RESUMO
Most available information on endoplasmic reticulum (ER)-plasma membrane (PM) contacts in cells of higher eukaryotes concerns proteins implicated in the regulation of Ca(2+) entry. However, growing evidence suggests that such contacts play more general roles in cell physiology, pointing to the existence of additionally ubiquitously expressed ER-PM tethers. Here, we show that the three extended synaptotagmins (E-Syts) are ER proteins that participate in such tethering function via C2 domain-dependent interactions with the PM that require PI(4,5)P2 in the case of E-Syt2 and E-Syt3 and also elevation of cytosolic Ca(2+) in the case of E-Syt1. As they form heteromeric complexes, the E-Syts confer cytosolic Ca(2+) regulation to ER-PM contact formation. E-Syts-dependent contacts, however, are not required for store-operated Ca(2+) entry. Thus, the ER-PM tethering function of the E-Syts (tricalbins in yeast) mediates the formation of ER-PM contacts sites, which are functionally distinct from those mediated by STIM1 and Orai1.
Assuntos
Cálcio/metabolismo , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Sinaptotagminas/metabolismo , Sequência de Aminoácidos , Linhagem Celular Tumoral , Membrana Celular/química , Membrana Celular/ultraestrutura , Retículo Endoplasmático/química , Retículo Endoplasmático/ultraestrutura , Células HeLa , Humanos , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Alinhamento de Sequência , Sinaptotagminas/química , Sinaptotagminas/genética , Leveduras/citologia , Leveduras/metabolismoRESUMO
The endoplasmic reticulum (ER) is the reservoir for calcium in cells. Luminal calcium levels are determined by calcium-sensing proteins that trigger calcium dynamics in response to calcium fluctuations. Here we report that Selenoprotein N (SEPN1) is a type II transmembrane protein that senses ER calcium fluctuations by binding this ion through a luminal EF-hand domain. In vitro and in vivo experiments show that via this domain, SEPN1 responds to diminished luminal calcium levels, dynamically changing its oligomeric state and enhancing its redox-dependent interaction with cellular partners, including the ER calcium pump sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA). Importantly, single amino acid substitutions in the EF-hand domain of SEPN1 identified as clinical variations are shown to impair its calcium-binding and calcium-dependent structural changes, suggesting a key role of the EF-hand domain in SEPN1 function. In conclusion, SEPN1 is a ER calcium sensor that responds to luminal calcium depletion, changing its oligomeric state and acting as a reductase to refill ER calcium stores.
Assuntos
Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas Sensoras de Cálcio Intracelular/metabolismo , Proteínas Musculares/metabolismo , Selenoproteínas/metabolismo , Células HeLa , Humanos , Proteínas Sensoras de Cálcio Intracelular/genética , Proteínas Musculares/genética , Oxirredução , Selenoproteínas/genéticaRESUMO
α3ß4 nicotinic acetylcholine receptors (nARs) are pentameric ligand-gated cation channels that function in peripheral tissue and in the peripheral and central nervous systems, where they are critical mediators of ganglionic synaptic transmission and modulators of reward-related behaviours. In the pentamer, two α3ß4 subunit couples provide ligand-binding sites, and the fifth single (accessory) subunit (α3 or ß4) regulates receptor trafficking from the endoplasmic reticulum to the cell surface. A number of rare missense variants of the human ß4 subunit have recently been linked to nicotine dependence and/or sporadic amyotrophic lateral sclerosis, and altered responses to nicotine have been reported for these variants; however, it is unknown whether the effects of mutations depend on the subunit within the ligand-binding couples and/or on the fifth subunit. Here, by expressing single populations of pentameric receptors with fixed stoichiometry in cultured cells, we investigated the effect of ß4 variants in the fifth position on the assembly and surface exposure of α3ß4 nAChRs. The results demonstrate that the missense mutations in the accessory subunit alone, despite not affecting the assembly of α3ß4 receptors, alter their trafficking and surface localisation. Thus, altered trafficking of an otherwise functional nAChR may underlie the pathogenic effects of these mutations.
