Biomedical subjects

Matthew J LaVoie

Publications and source records attributed to Matthew J LaVoie.

12 recordsLinked to original sources

PubMed2005

gamma-Secretase substrate selectivity can be modulated directly via interaction with a nucleotide-binding site.

gamma-Secretase is an unusual protease with an intramembrane catalytic site that cleaves many type I membrane proteins, including the amyloid beta-protein (Abeta) precursor (APP) and the Notch receptor. Genetic and biochemical studies have identified four membrane proteins as components of gamma-secretase: heterodimeric presenilin composed of its N- and C-terminal fragments, nicastrin, Aph-1, and Pen-2. Here we demonstrated that certain compounds, including protein kinase inhibitors and their derivatives, act directly on purified gamma-secretase to selectively block cleavage of APP- but not Notch-based substrates. Moreover, ATP activated the generation of the APP intracellular domain and Abeta, but not the generation of the Notch intracellular domain by the purified protease complex, and was a direct competitor of the APP-selective inhibitors, as were other nucleotides. In accord, purified gamma-secretase bound specifically to an ATP-linked resin. Finally, a photoactivable ATP analog specifically labeled presenilin 1-C-terminal fragments in purified gamma-secretase preparations; the labeling was blocked by ATP itself and APP-selective gamma-secretase inhibitors. We concluded that a nucleotide-binding site exists within gamma-secretase, and certain compounds that bind to this site can specifically modulate the generation of Abeta while sparing Notch. Drugs targeting the gamma-secretase nucleotide-binding site represent an attractive strategy for safely treating Alzheimer disease.

Adenosine Triphosphate↗

PubMed2005

Dopamine covalently modifies and functionally inactivates parkin.

Inherited mutations in PARK2, the gene encoding parkin, cause selective degeneration of catecholaminergic neurons in the substantia nigra and locus coeruleus of the brainstem, resulting in early-onset parkinsonism. But the role of parkin in common, sporadic forms of Parkinson disease remains unclear. Here we report that the neurotransmitter dopamine covalently modifies parkin in living dopaminergic cells, a process that increases parkin insolubility and inactivates its E3 ubiquitin ligase function. In the brains of individuals with sporadic Parkinson disease, we observed decreases in parkin solubility consistent with its functional inactivation. Using a new biochemical method, we detected catechol-modified parkin in the substantia nigra but not other regions of normal human brain. These findings show a vulnerability of parkin to modification by dopamine, the principal transmitter lost in Parkinson disease, suggesting a mechanism for the progressive loss of parkin function in dopaminergic neurons during aging and sporadic Parkinson disease.

Adult↗

PubMed2004

Purification and characterization of the human gamma-secretase complex.

Gamma-secretase is a member of an unusual class of proteases with intramembrane catalytic sites. This enzyme cleaves many type I membrane proteins, including the amyloid beta-protein (Abeta) precursor (APP) and the Notch receptor. Biochemical and genetic studies have identified four membrane proteins as components of gamma-secretase: heterodimeric presenilin (PS) composed of its N- and C-terminal fragments (PS-NTF/CTF), a mature glycosylated form of nicastrin (NCT), Aph-1, and Pen-2. Recent data from studies in Drosophila, mammalian, and yeast cells suggest that PS, NCT, Aph-1, and Pen-2 are necessary and sufficient to reconstitute gamma-secretase activity. However, many unresolved issues, in particular the possibility of other structural or regulatory components, would be resolved by actually purifying the enzyme. Here, we report a detailed, multistep purification procedure for active gamma-secretase and an initial characterization of the purified protease. Extensive mass spectrometry of the purified proteins strongly suggests that PS-NTF/CTF, mNCT, Aph-1, and Pen-2 are the components of active gamma-secretase. Using the purified gamma-secretase, we describe factors that modulate the production of specific Abeta species: (1) phosphatidylcholine and sphingomyelin dramatically improve activity without changing cleavage specificity within an APP substrate; (2) increasing CHAPSO concentrations from 0.1 to 0.25% yields a approximately 100% increase in Abeta42 production; (3) exposure of an APP-based recombinant substrate to 0.5% SDS modulates cleavage specificity from a disease-mimicking pattern (high Abeta42/43) to a physiological pattern (high Abeta40); and (4) sulindac sulfide directly and preferentially decreases Abeta42 cleavage within the purified complex. Taken together, our results define a procedure for purifying active gamma-secretase and suggest that the lipid-mediated conformation of both enzyme and substrate regulate the production of the potentially neurotoxic Abeta42 and Abeta43 peptides.

