RESUMEN
Amyloid precursor protein (APP), the precursor of amyloid beta peptide, plays a central role in Alzheimer's disease (AD), a pathology characterized by memory decline and synaptic loss upon aging. Understanding the physiological role of APP is fundamental in deciphering the progression of AD, and several studies suggest a synaptic function via protein-protein interactions. Nevertheless, it remains unclear whether and how these interactions contribute to memory. In Drosophila, we previously showed that APP-like (APPL), the fly APP homolog, is required for aversive associative memory in the olfactory memory center, the mushroom body (MB). In the present study, we show that APPL is required for appetitive long-term memory (LTM), another form of associative memory, in a specific neuronal subpopulation of the MB, the α'/ß' Kenyon cells. Using a biochemical approach, we identify the synaptic MAGUK (membrane-associated guanylate kinase) proteins X11, CASK, Dlgh2 and Dlgh4 as interactants of the APP intracellular domain (AICD). Next, we show that the Drosophila homologs CASK and Dlg are also required for appetitive LTM in the α'/ß' neurons. Finally, using a double RNAi approach, we demonstrate that genetic interactions between APPL and CASK, as well as between APPL and Dlg, are critical for appetitive LTM. In summary, our results suggest that APPL contributes to associative long-term memory through its interactions with the main synaptic scaffolding proteins CASK and Dlg. This function should be conserved across species.
Asunto(s)
Conducta Apetitiva/fisiología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de la Membrana/metabolismo , Memoria a Largo Plazo/fisiología , Cuerpos Pedunculados/fisiología , Proteínas del Tejido Nervioso/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Animales Modificados Genéticamente , Drosophila melanogaster/fisiologíaRESUMEN
L-leucyl-L-leucine methyl ester (LLOMe) induces apoptosis, which is thought to be mediated by release of lysosomal cysteine cathepsins from permeabilized lysosomes into the cytosol. Here, we demonstrated in HeLa cells that apoptotic as well as sub-apoptotic concentrations of LLOMe caused rapid and complete lysosomal membrane permeabilization (LMP), as evidenced by loss of the proton gradient and release into the cytosol of internalized lysosomal markers below a relative molecular mass of 10,000. However, there was no evidence for the release of cysteine cathepsins B and L into the cytosol; rather they remained within lysosomes, where they were rapidly inactivated and degraded. LLOMe-induced adverse effects, including LMP, loss of cysteine cathepsin activity, caspase activation and cell death could be reduced by inhibition of cathepsin C, but not by inhibiting cathepsins B and L. When incubated with sub-apoptotic LLOMe concentrations, lysosomes transiently lost protons but annealed and re-acidified within hours. Full lysosomal function required new protein synthesis of cysteine cathepsins and other hydrolyses. Our data argue against the release of lysosomal enzymes into the cytosol and their proposed proteolytic signaling during LLOMe-induced apoptosis.
Asunto(s)
Catepsinas/metabolismo , Cisteína/metabolismo , Citosol/metabolismo , Dipéptidos/farmacología , Lisosomas/metabolismo , Apoptosis/efectos de los fármacos , Citosol/efectos de los fármacos , Células HeLa , Humanos , Concentración de Iones de Hidrógeno , Hidrolasas/metabolismo , Membranas Intracelulares/efectos de los fármacos , Membranas Intracelulares/metabolismo , Membranas Intracelulares/ultraestructura , Lisosomas/ultraestructura , Modelos Biológicos , Permeabilidad/efectos de los fármacos , Biosíntesis de Proteínas/efectos de los fármacos , ProtonesRESUMEN
Bone is a dynamic tissue whose remodeling throughout life is orchestrated by repeated cycles of destruction mediated by osteoclasts and rebuilding by osteoblasts. Current understanding of osteoclast biology has largely relied on the generation of knockout mice exhibiting an abnormal bone phenotype. This has provided a better understanding of osteoclast biology and the key proteins that support osteoclast function. However, mouse models alone do not provide an integrated view on protein networks and post-translational modifications that might be important for osteoclast function. During the past years, a number of MS-based quantitative methods have been developed to investigate the complexity of biological systems. This review will summarize how such approaches have contributed to the understanding of osteoclast differentiation and function.
