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• W1580479524 abstract "Matrix metalloproteases (MMPs) MMP-2 and MMP-9 have been implicated in the physiological catabolism of Alzheimer's amyloid-β (Aβ). Conversely, their association with vascular amyloid deposits, blood-brain barrier disruption, and hemorrhagic transformations after ischemic stroke also highlights their involvement in pathological processes. To better understand this dichotomy, recombinant human (rh) MMP-2 and MMP-9 were incubated with Aβ40 and Aβ42, and the resulting proteolytic fragments were assessed via immunoprecipitation and quantitative mass spectrometry. Both MMPs generated Aβ fragments truncated only at the C terminus, ending at positions 34, 30, and 16. Using deuterated homologues as internal standards, we observed limited and relatively slow degradation of Aβ42 by rhMMP-2, although the enzyme cleaved >80% of Aβ40 during the 1st h of incubation. rhMMP-9 was significantly less effective, particularly in degrading Aβ(1–42), although the targeted peptide bonds were identical. Using Aβ(1–34) and Aβ(1–30), we demonstrated that these peptides are also substrates for both MMPs, cleaving Aβ(1–34) to produce Aβ(1–30) first and Aβ(1–16) subsequently. Consistent with the kinetics observed with full-length Aβ, rhMMP-9 degraded only a minute fraction of Aβ(1–34) and was even less effective in producing Aβ(1–16). Further degradation of Aβ(1–16) by either MMP-2 or MMP-9 was not observed even after prolonged incubation times. Notably, all MMP-generated C-terminally truncated Aβ fragments were highly soluble and did not exhibit fibrillogenic properties or induce cytotoxicity in human cerebral microvascular endothelial or neuronal cells supporting the notion that these truncated Aβ species are associated with clearance mechanisms rather than being key elements in the fibrillogenesis process. Matrix metalloproteases (MMPs) MMP-2 and MMP-9 have been implicated in the physiological catabolism of Alzheimer's amyloid-β (Aβ). Conversely, their association with vascular amyloid deposits, blood-brain barrier disruption, and hemorrhagic transformations after ischemic stroke also highlights their involvement in pathological processes. To better understand this dichotomy, recombinant human (rh) MMP-2 and MMP-9 were incubated with Aβ40 and Aβ42, and the resulting proteolytic fragments were assessed via immunoprecipitation and quantitative mass spectrometry. Both MMPs generated Aβ fragments truncated only at the C terminus, ending at positions 34, 30, and 16. Using deuterated homologues as internal standards, we observed limited and relatively slow degradation of Aβ42 by rhMMP-2, although the enzyme cleaved >80% of Aβ40 during the 1st h of incubation. rhMMP-9 was significantly less effective, particularly in degrading Aβ(1–42), although the targeted peptide bonds were identical. Using Aβ(1–34) and Aβ(1–30), we demonstrated that these peptides are also substrates for both MMPs, cleaving Aβ(1–34) to produce Aβ(1–30) first and Aβ(1–16) subsequently. Consistent with the kinetics observed with full-length Aβ, rhMMP-9 degraded only a minute fraction of Aβ(1–34) and was even less effective in producing Aβ(1–16). Further degradation of Aβ(1–16) by either MMP-2 or MMP-9 was not observed even after prolonged incubation times. Notably, all MMP-generated C-terminally truncated Aβ fragments were highly soluble and did not exhibit fibrillogenic properties or induce cytotoxicity in human cerebral microvascular endothelial or neuronal cells supporting the notion that these truncated Aβ species are associated with clearance mechanisms rather than being key elements in the fibrillogenesis process. Metalloproteases are a family of secreted and membrane-bound zinc-dependent endoproteases that are extrinsic regulators