Metalloprotease-Dependent S2'-Activation Promotes Cell-Cell Fusion and Syncytiation of SARS-CoV-2.

SARS-CoV-2 cell-cell fusion and syncytiation is an emerging pathomechanism in COVID-19, but the precise factors contributing to the process remain ill-defined. In this study, we show that metalloproteases promote SARS-CoV-2 spike protein-induced syncytiation in the absence of established serine proteases using in vitro cell-cell fusion assays. We also show that metalloproteases promote S2'-activation of the SARS-CoV-2 spike protein, and that metalloprotease inhibition significantly reduces the syncytiation of SARS-CoV-2 variants of concern. In the presence of serine proteases, however, metalloprotease inhibition does not reduce spike protein-induced syncytiation and a combination of metalloprotease and serine protease inhibition is necessitated. Moreover, we show that the spike protein induces metalloprotease-dependent ectodomain shedding of the ACE2 receptor and that ACE2 shedding contributes to spike protein-induced syncytiation. These observations suggest a benefit to the incorporation of pharmacological inhibitors of metalloproteases into treatment strategies for patients with COVID-19.

In Silico Identification of Potential Phosphorylation in the Cytoplasmic Domain of Epithelial Cell Adhesion Molecule.

The epithelial cell adhesion molecule (EpCAM) is a transmembrane cell adhesion glycoprotein, which primarily contributes to stemness, proliferation, and metastasis properties of tumor cells. Regulated intramembrane proteolysis by ADAM proteases and γ-secretase cleaves EpCAM into an ∼27 kDa soluble extracellular and an ∼4 kDa cytoplasmic domain (EpICD). After the EpICD fragment is released inside the cell, the formation of a nuclear signaling complex with the FHL2 molecule is critical for exerting its regulatory role. Trop-2, a homologous protein of EpCAM, undergoes phosphorylation in its cytoplasmic domain (Trop-IC). The phosphorylation of Trop-2 is reported to be crucial for its function. This led us to ask the fundamental question if EpCAM does undergo similar post-translational modification(PTM) like its homologous protein to carry out its diverse biological function. Here, we identify a putative phosphorylation site at Tyr297 located in the cytoplasmic domain of EpCAM. Molecular dynamic simulation (MDS) of 90 ns was carried out to understand the biological/functional relevance of the putative phosphorylation. It was observed that this phosphorylation stabilizes the α-helical structure of the EpICD. Though Tyr297 does not affect the γ-secretase mediated cleavage of EpCAM, it affects the binding of EpICD to FHL2. Docking analysis revealed that phosphorylation mediated structural stability of EpICD positively impacts its binding affinity with FHL2, which was further validated using 100 ns MDS. Phosphorylated EpICD forms higher numbers of hydrogen bonds, salt bridges, and other non-bonded interactions with FHL2, leading to enhanced interactions. This in silico study reveals a potential PTM in the EpICD, providing the basis for future research in understanding the mechanism behind the diverse biological function of EpCAM.

Price, Cost, and Value of Cancer Medicines: A Pharmaceutical Industry Perspective.

More than 1.8 million cancer diagnoses will be made in 2020 driving substantial health and economic burden for patients. The financial impact of out-of-pocket payments for hospital stays, outpatient services, physician appointments, and prescription drugs is a particular challenge. At the same time, the treatment of cancer is undergoing substantial transformation with growing benefits for patients. The complex factors contributing to the economic burden must be addressed so that patients have broad access to innovative oncology medicines both today and tomorrow. There are 2 parallel actions that are needed to drive broad reductions in costs while not putting at risk the incredible potential innovation awaiting these same patients: (i) the private sector must work together across the health care sector to accelerate innovative value-based partnerships; and (2) policymakers need to drive policy reforms that help ease out-of-pocket costs and remove barriers to and enable scaling of value-based care.

The anaphylatoxin C3a primes model colonic epithelial cells for expression of inflammatory mediators through Gαi.