Asunto(s)
Osteoclastos/metabolismo , Proteómica/métodos , Animales , Huesos/metabolismo , Humanos , Ratones , Osteoblastos/metabolismo , Procesamiento Proteico-PostraduccionalRESUMEN
The delivery of newly synthesized soluble lysosomal hydrolases to the endosomal system is essential for lysosome function and cell homeostasis. This process relies on the proper trafficking of the mannose 6-phosphate receptors (MPRs) between the trans-Golgi network (TGN), endosomes and the plasma membrane. Many transmembrane proteins regulating diverse biological processes ranging from virus production to the development of multicellular organisms also use these pathways. To explore how cell signaling modulates MPR trafficking, we used high-throughput RNA interference (RNAi) to target the human kinome and phosphatome. Using high-content image analysis, we identified 127 kinases and phosphatases belonging to different signaling networks that regulate MPR trafficking and/or the dynamic states of the subcellular compartments encountered by the MPRs. Our analysis maps the MPR trafficking pathways based on enzymes regulating phosphatidylinositol phosphate metabolism. Furthermore, it reveals how cell signaling controls the biogenesis of post-Golgi tubular carriers destined to enter the endosomal system through a SRC-dependent pathway regulating ARF1 and RAC1 signaling and myosin II activity.
Asunto(s)
Membrana Celular/enzimología , Endosomas/enzimología , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Interferencia de ARN , Receptor IGF Tipo 2/metabolismo , Red trans-Golgi/enzimología , Factor 1 de Ribosilacion-ADP/genética , Factor 1 de Ribosilacion-ADP/metabolismo , Análisis por Conglomerados , Regulación Enzimológica de la Expresión Génica , Redes Reguladoras de Genes , Células HeLa , Humanos , Fosfatos de Fosfatidilinositol/metabolismo , Mapas de Interacción de Proteínas , Transporte de Proteínas/genética , Receptor IGF Tipo 2/genética , Transducción de Señal , Transfección , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/metabolismo , Familia-src Quinasas/genética , Familia-src Quinasas/metabolismoRESUMEN
A major obstacle in defining the exact role of extracellular matrix (ECM) in stem cell niches is the lack of suitable in vitro methods that recapitulate complex ECM microenvironments. Here we describe a methodology that permits reliable anchorage of native cell-secreted ECM to culture carriers. We validated our approach by fabricating two types of human bone marrow-specific ECM substrates that were robust enough to support human mesenchymal stem cells (MSCs) and hematopoietic stem and progenitor cells in vitro. We characterized the molecular composition, structural features and nanomechanical properties of the MSC-derived ECM preparations and demonstrated their ability to support expansion and differentiation of bone marrow stem cells. Our methodology enables the deciphering and modulation of native-like multicomponent ECMs of tissue-resident stem cells and will therefore prepare the ground for a more rational design of engineered stem cell niches.
Asunto(s)
Células de la Médula Ósea/fisiología , Matriz Extracelular/fisiología , Células Madre Hematopoyéticas/fisiología , Células Madre Mesenquimatosas/fisiología , Nicho de Células Madre/fisiología , Animales , Células de la Médula Ósea/citología , Técnicas de Cultivo de Célula , Diferenciación Celular/fisiología , Células Madre Hematopoyéticas/citología , Humanos , Células Madre Mesenquimatosas/citología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Microscopía de Fuerza Atómica , Microscopía Electrónica de Rastreo , Organismos Libres de Patógenos EspecíficosRESUMEN
The analysis of glucose signaling in the Crabtree-positive eukaryotic model organism Saccharomyces cerevisiae has disclosed a dual role of its hexokinase ScHxk2, which acts as a glycolytic enzyme and key signal transducer adapting central metabolism to glucose availability. In order to identify evolutionarily conserved characteristics of hexokinase structure and function, the cellular response of the Crabtree-negative yeast Kluyveromyces lactis to rag5 null mutation and concomitant deficiency of its unique hexokinase KlHxk1 was analyzed by means of difference gel electrophoresis. In total, 2,851 fluorescent spots containing different protein species were detected in the master gel representing all of the K. lactis proteins that were solubilized from glucose-grown KlHxk1 wild-type and mutant cells. Mass spectrometric peptide analysis identified 45 individual hexokinase-dependent proteins related to carbohydrate, short-chain fatty acid and tricarboxylic acid metabolism as well as to amino acid and protein turnover, but also to general stress response and chromatin remodeling, which occurred as a consequence of KlHxk1 deficiency at a minimum 3-fold enhanced or reduced level in the mutant proteome. In addition, three proteins exhibiting homology to 2-methylcitrate cycle enzymes of S. cerevisiae were detected at increased concentrations, suggesting a stimulation of pyruvate formation from amino acids and/or fatty acids. Experimental validation of the difference gel electrophoresis approach by post-lysis dimethyl labeling largely confirmed the abundance changes detected in the mutant proteome via the former method. Taking into consideration the high proportion of identified hexokinase-dependent proteins exhibiting increased proteomic levels, KlHxk1 is likely to have a repressive function in a multitude of metabolic pathways. The proteomic alterations detected in the mutant classify KlHxk1 as a multifunctional enzyme and support the view of evolutionary conservation of dual-role hexokinases even in organisms that are less specialized than S. cerevisiae in terms of glucose utilization.
