Pascale Dauch

Molecular Cloning and Expression of Rat Brain Endopeptidase 3.4.24.16

We have isolated by immunological screening of a ZAPII cDNA library constructed from rat brain mRNAs a cDNA clone encoding endopeptidase 3.4.24.16. The longest open reading frame encodes a 704-amino acid protein with a theoretical molecular mass of 80,202 daltons and bears the consensus sequence of the zinc metalloprotease family. The sequence exhibits a 60.2% homology with those of another zinc metallopeptidase, endopeptidase 3.4.24.15. Northern blot analysis reveals two mRNA species of about 3 and 5 kilobases in rat brain, ileum, kidney, and testis. We have transiently transfected COS-7 cells with pcDNA containing the cloned cDNA and established the overexpression of a 70-75-kDa immunoreactive protein. This protein hydrolyzes QFS, a quenched fluorimetric substrate of endopeptidase 3.4.24.16, and cleaves neurotensin at a single peptide bond, leading to the formation of neurotensin(1, 2, 3, 4, 5, 6, 7, 8, 9, 10) and neurotensin (11, 12, 13). QFS and neurotensin hydrolysis are potently inhibited by the selective endopeptidase 3.4.24.16 dipeptide blocker Pro-Ile and by dithiothreitol, while the enzymatic activity remains unaffected by phosphoramidon and captopril, the specific inhibitors of endopeptidase 3.4.24.11 and angiotensin-converting enzyme, respectively. Altogether, these physicochemical, biochemical, and immunological properties unambiguously identify endopeptidase 3.4.24.16 as the protein encoded by the isolated cDNA clone.

α-Secretase-Derived Product of β-Amyloid Precursor Protein Is Decreased by Presenilin 1 Mutations Linked to Familial Alzheimer's Disease

Abstract: Recent reports indicate that missense mutations on presenilin (PS) 1 are likely responsible for the main early‐onset familial forms of Alzheimers disease (FAD). Consensual data obtained through distinct histopathological, cell biology, and molecular biology approaches have led to the conclusion that these PS1 mutations clearly trigger an increased production of the 42‐amino‐acid‐long species of β‐amyloid peptide (Aβ). Here we show that overexpression of wild‐type PS1 in HK293 cells increases Aβ40 secretion. By contrast, FAD‐linked mutants of PS1 trigger increased secretion of both Aβ40 and Aβ42 but clearly favor the production of the latter species. We also demonstrate that overexpression of the wild‐type PS1 augments the α‐secretase‐derived C‐terminally truncated fragment of β‐amyloid precursor protein (APPα) recovery, whereas transfectants expressing mutated PS1 secrete drastically lower amounts of APPα when compared with cells expressing wild‐type PS1. This decrease was also observed when comparing double transfectants overexpressing wild‐type β‐amyloid precursor protein and either PS1 or its mutated congener M146V‐PS1. Altogether, our data indicate that PS mutations linked to FAD not only trigger an increased ratio of Aβ42 over total Aβ secretion but concomitantly down‐regulate the production of APPα.

Stably Transfected Human Cells Overexpressing Rat Brain Endopeptidase 3.4.24.16: Biochemical Characterization of the Activity and Expression of Soluble and Membrane‐Associated Counterparts

