Carboxypeptidase E (CPE) is involved in the biosynthesis of most neuropeptides and peptide hormones. Until recently, CPE was the only intracellular carboxypeptidase thought to be involved in neuroendocrine peptide processing. However, the finding that fat/fat mice, which have a mutation within the CPE gene that inactivates the enzyme, are capable of a reduced amount of insulin processing suggests that another carboxypeptidase is present within the secretory pathway. We have detected a CPE-like enzyme, designated CPD, which has many properties in common with those of CPE. Like CPE, CPD is a metallocarboxypeptidase that has a pH optimum of 5.5-6. The Km and Kcat values for a series of short peptide substrates show only minor differences between CPD and CPE. Several active site-directed inhibitors also show generally similar potency toward the two enzymes, although guanidinoethylmercaptosuccinic acid is approximately 10-fold more potent, and hippuryl-Arg is approximately 100-fold more potent as an inhibitor of CPD than of CPE. A major difference between the two enzymes is the molecular masses; CPE is 50,000-56,000, whereas CPD is approximately 180,000. Also, CPD does not elute from a substrate affinity column when the pH is raised to 8, which elutes CPE, although CPD can subsequently be eluted by arginine. Both CPE and CPD are present in purified bovine anterior pituitary secretory vesicles, but the tissue distribution of CPD is more uniform than that of CPE. Antisera to the N- and C-terminal regions of CPE do not recognize CPD. The partial N-terminal amino acid sequence of bovine CPD shows 30-40% homology with an N-terminal region of bovine and rat CPE and 70% homology with a duck protein known as gp180, a hepatitis B virus particle binding protein that shows 47% homology to CPE. Taken together, these results suggest that CPD is a novel secretory pathway enzyme that may be the bovine homologue of gp180.

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BackgroundHyperphosphorylation of microtubule-associated protein tau is a distinct feature of neurofibrillary tangles (NFTs) that are the hallmark of neurodegenerative tauopathies. O-GlcNAcylation is a lesser known post-translational modification of tau that involves the addition of N-acetylglucosamine onto serine and threonine residues. Inhibition of O-GlcNAcase (OGA), the enzyme responsible for the removal of O-GlcNAc modification, has been shown to reduce tau pathology in several transgenic models. Clarifying the underlying mechanism by which OGA inhibition leads to the reduction of pathological tau and identifying translatable measures to guide human dosing and efficacy determination would significantly facilitate the clinical development of OGA inhibitors for the treatment of tauopathies.MethodsGenetic and pharmacological approaches are used to evaluate the pharmacodynamic response of OGA inhibition. A panel of quantitative biochemical assays is established to assess the effect of OGA inhibition on pathological tau reduction. A “click” chemistry labeling method is developed for the detection of O-GlcNAcylated tau.ResultsSubstantial (>80%) OGA inhibition is required to observe a measurable increase in O-GlcNAcylated proteins in the brain. Sustained and substantial OGA inhibition via chronic treatment with Thiamet G leads to a significant reduction of aggregated tau and several phosphorylated tau species in the insoluble fraction of rTg4510 mouse brain and total tau in cerebrospinal fluid (CSF). O-GlcNAcylated tau is elevated by Thiamet G treatment and is found primarily in the soluble 55 kD tau species, but not in the insoluble 64 kD tau species thought as the pathological entity.ConclusionThe present study demonstrates that chronic inhibition of OGA reduces pathological tau in the brain and total tau in the CSF of rTg4510 mice, most likely by directly increasing O-GlcNAcylation of tau and thereby maintaining tau in the soluble, non-toxic form by reducing tau aggregation and the accompanying panoply of deleterious post-translational modifications. These results clarify some conflicting observations regarding the effects and mechanism of OGA inhibition on tau pathology, provide pharmacodynamic tools to guide human dosing and identify CSF total tau as a potential translational biomarker. Therefore, this study provides additional support to develop OGA inhibitors as a treatment for Alzheimer’s disease and other neurodegenerative tauopathies.Electronic supplementary materialThe online version of this article (doi:10.1186/s13024-017-0181-0) contains supplementary material, which is available to authorized users.

