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0013-7227/91/1294-20ll$03.00/0 Endocrinology Copyright © 1991 by The Endocrine Society Vol. 129, No. 4 Printed in U.S.A. Characterization of a AT-Bromoacetyl-L-Thyroxine Affinity-Labeled 55-Kilodalton Protein as Protein Disulfide Isomerase in Cultured Glial Cells MARJORIE SAFRAN AND JACK L. LEONARD Molecular Endocrinology Laboratory, Departments of Medicine, Nuclear Medicine, and Molecular and Cellular Physiology, University of Massachusetts Medical School, Worcester, Massachusetts 01655 characterized glial-p55 and compared it with purified rat hepatic PDI. Glial-p55 appears to be identical to PDI by peptide fragmentation analysis, using multiple methods. The hydrodynamic properties of glial-p55, determined by molecular sieve chromatography and sucrose density centrifugation, showed that this protein has a native molecular mass of 115 kDa, which is in close agreement with that reported for PDI. Like PDI, glial-p55 is an acidic protein with a pi of 4.8 or less. Thus, the 55-kDa thyroid hormone-binding protein in glial cells appears to be PDI. (Endocrinology 129: 2011-2016,1991) ABSTRACT. In glial cells, thyroid hormone regulates the polymerization state of the actin cytoskeleton by a mechanism that does not require protein synthesis or the nuclear T3 receptor. Using the affinity label iV-bromoacetyl-L-T4, we identified a thyroid hormone-binding protein of 55 kDa (glial-p55) in cultured glial cells that is unique from the type II iodothyronine 5'-deiodinase and which exhibits a T4-dependent shift from a membrane-associated pool to the F-actin cytoskeleton. In a number of other cell types, a 55-kDa thyroid hormone-binding protein has been identified and shown to be a subunit of the enzyme protein disulfide isomerase (PDI). In this study we have I N THE brain, thyroid hormone regulates the levels of type II iodothyronine 5'-deiodinase (5'D-II) by modulating the turnover of the enzyme polypeptide (1). T4 increases the degradation of this short-lived membranebound protein in cultured glial cells by dynamically regulating the polymerization of the actin cytoskeleton through an energy-dependent mechanism that does not require protein synthesis or the nuclear T 3 receptor (2). This extranuclear action of thyroid hormone differs from the traditional pathway of thyroid hormone action, which requires access to a chromatin-bound receptor with subsequent modulation of gene expression and protein synthesis (3). Recently, several thyroid hormone-binding proteins in addition to the nuclear T 3 receptor have been identified in the cell (4) and form a group of candidate proteins that may mediate extranuclear effects of thyroid hormone (5, 6). These specific thyroid hormone-binding proteins have been described in mitochondria, cytoplasm, and plasma membranes of many tissues (4). Affinity labeling with bromoacetyl derivatives of T 4 and T 3 has proven to be a valuable method for identifying Received May 20,1991. Address all correspondence and requests for reprints to: Dr. Marjorie Safran, Division of Endocrinology and Metabolism, University of Massachusetts Medical Center, 55 Lake Avenue North, Worcester, Massachusetts 01655. * This work was supported by Grants DK-38772 and DK-32520 from the NIDDK, NIH (Bethesda, MD). many of these thyroid hormone-binding proteins (7-11). In glial cells, iV-bromoacetyl-L-T4 (BrAcT4) is found predominately in three proteins of 55,000, 29,000, and 18,000 mol wt (9). The 29-kDa protein has been identified as the substrate-binding subunit of 5'D-II (9, 12), while the identities of the 55- and 18-kDa proteins remain to be established. In other organs and cell lines, a 55-kDa affinity-labeled protein has been identified and proposed as a plasma membrane thyroid hormone transport protein (13). Subsequent analysis has shown this protein to be a subunit of the enzyme protein disulfide isomerase (PDI) (14, 15), which is localized to the endoplasmic reticulum and nuclear envelope (16). However, in glial cells, the affinity-labeled 55-kDa protein exhibits a T4dependent shift from a