Asunto(s)
Proteínas Fúngicas/metabolismo , Glucosa/farmacología , Hexoquinasa/deficiencia , Kluyveromyces/efectos de los fármacos , Kluyveromyces/enzimología , Proteoma/metabolismo , Proteómica , Carbono/farmacología , Electroforesis en Gel Bidimensional , Ontología de Genes , Hexoquinasa/metabolismo , Kluyveromyces/crecimiento & desarrollo , Redes y Vías Metabólicas/efectos de los fármacos , Mutación/genética , Fosforilación/efectos de los fármacos , Fosfoserina/metabolismoRESUMEN
The initial step of bone digestion is the adhesion of osteoclasts onto bone surfaces and the assembly of podosomal belts that segregate the bone-facing ruffled membrane from other membrane domains. During bone digestion, membrane components of the ruffled border also need to be recycled after macropinocytosis of digested bone materials. How osteoclast polarity and membrane recycling are coordinated remains unknown. Here, we show that the Cdc42-guanine nucleotide exchange factor FGD6 coordinates these events through its Src-dependent interaction with different actin-based protein networks. At the plasma membrane, FGD6 couples cell adhesion and actin dynamics by regulating podosome formation through the assembly of complexes comprising the Cdc42-interactor IQGAP1, the Rho GTPase-activating protein ARHGAP10, and the integrin interactors Talin-1/2 or Filamin A. On endosomes and transcytotic vesicles, FGD6 regulates retromer-dependent membrane recycling through its interaction with the actin nucleation-promoting factor WASH. These results provide a mechanism by which a single Cdc42-exchange factor controlling different actin-based processes coordinates cell adhesion, cell polarity, and membrane recycling during bone degradation.
Asunto(s)
Endosomas/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Membranas Intracelulares/metabolismo , Osteoclastos/citología , Osteoclastos/metabolismo , Animales , Huesos/metabolismo , Adhesión Celular , Línea Celular , Polaridad Celular , Factores de Intercambio de Guanina Nucleótido/genética , Ratones , Unión Proteica , Proteína de Unión al GTP cdc42/metabolismoRESUMEN
Numerous transport processes occur between the two mitochondrial (mt) membranes due to the diverse functions and metabolic processes of the mt organelle. The metabolite and ion transport through the mt outer membrane (OM) is widely assumed to be mediated by the porin pore, whereas in the mt inner membrane (IM) specific carriers are responsible for transport processes. Here, we provide evidence by means of Blue Native (BN)-PAGE analysis, co-immunoprecipitation, and tandem affinity purification that the two mt OM proteins Om14p and Om45p associate with the porin pore. Porin molecules seem to assemble independently to build the core unit. A subpopulation of these core units interacts with Om14p and Om45p. With preparative tandem affinity purification followed by MS analysis, we could identify interaction partners of this OM complex, which are mainly localized within the mt IM and function as carriers for diverse molecules. We propose a model for the role of the two OM proteins in addressing the porin pore to bind to specific channels in the mt IM to facilitate transport of metabolites.