of many biological and pathological processes extending from development, morphogenesis, and tissue remodeling to vascularization, wound healing, cell invasion, and tumor metastasis. To achieve these biological functions, metalloproteases proteolytically cleave growth factors, degrade a wide range of extracellular matrix proteins, and regulate cleavage of various receptors. The large family of metalloproteases includes, among others, matrix metalloproteases (MMPs) 4The abbreviations used are: MMPmatrix metalloproteaserhrecombinant humanAβamyloid βADAlzheimer diseaseCAPS3-(cyclohexylamino)propanesulfonic acidTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycineAPPamyloid precursor proteinBBBblood-brain barrierHFIP1,1,1,3,3,3-hexafluoro-2-propanolIPimmunoprecipitationCAAcerebral amyloid angiopathy. and a disintegrin-metalloproteases (ADAMs). Twenty six MMPs have been identified so far and are clustered into six major groups as follows: collagenases, gelatinases, stromelysins, matrilysins, membrane-type MMPs, and other MMPs according to their domain structure and substrate specificity (1.Sekhon B.S. Matrix metalloproteinases–an overview.Res. Rep. Biol. 2010; 2010: 1-20Google Scholar, 2.Sbardella D. Fasciglione G.F. Gioia M. Ciaccio C. Tundo G.R. Marini S. Coletta M. Human matrix metalloproteinases: an ubiquitarian class of enzymes involved in several pathological processes.Mol. Aspects Med. 2012; 33: 119-208Crossref PubMed Scopus (172) Google Scholar). They are secreted as inactive zymogens, requiring proteolytic removal of a propeptide domain before becoming enzymatically active, and are tightly regulated by specific tissue inhibitors of MMPs, a family of four endogenous protease inhibitors (3.Nagase H. Visse R. Murphy G. Structure and function of matrix metalloproteinases and TIMPs.Cardiovasc. Res. 2006; 69: 562-573Crossref PubMed Scopus (2392) Google Scholar). The ADAM family is composed of 21 members identified in the human genome, with only 12 of them exhibiting catalytic activity. They are type I transmembrane proteinases that also require the removal of their propeptide N-terminal domain to become enzymatically active. A large number of cell-surface proteins are known to undergo ectodomain shedding by proteolytic cleavage. Although some MMPs can act as “sheddases,” the best known shedding events are mediated by ADAMs, particularly ADAM-9, ADAM-10, and ADAM-17 (4.Endres K. Fahrenholz F. Regulation of α-secretase ADAM10 expression and activity.Exp. Brain Res. 2012; 217: 343-352Crossref PubMed Scopus (67) Google Scholar). matrix metalloprotease recombinant human amyloid β Alzheimer disease 3-(cyclohexylamino)propanesulfonic acid N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine amyloid precursor protein blood-brain barrier 1,1,1,3,3,3-hexafluoro-2-propanol immunoprecipitation cerebral amyloid angiopathy. Both MMPs and ADAMs have been reported to prevent the synthesis or to facilitate the clearance of Aβ, the 40–42-amino acid peptide composing vascular and parenchymal amyloid deposits in Alzheimer disease (AD). In mammalian cells and throughout life, Aβ is cleaved from its precursor APP by the sequential action of β- and γ-secretases, in a pathway dubbed “amyloidogenic” because the two main products, Aβ40 and Aβ42, are the central components of amyloid lesions in AD brains. APP is also processed by α-secretase in a “nonamyloidogenic” pathway that prevents the release of Aβ and results in the secretion of a soluble APPα fragment, reported to exhibit neuroprotective and memory-enhancing effects (5.Ghiso J. Frangione B. Amyloidosis and Alzheimer's disease.Adv. Drug Deliv. Rev. 2002; 54: 1539-1551Crossref PubMed Scopus (154) Google Scholar). Among the various candidates for the α-secretase activity, three members of the ADAM