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Histone H4 binds to secreted APP and is toxic to the human monocytic cell line U937
FOURTH INTERNATIONAL CONFERENCE ON ALZHEIMER’S DISEASE
Lieberburg, lun Zbao. Athena Neurosciences, Inc., South San Francisco, California 94080, USA. The factors which regulate p-secretase activity in relation to a-secretase activity are not well understood We have explored the separateeffects of phorbol ester stimulation, and APP ectodomain truncation, on the relative activities of a- and B-secretaseby using transfection of various APP constructs into cultured human 293 cells. The transfection of APP constructs lacking most of the ectodmain (truncated 25 amino-acids upstream from Met-595) released Aj3 into the conditioned medium, and the presence of the “Swedish” double mutation led, as with the full-length APP consfructs,lo an increase in AD release. Treatment of cells with phorbol myristyl acetate (PMA) led to a stimulated release of secreted APP (s-APP) with either wild-type or SW- full-length APP constructs, accompanied by a dccreasc of AP released into the conditioned medium lmmunoprecipitation analysis of the s-APP released in the presence of PMA with antibodies specific to a-s-APP and a-s-APP revealed a dramatic decrease in the P-sAPPla-s-APP ratio, primarily drnvenby a large increase in a-s-APP. Inhibition of AP release in the presenceof PMA was also seen with the ectodomain-truncated constructs, apparently also accompanied by a stimulation of a-secretase cleavage. Thus, very similar regulation of a-secretase activity occurs with either full-length APP or constructs lacking the bulk of the ectodomain, suggesting that the signal(s) mediating the phorbol ester stimulation of a-secretase activity probably do not reside in APP ectodomain. Additionally, p-secret% activity is stimulated by the presence of the Swedish mutation, and this phenomenon is again independent of the ectodomain. suggesting an effect probably driven by protease specificity alone. Lastly, unlike the strong positive regulation of a-secretase activity by phorbol esters, little parallel inhibition of bsecretaseactivity is apparent, suggesting that the transient drop in Ap is mediated by other factors.
348 HISTONE H4 BINDS TO SECRETED APP AND IS TOXIC TO THE HUMAN MONOCYTIC CELL LINE U937. J.R. Cur&, M.-C. Chen Hwang, A. Potempska, N. Ramakrishna, H.M. Wisniewski and D.L. Miier. New York State Institute for Basic Research in Developmental Disabilities. Staten Island, New York 10314 USA. In a search for Ugands of APP, Potempska et al. (Arch. Biochem. Biophys. 304: 448,, 1993) found that h&ones bind with highaffinity and specificity to secreted APP751 purified from recombinant baculovirusinfected cells. We have investigated the effects that purified histone H4 has on cells which secrete little APP (U937 cells) compared to those that secrete abundant APP (COS7 cells). The U937 cells were fmt adapted to a serumfree medium using insulin-transferrin-selinium (ITS) as a serum substitute. The COS7 cells were weaned to 2% serum plus ITS. Cells (104) were placed in the wells of a 96 weU plate. H4 was added to achieve final concentrations of 0 lo 8.85 PM in 100 ~1. The cells were allowed to survive for periods up to 24h. CeU viability was measured by 3H-thymidine incorporation and the alamar Blue assay. Generally each experiment was done in triplicate and repeated at least three times. The results indicate that a dose of about 1.5 fiM H4 kills half of the U937 ceUs by 4h. Cells swelling and death can be observed within 5 min. at the higher doses. Doses of 7 fiM H4 and above kiU aU the U937 cells. Doses below 1 MMhave little effect on the survival of the cells. Addition of secreted APP at 24 pg/mI partially protects the cells. COS7 cells are more resistant to the toxic effects of H4. The LDSO forCOS7 cells is about 3.5 PM H4. Very little effect is seen at 2 j&I or below. While 7 PM kills all the U937 ceUs, about 30% of the COS7 cells are still alive at this concentration. CeUswhich make less APP (U937) are more susceptible to the toxic effects of H4 than cells which make 10X as much (COS7). Histone H4 and derived peptides have multiple effects on cells such as increased glucose transport and oxidation, increased protein phosphotylation, osteogenic and opioid activity, and binding to hormone receptors. Soluble H4 has been isolated from milk and serum. Interestingly, serum amyloid P and q-macroglobulin, two other serum proteins involved with amyloidosls, also bind to histones. We are investigating the mechanism of cell-killing exhibited by H4. We suspect that it will involve H4 interactions at the ceU surface since its effects are so rapid. Whether it binds to full-length cell-surface APP remains to be determined. Supported by NYSOMRDD and NIH AG04220.