Amino Acid Sequence↗

PubMed2004

Detergent-dependent dissociation of active gamma-secretase reveals an interaction between Pen-2 and PS1-NTF and offers a model for subunit organization within the complex.

Gamma-secretase is a member of a new class of proteases with an intramembrane catalytic site and cleaves numerous type I membrane proteins, including the amyloid beta-protein precursor (APP) and the Notch receptor. Biochemical and genetic studies have identified four membrane proteins as components of gamma-secretase: a heterodimeric form of presenilin (PS), composed of its N- and C-terminal fragments (PS-NTF and PS-CTF, respectively), a highly glycosylated, mature form of nicastrin (NCT), Aph-1, and Pen-2. However, it is unclear how these components interact physically with each other and assemble into functional complexes. We and others recently found that Aph-1 interacts with a less glycosylated, immature form of nicastrin as an intermediate toward full assembly of gamma-secretase. Here we show that (1) the detergent dodecyl beta-d-maltoside (DDM) mediates the dissociation and inactivation of active gamma-secretase in a concentration-dependent manner, (2) DDM-dependent dissociation of the active gamma-secretase complex generates two major inactive complexes (Pen-2-PS1-NTF and mNCT-Aph-1) and two minor inactive complexes (mNCT-Aph1-PS1-CTF and PS1-NTF-PS1-CTF), and (3) Pen-2 can also associate with the PS holoprotein in complexes devoid of NCT and Aph-1. Taken together, our results demonstrate that Pen-2 interacts with PS-NTF within active gamma-secretase and offer a model for how the components of active gamma-secretase interact physically with each other.

Amyloid Precursor Protein Secretases↗

PubMed2004

Microglial activation precedes dopamine terminal pathology in methamphetamine-induced neurotoxicity.

Previous studies have demonstrated methamphetamine (METH)-induced toxicity to dopaminergic and serotonergic axons in rat striatum. Although several studies have identified the nature of reactive astrogliosis in this lesion model, the response of microglia has not been examined in detail. In this investigation, we characterized the temporal relationship of reactive microgliosis to neuropathological alterations of dopaminergic axons in striatum following exposure to methamphetamine. Adult male Sprague-Dawley rats were administered a neurotoxic regimen of methamphetamine and survived 12 h, or 1, 2, 4, and 6 days after treatment. Immunohistochemical methods were used to evaluate reactive changes in microglia throughout the brain of methamphetamine-treated rats, with a particular focus upon striatum. Pronounced morphological changes, indicative of reactive microgliosis, were evident in the brains of all methamphetamine-treated animals and were absent in saline-treated control animals. These included hyperplastic changes in cell morphology that substantially increased the size and staining intensity of reactive microglia. Quantitative analysis of reactive microglial changes in striatum demonstrated that these changes were most robust within the ventrolateral region and were maximal 2 days after methamphetamine administration. Analysis of tissue also revealed that microglial activation preceded the appearance of pathological changes in striatal dopamine fibers. Reactive microgliosis was also observed in extra-striatal regions (somatosensory and piriform cortices, and periaqueductal gray). These data demonstrate a consistent, robust, and selective activation of microglia in response to methamphetamine administration that, at least in striatum, precedes the appearance of morphological indicators of axon pathology. These observations raise the possibility that activated microglia may contribute to methamphetamine-induced neurotoxicity.

Animals↗

PubMed2004

Dimerization of Parkinson's disease-causing DJ-1 and formation of high molecular weight complexes in human brain.