Asunto(s)
Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/metabolismo , Modelos Biológicos , Porinas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Biológico/fisiología , Proteínas Mitocondriales/genética , Porinas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Metabolic disorders like diabetes mellitus and obesity may compromise the fertility of men and women. To unveil disease-associated proteomic changes potentially affecting male fertility, the proteomes of sperm cells from type-1 diabetic, type-2 diabetic, non-diabetic obese and clinically healthy individuals were comparatively analyzed by difference gel electrophoresis. The adaptation of a general protein extraction procedure to the solubilization of proteins from sperm cells allowed for the resolution of 3187 fluorescent spots in the difference gel electrophoresis image of the master gel, which contained the entirety of solubilized sperm proteins. Comparison of the pathological and reference proteomes by applying an average abundance ratio setting of 1.6 and a p ≤ 0.05 criterion resulted in the identification of 79 fluorescent spots containing proteins that were present at significantly changed levels in the sperm cells. Biometric evaluation of the fluorescence data followed by mass spectrometric protein identification revealed altered levels of 12, 71, and 13 protein species in the proteomes of the type-1 diabetic, type-2 diabetic, and non-diabetic obese patients, respectively, with considerably enhanced amounts of the same set of one molecular form of semenogelin-1, one form of clusterin, and two forms of lactotransferrin in each group of pathologic samples. Remarkably, ß-galactosidase-1-like protein was the only protein that was detected at decreased levels in all three pathologic situations. The former three proteins are part of the eppin (epididymal proteinase inhibitor) protein complex, which is thought to fulfill fertilization-related functions, such as ejaculate sperm protection, motility regulation and gain of competence for acrosome reaction, whereas the putative role of the latter protein to function as a glycosyl hydrolase during sperm maturation remains to be explored at the protein/enzyme level. The strikingly similar differences detected in the three groups of pathological sperm proteomes reflect a disease-associated enhanced formation of predominantly proteolytically modified forms of three eppin protein complex components, possibly as a response to enduring hyperglycemia and enhanced oxidative stress.
Asunto(s)
Diabetes Mellitus Tipo 1/patología , Diabetes Mellitus Tipo 2/patología , Infertilidad Masculina/patología , Obesidad/patología , Proteínas Inhibidoras de Proteinasas Secretoras/metabolismo , Espermatozoides/metabolismo , Adulto , Anciano , Biomarcadores/metabolismo , Estudios de Casos y Controles , Forma de la Célula , Clusterina/metabolismo , Diabetes Mellitus Tipo 1/complicaciones , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/metabolismo , Humanos , Infertilidad Masculina/etiología , Infertilidad Masculina/metabolismo , Lactoferrina/metabolismo , Masculino , Persona de Mediana Edad , Complejos Multiproteicos/metabolismo , Obesidad/complicaciones , Obesidad/metabolismo , Isoformas de Proteínas/metabolismo , Proteoma/metabolismo , Estándares de Referencia , Proteínas de Secreción de la Vesícula Seminal/metabolismo , Recuento de Espermatozoides , Motilidad Espermática , Electroforesis Bidimensional Diferencial en Gel/normas , Adulto JovenRESUMEN
Endospanin-1 is a negative regulator of the cell surface expression of leptin receptor (OB-R), and endospanin-2 is a homologue of unknown function. We investigated the mechanism for endospanin-1 action in regulating OB-R cell surface expression. Here we show that endospanin-1 and -2 are small integral membrane proteins that localize in endosomes and the trans-Golgi network. Antibody uptake experiments showed that both endospanins are transported to the plasma membrane and then internalized into early endosomes but do not recycle back to the trans-Golgi network. Overexpression of endospanin-1 or endospanin-2 led to a decrease of OB-R cell surface expression, whereas shRNA-mediated depletion of each protein increased OB-R cell surface expression. This increased cell surface expression was not observed with OB-Ra mutants defective in endocytosis or with transferrin and EGF receptors. Endospanin-1 or endospanin-2 depletion did not change the internalization rate of OB-Ra but slowed down its lysosomal degradation. Thus, both endospanins are regulators of postinternalization membrane traffic of the endocytic pathway of OB-R.
