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【摘要】
Amyloidosis is a disease of protein misfolding that ultimately impairs organ function. Previously, we demonstrated that amyloidogenic light chains (1, 6, and 3 subtypes), internalized by cardiac fibroblasts, enhanced sulfation of secreted glycosaminoglycans. In this study, we investigated the inter-nalization and cellular trafficking of urinary immunoglobulin light chains into cardiac fibroblasts. We demonstrate that these light chains have the ability to form annular rings in solution. Internalization was assessed by incubating cells in the presence of light chain conjugated to Oregon Green 488 followed by monitoring with live cell confocal imaging. The rate of light chain internalization was reduced by treatment with methyl-ß-cyclodextrin but not filipin. Amyloid light chain did co-localize with dextran-Texas Red. Once internalized, the light chains were detected in lysosomes and then secreted into the extracellular medium. The light chain detected in the cell lysate and medium possessed a lower hydrophobic species. Nocodazole, a microtubule inhibitor, did not disperse aggregates. In addition, internalization and retention of the light chain proteins was altered in the presence of the proteasomal inhibitor MG132. These results indicate that the cell internalizes light chain by a fluid phase endocytosis, which is then modified and ultimately compro-mises the cell.
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The dynamics of cellular processing of light chains (LCs) has been of great interest to investigators, but the cellular response is not well understood. Primary (AL) amyloidosis is a disease of protein misfolding. Plasma cell dyscrasia produces amyloidogenic immunoglobulin LCs that circulate through the vascular system and deposit as insoluble fibrils in tissues. The nonbranching fibrils are composed of filaments that form ß-pleated sheets, stain with Congo Red, and display an apple-green birefringence with polarizing microscopy.
The LCs are known to disrupt the normal physiology of organs such as heart, kidney, lungs, peripheral nerves, and intestines, with the most common involvement in kidney and heart. Although renal amyloidosis often results in nephrotic syndrome, cardiac involvement occurs in up to 50% of patients with AL amyloidosis and is a leading cause of morbidity.1,2 Congestive heart failure is associated with amyloidotic cardiomyopathy.2
Investigators have demonstrated that LCs induce oxidative stress responses in isolated cardiac cells and are associated with a decrease in contractility.3 These data indicate that LC toxicity may contribute to cardiac dysfunction as well as play a role in amyloid fibril deposition. Furthermore, internalization of LCs (1, 6, and 3 subtypes) by cardiac fibroblasts resulted in enhanced sulfation of secreted glycosaminoglycans (GAGs) with minimal heparan-sulfated proteoglycans localized within the cytoplasm.4 These results supported earlier studies demonstrating an increase in GAGs with amyloid deposition.5 However, the mechanism of internalization and cellular trafficking of LCs is not well understood.
Internalization and cellular trafficking has been studied using a number of amyloid systems. Models of AA and Aß amyloid suggest that endocytosis and/or pinocytosis may be critical to amyloid protein internalization.6,7 In addition, studies using mesangial cells demonstrate that internalization of LCs by cells utilizes a specific receptor complex pathway.8,9 In neuronal cells, investigators showed that receptors of advanced glycation end products function as signal-transducing cell surface acceptors for Aß.10 In fact, receptors of advanced glycation end products are proposed by investigators to act as a general mechanism for cellular uptake of misfolded proteins.11 These results may reflect differences in cell types.
Structural analyses of a number of amyloidogenic proteins suggest that internalization of these biomolecules may occur when they adopt pore-like conformations that can penetrate and disrupt the cellular membrane.12-18 The oligomers possess a number of morphologies, including annular rings that form on mica at concentrations less than 50 µg/ml.12 Pore-like structures such as these have been identified in other diseases with protein misfolding, ie, Parkinson??s,19 , Huntington??s, and Alzheimer??s14,20 diseases and on mitochondrial21 and lysosomal22 membranes.