The presenilins and nicastrin, a type 1 transmembrane glycoprotein, form high molecular weight complexes that are involved in cleaving the beta-amyloid precursor protein (betaAPP) and Notch in their transmembrane domains. The former process (termed gamma-secretase cleavage) generates amyloid beta-peptide (Abeta), which is involved in the pathogenesis of Alzheimer's disease. The latter process (termed S3-site cleavage) generates Notch intracellular domain (NICD), which is involved in intercellular signalling. Nicastrin binds both full-length betaAPP and the substrates of gamma-secretase (C99- and C83-betaAPP fragments), and modulates the activity of gamma-secretase. Although absence of the Caenorhabditis elegans nicastrin homologue (aph-2) is known to cause an embryonic-lethal glp-1 phenotype, the role of nicastrin in this process has not been explored. Here we report that nicastrin binds to membrane-tethered forms of Notch (substrates for S3-site cleavage of Notch), and that, although mutations in the conserved 312-369 domain of nicastrin strongly modulate gamma-secretase, they only weakly modulate the S3-site cleavage of Notch. Thus, nicastrin has a similar role in processing Notch and betaAPP, but the 312-369 domain may have differential effects on these activities. In addition, we report that the Notch and betaAPP pathways do not significantly compete with each other.

The calpain inhibitor N-acetyl-leucyl-leucyl-norleucinal (ALLN) has been reported to have complex effects on the production of the ␤-amyloid peptide (A␤). In this study, the effects of ALLN on the processing of the amyloid precursor protein (APP) to A␤ were examined in 293 cells expressing APP or the C-terminal 100 amino acids of APP (C100). In cells expressing APP or low levels of C100, ALLN increased A␤40 and A␤42 secretion at low concentrations, decreased A␤40 and A␤42 secretion at high concentrations, and increased cellular levels of C100 in a concentration-dependent manner by inhibiting C100 degradation. Low concentrations of ALLN increased A␤42 secretion more dramatically than A␤40 secretion. ALLN treatment of cells expressing high levels of C100 did not alter cellular C100 levels and inhibited A␤40 and A␤42 secretion with similar IC 50 values. These results suggest that C100 can be processed both by ␥-secretase and by a degradation pathway that is inhibited by low concentrations of ALLN. The data are consistent with inhibition of ␥-secretase by high concentrations of ALLN but do not support previous assertions that ALLN is a selective inhibitor of the ␥-secretase producing A␤40. Rather, A␤42 secretion may be more dependent on C100 substrate concentration than A␤40 secretion.The ␤-amyloid peptide (A␤) 1 is the major protein component of the senile plaques found in the brain of Alzheimer's disease (AD) patients. A␤ is produced by proteolysis of a single transmembrane domain protein known as the amyloid precursor protein (APP) (reviewed in Refs. 1 and 2). The first step in A␤ production involves the cleavage of APP by an uncharacterized protease termed ␤-secretase. Cleavage of APP by ␤-secretase produces a large ectodomain protein known as APPs␤, which is ultimately secreted, and a C-terminal 14-kDa membranebound fragment known as C100 (also termed C99 in some references). C100 is subsequently cleaved by ␥-secretase, another uncharacterized protease that cleaves within the transmembrane domain of C100 and produces the 39 -43-amino acid A␤ peptides. A␤40 is the dominant species of A␤ secreted from cultured cells and is also more abundant in cerebrospinal fluid of normal and AD patients. A␤42, which comprises about 5-10% of total A␤ secreted from cultured cells, is more amyloidogenic and is the major species of A␤ that is deposited at the early stage of senile plaques formation.Familial AD has thus far been associated with autosomal dominant mutations in the genes encoding APP, presenilin 1 (PS1), and presenilin 2 (2, 3). Multiple mutations in these three genes are associated with increased A␤42 production (4 -6). Collectively, these data suggest that excessive A␤42 production is critical for the development of AD. Whereas the locations of mutations in the APP gene suggest that the mutations lead to increased A␤42 production by increasing cleavage of APP by ␤-or ␥-secretase, the mechanism by which presenilin mutations increase A␤42 production remains unclear. Primary neuronal cultures derived from PS1 knock-ou...