Development of a fluorescence and quencher-based FRET assay for detection of endogenous peptide:N-glycanase/NGLY1 activity.

Cytosolic peptide:N-glycanase (PNGase/NGLY1 in mammals) catalyzes deglycosylation of N-glycans on glycoproteins. A genetic disorder caused by mutations in the NGLY1 gene leads to NGLY1 deficiency with symptoms including motor deficits and neurological problems. Effective therapies have not been established, though, a recent study used the administration of an adeno-associated viral vector expressing human NGLY1 to dramatically rescue motor functions in young Ngly1 rats.

Amino acid editing of NFE2L1 by PNGase causes abnormal mobility on SDS-PAGE.

NFE2L1 (also known as NRF1) is a member of the nuclear erythroid 2-like family of transcription factors and is critical for counteracting various types of cellular stress such as oxidative, proteotoxic or metabolic stress. This unique transcription factor is also known to undergo changes, including post-translational modifications, limited proteolysis or translocation into the nucleus, before it exerts full transcriptional activity. As a result, there are various molecular forms with distinct sizes for this protein, while the precise nature of each form remains elusive.

O-GalNAc glycosylation determines intracellular trafficking of APP and Aβ production.

A primary pathology of Alzheimer's disease (AD) is amyloid β (Aβ) deposition in brain parenchyma and blood vessels, the latter being called cerebral amyloid angiopathy (CAA). Parenchymal amyloid plaques presumably originate from neuronal Aβ precursor protein (APP). Although vascular amyloid deposits' origins remain unclear, endothelial APP expression in APP knock-in mice was recently shown to expand CAA pathology, highlighting endothelial APP's importance.

Endothelial expression of human amyloid precursor protein leads to amyloid β in the blood and induces cerebral amyloid angiopathy in knock-in mice.

The deposition of amyloid β (Aβ) in blood vessels of the brain, known as cerebral amyloid angiopathy (CAA), is observed in most patients with Alzheimer's disease (AD). Compared with the pathology of CAA in humans, the pathology in most mouse models of AD is not as evident, making it difficult to examine the contribution of CAA to the pathogenesis of AD. On the basis of biochemical analyses that showed blood levels of soluble amyloid precursor protein (APP) in rats and mice were markedly lower than those measured in human samples, we hypothesized that endothelial APP expression would be markedly lower in rodents and subsequently generated mice that specifically express human WT APP (APP770) in endothelial cells (ECs).

A method for assaying peptide: N-glycanase/N-glycanase 1 activities in crude extracts using an N-glycosylated cyclopeptide.

Cytosolic peptide: N-glycanase (PNGase; NGLY1), an enzyme responsible for de-glycosylation of N-glycans on glycoproteins, is known to play pivotal roles in a variety of biological processes. In 2012, NGLY1 deficiency, a rare genetic disorder, was reported and since then, more than 100 patients have now been identified worldwide. Patients with this disease exhibit several common symptoms that are caused by the dysfunction of NGLY1.

Regulation of BMP4/Dpp retrotranslocation and signaling by deglycosylation.

During endoplasmic reticulum-associated degradation (ERAD), the cytoplasmic enzyme -glycanase 1 (NGLY1) is proposed to remove -glycans from misfolded -glycoproteins after their retrotranslocation from the ER to the cytosol. We previously reported that NGLY1 regulates BMP signaling in a tissue-specific manner (Galeone et al., 2017).

Chlamydia trachomatis-infected human cells convert ceramide to sphingomyelin without sphingomyelin synthases 1 and 2.

The obligate intracellular bacterium Chlamydia trachomatis proliferates in the membranous compartment inclusion formed in host cells. The host ceramide transport protein CERT delivers ceramide from the endoplasmic reticulum to the Golgi complex for the synthesis of sphingomyelin (SM). Chlamydia trachomatis has been suggested to employ CERT to produce SM in the inclusion by host SM synthases (SMSs).

A CRISPR Screen Identifies LAPTM4A and TM9SF Proteins as Glycolipid-Regulating Factors.

Glycosphingolipids (GSLs) are produced by various GSL-synthesizing enzymes, but post-translational regulation of these enzymes is incompletely understood. To address this knowledge disparity, we focused on biosynthesis of globotriaosylceramide (Gb3), the Shiga toxin (STx) receptor, and performed a genome-wide CRISPR/CAS9 knockout screen in HeLa cells using STx1-mediated cytotoxicity. We identified various genes including sphingolipid-related genes and membrane-trafficking genes.

Excess APP O-glycosylation by GalNAc-T6 decreases Aβ production.

Alterations of the structure and/or amount of glycans present on proteins are associated with many diseases. We previously demonstrated that changes in N-glycans alter Aβ production. In the present study, we focused on the relationship between Alzheimer's disease (AD) and O-glycan, another type of glycan.

