Potential link between amyloid beta-protein 42 and C-terminal fragment gamma 49-99 of beta-amyloid precursor protein

A novel cleavage of beta-amyloid precursor protein (APP), referred to as epsilon-cleavage, occurs downstream of the gamma-cleavage and generates predominantly a C-terminal fragment (CTFgamma) that begins at Val-50, according to amyloid beta-protein (Abeta) numbering. Whether this cleavage occurs independently of, or is coordinated with, gamma-cleavage is unknown. Using a cell-free system, we show here that, although Abeta40 and CTFgamma 50-99 were the predominant species produced by membranes prepared from cells overexpressing wild-type (wt) APP and wt presenilin (PS) 1 or 2, the production of CTFgamma 49-99, which begins at Leu-49, was remarkably enhanced in membranes from cells overexpressing mutant (mt) APP or mtPS1/2 that increases the production of Abeta42. Furthermore, a gamma-secretase inhibitor, which suppresses Abeta40 production and paradoxically enhances Abeta42 production at low concentrations, caused the proportion of CTFgamma 50-99 to decrease and that of CTFgamma 49-99 to increase significantly. These results strongly suggest a link between the production of Abeta42 and CTFgamma 49-99 and provide an important insight into the mechanisms of altered gamma-cleavage caused by mtAPP and mtPS1/2.     

Presenilin 1 mutations activate gamma 42-secretase but reciprocally inhibit epsilon-secretase cleavage of amyloid precursor protein (APP) and S3-cleavage of notch

The presenilin 1 (PS1) and presenilin 2 (PS2) proteins are necessary for proteolytic cleavage of the amyloid precursor protein (APP) within its transmembrane domain. One of these cleavage events (termed gamma-secretase) generates the C-terminal end of the Abeta-peptide by proteolysis near residue 710 or 712 of APP(770). Another event (termed gamma-like or epsilon-secretase cleavage) cleaves near residue 721 at approximately 2-5 residues inside the cytoplasmic membrane boundary to generate a series of stable, C-terminal APP fragments. This latter cleavage is analogous to S3-cleavage of Notch. We report here that specific mutations in the N terminus, loop, or C terminus of PS1 all increase the production of Abeta(42) but cause inhibition of both epsilon-secretase cleavage of APP and S3-cleavage of Notch. These data support the hypothesis that epsilon-cleavage of APP and S3-cleavage of Notch are similar events. They also argue that, although both the gamma-site and the epsilon-site cleavage of APP are presenilin-dependent, they are likely to be independent catalytic events.     

Cross-linking of bacteriorhodopsin using specific carboxyl modifications and proteolytic cleavage

Specific carboxyl modification of purple membrane using a water-soluble carbodiimide yielded a mixture of oligomers, revealed by gel electrophoresis. Purple membrane pre-treated with papain or trypsin, cleaving the C-terminal tail, showed the same pattern of cross-linked products. Chymotryptic cleavage released amino acids 1-72 (7kD fragment) from the cross-linked products, as it did with native membrane. The tail and helices A and B are not, therefore, involved in carbodiimide-promoted cross-linking. Similar cleavage of a hydrophobic dihydroquinoline-modified sample showed that mainly intra-molecular cross-linking occurs, with little cross-linking between the large and small chymotryptic fragments.     

Caspase cleavage of members of the amyloid precursor family of proteins

The synapse loss and neuronal cell death characteristic of Alzheimer's disease (AD) are believed to result in large part from the neurotoxic effects of beta-amyloid peptide (Abeta), a 40-42 amino acid peptide(s) derived proteolytically from beta-amyloid precursor protein (APP). However, APP is also cleaved intracellularly to generate a second cytotoxic peptide, C31, and this cleavage event occurs in vivo as well as in vitro and preferentially in the brains of AD patients (Lu et al. 2000). Here we show that APPC31 is toxic to neurons in primary culture, and that like APP, the APP family members APLP1 and possibly APLP2 are cleaved by caspases at their C-termini. The carboxy-terminal peptide derived from caspase cleavage of APLP1 shows a degree of neurotoxicity comparable to APPC31. Our results suggest that even though APLP1 and APLP2 cannot generate Abeta, they may potentially contribute to the pathology of AD by generating peptide fragments whose toxicity is comparable to that of APPC31.     