family, ADAM-9, ADAM-10, and ADAM-17, have been proposed, with emerging consensus that ADAM-10 is largely responsible for the cleavage of APP at the Lys687–Leu688 peptide bond (amino acids 16 and 17 of the Aβ sequence) and the generation of the soluble APPα fragment (6.Vardy E.R. Catto A.J. Hooper N.M. Proteolytic mechanisms in amyloid-β metabolism: therapeutic implications for Alzheimer's disease.Trends Mol. Med. 2005; 11: 464-472Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 7.De Strooper B. Proteases and proteolysis in Alzheimer disease: a multifactorial view on the disease process.Physiol. Rev. 2010; 90: 465-494Crossref PubMed Scopus (351) Google Scholar). Members of the MMP family with gelatinase activity, MMP-2 and MMP-9, exhibit the ability to degrade Aβ peptides in vitro (8.Backstrom J.R. Lim G.P. Cullen M.J. Tökés Z.A. Matrix metalloproteinase-9 (MMP-9) is synthesized in neurons of the human hippocampus and is capable of degrading the amyloid-β peptide (1–40).J. Neurosci. 1996; 16: 7910-7919Crossref PubMed Google Scholar, 9.Yan P. Hu X. Song H. Yin K. Bateman R.J. Cirrito J.R. Xiao Q. Hsu F.F. Turk J.W. Xu J. Hsu C.Y. Holtzman D.M. Lee J.M. Matrix metalloproteinase-9 degrades amyloid-β fibrils in vitro and compact plaques in situ.J. Biol. Chem. 2006; 281: 24566-24574Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar, 10.Roher A.E. Kasunic T.C. Woods A.S. Cotter R.J. Ball M.J. Fridman R. Proteolysis of Aβ peptide from Alzheimer disease brain by gelatinase A.Biochem. Biophys. Res. Commun. 1994; 205: 1755-1761Crossref PubMed Scopus (124) Google Scholar, 11.Crouch P.J. Tew D.J. Du T. Nguyen D.N. Caragounis A. Filiz G. Blake R.E. Trounce I.A. Soon C.P. Laughton K. Perez K.A. Li Q.X. Cherny R.A. Masters C.L. Barnham K.J. White A.R. Restored degradation of the Alzheimer's amyloid-β peptide by targeting amyloid formation.J. Neurochem. 2009; 108: 1198-1207Crossref PubMed Scopus (75) Google Scholar, 12.Yin K.J. Cirrito J.R. Yan P. Hu X. Xiao Q. Pan X. Bateman R. Song H. Hsu F.F. Turk J. Xu J. Hsu C.Y. Mills J.C. Holtzman D.M. Lee J.M. Matrix metalloproteinases expressed by astrocytes mediate extracellular amyloid-β peptide catabolism.J. Neurosci. 2006; 26: 10939-10948Crossref PubMed Scopus (277) Google Scholar) and have been implicated as central players in the process of Aβ catabolism. A role for the increased expression of MMPs in hemorrhagic complications associated with vascular amyloid deposits in cerebral amyloid angiopathy (CAA) has been suggested by the known association of these proteases with blood-brain barrier (BBB) disruption in many vasculopathies and with hemorrhagic transformations after ischemic stroke (13.Montaner J. Molina C.A. Monasterio J. Abilleira S. Arenillas J.F. Ribó M. Quintana M. Alvarez-Sabín J. Matrix metalloproteinase-9 pretreatment level predicts intracranial hemorrhagic complications after thrombolysis in human stroke.Circulation. 2003; 107: 598-603Crossref PubMed Scopus (479) Google Scholar, 14.Alvarez-Sabín J. Delgado P. Abilleira S. Molina C.A. Arenillas J. Ribó M. Santamarina E. Quintana M. Monasterio J. Montaner J. Temporal profile of matrix metalloproteinases and their inhibitors after spontaneous intracerebral hemorrhage: relationship to clinical and radiological outcome.Stroke. 2004; 35: 1316-1322Crossref PubMed Scopus (186) Google Scholar, 15.Abilleira S. Montaner J. Molina C.A. Monasterio J. Castillo J. Alvarez-Sabín J. Matrix metalloproteinase-9 concentration after spontaneous intracerebral hemorrhage.J. Neurosurg. 