Our goal was to evaluate the internalization and processing of soluble LCs by cardiac fibroblasts. Cardiac fibroblasts were chosen because of their role in the enhanced production of glycosaminoglycans in response to LCs, and the known association of GAGs and amyloid deposits.4,23,24 Rat primary cardiac fibroblasts were treated with fluorescently labeled LCs in the presence of endocytic inhibitors, proteasomal inhibitors, and cellular probes. The response of the cells and the localization of the LCs were evaluated. We established that the rate of internalization was concentration and mass-dependent and was reduced by treatment with methyl-ß-cyclodextrin (MßCD) but not by filipin. Co-localization is detected with dextran-Texas Red, a marker of pinocytosis. In addition, proteasomal inhibitors altered processing of the LC, resulting in increased retention.25,26
【关键词】 endocytic inhibitory internalization amyloidogenic fibroblasts
Materials and Methods
LC Purification
LCs were purified from urine collected from patients with AL amyloidosis with the approval of the Institutional Review Board of Boston University Medical Center. A total of four 1 and one 6 LCs were evaluated. The LCs included two monomers, a truncated form, and a dimer. The 6 LC was a monomer. All LCs examined here had been evaluated previously and had demonstrated internalization.4 Urine samples were dialyzed, lyophilized, and treated with Affi-Gel Blue (Bio-Rad Laboratories, Hercules, CA) to remove albumin. LC proteins were purified by chromatographic separation on a Sephacryl S-200 column (Amersham Pharmacia Bio-Tech, Buckingham, UK). The immunoglobulin LC proteins were subtyped using a number of cellular and molecular analyses and the sequence examined using both molecular and mass spectrometry.27
Cell Culture
Primary rat cardiac fibroblasts were isolated as previously described.4 In brief, ventricles were subjected to digestion in a buffer containing collagenase, hyaluronidase, and trypsin. Cells were cultured in Dulbecco??s low-glucose modified Eagle??s medium supplemented with 7% calf serum, 1% nonessential amino acids, 100 U/ml penicillin, and 100 µg/ml streptomycin (Life Technologies, Grand Island, NY).4 Cells were used in either the first or second passage. For live cell imaging experiments cells were plated at a concentration of 5 x 103 cells/ml on eight-well coverslip chamber slides (Nalge Nunc, Rochester, NY) and cultured for 48 hours. For other assays, cells were plated on P-100s at an equivalent cell density.
Confocal Laser-Scanning Microscopy
Imaging was performed on a Zeiss LSM 510 confocal microscope (Thornwood, NY) as described previously.4,28,29 Images were taken using a x63 objective with an optical slice of 3 µm. Live cell imaging was performed with a x40 objective with an optical slice ranging from 1 to 3 µm. Z-stacks were taken with an optical slice of 1 µm at an interval of 0.5 µm. Images were collected with x4 averaging. Detector gain and amplitude offset were determined for each experiment to maximize the linear range without saturation and were kept consistent for comparable experiments. Average fluorescence intensities of internalized LCs were measured in individual cells using Zeiss LSM software for region of interest and graphed throughout time.
Localization of LC
Live cell imaging was performed to monitor the localization and internalization rate of LC proteins. Amyloidogenic LCs were conjugated to Oregon Green 488 (Molecular Probes, Eugene, OR) and purified over a sizing column using the suggested methodology from Molecular Probes and Trinkaus-Randall and colleagues.4 The ratio of Oregon Green molecules/LC was determined for each conjugation and used to calculate comparative internalization rates between different LCs. OG488:LC = A496 ?
