Association of a novel human FE65-like protein with the cytoplasmic domain of the beta-amyloid precursor protein.

We identified a novel human homologue of the rat FE65 gene, hFE65L, by screening the cytoplasmic domain of beta-amyloid precursor protein (beta PP) with the "interaction trap." The cytoplasmic domains of the beta PP homologues, APLP1 and APLP2 (amyloid precursor-like proteins), were also tested for interaction with hFE65L. APLP2, but not APLP1, was found to interact with hFE65L. We confirmed these interactions in vivo by successfully coimmunoprecipatating endogenous beta PP and APLP2 from mammalian cells overexpressing a hemagglutinin-tagged fusion of the C-terminal region of hFE65L. We report the existence of a human FE65 gene family and evidence supporting specific interactions between members of the beta PP and FE65 protein families. Sequence analysis of the FE65 human gene family reveals the presence of two phosphotyrosine interaction (PI) domains. Our data show that a single PI domain is sufficient for binding of hFE65L to the cytoplasmic domain of beta PP and APLP2. The PI domain of the protein, Shc, is known to interact with the NPXYp motif found in the cytoplasmic domain of a number of different growth factor receptors. Thus, it is likely that the PI domains present in the C-terminal moiety of the hFE65L protein bind the NPXY motif located in the cytoplasmic domain of beta PP and APLP2.

Apoptosis mediated by HIV protease is preceded by cleavage of Bcl-2.

Expression of the human immunodeficiency virus type 1 (HIV) protease in cultured cells leads to apoptosis, preceded by cleavage of bcl-2, a key negative regulator of cell death. In contrast, a high level of bcl-2 protects cells in vitro and in vivo from the viral protease and prevents cell death following HIV infection of human lymphocytes, while reducing the yields of viral structural proteins, infectivity, and tumor necrosis factor alpha. We present a model for HIV replication in which the viral protease depletes the infected cells of bcl-2, leading to oxidative stress-dependent activation of NF kappa B, a cellular factor required for HIV transcription, and ultimately to cell death. Purified bcl-2 is cleaved by HIV protease between phenylalanine 112 and alanine 113. The results suggest a new option for HIV gene therapy; bcl-2 muteins that have noncleavable alterations surrounding the HIV protease cleavage site.

Effects of inefficient cleavage of the signal sequence of HIV-1 gp 120 on its association with calnexin, folding, and intracellular transport.

The HIV-1 envelope glycoprotein gp120 displays inefficient intracellular transport, which is caused by its retention in the endoplasmic reticulum. Coexpression in insect cells (Sf9) of HIV-1 gp120 with calnexin has shown that their interaction was modulated by the signal sequence of HIV-1 gp120. gp120, with its natural signal sequence, showed a prolonged association with calnexin with a t1/2 of greater than 20 min. Replacement of the natural signal sequence with the signal sequence from mellitin led to a decreased time of association of gp120 with calnexin (t1/2 < 10 min). These different times of calnexin association coincided both with the folding of gp120 as measured by the ability of bind CD4 and with endoplasmic reticulum to Golgi transport as analyzed by the acquisition of partial endoglycosidase H resistance. Using a monospecific antibody to the HIV-1 gp120 natural signal peptide, we showed that calnexin associated with N-glycosylated but uncleaved gp120. Only after dissociation from calnexin was gp120 cleaved, but very inefficiently. Only the small proportion of signal-cleaved gp120 molecules acquired transport competence and were secreted. This is the first report demonstrating the effect of the signal sequence on calnexin association.

Trypanosome U-deletional RNA editing involves guide RNA-directed endonuclease cleavage, terminal U exonuclease, and RNA ligase activities.