2003; 99: 65-70Crossref PubMed Scopus (168) Google Scholar). MMP-9 has been shown to degrade fibrillar structures and compact plaques in ex vivo experiments (9.Yan P. Hu X. Song H. Yin K. Bateman R.J. Cirrito J.R. Xiao Q. Hsu F.F. Turk J.W. Xu J. Hsu C.Y. Holtzman D.M. Lee J.M. Matrix metalloproteinase-9 degrades amyloid-β fibrils in vitro and compact plaques in situ.J. Biol. Chem. 2006; 281: 24566-24574Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar) and to co-localize in vivo with neuritic plaques, vascular amyloid deposits, and neurofibrillary tangles. MMP-2 has been demonstrated to be a key element in the disruption of tight junction proteins and induction of BBB permeability alterations. It is secreted by endothelial cells in culture upon stimulation with Aβ peptides, being particularly responsive to vasculotropic Aβ mutants (16.Hernandez-Guillamon M. Mawhirt S. Fossati S. Blais S. Pares M. Penalba A. Boada M. Couraud P.O. Neubert T.A. Montaner J. Ghiso J. Rostagno A. Matrix metalloproteinase 2 (MMP-2) degrades soluble vasculotropic amyloid-β E22Q and L34V mutants, delaying their toxicity for human brain microvascular endothelial cells.J. Biol. Chem. 2010; 285: 27144-27158Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Through their ability to degrade Aβ, MMP-2 and MMP-9 likely contribute to the brain homeostasis of Aβ and to the maintenance of Aβ levels in biological fluids. Both enzymes are able to generate in vitro Aβ fragments that are found in vivo in cerebrospinal fluid (CSF) (17.Hampel H. Shen Y. Walsh D.M. Aisen P. Shaw L.M. Zetterberg H. Trojanowski J.Q. Blennow K. Biological markers of amyloid β-related mechanisms in Alzheimer's disease.Exp. Neurol. 2010; 223: 334-346Crossref PubMed Scopus (129) Google Scholar). However, no systematic studies have been performed identifying the steps through which these truncated species are generated. Using mass spectrometry (MS) approaches, the studies presented herein compare the kinetics of Aβ proteolysis by both MMP-2 and MMP-9 using Aβ40 and Aβ42 homologues as substrates. The data indicate that both enzymes generate sequential intermediates that also serve as self-substrates and identify the end point peptide fragments as molecules of high solubility, lacking fibrillogenic propensity and neurotoxicity. The data support the notion that the proteolytically generated fragments are related to clearance mechanisms rather than being key elements in the amyloidogenesis process. Synthetic homologues of Aβ42, Aβ40, the C-terminal truncated fragments Aβ(1–34), Aβ(1–30), and Aβ(1–16), as well as the Aβ40 genetic variant containing the L34V substitution were synthesized using N-tert-butyloxycarbonyl chemistry by James I. Elliott at Yale University. For quantitative MS, isotopically labeled peptide standards of Aβ42, Aβ40, and the C-terminal truncated fragments Aβ(1–34), Aβ(1–30), and Aβ(1–16) were synthesized with deuterated [2H]phenylalanine residues; labels were introduced at positions 4, 19, and 20 in Aβ42, Aβ40, Aβ34, and Aβ30, whereas Aβ(1–16) contained a single labeled residue at position 4. All synthetic peptides were purified by reverse phase-high performance liquid chromatography, molecular masses corroborated by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS, and concentrations assessed by amino acid analysis, as described previously (16.Hernandez-Guillamon M. Mawhirt S. Fossati S. Blais S. Pares M. Penalba A. Boada M. Couraud P.O. Neubert T.A. Montaner J. Ghiso J. Rostagno A. Matrix metalloproteinase 2 (MMP-2) degrades soluble vasculotropic amyloid-β E22Q and L34V mutants, delaying their toxicity for human brain microvascular endothelial cells.J. Biol. Chem. 2010; 285: 27144-27158Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 18.Fossati S. Cam J. Meyerson J. Mezhericher E. Romero I.A. Couraud P.