Multiple studies have identified that complement becomes activated during inflammation of the intestines yet it is unclear what roles the split complement molecules play. The epithelium, in particular, may be impacted and accordingly, we first discovered that colonic cell lines indeed possess the C5aR. Here we examined whether these cells also possess the C3aR. We determined that T84, HT-29 and Caco2 all possess C3aR mRNA and protein; T84 and HT29 were used to further explore the consequence of C3a binding the C3aR. C3a led to increased mRNA for CXCL2, CXCL8 and CXCL11. Polarized T84 monolayers responded to apically applied C3a with increased CXCL8 mRNA more rapidly than if the C3a was applied basolaterally. Polarized monolayers also increased permeability when treated with C3a. ERK1/2 was activated by C3a and the increase in CXCL8 mRNA was ERK-dependent in both T84 and HT-29. C3a resulted in activation of Gαi, determined by the ERK1/2 signal showing sensitivity to pertussis toxin. The transmembrane signal was further mapped to include Ras and c-Raf. Finally, we show that the C3aR is expressed by primary cells in mouse enteroids. We conclude that complement activation will contribute to the epithelial response during inflammation through C3a binding to the C3aR including by priming the cells to upregulate mRNA for selected chemokines.

Enhancing the Cancer Cell Growth Inhibitory Effects of Table Grape Anthocyanins.

The risk for breast and colon cancer may be lowered in part by high intake of fruits and vegetables. Fruits such as grapes are abundant in bioactive compounds such as anthocyanins. The potential anticancer activity of anthocyanins may be limited by their metabolism in the gut and liver. One metabolic transformation is due to the enzyme catechol-O-methyltransferase (COMT), which methylates polyphenols such as anthocyanins. Entacapone is a clinically used inhibitor of COMT, and has been shown to modulate the methylation of food-derived polyphenols. In this study, we compared the effect of entacapone on the cell viability of colon (Caco-2 and HT-29) and breast (MDA-MB-231) cancer cell lines treated with anthocyanins. Cells were treated with either cyanidin-3-glucoside, delphinidin-3-glucoside, or an anthocyanin-rich grape extract, in the absence or presence of entacapone. Cell viability was assessed using the thiazolyl blue tetrazolium bromide (MTT) assay. Entacapone in combination with the anthocyanins had a greater than additive effect on growth inhibition of the Caco-2 cells. In the MDA-MB-231 cell line, entacapone similarly enhanced the growth inhibitory activity of the anthocyanin extract. Entacapone also had antiproliferative effects when used as a single treatment. Total hydroperoxides was quantified in the cell culture media. Greater concentrations of the treatments resulted in higher levels of total hydroperoxides, indicating that oxidative stress may be an important mechanism for growth inhibition. In conclusion, the antiproliferative activity of fruit-derived anthocyanins was improved in human cancer cell lines by the clinically used drug entacapone. The efficacy and mechanisms of entacapone/anthocyanin combinations should be carefully studied in vivo. PRACTICAL APPLICATION: Chemical components of grapes are good for our health and have been shown to lower risk for certain cancers. Their beneficial health effects could also be enhanced by consuming other molecules that improve their bioavailability.

Regulated intramembrane proteolysis: emergent role in cell signalling pathways.

Receptor signalling events including those initiated following activation of cytokine and growth factor receptors and the well-characterised death receptors (tumour necrosis factor receptor, type 1, FasR and TRAIL-R1/2) are initiated at the cell surface through the recruitment and formation of intracellular multiprotein signalling complexes that activate divergent signalling pathways. Over the past decade, research studies reveal that many of these receptor-initiated signalling events involve the sequential proteolysis of specific receptors by membrane-bound proteases and the γ-secretase protease complexes. Proteolysis enables the liberation of soluble receptor ectodomains and the generation of intracellular receptor cytoplasmic domain fragments. The combined and sequential enzymatic activity has been defined as regulated intramembrane proteolysis and is now a fundamental signal transduction process involved in the termination or propagation of receptor signalling events. In this review, we discuss emerging evidence for a role of the γ-secretase protease complexes and regulated intramembrane proteolysis in cell- and immune-signalling pathways.

How Should We Intervene on the Financial Toxicity of Cancer Care? One Shot, Four Perspectives.

The median price of a month of chemotherapy has increased by an order of magnitude during the past 20 years, far exceeding inflation over the same period. Along with rising prices, increases in cost sharing have forced patients to directly shoulder a greater portion of those costs, resulting in undue financial burden and, in some cases, cost-related nonadherence to treatment. What can we do to intervene on treatment-related financial toxicity of patients? No one party can single-handedly solve the problem, and the solution must be multifaceted and creative. A productive discussion of the problem must avoid casting blame and, instead, must look inward for concrete starting points toward improvement in the affordability and value of cancer care. With these points in mind, the authors-representatives from the pharmaceutical industry, insurance providers, oncologists, and patient advocacy-have each been asked to respond with a practical answer to the provocative hypothetical question, "If you could propose one thing, and one thing only, in terms of an action or change by the constituency you represent in this discussion, what would that be?"