Purification of limited tryptic fragments of Ca2+,Mg2+-adenosine triphosphatase of the sarcoplasmic reticulum and identification of conformation-sensitive cleavage sites

Sarcoplasmic reticulum membranes were treated with trypsin, and samples enriched with A1a, A1b, and C fragments (Saito, K. et al. (1984) J. Biochem. 95, 1297-1304), respectively, were prepared. A1b and C fragments were purified to apparent homogeneity, and an approximately equimolar mixture of A1(Met1-Arg198), A1a, and A1b fragments free from other contaminants was also obtained through gel permeation and hydroxylapatite chromatography in the presence of sodium dodecyl sulfate. N- and C-terminal amino acid sequence analyses of these peptides were carried out in order to identify the tryptic cleavage sites responsible for the formation of these fragments. Both A1a and A1b fragments had the same C-terminal sequence as A1 fragment. Single cleavage of A1 at T3a (Lys218-Ala219) yielded A1a, while a cleavage between either Lys234-Ile235 or Arg236-Asp237 (collectively designated as T3b) resulted in A1b fragment. Thus, A1a and A1b fragments differed from A1 fragment only by their loss of short stretches corresponding to the N-terminal region of the latter. On the other hand, C fragment represented the C-terminal half of B fragment (Ala506-Gly994). It had the same C-terminal sequence as B fragment and was produced by cleavage at T4 (Lys728-Thr729). Cleavages at T3a and T3b profoundly affected the catalytic properties of SR-ATPase (Imamura, Y. and Kawakita, M. (1986) J. Biochem. 100, 133-141), and it was suggested that the segment of the ATPase molecule including the region between Ala199 and Arg236 is important in mediating the coupling between ATP splitting and Ca2+-transport.     

The influence of endoproteolytic processing of familial Alzheimer's disease presenilin 2 on abeta42 amyloid peptide formation

Mutant presenilins (PS) contribute to the pathogenesis of familial Alzheimer's disease (FAD) by enhancing the production of Abeta42 from beta-amyloid precursor protein. Presenilins are endoproteolytically processed to N-terminal and C-terminal fragments, which together form a stable 1:1 complex. We have mapped the cleavage site in the PS2 protein by direct sequencing of its C-terminal fragment isolated from mouse liver. Three different N-terminal residues were identified starting at Val-299, Thr-301, and Leu-307 that correspond closely to the previously described N termini of the C-terminal fragment of human PS1. Mutational analysis of the PS2 cleavage site indicates that the principal endoproteolytic cleavage occurs at residues Met-298/Val-299 and that the N terminus is subsequently modified by secondary proteolytic cleavages. We have generated cleavage defective PS2 constructs, which accumulate exclusively as full-length polypeptides in transfected Neuro2a cells. Functional analysis of such cleavage defective PS2 carrying the FAD mutation Asn-141 --> Ile showed that its Abeta42 producing activity was strongly reduced compared with cleavage-competent FAD PS2. In contrast, cleavage defective PS2 was active in rescuing the egg-laying defect of a sel-12 mutant in Caenorhabditis elegans. We conclude that PS2 endoproteolytic cleavage is not an absolute requirement for its activities but may rather selectively enhance or stabilize its functions.     

Characterization of beta-N-acetylglucosaminidase cleavage by caspase-3 during apoptosis

Beta-O-linked N-acetylglucosamine is a dynamic post-translational modification involved in protein regulation in a manner similar to phosphorylation. Removal of N-acetylglucosamine is regulated by beta-N-acetylglucosaminidase (O-GlcNAcase), which was previously shown to be a substrate of caspase-3 in vitro. Here we show that O-GlcNAcase is cleaved by caspase-3 into two fragments during apoptosis, an N-terminal fragment containing the O-GlcNAcase active site and a C-terminal fragment containing a region with homology to GCN5 histone acetyl-transferases. The caspase-3 cleavage site of O-GlcNAcase, mapped by Edman sequencing, is a noncanonical recognition site that occurs after Asp-413 of the SVVD sequence in human O-GlcNAcase. A point mutation, D413A, abrogates cleavage by caspase-3 both in vitro and in vivo. Finally, we show that O-GlcNAcase activity is not affected by caspase-3 cleavage because the N- and C-terminal O-GlcNAcase fragments remain associated after the cleavage. Furthermore, when co-expressed simultaneously in the same cell, the N-terminal and C-terminal caspase fragments associate to reconstitute O-GlcNAcase enzymatic activity. These studies support the identification of O-GlcNAcase as a caspase-3 substrate with a novel caspase-3 cleavage site and provide insight about O-GlcNAcase regulation during apoptosis.     