-O. Weksler B.B. Ghiso J. Rostagno A. Differential activation of mitochondrial apoptotic pathways by vasculotropic amyloid-β variants in cells composing the cerebral vessel walls.FASEB J. 2010; 24: 229-241Crossref PubMed Scopus (62) Google Scholar). To remove pre-formed aggregates, peptides were pretreated with 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) for at least 24 h at room temperature with intermittent vigorous vortexing. After lyophilization, peptides were either reconstituted in deionized water to a 1 μg/μl concentration (stock solution) for immediate use or stored at −80 °C for up to 2 months. The various Aβ homologues were proteolytically cleaved by incubation with activated recombinant human MMP-2 or MMP-9 (R&D Systems, Minneapolis, MN). Prior to the enzymatic assay, and following the supplier's protocol, both rhMMP-2 and rhMMP-9 were activated by 37 °C incubation with 1 mm para-aminophenylmercuric acetate (Sigma) in 50 mm Tris, pH 7.5, containing 10 mm CaCl2, 150 mm NaCl, and 0.05% Brij-35 (TCNB; rhMMP-2 for 1 h and rhMMP-9 for 24 h). After activation, MMP-2- and MMP-9-specific activities were assessed by their ability to cleave the specific fluorescent peptide substrate Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 (R&D Systems; where Mca is 7-methoxycoumarin-4-yl acetyl and Dpa is N-3–2,4-dinitrophenyl-l-2–3-diaminopropionyl (19.Knight C.G. Willenbrock F. Murphy G. A novel coumarin-labelled peptide for sensitive continuous assays of the matrix metalloproteinases.FEBS Lett. 1992; 296: 263-266Crossref PubMed Scopus (678) Google Scholar)) using the manufacturer's protocol. In all cases both enzymes exhibited comparable specific activity (consistently ranging between 1250 and 1400 pmol/min/μg, depending on the lot). Proteolytic cleavage of Aβ was performed at 37 °C at an enzyme/peptide ratio of 1:25 (100 nm enzyme, 2.5 μm Aβ in TCNB), as described previously (16.Hernandez-Guillamon M. Mawhirt S. Fossati S. Blais S. Pares M. Penalba A. Boada M. Couraud P.O. Neubert T.A. Montaner J. Ghiso J. Rostagno A. Matrix metalloproteinase 2 (MMP-2) degrades soluble vasculotropic amyloid-β E22Q and L34V mutants, delaying their toxicity for human brain microvascular endothelial cells.J. Biol. Chem. 2010; 285: 27144-27158Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Aliquots were retrieved at different time points for up to 24 h; in the case of Aβ40L34V, incubation with MMP-9 was further extended up to 10 days. In all cases, protease activity was stopped by addition of 5 μl of 15 mm EDTA, pH 8.0, followed by freezing at −80 °C. In all cases, assessment of proteolytic degradation was evaluated by Western blot analysis and by the identification of specific Aβ degradation products via immunoprecipitation (IP) followed by MS analysis, as described below. Intact Aβ peptides and newly generated proteolytic fragments were immunoprecipitated by incubation with paramagnetic beads (Dynabeads M-280, Invitrogen) coated with a combination of 4G8 and 6E10 antibodies (Covance, Princeton, NJ), as described previously (16.Hernandez-Guillamon M. Mawhirt S. Fossati S. Blais S. Pares M. Penalba A. Boada M. Couraud P.O. Neubert T.A. Montaner J. Ghiso J. Rostagno A. Matrix metalloproteinase 2 (MMP-2) degrades soluble vasculotropic amyloid-β E22Q and L34V mutants, delaying their toxicity for human brain microvascular endothelial cells.J. Biol. Chem. 2010; 285: 27144-27158Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 20.Tomidokoro Y. Lashley T. Rostagno A. Neubert T.A. Bojsen-Møller M. Braendgaard H. Plant G. Holton J. Frangione B. Révész T. Ghiso J. Familial Danish dementia: co-existence of ADan and Aβ amyloid subunits in the absence of compact plaques.J. Biol. Chem. 2005; 280: 36883-36894Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 21.Tomidokoro Y. Rostagno A. Neubert T.A. Lu Y. Rebeck G.W. Frangione B. Greenberg S.M. Ghiso J. Iowa variant of familial Alzheimer's disease: accumulation of posttranslationally modified AβD23N in parenchymal and cerebrovascular amyloid deposits.Am. J. Pathol. 