Enzymatic generation of the amino terminus of the beta-amyloid peptide.

The major pathological change in Alzheimer's disease is the deposition of 39-42-amino acid beta-amyloid peptide (BAP) in the brain. Since BAP begins at the aspartate residue (Asp1, or codon 672 of the amyloid precursor protein (APP)770 transcript), the ability of several proteases to cleave the peptide bond methionine-Asp1 (M/D) was evaluated by using peptides and recombinant APP molecules as substrates. Cathepsin G and chymotrypsin cleave the synthetic peptide HSEVKMDAEF at M/D under acidic conditions, whereas cleavage at lysine-methionine (K/M) predominates when the pH is alkaline. Trypsin and cathepsins B, D, and L are unable to cleave the synthetic peptide at M/D. Peptide SEVNLDAEF, representing the mutation found in early onset Alzheimer's disease families from Sweden, is cleaved by cathepsin G and chymotrypsin at leucine-aspartate (L/D). Incubation of cathepsin G with soluble protease nexin-2 obtained from recombinant APP (APP-REP) derivatives resulted in proteolytic cleavage at or near the amino terminus of BAP. Cathepsin G-mediated cleavage was also observed in the domain representing the amino terminus of BAP when mature plasma membrane-associated APP-REP molecules were used as substrates. Our results strongly suggest the involvement of a chymotrypsin-like serine protease in the generation of the amino terminus of BAP beginning at Asp1.

Calcium binding protein (calbindin-D28k) and glutamate decarboxylase gene expression after kindling induced seizures.

In order to determine whether calcium binding protein (calbindin-D28k or CaBP) and glutamate decarboxylase (GAD) may be involved in the process underlying the generation of seizure activity, changes in CaBP protein and mRNA and in GAD mRNA were examined in the kindling model of epilepsy. Following amygdaloid (AK) and commissure (CK) kindling significant decreases in the concentration of CaBP of 20% and 30%, respectively, were specifically observed in the hippocampal formation. However, using a cDNA specific to mammalian CaBP, Northern analysis of poly(A+) RNA and slot blot analysis of total RNA revealed no changes in the levels of CaBP mRNA in hippocampus, subcortical area (including amygdala, substantia nigra and striatum) or cerebellum of rats sacrificed 30 min, 1 h, 6 h or 24 h after the last kindled seizure. Similarly when these blots were reprobed with a cDNA specific to mammalian GAD, no changes in GAD gene expression were observed. However, fos gene expression was markedly enhanced at 1 h after seizure. We also tested whether changes in CaBP or GAD mRNA could be detected at any of the various stages of the kindling process. Slot blot analysis of cortex, subcortical structures and hippocampus revealed no changes in CaBP or GAD mRNA during the course of commissure kindling. In situ hybridization studies with GAD and CaBP 35S-labeled antisense probes also indicated no obvious changes upon visual analysis of autoradiographs. However, when silver grains were counted, significant changes in GAD mRNA in individual cells in hippocampus and substantia nigra were noted after kindling induced epilepsy. Our results indicate that, unlike fos gene expression, prominent alterations in GAD and CaBP mRNA in gross brain regions (as measured by slot blot and Northern blot analyses) are not observed in the kindling process. However, our in situ hybridization studies suggest that changes in GAD mRNA in individual cells may be involved in the process underlying kindling induced seizure activity.

The biological activity of atrial natriuretic factor cleaved by endoprotease 3.4.24.11.

Ring cleavage of atrial natriuretic peptide (ANF) between Cys7 and Phe8 by endoprotease 3.4.24.11 yields X-ANF. Since endoprotease 3.4.24.11 may contribute to ANF metabolism in vivo, the present study determined if X-ANF exhibits reduced biological activity in comparison to the parent molecule.

Regulation of proenkephalin by Fos and Jun.

Fos and Jun form a heterodimeric complex that associates with the nucleotide sequence motif known as the AP-1 binding site. Although this complex has been proposed to function as a transcriptional regulator in neurons, no specific target gene has yet been identified. Proenkephalin mRNA increased in the hippocampus during seizure just after an increase in c-fos and c-jun expression was detected. Fos-Jun complexes bound specifically to a regulatory sequence in the 5' control region of the proenkephalin gene. Furthermore, c-fos and c-jun stimulated transcription from this control region synergistically in transactivation assays. These data suggest that the proenkephalin gene may be a physiological target for Fos and Jun in the hippocampus and indicate that these proto-oncogene transcription factors may play a role in neuronal responses to stimulation.