Presenilin-1 and -2 are molecular targets for gamma-secretase inhibitors

Presenilins are integral membrane protein involved in the production of amyloid beta-protein. Mutations of the presenilin-1 and -2 gene are associated with familial Alzheimer's disease and are thought to alter gamma-secretase cleavage of the beta-amyloid precursor protein, leading to increased production of longer and more amyloidogenic forms of A beta, the 4-kDa beta-peptide. Here, we show that radiolabeled gamma-secretase inhibitors bind to mammalian cell membranes, and a benzophenone analog specifically photocross-links three major membrane polypeptides. A positive correlation is observed among these compounds for inhibition of cellular A beta formation, inhibition of membrane binding and cross-linking. Immunological techniques establish N- and C-terminal fragments of presenilin-1 as specifically cross-linked polypeptides. Furthermore, binding of gamma-secretase inhibitors to embryonic membranes derived from presenilin-1 knockout embryos is reduced in a gene dose-dependent manner. In addition, C-terminal fragments of presenilin-2 are specifically cross-linked. Taken together, these results indicate that potent and selective gamma-secretase inhibitors block A beta formation by binding to presenilin-1 and -2.     

The presence and in vivo biosynthesis of fragments of CPP (the C-terminal glycopeptide of the rat vasopressin precursor) in the hypothalamo-neurohypophyseal system

The existence and rate of formation of fragments of the 39-residue C-terminal glycopeptide of propressophysin (CPP1-39) was investigated in the hypothalamo-neurohypophyseal system. Newly-prepared antisera to CPP were used to confirm the existence of smaller C-terminal fragments derived from CPP1-39. Radiolabelled fucose was injected into rats in vivo into the area of the supraoptic nucleus, and the labelled peptides formed in the neurohypophysis were examined at various time intervals up to five weeks after the injection. The products derived from the neurohypophyseal hormone precursors were separated by high-performance liquid chromatography. The level of the major immunoreactive C-terminal fragment (CPP22-39) was constant and represented about 5% of the intact CPP1-39 in the neurohypophysis. The appearance of newly-synthesized N-terminal fragment of CPP1-39 occurred only after 3 or 4 days. This fucose labelled fragment increased slowly thereafter until it reached the same level as the CPP C-terminal fragment immunoreactivity between 21 and 28 days after injection. The results suggest that CPP1-39 is extremely stable in the hypothalamo-neurohypophyseal neurons, and that the cleavage at Arg21-Leu22 is a delayed proteolytic event in the magnocellular neurons of the SON.     

Comparative studies of the neurofilament triplet protein peptide mapping by chemical cleavage

Peptide mapping of the three neurofilament protein subunits with apparent mol. weights of 210 kDa, 160 kDa and 70 kDa was performed with two different reagents: CNBr, BNPS-Skatole leading to the cleavage of methionyl and tryptophanyl bonds respectively. With BrCN we obtained two large fragments resistant to the cleavage, with mol. wts of 85 kDa for the 160 kDa and 135 kDa for the 210 kDa neurofilament proteins respectively. These fragments were located on the C-terminal part of the proteins (the tails) and correspond to specific regions responsible for their physiological identity. On the other hand, the cleavage with BNPS-Skatole at the tryptophanyl bonds gave similar patterns. The 210 kDa and 160 kDa neurofilament proteins gave a doublet of high mol. wt resistant to the cleavage, corresponding very likely to the C-terminal part and 4 fragments of mol. wt between 30 and 40 kDa corresponding to the N-terminal part. The neurofilament triplet share a common 30.5 kDa fragment located on the N-terminal part. From these peptide mapping studies, we conclude that the two neurofilament subunit proteins with mol. wts of 160 kDa and 210 kDa are different but related structures and that the CNBr characterized cleavage fragments of mol. wt 85,000 and 135 kDa are suitable polypeptides for sequence and immunological studies of the C-terminal part of these proteins.     

Identification of a heparin binding domain in the N-terminal cleavage site of pro-islet amyloid polypeptide. Implications for islet amyloid formation

Islet amyloid deposits are a characteristic pathologic lesion of the pancreas in type 2 diabetes and are composed primarily of the islet beta cell peptide islet amyloid polypeptide (IAPP or amylin) as well as the basement membrane heparan sulfate proteoglycan perlecan. Impaired processing of the IAPP precursor has been implicated in the mechanism of islet amyloid formation. The N- and C-terminal cleavage sites where pro-IAPP is processed by prohormone convertases contain a series of basic amino acid residues that we hypothesized may interact with heparan sulfate proteoglycans. This possibility was tested using affinity chromatography by applying synthetic fragments of pro-IAPP to heparin-agarose and heparan sulfate-Sepharose. An N-terminal human pro-IAPP fragment (residues 1-30) was retained by both heparin-agarose and heparan sulfate-Sepharose, eluting at 0.18 m NaCl at pH 7.5. Substitution of alanine residues for two basic residues in the N-terminal cleavage site abolished heparin and heparan sulfate binding activity. At pH 5.5, the affinity of the wild-type peptide for heparin/heparan sulfate was increased, implying a role for histidine residues at positions 6 and 28 of pro-IAPP. A C-terminal pro-IAPP fragment (residues 41-67) had no specific affinity for either heparin or heparan sulfate, and the N- or C-terminal fragments had only weak affinity for chondroitin sulfate. These data suggest that monomeric N-terminal human pro-IAPP contains a heparin binding domain that is lost during normal processing of pro-IAPP.     