Triton X-100-soluble (membranes/cytosol) to a Triton X-100-insoluble pool (cytoskeleton), presumably due to an association with F-actin (17). Thus, the observation that the BrAcT4-labeled 55kDa protein (glial-p55) becomes associated with the actin cytoskeleton in the presence of thyroid hormone suggests that glial-p55 in glial cells may play a role in ^-dependent actin polymerization. In this study we have characterized the 55-kDa thyroid hormone-binding protein present in cultured astrocytes and compared it with purified liver PDI. Glial-p55 appears to be identical to PDI by peptide fragmentation analysis. The hydrodynamic properties of glial-p55, de- 2011 2012 BrAcT4-LABELED 55-kDa PROTEIN IN GLIAL CELLS termined by sucrose density centrifugation and molecular sieve chromatography, showed that the taurodeoxycholate-solubilized glial-p55 has a sedimentation coefficient of 3.94S, a Stokes radius of 4.14 nm, and a native molecular mass of 115 kDa, in good agreement with the mol wt reported for PDI (18). Glial-p55 has a pi of 4.8 by isoelectric focusing, which is also similar to the pi reported for PDI (19). Thus, the 55-kDa thyroid hormone-binding protein in glial cells appears to be PDI. Since previous work in our laboratory has demonstrated that affinity-labeled glial-p55 becomes actin associated in a T4-dependent manner (17), PDI may play a role in the thyroid hormone-dependent internalization of shortlived membrane proteins. Materials and Methods Materials Dulbecco's Modified Eagle's Medium, antibiotics, Hanks' Salt Solution, glucose, and trypsin were obtained from Gibco (Grand Island, NY); supplemented bovine calf serum from Hyclone, Inc. (Logan, UT); (Bu)2cAMP, Staphlococcal aureus V8protease (V8-protease), and hydrocortisone from Sigma (St. Louis, MO); cyanogen bromide (CNBr) from Fluka (Buchs, Switzerland); acrylamide and iV,JV'-methylene-bis-acrylamide from U.S. Biochemical Corp. (Cleveland, OH); ammonium persulfate and AT,iV,A^iV'-tetramethylethylenediamine (TEMED) from Bio-Rad Laboratories (Richmond, CA); Ampholines from LKB (Rockville, MD); and dithiothreitol (DTT) from Calbiochem (La Jolla, CA). BrAc-[125I]T4 was synthesized as described previously (7). Its purity was greater than 90%, as determined by reverse phase HPLC analysis (8). Culture conditions and affinity labeling of astrocytes Neonatal rats were decapitated, and glial cells were prepared as previously described (20). This study was approved by the Animal Research Committee and complies with the institutional assurance certification of the University of Massachusetts Medical Center. Cells were seeded at 4 X 105 cells/cm2 in 75-cm2 flasks and used during passages 2-6. The cells were cultured in Dulbecco's Modified Eagle's Medium containing 10% supplemented bovine calf serum, 50 U/ml penicillin, and 90 U/ml streptomycin in a humidified atmosphere of 5% CO2 and 95% air at 37 C. Cells were used at confluence in 25- or 75-cm2 flasks. Glial cells were changed to serum-free medium 24 h before affinity labeling. Sixteen hours before affinity labeling, 1 mM (Bu)2cAMP and 100 nM hydrocortisone were added to induce 5'D-II activity. At the time of labeling, medium was removed, and the cells were incubated with 2.7 nM BrAc-[125I]T4 (5 X 107 cpm/75-cm2 flask) in 3 ml serum-free Hanks' solution for 20 min at 37 C. After washing twice with 5 ml ice-cold 20 mM PBS (pH 7.4), the cells were harvested by scraping and then collected by centrifugation at 500 X g for 10 min. Cell pellets were sonicated in 10 mM HEPES (pH 7.0) containing 10 mM DTT, 0.1 mM EDTA, and 0.1 mM phenylmethylsulfonylfluoride (lysis buffer) and kept frozen at -20 C until use. Samples used Endo • 1991 Vol 129 »No 4 for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis were treated by the addition of 0.25 vol 5X SDS-PAGE sample buffer [250 mM Tris-HCl (pH 6.8), 50% (vol/vol) glycerol, 5% (wt/vol) SDS, 5% (vol/vol) mercaptoethanol, and 0.01% (wt/vol) bromphenol blue] and denatured by heating at 70 C for 5 min. Purification of PDI PDI was isolated from rat liver using an adaptation of the procedure described by