Role of Intracellular Lipid Logistics in the Preferential Usage of Very Long Chain-Ceramides in Glucosylceramide.

Ceramide is a common precursor of sphingomyelin (SM) and glycosphingolipids (GSLs) in mammalian cells. Ceramide synthase 2 (CERS2), one of the six ceramide synthase isoforms, is responsible for the synthesis of very long chain fatty acid (C20-26 fatty acids) (VLC)-containing ceramides (VLC-Cer). It is known that the proportion of VLC species in GSLs is higher than that in SM.

Soluble amyloid precursor protein 770 is a novel biomarker candidate for acute coronary syndrome.

Most Alzheimer disease patients show deposition of amyloid β (Aβ) peptide in blood vessels as well as the brain parenchyma. We previously found that vascular endothelial cells express amyloid β precursor protein (APP) 770, a different APP isoform from neuronal APP695, and that they produce amyloid β peptide. We analyzed the glycosylation of APP770 and found that O-glycosylated sAPP770 is preferentially processed by proteases for Aβ production.

Soluble amyloid precursor protein 770 is released from inflamed endothelial cells and activated platelets: a novel biomarker for acute coronary syndrome.

Background: Separate monitoring of the cleavage products of different amyloid β precursor protein (APP) variants may provide useful information.

Results: We found that soluble APP770 (sAPP770) is released from inflamed endothelial cells and activated platelets as judged by ELISA.

Conclusion: sAPP770 is an indicator for endothelial and platelet dysfunctions.

Brain endothelial cells produce amyloid {beta} from amyloid precursor protein 770 and preferentially secrete the O-glycosylated form.

Deposition of amyloid β (Aβ) in the brain is closely associated with Alzheimer disease (AD). Aβ is generated from amyloid precursor protein (APP) by the actions of β- and γ-secretases. In addition to Aβ deposition in the brain parenchyma, deposition of Aβ in cerebral vessel walls, termed cerebral amyloid angiopathy, is observed in more than 80% of AD individuals.

Interleukin-1 beta up-regulates TACE to enhance alpha-cleavage of APP in neurons: resulting decrease in Abeta production.

The proinflammatory cytokine interleukin (IL)-1beta is up-regulated in microglial cells surrounding amyloid plaques, leading to the hypothesis that IL-1beta is a risk factor for Alzheimer's disease. However, we unexpectedly found that IL-1beta significantly enhanced alpha-cleavage, indicated by increases in sAPPalpha and C83, but reduced beta-cleavage, indicated by decreases in sAPPbeta and Abeta40/42, in human neuroblastoma SK-N-SH cells. IL-1beta did not significantly alter the mRNA levels of BACE1, ADAM-9, and ADAM-10, but up-regulated that of TACE by threefold.

Screening a series of sialyltransferases for possible BACE1 substrates.

Deposition of amyloid beta-peptide (Abeta) and neurofibrillary tangles in the brain are hallmarks of Alzheimer's disease (AD) pathogenesis. BACE1, a membrane-bound aspartic protease that cleaves amyloid precursor protein (APP) to produce Abeta, has been implicated in triggering the pathogenesis of the disease. We previously reported that BACE1 also cleaved alpha2,6-sialyltransferase (ST6Gal I) in the Golgi apparatus and induced its secretion from the cell.

In vivo cleavage of alpha2,6-sialyltransferase by Alzheimer beta-secretase.

beta-Site amyloid precursor protein-cleaving enzyme 1 (BACE1) is a membrane-bound aspartic protease that cleaves amyloid precursor protein to produce a neurotoxic peptide, Abeta, and is implicated in triggering the pathogenesis of Alzheimer disease. We previously reported that BACE1 cleaved rat beta-galactoside alpha2,6-sialyltransferase (ST6Gal I) that was overexpressed in COS cells and that the NH(2) terminus of ST6Gal I secreted from the cells (E41 form) was Glu(41). Here we report that BACE1 gene knock-out mice have one third as much plasma ST6Gal I as control mice, indicating that BACE1 is a major protease which is responsible for cleaving ST6Gal I in vivo.

Characterization of alpha 2,6-sialyltransferase cleavage by Alzheimer's beta -secretase (BACE1).

BACE1 is a membrane-bound aspartic protease that cleaves the amyloid precursor protein (APP) at the beta-secretase site, a critical step in the Alzheimer disease pathogenesis. We previously found that BACE1 also cleaved a membrane-bound sialyltransferase, ST6Gal I. By BACE1 overexpression in COS cells, the secretion of ST6Gal I markedly increased, and the amino terminus of the secreted ST6Gal I started at Glu(41).