共查询到20条相似文献,搜索用时 31 毫秒
1.
Chinese hamster ovary (CHO) cells are the most commonly used host cell line for the production of recombinant biopharmaceuticals. These biopharmaceuticals are typically secreted from CHO cells and purified from harvested cell culture media. The purpose of this study was to investigate changes in the secreted proteome of CHO cells over the various stages of the growth cycle using Surface Enhanced Laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF MS). Conditioned media samples were collected each day over a 6 day growth period from CHO-K1 cells grown in low serum (0.5% FBS) conditions in monolayer culture. Samples were profiled on a number of ProteinChip arrays with different chromatographic surfaces. From this study, 24 proteins were found to be differentially regulated at different phases of the growth cycle in CHO-K1 cells, when profiled on two chromatographic surfaces, Q10 (anionic) and IMAC30 (metal affinity) ProteinChip arrays. 相似文献
2.
J M Pasternack M R Palmert M Usiak R Wang H Zurcher-Neely P A Gonzalez-De Whitt M B Fairbanks T Cheung D Blades R L Heinrikson 《Biochemistry》1992,31(44):10936-10940
The 39-43 amino acid beta amyloid protein (A beta) that deposits as amyloid in the brains of patients with Alzheimer's disease (AD) is encoded as an internal sequence within a larger membrane-associated protein known as the amyloid protein precursor (APP). In cultured cells, the APP is normally cleaved within the A beta to generate a large secreted derivative and a small membrane-associated fragment. Neither of these derivatives can produce amyloid because neither contains the entire A beta. Our study was designed to determine whether the soluble APP derivatives in human brain end within the A beta as described in cell culture or whether AD brain produces potentially amyloidogenic soluble derivatives that contain the entire A beta. We find that both AD and control brain contain nonamyloidogenic soluble derivatives that end at position 15 of the A beta. We have been unable to detect any soluble derivatives that contain the entire A beta in either the AD or control brain. 相似文献
3.
4.
S L Gillespie T E Golde S G Younkin 《Biochemical and biophysical research communications》1992,187(3):1285-1290
The 39-43 residue polypeptide (amyloid beta protein, beta A4) deposited as amyloid in Alzheimer's disease (AD) is derived from a set of 695-770 residue precursors referred to as the amyloid beta A4 protein precursor (beta APP). In each of the 695, 751, and 770 residue precursors, the 43 residue beta A4 is an internal peptide that begins 99 residues from the COOH-terminus of the beta APP. Each holoform is normally cleaved within the beta A4 to produce a large secreted derivative as well as a small membrane associated fragment. Neither of these derivatives can produce amyloid because neither contains the entire beta A4 peptide. In this study, we employ cells stably transfected with full length beta APP695, beta APP751, or beta APP770 expression constructs to show that phorbol ester activation of protein kinase C substantially increases the production of secreted forms from each isoform. By increasing processing of beta APP in the secretory pathway, PKC phosphorylation may help to prevent amyloid deposition. 相似文献
5.
Glu11 site cleavage and N-terminally truncated A beta production upon BACE overexpression 总被引:6,自引:0,他引:6
Amyloid beta peptides (A beta) are generated by the proteolytic processing of the amyloid beta precursor protein (APP). The newly identified beta-site APP-cleaving enzyme (BACE) cleaves APP at Asp1 as well as between Tyr10 and Glu11 of A beta, producing C-terminal fragments (CTFs) C99 and C89, respectively. Subsequent cleavage by gamma-secretase gives rise to A beta 1-40/42 and A beta 11-40/42. Although both full-length and A beta peptides truncated at residue 11 have been identified in amyloid plaques in the AD brain, the relative proportion of these two cleavage products produced by BACE and secreted into the medium by cultured cells is unknown. Using cell lines stably overexpressing BACE, we found that A beta 11-40 and A beta 11-42 are major A beta cleavage products generated by BACE. We further showed that BACE utilizes both full-length APP as well as C99 as substrates for the production of C89, and that A beta 11-40/42 can be generated by sequential cleavage of single APP molecules by BACE and gamma-secretase. Taken together, the abundance of A beta 11-40/42 produced by BACE suggests that their roles in AD pathogenesis may be underestimated. 相似文献
6.