Assuntos
Mutação de Sentido Incorreto , Receptores Nicotínicos , Humanos , Ligantes , Receptores Nicotínicos/metabolismo , Nicotina/metabolismo , Membrana Celular/metabolismoRESUMO
VAPB and VAPA are ubiquitously expressed endoplasmic reticulum membrane proteins that play key roles in lipid exchange at membrane contact sites. A mutant, aggregation-prone, form of VAPB (P56S) is linked to a dominantly inherited form of amyotrophic lateral sclerosis; however, it has been unclear whether its pathogenicity is due to toxic gain of function, to negative dominance, or simply to insufficient levels of the wild-type protein produced from a single allele (haploinsufficiency). To investigate whether reduced levels of functional VAPB, independently from the presence of the mutant form, affect the physiology of mammalian motoneuron-like cells, we generated NSC34 clones, from which VAPB was partially or nearly completely depleted. VAPA levels, determined to be over fourfold higher than those of VAPB in untransfected cells, were unaffected. Nonetheless, cells with even partially depleted VAPB showed an increase in Golgi- and acidic vesicle-localized phosphatidylinositol-4-phosphate (PI4P) and reduced neurite extension when induced to differentiate. Conversely, the PI4 kinase inhibitors PIK93 and IN-10 increased neurite elongation. Thus, for long-term survival, motoneurons might require the full dose of functional VAPB, which may have unique function(s) that VAPA cannot perform.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Retículo Endoplasmático/metabolismo , Neurônios Motores/metabolismo , Neuritos/metabolismo , Fosfatidilinositóis/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Animais , Complexo de Golgi/metabolismo , Células HeLa , Humanos , Neurônios Motores/patologia , Mutação , Neuritos/patologia , Ratos , Proteínas de Transporte Vesicular/genéticaRESUMO
Tail-anchored (TA) proteins insert into their target organelles by incompletely elucidated posttranslational pathways. Some TA proteins spontaneously insert into protein-free liposomes, yet target a specific organelle in vivo. Two spontaneously inserting cytochrome b5 forms, b5-ER and b5-RR, which differ only in the charge of the C-terminal region, target the endoplasmic reticulum (ER) or the mitochondrial outer membrane (MOM), respectively. To bridge the gap between the cell-free and in cellula results, we analyzed targeting in digitonin-permeabilized adherent HeLa cells. In the absence of cytosol, the MOM was the destination of both b5 forms, whereas in cytosol the C-terminal negative charge of b5-ER determined targeting to the ER. Inhibition of the transmembrane recognition complex (TRC) pathway only partially reduced b5 targeting, while strongly affecting the classical TRC substrate synaptobrevin 2 (Syb2). To identify additional pathways, we tested a number of small inhibitors, and found that Eeyarestatin I (ESI ) reduced insertion of b5-ER and of another spontaneously inserting TA protein, while not affecting Syb2. The effect was independent from the known targets of ESI , Sec61 and p97/VCP. Our results demonstrate that the MOM is the preferred destination of spontaneously inserting TA proteins, regardless of their C-terminal charge, and reveal a novel, substrate-specific ER-targeting pathway.
Assuntos
Citocromos b5/metabolismo , Retículo Endoplasmático/metabolismo , Membranas Mitocondriais/metabolismo , Animais , Linhagem Celular , Chlorocebus aethiops , Citocromos b5/química , Células HeLa , Humanos , Domínios Proteicos , Transporte Proteico , Proteínas R-SNARE/metabolismoRESUMO
Primitive myxoid mesenchymal tumor of infancy is a rare soft tissue tumor. The present case is one of the most invasive primitive myxoid mesenchymal tumor of infancy reported to date. To our knowledge, it is the first case described with extensive involvement of pelvis and the third described developing metastasis and with an invasion of the spinal canal without evidence of transformation into undifferentiated sarcoma. The patient failed to respond to chemotherapy (CHT). According to the few available data, CHT seems to be more effective in the presence of metastatic disease or increased cellularity. However, CHT, including high-dose ifosfamide, resulted ineffective even after lung metastasis development with pathologic evidence of increased mitotic rate. The management of this case and the data in the literature confirm surgery as the gold standard treatment in this pathology.