2010; 176: 1841-1854Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). After overnight incubation at 4 °C with the anti-Aβ-coated beads, samples were resuspended in PBS containing 0.025% Tween 20 (PBS-T), washed three times with PBS-T and once with 50 mm NH4HCO3, followed by elution with 0.5% formic acid. The eluted IP material was dried down in a Savant SpeedVac concentrator (Thermo Fisher) and further reconstituted in 5 μl of 0.1% formic acid in 50% acetonitrile for MS. One-fifth of the reconstituted sample (1 μl) was combined with an equal volume of α-4-hydroxycinnamic acid matrix (Agilent Technologies) reconstituted in 0.1% trifluoroacetic acid (TFA) and 100% acetonitrile at a concentration of 15 g/liter and 1 μl of the resulting mixture spotted onto a Bruker Daltonics MTP 384 massive target T aluminum plate pre-seeded with 0.5 μl of α-4-hydroxycinnamic acid (1 g/liter). All samples were spotted in duplicate and analyzed at the New York University Mass Spectrometry Core for Neuroscience using a Bruker Daltonics Autoflex MALDI-TOF mass spectrometer (Bremen, Germany) in linear mode using standard instrument settings. For quantitative MS assessments, known amounts of the corresponding deuterated standards were added to the digestion mixture prior to the IP and MS analysis. At least two sets of measurements containing different amounts of the same standard were used for each experiment to estimate the concentration of the truncated peptides generated. In all cases, MS spectra were processed and analyzed by FlexAnalysis, and quantitation was performed by comparison with peak intensities of the pertinent labeled standards. Detection of Aβ by Western blot was performed following previously described protocols (22.Rostagno A. Ghiso J. Isolation and biochemical characterization of amyloid plaques and paired helical filaments.Curr. Protoc. Cell Biol. 2009: 3.33.31-33.33.33Crossref Scopus (30) Google Scholar). Briefly, MMP digestion samples were separated on 16.5% Tris/Tricine SDS-PAGE under reducing conditions and transferred onto 0.45-μm pore size polyvinylidene difluoride membranes (PVDF; Millipore Corp, Billerica, MA) for 45 min at 400 mA using CAPS buffer, pH 11. After blocking for 1 h with 5% nonfat milk in PBS-T, membranes were probed with rabbit polyclonal antibodies anti-Aβ40 (1:500, Invitrogen) or anti-Aβ42 (1:500, Invitrogen) recognizing specifically the fragments ending at positions 40 or 42, respectively, without immunoreacting wit the C-terminal truncated species. This was followed by incubation with HRP-conjugated anti-rabbit IgG (1:3000, BioSource-Invitrogen) for 1 h, development of fluorograms by enhanced chemiluminescence using Supersignal West Pico Luminol/Enhancer and Stable Peroxide solutions (Thermo Scientific), and image analysis with ImageJ software (rsbweb.nih.gov). Human neuroblastoma cells SH-SY5Y were obtained from the ATCC and grown in DMEM/F-12 (Life Technologies/Gibco) containing 15% (v/v) fetal bovine serum (FBS, Sigma) and 100 units/ml penicillin, 100 g/ml streptomycin (Life Technologies/Gibco). Human cerebral microvascular endothelial cells (hCMEC/D3), provided by B. Weksler, Weill Medical College, Cornell University, New York, NY (23.Weksler B.B. Subileau E.A. Perrière N. Charneau P. Holloway K. Leveque M. Tricoire-Leignel H. Nicotra A. Bourdoulous S. Turowski P. Male D.K. Roux F. Greenwood J. Romero I.A. Couraud P.O. Blood-brain barrier-specific properties of a human adult brain endothelial cell line.FASEB J. 2005; 19: 1872-1874Crossref PubMed Scopus (1033) Google Scholar), were grown in EGM-2 medium (Lonza, Allendale, NJ) supplemented with VEGF, IGF, bFGF, hydrocortisone, ascorbate, and 2.5% FBS, as described (18.Fossati S. Cam J. Meyerson J. Mezhericher E. Romero I.A. Couraud P.