Pietrak BL Crouthamel MC Tugusheva K Lineberger JE Xu M DiMuzio JM Steele T Espeseth AS Stachel SJ Coburn CA Graham SL Vacca JP Shi XP Simon AJ Hazuda DJ Lai MT 《Analytical biochemistry》2005,342(1):144-151
The deposition of beta-amyloid peptides (A beta42 and A beta40) in neuritic plaques is one of the hallmarks of Alzheimer's disease (AD). A beta peptides are derived from sequential cleavage of amyloid precursor protein (APP) by beta- and gamma-secretases. BACE-1 has been shown to be the major beta-secretase and is a primary therapeutic target for AD. In this article, two novel assays for the characterization of BACE-1 inhibitors are reported. The first is a sensitive 96-well HPLC biochemical assay that uses a unique substrate containing an optimized peptide cleavage sequence, NFEV, spanning from the P2-P2' positions This substrate was processed by BACE-1 approximately 10 times more efficiently than was the widely used substrate containing the Swedish (NLDA) sequence. As a result, the concentration of the enzyme required for the assay can be as low as 100 pM, permitting the evaluation of inhibitors with subnanomolar potency. The assay has also been applied to related aspartyl proteases such as cathepsin D (Cat D) and BACE-2. The second assay is a homogeneous electrochemiluminescence assay for the evaluation of BACE-1 inhibition in cultured cells that assesses the level of secreted amyloid EV40_NF from HEK293T cells stably transfected with APP containing the novel NFEV sequence. To illustrate the use of these assays, the properties of a potent, cell-active BACE-1 inhibitor are described. 相似文献
7.
The amyloid beta peptide (A beta P) is the major constituent of the amyloid deposits that accumulate extracellularly in the brain of patients with Alzheimer's disease. This peptide is obtained from transmembrane amyloid protein precursors (APP) which sometimes contain a Kunitz protease inhibitor (KPI) insert in their extracellular domain and therefore are able to inhibit serine proteases. Expression of the transmembrane and the secreted APP containing the KPI domain was obtained by transient transfection of COS-1 cells. The overexpressed proteins were detected in immunoblotting experiments and inhibition of trypsin was analyzed using reverse enzymography. Our results indicate that post-translational modifications including glycosylation improve the inhibition of trypsin by the APP containing the KPI domain. 相似文献
8.
Sagi SA Weggen S Eriksen J Golde TE Koo EH 《The Journal of biological chemistry》2003,278(34):31825-31830
Epidemiological evidence suggests that chronic use of non-steroidal anti-inflammatory drugs (NSAIDs) reduces the risk of Alzheimer's disease. Recently, NSAIDs have been shown to decrease amyloid pathology in a transgenic mouse model of Alzheimer's disease. This benefit may be partially attributable to the ability of NSAIDs to selectively reduce production of the amyloidogenic A beta 42 peptide in both cultured cells and transgenic mice. Although this activity does not appear to require the action of cyclooxygenases in cultured cells, it is not known whether other NSAID-sensitive targets contribute to this A beta 42 effect. In this study, we have used both pharmacological and genetic means to determine if other known cellular targets of NSAIDs could mediate the reduction in A beta 42 secretion from cultured cells. We find that altered arachidonic acid metabolism via NSAID action on cyclooxygenases and lipoxygenases does not alter A beta 42 production. Furthermore, we demonstrate that alterations in activity of peroxisome proliferator-activated receptors, I kappa B kinase beta or nuclear factor kappa B do not affect A beta 42 production. Thus, NSAIDs do not appear to alter A beta 42 production indirectly through previously identified cellular targets and may interact directly with the gamma-secretase complex itself to affect amyloid production. 相似文献
9.
Bhattacharya S Latha JN Kumresan R Singh S 《Biochemical and biophysical research communications》2007,356(3):622-628
Efforts to clone amyloidogenic proteins in the cells often have resulted in cell death. We report successful cloning and expression of recombinant human islet amyloid polypeptide (hIAPP) in cultured mammalian cells. Amylin gets secreted, forms fibrils that are toxic to target cells like beta cells of rat and human. The study involves cloning of full-length amylin in fluorescent protein vector followed by transfection into mammalian cells. The transfected cells with recombinant human amylin, secrete the translated protein corresponding to 37-amino acid native mature IAPP. The mature IAPP secreted out of the cell is purified and characterized by MALDI-TOF/TOF-MS and Western blotting. Purified IAPP forms fibrils as seen by Thioflavin-T fluorescence and AFM, and these fibrils were cytotoxic towards pancreatic cell line RIN5mf cells. 相似文献
10.