Assuntos
Neoplasias de Tecidos Moles/patologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Evolução Fatal , Humanos , Lactente , Masculino , Neoplasias de Tecidos Moles/tratamento farmacológico , Neoplasias de Tecidos Moles/cirurgiaRESUMO
The α3ß4 subtype is the predominant neuronal nicotinic acetylcholine receptor present in the sensory and autonomic ganglia and in a subpopulation of brain neurons. This subtype can form pentameric receptors with either 2 or 3 ß4 subunits that have different pharmacologic and functional properties. To further investigate the role of the fifth subunit, we coexpressed a dimeric construct coding for a single polypeptide containing the ß4 and α3 subunit sequences, with different monomeric subunits. With this strategy, which allowed the formation of single populations of receptors with unique stoichiometry, we demonstrated with immunofluorescence and biochemical and functional assays that only the receptors with 3 ß4 subunits are efficiently expressed at the plasma membrane. Moreover, the LFM export motif of ß4 subunit in the fifth position exerts a unique function in the regulation of the intracellular trafficking of the receptors, their exposure at the cell surface, and consequently, their function, whereas the same export motif present in the ß4 subunits forming the acetylcholine binding site is dispensable.-Crespi, A., Plutino, S., Sciaccaluga, M., Righi, M., Borgese, N., Fucile, S., Gotti, C., Colombo, S. F. The fifth subunit in α3ß4 nicotinic receptor is more than an accessory subunit.
Assuntos
Subunidades Proteicas/metabolismo , Receptores Nicotínicos/metabolismo , Sítios de Ligação/fisiologia , Membrana Celular/metabolismo , Células Cultivadas , HumanosRESUMO
The Golgi complex and ER are dynamically connected by anterograde and retrograde trafficking pathways. To what extent and by what mechanism outward-bound cargo proteins escape retrograde trafficking has been poorly investigated. Here, we analysed the behaviour of several membrane proteins at the ER/Golgi interface in live cells. When Golgi-to-plasma membrane transport was blocked, vesicular stomatitis virus glycoprotein (VSVG), which bears an ER export signal, accumulated in the Golgi, whereas an export signal-deleted version of VSVG attained a steady state determined by the balance of retrograde and anterograde traffic. A similar behaviour was displayed by EGF receptor and by a model tail-anchored protein, whose retrograde traffic was slowed by addition of VSVG's export signal. Retrograde trafficking was energy- and Rab6-dependent, and Rab6 inhibition accelerated signal-deleted VSVG's transport to the cell surface. Our results extend the dynamic bi-directional relationship between the Golgi and ER to include surface-directed proteins, uncover an unanticipated role for export signals at the Golgi complex, and identify recycling as a novel factor that regulates cargo transport out of the early secretory pathway.
Assuntos
Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Sinais Direcionadores de Proteínas , Transporte Proteico , Vesículas Secretórias/metabolismo , Animais , Linhagem Celular , Glicoproteínas/metabolismo , Humanos , Ratos , Proteínas Virais/metabolismoRESUMO
The GET (guided entry of tail-anchored proteins)/TRC (transmembrane recognition complex) pathway for tail-anchored protein targeting to the endoplasmic reticulum (ER) has been characterized in detail in yeast and is thought to function similarly in mammals, where the orthologue of the central ATPase, Get3, is known as TRC40 or Asna1. Get3/TRC40 function requires an ER receptor, which in yeast consists of the Get1/Get2 heterotetramer and in mammals of the WRB protein (tryptophan-rich basic protein), homologous to yeast Get1, in combination with CAML (calcium-modulating cyclophilin ligand), which is not homologous to Get2. To better characterize the mammalian receptor, we investigated the role of endogenous WRB and CAML in tail-anchored protein insertion as well as their association, concentration, and stoichiometry in rat liver microsomes and cultured cells. Functional proteoliposomes, reconstituted from a microsomal detergent extract, lost their activity when made with an extract depleted of TRC40-associated proteins or of CAML itself, whereas in vitro synthesized CAML and WRB together were sufficient to confer insertion competence to liposomes. CAML was found to be in â¼5-fold excess over WRB, and alteration of this ratio did not inhibit insertion. Depletion of each subunit affected the levels of the other one; in the case of CAML silencing, this effect was attributable to destabilization of the WRB transcript and not of WRB protein itself. These results reveal unanticipated complexity in the mutual regulation of the TRC40 receptor subunits and raise the question as to the role of the excess CAML in the mammalian ER.