Mutations in the DJ-1 gene have been implicated in the PARK7-linked autosomal recessive form of Parkinson's disease (PD). The molecular properties of DJ-1WT, DJ-1L166P, and a newly identified disease-causing mutant DJ-1M26I were explored after they were transiently expressed in mammalian cells. Treatment of intact, living cells with the chemical crosslinker disuccinimidyl suberate (DSS) revealed that DJ-1WT and mutant DJ-1M26I were present as stable homodimers; DJ-1L166P in particular tended to form high-order complexes as well. In contrast to DJ-1L166P that is quickly degraded by the proteasome, DJ-1M26I was found to be an efficiently expressed and stable variant of DJ-1, suggesting that these mutations have distinct biochemical effects on DJ-1. We further provide evidence that in human brain, under nondenaturing conditions, DJ-1 is present in high molecular weight (HMW) complexes of approximately 250-700 kDa containing parkin, another PD-associated protein.

Animals↗

PubMed2003

Assembly of the gamma-secretase complex involves early formation of an intermediate subcomplex of Aph-1 and nicastrin.

The gamma-secretase complex is an unusual multimeric protease responsible for the intramembrane cleavage of a variety of type 1 transmembrane proteins, including the beta-amyloid precursor protein and Notch. Genetic and biochemical data have revealed that this protease consists of the presenilin heterodimer, a highly glycosylated form of nicastrin, and the recently identified gene products, Aph-1 and Pen-2. Whereas current evidence supports the notion that presenilin comprises the active site of the protease and that the other three components are members of the active complex required for proteolytic activity, the individual roles of the three co-factors remain unclear. Here, we demonstrate that endogenous Aph-1 interacts with an immature species of nicastrin, forming a stable intermediate early in the assembly of the gamma-secretase complex, prior to the addition of presenilin and Pen-2. Our data suggest 1) that Aph-1 is involved in the early stages of gamma-secretase assembly through the stabilization and perhaps glycosylation of nicastrin and by scaffolding nicastrin to the immature gamma-secretase complex, and 2) that presenilin, and later Pen-2, bind to this intermediate during the formation of the mature protease.

Amyloid Precursor Protein Secretases↗

PubMed2003

The Notch ligands, Jagged and Delta, are sequentially processed by alpha-secretase and presenilin/gamma-secretase and release signaling fragments.

The cleavage of Notch by presenilin (PS)/gamma-secretase is a salient example of regulated intramembrane proteolysis, an unusual mechanism of signal transduction. This cleavage is preceded by the binding of protein ligands to the Notch ectodomain, activating its shedding. We hypothesized that the Notch ligands, Delta and Jagged, themselves undergo PS-mediated regulated intramembrane proteolysis. Here, we show that the ectodomain of mammalian Jagged is cleaved by an A disintegrin and metalloprotease (ADAM) 17-like activity in cultured cells and in vivo, similar to the known cleavage of Drosophila Delta by Kuzbanian. The ectodomain shedding of ligand can be stimulated by Notch and yields membrane-tethered C-terminal fragments (CTFs) of Jagged and Delta that accumulate in cells expressing a dominant-negative form of PS or treated with gamma-secretase inhibitors. PS forms stable complexes with Delta and Jagged and with their respective CTFs. PS/gamma-secretase then mediates the cleavage of the latter to release the Delta and Jagged intracellular domains, a portion of which can enter the nucleus. The ligand CTFs compete with an activated form of Notch for cleavage by gamma-secretase and can thus inhibit Notch signaling in vitro. The soluble Jagged intracellular domain can activate gene expression via the transcription factor AP1, and this effect is counteracted by the co-expression of the gamma-secretase-cleaved product of Notch, Notch intracellular domain. We conclude that Delta and Jagged undergo ADAM-mediated ectodomain processing followed by PS-mediated intramembrane proteolysis to release signaling fragments. Thus, Notch and its cognate ligands are processed by the same molecular machinery and may antagonistically regulate each other's signaling.

ADAM Proteins↗

PubMed2003

gamma-Secretase cleavage and binding to FE65 regulate the nuclear translocation of the intracellular C-terminal domain (ICD) of the APP family of proteins.

