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1.
Cell ; 166(1): 193-208, 2016 Jun 30.
Artículo en Inglés | MEDLINE | ID: mdl-27293189

RESUMEN

γ-Secretases are a family of intramembrane-cleaving proteases involved in various signaling pathways and diseases, including Alzheimer's disease (AD). Cells co-express differing γ-secretase complexes, including two homologous presenilins (PSENs). We examined the significance of this heterogeneity and identified a unique motif in PSEN2 that directs this γ-secretase to late endosomes/lysosomes via a phosphorylation-dependent interaction with the AP-1 adaptor complex. Accordingly, PSEN2 selectively cleaves late endosomal/lysosomal localized substrates and generates the prominent pool of intracellular Aß that contains longer Aß; familial AD (FAD)-associated mutations in PSEN2 increased the levels of longer Aß further. Moreover, a subset of FAD mutants in PSEN1, normally more broadly distributed in the cell, phenocopies PSEN2 and shifts its localization to late endosomes/lysosomes. Thus, localization of γ-secretases determines substrate specificity, while FAD-causing mutations strongly enhance accumulation of aggregation-prone Aß42 in intracellular acidic compartments. The findings reveal potentially important roles for specific intracellular, localized reactions contributing to AD pathogenesis.


Asunto(s)
Enfermedad de Alzheimer/patología , Secretasas de la Proteína Precursora del Amiloide/análisis , Péptidos beta-Amiloides/metabolismo , Fragmentos de Péptidos/metabolismo , Presenilina-2/análisis , Complejo 1 de Proteína Adaptadora/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Secuencias de Aminoácidos , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Animales , Línea Celular Tumoral , Endosomas/química , Humanos , Lisosomas/química , Ratones , Presenilina-1/análisis , Presenilina-1/química , Presenilina-1/genética , Presenilina-1/metabolismo , Presenilina-2/química , Presenilina-2/genética , Presenilina-2/metabolismo , Ratas , Especificidad por Sustrato
2.
Semin Cell Dev Biol ; 105: 12-26, 2020 09.
Artículo en Inglés | MEDLINE | ID: mdl-32146031

RESUMEN

γ-Secretase cleavage is essential for many biological processes and its dysregulation is linked to disease, including cancer and Alzheimer's disease. Therefore, understanding the regulation of its activity is of major importance to improve drug design and develop novel therapeutics. γ-Secretase belongs to the family of intramembrane cleaving proteases (i-CLiPs), which cleaves its substrates in a process termed regulated intramembrane proteolysis (RIP). During RIP, type-I transmembrane proteins are first cleaved within their ectodomain by a sheddase and then within their transmembrane domain by γ-secretase. γ-Secretase is composed of four integral membrane proteins that are all essential for its function: presenilin (PSEN), anterior pharynx defective 1 (APH1), nicastrin (NCT) and presenilin enhancer 2 (PEN-2). Given the presence of two PSEN homologues (PSEN1 & 2) and several APH1 isoforms, a heterogeneity exists in cellular γ-secretase complexes. It is becoming clear that each of these complexes has overlapping as well as distinct biological characteristics. This review summarizes our current knowledge on complex formation, trafficking, subcellular localization, interactors and the structure of γ-secretase, with a focus, when possible or known, on the contribution of PSEN1 and PSEN2 herein.


Asunto(s)
Enfermedad de Alzheimer/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Biología Celular/normas , Presenilinas/metabolismo , Humanos
3.
J Biol Chem ; 296: 100182, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33310703