Assuntos
ATPases Transportadoras de Arsenito/química , ATPases Transportadoras de Arsenito/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Animais , ATPases Transportadoras de Arsenito/genética , Linhagem Celular , Células Cultivadas , Síndrome de Down/genética , Síndrome de Down/metabolismo , Retículo Endoplasmático/metabolismo , Regulação da Expressão Gênica , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células HeLa , Humanos , Microssomos Hepáticos/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Subunidades Proteicas , Transporte Proteico , Proteolipídeos/metabolismo , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
UNLABELLED: Sepsis is the major cause of morbidity and mortality in children, especially in immunocompromised patients, and a rapid identification of causative pathogen is strongly related with a better outcome. This prospective study analyzes the role of a multiplex real-time polymerase chain reaction in sepsis' etiological diagnosis. Magicplex(TM) Sepsis Real-Time tests were performed in tertiary Regina Margherita Children's Hospital (Turin, Italy), and the medical records of children who underwent a Magicplex test were prospectively evaluated. Results of the Magicplex test were compared with those of blood culture collected at a close time point. One hundred fifty Magicplex tests were collected from 89 patients (54 males and 35 females, age interquartile range: 2.6-12.1 years). Etiological definition was achieved in 60 bloodstream infection cases (40 %). In 32 episodes, Magicplex test alone gave a positive result, and blood culture alone permitted the etiological diagnosis in 5 septic episodes. Magicplex test allowed a 143 % increase in the diagnostic value of blood cultures. CONCLUSION: These results suggest that molecular biology can be useful for rapid pathogen's identification also in children. WHAT IS KNOWN: ⢠Sepsis represents a major cause of morbidity and mortality in children. ⢠Sepsis outcome is strongly related to rapid microbiological identification and prompt initiation of an appropriate chemotherapy. What is New: ⢠This manuscript is the first that describes the use of Magicplex (TM) Sepsis Real-Time test in children. ⢠The results suggest that molecular biology can be useful for rapid pathogen's identification also in children.
Assuntos
Bacteriemia/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real/métodos , Criança , Pré-Escolar , Feminino , Humanos , Hospedeiro Imunocomprometido , Masculino , Estudos Prospectivos , Sensibilidade e Especificidade , Fatores de TempoRESUMO
Allotypes of the natural killer (NK) cell receptor KIR3DL1 vary in both NK cell expression patterns and inhibitory capacity upon binding to their ligands, HLA-B Bw4 molecules, present on target cells. Using a sample size of over 1,500 human immunodeficiency virus (HIV)+ individuals, we show that various distinct allelic combinations of the KIR3DL1 and HLA-B loci significantly and strongly influence both AIDS progression and plasma HIV RNA abundance in a consistent manner. These genetic data correlate very well with previously defined functional differences that distinguish KIR3DL1 allotypes. The various epistatic effects observed here for common, distinct KIR3DL1 and HLA-B Bw4 combinations are unprecedented with regard to any pair of genetic loci in human disease, and indicate that NK cells may have a critical role in the natural history of HIV infection.
Assuntos
Infecções por HIV/imunologia , HIV-1/genética , Antígenos HLA-B/metabolismo , Receptores Imunológicos/metabolismo , Alelos , Estudos de Coortes , Progressão da Doença , Infecções por HIV/genética , Infecções por HIV/metabolismo , Antígenos HLA-B/genética , Antígenos HLA-B/imunologia , Humanos , Imunidade Inata , Células Matadoras Naturais/imunologia , RNA Viral/sangue , RNA Viral/genética , Receptores Imunológicos/genética , Receptores Imunológicos/imunologia , Receptores KIR , Receptores KIR3DL1 , Carga ViralRESUMO
Human genetic variation contributes to differences in susceptibility to HIV-1 infection. To search for novel host resistance factors, we performed a genome-wide association study (GWAS) in hemophilia patients highly exposed to potentially contaminated factor VIII infusions. Individuals with hemophilia A and a documented history of factor VIII infusions before the introduction of viral inactivation procedures (1979-1984) were recruited from 36 hemophilia treatment centers (HTCs), and their genome-wide genetic variants were compared with those from matched HIV-infected individuals. Homozygous carriers of known CCR5 resistance mutations were excluded. Single nucleotide polymorphisms (SNPs) and inferred copy number variants (CNVs) were tested using logistic regression. In addition, we performed a pathway enrichment analysis, a heritability analysis, and a search for epistatic interactions with CCR5 Δ32 heterozygosity. A total of 560 HIV-uninfected cases were recruited: 36 (6.4%) were homozygous for CCR5 Δ32 or m303. After quality control and SNP imputation, we tested 1 081 435 SNPs and 3686 CNVs for association with HIV-1 serostatus in 431 cases and 765 HIV-infected controls. No SNP or CNV reached genome-wide significance. The additional analyses did not reveal any strong genetic effect. Highly exposed, yet uninfected hemophiliacs form an ideal study group to investigate host resistance factors. Using a genome-wide approach, we did not detect any significant associations between SNPs and HIV-1 susceptibility, indicating that common genetic variants of major effect are unlikely to explain the observed resistance phenotype in this population.