Polarized sorting of beta-amyloid precursor protein and its proteolytic products in MDCK cells is regulated by two independent signals 总被引:6,自引:1,他引:5
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《The Journal of cell biology》1995,128(4):537-547
Progressive cerebral deposition of the amyloid (A beta) beta-protein is an early and invariant feature of Alzheimer's disease. A beta is derived by proteolysis from the membrane-spanning beta-amyloid precursor protein (beta APP). beta APP is processed into various secreted products, including soluble beta APP (APPs), the 4-kD A beta peptide, and a related 3-kD peptide (p3). We analyzed the mechanisms regulating the polarized basolateral sorting of beta APP and its proteolytic derivatives in MDCK cells. Deletion of the last 32 amino acids (residues 664-695) of the beta APP cytoplasmic tail had no influence on either the constitutive approximately 90% level of basolateral sorting of surface beta APP, or the strong basolateral secretion of APPs, A beta, and p3. However, deleting the last 42 amino acids (residues 654-695) or changing tyrosine 653 to alanine altered the distribution of cell surface beta APP so that approximately 40-50% of the molecules were inserted apically. In parallel, A beta was now secreted from both surfaces. Surprisingly, this change in surface beta APP had no influence on the basolateral secretion of APPs and p3. This result suggests that most beta APP molecules which give rise to APPs in MDCK cells are cleaved intracellularly before reaching the surface. Consistent with this conclusion, we readily detected intracellular APPs in carbonate extracts of isolated membrane vesicles. Moreover, ammonium chloride treatment resulted in the equal secretion of APPs into both compartments, as occurs with other non-membranous, basolaterally secreted proteins, but it did not influence the polarity of cell surface beta APP. These results demonstrate that in epithelial cells two independent mechanisms mediate the polarized trafficking of beta APP holoprotein and its major secreted derivative (APPs) and that A beta peptides are derived in part from beta APP holoprotein targeted to the cell surface by a signal that includes tyrosine 653. 相似文献
11.
Fluhrer R Multhaup G Schlicksupp A Okochi M Takeda M Lammich S Willem M Westmeyer G Bode W Walter J Haass C 《The Journal of biological chemistry》2003,278(8):5531-5538
The beta-amyloid precursor protein (beta APP) is proteolytically processed by two secretase activities to produce the pathogenic amyloid beta-peptide (A beta). N-terminal cleavage is mediated by beta-secretase (BACE) whereas C-terminal intramembraneous cleavage is exerted by the presenilin (PS) gamma-secretase complex. The A beta-generating gamma-secretase cleavage principally occurs after amino acid 40 or 42 and results in secretion of A beta-(1-40) or A beta-(1-42). Upon overexpression of BACE in cultured cells we unexpectedly noticed a reduction of secreted A beta-(1-40/42). However, mass spectrometry revealed a truncated A beta species, which terminates at amino acid 34 (A beta-(1-34)) suggesting an alternative gamma-secretase cut. Indeed, expression of a loss-of-function variant of PS1 inhibited not only the production of A beta-(1-40) and A beta-(1-42) but also that of A beta-(1-34). However, expression levels of BACE correlate with the amount of A beta-(1-34), and A beta-(1-34) is produced at the expense of A beta-(1-40) and A beta-(1-42). Since this suggested that BACE is involved in a C-terminal truncation of A beta, we incubated purified BACE with A beta-(1-40) in vitro. Under these conditions A beta-(1-34) was generated. Moreover, when conditioned media containing Abeta-(1-40) and A beta-(1-42) were incubated with cells expressing a loss-of-function PS1 variant together with BACE, A beta-(1-34) was efficiently produced in vivo. These data demonstrate that an apparently gamma-secretase-dependent A beta derivative is produced after the generation of the non-truncated A beta via an additional and unexpected activity of BACE. 相似文献
12.
C C Overly L C Fritz I Lieberburg L McConlogue 《Biochemical and biophysical research communications》1991,181(2):513-519
The beta-amyloid peptide is derived from a larger membrane bound protein and accumulates as amyloid in Alzheimer's diseased brains. beta-amyloid precursor protein (beta APP) proteolytically processed during constitutive secretion cannot be a source of deposited amyloid because this processing results in cleavage within the amyloidogenic peptide. To see if other secretory pathways could be responsible for generating potentially amyloidogenic molecules we tested the possibility that beta APP is targeted to the regulated secretory pathway. Stable AtT20 cell lines expressing exogenous human beta APP were genetically engineered. These cells were labeled with [35S]-methionine, and chased in the presence or absence of secretagogue. The beta APP both inside the cells and released from the cells was analyzed by immunoprecipitation and gel analysis. Quantitation of autoradiograms showed that virtually all of the synthesized beta APP was secreted by the constitutive pathway, and that no detectable (less than 1%) beta APP was targeted to the regulated secretory pathway. 相似文献
13.