【参考文献】
Pascali E: Diagnosis and treatment of primary amyloidosis. Crit Rev Oncol Hematol 1995, 19:149-181
Falk RH, Comenzo RL, Skinner M: The systemic amyloidoses. N Engl J Med 1997, 337:898-909
Brenner DA, Jain M, Pimentel DR, Wang B, Connors LH, Skinner M, Apstein CS, Liao R: Human amyloidogenic light chains directly impair cardiomyocyte function through an increase in cellular oxidant stress. Circ Res 2004, 94:1008-1010
Trinkaus-Randall V, Walsh MT, Steeves S, Monis G, Connors LH, Skinner M: Cellular response of cardiac fibroblasts to amyloidogenic light chains. Am J Pathol 2005, 166:197-208
Ancsin JB: Amyloidogenesis: historical and modern observations point to heparan sulfate proteoglycans as a major culprit. Amyloid 2003, 10:67-79
Kluve-Beckerman B, Manaloor JJ, Liepnieks JJ: A pulse-chase study tracking the conversion of macrophage-endocytosed serum amyloid A into extracellular amyloid. Arthritis Rheum 2002, 46:1905-1913
Bamberger ME, Harris ME, McDonald DR, Husemann J, Landreth GE: A cell surface receptor complex for fibrillar beta-amyloid mediates microglial activation. J Neurosci 2003, 23:2665-2674
Russell WJ, Cardelli J, Harris E, Baier RJ, Herrera GA: Monoclonal light chainCmesangial cell interactions: early signaling events and subsequent pathologic effects. Lab Invest 2001, 81:689-703
Teng J, Russell WJ, Gu X, Cardelli J, Jones ML, Herrera GA: Different types of glomerulopathic light chains interact with mesangial cells using a common receptor but exhibit different intracellular trafficking patterns. Lab Invest 2004, 84:440-451
Arancio O, Zhang HP, Chen X, Lin C, Trinchese F, Puzzo D, Liu S, Hegde A, Yan SF, Stern A, Luddy JS, Lue LF, Walker DG, Roher A, Buttini M, Mucke L, Li W, Schmidt AM, Kindy M, Hyslop PA, Stern DM, Du Yan SS: RAGE potentiates Abeta-induced perturbation of neuronal function in transgenic mice. EMBO J 2004, 23:4096-4105
Valencia JV, Weldon SC, Quinn D, Kiers GH, DeGroot J, TeKoppele JM, Hughes TE: Advanced glycation end product ligands for the receptor for advanced glycation end products: biochemical characterization and formation kinetics. Anal Biochem 2004, 324:68-78
Zhu M, Han S, Zhou F, Carter SA, Fink AL: Annular oligomeric amyloid intermediates observed by in-situ AFM. J Biol Chem 2004, 279:24452-24459
Lashuel HA, Hartley DM, Petre BM, Wall JS, Simon MN, Walz T, Lansbury PT, Jr: Mixtures of wild-type and a pathogenic (E22G) form of Abeta40 in vitro accumulate protofibrils, including amyloid pores. J Mol Biol 2003, 332:795-808
Arispe N, Pollard HB, Rojas E: Zn2+ interaction with Alzheimer amyloid beta protein calcium channels. Proc Natl Acad Sci USA 1996, 93:1710-1715
Shtilerman MD, Ding TT, Lansbury PT, Jr: Molecular crowding accelerates fibrillization of alpha-synuclein: could an increase in the cytoplasmic protein concentration induce Parkinson??s disease? Biochemistry 2002, 41:3855-3860
Quist A, Doudevski I, Lin H, Azimova R, Ng D, Frangione B, Kagan B, Ghiso J, Lal R: Amyloid ion channels: a common structural link for protein-misfolding disease. Proc Natl Acad Sci USA 2005, 102:10427-10432
Wall JS, Ayoub FM, O??Shea PS: A study of the interactions of an immunoglobulin light chain with artificial and B-lymphocyte membranes. Front Biosci 1996, 1:a46-a58
Wimley WC, Hristova K, Ladokhin AS, Silvestro L, Axelsen PH, White SH: Folding of beta-sheet membrane proteins: a hydrophobic hexapeptide model. J Mol Biol 1998, 277:1091-1110
