in any positive quinone staining, eliminating the possibility that there were any SDS–PAGE stable chemical interactions between DOPA and cell proteins. The staining patterns obtained from THP-1 cell proteins by measuring PB-[14C]DOPA by phosphorimage analysis and PB-DOPA by redox staining were very similar, supporting the validity of the redox staining as a sensitive and selective method to detect DOPA-containing proteins. In addition, a linear correlation was demonstrated between the intensity of the redox staining and the amount of sample examined. The levels of [14C]DOPA used in these studies required 28 d to detect DOPA-containing proteins, whereas redox staining could be performed in 30 min. The redox staining was therefore shown to be a fast and quantifiable method for the detection of DOPA-containing proteins. The range of the newly synthesized proteins which contained DOPA was shown to be similar to the total protein staining, suggesting that many proteins contained biosynthetically incorporated DOPA and that many of these proteins were synthesized as the fulllength forms.
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Most of the DOPA-containing proteins were removed by the THP-1 cells after 24 h; this process could be delayed by inhibitors of both the proteasomal and the lysosomal pathways, suggesting that both pathways are involved in their degradation. The proteasome seemed to be the primary pathway for the removal of DOPAcontaining proteins. Inhibitors of the lysosomal proteinases reduced the degradation of DOPA-containing proteins by around 30%, consistent with our previous studies using a mouse macrophage cell line. These data support a role for the endosomal–lysosomal system in the turnover of these endogenously generated DOPA-containing proteins but indicate that the proteasome is the primary pathway responsible for their turnover. At the lower levels of DOPA incorporation into proteins their turnover was greatly accelerated without any detectable increases in the activity of proteasomes or the lysosomal enzymes, suggesting that the increased turnover rate of the modified proteins did not require upregulation in either of the major proteolytic pathways. A small but significant decrease in cathepsin B and L activity was found at lower levels of DOPA incorporation. The ability of DOPA to transfer damage to other proteins has been described and this process may be amplified in the acid environment of the lysosome, which may also contain redox-active iron [21,22]. DOPA could also directly inactivate lysosomal cysteine proteinases by cross-linking with the active-site cysteine residues to form cysteinyl-DOPA, a reaction which is known to occur in vivo [23]. No decrease in cathepsin activity was found when protein synthesis was blocked; this would suggest that inhibition was from protein-bound DOPA rather than free DOPA. As we reported previously, at higher levels of DOPA incorporation into proteins their turnover rate begins to decrease; this may reflect the situation in pathological tissues in which these proteins begin to accumulate. The activity of the proteasome in THP-1 cells was not altered when DOPA-containing proteins were present at higher levels of incorporation, suggesting that there was no direct inhibition of proteasomes by DOPA-containing proteins; this is consistent with our previous studies, which showed that the turnover of DOPA-containing proteins did not alter the degradation rates of native proteins, which are thought to be degraded mainly by the proteasomal pathway [13]. The decrease in turnover of DOPA-containing proteins was accompanied by a substantial increase in the activity of the lysosomal proteinases cathepsins B and L. A similar increase in cathepsin activity (or immunereactive cathepsin) has been reported in response to aggregated protein accumulation in both experimental systems [24] and pathological tissues as a response to protein aggregation [25]. Here we demonstrate that an
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increase in lysosomal proteinase activity may also be a compensatory response to the presence of oxidized amino-acid-containing proteins, which are less readily degraded by the cells. Cathepsin S activity was significantly increased at lower levels of DOPA incorporation but returned to basal levels at higher levels of DOPA incorporation, which may suggest a more specialized role for cathepsin S in the cell, such as in the generation of MHC class II epitopes. DOPA-containing proteins were also generated in human monocyte-derived macrophages on incubation of the cells with DOPA. The inclusion of an inhibitor of protein synthesis blocked the incorporation of DOPA, demonstrating that incorporation was by biosynthesis. As was observed for the THP-1 cells, a range of DOPAcontaining proteins was generated by the monocytederived macrophages and could be visualized by redoxcycling staining. These data raise the possibility that DOPA could be biosynthetically incorporated into human proteins in vivo. In summary, we demonstrate that DOPA can be biosynthetically incorporated into a broad range of proteins by a human monocyte cell line (THP-1) and primary human cells (monocyte-derived macrophages). The cellular removal of DOPA-containing proteins by THP-1 cells is by proteolysis, involving both the proteasomal and the lysosomal systems. At higher levels of incorporation the activity of certain lysosomal cathepsins is selectively increased but the rate of removal of the proteins is reduced. These studies raise the possibility that DOPA-containing proteins can be generated in vivo from l-DOPA (levodopa), which is the primary treatment for Parkinson disease. Acknowledgments — This study was supported by the National Health and Medical Research Council of Australia (K.R., P.H., R.T.D.) and by an Australian Postgraduate Award (R.A.D.). REFERENCES [1] Ames, B. N.; Shigenaga, M. K.; Hagen, T. M. Oxidants, antioxidants, and the degenerative diseases of aging. Proc. Natl. Acad. Sci. USA 90:7915 – 7922; 1993. [2] Dean, R. T.; Fu, S.; Stocker, R.; Davies, M. J. The biochemistry and pathology of radical-mediated protein oxidation. Biochem. J. 324:1 – 18; 1997. [3] Dean, R. T.; Dunlop, R.; Hume, P.; Rodgers, K. J. Proteolytic ddefencesT and the accumulation of oxidized polypeptides in cataractogenesis and atherogenesis. Biochem. Soc. Symp. 70: 135 – 146; 2003. [4] Linton, S.; Davies, M. J.; Dean, R. T. Protein oxidation and ageing. Exp. Gerontol. 36:1503 – 1518; 2001. [5] Dunlop, R. A.; Rodgers, K. J.; Dean, R. T. Recent developments in the intracellular degradation of oxidized proteins. Free Radic. Biol. Med. 33:894 – 906; 2002. [6] Rodgers, K. J.; Dean, R. T. The metabolism of protein-bound DOPA in mammals. Int. J. Biochem. Cell Biol. 32:945 – 955; 2000. [7] Berlett, B. S.; Stadtman, E. R. Protein oxidation in aging, disease, and oxidative stress. J. Biol. Chem. 272:20313 – 20316; 1997.