Lambert and Freedman (18). Briefly, liver microsomes were prepared as previously described (21), solubilized in 0.1 M sodium phosphate buffer (pH 7.5) containing 5 mM EDTA and 1% Triton X-100 (vol/vol), heated to 54 C for 5 min, cooled on ice, and centrifuged for 7.2 x 106 g-min. The clarified supernatant was fractionated by NH4SO4 and the 55-85% saturated NH4SO4 fraction was dissolved in 25 mM sodium citrate buffer (pH 5.3) and diafiltered using a Centricon30 microconcentrator (Amicon, Danvers, MA) with three changes of the same buffer. The dialyzed sample was applied to a 1 x 5-cm Sephadex C-50 column at 4 C and eluted with the 25 mM sodium citrate buffer (pH 5.3). The unretarded fraction was collected, and the buffer was changed to 20 mM sodium phosphate (pH 6.3) by diafiltration using three changes of buffer. This sample was applied to a 1 x 5-cm DEAESephacel column equilibrated in 20 mM sodium phosphate (pH 6.3). Proteins were eluted at a flow rate of 20 ml/h with a 0- to 700-mM NaCl gradient in equilibration buffer and monitored at OD28o- Two-milliliter fractions were collected, and the identity of the eluted proteins was determined by SDS-PAGE. SDS-PAGE analysis Heat-denatured samples (70 C for 5 min) were electrophoresed on a 12.5% SDS-PAGE gel according to the method of Laemmli (22). Gels were fixed in 25% isopropyl alcohol (vol/ vol) and 10% acetic acid (vol/vol), dried, and exposed to XAR5 film (Eastman Kodak, Rochester, NY) with intensifying screens for 3-7 seven days at -70 C. Radioautograms were used to identify affinity-labeled proteins. When indicated, the affinity-labeled proteins were cut from the dried gels and cleaved directly in the gel slices, as described previously (12). Affinity labeling of purified PDI Purified PDI (10 fig) was affinity labeled with 130 pM BrAc[125I]T4 (5 X 105 cpm) in a total volume of 100 /A containing 100 mM potassium phosphate buffer (pH 7), 1 mM EDTA, and 10 mM DTT by incubating at 37 C for 20 min. Labeling was stopped by the addition of 0.25 vol 5x SDS-PAGE sample buffer, and the affinity-labeled PDI was stored at -20 C. N-Terminal sequencing was used to confirm the identity of the purified PDI. Affinity-labeled PDI was heat denatured, electrophoresed on SDS-PAGE, and transferred to Imobilon-P (Millipore, Bedford, MA), using the method of Towbin et al. (23). Membranes were stained with 0.1% Coomassie blue (wt/ vol) in 50% methanol (vol/vol) for 5 min, followed by destaining for 10 min in 50% methanol (vol/vol) and 10% acetic acid (vol/ vol) to identify the 55-kDa protein. N-Terminal sequencing was determined by automated Edman degradation in the Protein Sequencing Core Facility at the University of Massachusetts Medical School. BrAcT4-LABELED 55-kDa PROTEIN IN GLIAL CELLS Molecular sieve chromatography and sucrose density centrifugation Procedures have been previously described in detail (12). In brief, taurodeoxycholate-solubilized BrAc-[125I]T4-labeled glial p55 was analyzed by molecular sieve chromatography, using an LKB TSK 3000 column (Rockville, MD), and by isopynic centrifugation on 5-20% linear sucrose gradients in H2O and 2H2O. Fractions were analyzed by SDS-PAGE, and the resulting radioautographs were analyzed by scanning densitometry. 1.0 800 0.8 -- eod 3 0.6 -- 3 . CM 1 > 0.4- 200 0.2 -- 0.0 -h Isoelectric focusing and two-dimensional gel analysis Isoelectric focusing of affinity-labeled glial-p55 was performed using the Mini-Protean II (Bio-Rad Laboratories, Richmond, CA). Samples were mixed with an equal volume of sample buffer (9.5 M urea, 1% Nonidet P-40, 5% j8-mercaptoethanol, and 0.4% pH 3.5-10 and 1.6% pH 5-8 Ampholines) and applied to gels containing 4% polyacrylamide, 9 M urea, and 0.4% pH 3.5-10 and 1.6% pH 5-8 Ampholines. Gels were focused for 3 h using 10 mM H3PO4 as the anode buffer and 20 mM NaOH as the cathode buffer. Focused gels were then separated on a 12.5% SDS-PAGE gel, which was analyzed by radioautography. The pH gradient was determined separately on concurrently focused gels by allowing 0.5-cm fragments to equilibrate for 10 min in water and then measuring