-O. Weksler B.B. Ghiso J. Rostagno A. Differential activation of mitochondrial apoptotic pathways by vasculotropic amyloid-β variants in cells composing the cerebral vessel walls.FASEB J. 2010; 24: 229-241Crossref PubMed Scopus (62) Google Scholar). Both cell types were challenged for 3 days with the different Aβ peptides pretreated as above and reconstituted in EGM-2, 1% FBS or DMEM, 1% FBS at 50 μm final concentration. Apoptosis induced by the treatment with different Aβ homologues was evaluated by quantitation of DNA fragmentation using Cell Death ELISA (Roche Applied Science) in accordance with the manufacturer's specifications, as we described previously (18.Fossati S. Cam J. Meyerson J. Mezhericher E. Romero I.A. Couraud P.-O. Weksler B.B. Ghiso J. Rostagno A. Differential activation of mitochondrial apoptotic pathways by vasculotropic amyloid-β variants in cells composing the cerebral vessel walls.FASEB J. 2010; 24: 229-241Crossref PubMed Scopus (62) Google Scholar, 24.Fossati S. Todd K. Sotolongo K. Ghiso J. Rostagno A. Differential contribution of isoaspartate post-translational modifications to the fibrillization and toxic properties of amyloid β and the Asn23 Iowa mutation.Biochem. J. 2013; 456: 347-360Crossref PubMed Scopus (34) Google Scholar). After amyloid treatment, culture plates were centrifuged in a Beckman J-6B centrifuge (10 min; 1,000 rpm) to collect floaters; cells were lysed, and quantitation of DNA-histone complexes (nucleosomes) was assessed by absorbance at 405 nm. Results are expressed as fold-change compared with no-peptide controls. Cell viability after Aβ treatments was evaluated by assessing the release to the culture supernatants of the cytoplasmic enzyme lactate dehydrogenase (cytotoxicity detection kit plus, Roche Applied Science), following the manufacturer's instructions. Results are expressed as fold-change compared with no-peptide controls. The presence of fibrillar components in culture supernatants challenged with the different Aβ peptides was assessed by thioflavin T binding assays. Briefly, 24-μl aliquots of the respective culture supernatants were added to 10 μl of freshly prepared thioflavin T (Sigma; 0.1 mm) and 50 mm Tris buffer, pH 8.5, to a final volume of 200 μl, as described previously (16.Hernandez-Guillamon M. Mawhirt S. Fossati S. Blais S. Pares M. Penalba A. Boada M. Couraud P.O. Neubert T.A. Montaner J. Ghiso J. Rostagno A. Matrix metalloproteinase 2 (MMP-2) degrades soluble vasculotropic amyloid-β E22Q and L34V mutants, delaying their toxicity for human brain microvascular endothelial cells.J. Biol. Chem. 2010; 285: 27144-27158Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Fluorescence was recorded after 300 s in a LS-50B luminescence spectrometer (PerkinElmer Life Sciences) with excitation and emission wavelengths of 435 nm (slit width = 10 nm) and 490 nm (slit width = 10 nm), respectively. Each sample was analyzed in duplicate. Curve fitting for quantitative MS data were performed by spline/lowess analysis using Graph Pad Prism (GraphPad Software, La Jolla, CA). Statistical analyses of cell death ELISA, lactate dehydrogenase release, and thioflavin T binding data were performed by one-way analysis of variance with Newman-Keuls Multiple Comparison post hoc test (Graph-Pad Prism); values of p < 0.05 were considered significant. IP/MS analysis of the time course degradation of full-length Aβ40 (average mass M + H = 4,330.86 Da) and Aβ42 (M + H 4,515.10 Da) peptides by rhMMP-2 and rhMMP-9 revealed the generation of three major proteolytic products, all truncated at the C terminus as follows: Aβ(1–34) (M + H 3,788.16 Da); Aβ(1–30) (M + H 3,391.63 Da); and Aβ(1–16) (M + H 1,956.03 Da). Although the resulting degradation fragments were basically the same, Aβ40 and Aβ42 showed different susceptibility to each MMP despite