349 APP BINDS WITH HIGH AFFINITY TO A SPECIFIC HEPARAN SULPHATE PROTEOGLYCANAND STIMULATES NEURlTE OUTGROWTH. T.G. Williamson, D.H. Small, V. Nurcombe+, K. Beyreuthefi and C.L. Masters. Department of Pathology and +Department of Anatomy and Cell Biology, University of Melbourne, Parkville Victoria, Australia 3052; #Center for Molecular Biology, Heidelberg University, Germany.
S85
The amyloid protein precursor (APP) has been implicated in the modulation of neurite outgrowth through an interaction with substratum-bound heparan sulphate proteoglycans (HSPG) (Small et al., 1992). To investigate the relative affinities of HSPGs for binding APP, an affinity column was made using a peptide homologous to a heparinbinding domain of APP (residues 96 - 110) . Conditioned medium from dissociated cultures of post-natal day three mouse brain was applied to the column. Using this approach, a proteoglycan was purified from the conditioned medium. APP bound with high affinity (K,t = 0.6 nM) to the purified HSPG. APP was found to promote neurite-outgrowth with the purified HSPG as a cofactor. These results suggest that a specific HSPG binds APP and modulates the neurotrophic activity of APP. A change in the binding of APP to a specific HSPG may play a role in the pathogenesis of Alzheimer’s disease.
350 A CHOLERA TOXIN SENSITIVE PROTEIN UODULATES PHORBOL CLEAVAGE AND SECRETION OF ESTER REGULATED THE ANYMID PRECURSOR. ALZHEINER'S S.EFTHIMIOPOULQS, N.K.ROBAUIS, L.M.REPOLo,. Dept. Psychiatry, Fishberg Res. Cntr. Neurobiol., Mount Sinai Med.Cntr.,NY,NY 10029 Phorbol esters regulate the cleavage and secretion of the Alzheimer's Amyloid Precursor Protein (APP). The mechanism of the phorbol ester effect is unknown. Furthermore, there is no evidence that the phorbol ester effect on APP processing iS due to a change in the phosphorylation of APP. Therefore, we initiated a study to further elucidate the mechanism of phorbol ester action on APP processing. Recently, we have data (S.Efthimiopoulos et al., 1994; obtained J.Neurosc. Res.in press.) suggesting the involvement of a cholera toxin sensitive protein in the phorbol ester One of the activities of cholera toxin is response. the ribosylation and activation of the G:a subunit of heterotrimeric G-proteins. Cholera toxin, but not pertussis toxin inhibited the phorbol ester effect by more than two fold. Neither of these toxins had any affect on the constitutive cleavage and secretion of
APP. Furthermore, cholera toxin had no affect on the intracellular levels of APP. These data suggest that a cholera toxin sensitive protein modulates the phorbol ester effect on APP processing. We will discuss the G-proteins and the involvement of heterotrimeric components of the signal transduction pathway which interact with the putative cholera toxin sensitive Gprotein, including cANP and phospholipases A,C and D. suggest that heterotrimeric GRecent reports regulate protein proteins trafficking. Moreover, alterations in G-protein levels have been reported in AD. Further studies may clarify the role of G-proteins in APP processing and provide a connection regarding alterations of G-protein levels in the AD brain and amyloidogenic processing of APP .
351 DETECTION AND CHARACTERIZATION OF A CELLULAR PROTEIN WHICH BINDS TO THE CYTOPLASMIC DOMAIN OF APP. I.P. Shapiro, G.H. Murdoch. Department of Pathology, L-113, Oregon Health Sciences University. 3181 SW Sam Jackson Park Rd., Portland, OR 97201 USA.
Our goal was to identify cellular proteins that interact with and mediate the effects of the APP cytoplasmic domain (APPc). Evidence in the literature suggests that APPc may be involved in the processing, or/and subcellular localization, or/and function of APP, since: I) truncations or mutations of APPc reportedly change the levels of secretion of amyloid p-peptide (AP)and APPso (soluble secret&se product) and the N-terminal sequence of Ap; 2) APP has heen shown to interact with the cytoskeleton, presumahly via the APP cytoplasmic domain;and 3) there are analogous functions of the cytoplasmic domains of other transmemhrane proteins and interacting cellular proteins. Also, by analogy with other transmembrane proteins, more than one “signal” or functional domain may be. present in APPc, in addition to the “NPTY” clathrin-mediated mtemalization signal.
