Yoke Peng Loh

Comparison of the Molecular Forms of the Kex2/Subtilisin-Like Serine Proteases SPC2, SPC3, and Furin in Neuroendocrine Secretory Vesicles Reveals Differences in Carboxyl-Terminus Truncation and Membrane Association

Abstract: The molecular forms and membrane association of SPC2, SPC3, and furin were investigated in neuroendocrine secretory vesicles from the anterior, intermediate, and neural lobes of bovine pituitary and bovine adrenal medulla. The major immunoreactive form of SPC2 was the full‐length enzyme with a molecular mass of 64 kDa. The major immunoreactive form of SPC3 was truncated at the carboxyl terminus and had a molecular mass of 64 kDa. Full‐length 86‐kDa SPC3 with an intact carboxyl terminus was found only in bovine chromaffin granules. Immunoreactive furin was also detected in secretory vesicles. The molecular masses of 80 and 76 kDa were consistent with carboxyl‐terminal truncation of furin to remove the transmembrane domain. All three enzymes were distributed between the soluble and membrane fractions of secretory vesicles although the degree of membrane association was tissue specific and, in the case of SPC3, dependent on the molecular form of the enzyme. Significant amounts of membrane‐associated and soluble forms of SPC2, SPC3, and furin were found in pituitary secretory vesicles, whereas the majority of the immunoreactivity in chromaffin granules was membrane associated. More detailed analyses of chromaffin granule membranes revealed that 86‐kDa SPC3 was more tightly associated with the membrane fraction than the carboxyl terminus‐truncated 64‐kDa form.

Rosiglitazone‐activated PPARγ induces neurotrophic factor‐α1 transcription contributing to neuroprotection

Brain peroxisome proliferator‐activated receptor gamma (PPARγ), a member of the nuclear receptor superfamily of ligand‐dependent transcription factors, is involved in neuroprotection. It is activated by the drug rosiglitazone, which then can increase the pro‐survival protein B‐cell lymphoma 2 (BCL‐2), to mediate neuroprotection. However, the mechanism underlying this molecular cascade remains unknown. Here, we show that the neuroprotective protein neurotrophic factor‐α1 (NF‐α1), which also induces the expression of BCL‐2, has a promoter that contains PPARγ‐binding sites that are activated by rosiglitazone. Treatment of Neuro2a cells and primary hippocampal neurons with rosiglitazone increased endogenous NF‐α1 expression and prevented H2O2‐induced cytotoxicity. Concomitant with the increase in NF‐α1, BCL‐2 was also increased in these cells. When siRNA against NF‐α1 was used, the induction of BCL‐2 by rosiglitazone was prevented, and the neuroprotective effect of rosiglitazone was reduced. These results demonstrate that rosiglitazone‐activated PPARγ directly induces the transcription of NF‐α1, contributing to neuroprotection in neurons.

Carboxypeptidase E Protects Hippocampal Neurons During Stress in Male Mice by Up-regulating Pro-survival BCL2 Protein Expression

Prolonged chronic stress causing elevated plasma glucocorticoids leads to neurodegeneration. Adaptation to stress (allostasis) through neuroprotective mechanisms can delay this process. Studies on hippocampal neurons have identified carboxypeptidase E (CPE) as a novel neuroprotective protein that acts extracellularly, independent of its enzymatic activity, although the mechanism of action is unclear. Here, we aim to determine if CPE plays a neuroprotective role in allostasis in mouse hippocampus during chronic restraint stress (CRS), and the molecular mechanisms involved. Quantitative RT-PCR/in situ hybridization and Western blots were used to assay for mRNA and protein. After mild CRS (1 h/d for 7 d), CPE protein and mRNA were significantly elevated in the hippocampal CA3 region, compared to naïve littermates. In addition, luciferase reporter assays identified a functional glucocorticoid regulatory element within the cpe promoter that mediated the up-regulation of CPE expression in primary hippocampal neurons following dexamethasone treatment, suggesting that circulating plasma glucocorticoids could evoke a similar effect on CPE in the hippocampus in vivo. Overexpression of CPE in hippocampal neurons, or CRS in mice, resulted in elevated prosurvival BCL2 protein/mRNA and p-AKT levels in the hippocampus; however, CPE(-/-) mice showed a decrease. Thus, during mild CRS, CPE expression is up-regulated, possibly contributed by glucocorticoids, to mediate neuroprotection of the hippocampus by enhancing BCL2 expression through AKT signaling, and thereby maintaining allostasis.

Carboxypeptidase E cytoplasmic tail mediates localization of synaptic vesicles to the pre‐active zone in hypothalamic pre‐synaptic terminals

J. Neurochem. (2010) 114, 886–896.

Evidence for a receptor-mediated mechanism for sorting proopiomelanocortin to the regulated secretory pathway

A sorting signal which directs the prohormone pro-opiomelanocortin (POMC) to the regulated secretory pathway was identified. This sorting signal motif consists of a 13 amino acid (POMC8–20) amphipathic loop stabilized by a disulfide bridge (Cys8–Cys20) located at the N-terminus of pro-opiomelanocortin. It binds at an acidic pH specifically to an ~64 kD sorting receptor protein present in Golgi and secretory granule membranes of neuroendocrine cells. These findings support the hypothesis that pro-opiomelanocortin is targeted to the regulated secretory pathway by a receptor-mediated mechanism.