RESUMEN

Polyamines, such as putrescine, spermidine, and spermine, are physiologically important polycations, but the transporters responsible for their uptake in mammalian cells remain poorly characterized. Here, we reveal a new component of the mammalian polyamine transport system using CHO-MG cells, a widely used model to study alternative polyamine uptake routes and characterize polyamine transport inhibitors for therapy. CHO-MG cells present polyamine uptake deficiency and resistance to a toxic polyamine biosynthesis inhibitor methylglyoxal bis-(guanylhydrazone) (MGBG), but the molecular defects responsible for these cellular characteristics remain unknown. By genome sequencing of CHO-MG cells, we identified mutations in an unexplored gene, ATP13A3, and found disturbed mRNA and protein expression. ATP13A3 encodes for an orphan P5B-ATPase (ATP13A3), a P-type transport ATPase that represents a candidate polyamine transporter. Interestingly, ATP13A3 complemented the putrescine transport deficiency and MGBG resistance of CHO-MG cells, whereas its knockdown in WT cells induced a CHO-MG phenotype demonstrated as a decrease in putrescine uptake and MGBG sensitivity. Taken together, our findings identify ATP13A3, which has been previously genetically linked with pulmonary arterial hypertension, as a major component of the mammalian polyamine transport system that confers sensitivity to MGBG.


Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Poliaminas/metabolismo , Putrescina/metabolismo , Adenosina Trifosfatasas/genética , Animales , Transporte Biológico , Células CHO , Cricetinae , Cricetulus , Inhibidores Enzimáticos/farmacología , Mitoguazona/farmacología , Mutación , Secuenciación Completa del Genoma/métodos
4.
J Cell Sci ; 132(5)2019 02 28.
Artículo en Inglés | MEDLINE | ID: mdl-30709920

RESUMEN

The metabolism of PI(3,5)P2 is regulated by the PIKfyve, VAC14 and FIG4 complex, mutations in which are associated with hypopigmentation in mice. These pigmentation defects indicate a key, but as yet unexplored, physiological relevance of this complex in the biogenesis of melanosomes. Here, we show that PIKfyve activity regulates formation of amyloid matrix composed of PMEL protein within the early endosomes in melanocytes, called stage I melanosomes. PIKfyve activity controls the membrane remodeling of stage I melanosomes, which regulates PMEL abundance, sorting and processing. PIKfyve activity also affects stage I melanosome kiss-and-run interactions with lysosomes, which are required for PMEL amyloidogenesis and the establishment of melanosome identity. Mechanistically, PIKfyve activity promotes both the formation of membrane tubules from stage I melanosomes and their release by modulating endosomal actin branching. Taken together, our data indicate that PIKfyve activity is a key regulator of the melanosomal import-export machinery that fine tunes the formation of functional amyloid fibrils in melanosomes and the maintenance of melanosome identity.This article has an associated First Person interview with the first author of the paper.


Asunto(s)
Flavoproteínas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lisosomas/metabolismo , Melanocitos/metabolismo , Melanosomas/metabolismo , Proteínas de la Membrana/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfoinosítido Fosfatasas/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Amiloide/metabolismo , Animales , Células Cultivadas , Flavoproteínas/genética , Homeostasis , Péptidos y Proteínas de Señalización Intracelular/genética , Melanocitos/patología , Melanosomas/ultraestructura , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Fosfatidilinositol 3-Quinasas/genética , Fosfoinosítido Fosfatasas/genética , Transporte de Proteínas , Epitelio Pigmentado de la Retina/patología , Antígeno gp100 del Melanoma/metabolismo
5.
PLoS Comput Biol ; 12(9): e1005095, 2016 09.
Artículo en Inglés | MEDLINE | ID: mdl-27603951

RESUMEN

Cell surface protein and lipid molecules are organized in various patterns: randomly, along gradients, or clustered when segregated into discrete micro- and nano-domains. Their distribution is tightly coupled to events such as polarization, endocytosis, and intracellular signaling, but challenging to quantify using traditional techniques. Here we present a novel approach to quantify the distribution of plasma membrane proteins and lipids. This approach describes spatial patterns in degrees of inhomogeneity and incorporates an intensity-based correction to analyze images with a wide range of resolutions; we have termed it Quantitative Analysis of the Spatial distributions in Images using Mosaic segmentation and Dual parameter Optimization in Histograms (QuASIMoDOH). We tested its applicability using simulated microscopy images and images acquired by widefield microscopy, total internal reflection microscopy, structured illumination microscopy, and photoactivated localization microscopy. We validated QuASIMoDOH, successfully quantifying the distribution of protein and lipid molecules detected with several labeling techniques, in different cell model systems. We also used this method to characterize the reorganization of cell surface lipids in response to disrupted endosomal trafficking and to detect dynamic changes in the global and local organization of epidermal growth factor receptors across the cell surface. Our findings demonstrate that QuASIMoDOH can be used to assess protein and lipid patterns, quantifying distribution changes and spatial reorganization at the cell surface. An ImageJ/Fiji plugin of this analysis tool is provided.