[8] Simpson, J. A.; Narita, S.; Gieseg, S.; Gebicki, S.; Gebicki, J. M.; Dean, R. T. Long-lived reactive species on free-radical-damaged proteins. Biochem. J. 282:621 – 624; 1992. [9] Gieseg, S. P.; Simpson, J. A.; Charlton, T. S.; Duncan, M. W.; Dean, R. T. Protein-bound 3,4-dihydroxyphenylalanine is a major reductant formed during hydroxyl radical damage to proteins. Biochemistry 32:4780 – 4786; 1993. [10] Fu, S.; Davies, M. J.; Stocker, R.; Dean, R. T. Evidence for roles of radicals in protein oxidation in advanced human atherosclerotic plaque. Biochem. J. 333:519 – 525; 1998. [11] Fu, S.; Dean, R.; Southan, M.; Truscott, R. The hydroxyl radical in lens nuclear cataractogenesis. J. Biol. Chem. 273:28603 – 28609; 1998. [12] Ito, S.; Kato, T.; Shinpo, K.; Fujita, K. Oxidation of tyrosine residues by tyrosinase—formation of protein-bound 3,4-dihydroxyphenylalanine and 5-S-cysteinyl-3,4-dihydroxyphenylalanine. Biochem. J. 222:407 – 411; 1984. [13] Rodgers, K. J.; Wang, H.; Fu, S.; Dean, R. T. Biosynthetic incorporation of oxidized amino acids into proteins and their cellular proteolysis. Free Radic. Biol. Med. 32:766 – 775; 2002. [14] Garner, B.; Dean, R. T.; Jessup, W. Human macrophage-mediated oxidation of low-density lipoprotein is delayed and independent of superoxide production. Biochem. J. 301:421 – 428; 1994. [15] Paz, M. A.; Fluckiger, R.; Boak, A.; Kagan, H. M.; Gallop, P. M. Specific detection of quinoproteins by redox-cycling staining. J. Biol. Chem. 266:689 – 692; 1991. [16] Kirschke, H.; Wiederanders, B.; Bromme, D.; Rinne, A. Cathepsin S from bovine spleen: purification, distribution, intracellular localization and action on proteins. Biochem. J. 264:467 – 473; 1989. [17] Sitte, N.; Huber, M.; Grune, T.; Ladhoff, A.; Doecke, W. D.; Von Zglinicki, T.; Davies, K. J. Proteasome inhibition by lipofuscin/ ceroid during postmitotic aging of fibroblasts. FASEB J. 14:1490 – 1498; 2000. [18] Orange, R. P.; Moore, E. G. Functional characterization of rat mast cell arylsulfatase activity. J. Immunol. 117:2191 – 2196; 1976. [19] Rodgers, K. J.; Dean, R. T. Assessment of proteasome activity in cell lysates and tissue homogenates using peptide substrates. Int. J. Biochem. Cell Biol. 35:716 – 727; 2003. [20] Rivett, A. J.; Bose, S.; Pemberton, A. J.; Brooks, P.; Onion, D.; Shirley, D.; Stratford, F. L.; Forti, K. Assays of proteasome activity in relation to aging. Exp. Gerontol. 37:1217 – 1222; 2002. [21] Marzabadi, M. R.; Sohal, R. S.; Brunk, U. T. Effect of ferric iron and desferrioxamine on lipofuscin accumulation in cultured rat heart myocytes. Mech. Ageing Dev. 46:145 – 157; 1988. [22] Persson, H. L.; Yu, Z.; Tirosh, O.; Eaton, J. W.; Brunk, U. T. Prevention of oxidant-induced cell death by lysosomotropic iron chelators. Free Radic. Biol. Med. 34:1295 – 1305; 2003. [23] Ito, S.; Kato, T.; Fujita, K. Covalent binding of catechols to protein through the sulphydryl group. Biochem. Pharmacol. 37:1707 – 1710; 1988. [24] Bendiske, J.; Bahr, B. A. Lysosomal activation is a compensatory response against protein accumulation and associated synaptopathogenesis—an approach for slowing Alzheimer disease? J. Neuropathol. Exp. Neurol. 62:451 – 463; 2003. [25] Ii, K.; Ito, H.; Kominami, E.; Hirano, A. Abnormal distribution of cathepsin proteinases and endogenous inhibitors (cystatins) in the hippocampus of patients with AlzheimerTs disease, parkinsonismdementia complex on Guam, and senile dementia and in the aged. Virchows Arch. A Pathol. Anat. Histopathol. 423:185 – 194; 1993. ABBREVIATIONS
DMEM — DulbeccoTs minimal essential medium DOPA — 3,4-dihydroxyphenylalanine E64 — l-transepoxysuccinylleucylamide-(4-guanido) butane FCS — fetal calf serum PGPH — peptidylglutamyl peptide hydrolyzing TCA — trichloroacetic acid