Two amyloid precursor protein transgenic mouse models with Alzheimer disease-like pathology

Mutations in the amyloid precursor protein (APP) gene cause early-onset familial Alzheimer disease (AD) by affecting the formation of the amyloid β (Aβ) peptide, the major constituent of AD plaques. We expressed human APP751 containing these mutations in the brains of transgenic mice. Two transgenic mouse lines develop pathological features reminiscent of AD. The degree of pathology depends on expression levels and specific mutations. A 2-fold overexpression of human APP with the Swedish double mutation at positions 670/671 combined with the V717I mutation causes Aβ deposition in neocortex and hippocampus of 18-month-old transgenic mice. The deposits are mostly of the diffuse type; however, some congophilic plaques can be detected. In mice with 7-fold overexpression of human APP harboring the Swedish mutation alone, typical plaques appear at 6 months, which increase with age and are Congo Red-positive at first detection. These congophilic plaques are accompanied by neuritic changes and dystrophic cholinergic fibers. Furthermore, inflammatory processes indicated by a massive glial reaction are apparent. Most notably, plaques are immunoreactive for hyperphosphorylated tau, reminiscent of early tau pathology. The immunoreactivity is exclusively found in congophilic senile plaques of both lines. In the higher expressing line, elevated tau phosphorylation can be demonstrated biochemically in 6-month-old animals and increases with age. These mice resemble major features of AD pathology and suggest a central role of Aβ in the pathogenesis of the disease.

Inhibition of tumorigenicity of the teratoma PC cell line by transfection with antisense cDNA for PC cell-derived growth factor (PCDGF, epithelin/granulin precursor)

The PC cell line is a highly tumorigenic, insulin-independent, teratoma-derived cell line isolated from the nontumorigenic, insulin-dependent 1246 cell line. Studies of the PC cell growth properties have led to the purification of an 88-kDa secreted glycoprotein called PC cell-derived growth factor (PCDGF), which has been shown to stimulate the growth of PC cells as well as 3T3 fibroblasts. Sequencing of PCDGF cDNA demonstrated its identity to the precursor of a family of 6-kDa double-cysteine-rich polypeptides called epithelins or granulins (epithelin/granulin precursor). Since PCDGF was isolated from highly tumorigenic cells, its level of expression was examined in PC cells as well as in nontumorigenic and moderately tumorigenic cells from which PC cells were derived. Northern blot and Western blot analyses indicate that the levels of PCDGF mRNA and protein were very low in the nontumorigenic cells and increased in tumorigenic cell lines in a positive correlation with their tumorigenic properties. Experiments were performed to determine whether the autocrine production of PCDGF was involved in the tumorigenicity of PC cells. For this purpose, we examined the in vivo growth properties in syngeneic C3H mice of PC cells where PCDGF expression had been inhibited by transfection of antisense PCDGF cDNA. The results show that inhibition of PCDGF expression resulted in a dramatic inhibition of tumorigenicity of the transfected cells when compared with empty-vector control cells. These data demonstrate the importance in tumor formation of overexpression of the novel growth factor PCDGF.

PAT1, a microtubule-interacting protein, recognizes the basolateral sorting signal of amyloid precursor protein

In epithelial cells, sorting of membrane proteins to the basolateral surface depends on the presence of a basolateral sorting signal (BaSS) in their cytoplasmic domain. Amyloid precursor protein (APP), a basolateral protein implicated in the pathogenesis of Alzheimer’s disease, contains a tyrosine-based BaSS, and mutation of the tyrosine residue results in nonpolarized transport of APP. Here we report identification of a protein, termed PAT1 (protein interacting with APP tail 1), that interacts with the APP-BaSS but binds poorly when the critical tyrosine is mutated and does not bind the tyrosine-based endocytic signal of APP. PAT1 shows homology to kinesin light chain, which is a component of the plus-end directed microtubule-based motor involved in transporting membrane proteins to the basolateral surface. PAT1, a cytoplasmic protein, associates with membranes, cofractionates with APP-containing vesicles, and binds microtubules in a nucleotide-sensitive manner. Cotransfection of PAT1 with a reporter protein shows that PAT1 is functionally linked with intracellular transport of APP. We propose that PAT1 is involved in the translocation of APP along microtubules toward the cell surface.