γ-Secretase Activity Is Required for Regulated Intramembrane Proteolysis of Tumor Necrosis Factor (TNF) Receptor 1 and TNF-mediated Pro-apoptotic Signaling.

The γ-secretase protease and associated regulated intramembrane proteolysis play an important role in controlling receptor-mediated intracellular signaling events, which have a central role in Alzheimer disease, cancer progression, and immune surveillance. An increasing number of γ-secretase substrates have a role in cytokine signaling, including the IL-6 receptor, IL-1 receptor type I, and IL-1 receptor type II. In this study, we show that following TNF-converting enzyme-mediated ectodomain shedding of TNF type I receptor (TNFR1), the membrane-bound TNFR1 C-terminal fragment is subsequently cleaved by γ-secretase to generate a cytosolic TNFR1 intracellular domain. We also show that clathrin-mediated internalization of TNFR1 C-terminal fragment is a prerequisite for efficient γ-secretase cleavage of TNFR1. Furthermore, using in vitro and in vivo model systems, we show that in the absence of presenilin expression and γ-secretase activity, TNF-mediated JNK activation was prevented, assembly of the TNFR1 pro-apoptotic complex II was reduced, and TNF-induced apoptosis was inhibited. These observations demonstrate that TNFR1 is a γ-secretase substrate and suggest that γ-secretase cleavage of TNFR1 represents a new layer of regulation that links the presenilins and the γ-secretase protease to pro-inflammatory cytokine signaling.

Loss of Presenilin 2 Function Is Associated with Defective LPS-Mediated Innate Immune Responsiveness.

The importance of presenilin-dependent γ-secretase protease activities in the development, neurogenesis, and immune system is highlighted by the diversity of its substrates and characterization of Psen1- and Psen2-deficient transgenic animals. Functional differences between presenilin 1 (PS1) and presenilin 2 (PS2) are incompletely understood. In this study, we have identified a Psen2-specific function, not shared by Psen1 in Toll-like receptor signaling. We show that immortalized fibroblasts and bone marrow-derived macrophages from Psen2- but not Psen1-deficient mice display reduced responsiveness to lipopolysaccharide (LPS) with decreased nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and mitogen-activated protein kinase (MAPK) activity and diminished pro-inflammatory cytokine production. In whole animal in vivo responses, Psen2-deficient animals have abnormal systemic production of LPS-stimulated pro-inflammatory cytokines. Mechanistically, we demonstrate that Psen2 deficiency is paralleled by reduced transcription of tlr4 mRNA and loss of LPS-induced tlr4 mRNA transcription regulation. These observations illustrate a novel PS2-dependent means of modulating LPS-mediated immune responses and identify a functional distinction between PS1 and PS2 in innate immunity.

Beyond γ-secretase activity: The multifunctional nature of presenilins in cell signalling pathways.

The presenilins are the catalytic subunit of the membrane-embedded tetrameric γ-secretase protease complexes. More that 90 transmembrane proteins have been reported to be γ-secretase substrates, including the widely studied amyloid precursor protein (APP) and the Notch receptor, which are precursors for the generation of amyloid-ß peptides and biologically active APP intracellular domain (AICD) and Notch intracellular domain (NICD). The diversity of γ-secretase substrates highlights the importance of presenilin-dependent γ-secretase protease activities as a regulatory mechanism in a range of biological systems. However, there is also a growing body of evidence that supports the existence of γ-secretase-independent functions for the presenilins in the regulation and progression of an array of cell signalling pathways. In this review, we will present an overview of current literature that proposes evolutionarily conserved presenilin functions outside of the γ-secretase complex, with a focus on the suggested role of the presenilins in the regulation of Wnt/ß-catenin signalling, protein trafficking and degradation, calcium homeostasis and apoptosis.

Gamma-secretase-independent role for cadherin-11 in neurotrophin receptor p75 (p75(NTR)) mediated glioblastoma cell migration.