Asunto(s)
Membrana Celular/química , Biología Computacional/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Lípidos/análisis , Proteínas de la Membrana/análisis , Algoritmos , Animales , Células Cultivadas , Lípidos/química , Proteínas de la Membrana/química , Ratones , Microscopía Fluorescente , Programas Informáticos
6.
EMBO J ; 31(15): 3252-69, 2012 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-22781129

RESUMEN

Dendritic filopodia are dynamic structures thought to be the precursors of spines during synapse development. Morphological maturation to spines is associated with the stabilization and strengthening of synapses, and can be altered in various neurological disorders. Telencephalin (TLN/intercellular adhesion molecule-5 (ICAM5)) localizes to dendritic filopodia, where it facilitates their formation/maintenance, thereby slowing spine morphogenesis. As spines are largely devoid of TLN, its exclusion from the filopodia surface appears to be required in this maturation process. Using HeLa cells and primary hippocampal neurons, we demonstrate that surface removal of TLN involves internalization events mediated by the small GTPase ADP-ribosylation factor 6 (ARF6), and its activator EFA6A. This endocytosis of TLN affects filopodia-to-spine transition, and requires Rac1-mediated dephosphorylation/release of actin-binding ERM proteins from TLN. At the somato-dendritic surface, TLN and EFA6A are confined to distinct, flotillin-positive membrane subdomains. The co-distribution of TLN with this lipid raft marker also persists during its endosomal targeting to CD63-positive late endosomes. This suggests a specific microenvironment facilitating ARF6-mediated mobilization of TLN that contributes to promotion of dendritic spine development.


Asunto(s)
Factores de Ribosilacion-ADP/fisiología , Moléculas de Adhesión Celular/metabolismo , Dendritas/fisiología , Espinas Dendríticas/metabolismo , Endosomas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Seudópodos/metabolismo , Factor 6 de Ribosilación del ADP , Factores de Ribosilacion-ADP/química , Factores de Ribosilacion-ADP/genética , Factores de Ribosilacion-ADP/metabolismo , Secuencia de Aminoácidos , Animales , Moléculas de Adhesión Celular/química , Moléculas de Adhesión Celular/genética , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Células Cultivadas , Microambiente Celular/genética , Microambiente Celular/fisiología , Dendritas/genética , Dendritas/metabolismo , Espinas Dendríticas/genética , Espinas Dendríticas/fisiología , Células HeLa , Humanos , Modelos Biológicos , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Cultivo Primario de Células , Transporte de Proteínas/genética , Seudópodos/genética , Seudópodos/fisiología , Homología de Secuencia de Aminoácido
8.
Biochim Biophys Acta ; 1828(12): 2815-27, 2013 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-24099003

RESUMEN

γ-secretase, which assembles as a tetrameric complex, is an aspartyl protease that proteolytically cleaves substrate proteins within their membrane-spanning domain; a process also known as regulated intramembrane proteolysis (RIP). RIP regulates signaling pathways by abrogating or releasing signaling molecules. Since the discovery, already >15 years ago, of its catalytic component, presenilin, and even much earlier with the identification of amyloid precursor protein as its first substrate, γ-secretase has been commonly associated with Alzheimer's disease. However, starting with Notch and thereafter a continuously increasing number of novel substrates, γ-secretase is becoming linked to an equally broader range of biological processes. This review presents an updated overview of the current knowledge on the diverse molecular mechanisms and signaling pathways controlled by γ-secretase, with a focus on organ development, homeostasis and dysfunction. This article is part of a Special Issue entitled: Intramembrane Proteases.


Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/metabolismo , Regulación del Desarrollo de la Expresión Génica , Morfogénesis/genética , Presenilina-1/metabolismo , Subunidades de Proteína/metabolismo , Transducción de Señal , Enfermedad de Alzheimer/enzimología , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Secretasas de la Proteína Precursora del Amiloide/química , Secretasas de la Proteína Precursora del Amiloide/genética , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Embrión de Mamíferos , Homeostasis , Humanos , Presenilina-1/química , Presenilina-1/genética , Multimerización de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/genética , Proteolisis , Receptores Notch/genética , Receptores Notch/metabolismo , Especificidad por Sustrato
9.
J Cell Sci ; 125(Pt 9): 2257-66, 2012 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-22357945

RESUMEN

Visualization of organelles and molecules at nanometer resolution is revolutionizing the biological sciences. However, such technology is still limited for many cell biologists. We present here a novel approach using photobleaching microscopy with non-linear processing (PiMP) for sub-diffraction imaging. Bleaching of fluorophores both within the single-molecule regime and beyond allows visualization of stochastic representations of sub-populations of fluorophores by imaging the same region over time. Our method is based on enhancing the probable positions of the fluorophores underlying the images. The random nature of the bleached fluorophores is assessed by calculating the deviation of the local actual bleached fluorescence intensity to the average bleach expectation as given by the overall decay of intensity. Subtracting measured from estimated decay images yields differential images. Non-linear enhancement of maxima in these diffraction-limited differential images approximates the positions of the underlying structure. Summing many such processed differential images yields a super-resolution PiMP image. PiMP allows multi-color, three-dimensional sub-diffraction imaging of cells and tissues using common fluorophores and can be implemented on standard wide-field or confocal systems.


Asunto(s)
Fibroblastos/ultraestructura , Microscopía Fluorescente/métodos , Imagen Molecular/métodos , Animales , Colorantes Fluorescentes , Células HeLa , Humanos , Procesamiento de Imagen Asistido por Computador , Ratones , Microscopía Confocal , Microscopía Fluorescente/instrumentación , Imagen Molecular/instrumentación , Fotoblanqueo , Procesos Estocásticos
11.
Proc Natl Acad Sci U S A ; 108(34): E559-68, 2011 Aug 23.
Artículo en Inglés | MEDLINE | ID: mdl-21825135

RESUMEN

Amyloid ß (Aß) peptides, the primary constituents of senile plaques and a hallmark in Alzheimer's disease pathology, are generated through the sequential cleavage of amyloid precursor protein (APP) by ß-site APP cleaving enzyme 1 (BACE1) and γ-secretase. The early endosome is thought to represent a major compartment for APP processing; however, the mechanisms of how BACE1 encounters APP are largely unknown. In contrast to APP internalization, which is clathrin-dependent, we demonstrate that BACE1 is sorted to early endosomes via a route controlled by the small GTPase ADP ribosylation factor 6 (ARF6). Altering ARF6 levels or its activity affects endosomal sorting of BACE1, and consequently results in altered APP processing and Aß production. Furthermore, sorting of newly internalized BACE1 from ARF6-positive towards RAB GTPase 5 (RAB5)-positive early endosomes depends on its carboxyterminal short acidic cluster-dileucine motif. This ARF6-mediated sorting of BACE1 is confined to the somatodendritic compartment of polarized neurons in agreement with Aß peptides being primarily secreted from here. These results demonstrate a spatial separation between APP and BACE1 during surface-to-endosome transport, suggesting subcellular trafficking as a regulatory mechanism for this proteolytic processing step. It thereby provides a novel avenue to interfere with Aß production through a selective modulation of the distinct endosomal transport routes used by BACE1 or APP.


Asunto(s)
Factores de Ribosilacion-ADP/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Endosomas/enzimología , Procesamiento Proteico-Postraduccional , Factor 6 de Ribosilación del ADP , Secuencias de Aminoácidos , Secretasas de la Proteína Precursora del Amiloide/química , Animales , Antígenos CD59/metabolismo , Compartimento Celular , Polaridad Celular , Dendritas/metabolismo , Endocitosis , Células HeLa , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Leucina/metabolismo , Ratones , Modelos Biológicos , Transporte de Proteínas , Ratas , Receptores de Transferrina/metabolismo , Proteínas de Unión al GTP rab/metabolismo
12.
Dev Cell ; 59(12): 1571-1592.e9, 2024 Jun 17.
Artículo en Inglés | MEDLINE | ID: mdl-38626765