Asunto(s)
Proteínas Portadoras/metabolismo , Endocitosis/fisiología , Receptores de Leptina/metabolismo , Animales , Proteínas Portadoras/genética , Regulación de la Expresión Génica/fisiología , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular , Lisosomas/genética , Lisosomas/metabolismo , Mutación , Transporte de Proteínas/fisiología , Ratas , Receptores de Leptina/genética , Red trans-Golgi/genética , Red trans-Golgi/metabolismoRESUMEN
Bone digestion occurs when osteoclasts adhere onto bone surfaces and polarize to form acidic, hydrolase-rich resorption lacunae. For this process, they condense their actin-rich podosomes in tight belts to establish sealing zones, which segregate their basal membranes from those facing resorption lacunae. This polarization process remains poorly understood. Here, we combined quantitative proteomics and gene silencing to identify new substrates of the Src tyrosine kinase, a key regulator of osteoclast function. We now report that a depletion of the ARF GTPase-activating protein GIT2, which localizes to sealing zones upon Src phosphorylation, or a lack of GTP hydrolysis on ARF6 impairs sealing zone formation and polarized membrane traffic. Surprisingly, the Rho guanine nucleotide exchange factors alpha and beta PIX, which usually coordinate ARF and Rho signaling, were found to be dispensable. We conclude that the Src-dependent localization of GIT2 is essential for down-regulating ARF6 activity at sealing zones, and thus for maintaining osteoclast polarity.
Asunto(s)
Factores de Ribosilacion-ADP/genética , Huesos/metabolismo , Osteoclastos/metabolismo , Familia-src Quinasas/metabolismo , Factor 6 de Ribosilación del ADP , Animales , Resorción Ósea , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Cromatografía Liquida , Regulación hacia Abajo , Proteínas Activadoras de GTPasa , Guanosina Trifosfato/metabolismo , Hidrólisis , Péptidos y Proteínas de Señalización Intercelular , Ratones , Osteoclastos/enzimología , Fosfoproteínas/metabolismo , Fosforilación , Unión Proteica , Espectrometría de Masas en TándemRESUMEN
Glucose and cAMP-inducing agents such as 3-isobutyl-1-methylxanthine (IBMX) rapidly change the expression profile of insulin-producing pancreatic beta-cells mostly through post-transcriptional mechanisms. A thorough analysis of these changes, however, has not yet been performed. By combining two-dimensional differential gel electrophoresis and mass spectrometry, we identified 165 spots, corresponding to 78 proteins, whose levels significantly change after stimulation of the beta-cell model INS-1 cells with 25 mM glucose + 1 mM IBMX for 2 h. Changes in the expression of selected proteins were verified by one- and two-dimensional immunoblotting. Most of the identified proteins are novel targets of rapid regulation in beta-cells. The transcription inhibitor actinomycin D failed to block changes in two-thirds of the spots, supporting their post-transcriptional regulation. More spots changed in response to IBMX than to glucose alone conceivably because of phosphorylation. Fourteen mRNA- binding proteins responded to stimulation, thus representing the most prominent class of rapidly regulated proteins. Bioinformatics analysis indicated that the mRNA 5'- and 3'-untranslated regions of 22 regulated proteins contain potential binding sites for polypyrimidine tract-binding protein 1, which promotes mRNA stability and translation in stimulated beta-cells. Overall our findings support the idea that mRNA-binding proteins play a major role in rapid adaptive changes in insulin-producing cells following their stimulation with glucose and cAMP-elevating agents.