The p75 neurotrophin receptor (p75(NTR)) undergoes γ-secretase-mediated regulated intramembrane proteolysis and is involved in glioblastoma cell migration and invasion. Consistent with previous reports, in this study we show that p75NTR increases U87-MG glioblastoma cell migration, which is reversed by inhibition of γ-secretase activity. However, we show that expression or stabilization of the γ-secretase-generated p75(NTR) intracellular domain (ICD) is not sufficient to induce U87-MG glioblastoma cell migration, and that exogenous expression of p75(NTR) ICD inhibits p75(NTR)-mediated glioblastoma cell (U87-MG and U373-MG) migration. To identify pathways and to determine how p75(NTR) mediates glioblastoma migration we utilized a microarray approach to assess differential gene expression profiles between parental U87-MG and cells stably expressing wild-type p75(NTR), a γ-secretase cleavage-resistant chimeric p75(NTR) mutant (p75FasTM) and the γ-secretase-generated p75(NTR)-ICD, which mimics constitutively cleaved p75(NTR) receptor. In our microarray data analysis we identified a subset of genes that were constitutively up-regulated in wild-type p75(NTR) cells, which were also repressed in p75(NTR) ICD expressing cells. Furthermore, our data revealed among the many differentially expressed genes, cadherin-11 (Cdh-11), matrix metalloproteinase 12 and relaxin/insulin-like family peptide receptor 2 as constitutively up-regulated in wild-type p75(NTR) cells, independent of γ-secretase activity. Consistent with a role in glioblastoma migration, we found that U87-p75(NTR) cells express higher levels of Cdh-11 protein and that siRNA-mediated knockdown of Cdh-11 resulted in a significant decrease in p75(NTR)-mediated glioblastoma cell migration. Therefore, we hypothesize that p75(NTR) can impact U87-MG glioblastoma cell migration in a γ-secretase-independent manner through modulation of specific genes, including Cdh-11, and that both γ-secretase-independent and -dependent mechanisms are involved in p75(NTR)-mediated U87-MG glioblastoma cell migration.

A ubiquitin-binding CUE domain in presenilin-1 enables interaction with K63-linked polyubiquitin chains.

The presenilins (PS1 and PS2) are the catalytic component of the γ-secretase intramembrane protease complex, involved in the regulated intramembrane proteolysis of numerous type I transmembrane proteins, including amyloid precursor protein (APP) and Notch. Herein, we describe the identification and characterization of a CUE (coupling of ubiquitin conjugation to endoplasmic reticulum degradation) ubiquitin-binding domain (UBD) in PS1, and demonstrate that the CUE domain of PS1 mediates non-covalent binding to Lysine 63-linked polyubiquitin chains. Our results highlight a γ-secretase-independent function for non-covalent ubiquitin signaling in the regulation of PS1, and add new insights into the structure and function of the presenilin proteins.

Presenilins are novel substrates for TRAF6-mediated ubiquitination.

Mutations in presenilins (PS1 and PS2) have been linked to the pathogenesis of early onset familial Alzheimer's disease. Presenilins function as the catalytic component of the γ-secretase protease complexes responsible for the cleavage of the amyloid precursor protein (APP), subsequent generation of amyloid-ß and associated amyloid plaques in Alzheimer's disease. Biochemical and genetic studies have revealed that through interactions with several proteins, the presenilins are functionally involved in a range of cellular processes, including the regulation of intracellular calcium homeostasis. Our group has previously reported an association between presenilins and members of the tumour necrosis factor receptor-associated factor (TRAF) family of proteins. In this study we further investigated the association between TRAF6, an E3 ubiquitin ligase, and the presenilins. Here we show that the presenilin full-length holoproteins are novel substrates of TRAF6-mediated Lysine-63-linked ubiquitination. Interestingly, co-expression of catalytically active TRAF6 with the presenilins leads to decreased turnover of PS1 full-length holoprotein accompanying elevated presenilin protein levels. Similarly, while overexpression of TRAF6 increases presenilin holoprotein levels and ubiquitination in HEK293 cells, expression of catalytically deficient TRAF6 or TRAF6-deficiency leads to a reduction in presenilin protein levels and reduced PS1 ubiquitination. We also demonstrate that TRAF6 induces PS1 gene transcription in a JNK-dependent manner. Notably, we reveal that TRAF6-mediated ubiquitination of presenilin does not affect γ-secretase enzyme activity, but may regulate presenilin function in calcium signalling. Taken together, we propose that presenilins are novel substrates for TRAF6-mediated K63-linked ubiquitination and that ubiquitination of presenilins by TRAF6 increases presenilin holoprotein levels and in conditions in which TRAF6 ubiquitination of presenilins is reduced results in reduction of calcium release from the endoplasmic reticulum.

Semagacestat, a γ-secretase inhibitor, activates the growth hormone secretagogue (GHS-R1a) receptor.