RESUMEN

Neuronal endosomal and lysosomal abnormalities are among the early changes observed in Alzheimer's disease (AD) before plaques appear. However, it is unclear whether distinct endolysosomal defects are temporally organized and how altered γ-secretase function or amyloid precursor protein (APP) metabolism contribute to these changes. Inhibiting γ-secretase chronically, in mouse embryonic fibroblast and hippocampal neurons, led to a gradual endolysosomal collapse initiated by decreased lysosomal calcium and increased cholesterol, causing downstream defects in endosomal recycling and maturation. This endolysosomal demise is γ-secretase dependent, requires membrane-tethered APP cytoplasmic domains, and is rescued by APP depletion. APP C-terminal fragments (CTFs) localized to late endosome/lysosome-endoplasmic reticulum contacts; an excess of APP-CTFs herein reduced lysosomal Ca2+ refilling from the endoplasmic reticulum, promoting cholesterol accretion. Tonic regulation by APP-CTFs provides a mechanistic explanation for their cellular toxicity: failure to timely degrade APP-CTFs sustains downstream signaling, instigating lysosomal dyshomeostasis, as observed in prodromal AD. This is the opposite of substrates such as Notch, which require intramembrane proteolysis to initiate signaling.


Asunto(s)
Enfermedad de Alzheimer , Secretasas de la Proteína Precursora del Amiloide , Precursor de Proteína beta-Amiloide , Retículo Endoplásmico , Endosomas , Lisosomas , Neuronas , Lisosomas/metabolismo , Animales , Endosomas/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Ratones , Retículo Endoplásmico/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Neuronas/metabolismo , Colesterol/metabolismo , Hipocampo/metabolismo , Hipocampo/patología , Calcio/metabolismo , Humanos , Fibroblastos/metabolismo , Transducción de Señal , Proteolisis
13.
FASEB J ; 26(9): 3765-78, 2012 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-22661005

RESUMEN

Presenilins (PSENs) form the catalytic component of the γ-secretase complex, responsible for intramembrane proteolysis of amyloid precursor protein (APP) and Notch, among many other membrane proteins. Previously, we identified a PSEN1-binding domain in APP, encompassing half of the transmembrane domain following the amyloid ß (Aß) sequence. Based on this, we designed peptides mimicking this interaction domain with the aim to selectively block APP processing and Aß generation through interfering with enzyme-substrate binding. We identified a peptide sequence that, when fused to a virally derived translocation peptide, significantly lowered Aß production (IC(50): 317 nM) in cell-free and cell-based assays using APP-carboxy terminal fragment as a direct γ-secretase substrate. Being derived from the APP sequence, this inhibitory peptide did not affect NotchΔE γ-cleavage, illustrating specificity and potential therapeutic value. In cell-based assays, the peptide strongly suppressed APP shedding, demonstrating that it exerts the inhibitory effect already upstream of γ-secretase, most likely through steric hindrance.


Asunto(s)
Péptidos beta-Amiloides/biosíntesis , Precursor de Proteína beta-Amiloide/metabolismo , Proteínas de la Membrana/metabolismo , Péptidos/metabolismo , Presenilinas/metabolismo , Procesamiento Proteico-Postraduccional , Secuencia de Aminoácidos , Precursor de Proteína beta-Amiloide/química , Células HEK293 , Células HeLa , Humanos , Microscopía Confocal , Datos de Secuencia Molecular , Resonancia por Plasmón de Superficie
14.
Nat Commun ; 14(1): 2847, 2023 05 24.
Artículo en Inglés | MEDLINE | ID: mdl-37225734