We have studied the mechanism of accurate in vitro RNA editing of Trypanosoma brucei ATPase 6 mRNA, using four mRNA-guide RNA (gRNA) pairs that specify deletion of 2, 3, or 4 U residues at editing site 1 and mitochondrial extract. This extract not only catalyzes deletion of the specified number of U residues but also exhibits a novel endonuclease activity that cleaves the input pre-mRNA in a gRNA-directed manner, precisely at the phosphodiester bond predicted in a simple enzymatic model of RNA editing. This cleavage site is inconsistent with a chimera-based editing mechanism. The U residues to be deleted, present at the 3' end of the upstream cleavage product, are then removed evidently by a 3' U-specific exonuclease and not by a reverse reaction of terminal U transferase. RNA ligase can then join the mRNA halves through their newly formed 5' P and 3' OH termini, generating mRNA faithfully edited at the first editing site. This resultant, partially edited mRNA can then undergo accurate, gRNA-directed cleavage at editing site 2, again precisely as predicted by the enzymatic editing model. All of these enzymatic activities cofractionate with the U-deletion activity and may reside in a single complex. The data imply that each round of editing is a four-step process, involving (i) gRNA-directed cleavage of the pre-mRNA at the bond immediately 5' of the region base paired to the gRNA, (ii) U deletion from or U addition to the 3' OH of the upstream mRNA half, (iii) ligation of the mRNA halves, and (iv) formation of additional base pairing between the correctly edited site and the gRNA that directs subsequent nuclease cleavage at the next editing site.

The methylotrophic yeast Pichia pastoris synthesizes a functionally active chromophore precursor of the plant photoreceptor phytochrome.

Induction of the expression of an algal phytochrome cDNA in the methylotrophic yeast Pichia pastoris led to time-dependent formation of photoactive holophytochrome without the addition of exogenous bilins. Both in vivo and in vitro difference spectra of this phytochromic species are very similar to those of higher plant phytochrome A, supporting the conclusion that this species possesses a phytochromobilin prosthetic group. Zinc blot analyses confirm that a bilin chromophore is covalently bound to the algal phytochrome apoprotein. The hypothesis that P. pastoris contains phytochromobilin synthase, the enzyme that converts biliverdin IX alpha to phytochromobilin, was also addressed in this study. Soluble extracts from P. pastoris were able to convert biliverdin to a bilin pigment, which produced a native difference spectrum upon assembly with oat apophytochrome A. HPLC analyses confirm that biliverdin is converted to both 3E- and 3Z-isomers of phytochromobilin. These investigations demonstrate that the ability to synthesize phytochromobilin is not restricted to photosynthetic organisms and support the hypothesis of a more widespread distribution of the phytochrome photoreceptor.

Cleavage of lamin A by Mch2 alpha but not CPP32: multiple interleukin 1 beta-converting enzyme-related proteases with distinct substrate recognition properties are active in apoptosis.

Although proteases related to the interleukin 1 beta-converting enzyme (ICE) are known to be essential for apoptotic execution, the number of enzymes involved, their substrate specificities, and their specific roles in the characteristic biochemical and morphological changes of apoptosis are currently unknown. These questions were addressed using cloned recombinant ICE-related proteases (IRPs) and a cell-free model system for apoptosis (S/M extracts). First, we compared the substrate specificities of two recombinant human IRPs, CPP32 and Mch2 alpha. Both enzymes cleaved poly-(ADP-ribose) polymerase, albeit with different efficiencies. Mch2 alpha also cleaved recombinant and nuclear lamin A at a conserved VEID decreases NG sequence located in the middle of the coiled-coil rod domain, producing a fragment that was indistinguishable from the lamin A fragment observed in S/M extracts and in apoptotic cells. In contrast, CPP32 did not cleave lamin A. The cleavage of lamin A by Mch2 alpha and by S/M extracts was inhibited by millimolar concentrations of Zn2+, which had a minimal effect on cleavage of poly (ADP-ribose) polymerase by CPP32 and by S/M extracts. We also found that N-(acetyltyrosinylvalinyl-N epsilon-biotinyllysyl)aspartic acid [(2,6-dimethylbenzoyl)oxy]methyl ketone, which derivatizes the larger subunit of active ICE, can affinity label up to five active IRPs in S/M extracts. Together, these observations indicate that the processing of nuclear proteins in apoptosis involves multiple IRPs having distinct preferences for their apoptosis-associated substrates.

Primary structure and tissue distribution of the orphanin FQ precursor.