Specific intercellular binding of the β-amyloid precursor protein to the presenilins induces intercellular signaling: Its significance for Alzheimer’s disease

Genetic evidence has implicated three proteins, the β-amyloid precursor protein (β-APP) and the two homologous presenilins (PS-1 and PS-2), in the etiology of Alzheimer’s disease (AD). How these three proteins jointly contribute to AD, however, is not clear. Nor is any of their normal physiological functions known. Herein, we demonstrate, confirming a prediction made earlier, that β-APP and either PS-1 or PS-2 act as a specific membrane-bound ligand binding intercellularly with either of its two membrane receptors. This results in a cell–cell adhesion, after which rapid transient increases in protein tyrosine kinase activity and protein tyrosine phosphorylation occur coordinately inside one or both of the two adherent cells. The spectrum of proteins modified by tyrosine phosphorylation differs depending on whether PS-1 or PS-2 is involved in the specific intercellular binding to β-APP, which implies that PS-1 and PS-2 have distinct, rather than redundant, functions in normal physiology. The relevance of this intercellular interaction and signaling process to AD is discussed.

Three small nucleolar RNAs that are involved in ribosomal RNA precursor processing

Three small nucleolar RNAs (snoRNAs), E1, E2 and E3, have been described that have unique sequences and interact directly with unique segments of pre-rRNA in vivo. In this report, injection of antisense oligodeoxynucleotides into Xenopus laevis oocytes was used to target the specific degradation of these snoRNAs. Specific disruptions of pre-rRNA processing were then observed, which were reversed by injection of the corresponding in vitro-synthesized snoRNA. Degradation of each of these three snoRNAs produced a unique rRNA maturation phenotype. E1 RNA depletion shut down 18 rRNA formation, without overaccumulation of 20S pre-rRNA. After E2 RNA degradation, production of 18S rRNA and 36S pre-rRNA stopped, and 38S pre-rRNA accumulated, without overaccumulation of 20S pre-rRNA. E3 RNA depletion induced the accumulation of 36S pre-rRNA. This suggests that each of these snoRNAs plays a different role in pre-rRNA processing and indicates that E1 and E2 RNAs are essential for 18S rRNA formation. The available data support the proposal that these snoRNAs are at least involved in pre-rRNA processing at the following pre-rRNA cleavage sites: E1 at the 5′ end and E2 at the 3′ end of 18S rRNA, and E3 at or near the 5′ end of 5.8S rRNA.

Molecular cloning, sequence, expression, and processing of the interleukin 16 precursor

Interleukin 16 (IL-16) has been shown to function as chemoattractant factor, as a modulator of T-cell activation, and as an inhibitor of immunodeficiency virus replication. The recent identification of inconsistencies in published IL-16 cDNA nucleotide sequences led to the proposal that IL-16 is synthesized in the form of a large precursor protein (pro-IL-16). To identify the true transcriptional start of the IL-16 mRNA rapid amplification of cDNA ends methods were applied. The complete pro-IL-16 cDNA was subsequently molecularly cloned, sequenced, and expressed in COS-7 cells. We report here that pro-IL-16 is most likely synthesized as a 67-kDa protein and is encoded from a major 2.6-kb transcript. Recombinant pro-IL-16 polypeptides are specifically cleaved in lysates of CD8(+) cells, suggesting that the naturally secreted bioactive form of IL-16 is smaller than the originally published 130 amino acids fragment. Moreover, in contrast to other interleukins such as IL-15, IL-16 mRNA expression is almost exclusively limited to lymphatic tissues underlining the potential of IL-16 as an immune regulatory molecule.