Nerve growth factor-induced differentiation in PC-12 cells is blocked by fos oncogene.

Rat pheochromocytoma (PC-12) cells respond to nerve growth factor (NGF) by cessation of cell division and by expression of several properties resembling those of differentiated sympathetic neurons. Within minutes after addition, NGF rapidly stimulates transient expression of c-fos. To investigate the possible role of c-fos in NGF-induced differentiation, activated mouse c-fos genes were introduced into PC-12 cells by electroporation. Constitutive expression of fos inhibited NGF-induced differentiation although transfected cells harbored intact NGF receptors. Dibutyryl cyclic AMP (db cAMP) and basic fibroblast growth factor (b-FGF)-induced differentiation were also inhibited. Transcriptional activation of c-fos, c-jun, and ornithine decarboxylase (ODC) by NGF was down-regulated, whereas expression of egr-1 was unaffected in PC-12 fos clones. These results suggest that deregulated expression of fos can interfere with the normal role of NGF in neuronal differentiation.

Dynamic alterations occur in the levels and composition of transcription factor AP-1 complexes after seizure.

Seizure causes a rapid and protracted increase in transcription factor AP-1 levels in the brain. The composition of AP-1 nucleoprotein complexes changes with time after seizure as a result of the sequential appearance and disappearance of Fos and several Fos-related proteins. Although these changes occur over an 8 hr time period, they are triggered by 15 min of seizure. Alterations in the levels and composition of transcription factors may represent one of the molecular mechanisms underlying neuronal adaptation.

Glutamate receptor agonists increase the expression of Fos, Fra, and AP-1 DNA binding activity in the mammalian brain.

Administration of convulsant doses of Metrazole (pentylenetetrazol) and picrotoxin, as well as maximal electroshock, results in a rapid but transient increase in c-fos mRNA in mouse brain. Elevation of c-fos mRNA is followed by the accumulation and subsequent disappearance of Fos, the protein encoded by c-fos. In addition, immunoblots reveal the induction of two additional proteins that are antigenically related to Fos (Fra, Fos-related antigens). Fos and the various Fra appear and disappear in a staggered manner over an 8 hour period, such that at longer times after stimulation the brain contains no Fos but relatively large amounts of Fra (the latter being designated here by their apparent molecular weights, Fra-46K and Fra-35K). Previous studies have established that Fos, as well as several Fra, contribute to transcription factor AP-1 nucleoprotein complexes along with Jun, the product of the jun proto-oncogene. The appearance in brain of Fos and Fra coincides with a protracted increase in total AP-1 DNA binding activity, indicating that all the Fos-like proteins can participate in AP-1 complexes. Furthermore, the molecular composition of these complexes alters with time after stimulation. The induction of c-fos by Metrazole is blocked or attenuated by known anticonvulsants such as diazepam and valproate as well as the N-methyl-D-aspartate (NMDA) receptor antagonists, 2-amino-5-phosphonovaleric acid (APV) and MK-801. This suggests that fos induction might involve stimulation of a glutamate receptor. This conclusion was strengthened by the observations that two glutamate receptor agonists, kainic acid and NMDA, induced c-fos expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of protease 3.4.24.11 as the major atrial natriuretic factor degrading enzyme in the rat kidney.

We have identified a metalloendoprotease from rat kidney cortex that cleaves the cysteine-phenylalanine bond (Cys7-Phe8) within the 17 amino acid ring structure of atrial natriuretic factor (ANF). Cleavage at this site represents the major ANF degradative activity in rat kidney, and is inhibited by the known metalloendoprotease inhibitors, thiorphan, phosphoramidon and zincov with IC50 values in the nanomolar range. Since these are specific inhibitors of protease 3.4.24.11, both protease 3.4.24.11 and ANF degrading activities were monitored during purification. Both activities copurified at each chromatographic step. Furthermore, purified protease 3.4.24.11 cleaved ANF specifically at the Cys7-Phe8 bond. It is concluded from this work that the major ANF degrading enzyme in rat kidney is protease 3.4.24.11.