Assuntos
Resistência à Doença/genética , Estudo de Associação Genômica Ampla , Infecções por HIV/genética , Hemofilia A/genética , Adulto , Variações do Número de Cópias de DNA , Epistasia Genética , Fator VIII/uso terapêutico , Feminino , Deleção de Genes , Predisposição Genética para Doença , Soropositividade para HIV/genética , Heterozigoto , Homozigoto , Humanos , Modelos Logísticos , Masculino , Metanálise como Assunto , Pessoa de Meia-Idade , Fenótipo , Polimorfismo de Nucleotídeo Único , Estudos Prospectivos , Receptores CCR5/genética , Receptores CCR5/metabolismoRESUMO
Heteromeric nAChRs are pentameric cation channels, composed of combinations of two or three α and three or two ß subunits, which play key physiological roles in the central and peripheral nervous systems. The prototypical agonist nicotine acts intracellularly to upregulate many nAChR subtypes, a phenomenon that is thought to contribute to the nicotine dependence of cigarette smokers. The α3ß4 subtype has recently been genetically linked to nicotine dependence and lung cancer; however, the mode of action of nicotine on this receptor subtype has been incompletely investigated. Here, using transfected mammalian cells as model system, we characterized the response of the human α3ß4 receptor subtype to nicotine and the mechanism of action of the drug. Nicotine, when present at 1 mm concentration, elicited a â¼5-fold increase of cell surface α3ß4 and showed a more modest upregulatory effect also at concentrations as low as 10 µM. Upregulation was obtained if nicotine was present during, but not after, pentamer assembly and was caused by increased stability and trafficking of receptors assembled in the presence of the drug. Experimental determinations as well as computational studies of subunit stoichiometry showed that nicotine favors assembly of pentamers with (α3)2(ß4)3 stoichiometry; these are less prone than (α3)3(ß4)2 receptors to proteasomal degradation and, because of the presence in the ß subunit of an endoplasmic reticulum export motif, more efficiently transported to the plasma membrane. Our findings uncover a novel mechanism of nicotine-induced α3ß4 nAChR upregulation that may be relevant also for other nAChR subtypes.
Assuntos
Nicotina/farmacologia , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , Receptores Nicotínicos/metabolismo , Fumar/fisiopatologia , Animais , Anticorpos/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Células HeLa , Humanos , Masculino , Modelos Químicos , Mutagênese/fisiologia , Neuroblastoma , Agonistas Nicotínicos/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Coelhos , Receptores Nicotínicos/química , Receptores Nicotínicos/genética , Receptores Nicotínicos/imunologia , Fumar/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologiaRESUMO
The newly synthesized mutant L501fsX533 Frizzled-4 form and the alpha3beta4 nicotinic acetylcholine receptor expressed in the absence of nicotine accumulate in the endoplasmic reticulum of COS-7 cells and induce the formation of large areas of smooth and highly convoluted cisternae. This results in a generalized block of the transport to the Golgi complex of newly synthesized proteins. Intriguingly, both effects happen peculiarly in COS-7 cells; HeLa, Huh-7, and HEK293 cells expressing the two receptors at similar level than COS-7 cells show normal ER and normal transport toward the plasma membrane. These results question the conclusion that a dominant-negative mechanism would explain the dominance of the mutant L501fsX533 Fz4 allele in the transmission of a form of Familial exudative vitreoretinopathy. Moreover, they indicate that the coordination of endoplasmic reticulum homeostasis in COS-7 cells is particularly error prone. This finding suggests that COS-7 cells may be extremely useful to study the molecular mechanisms regulating endoplasmic reticulum size and architecture.