Stimulation of amyloid precursor protein synthesis by adrenergic receptors coupled to cAMP formation

Amyloid plaques in Alzheimer disease are primarily aggregates of Aβ peptides that are derived from the amyloid precursor protein (APP). Neurotransmitter agonists that activate phosphatidylinositol hydrolysis and protein kinase C stimulate APP processing and generate soluble, non-amyloidogenic APP (APPs). Elevations in cAMP oppose this stimulatory effect and lead to the accumulation of cell-associated APP holoprotein containing amyloidogenic Aβ peptides. We now report that cAMP signaling can also increase cellular levels of APP holoprotein by stimulating APP gene expression in astrocytes. Treatment of astrocytes with norepinephrine or isoproterenol for 24 h increased both APP mRNA and holoprotein levels, and these increases were blocked by the β-adrenergic antagonist propranolol. Treatment with 8-bromo-adenosine 3′,5′-cyclic monophosphate or forskolin for 24 h similarly increased APP holoprotein levels; astrocytes were also transformed into process-bearing cells expressing increased amounts of glial fibrillary acidic protein, suggesting that these cells resemble reactive astrocytes. The increases in APP mRNA and holoprotein in astrocytes caused by cAMP stimulation were inhibited by the immunosuppressant cyclosporin A. Our study suggests that APP overexpression by reactive astrocytes during neuronal injury may contribute to Alzheimer disease neuropathology, and that immunosuppressants can inhibit cAMP activation of APP gene transcription.

Chemical synthesis of the precursor molecule of the Aequorea green fluorescent protein, subsequent folding, and development of fluorescence

The present paper describes the total chemical synthesis of the precursor molecule of the Aequorea green fluorescent protein (GFP). The molecule is made up of 238 amino acid residues in a single polypeptide chain and is nonfluorescent. To carry out the synthesis, a procedure, first described in 1981 for the synthesis of complex peptides, was used. The procedure is based on performing segment condensation reactions in solution while providing maximum protection to the segment. The effectiveness of the procedure has been demonstrated by the synthesis of various biologically active peptides and small proteins, such as human angiogenin, a 123-residue protein analogue of ribonuclease A, human midkine, a 121-residue protein, and pleiotrophin, a 136-residue protein analogue of midkine. The GFP precursor molecule was synthesized from 26 fully protected segments in solution, and the final 238-residue peptide was treated with anhydrous hydrogen fluoride to obtain the precursor molecule of GFP containing two Cys(acetamidomethyl) residues. After removal of the acetamidomethyl groups, the product was dissolved in 0.1 M Tris⋅HCl buffer (pH 8.0) in the presence of DTT. After several hours at room temperature, the solution began to emit a green fluorescence (λmax = 509 nm) under near-UV light. Both fluorescence excitation and fluorescence emission spectra were measured and were found to have the same shape and maxima as those reported for native GFP. The present results demonstrate the utility of the segment condensation procedure in synthesizing large protein molecules such as GFP. The result also provides evidence that the formation of the chromophore in GFP is not dependent on any external cofactor.

Transgenic Drosophila expressing human amyloid precursor protein show γ-secretase activity and a blistered-wing phenotype

The importance of the amyloid precursor protein (APP) in the pathogenesis of Alzheimer’s disease (AD) became apparent through the identification of distinct mutations in the APP gene, causing early onset familial AD with the accumulation of a 4-kDa peptide fragment (βA4) in amyloid plaques and vascular deposits. However, the physiological role of APP is still unclear. In this work, Drosophila melanogaster is used as a model system to analyze the function of APP by expressing wild-type and various mutant forms of human APP in fly tissue culture cells as well as in transgenic fly lines. After expression of full-length APP forms, secretion of APP but not of βA4 was observed in both systems. By using SPA4CT, a short APP form in which the signal peptide was fused directly to the βA4 region, transmembrane domain, and cytoplasmic tail, we observed βA4 release in flies and fly-tissue culture cells. Consequently, we showed a γ-secretase activity in flies. Interestingly, transgenic flies expressing full-length forms of APP have a blistered-wing phenotype. As the wing is composed of interacting dorsal and ventral epithelial cell layers, this phenotype suggests that human APP expression interferes with cell adhesion/signaling pathways in Drosophila, independently of βA4 generation.