The control of rat hepatocyte DNA synthesis in vitro by Kupffer cells and transformed perisinusoidal lipocytes, i.e. myofibroblast-like cells was studied. Conditioned media from Kupffer cells inhibit the replicative (hydroxyurea-sensitive) DNA synthesis dose-dependently in primary cultures of hepatocytes stimulated by epidermal growth factor (EGF). The cytokine responsible for the inhibition was identified as transforming growth factor beta (TGF beta). After neutralization of activated TGF beta in these media, DNA synthesis is stimulated in quiescent hepatocytes via transforming growth factor alpha (TGF alpha) demonstrated by competitive TGF alpha/EGF-receptor blocking on hepatocytes. Results similar to those obtained with Kupffer cells were found with conditioned media of myofibroblast-like cells. Northern blot hybridization confirms the expression of both TGF beta and TGF alpha in Kupffer cells and myofibroblast-like cells, respectively. These findings support the notion that Kupffer cells and myofibroblast-like cells might regulate in both directions liver regeneration depending on the proportion of secreted TGF alpha and TGF beta and on the activation status of TGF beta, of which a significant fraction is secreted in the latent form. 相似文献
14.
Biosynthesis of islet amyloid polypeptide. Elevated expression in mouse beta TC3 cells 总被引:2,自引:0,他引:2
Islet amyloid polypeptide (IAPP) messenger RNA levels, biosynthesis, processing, and secretion were studied in cultured mouse beta TC3 insulinoma cells. Northern blot analysis revealed that the size of IAPP mRNA (0.9 kb) in beta TC3 cells was the same as that in normal mouse islets; IAPP mRNA was approximately 60% of the level of insulin mRNA in beta TC3 cells. However, the ratio of synthesis of insulin to IAPP was approximately 6:1, suggesting that IAPP mRNA is not translated efficiently in these cells. Metabolic labeling of beta TC3 cells with [3H]leucine revealed the synthesis of both a precursor form of IAPP (pro-IAPP) of apparent Mr 7400 and a mature form (IAPP) of apparent Mr 3900. In pulse-chase experiments, pro-IAPP could be shown to be processed to IAPP in a manner similar to proinsulin. The t1/2 for conversion of pro-IAPP to IAPP was about 25 min, faster than the t1/2 for proinsulin to insulin of 70 min. A significant proportion of newly synthesized IAPP and insulin precursors were secreted via a constitutive pathway from beta TC3 cells. Possible effects of dexamethasone and forskolin on IAPP mRNA levels and biosynthesis were examined but no effects were observed. In conclusion, the IAPP gene is strongly expressed in beta TC3 cells leading to the biosynthesis, proteolytic processing, and secretion of IAPP, a putative islet hormone. 相似文献
15.
Comparison of proteins synthesized by polarized caprine oviductal epithelial cells and oviductal explants in vitro 总被引:1,自引:0,他引:1
Our objectives were to compare proteins secreted by caprine oviductal explants and oviductal epithelial (OE) cells in vitro. Oviducts were collected from goats on Days 1 (n=5) and 5 (n=5) of the estrous cycle. Radiolabeled secretory proteins from tissue segments and cell cultures were visualized using SDS-PAGE and fluorography. After culture, media from ampulla oviduct segments collected on Days 1 and 5 of the estrous cycle contained an acidic 97 kDa protein, which was greatly reduced in culture medium obtained from infundibulum and isthmus oviduct segments. A complex of low molecular weight proteins (14-26 kDa) could be modulated by estradiol when OE cells were cultured on plastic. This complex was constitutively expressed when OE cells were cultured on Matrigel-coated filters. Polarized OE cells were also capable of compartment-specific secretion of [L-(35)S]-methionine-labeled proteins. A 45 kDa acidic protein was predominantly secreted into the apical compartment while a 66 kDa acidic protein was preferentially localized in the basal compartment. Proteins secreted by OE cells were similar to proteins secreted by tissue segments in vitro. Therefore, under well-defined culture conditions OE cells may be useful in enhancing in vitro fertilization or early embryonic development. 相似文献
16.