Caspase cleavage of the Golgi stacking factor GRASP65 is required for Fas/CD95-mediated apoptosis

GRASP65 (Golgi reassembly and stacking protein of 65 KDa) is a cis-Golgi protein with roles in Golgi structure, membrane trafficking and cell signalling. It is cleaved by caspase-3 early in apoptosis, promoting Golgi fragmentation. We now show that cleavage is needed for Fas-mediated apoptosis: expression of caspase-resistant GRASP65 protects cells, whereas expression of membrane proximal caspase-cleaved GRASP65 fragments dramatically sensitises cells. GRASP65 coordinates passage through the Golgi apparatus of proteins containing C-terminal hydrophobic motifs, via its tandem PDZ type ‘GRASP'' domains. Fas/CD95 contains a C-terminal leucine–valine pairing so its trafficking might be coordinated by GRASP65. Mutagenesis of the Fas/CD95 LV motif reduces the number of cells with Golgi-associated Fas/CD95, and generates a receptor that is more effective at inducing apoptosis; however, siRNA-mediated silencing or expression of mutant GRASP65 constructs do not alter the steady state distribution of Fas/CD95. We also find no evidence for a GRASP65–Fas/CD95 interaction at the molecular level. Instead, we find that the C-terminal fragments of GRASP65 produced following caspase cleavage are targeted to mitochondria, and ectopic expression of these sensitises HeLa cells to Fas ligand. Our data suggest that GRASP65 cleavage promotes Fas/CD95-mediated apoptosis via release of C-terminal fragments that act at the mitochondria, and we identify Bcl-X_L as a candidate apoptotic binding partner for GRASP65.     

TRP-ML1 is a lysosomal monovalent cation channel that undergoes proteolytic cleavage

Mutations in the gene MCOLN1 coding for the TRP (transient receptor potential) family ion channel TRP-ML1 lead to the lipid storage disorder mucolipidosis type IV (MLIV). The function and role of TRP-ML1 are not well understood. We report here that TRP-ML1 is a lysosomal monovalent cation channel. Both native and recombinant TRP-ML1 are cleaved resulting in two products. Recombinant TRP-ML1 is detected as the full-length form and as short N- and C-terminal forms, whereas in native cells mainly the cleaved N and C termini are detected. The N- and C-terminal fragments of TRP-ML1 were co-immunoprecipitated from cell lysates and co-eluted from a Ni2+ column. TRP-ML1 undergoes proteolytic cleavage that is inhibited by inhibitors of cathepsin B (CatB) and is altered when TRP-ML1 is expressed in CatB-/- cells. N-terminal sequencing of purified C-terminal fragment of TRP-ML1 expressed in Sf9 cells indicates a cleavage site at Arg200 downward arrow Pro201. Consequently, the conserved R200H mutation changed the cleavage pattern of TRP-ML1. The cleavage inhibited TRP-ML1 channel activity. This work provides the first example of inactivation by cleavage of a TRP channel. The significance of the cleavage to the function of TRP-ML1 is under investigation.     

Glu11 site cleavage and N-terminally truncated A beta production upon BACE overexpression

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.     

