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- Lindholm, D, et al.
(författare)
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Neuronal apoptosis inhibitory protein : structural requirements for hippocalcin binding and effects on survival of NGF-denendent sympathetic neurons
- 2002
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Ingår i: Biochimica et Biophysica Acta - Proteins and Proteomics. - : ELSEVIER SCIENCE BV. - 1570-9639 .- 1878-1454. ; 1600:1-2, s. 138-147
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Tidskriftsartikel (refereegranskat)abstract
- Neuronal apoptosis inhibitory protein (NAIP) has been linked to the inherited disease, spinal muscular atrophy (SMA), which occurs in children with degeneration of the motorneurons. In the nervous system, NAIP is expressed by specific classes of neurons including spinal -motorneurons. Recently, NAIP was shown to interact with hippocalcin, which belongs to the neuronal calcium sensor (NCS) protein family. Here we-have studied this interaction in more detail, using deletions and a mutagenesis of the third baculovirus inhibitory repeat (BIR) motif in NAIP, and functional assays for neuronal death. The results showed that specific amino acids and the zinc finger domain in BIR3 are needed for efficient interaction of NAIP with hippocalcin. Cotransfections of NAIP-BIR3 and hippocalcin resulted in translocation and colocalisation of the two proteins in neuroblastoma cells. This was accompanied by an enhanced resistance towards cell death induced by high levels of calcium. In contrast, expression of NAIP-BIR3 and hippocalcin in sympathetic neurons did not protect against death induced by nerve growth factor (NGF) withdrawal. The results demonstrate a functional interaction of hippocalcin with NAIP-BIR3, which in neuroblastoma cells leads to rescue of cells after high intracellular calcium, but which in sympathetic neurons had no significant effect. The results indicate that NAIP in conjunction with hippocalcin can affect the survival of some, but not all neural cells, and this interaction may play a role in the neurodegenerative processes in SMA, and possible other human disorders. (C) 2002 Elsevier Science B.V All rights reserved.
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- Lund, P E, et al.
(författare)
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The orexin OX1 receptor activates a novel Ca2+ influx pathway necessary for coupling to phospholipase C.
- 2000
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 275:40, s. 30806-12
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Tidskriftsartikel (refereegranskat)abstract
- Ca(2+) elevations in Chinese hamster ovary cells stably expressing OX(1) receptors were measured using fluorescent Ca(2+) indicators fura-2 and fluo-3. Stimulation with orexin-A led to pronounced Ca(2+) elevations with an EC(50) around 1 nm. When the extracellular [Ca(2+)] was reduced to a submicromolar concentration, the EC(50) was increased 100-fold. Similarly, the inositol 1,4,5-trisphosphate production in the presence of 1 mm external Ca(2+) was about 2 orders of magnitude more sensitive to orexin-A stimulation than in low extracellular Ca(2+). The shift in the potency was not caused by depletion of intracellular Ca(2+) but by a requirement of extracellular Ca(2+) for production of inositol 1,4,5-trisphosphate. Fura-2 experiments with the "Mn(2+)-quench technique" indicated a direct activation of a cation influx pathway by OX(1) receptor independent of Ca(2+) release or pool depletion. Furthermore, depolarization of the cells to +60 mV, which almost nullifies the driving force for Ca(2+) entry, abolished the Ca(2+) response to low concentrations of orexin-A. The results thus suggest that OX(1) receptor activation leads to two responses, (i) a Ca(2+) influx and (ii) a direct stimulation of phospholipase C, and that these two responses converge at the level of phospholipase C where the former markedly enhances the potency of the latter.
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