The heptadecapeptide orphanin FQ (OFQ) is a recently discovered neuropeptide that exhibits structural features reminiscent of the opioid peptides and that is an endogenous ligand to a G protein-coupled receptor sequentially related to the opioid receptors. We have cloned both the human and rat cDNAs encoding the OFQ precursor proteins, to investigate whether the sequence relationships existing between the opioid and OFQ systems are also found at the polypeptide precursor level, in particular whether the OFQ precursor would encode several bioactive peptides as do the opioid precursors, and to study the regional distribution of OFQ sites of synthesis. The entire precursor protein displays structural homology to the opioid peptide precursors, especially preprodynorphin and preproenkephalin. The predicted amino acid sequence of the OFQ precursor contains a putative signal peptide and one copy of the OFQ sequence flanked by pairs of basic amino acid residues. Carboxyl-terminal to the OFQ sequence, the human and rat precursors contain a stretch of 28 amino acids that is 100% conserved and thus may encode novel bioactive peptides. Two peptides derived from this stretch were synthesized but were found to be unable to activate the OFQ receptor, suggesting that if they are produced in vivo, these peptides would likely recognize receptors different from the OFQ receptor. To begin analyzing the sites of OFQ mRNA synthesis, Northern analysis of human and rat tissues were carried out and showed that the OFQ precursor mRNA is mainly expressed in the brain. In situ hybridization of rat brain slices demonstrated a regional distribution pattern of the OFQ precursor mRNA, which is distinct from that of the opioid peptide precursors. These data confirm that the OFQ system differs from the opioid system at the molecular level, although the OFQ and opioid precursors may have arisen from a common ancestral gene.

Exon shuffling and the origin of the mitochondrial targeting function in plant cytochrome c1 precursor.

Since most of the examples of "exon shuffling" are between vertebrate genes, the view is often expressed that exon shuffling is limited to the evolutionarily recent lineage of vertebrates. Although exon shuffling in plants has been inferred from the analysis of intron phases of plant genes [Long, M., Rosenberg, C. & Gilbert, W. (1995) Proc. Natl. Acad. Sci. USA 92, 12495-12499] and from the comparison of two functionally unknown sunflower genes [Domon, C. & Steinmetz, A. (1994) Mol. Gen. Genet. 244, 312-317], clear cases of exon shuffling in plant genes remain to be uncovered. Here, we report an example of exon shuffling in two important nucleus-encoded plant genes: cytosolic glyceraldehyde-3-phosphate dehydrogenase (cytosolic GAPDH or GapC) and cytochrome c1 precursor. The intron-exon structures of the shuffled region indicate that the shuffling event took place at the DNA sequence level. In this case, we can establish a donor-recipient relationship for the exon shuffling. Three amino terminal exons of GapC have been donated to cytochrome c1, where, in a new protein environment, they serve as a source of the mitochondrial targeting function. This finding throws light upon an old important but unsolved question in gene evolution: the origin of presequences or transit peptides that generally exist in nucleus-encoded organelle genes.

Cholinergic agonists stimulate secretion of soluble full-length amyloid precursor protein in neuroendocrine cells.

The Abeta peptide of Alzheimer disease is derived from the proteolytic processing of the amyloid precursor proteins (APP), which are considered type I transmembrane glycoproteins. Recently, however, soluble forms of full-length APP were also detected in several systems including chromaffin granules. In this report we used antisera specific for the cytoplasmic sequence of APP to show that primary bovine chromaffin cells secrete a soluble APP, termed solAPPcyt, of an apparent molecular mass of 130 kDa. This APP was oversecreted from Chinese hamster ovary cells transfected with a full-length APP cDNA indicating that solAPPcyt contained both the transmembrane and Abeta sequence. Deglycosylation of solAPPcyt showed that it contained both N- and O-linked sugars, suggesting that this APP was transported through the endoplasmic reticulum-Golgi pathway. Secretion of solAPPcyt from primary chromatin cells was temperature-, time-, and energy-dependent and was stimulated by cell depolarization in a Ca2+-dependent manner. Cholinergic receptor agonists, including acetylcholine, nicotine, or carbachol, stimulated the rapid secretion of solAPPcyt, a process that was inhibited by cholinergic antagonists. Stimulation of solAPPcyt secretion was paralleled by a stimulation of secretion in catecholamines and chromogranin A, indicating that secretion of solAPPcyt was mediated by chromaffin granule vesicles. Taken together, our results show that release of the potentially amyloidogenic solAPPcyt is an active cellular process mediated by both the constitutive and regulated pathways. solAPPcyt was also detected in human cerebrospinal fluid. Combined with the neuronal physiology of chromaffin cells, our data suggest that cholinergic agonists may stimulate the release of this APP in neuronal synapses where it may exert its biological functions. Moreover, vesicular or secreted solAPPcyt may serve as a soluble precursor of Abeta.