Zhu M, Souillac PO, Ionescu-Zanetti C, Carter SA, Fink AL: Surface-catalyzed amyloid fibril formation. J Biol Chem 2002, 277:50914-50922
Lashuel HA, Hartley D, Petre BM, Walz T, Lansbury PT, Jr: Neurodegenerative disease: amyloid pores from pathogenic mutations. Nature 2002, 418:291
Hirakura Y, Kagan BL: Pore formation by beta-2-microglobulin: a mechanism for the pathogenesis of dialysis associated amyloidosis. Amyloid 2001, 8:94-100
Mingeot-Leclercq MP, Lins L, Bensliman M, Van Bambeke F, Van Der Smissen P, Peuvot J, Schanck A, Brasseur R: Membrane destabilization induced by beta-amyloid peptide 29-42: importance of the amino-terminus. Chem Phys Lipids 2002, 120:57-74
Yang AJ, Chandswangbhuvana D, Margol L, Glabe CG: Loss of endosomal/lysosomal membrane impermeability is an early event in amyloid Abeta1-42 pathogenesis. J Neurosci Res 1998, 52:691-698
Long CS, Henrich CJ, Simpson PC: A growth factor for cardiac myocytes is produced by cardiac nonmyocytes. Cell Regul 1991, 2:1081-1095
Goldsmith EC, Hoffman A, Morales MO, Potts JD, Price RL, McFadden A, Rice M, Borg TK: Organization of fibroblasts in the heart. Dev Dyn 2004, 230:787-794
Voorhees PM, Orlowski RZ: The proteasome and proteasome inhibitors in cancer therapy. Annu Rev Pharmacol Toxicol 2006, 46:189-213
Orlowski RZ, Voorhees PM, Garcia RA, Hall MD, Kudrik FJ, Allred T, Johri AR, Jones PE, Ivanova A, Van Deventer HW, Gabriel DA, Shea TC, Mitchell BS, Adams J, Esseltine DL, Trehu EG, Green M, Lehman MJ, Natoli S, Collins JM, Lindley CM, Dees EC: Phase 1 trial of the proteasome inhibitor bortezomib and pegylated liposomal doxorubicin in patients with advanced hematologic malignancies. Blood 2005, 105:3058-3065
Lim A, Wally J, Walsh MT, Skinner M, Costello CE: Identification and location of a cysteinyl posttranslational modification in an amyloidogenic kappa1 light chain protein by electrospray ionization and matrix-assisted laser desorption/ionization mass spectrometry. Anal Biochem 2001, 295:45-56
Klepeis VE, Weinger I, Kaczmarek E, Trinkaus-Randall V: P2Y receptors play a critical role in epithelial cell communication and migration. J Cell Biochem 2004, 93:1115-1133
Richardson TP, Trinkaus-Randall V, Nugent MA: Regulation of heparan sulfate proteoglycan nuclear localization by fibronectin. J Cell Sci 2001, 114:1613-1623
Song QH, Gong H, Trinkaus-Randall V: Role of epidermal growth factor and epidermal growth factor receptor on hemidesmosome complex formation and integrin subunit beta4. Cell Tissue Res 2003, 312:203-220
Trinkaus-Randall V, Tong M, Thomas P, Cornell-Bell A: Confocal imaging of the alpha 6 and beta 4 integrin subunits in the human cornea with aging. Invest Ophthalmol Vis Sci 1993, 34:3103-3109
Trinkaus-Randall V, Kewalramani R, Payne J, Cornell-Bell A: Calcium signaling induced by adhesion mediates protein tyrosine phosphorylation and is independent of pHi. J Cell Physiol 2000, 184:385-399
Ghosh RN, Gelman DL, Maxfield FR: Quantification of low density lipoprotein and transferrin endocytic sorting HEp2 cells using confocal microscopy. J Cell Sci 1994, 107:2177-2189
Manders EM, Verbeek FJ, Aten JA: Measurement of co-localization of objects in dual-colour images. J Microsc 1993, 169:375-382
Besterman JM, Low RB: Endocytosis: a review of mechanisms and plasma membrane dynamics. Biochem J 1983, 210:1-13
Vrljic M, Nishimura SY, Moerner WE, McConnell HM: Cholesterol depletion suppresses the translational diffusion of class II major histocompatibility complex proteins in the plasma membrane. Biophys J 2005, 88:334-347