Lieberburg, lun Zbao. Athena Neurosciences, Inc., South San Francisco, California 94080, USA. The factors which regulate p-secretase activity in relation to a-secretase activity are not well understood We have explored the separateeffects of phorbol ester stimulation, and APP ectodomain truncation, on the relative activities of a- and B-secretaseby using transfection of various APP constructs into cultured human 293 cells. The transfection of APP constructs lacking most of the ectodmain (truncated 25 amino-acids upstream from Met-595) released Aj3 into the conditioned medium, and the presence of the “Swedish” double mutation led, as with the full-length APP consfructs,lo an increase in AD release. Treatment of cells with phorbol myristyl acetate (PMA) led to a stimulated release of secreted APP (s-APP) with either wild-type or SW- full-length APP constructs, accompanied by a dccreasc of AP released into the conditioned medium lmmunoprecipitation analysis of the s-APP released in the presence of PMA with antibodies specific to a-s-APP and a-s-APP revealed a dramatic decrease in the P-sAPPla-s-APP ratio, primarily drnvenby a large increase in a-s-APP. Inhibition of AP release in the presenceof PMA was also seen with the ectodomain-truncated constructs, apparently also accompanied by a stimulation of a-secretase cleavage. Thus, very similar regulation of a-secretase activity occurs with either full-length APP or constructs lacking the bulk of the ectodomain, suggesting that the signal(s) mediating the phorbol ester stimulation of a-secretase activity probably do not reside in APP ectodomain. Additionally, p-secret% activity is stimulated by the presence of the Swedish mutation, and this phenomenon is again independent of the ectodomain. suggesting an effect probably driven by protease specificity alone. Lastly, unlike the strong positive regulation of a-secretase activity by phorbol esters, little parallel inhibition of bsecretaseactivity is apparent, suggesting that the transient drop in Ap is mediated by other factors.
348 HISTONE H4 BINDS TO SECRETED APP AND IS TOXIC TO THE HUMAN MONOCYTIC CELL LINE U937. J.R. Cur&, M.-C. Chen Hwang, A. Potempska, N. Ramakrishna, H.M. Wisniewski and D.L. Miier. New York State Institute for Basic Research in Developmental Disabilities. Staten Island, New York 10314 USA. In a search for Ugands of APP, Potempska et al. (Arch. Biochem. Biophys. 304: 448,, 1993) found that h&ones bind with highaffinity and specificity to secreted APP751 purified from recombinant baculovirusinfected cells. We have investigated the effects that purified histone H4 has on cells which secrete little APP (U937 cells) compared to those that secrete abundant APP (COS7 cells). The U937 cells were fmt adapted to a serumfree medium using insulin-transferrin-selinium (ITS) as a serum substitute. The COS7 cells were weaned to 2% serum plus ITS. Cells (104) were placed in the wells of a 96 weU plate. H4 was added to achieve final concentrations of 0 lo 8.85 PM in 100 ~1. The cells were allowed to survive for periods up to 24h. CeU viability was measured by 3H-thymidine incorporation and the alamar Blue assay. Generally each experiment was done in triplicate and repeated at least three times. The results indicate that a dose of about 1.5 fiM H4 kills half of the U937 ceUs by 4h. Cells swelling and death can be observed within 5 min. at the higher doses. Doses of 7 fiM H4 and above kiU aU the U937 cells. Doses below 1 MMhave little effect on the survival of the cells. Addition of secreted APP at 24 pg/mI partially protects the cells. COS7 cells are more resistant to the toxic effects of H4. The LDSO forCOS7 cells is about 3.5 PM H4. Very little effect is seen at 2 j&I or below. While 7 PM kills all the U937 ceUs, about 30% of the COS7 cells are still alive at this concentration. CeUswhich make less APP (U937) are more susceptible to the toxic effects of H4 than cells which make 10X as much (COS7). Histone H4 and derived peptides have multiple effects on cells such as increased glucose transport and oxidation, increased protein phosphotylation, osteogenic and opioid activity, and binding to hormone receptors. Soluble H4 has been isolated from milk and serum. Interestingly, serum amyloid P and q-macroglobulin, two other serum proteins involved with amyloidosls, also bind to histones. We are investigating the mechanism of cell-killing exhibited by H4. We suspect that it will involve H4 interactions at the ceU surface since its effects are so rapid. Whether it binds to full-length cell-surface APP remains to be determined. Supported by NYSOMRDD and NIH AG04220.