Asunto(s)
1-Metil-3-Isobutilxantina/farmacología , Glucosa/farmacología , Insulinoma/metabolismo , Insulinoma/patología , Proteínas de Unión al ARN/metabolismo , Secuencias de Aminoácidos , Animales , Sitios de Unión , Western Blotting , Biología Computacional , Secuencia Conservada , Electroforesis en Gel Bidimensional , Ribonucleoproteínas Nucleares Heterogéneas , Humanos , Espectrometría de Masas , Ratones , Proteínas Musculares/metabolismo , Proteínas de Neoplasias/metabolismo , Proteína de Unión al Tracto de Polipirimidina , Proteómica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/química , Ratas , Reproducibilidad de los Resultados , Vesículas Secretoras/efectos de los fármacos , Vesículas Secretoras/metabolismo , Regiones no Traducidas/genéticaRESUMEN
The exchange of proteins and lipids between the trans-Golgi network (TGN) and the endosomal system requires multiple cellular machines, whose activities are coordinated in space and time to generate pleomorphic, tubulo-vesicular carriers that deliver their content to their target compartments. These machines and their associated protein networks are recruited and/or activated on specific membrane domains where they select proteins and lipids into carriers, contribute to deform/elongate and partition membrane domains using the mechanical forces generated by actin polymerization or movement along microtubules. The coordinated action of these protein networks contributes to regulate the dynamic state of multiple receptors recycling between the cell surface, endosomes and the TGN, to maintain cell homeostasis as exemplified by the biogenesis of lysosomes and related organelles, and to establish/maintain cell polarity. The dynamic assembly and disassembly of these protein networks mediating the exchange of membrane domains between the TGN and endosomes regulates cell-cell signalling and thus the development of multi-cellular organisms. Somatic mutations in single network components lead to changes in transport dynamics that may contribute to pathological modifications underlying several human diseases such as mental retardation.
Asunto(s)
Endosomas/metabolismo , Red trans-Golgi/metabolismo , Animales , Humanos , Transporte de ProteínasRESUMEN
Difference gel electrophoresis (DIGE) of fluorescently labelled human sperm proteins was used to identify diabetes- and obesity-associated changes of the sperm proteome. Semen samples from type 1 diabetics, non-diabetic obese individuals and a reference group of clinically healthy fertile donors were evaluated in a comparative study. The adaptation of a general protein extraction procedure to the solubilization of proteins from isolated progressively motile human spermatozoa resulted in the detection of approximately 2700 fluorescent protein spots in the DIGE images. Comparison of the patients' sperm proteomes with those of the reference group allowed the identification of 20 spots containing proteins that were present in the sperm lysates at significantly increased or decreased concentrations. In detail, eight of these spots were apparently related to type 1 diabetes while 12 spots were apparently related to obesity. Tryptic digestion of the spot proteins and mass spectrometric analysis of the corresponding peptides identified seven sperm proteins apparently associated with type 1 diabetes and nine sperm proteins apparently associated with obesity, three of which existing in multiple molecular forms. The established proteomic approach is expected to function as a non-invasive experimental tool in the diagnosis of male infertility and in monitoring any fertility-restoring therapy.
Asunto(s)
Diabetes Mellitus Tipo 1/metabolismo , Obesidad/metabolismo , Proteoma/metabolismo , Proteómica/métodos , Espermatozoides/metabolismo , Diabetes Mellitus Tipo 1/complicaciones , Diabetes Mellitus Tipo 1/genética , Electroforesis en Gel de Poliacrilamida , Humanos , Procesamiento de Imagen Asistido por Computador , Infertilidad Masculina/complicaciones , Infertilidad Masculina/metabolismo , Masculino , Espectrometría de Masas , Obesidad/complicaciones , Obesidad/genética , Semen/metabolismoRESUMEN
Bone-resorbing osteoclasts play a central role in bone remodeling and its pathology. To digest bone, osteoclasts re-organize both F-actin, to assemble podosomes/sealing zones, and membrane traffic, to form bone-facing ruffled borders enriched in lysosomal membrane proteins. It remains elusive how these processes are coordinated. Here, we show that ARAP1 (ArfGAP with RhoGAP domain, ankyrin repeat and PH domain-containing protein 1) fulfills this function. At podosomes/sealing zones, ARAP1 is part of a protein complex where its RhoGAP domain regulates actin dynamics. At endosomes, ARAP1 interacts with AP-3 adaptor complexes where its Arf-GAP domain regulates the Arf1-dependent AP-3 binding to membranes and, consequently lysosomal membrane protein transport to ruffled borders. Accordingly, ARAP1 or AP-3 depletion in osteoclasts alters their capacity to digest bone in vitro. and AP-3δ-deficient mocha mice, a model of the Hermansky-Pudlak storage pool syndrome, develop osteoporosis. Thus, ARAP1 bridges F-actin and membrane dynamics in osteoclasts for proper bone homeostasis.