the comparable specific activity of both enzymes. Fig. 1 depicts the time-resolved MALDI-TOF MS spectra and relative ion counts at different time points, illustrating the progression of the enzymatic reaction with the Aβ(1–40) peptide. As illustrated, rhMMP-9 required 30 times longer incubation than rhMMP2 (5 h versus 10 min) to generate Aβ(1–34) and Aβ(1–30) fragments with comparable intensity ratios. The smaller fragment Aβ(1–16) required even longer degradation time to be detected, ∼30 min with rhMMP-2 versus more than 5 h with rhMMP-9, suggesting that either the Aβ peptide bond Lys16–Leu17 is less susceptible to proteolysis (e.g. through a steric hindrance-like mechanism) or it is generated from a substrate different from the initial intact peptide (e.g. one of the cleavage products) and has, therefore, a delayed appearance. By assessing the relative intensity ratios between the intact peptide and each of the proteolytically generated fragments at all the tested time points, the data indicate that by 30 min of incubation with Aβ40, rhMMP-2 reaches a degradation plateau (Fig. 1A), and similar levels of the remaining intact peptide are achieved only after 24 h of incubation with rhMMP-9 (Fig. 1B). Judged by the relative intensity ratios produced by rhMMP-2 at the various time points, it seemed that the smaller fragments were generated at the expense of intermediate degradation products rather than the full-length Aβ40 (Fig. 1A). Notably, the longest incubation (24 h) with MMP-9, but not with MMP2, generated an additional fragment with a mass compatible with the fragment Aβ(1–22) (m/z 2661.87 theoretical; 2661.76 experimental). Further validation for the Aβ(1–40) degradation by rhMMP-2 and rhMMP-9 was obtained via Western blot analysis utilizing an antibody specific to the C terminus of the peptide (anti-Aβ40) that does not react with the proteolytic fragments ending at positions 34, 30, and 16. As shown for selected time points (0, 1, and 24 h), Aβ(1–40) was more efficiently cleaved by rhMMP-2 than by rhMMP-9. Loss of intact Aβ(1–40) as a result of proteolytic degradation was clearly visualized at the 1-h incubation time point with rhMMP-2 (Fig. 1A), whereas rhMMP-9 required a longer incubation time (Fig. 1B). No obvious Aβ oligomerization was visualized in the time frame of the experiments under the current experimental conditions that could account for the different enzymatic susceptibility. A similar degradation profile illustrated in Fig. 1 for Aβ40 was obtained for Aβ42. As indicated in Fig. 2, the same three fragments ending at positions 34, 30, and 16 were generated with both enzymes, although for rhMMP-9 the degradation process was much slower than with rhMMP-2 (Fig. 2A) despite the comparable specific activities of both enzymes, and the Aβ(1–16) fragment was hardly detected even after the 24-h incubation of Aβ(1–42) with rhMMP-9 (Fig. 2B). Time-resolved MALDI-TOF spectra revealed that the rhMMP-2 degradation products Aβ(1–34), Aβ(1–30), and Aβ(1–16) were generated from Aβ42 within a comparable time frame as those originating from Aβ40; however, the reaction reached a plateau at a higher Aβ42/degradation products relative intensity ratio, a clear indication that m" @default.
• W1580479524 created "2016-06-24" @default.
• W1580479524 creator A5031100058 @default.
• W1580479524 creator A5031727434 @default.
• W1580479524 creator A5048009605 @default.
• W1580479524 creator A5065356048 @default.
• W1580479524 creator A5081547926 @default.
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• W1580479524 date "2015-06-01" @default.
• W1580479524 modified "2023-10-15" @default.
• W1580479524 title "Sequential Amyloid-β Degradation by the Matrix Metalloproteases MMP-2 and MMP-9" @default.
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