349 APP BINDS WITH HIGH AFFINITY TO A SPECIFIC HEPARAN SULPHATE PROTEOGLYCANAND STIMULATES NEURlTE OUTGROWTH. T.G. Williamson, D.H. Small, V. Nurcombe+, K. Beyreuthefi and C.L. Masters. Department of Pathology and +Department of Anatomy and Cell Biology, University of Melbourne, Parkville Victoria, Australia 3052; #Center for Molecular Biology, Heidelberg University, Germany.
S85
The amyloid protein precursor (APP) has been implicated in the modulation of neurite outgrowth through an interaction with substratum-bound heparan sulphate proteoglycans (HSPG) (Small et al., 1992). To investigate the relative affinities of HSPGs for binding APP, an affinity column was made using a peptide homologous to a heparinbinding domain of APP (residues 96 - 110) . Conditioned medium from dissociated cultures of post-natal day three mouse brain was applied to the column. Using this approach, a proteoglycan was purified from the conditioned medium. APP bound with high affinity (K,t = 0.6 nM) to the purified HSPG. APP was found to promote neurite-outgrowth with the purified HSPG as a cofactor. These results suggest that a specific HSPG binds APP and modulates the neurotrophic activity of APP. A change in the binding of APP to a specific HSPG may play a role in the pathogenesis of Alzheimer’s disease.
350 A CHOLERA TOXIN SENSITIVE PROTEIN UODULATES PHORBOL CLEAVAGE AND SECRETION OF ESTER REGULATED THE ANYMID PRECURSOR. ALZHEINER'S S.EFTHIMIOPOULQS, N.K.ROBAUIS, L.M.REPOLo,. Dept. Psychiatry, Fishberg Res. Cntr. Neurobiol., Mount Sinai Med.Cntr.,NY,NY 10029 Phorbol esters regulate the cleavage and secretion of the Alzheimer's Amyloid Precursor Protein (APP). The mechanism of the phorbol ester effect is unknown. Furthermore, there is no evidence that the phorbol ester effect on APP processing iS due to a change in the phosphorylation of APP. Therefore, we initiated a study to further elucidate the mechanism of phorbol ester action on APP processing. Recently, we have data (S.Efthimiopoulos et al., 1994; obtained J.Neurosc. Res.in press.) suggesting the involvement of a cholera toxin sensitive protein in the phorbol ester One of the activities of cholera toxin is response. the ribosylation and activation of the G:a subunit of heterotrimeric G-proteins. Cholera toxin, but not pertussis toxin inhibited the phorbol ester effect by more than two fold. Neither of these toxins had any affect on the constitutive cleavage and secretion of
APP. Furthermore, cholera toxin had no affect on the intracellular levels of APP. These data suggest that a cholera toxin sensitive protein modulates the phorbol ester effect on APP processing. We will discuss the G-proteins and the involvement of heterotrimeric components of the signal transduction pathway which interact with the putative cholera toxin sensitive Gprotein, including cANP and phospholipases A,C and D. suggest that heterotrimeric GRecent reports regulate protein proteins trafficking. Moreover, alterations in G-protein levels have been reported in AD. Further studies may clarify the role of G-proteins in APP processing and provide a connection regarding alterations of G-protein levels in the AD brain and amyloidogenic processing of APP .
351 DETECTION AND CHARACTERIZATION OF A CELLULAR PROTEIN WHICH BINDS TO THE CYTOPLASMIC DOMAIN OF APP. I.P. Shapiro, G.H. Murdoch. Department of Pathology, L-113, Oregon Health Sciences University. 3181 SW Sam Jackson Park Rd., Portland, OR 97201 USA.
Our goal was to identify cellular proteins that interact with and mediate the effects of the APP cytoplasmic domain (APPc). Evidence in the literature suggests that APPc may be involved in the processing, or/and subcellular localization, or/and function of APP, since: I) truncations or mutations of APPc reportedly change the levels of secretion of amyloid p-peptide (AP)and APPso (soluble secret&se product) and the N-terminal sequence of Ap; 2) APP has heen shown to interact with the cytoskeleton, presumahly via the APP cytoplasmic domain;and 3) there are analogous functions of the cytoplasmic domains of other transmemhrane proteins and interacting cellular proteins. Also, by analogy with other transmembrane proteins, more than one “signal” or functional domain may be. present in APPc, in addition to the “NPTY” clathrin-mediated mtemalization signal.