Protein C-Mannosylation Is Enzyme-catalysed and Uses Dolichyl-Phosphate-Mannose as a Precursor

C-mannosylation of Trp-7 in human ribonuclease 2 (RNase 2) is a novel kind of protein glycosylation that differs fundamentally from N- and O-glycosylation in the protein-sugar linkage. Previously, we established that the specificity determinant of the acceptor substrate (RNase 2) consists of the sequence W-x-x-W, where the first Trp becomes C-mannosylated. Here we investigated the reaction with respect to the mannosyl donor and the involvement of a glycosyltransferase. C-mannosylation of Trp-7 was reduced 10-fold in CHO (Chinese hamster ovary) Lec15 cells, which are deficient in dolichyl-phosphate-mannose (Dol-P-Man) synthase activity, compared with wild-type cells. This was not a result of a decrease in C-mannosyltransferase activity. Rat liver microsomes were used to C-mannosylate the N-terminal dodecapeptide from RNase 2 in vitro, with Dol-P-Man as the donor. This microsomal transferase activity was destroyed by heat and protease treatment, and displayed the same acceptor substrate specificity as the in vivo reaction studied previously. The C-C linkage between the indole and the mannosyl moiety was demonstrated by tandem electrospray mass spectrometry analysis of the product. GDP-Man, in the presence of Dol-P, functioned as a precursor in vitro with membranes from wild-type but not CHO Lec15 cells. In contrast, with Dol-P-Man both membrane preparations were equally active. It is concluded that a microsomal transferase catalyses C-mannosylation of Trp-7, and that the minimal biosynthetic pathway can be defined as: Man –> –> GDP-Man –> Dol-P-Man –> (C2-Man-)Trp.

Sec61p Serves Multiple Roles in Secretory Precursor Binding and Translocation into the Endoplasmic Reticulum Membrane

The evolutionarily conserved Sec61 protein complex mediates the translocation of secretory proteins into the endoplasmic reticulum. To investigate the role of Sec61p, which is the main subunit of this complex, we generated recessive, cold-sensitive alleles of sec61 that encode stably expressed proteins with strong defects in translocation. The stage at which posttranslational translocation was blocked was probed by chemical crosslinking of radiolabeled secretory precursors added to membranes isolated from wild-type and mutant strains. Two classes of sec61 mutants were distinguished. The first class of mutants was defective in preprotein docking onto a receptor site of the translocon that included Sec61p itself. The second class of mutants allowed docking of precursors onto the translocon but was defective in the ATP-dependent release of precursors from this site that in wild-type membranes leads to pore insertion and full translocation. Only mutants of the second class were partially suppressed by overexpression of SEC63, which encodes a subunit of the Sec61 holoenzyme complex responsible for positioning Kar2p (yeast BiP) at the translocation channel. These mutants thus define two early stages of translocation that require SEC61 function before precursor protein transfer across the endoplasmic reticulum membrane.

In vitro and in vivo differentiation into B cells, T cells, and myeloid cells of primitive yolk sac hematopoietic precursor cells expanded >100-fold by coculture with a clonal yolk sac endothelial cell line