Pichler H, Riezman H: Where sterols are required for endocytosis. Biochim Biophys Acta 2004, 1666:51-61
Hansen GH, Dalskov SM, Rasmussen CR, Immerdal L, Niels-Christiansen LL, Danielsen EM: Cholera toxin entry into pig enterocytes occurs via a lipid raft- and clathrin-dependent mechanism. Biochemistry 2005, 44:873-882
Watts C, Marsh M: Endocytosis: what goes in and how? J Cell Sci 1992, 103:1-8
Lidke DS, Nagy P, Heintzmann R, Arndt-Jovin DJ, Post JN, Grecco HE, Jares-Erijman EA, Jovin TM: Quantum dot ligands provide new insights into erbB/HER receptor-mediated signal transduction. Nat Biotechnol 2004, 22:198-203
Besterman JM, Airhart JA, Low RB, Rannels DE: Pinocytosis and intracellular degradation of exogenous protein: modulation by amino acids. J Cell Biol 1983, 96:1586-1591
Besterman JM, Airhart JA, Woodworth RC, Low RB: Exocytosis of pinocytosed fluid in cultured cells: kinetic evidence for rapid turnover and compartmentation. J Cell Biol 1981, 91:716-727
Williams KE, Kidston EM, Beck F, Lloyd JB: Quantitative studies of pinocytosis. II. Kinetics of protein uptake and digestion by rat yolk sac cultured in vitro. J Cell Biol 1975, 64:123-134
Anguiano M, Nowak RJ, Lansbury PT, Jr: Protofibrillar islet amyloid polypeptide permeabilizes synthetic vesicles by a pore-like mechanism that may be relevant to type II diabetes. Biochemistry 2002, 41:11338-11343
Zhu M, Li J, Fink AL: The association of alpha-synuclein with membranes affects bilayer structure, stability, and fibril formation. J Biol Chem 2003, 278:40186-40197
Durell SR, Guy HR, Arispe N, Rojas E, Pollard HB: Theoretical models of the ion channel structure of amyloid beta-protein. Biophys J 1994, 67:2137-2145
Dul JL, Davis DP, Williamson EK, Stevens FJ, Argon Y: Hsp70 and antifibrillogenic peptides promote degradation and inhibit intracellular aggregation of amyloidogenic light chains. J Cell Biol 2001, 152:705-716
Merrifield CJ: Seeing is believing: imaging actin dynamics at single sites of endocytosis. Trends Cell Biol 2004, 14:352-358
Goldman RD, Clement S, Khuon S, Moir R, Trejo-Skalli A, Spann T, Yoon M: Intermediate filament cytoskeletal system: dynamic and mechanical properties. Biol Bull 1998, 194:361-363
Fratta P, Engel WK, McFerrin J, Davies KJ, Lin SW, Askanas V: Proteasome inhibition and aggresome formation in sporadic inclusion-body myositis and in amyloid-beta precursor protein-overexpressing cultured human muscle fibers. Am J Pathol 2005, 167:517-526
Ditaranto K, Tekirian TL, Yang AJ: Lysosomal membrane damage in soluble Abeta-mediated cell death in Alzheimer??s disease. Neurobiol Dis 2001, 8:19-31
Andersen OM, Reiche J, Schmidt V, Gotthardt M, Spoelgen R, Behlke J, von Arnim CA, Breiderhoff T, Jansen P, Wu X, Bales KR, Cappai R, Masters CL, Gliemann J, Mufson EJ, Hyman BT, Paul SM, Nykjaer A, Willnow TE: Neuronal sorting protein-related receptor sorLA/LR11 regulates processing of the amyloid precursor protein. Proc Natl Acad Sci USA 2005, 102:13461-13466
Rodrigues CM, Sola S, Brito MA, Brondino CD, Brites D, Moura JJ: Amyloid beta-peptide disrupts mitochondrial membrane lipid and protein structure: protective role of tauroursodeoxycholate. Biochem Biophys Res Commun 2001, 281:468-474
作者单位：From the Departments of Pathology,* Biochemistry, Ophthalmology,¶ and Medicine, The Mass Spectrometry Resource 224 and the Amyloid Treatment and Research Program and Gerry Amyloid Research Laboratory, Boston University School of Medicine, Boston, Massachusetts