RESUMEN
The Saccharomyces cerevisiae VPS55 (YJR044c) gene encodes a small protein of 140 amino acids with four potential transmembrane domains. VPS55 belongs to a family of genes of unknown function, including the human gene encoding the obesity receptor gene-related protein (OB-RGRP). Yeast cells with a disrupted VPS55 present normal vacuolar morphology, but exhibit an abnormal secretion of the Golgi form of the soluble vacuolar carboxypeptidase Y. However, trafficking of the membrane-bound vacuolar alkaline phosphatase remains normal. The endocytosis of uracil permease, used as an endocytic marker, is normal in vps55Delta cells, but its degradation is delayed and this marker transiently accumulates in late endosomal compartments. We also found that Vps55p is mainly localized in the late endosomes. Collectively, these results indicate that Vps55p is involved in late endosome to vacuole trafficking. Finally, we show that human OB-RGRP displays the same distribution as Vps55p and corrects the phenotypic defects of the vps55Delta strain. Therefore, the function of Vps55p has been conserved throughout evolution. This study highlights the importance of the multispanning Vps55p and OB-RGRP in membrane trafficking to the vacuole/lysosome of eukaryotic cells.
Asunto(s)
Endosomas/metabolismo , Factores de Intercambio de Guanina Nucleótido , Proteínas de la Membrana/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Vacuolas/metabolismo , Carboxipeptidasas/biosíntesis , Carboxipeptidasas/metabolismo , Proteínas Portadoras/genética , Catepsina A , Proteínas Fúngicas/metabolismo , Aparato de Golgi/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Isoquinolinas/metabolismo , Proteínas de la Membrana/metabolismo , Familia de Multigenes , Mutación , Proteínas de Transporte de Nucleótidos/metabolismo , Transporte de Proteínas , Saccharomyces cerevisiae/genética , Uracilo/metabolismo , Proteínas de Transporte VesicularRESUMEN
We have stably expressed in HeLa cells a chimeric protein made of the green fluorescent protein (GFP) fused to the transmembrane and cytoplasmic domains of the mannose 6-phosphate/insulin like growth factor II receptor in order to study its dynamics in living cells. At steady state, the bulk of this chimeric protein (GFP-CI-MPR) localizes to the trans-Golgi network (TGN), but significant amounts are also detected in peripheral, tubulo-vesicular structures and early endosomes as well as at the plasma membrane. Time-lapse videomicroscopy shows that the GFP-CI-MPR is ubiquitously detected in tubular elements that detach from the TGN and move toward the cell periphery, sometimes breaking into smaller tubular fragments. The formation of the TGN-derived tubules is temperature dependent, requires the presence of intact microtubule and actin networks, and is regulated by the ARF-1 GTPase. The TGN-derived tubules fuse with peripheral, tubulo-vesicular structures also containing the GFP-CI-MPR. These structures are highly dynamic, fusing with each other as well as with early endosomes. Time-lapse videomicroscopy performed on HeLa cells coexpressing the CFP-CI-MPR and the AP-1 complex whose gamma-subunit was fused to YFP shows that AP-1 is present not only on the TGN and peripheral CFP-CI-MPR containing structures but also on TGN-derived tubules containing the CFP-CI-MPR. The data support the notion that tubular elements can mediate MPR transport from the TGN to a peripheral, tubulo-vesicular network dynamically connected with the endocytic pathway and that the AP-1 coat may facilitate MPR sorting in the TGN and endosomes.