OBJECTIVES: Semagacestat, is a γ-secretase inhibitor, which belongs to a class of drugs that are being developed as therapeutic agents for Alzheimer's disease (AD). This study aims to evaluate another potential effect of semagacestat, namely its ability to stimulate the growth hormone secretagogue receptor (GHS-R1a), which may also contribute to its therapeutic efficacy. METHODS: The GHS-R1a-activating potential of semagacestat and its synthetic precursor was assessed in an in vitro calcium mobilization assay in cells expressing the GHS-R1a receptor and compared with that of the endogenous peptide GHS-R1a agonist, acyl-ghrelin, as well as the non-peptidyl synthetic GHS-R1a agonist, MK0677. In addition, semagacestat-mediated cellular trafficking of the GHS-R1a receptor, expressed as an enhanced green fluorescent protein tagged fusion protein, was analysed. KEY FINDINGS: Semagacestat and its precursor were shown to activate the GHS-R1a receptor, as demonstrated by an increased GHS-R1a-mediated intracellular calcium influx. Moreover, a synergistic GHS-R1a receptor activation was shown following a combined exposure to ghrelin and semagacestat. In addition, GHS-R1a receptor internalization was observed upon exposure to semagacestat and its precursor. CONCLUSION: These data suggest a novel molecular mechanism of action for semagacestat via modest GHS-R1a receptor activation. Studies focusing on the relative functional consequence of such effects in vivo are now warranted.

TRAF6 promotes ubiquitination and regulated intramembrane proteolysis of IL-1R1.

It has recently been shown that Interleukin-1 receptor, type 1, an essential regulator of inflammation and inate immunity, undergoes regulated intramembrane proteolysis (RIP). Although IL-1R1-mediated intracellular signalling has been well studied, very little is known about how RIP of IL-1R1 is modulated. In this study, by using wild-type TRAF6 and TRAF6 mutants that are defective in its ubiquitin ligase activity, we show for the first time that TRAF6 induces ubiquitination of IL-1R1. We further demonstrate that of all TRAF family members examined, TRAF6 preferentially ubiquitinates IL-1R1. Moreover, we show that TRAF6 ubiquitin ligase activity and ubiquitination of IL-1R1 are positively correlated with IL-1R1 ectodomain shedding and subsequent gamma-secretase cleavage. Our results indicate that TRAF6-mediated ubiquitination of IL-1R1 has a decisive role in IL-1R1 signalling and propose a molecular mechanism whereby TRAF6 promotes ubiquitination and RIP of IL-1R1 through its ubiquitin ligase activity.

Interleukin-1 receptor type 1 is a substrate for gamma-secretase-dependent regulated intramembrane proteolysis.

Biochemical and genetic studies have revealed that the presenilins interact with several proteins and are involved in the regulated intramembrane proteolysis of numerous type 1 membrane proteins, thereby linking presenilins to a range of cellular processes. In this study, we report the characterization of a highly conserved tumor necrosis factor receptor-associated factor-6 (TRAF6) consensus-binding site within the hydrophilic loop domain of presenilin-1 (PS-1). In coimmunoprecipitation studies we indicate that presenilin-1 interacts with TRAF6 and interleukin-1 receptor-associated kinase 2. Substitution of presenilin-1 residues Pro-374 and Glu-376 by site-directed mutagenesis greatly reduces the ability of PS1 to associate with TRAF6. By studying these interactions, we also demonstrate that the interleukin-1 receptor type 1 (IL-1R1) undergoes intramembrane proteolytic processing, mediated by presenilin-dependent gamma-secretase activity. A metalloprotease-dependent proteolytic event liberates soluble IL-1R1 ectodomain and produces an approximately 32-kDa C-terminal domain. This IL-1R1 C-terminal domain is a substrate for subsequent gamma-secretase cleavage, which generates an approximately 26-kDa intracellular domain. Specific pharmacological gamma-secretase inhibitors, expression of dominant negative presenilin-1, or presenilin deficiency independently inhibit generation of the IL-1R1 intracellular domain. Attenuation of gamma-secretase activity also impairs responsiveness to IL-1beta-stimulated activation of the MAPKs and cytokine secretion. Thus, TRAF6 and interleukin receptor-associated kinase 2 are novel binding partners for PS1, and IL-1R1 is a new substrate for presenilin-dependent gamma-secretase cleavage. These findings also suggest that regulated intramembrane proteolysis may be a control mechanism for IL-1R1-mediated signaling.

Association between Presenilin-1 and TRAF6 modulates regulated intramembrane proteolysis of the p75NTR neurotrophin receptor.