RESUMEN

Phospholipase D3 (PLD3) polymorphisms are linked to late-onset Alzheimer's disease (LOAD). Being a lysosomal 5'-3' exonuclease, its neuronal substrates remained unknown as well as how a defective lysosomal nucleotide catabolism connects to AD-proteinopathy. We identified mitochondrial DNA (mtDNA) as a major physiological substrate and show its manifest build-up in lysosomes of PLD3-defective cells. mtDNA accretion creates a degradative (proteolytic) bottleneck that presents at the ultrastructural level as a marked abundance of multilamellar bodies, often containing mitochondrial remnants, which correlates with increased PINK1-dependent mitophagy. Lysosomal leakage of mtDNA to the cytosol activates cGAS-STING signaling that upregulates autophagy and induces amyloid precursor C-terminal fragment (APP-CTF) and cholesterol accumulation. STING inhibition largely normalizes APP-CTF levels, whereas an APP knockout in PLD3-deficient backgrounds lowers STING activation and normalizes cholesterol biosynthesis. Collectively, we demonstrate molecular cross-talks through feedforward loops between lysosomal nucleotide turnover, cGAS-STING and APP metabolism that, when dysregulated, result in neuronal endolysosomal demise as observed in LOAD.


Asunto(s)
ADN Mitocondrial , Nucleótidos , Mitocondrias , Nucleotidiltransferasas , Proteínas Amiloidogénicas , Cromogranina A , Fosfolipasas
15.
J Biol Chem ; 286(10): 8677-8687, 2011 Mar 11.
Artículo en Inglés | MEDLINE | ID: mdl-21209097

RESUMEN

ß-Secretase (BACE1) is an attractive drug target for Alzheimer disease. However, the design of clinical useful inhibitors targeting its active site has been extremely challenging. To identify alternative drug targeting sites we have generated a panel of BACE1 monoclonal antibodies (mAbs) that interfere with BACE1 activity in various assays and determined their binding epitopes. mAb 1A11 inhibited BACE1 in vitro using a large APP sequence based substrate (IC(50) ∼0.76 nm), in primary neurons (EC(50) ∼1.8 nm), and in mouse brain after stereotactic injection. Paradoxically, mAb 1A11 increased BACE1 activity in vitro when a short synthetic peptide was used as substrate, indicating that mAb 1A11 does not occupy the active-site. Epitope mapping revealed that mAb 1A11 binds to adjacent loops D and F, which together with nearby helix A, distinguishes BACE1 from other aspartyl proteases. Interestingly, mutagenesis of loop F and helix A decreased or increased BACE1 activity, identifying them as enzymatic regulatory elements and as potential alternative sites for inhibitor design. In contrast, mAb 5G7 was a potent BACE1 inhibitor in cell-free enzymatic assays (IC(50) ∼0.47 nm) but displayed no inhibitory effect in primary neurons. Its epitope, a surface helix 299-312, is inaccessible in membrane-anchored BACE1. Remarkably, mutagenesis of helix 299-312 strongly reduced BACE1 ectodomain shedding, suggesting that this helix plays a role in BACE1 cellular biology. In conclusion, this study generated highly selective and potent BACE1 inhibitory mAbs, which recognize unique structural and functional elements in BACE1, and uncovered interesting alternative sites on BACE1 that could become targets for drug development.


Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Secretasas de la Proteína Precursora del Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Anticuerpos Monoclonales de Origen Murino/farmacología , Anticuerpos Neutralizantes/farmacología , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/genética , Ácido Aspártico Endopeptidasas/metabolismo , Encéfalo/enzimología , Neuronas/enzimología , Inhibidores de Proteasas/farmacología , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/enzimología , Enfermedad de Alzheimer/inmunología , Secretasas de la Proteína Precursora del Amiloide/inmunología , Animales , Anticuerpos Monoclonales de Origen Murino/inmunología , Anticuerpos Neutralizantes/inmunología , Ácido Aspártico Endopeptidasas/inmunología , Encéfalo/inmunología , Células HEK293 , Humanos , Ratones , Ratones Transgénicos , Mutagénesis , Inhibidores de Proteasas/inmunología , Estructura Secundaria de Proteína
16.
Curr Opin Cell Biol ; 15(4): 438-45, 2003 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-12892784

RESUMEN

In the secretory pathway, the forward (anterograde) membrane flow is compensated by retrograde transport of proteins and lipids. Membrane recycling is required for the maintenance of organelle homeostasis and the re-use of components of the transport machineries for the generation of new transport intermediates. However, the molecular mechanisms and other cellular functions of retrograde traffic are still poorly understood. In recent years, a multitude of protein factors that function in the secretory pathway have been discovered, most of them originally suggested to play a role in forward trafficking. However, in many cases subsequent studies have revealed that these proteins participate (also) in retrograde traffic. It is likely that this shift will continue, reflecting the fact that the two pathways are intimately connected.