Asunto(s)
Complejo 1 de Proteína Adaptadora/metabolismo , Receptor IGF Tipo 2/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Red trans-Golgi/metabolismo , Complejo 1 de Proteína Adaptadora/genética , Transporte Biológico , Genes Reporteros , Células HeLa , Humanos , Microtúbulos/metabolismo , Receptor IGF Tipo 2/genética , Proteínas Recombinantes de Fusión/genéticaRESUMEN
Clathrin/adaptor protein-1-coated carriers connect the secretory and the endocytic pathways. Carrier biogenesis relies on distinct protein networks changing membrane shape at the trans-Golgi network, each regulating coat assembly, F-actin-based mechanical forces, or the biophysical properties of lipid bilayers. How these different hubs are spatiotemporally coordinated remains largely unknown. Using in vitro reconstitution systems, quantitative proteomics, and lipidomics, as well as in vivo cell-based assays, we characterize the protein networks controlling membrane lipid composition, membrane shape, and carrier scission. These include PIP5K1A and phospholipase C-beta 3 controlling the conversion of PI[4]P into diacylglycerol. PIP5K1A binding to RAC1 provides a link to F-actin-based mechanical forces needed to tubulate membranes. Tubular membranes then recruit the BAR-domain-containing arfaptin-1/2 guiding carrier scission. These findings provide a framework for synchronizing the chemical/biophysical properties of lipid bilayers, F-actin-based mechanical forces, and the activity of proteins sensing membrane shape during clathrin/adaptor protein-1-coated carrier biogenesis.
Asunto(s)
Actinas/metabolismo , Complejo 1 de Proteína Adaptadora/metabolismo , Vesículas Cubiertas por Clatrina/metabolismo , Metabolismo de los Lípidos , Animales , Fenómenos Biomecánicos , Proteínas Portadoras/metabolismo , Clatrina/metabolismo , Diglicéridos/biosíntesis , Células HeLa , Humanos , Ratones , Fosfatos de Fosfatidilinositol/metabolismo , Fosfolipasa C beta/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Polimerizacion , Proteína de Unión al GTP rac1/metabolismoRESUMEN
Multipotent mesenchymal stromal cells (MSCs) are promising tools for regenerative medicine. They can be isolated from different sources based on their plastic-adherence property. The identification of reliable cell surface markers thus becomes the Holy Grail for their prospective isolation. Here, we determine the cell surface proteomes of human dental pulp-derived MSCs isolated from single donors after culture expansion in low (2%) or high (10%) serum-containing media. Cell surface proteins were tagged on intact cells using cell impermeable, cleavable sulfo-NHS-SS-biotin, which allows their enrichment by streptavidin pull-down. For the proteomic analyses, we first compared label-free methods to analyze cell surface proteomes i.e. composition, enrichment and proteomic differences, and we developed a new mathematical model to determine cell surface protein enrichment using a combinatorial gene ontology query. Using this workflow, we identified 101 cluster of differentiation (CD) markers and 286 non-CD cell surface proteins. Based on this proteome profiling, we identified 14 cell surface proteins, which varied consistently in abundance when cells were cultured under low or high serum conditions. Collectively, our analytical methods provide a basis for identifying the cell surface proteome of dental pulp stem cells isolated from single donors and its evolution during culture or differentiation. Our data provide a comprehensive cell surface proteome for the precise identification of dental pulp-derived MSC populations and their isolation for potential therapeutic intervention.
Asunto(s)
Pulpa Dental/metabolismo , Espectrometría de Masas , Proteoma/análisis , Adolescente , Antígenos CD/análisis , Antígenos CD/genética , Biotina/química , Células Cultivadas , Pulpa Dental/citología , Citometría de Flujo , Humanos , Immunoblotting , Inmunohistoquímica , Proteínas de la Membrana/análisis , Proteínas de la Membrana/genética , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Proteoma/genética , Proteómica , Reacción en Cadena en Tiempo Real de la Polimerasa , Adulto JovenRESUMEN
The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) facilitates fast axonal transport in neurons. However, given that GAPDH does not produce ATP, it is unclear whether glycolysis per se is sufficient to propel vesicles. Although many proteins regulating transport have been identified, the molecular composition of transported vesicles in neurons has yet to be fully elucidated. Here we selectively enrich motile vesicles and perform quantitative proteomic analysis. In addition to the expected molecular motors and vesicular proteins, we find an enrichment of all the glycolytic enzymes. Using biochemical approaches and super-resolution microscopy, we observe that most glycolytic enzymes are selectively associated with vesicles and facilitate transport of vesicles in neurons. Finally, we provide evidence that mouse brain vesicles produce ATP from ADP and glucose, and display movement in a reconstituted in vitro transport assay of native vesicles. We conclude that transport of vesicles along microtubules can be autonomous.