the pH. 2013 40 60 80 Fraction FIG. 1. OD280 absorbance of fractions obtained by gradient elution (0700 mM NaCl) of DEAE-Sephacel during purification of PDI from rat liver microsomes. Fractions 37-41, 57-63, and 80-94 were pooled to make peaks 1, 2, and 3, respectively. B Mr x10"3 55 — Other procedures Protein was determined by the method of Bradford (24). Results Affinity-labeled glial cells contain three proteins, of 55,000, 29,000, and 18,000 mol wt, that contained greater than 90% of the affinity label (9). The 29-kDa protein has been identified as the substrate-binding subunit of the type II iodothyronine 5'D-II (9,12). To confirm that the 55-kDa protein was not a dimer of the 5'D-II substrate-binding subunit, the two proteins were compared by peptide fragmentation. Peptide fingerprinting of the glial 29-kDa protein and glial-p55 using CNBr fragmentation and V8-protease digests confirmed that the two proteins are unique (data not shown). These data are consistent with earlier work demonstrating that the affinity-labeled 55-kDa protein in rat liver and kidney microsomes differs from the 27-kDa substrate-binding subunit of 5'D-I (7). Since the BrAcTVlabeled 55-kDa protein in other cell types had previously been identified as PDI (14, 15), we compared glial-p55 to purified PDI. The procedure of Lambert and Freedman (18) was used to isolate PDI from rat liver microsomes. As shown in Fig. 1, 3 peaks of proteins were eluted from the DEAE-Sephacel column when monitored by absorbance at OD28o- When analyzed by SDS-PAGE, protein was identified by Coomassie staining of the gels only in peaks 1 and 2. Peak 1 contained a protein of 65 kDa and was labeled very poorly with BrAcT4 (Fig. 2). Greater than 99% of peak 2 was 36 Peak 1 1 FIG. 2. Protein staining and affinity labeling of proteins eluting off the DEAE-Sephacel column during purification of PDI from rat liver microsomes. Proteins were affinity labeled and separated by SDSPAGE, as described in Materials and Methods. A, Coomassie staining of peaks 1 and 2. B, The radioautograph of BrAc-[125I]T4-labeled peaks 1 and 2 in the same gel as A. Mr, Mol wt. comprised of a 55-kDa protein that incorporated 0.1 pmol BrAcT4/mg protein (Fig. 2). N-Terminal sequencing of the peak 2 protein showed that the first 13 amino acids were identical to those of the N-terminus of PDI. This PDI was used without further purification. Glial-p55 was compared to PDI by peptide fingerprinting of the isolated proteins. Partial digests of glial-p55 and PDI using V8-protease showed 11 labeled fragments of 5,700-39,000 mol wt (Fig. 3). While the intensity of the fragments differed between the two preparations, fragment mol wt were identical. Cyanogen bromide digests of glial-p55 and affinity-labeled PDI revealed identical fragmentation patterns, with fragments of 45,000, 40,000, and 35,000 mol wt (Fig. 4). Similarly, partial digests of the two proteins using 2 ng trypsin also yielded identical fragmentation patterns (data not shown). Detergent-solubilized membranes from BrAc-[125I]T4- BrAcT4-LABELED 55-kDa PROTEIN IN GLIAL CELLS 2014 Glial-p55 Mr x10"3 0 0.04 PDI 0.2 0 0.04 Endo • 1991 Vol 129 • No 4 1.0 0.2 1 0.9 ug V8 0.8 pSS-II - <3)O 0.7 i 0.6 2 3 St okes Radius 55 — 4 ^ \ 5 o 29 30 40 50 Fraction Number FIG. 5. Molecular sieve chromatography of glial-p55. Taurodeoxycholate-solubilized BrAcT4 affinity-labeled glial membranes were chromatographed on a LKB TSK 3000 column. Glial-p55 was identified by the distribution of the affinity-labeled 55-kDa protein. Data shown are representative of three experiments. The Stokes radius of the protein was determined using the following protein standards: 1) cytochromec, 2) a-chymotrypsin, 3) ovalbumin, 4) BSA, and 5) alcohol dehydrogenase. The data reported in the inset represent the mean of four experiments. 