U1 small nuclear RNA chimeric ribozymes with substrate specificity for the Rev pre-mRNA of human immunodeficiency virus.

The in vivo effectiveness of ribozymes strongly depends on the correct choice of the vector molecule. High levels of expression, stability, active conformation, and correct cellular localization are the most important features for a ribozyme vector. We have exploited the utilization of the U1 small nuclear RNA (snRNA) as a vector for specifically targeting a ribozyme into the nucleus. The Rev pre-mRNA of human immunodeficiency virus type 1 was chosen as target for testing the activity of the Ul-ribozyme. The catalytic core of the hammerhead motif, plus the recognition sequences, substituted the stem-loop III of the U1 snRNA. The resulting construct displays efficient cleavage activity in vitro. In addition, in the in vivo system of Xenopus laevis oocytes, the Ul-chimeric ribozyme accumulates in large amounts in the nucleus and produces a considerable reduction of Rev pre-mRNA levels. The Rev-specific ribozyme was also inserted in a derivative of the Ul snRNA mutated in the region of pairing with the 5' splice site, such as to match it with the suboptimal splice junction of the Rev precursor. This construct shows more efficient reduction of Rev pre-mRNA in vivo than the wild-type U1 vector.

The precursor of PsaD assembles into the photosystem I complex in two steps.

The present study addresses the assembly in the chloroplast thylakoid membranes of PsaD, a peripheral membrane protein of the photosystem I complex. Located on the stromal side of the thylakoids, PsaD was found to assemble in vitro into the membranes in its precursor (pre-PsaD) and also in its mature (PsaD) form. Newly assembled unprocessed pre-PsaD was resistant to NaBr and alkaline wash. Yet it was sensitive to proteolytic digestion. In contradistinction, when the assembled precursor was processed, the resulting mature PsaD was resistant to proteases to the same extent as endogenous [correction of endogeneous] PsaD. The accumulation of protease-resistant PsaD in the thylakoids correlated with the increase of mature-PsaD in the membranes. This protection of mature PsaD from proteolysis could not be observed when PsaD was in a soluble form-i.e. not assembled within the thylakoids. The data suggest that pre-PsaD assembles to the membranes and only in a second step processing takes place. The observation that the assembly of pre-PsaD is affected by salts to a much lesser extent than that of mature-PsaD supports a two-step assembly of pre-PsaD.

Generation of mice with a 200-kb amyloid precursor protein gene deletion by Cre recombinase-mediated site-specific recombination in embryonic stem cells.

Gene disruptions and deletions of up to 20kb have been generated by homologous recombination with appropriate targeting vectors in murine embryonic stem (ES) cells. Because we could not obtain a deletion of about 200 kb in the mouse amyloid precursor protein gene by the classical technique, we employed strategies involving the insertion of loxP sites upstream and downstream of the region to be deleted by homologous recombination and elicited excision of the loxP-flanked region by introduction of a Cre expression vector into the ES cells. In the first approach, the loxP sequences were inserted in two successive steps and after each step, ES cell clones were isolated and characterized. Deletion of the loxP-flanked sequence was accomplished by introducing the cre gene in a third step. In the second approach, ES cells containing the upstream loxP cassette were electroporated simultaneously with the downstream loxP targeting vector and the Cre expression plasmid. ES cells were obtained that gave rise to chimeric mice capable of germ-line transmission of the deleted amyloid precursor protein allele.

Subunit cleavage of mosquito pro-vitellogenin by a subtilisin-like convertase.