The p75 neurotrophin receptor (p75(NTR)) is a member of the tumour necrosis factor superfamily, which relies on the recruitment of cytosolic protein partners including the tumour necrosis factor receptor-associated factor 6 (TRAF6) E3 ubiquitin ligase to produce cellular responses. Recently, p75(NTR) was also shown to undergo presenilin-dependent, gamma-secretase-mediated regulated intramembrane proteolysis. In this study, we report the characterization of a highly conserved TRAF6-binding site (PxExxAr/Ac) in presenilin-1 (PS1) that mediates nerve growth factor (NGF)-induced association between PS1 and TRAF6. We demonstrate that disruption of this interaction between PS1 and TRAF6 inhibits TRAF6 autoubiquitination and gamma-secretase cleavage of p75(NTR). Additionally, we show that PS1-deficiency antagonizes NGF-induced I-kappaB degradation. Finally, we also show that p75(NTR) is a substrate for TRAF6-mediated ubiquitination and that TRAF6 E3 ligase activity is required for regulated intramembrane proteolysis of p75(NTR). In summary, our data suggest that an NGF-induced association between PS1 and TRAF6 influences regulated intramembrane proteolysis of p75(NTR).

The insulin-like growth factor 1 (IGF-1) receptor is a substrate for gamma-secretase-mediated intramembrane proteolysis.

Several type-1 membrane proteins undergo regulated intramembrane proteolysis resulting in the generation of biologically active protein fragments. Presenilin-dependant gamma-secretase activity is central to this event and includes amyloid precursor protein (APP), Notch and ErbB4 as substrates. Here we show that the insulin-like growth factor 1 receptor (IGF-IR) undergoes regulated intramembrane proteolysis. A metalloprotease-dependant ectodomain-shedding event generates a approximately 52 kDa IGF-IR-carboxyl terminal domain (CTD). The IGF-IR-CTD is consequentially a substrate for gamma-secretase cleavage, liberating a approximately 50 kDa intracellular domain (ICD) that can be inhibited by a specific gamma-secretase inhibitor. This study suggests that the IGF-IR is a substrate for gamma-secretase and may mediate a function independent of its role as a receptor tyrosine kinase.

Presenilin-1 is an unprimed glycogen synthase kinase-3beta substrate.

Previously we described presenilin-1 (PS1) as a GSK-3beta substrate [Kirschenbaum, F., Hsu, S.C., Cordell, B. and McCarthy, J.V. (2001) Substitution of a glycogen synthase kinase-3beta phosphorylation site in presenilin 1 separates presenilin function from beta-catenin signalling. J. Biol. Chem. 276, 7366-7375; Kirschenbaum, F., Hsu, S.C., Cordell, B. and McCarthy, J.V. (2001) Glycogen synthase kinase-3beta regulates presenilin 1 C-terminal fragment levels. J. Biol. Chem. 276, 30701-30707], though it has not been determined whether PS1 is a primed or unprimed GSK-3beta substrate. A means of separating GSK-3beta activity toward primed and unprimed substrates was identified in the GSK-3beta-R96A phosphate binding pocket mutant [Frame, S., Cohen, P. and Biondi, R.M. (2001) A common phosphate binding site explains the unique substrate specificity of GSK3 and its inactivation by phosphorylation. Mol. Cell 7, 1321-1327], which is unable to phosphorylate primed but retains the ability to phosphorylate unprimed GSK-3beta substrates. By using wild type GSK-3beta, GSK-3beta-R96A, and a pharmacological modulator of GSK-3beta activity, we demonstrate that PS1 is an unprimed GSK-3beta substrate. These findings have important implications for regulation of PS1 function and the pathogenesis of Alzheimer's disease.

Apoptosis and development.

Apoptosis is an evolutionarily conserved process used by multicellular organisms to developmentally regulate cell number or to eliminate cells that are potentially detrimental to the organism. The large diversity of regulators of apoptosis in mammalian cells and their numerous interactions complicate the analysis of their individual functions, particularly in development. The remarkable conservation of apoptotic mechanisms across species has allowed the genetic pathways of apoptosis determined in lower species, such as the nematode Caenorhabditis elegans and the fruitfly Drosophila melanogaster, to act as models for understanding the biology of apoptosis in mammalian cells. Though many components of the apoptotic pathway are conserved between species, the use of additional model organisms has revealed several important differences and supports the use of model organisms in deciphering complex biological processes such as apoptosis.