Asunto(s)
Proteínas Portadoras/metabolismo , Endosomas/metabolismo , Membranas Intracelulares/metabolismo , Red trans-Golgi/metabolismo , Animales , Vesículas Cubiertas por Proteínas de Revestimiento/metabolismo , Retículo Endoplásmico/metabolismo , Humanos , Transporte de Proteínas/fisiología , Proteínas de Unión al GTP rab/metabolismo
17.
J Cell Biol ; 220(9)2021 09 06.
Artículo en Inglés | MEDLINE | ID: mdl-34292306

RESUMEN

γ-Secretase affects many physiological processes through targeting >100 substrates; malfunctioning links γ-secretase to cancer and Alzheimer's disease. The spatiotemporal regulation of its stoichiometric assembly remains unresolved. Fractionation, biochemical assays, and imaging support prior formation of stable dimers in the ER, which, after ER exit, assemble into full complexes. In vitro ER budding shows that none of the subunits is required for the exit of others. However, knockout of any subunit leads to the accumulation of incomplete subcomplexes in COPII vesicles. Mutating a DPE motif in presenilin 1 (PSEN1) abrogates ER exit of PSEN1 and PEN-2 but not nicastrin. We explain this by the preferential sorting of PSEN1 and nicastrin through Sec24A and Sec24C/D, respectively, arguing against full assembly before ER exit. Thus, dimeric subcomplexes aided by Sec24 paralog selectivity support a stepwise assembly of γ-secretase, controlling final levels in post-Golgi compartments.


Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/metabolismo , Vesículas Cubiertas por Proteínas de Revestimiento/metabolismo , Endopeptidasas/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/metabolismo , Neuronas/metabolismo , Presenilina-1/metabolismo , Secretasas de la Proteína Precursora del Amiloide/química , Secretasas de la Proteína Precursora del Amiloide/genética , Animales , Transporte Biológico , Vesículas Cubiertas por Proteínas de Revestimiento/química , Vesículas Cubiertas por Proteínas de Revestimiento/genética , Línea Celular , Línea Celular Tumoral , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Endopeptidasas/química , Endopeptidasas/genética , Fibroblastos/citología , Fibroblastos/metabolismo , Regulación de la Expresión Génica , Aparato de Golgi/metabolismo , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Ratones , Modelos Moleculares , Neuronas/citología , Presenilina-1/química , Presenilina-1/genética , Cultivo Primario de Células , Unión Proteica , Conformación Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Multimerización de Proteína , Ratas , Ratas Wistar , Transducción de Señal , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo
18.
Elife ; 92020 07 07.
Artículo en Inglés | MEDLINE | ID: mdl-32631487

RESUMEN

γ-Secretase is a multi-subunit enzyme whose aberrant activity is associated with Alzheimer's disease and cancer. While its structure is atomically resolved, γ-secretase localization in the membrane in situ relies mostly on biochemical data. Here, we combined fluorescent tagging of γ-secretase subunits with super-resolution microscopy in fibroblasts. Structured illumination microscopy revealed single γ-secretase complexes with a monodisperse distribution and in a 1:1 stoichiometry of PSEN1 and nicastrin subunits. In living cells, sptPALM revealed PSEN1/γ-secretase mainly with directed motility and frequenting 'hotspots' or high track-density areas that are sensitive to γ-secretase inhibitors. We visualized γ-secretase association with substrates like amyloid precursor protein and N-cadherin, but not with its sheddases ADAM10 or BACE1 at the cell surface, arguing against pre-formed megadalton complexes. Nonetheless, in living cells PSEN1/γ-secretase transiently visits ADAM10 hotspots. Our results highlight the power of super-resolution microscopy for the study of γ-secretase distribution and dynamics in the membrane.


Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/genética , Presenilina-1/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Animales , Línea Celular , Membrana Celular/metabolismo , Fibroblastos , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Microscopía , Presenilina-1/metabolismo
19.
Mol Biol Cell ; 17(4): 1514-26, 2006 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-16421253

RESUMEN

The function of the pre-Golgi intermediate compartment (IC) and its relationship with the endoplasmic reticulum (ER) and Golgi remain only partially understood. Here, we report striking segregation of IC domains in polarized PC12 cells that develop neurite-like processes. Differentiation involves expansion of the IC and movement of Rab1-containing tubules to the growth cones of the neurites, whereas p58- and COPI-positive IC elements, like rough ER and Golgi, remain in the cell body. Exclusion of Rab1 effectors p115 and GM130 from the neurites further indicated that the centrifugal, Rab1-mediated pathway has functions that are not directly related to ER-to-Golgi trafficking. Disassembly of COPI coats did not affect this pathway but resulted in missorting of p58 to the neurites. Live cell imaging showed that green fluorescent protein (GFP)-Rab1A-containing IC elements move bidirectionally both within the neurites and cell bodies, interconnecting different ER exit sites and the cis-Golgi region. Moreover, in nonpolarized cells GFP-Rab1A-positive tubules moved centrifugally towards the cell cortex. Hydroxymethylglutaryl-CoA reductase, the key enzyme of cholesterol biosynthesis, colocalized with slowly sedimenting, Rab1-enriched membranes when the IC subdomains were separated by velocity sedimentation. These results reveal a novel pathway directly connecting the IC with the cell periphery and suggest that this Rab1-mediated pathway is linked to the dynamics of smooth ER.


Asunto(s)
Compartimento Celular , Polaridad Celular , Aparato de Golgi/metabolismo , Lectinas de Unión a Manosa/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Unión al GTP rab1/metabolismo , Animales , Perros , Retículo Endoplásmico/metabolismo , Chaperón BiP del Retículo Endoplásmico , Proteínas de Choque Térmico/análisis , Proteínas de Choque Térmico/metabolismo , Humanos , Lectinas de Unión a Manosa/análisis , Proteínas de la Membrana/análisis , Chaperonas Moleculares/análisis , Chaperonas Moleculares/metabolismo , Factor de Crecimiento Nervioso/farmacología , Neuritas/química , Neuritas/metabolismo , Neuritas/fisiología , Neuronas/química , Neuronas/citología , Neuronas/efectos de los fármacos , Células PC12 , Fosfoproteínas Fosfatasas/análisis , Fosfoproteínas Fosfatasas/metabolismo , Transporte de Proteínas , Ratas , Transfección , Proteínas de Unión al GTP rab1/análisis
20.
Methods Mol Biol ; 457: 253-65, 2008.
Artículo en Inglés | MEDLINE | ID: mdl-19066033

RESUMEN

A prerequisite for understanding the cellular functions of an unknown protein is the establishment of its subcellular localization. As increasing numbers of novel proteins of the biosynthetic pathway are currently being identified, accessible new methods are required to facilitate their localization. Differentiating rat pheochromocytoma (PC12) cells reorganize their biosynthetic membrane compartments as they develop neurite-like processes. The authors recently showed that polarization of these cells involves the expansion of the intermediate compartment (IC) between the rough endoplasmic reticulum (RER) and the Golgi apparatus. Tubules emerging from the vacuolar parts of the IC move to the developing neurites accumulating in their growth cones, whereas the vacuoles, like RER and Golgi, remain in the cell body. Thus, polarized PC12 cells enhance the resolution for immunofluorescence microscopic mapping of protein localization in the early biosynthetic pathway. The authors also describe here a rapid cell fractionation protocol employing velocity sedimentation in iodixanol gradients that allows one-step separation of the pre-Golgi vacuoles, tubules, and RER.


Asunto(s)
Polaridad Celular , Biología Molecular/métodos , Biosíntesis de Proteínas , Proteínas/metabolismo , Animales , Compartimento Celular , Diferenciación Celular , Proliferación Celular , Centrifugación por Gradiente de Densidad , Técnica del Anticuerpo Fluorescente , Células PC12 , Transporte de Proteínas , Ratas , Ácidos Triyodobenzoicos
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