12.4 — FlG. 3. Peptide fragmentation patterns obtained with Staphfococcal V8-protease (V8) digests of glial-p55 and PDI. Gel slices containing affinity-labeled glial-p55 and PDI were loaded along with the indicated quantity of V8-protease onto a 15% SDS-PAGE gel with a 4% stacking gel. Proteolysis was allowed to occur within the stacking gel with the current turned off for 30 min. Electrophoresis was then allowed to continue as usual. The peptide map shown is representative of three experiments. Mr, Mol wt. Mr GlialPDI p55 XKT3 55 36 FlG. 4. Peptide fragmentation patterns obtained with cyanogen bromide (CNBr) digests of glial-p55 and PDI. Gel slices containing affinity-labeled glial-p55 and PDI were treated with 0.8 M cyanogen bromide for 2 h at room temperature. Treated gel slices were then loaded onto a 15% SDS-PAGE gel with a 4% stacking gel, and electrophoresis was performed as usual. The peptide map shown is representative of three experiments. Mr, Mol wt. labeled glial cells were applied to a TSK 3000 column, and the distribution of the 55-kDa protein was determined by SDS-PAGE. As shown in Fig. 5, the majority (73%) of glial-p55 eluted in fractions 31-32 (p55-I). The Stokes radius of this peak, determined graphically from the Kd1/3 was 4.14 ± 0.1 nm (Fig. 5, inset). A second smaller peak (27%) present in fractions 35-37 (p55-II) migrated with an estimated mol wt of ~54,000 and a Stokes radius of 3.07 ± 0.09 nm, and most likely represents a protein monomer. The hydrodynamic properties of glial-p55 were determined from the fractional radial migration of detergentsolubilized proteins in the presence of H2O and 2H2O. Solubilized glial membranes were centrifuged on 5-20% linear sucrose gradients prepared in both H2O and 2H2O. The migration of glial-p55 was determined from the distribution of the affinity-labeled protein and is shown in Fig. 6. Glial-p55 migrated with a r/r 0 = 1.392 ± 0.034 in H2O and r/r 0 = 1.302 in 2H2O. Occasionally, a second peak of affinity-labeled p55 was present which sedimented faster than glial-p55, most likely secondary to protein aggregates. The S2o,w of glial-p55, as determined by interpolation from the sedimentation in H2O, was 3.94 ± 0.40S (mean ± SE; n = 6) and was similar in 2H2O (4.05S). The calculated partial specific volume of glialp55 was 0.74 ± 0.07 cm3/g, which is consistent with it being a globular protein. The mol wt of p55-I, calculated from the Stokes radius of the major peak, partial specific volume, and sedimentation coefficient, was determined to be 114,800 ± 39,000. The pi of BrAcT4-labeled glial-p55 was 4.8 ± 0.2 (mean ± SE; n = 7), and the protein focused as a single spot, with no microheterogeneity (data not shown). Discussion The affinity labels BrAcT4 and BrAcT3 have identified a widely distributed 55-kDa thyroid hormone-binding BrAcT4-LABELED 55-kDa PROTEIN IN GLIAL CELLS I-LO Ho0 CD 2.0 FlG. 6. Sucrose density centrifugation of glial-p55. Taurodeoxycholate-solubilized BrAc-[125I]T4 affinity-labeled glial membranes were sedimented on 5-20% linear sucrose gradients in the presence of H2O and 2H2O. The migration of glial-p55 was determined from radioautographs of the sucrose gradient fractions after analysis by SDS-PAGE. The results shown are representative of individual gradients repeated six (H2O) or four (2H2O) times. The protein standards used were carbonic anhydrase, BSA, and alcohol dehydrogenase. protein (7-11). Cheng and co-workers (10, 11, 14) and Yamauchi etal. (15) purified a 55-kDa protein from GH3 (a rat pituitary tumor cell line), A431 (a human epithelioid carcinoma), and 3T3-4 (Swiss mouse fibroblast cell line) cells and isolated a cDNA encoding this protein that showed 85% homology at the nucleotide level to the endoplasmic reticulum luminal protein, PDI (14). In glial cells, a 55-kDa protein contains ~25% of the total BrAcT4 incorporated into cell proteins (9). Since p55 has been identified as PDI in other cells, we sought to determine whether glial-p55 was also PDI. The current study indicates that the 55-kDa thyroid hormone-binding protein in glial cells is similar if not identical to PDI. Partial peptide fragmentation of PDI isolated from rat liver and glial-p55, using multiple methods yielded identical peptide fingerprints. The native undenatured mol wt of detergent-solubilized glial-p55 is 115,000, in close agreement with that of