The eukaryotic convertase family plays an important role in posttranslational proteolytic processing and activation of many pro- and polypeptides that have at their cleavage sites the paired basic motif, RX(K/R)R. Recent studies have revealed that the cleavage site of insect pro-vitellogenins (pro-Vg) also contains this motif. To identify and characterize the insect pro-Vg processing enzyme, Vg convertase (VC), its cDNA was cloned from a vitellogenic female fat body cDNA library of the mosquito, Aedes aegypti. The 3735-bp-long VC cDNA has an open reading frame encoding a 115-kDa protein. In vitro transcription/translation of VC cDNA revealed that this 115-kDa protein becomes 140 kDa after co- and posttranslational modifications. The VC deduced amino acid sequence has high similarity to and a domain structure characteristic of furin-like convertases. Northern blot analysis showed that a single 4.2-kb transcript was expressed in the fat body during the first 18 hr of the Vg synthetic period. Coexpression of VC cDNA with mosquito Vg cDNA resulted in correct cleavage of pro-Vg. Thus, this newly identified convertase is, indeed, a functional fat body-specific enzyme for pro-Vg cleavage.

Internal cleavage of the inhibitory 7B2 carboxyl-terminal peptide by PC2: a potential mechanism for its inactivation.

The neuroendocrine protein 7B2 contains two domains, a 21-kDa protein required for prohormone convertase 2 (PC2) maturation and a carboxyl-terminal (CT) peptide that inhibits PC2 at nanomolar concentrations. To determine how the inhibition of PC2 is terminated, we studied the metabolic fate of the 7B2 CT peptide in RinPE-7B2, AtT-20/PC2-7B2, and alphaTC1-6 cells. Extracts obtained from cells labeled for 6 h with [3H]valine were subjected to immunoprecipitation using an antibody raised against the extreme carboxyl terminus of r7B2, and immunoprecipitated peptides were separated by gel filtration. All three cell lines yielded two distinct peaks at about 3.5 kDa and 1.5 kDa, corresponding to the CT peptide and a smaller fragment consistent with cleavage at an interior Lys-Lys site. These results were corroborated using a newly developed RIA against the carboxyl terminus of the CT peptide which showed that the intact CT peptide represented only about half of the stored CT peptide immunoreactivity, with the remainder present as the 1.5-kDa peptide. Both peptides could be released upon phorbol 12-myristate 13-acetate stimulation. We investigated the possibility that PC2 itself could be responsible for this cleavage by performing in vitro experiments. When 125I-labeled CT peptide was incubated with purified recombinant PC2, a smaller peptide was generated. Analysis of CT peptide derivatives for their inhibitory potency revealed that CT peptide 1-18 (containing Lys-Lys at the carboxyl terminus) represented a potent inhibitor, but that peptide 1-16 was inactive. Inclusion of carboxypeptidase E (CPE) in the reaction greatly diminished the inhibitory potency of the CT peptide against PC2, in line with the notion that the CT peptide cleavage product is not inhibitory after the removal of terminal lysines by CPE. In summary, our data support the idea that PC2 cleaves the 7B2 CT peptide at its internal Lys-Lys site within secretory granules; deactivation of the cleavage product is then accomplished by CPE, thus providing an efficient mechanism for intracellular inactivation of the CT peptide.

Metabolism of Alzheimer beta-amyloid precursor protein: regulation by protein kinase A in intact cells and in a cell-free system.

Various compounds that affect signal transduction regulate the relative utilization of alternative processing pathways for the beta-amyloid precursor protein (beta APP) in intact cells, increasing the production of nonamyloidogenic soluble beta APP (s beta APP) and decreasing that of amyloidogenic beta-amyloid peptide. In a recent study directed toward elucidating the mechanisms underlying phorbol ester-stimulated s beta APP secretion from cells, it was demonstrated that protein kinase C increases the formation from the trans-Golgi network (TGN) of beta APP-containing secretory vesicles. Here we present evidence that forskolin increases s beta APP production from intact PC12 cells, and protein kinase A stimulates formation from the TGN of beta APP-containing vesicles. Although protein kinase A and protein kinase C converge at the level of formation from the TGN of beta APP-containing vesicles, additional evidence indicates that the regulatory mechanisms involved are distinct.