Assuntos
Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Receptores Frizzled/metabolismo , Receptores Nicotínicos/metabolismo , Animais , Células COS , Chlorocebus aethiops , Receptores Frizzled/genética , Células HEK293 , Células HeLa , Humanos , Mutação/genética , Receptores Nicotínicos/genéticaRESUMO
A large group of diverse, functionally important, and differently localized transmembrane proteins shares a particular membrane topology, consisting of a cytosolic N-terminal region, followed by a transmembrane domain close to the C-terminus. Because of their structure, these C-tail-anchored (TA) proteins must insert into all their target membranes by post-translational pathways. Recent work, based on the development of stringent and sensitive biochemical assays, has demonstrated that novel unexplored mechanisms underlie these post-translational targeting and membrane insertion pathways. Unravelling these pathways will shed light on the biosynthesis and regulation of an important group of membrane proteins and is likely to lead to new concepts in the field of membrane biogenesis.
Assuntos
Proteínas de Membrana/biossíntese , Modelos Biológicos , Animais , Retículo Endoplasmático/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Peroxissomos/metabolismo , Processamento de Proteína Pós-Traducional , Sinais Direcionadores de Proteínas , Transporte Proteico , Transdução de SinaisRESUMO
A synthetic protocol for the preparation of a new class of morpholino homonucleosides in enantiopure form starting from readily available 1,2-aminoalcohols or glycidol has been developed. Key intermediates of the synthetic sequence are 2-bromomethyl morpholines, diastereoselectively achieved from the corresponding alkenols by palladaelectro-catalyzed alkoxybromination of unactivated alkenes. The so obtained bromo derivatives are in turn susceptible to functionalization with nucleic bases for easy access to morpholino homonucleosides.
RESUMO
SOD1 gene is associated with progressive motor neuron degeneration in the familiar forms of amyotrophic lateral sclerosis. Although studies on mutant human SOD1 transgenic rodent models have provided important insights into disease pathogenesis, they have not led to the discovery of early biomarkers or effective therapies in human disease. The recent generation of a transgenic swine model expressing the human pathological hSOD1G93A gene, which recapitulates the course of human disease, represents an interesting tool for the identification of early disease mechanisms and diagnostic biomarkers. Here, we analyze the activation state of CNS cells in transgenic pigs during the disease course and investigate whether changes in neuronal and glial cell activation state can be reflected by the amount of extracellular vesicles they release in biological fluids. To assess the activation state of neural cells, we performed a biochemical characterization of neurons and glial cells in the spinal cords of hSOD1G93A pigs during the disease course. Quantification of EVs of CNS cell origin was performed in cerebrospinal fluid and plasma of transgenic pigs at different disease stages by Western blot and peptide microarray analyses. We report an early activation of oligodendrocytes in hSOD1G93A transgenic tissue followed by astrocyte and microglia activation, especially in animals with motor symptoms. At late asymptomatic stage, EV production from astrocytes and microglia is increased in the cerebrospinal fluid, but not in the plasma, of transgenic pigs reflecting donor cell activation in the spinal cord. Estimation of EV production by biochemical analyses is corroborated by direct quantification of neuron- and microglia-derived EVs in the cerebrospinal fluid by a Membrane Sensing Peptide enabled on-chip analysis that provides fast results and low sample consumption. Collectively, our data indicate that alteration in astrocytic EV production precedes the onset of disease symptoms in the hSODG93A swine model, mirroring donor cell activation in the spinal cord, and suggest that EV measurements from the cells first activated in the ALS pig model, i.e. OPCs, may further improve early disease detection.
