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1.
Cell ; 152(5): 1119-33, 2013 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-23452857

RESUMO

The activation of N-methyl-D-aspartate-receptors (NMDARs) in synapses provides plasticity and cell survival signals, whereas NMDARs residing in the neuronal membrane outside synapses trigger neurodegeneration. At present, it is unclear how these opposing signals are transduced to and discriminated by the nucleus. In this study, we demonstrate that Jacob is a protein messenger that encodes the origin of synaptic versus extrasynaptic NMDAR signals and delivers them to the nucleus. Exclusively synaptic, but not extrasynaptic, NMDAR activation induces phosphorylation of Jacob at serine-180 by ERK1/2. Long-distance trafficking of Jacob from synaptic, but not extrasynaptic, sites depends on ERK activity, and association with fragments of the intermediate filament α-internexin hinders dephosphorylation of the Jacob/ERK complex during nuclear transit. In the nucleus, the phosphorylation state of Jacob determines whether it induces cell death or promotes cell survival and enhances synaptic plasticity.


Assuntos
Núcleo Celular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapses/metabolismo , Animais , Sobrevivência Celular , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Regulação da Expressão Gênica , Hipocampo/citologia , Hipocampo/metabolismo , Proteínas de Filamentos Intermediários/metabolismo , Potenciação de Longa Duração , Depressão Sináptica de Longo Prazo , Sistema de Sinalização das MAP Quinases , Camundongos , Neurônios/citologia , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação , Ratos
2.
J Nanobiotechnology ; 21(1): 376, 2023 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-37838659

RESUMO

BACKGROUND: Nanoparticles represent one of the most important innovations in the medical field. Among nanocarriers, polymeric nanoparticles (PNPs) attracted much attention due to their biodegradability, biocompatibility, and capacity to increase efficacy and safety of encapsulated drugs. Another important improvement in the use of nanoparticles as delivery systems is the conjugation of a targeting agent that enables the nanoparticles to accumulate in a specific tissue. Despite these advantages, the clinical translation of therapeutic approaches based on nanoparticles is prevented by their interactions with blood proteins. In fact, the so-formed protein corona (PC) drastically alters the biological identity of the particles. Adsorbed activated proteins of the complement cascade play a pivotal role in the clearance of nanoparticles, making them more easily recognized by macrophages, leading to their rapid elimination from the bloodstream and limiting their efficacy. Since the mouse is the most used preclinical model for human disease, this work compared human and mouse PC formed on untargeted PNPs (uPNPs) and targeted PNPs (tPNPs), paying particular attention to complement activation. RESULTS: Mouse and human serum proteins adsorbed differently to PNPs. The differences in the binding of mouse complement proteins are minimal, whereas human complement components strongly distinguish the two particles. This is probably due to the human origin of the Fc portion of the antibody used as targeting agent on tPNPs. tPNPs and uPNPs mainly activate complement via the classical and alternative pathways, respectively, but this pattern did not affect their binding and internalization in macrophages and only a limited consumption of the activity of the human complement system was documented. CONCLUSIONS: The results clearly indicate the presence of complement proteins on PNPs surface but partially derived from an unspecific deposition rather than an effective complement activation. The presence of a targeting antibody favors the activation of the classical pathway, but its absence allows an increased activation of the alternative pathway. This results in similar opsonization of both PNPs and similar phagocytosis by macrophages, without an impairment of the activity of circulating complement system and, consequently, not enhancing the susceptibility to infection.


Assuntos
Nanopartículas , Coroa de Proteína , Humanos , Camundongos , Animais , Opsonização , Proteínas do Sistema Complemento/metabolismo , Anticorpos , Polímeros
3.
Nucleic Acids Res ; 49(11): 6437-6455, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34096600

RESUMO

The biogenesis of small uridine-rich nuclear ribonucleoproteins (UsnRNPs) depends on the methylation of Sm proteins catalyzed by the methylosome and the subsequent action of the SMN complex, which assembles the heptameric Sm protein ring onto small nuclear RNAs (snRNAs). In this sophisticated process, the methylosome subunit pICln (chloride conductance regulatory protein) is attributed to an exceptional key position as an 'assembly chaperone' by building up a stable precursor Sm protein ring structure. Here, we show that-apart from its autophagic role-the Ser/Thr kinase ULK1 (Uncoordinated [unc-51] Like Kinase 1) functions as a novel key regulator in UsnRNP biogenesis by phosphorylation of the C-terminus of pICln. As a consequence, phosphorylated pICln is no longer capable to hold up the precursor Sm ring structure. Consequently, inhibition of ULK1 results in a reduction of efficient UsnRNP core assembly. Thus ULK1, depending on its complex formation, exerts different functions in autophagy or snRNP biosynthesis.