The yolk sac, first site of hematopoiesis during mammalian development, contains not only hematopoietic stem cells but also the earliest precursors of endothelial cells. We have previously shown that a nonadherent yolk sac cell population (WGA+, density <1.077, AA4.1+) can give rise to B cells, T cells, and myeloid cells both in vitro and in vivo. We now report on the ability of a yolk sac-derived cloned endothelial cell line (C166) to provide a suitable microenvironment for expansion of these early precursor cells. Single day 10 embryonic mouse yolk sac hematopoietic stem cells were expanded >100 fold within 8 days by coculture with irradiated C166 cells. Colony-forming ability was retained for at least three passages in vitro, with retention of the ability to differentiate into T-cell, B-cell, and myeloid lineages. Stem cell properties were maintained by a significant fraction of nonadherent cells in the third passage, although these stem cells expressed a somewhat more mature cell surface phenotype than the initial yolk sac stem cells. When reintroduced into adult allogeneic immunocompromised (scid) hosts, they were able to give rise to all of the leukocyte lineages, including T cells, B cells, and myeloid cells. We conclude that yolk sac endothelial cells can support the stable proliferation of multipotential hematopoietic stem cells, thus generating adequate numbers of cells for study of the mechanisms involved in their subsequent development and differentiation, for in vivo hematopoietic restitution, and for potential use as a vehicle for gene transfer.

Human aspartic protease memapsin 2 cleaves the β-secretase site of β-amyloid precursor protein

The cDNAs of two new human membrane-associated aspartic proteases, memapsin 1 and memapsin 2, have been cloned and sequenced. The deduced amino acid sequences show that each contains the typical pre, pro, and aspartic protease regions, but each also has a C-terminal extension of over 80 residues, which includes a single transmembrane domain and a C-terminal cytosolic domain. Memapsin 2 mRNA is abundant in human brain. The protease domain of memapsin 2 cDNA was expressed in Escherichia coli and was purified. Recombinant memapsin 2 specifically hydrolyzed peptides derived from the β-secretase site of both the wild-type and Swedish mutant β-amyloid precursor protein (APP) with over 60-fold increase of catalytic efficiency for the latter. Expression of APP and memapsin 2 in HeLa cells showed that memapsin 2 cleaved the β-secretase site of APP intracellularly. These and other results suggest that memapsin 2 fits all of the criteria of β-secretase, which catalyzes the rate-limiting step of the in vivo production of the β-amyloid (Aβ) peptide leading to the progression of Alzheimer's disease. Recombinant memapsin 2 also cleaved a peptide derived from the processing site of presenilin 1, albeit with poor kinetic efficiency. Alignment of cleavage site sequences of peptides indicates that the specificity of memapsin 2 resides mainly at the S1′ subsite, which prefers small side chains such as Ala, Ser, and Asp.

A novel precursor recognition element facilitates posttranslational binding to the signal recognition particle in chloroplasts

Signal recognition particles (SRPs) in the cytosols of prokaryotes and eukaryotes are used to target proteins to cytoplasmic membranes and the endoplasmic reticulum, respectively. The mechanism of targeting relies on cotranslational SRP binding to hydrophobic signal sequences. An organellar SRP identified in chloroplasts (cpSRP) is unusual in that it functions posttranslationally to localize a subset of nuclear-encoded thylakoid proteins. In assays that reconstitute thylakoid integration of the light harvesting chlorophyll-binding protein (LHCP), stromal cpSRP binds LHCP posttranslationally to form a cpSRP/LHCP transit complex, which is believed to represent the LHCP form targeted to thylakoids. In this investigation, we have identified an 18-aa sequence motif in LHCP (L18) that, along with a hydrophobic domain, is required for transit complex formation. Fusion of L18 to the amino terminus of an endoplasmic reticulum-targeted protein, preprolactin, led to transit complex formation whereas wild-type preprolactin exhibited no ability to form a transit complex. In addition, a synthetic L18 peptide, which competed with LHCP for transit complex formation, caused a parallel inhibition of LHCP integration. Translocation of proteins by the thylakoid Sec and Delta pH transport systems was unaffected by the highest concentration of L18 peptide examined. Our data indicate that a motif contained in L18 functions in precursor recruitment to the posttranslational SRP pathway, one of at least four different thylakoid sorting pathways used by chloroplasts.