PDI determined by gel filtration on Sephadex G-200 (18). These data indicate that both glial-p55 and PDI are homodimers. In addition, both PDI and affinity-labeled glial-p55 are acidic proteins with pi values of 4.8 or less, and both proteins show little or no microheterogeneity. Up to 27% of glial-p55 was present as a monomer. PDI has been shown to act as a molecular chaparone catalyzing native disulfide bond formation and is abundant in tissues that secrete proteins (16). In addition, PDI has also been demonstrated to be a component of other multimeric enzymes and proteins, such as prolyl4-hydroxylase (25) and the microsomal triglyceride transfer protein complex (26). By both subcellular fractionation and ultrastructure analyses, PDI has been shown to be an extrinsic membrane protein localized to the lumen of the endoplasmic reticulum (16) and the 2015 pore complex of the nuclear envelope (27). However, ~ 5 10% of PDI has been found on the external surface of the plasma membranes of rat exocrine pancreatic cells, suggesting that the protein may be deposited on the cell surface during secretion (28). The presence of the small amount of PDI on the cell surface raises the possibility that the molecule is missorted and carried to this site by secretory vesicles. The rescue of this misdirected protein may explain the observation of Cheng and co-workers that PDI functioned as a thyroid hormone transporter. They followed the migration of endocytic vesicles containing a rhodamine-T3PDI conjugate from the cell surface to the perinuclear space (29). Since endocytic vesicles are initially targeted to the perinuclear space for sorting (30), the movement of T 3 bound to PDI in these vesicles would mimic nuclear translocation of the hormone. Other cell surface receptors, such as the apolipoprotein receptor, may also nonspecifically carry thyroid hormone to the perinuclear space, since T 3 bound to apolipoprotein is internalized into cells by endocytosis (31). However, the thyroid hormone still has to cross the vesicle bilipid layer and the nuclear envelope to attain access to its chromatinbound receptor. The ability of cell surface receptors to passively carry thyroid hormone to the perinuclear space implies that PDI may not be a specific thyroid hormone transport protein. The identification of two peaks of glial-p55 by molecular sieve chromatography suggests that PDI may exist in two different forms in the glial cell. Since the majority of PDI is confined to the lumen of the endoplasmic reticulum, it is unlikely to be accessible to the actin cytoskeleton. Monomeric glial-p55 comprises more than 25% of the total glial PDI. The presence of two forms of PDI raises the possibility that one of these may be localized in a different subcellular site, possibly the cytosol, where it could act to modulate actin polymerization. More work is needed to evaluate this possibility. The ability of PDI to associate with the actin cytoskeleton in a hormone-dependent manner in cells derived from the brain has not been observed previously. The addition of T 4 to thyroid hormone-deficient glial cells causes the percentage of polymerized actin to increase from 65% to 90% of the total cellular actin (17). The observation that this treatment also resulted in the rapid movement of glial-p55 from a membrane-associated pool to the F-actin cytoskeleton (17) implied that glial-p55 may participate in T4-dependent regulation of actin polymerization. The current demonstration that glial-p55 is PDI may represent a new role for this multifunctional polypeptide in cells of the central nervous system. Acknowledgments We thank Ms. Susan Dubord for technical assistance, and Dr. Jacqueline Anderson for performing the protein sequencing. 2016 BrAcT4-LABELED 55-kDa PROTEIN IN GLIAL CELLS References 1. Leonard JL, Siegrist-Kaiser CA, Zuckerman CJ 1990 Regulation of type II iodothyronine 5'-deiodinase by thyroid hormone: inhibition of actin polymerization blocks enzyme inactivation in cAMP-stimulated glial cells. J Biol Chem 265:940-946 2. 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