Regulated intramembrane proteolysis (RIP) of the amyloid precursor protein (APP) produces amyloid beta-protein (Abeta), the probable causative agent of Alzheimer's disease (AD), and is therefore an important target for therapeutic intervention. However, there is a burgeoning consensus that gamma-secretase, one of the proteases that generates Abeta, is also critical for the signal transduction of APP and a growing list of other receptors. APP is a member of a gene family that includes two amyloid precursor-like proteins, APLP1 and APLP2. Although APP and the APLPs undergo similar proteolytic processing, there is little information about the role of their gamma-secretase-generated intracellular domains (ICDs). Here, we show that APLP1 and 2 undergo presenilin-dependent RIP similar to APP, resulting in the release of a approximately 6 kDa ICD for each protein. Each of the ICDs are degraded by an insulin degrading enzyme-like activity, but they can be stabilized by members of the FE65 family and translocate to the nucleus. Given that modulation of APP processing is a therapeutic target and that the APLPs are processed in a manner similar to APP, any strategy aimed at altering APP proteolysis will have to take into account possible effects on signaling by APLP 1 and 2.

Amino Acid Sequence↗

PubMed2003

Gamma-secretase is a membrane protein complex comprised of presenilin, nicastrin, Aph-1, and Pen-2.

gamma-Secretase catalyzes the intramembrane proteolysis of Notch, beta-amyloid precursor protein, and other substrates as part of a new signaling paradigm and as a key step in the pathogenesis of Alzheimer's disease. This unusual protease has eluded identification, though evidence suggests that the presenilin heterodimer comprises the catalytic site and that a highly glycosylated form of nicastrin associates with it. The formation of presenilin heterodimers from the holoprotein is tightly gated by unknown limiting cellular factors. Here we show that Aph-1 and Pen-2, two recently identified membrane proteins genetically linked to gamma-secretase, associate directly with presenilin and nicastrin in the active protease complex. Coexpression of all four proteins leads to marked increases in presenilin heterodimers, full glycosylation of nicastrin, and enhanced gamma-secretase activity. These findings suggest that the four membrane proteins comprise the limiting components of gamma-secretase and coassemble to form the active enzyme in mammalian cells.

Amyloid Precursor Protein Secretases↗

PubMed2003

Functional gamma-secretase complex assembly in Golgi/trans-Golgi network: interactions among presenilin, nicastrin, Aph1, Pen-2, and gamma-secretase substrates.

Gamma-secretase is a proteolytic complex whose substrates include Notch, beta-amyloid precursor protein (APP), and several other type I transmembrane proteins. Presenilin (PS) and nicastrin are known components of this high-molecular-weight complex, and recent genetic screens in invertebrates have identified two additional gene products, Aph1 and Pen-2, as key factors in gamma-secretase activity. Here, we examined the interaction of the components of the gamma-secretase complex in Chinese hamster ovary cells stably expressing human forms of APP, PS1, Aph1, and Pen-2. Subcellular fractionation of membrane vesicles and subsequent coimmunoprecipitation of individual gamma-secretase components revealed that interactions among all proteins occurred in the Golgi/trans-Golgi network (TGN) compartments. Furthermore, incubation of the Golgi/TGN-enriched vesicles resulted in de novo generation of amyloid beta-protein and APP intracellular domain. Immunofluorescent staining of the individual gamma-secretase components supported our biochemical evidence that the gamma-secretase components assemble into the proteolytically active gamma-secretase complex in the Golgi/TGN compartment.

Amyloid Precursor Protein Secretases↗

PubMed2002

Complex N-linked glycosylated nicastrin associates with active gamma-secretase and undergoes tight cellular regulation.

The intramembranous proteolysis of Notch and the amyloid precursor protein by gamma-secretase exemplifies an unusual and newly recognized mechanism of signal transduction in multicellular organisms. Here, we show that only a form of nicastrin (NCT) containing N-linked complex oligosaccharides is present in active gamma-secretase complexes. Overexpression of NCT does not generate more of this mature protein, a phenomenon analogous to the strictly regulated formation of mature presenilin heterodimers from immature holoprotein. The absence of presenilin severely limits the maturation of NCT, yet combined overexpression of both proteins does not increase respective mature types. Taken together, our findings describe unusual regulatory features of this key signaling protease: the association of NCT with gamma-secretase is tightly regulated via glycosylation; at least one other cofactor exists; the least abundant member of the complex becomes limiting; and the cofactor that serves this role may vary by cell type.

Amyloid Precursor Protein Secretases↗