Abstract: We recently cloned endopeptidase‐24.16 (neurolysin; EC 3.4.24.16), a neurotensin‐degrading peptidase likely involved in the physiological termination of the neurotensinergic signal in the central nervous system and in the gastrointestinal tract. We stably transfected human kidney cells with the pcDNA3‐λ7aB1 construction bearing the whole open reading frame encoding the rat brain peptidase. Transfectants displayed endopeptidase‐24.16 immunoreactivity and exhibited QFS‐ and neurotensin‐hydrolyzing activities, the biochemical and specificity properties of which fully matched those observed with the purified murine enzyme. Cryoprotection experiments and substrate degradation by intact plated cells indicated that transfectants exhibited a membrane‐associated form of endopeptidase‐24.16, the catalytic site of which clearly faced the extracellular domain. Transfected cells were unable to secrete the enzyme. Overall, our experiments indicate that we have obtained stably transfectant cells that overexpress an enzymatic activity displaying biochemical properties identical to those of purified endopeptidase‐24.16. The membrane‐associated counterpart and lack of secretion of the enzyme were clearly reminiscent of what was observed with pure cultured neurons, but not with astrocytes. Therefore, the transfected cell model described here could prove useful for establishing, by a mutagenesis approach, the structural elements responsible for the “neuronal” phenotype exhibited by the enzyme in transfected cells.

Neurotensin and Neuromedin N Are Differently Metabolized in Ventral Tegmental Area and Nucleus Accumbens

Abstract: Whole homogenates and membrane‐bound and cytosoluble fractions prepared from rat ventral tegmental area (VTA) and nucleus accumbens were examined for their content of peptidasic activities and for their ability to metabolize neurotensin and its natural related hexapeptide neuromedin N. No qualitative differences were observed between these two brain regions concerning the presence and the subcellular distribution of a series of activities able to hydrolyze various specific fluorimetric enzymatic substrates. However, aminopeptidase B, endopeptidase 24‐15, and endopeptidase 24‐11 were significantly lower in the VTA than in the nucleus accumbens membrane preparations, while proline endopeptidase was detected in significantly higher amount only in the cytosolic fraction prepared from nucleus accumbens. Both neurotensin and neuromedin N were metabolized more rapidly in the nucleus accumbens than in the VTA. Furthermore, the degradation rate of neuromedin N was considerably faster than that of neurotensin whatever the cerebral area examined. Studies carried out with highly specific peptidase inhibitors revealed that endopeptidase 24–15 mainly contributed to the catabolism of neurotensin in homogenates and membrane‐bound preparations of nucleus accumbens and VTA, while aminopeptidase B appeared predominantly responsible for the rapid disappearance of neuromedin N in both cerebral tissues. The possibility that the different metabolic processes of the two peptide congeners could explain their distinct pharmacological profiles observed after their microinjection in the nucleus accumbens and in the VTA is discussed.

[21] Identification and distribution of endopeptidase 24.16 in central nervous system

Publisher Summary This chapter describes the development and use of a fluorimetric substrate, specific inhibitors, and a polyclonal antiserum directed toward endopeptidase 24.16. These new tools have permitted further investigation of the biochemical properties of the enzyme. It is now possible to monitor the activity in a complex mixture of enzymes such as crude extracts, provided that some precautions are taken. Furthermore, the development of specific inhibitors has established, for the first time, that endopeptidase 24.16 indeed participates in the physiological metabolism of neurotensin. These inhibitors will be of further help in assessing whether the enzyme also contributes to the catabolic pathways of other neuropeptides. Finally, the development of a monospecific antiserum directed toward endopeptidase 24.16 has rendered possible the immunological screening of a rat brain λgt 11 cDNA library and will allow the molecular cloning of the enzyme, which is in progress in the laboratories.

Lack of effect of Presenilin 1, βAPP and their Alzheimer's disease-related mutated forms on Xenopus oocytes membrane currents

The effect of the microinjection of Xenopus oocytes with various cRNAs coding for Presenilin 1 and four mutated presenilins linked to early onset familial forms of Alzheimers disease was examined. These cRNAs were injected either alone or in combination with the cRNA encoding betaAPP751 and the Swedish mutated form of betaAPP751 known to produce exacerbated amount of Abeta. Current-voltage relationships generated by voltage step were recorded. None of the cRNA injected alone or in combination displayed the ability to modify the current recorded with naive cells. Altogether, this study shows that Presenilin 1 does not mediate membrane currents and is more likely involved in the physiopathological maturation of betaAPP.