Abnormal proteinaceous deposits are found in the brain of patients with many different neurodegenerative diseases. In many of these diseases, the production of the deposits is probably associated with disease pathogenesis. In Alzheimer's disease (AD), the amyloid protein (A beta), is produced by the action of enzymes known as secretases, which cleave the beta-amyloid protein precursor. A beta is secreted from cells in the brain, after which it oligomerizes and is deposited in the extracellular compartment of the brain to form amyloid plaques and amyloid angiopathy. Targeting the production of A beta and its aggregation is now a key strategy in the development of novel therapeutic agents for the treatment of AD. This review examines the potential of immunization strategies, cholesterol-lowering drugs, protease inhibitors and nicotinic drugs for the treatment of AD. 相似文献
17.
Recently we reported that the soluble form of amyloid beta protein (sAβ) in normal human plasma and cerebrospinal fluid is associated with lipoprotein (LP) particles. In this paper we tested the sAβ secretion by cells in association with LP in the model of the human hepatoma HepG2 cell line. These cells secreted sAβ to the culture media and expressed intracellular sAβ immunoreactivity. Soluble Aβ in the cell supernatant was detected in 200–300kDa LP complexes in association with apoA-I, apoJ, transthyrethin and phospholipids, triglycerides and free and esterified cholesterol. This was assessed by size exclusion HPLC, immunoprecipitation with corresponding antibodies and by analysis of sAβ associated metabolically-labeled lipids, respectively. Our results suggest that sAβ to LP association represents a unique mechanism, governing the normal biology of sAβ. 相似文献
18.
Use of surface-enhanced laser desorption ionization-time-of-flight to identify heat shock protein 70 isoforms in closely related species of the virilis group of Drosophila
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Zatsepina OG Karavanov AA Garbuz DG Shilova V Tornatore P Evgen'ev MB 《Cell stress & chaperones》2005,10(1):12-16
The 70-kDa heat shock protein (Hsp) family in all Drosophila species includes 2 environmentally inducible family members, Hsp70 and Hsp68. Two-dimensional gel electrophoresis revealed an unusual pattern of heat shock-inducible proteins in the species of the virilis group. Trypsin fingerprinting and microsequencing of tryptic peptides using ProteinChip Array technology identified the major isoelectric variants of Hsp70 family, including Hsp68 isoforms that differ in both molecular mass and isoelectric point from those in Drosophila melanogaster. The peculiar electrophoretic mobility is consistent with the deduced amino acid sequence of corresponding hsp genes from the species of the virilis group. 相似文献
19.
S L Wagner R S Siegel T S Vedvick W C Raschke W E Van Nostrand 《Biochemical and biophysical research communications》1992,186(2):1138-1145
The protease inhibitor, protease nexin-2 (PN-2), is the secreted isoform of the Alzheimer's amyloid beta-protein precursor (A beta PP) that contains the Kunitz-type protease inhibitor (KPI) domain. Here we describe the use of the methylotrophic industrial yeast Pichia pastoris as a host system for the large scale production of the KPI domain of PN-2/A beta PP. In addition to the 57 amino acid KPI domain, the expression product contained an additional four amino acid residues at its amino terminus that correspond to amino acids 285-288 of A beta PP (Ponte et al. 1988 Nature 311:525-527). This expression system generated yields of greater than 1.0 gram of KPI domain per liter of fermentation media. The secreted 61 amino acid product was purified to homogeneity and biochemically characterized. Amino acid analysis and sequencing of the entire expressed KPI domain verified its integrity. Similar to native PN-2/A beta PP, the purified KPI domain potently inhibited trypsin, chymotrypsin, and coagulation factor XIa. Although heparin augments the inhibition of factor XIa by native PN-2/A beta PP it had no effect on the inhibition of factor XIa by expressed KPI domain suggesting that heparin binds to regions on native PN-2/A beta PP outside of the protease inhibitory domain. This KPI domain expression product should be useful in studying the physiologic and pathophysiologic functions of PN-2/A beta PP. 相似文献
20.
Lefterov IM Koldamova RP Lefterova MI Schwartz DR Lazo JS 《Biochemical and biophysical research communications》2001,283(4):994-999
Human bleomycin hydrolase (hBH) is a neutral cysteine protease that may regulate the secretion of soluble amyloid precursor protein (APP) and amyloid beta (A(beta)), which is a major constituent of the Alzheimer's disease-associated amyloid plaques. We have now determined that APP interacts with hBH by using yeast two hybrid methods and in vitro binding studies revealed that APP interacted with a 68 amino acid region that includes the catalytic domain of hBH. Ectopic expression of hBH increased the secretion of A(beta) but not of a second secreted protein, apolipoprotein A-I. Expression of hBH in which the catalytic cysteine 73 was mutated to serine failed to increase A(beta) secretion. These results indicate a critical role for cysteine 73 of hBH in mediating APP processing. 相似文献