Inhibition of MEPE cleavage by Phex

X-linked hypophosphatemia (XLH) and the Hyp-mouse disease homolog are caused by inactivating mutations of Phex which results in the local accumulation of an unknown autocrine/paracrine factor in bone that inhibits mineralization of extracellular matrix. In these studies, we evaluated whether the matrix phosphoglycoprotein MEPE, which is increased in calvaria from Hyp mice, is a substrate for Phex. Using recombinant full-length Phex (rPhexWT) produced in Sf9 cells, we failed to observe Phex-dependent hydrolysis of recombinant human MEPE (rMEPE). Rather, we found that rPhex-WT inhibited cleavage of rMEPE by endogenous cathepsin-like enzyme activity present in Sf9 membrane. Sf9 membranes as well as purified cathepsin B cleaved MEPE into two major fragments of approximately 50 and approximately 42kDa. rPhexWT protein in Sf9 membrane fractions, co-incubation of rPhexWT and cathepsin B, and pre-treatment of Sf9 membranes with leupeptin prevented the hydrolysis of MEPE in vitro. The C-terminal domain of Phex was required for inhibition of MEPE cleavage, since the C-terminal deletion mutant rPhex (1-433) [rPhex3(')M] failed to inhibit Sf9-dependent metabolism of MEPE. Phex-dependent inhibition of MEPE degradation, however, did not require Phex enzymatic activity, since EDTA, an inhibitor of rPhex, failed to block rPhexWT inhibition of MEPE cleavage by Sf9 membranes. Since we were unable to identify interactions of Phex with MEPE or actions of Phex to metabolize cathepsin B, Phex may be acting to interfere with the actions of other enzymes that degrade extracellular matrix proteins. Although the molecular mechanism and biological relevance of non-enzymatic actions of Phex need to be established, these findings indicate that MEPE may be involved in the pathogenesis defective mineralization due to Phex deficiency in XLH and the Hyp-mouse.     

A 39 kDa fragment of endogenous ASK1 suggests specific cleavage not degradation by the proteasome

Transfected human apoptosis signal-regulating kinase 1 (ASK1) produces a 150 kDa protein. However, we have detected endogenous ASK1 predominantly as 39 and 50 kDa C-terminal and 75 and 110 kDa N-terminal fragments in a panel of nontransfected cancer cell lines and HUVEC endothelial cells. This suggests that in nonapoptotic cells, endogenous ASK1 protein is normally cleaved at a number of specific sites, some of which are in the kinase domain. Transfected ASK1 protein is known to be degraded by the proteasome. In contrast, the cleavage of endogenous ASK1 is independent of the proteasome as treatment with the proteasome inhibitor, lactacystin did not inhibit cleavage. Cisplatin treatment decreased the amount of 39 kDa C-terminal ASK1 fragments in mutant p53 cell lines suggesting a decrease in cleavage associated with apoptosis. Transfected ASK1 may, therefore, not accurately reflect the role of endogenous ASK1.     

Genetic analyses of processing involving C-terminal cleavage in penicillin-binding protein 3 of Escherichia coli.

The processing of Escherichia coli penicillin-binding protein 3 (PBP 3) was investigated by gene manipulation for producing hybrid and truncated PBP 3 molecules. The hybrid PBP 3 was processed when the N-terminal 40 residues of PBP 3 were replaced by the murein lipoprotein signal peptide which lacked the cysteine residue for processing and followed by seven extra linker residues. In contrast, the PBP 3 molecules truncated at Thr-560 (28-residue deletion) or at Thr-497 (91-residue deletion) were not processed, and those truncated at Phe-576 (12-residue deletion) were processed at a greatly reduced rate. The results indicate that the C-terminal part, rather than the N-terminal part, is involved in the processing. This was supported by the result that the purified mature PBP 3 retained the complete N-terminal sequence with Met for translation initiation. The cleavage at the C-terminal region was shown by the loss of [35S]cysteine label when the cysteine-free hybrid PBP 3 joined to a cysteine-rich extra peptide tail was processed into the mature form. Confirmative assays for processing of PBP 3 were aided by a newly found prc mutant, defective in the processing involving the C-terminal region. A plasmid that directs PBP 3 truncated at Thr-560 complemented a thermosensitive PBP 3 mutation, but the truncated product was unstable in vivo. This suggests the importance of C-terminal hydrophobic regions that terminate at Leu-558 to PBP 3 functioning and the requirement of further-distal peptides for the stability of PBP 3.     

In the uncoupling protein from brown adipose tissue the C-terminus protrudes to the c-side of the membrane as shown by tryptic cleavage

Limited proteolytic digestion of the uncoupling protein (UCP) with trypsin yielded a cleavage product only about 2 kDa smaller than the original UCP (33 kDa). This cleavage can be obtained with the solubilized isolated protein detergent micelle as well as in original brown adipose mitochondria. The cleavage site is identified by C-terminal sequence to be located near the C-terminus at lysine 292. This C-terminus, a 10 residue long peptide, is strongly hydrophilic and can be expected to be localized outside the membrane. In UCP this C-terminal stretch represents a structural difference to the similarly folded ADP/ATP carrier which does not form a corresponding cleavage product. Comparison of tryptic cleavage of UCP in mitochondria with differently broken outer membrane, in sonic particles of mitochondria, as well as in UCP proteoliposomes, indicate that the C-terminus is directed versus the cytosolic site of the membrane. Because of the easy susceptibility to trypsin, the cleavage site must be surface-exposed and the C-terminal section unusually mobile.