Assuntos
Esclerose Lateral Amiotrófica , Vesículas Extracelulares , Camundongos , Animais , Humanos , Suínos , Superóxido Dismutase-1/genética , Neurônios Motores/metabolismo , Superóxido Dismutase/genética , Camundongos Transgênicos , Esclerose Lateral Amiotrófica/patologia , Medula Espinal/patologia , Neuroglia/patologia , Biomarcadores/metabolismo , Peptídeos/metabolismo , Modelos Animais de DoençasRESUMO
Genome-wide association studies have led to the identification of numerous susceptibility genes for type 2 diabetes. Among them is Cdkal1, which is associated with reduced ß-cell function and insulin release. Recently, CDKAL1 has been shown to be a methylthiotransferase that modifies tRNA(Lys) to enhance translational fidelity of transcripts, including the one encoding proinsulin. Here, we report that out of several CDKAL1 isoforms deposited in public databases, only isoform 1, which migrates as a 61-kDa protein by SDS-PAGE, is expressed in human islets and pancreatic insulinoma INS-1 and MIN6 cells. We show that CDKAL1 is a novel member of the tail-anchored protein family and exploits the TCR40/Get3-assisted pathway for insertion of its C-terminal transmembrane domain into the endoplasmic reticulum. Using endo-ß-N-acetylglucosaminidase H and peptide:N-glycosidase F sensitivity assays on CDKAL1 constructs carrying an N-glycosylation site within the luminal domain, we further established that CDKAL1 is an endoplasmic reticulum-resident protein. Moreover, we observed that silencing CDKAL1 in INS-1 cells reduces the expression of secretory granule proteins prochromogranin A and proICA512/ICA512-TMF, in addition to proinsulin and insulin. This correlated with reduced glucose-stimulated insulin secretion. Taken together, our findings provide new insight into the role of CDKAL1 in insulin-producing cells and help to understand its involvement in the pathogenesis of diabetes.
Assuntos
Quinase 5 Dependente de Ciclina/metabolismo , Retículo Endoplasmático/metabolismo , Insulinoma/metabolismo , Proteínas de Neoplasias/metabolismo , Animais , Sequência de Bases , Linhagem Celular Tumoral , Quinase 5 Dependente de Ciclina/genética , Diabetes Mellitus/genética , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patologia , Retículo Endoplasmático/genética , Retículo Endoplasmático/patologia , Inativação Gênica , Humanos , Insulinoma/genética , Insulinoma/patologia , Dados de Sequência Molecular , Proteínas de Neoplasias/genética , Proinsulina/genética , Proinsulina/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Ratos , Proteínas Tirosina Fosfatases Classe 8 Semelhantes a Receptores/genética , Proteínas Tirosina Fosfatases Classe 8 Semelhantes a Receptores/metabolismo , tRNA MetiltransferasesRESUMO
BACKGROUND: Persons infected with human immunodeficiency virus (HIV) have increased rates of coronary artery disease (CAD). The relative contribution of genetic background, HIV-related factors, antiretroviral medications, and traditional risk factors to CAD has not been fully evaluated in the setting of HIV infection. METHODS: In the general population, 23 common single-nucleotide polymorphisms (SNPs) were shown to be associated with CAD through genome-wide association analysis. Using the Metabochip, we genotyped 1875 HIV-positive, white individuals enrolled in 24 HIV observational studies, including 571 participants with a first CAD event during the 9-year study period and 1304 controls matched on sex and cohort. RESULTS: A genetic risk score built from 23 CAD-associated SNPs contributed significantly to CAD (P = 2.9 × 10(-4)). In the final multivariable model, participants with an unfavorable genetic background (top genetic score quartile) had a CAD odds ratio (OR) of 1.47 (95% confidence interval [CI], 1.05-2.04). This effect was similar to hypertension (OR = 1.36; 95% CI, 1.06-1.73), hypercholesterolemia (OR = 1.51; 95% CI, 1.16-1.96), diabetes (OR = 1.66; 95% CI, 1.10-2.49), ≥ 1 year lopinavir exposure (OR = 1.36; 95% CI, 1.06-1.73), and current abacavir treatment (OR = 1.56; 95% CI, 1.17-2.07). The effect of the genetic risk score was additive to the effect of nongenetic CAD risk factors, and did not change after adjustment for family history of CAD. CONCLUSIONS: In the setting of HIV infection, the effect of an unfavorable genetic background was similar to traditional CAD risk factors and certain adverse antiretroviral exposures. Genetic testing may provide prognostic information complementary to family history of CAD.