Assuntos
Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ribonucleoproteínas Nucleares Pequenas/biossíntese , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/antagonistas & inibidores , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/fisiologia , Linhagem Celular , Corpos Enovelados , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Canais Iônicos/metabolismo , Fosforilação , Proteína-Arginina N-Metiltransferases/metabolismo
4.
Arch Pharm (Weinheim) ; 356(7): e2200661, 2023 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-37196427

RESUMO

Ubiquitin-specific proteases represent a family of enzymes that catalyze the cleavage of ubiquitin from specific substrate proteins to regulate their activity. USP48 is a rarely studied USP, which has recently been linked to inflammatory signaling via regulation of the transcription factor nuclear factor kappa B. Nonetheless, a crystal structure of USP48 has not yet been resolved and potent inhibitors are not known. We screened a set of 14 commercially available USP inhibitors for their activity against USP48 and identified the USP2 inhibitor "ML364" as a candidate for further optimization. Using a ligand-based approach, we derived and synthesized a series of ML364 analogs. The IC50 concentrations of the new compounds to inhibit USP48 were determined in a deubiquitinylase activity assay by measuring the fluorescence intensity using tetra-ubiquitin rhodamine110 as substrate. A compound containing a carboxylic acid functionalization (17e) inhibited USP48 activity toward tetra-ubiquitin rhodamine110 with an IC50 of 12.6 µM. Further structure-based refinements are required to improve the inhibition activity and specificity.


Assuntos
Transdução de Sinais , Proteases Específicas de Ubiquitina , Relação Estrutura-Atividade , Proteases Específicas de Ubiquitina/química , Proteases Específicas de Ubiquitina/metabolismo , Fatores de Transcrição , Ubiquitinas
5.
Int J Mol Sci ; 24(21)2023 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-37958537

RESUMO

The survival motor neuron (SMN) complex is a multi-megadalton complex involved in post-transcriptional gene expression in eukaryotes via promotion of the biogenesis of uridine-rich small nuclear ribonucleoproteins (UsnRNPs). The functional center of the complex is formed from the SMN/Gemin2 subunit. By binding the pentameric ring made up of the Sm proteins SmD1/D2/E/F/G and allowing for their transfer to a uridine-rich short nuclear RNA (UsnRNA), the Gemin2 protein in particular is crucial for the selectivity of the Sm core assembly. It is well established that post-translational modifications control UsnRNP biogenesis. In our work presented here, we emphasize the crucial role of Gemin2, showing that the phospho-status of Gemin2 influences the capacity of the SMN complex to condense in Cajal bodies (CBs) in vivo. Additionally, we define Gemin2 as a novel and particular binding partner and phosphorylation substrate of the mTOR pathway kinase ribosomal protein S6 kinase beta-1 (p70S6K). Experiments using size exclusion chromatography further demonstrated that the Gemin2 protein functions as a connecting element between the 6S complex and the SMN complex. As a result, p70S6K knockdown lowered the number of CBs, which in turn inhibited in vivo UsnRNP synthesis. In summary, these findings reveal a unique regulatory mechanism of UsnRNP biogenesis.


Assuntos
Proteínas de Ligação a RNA , Proteínas Quinases S6 Ribossômicas 70-kDa , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Fosforilação , Ribonucleoproteínas Nucleares Pequenas/genética , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas do Complexo SMN/genética , Uridina/metabolismo
6.
J Neurochem ; 161(1): 20-39, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-35050500

RESUMO

Vitamins B1 (thiamine) and B6 (pyridox (al/ine/amine)) are crucial for central nervous system (CNS) function and neurogenesis due to the coenzyme action of their phosphorylated derivatives in the brain metabolism of glucose and neurotransmitters. Here, the non-coenzyme action of thiamine on the major mammalian producers of pyridoxal-5'-phosphate (PLP), such as pyridoxal kinase (PdxK) and pyridoxine 5'-phosphate oxidase (PNPO), is characterized. Among the natural thiamine compounds, thiamine triphosphate (ThTP) is the best effector of recombinant human PdxK (hPdxK) in vitro, inhibiting hPdxK in the presence of Mg2+ but activating the Zn2+ -dependent reaction. Inhibition of hPdxK by thiamine antagonists decreases from amprolium to pyrithiamine to oxythiamine, highlighting possible dysregulation of both the B1 - and B6 -dependent metabolism in the chemical models of thiamine deficiency. Compared with the canonical hPdxK, the D87H and V128I variants show a twofold increase in Kapp of thiamine inhibition, and the V128I and H246Q variants show a fourfold and a twofold decreased Kapp of thiamine diphosphate (ThDP), respectively. Thiamine administration changes diurnal regulation of PdxK activity and phosphorylation at Ser213 and Ser285, expression of the PdxK-related circadian kinases/phosphatases in the rat brain, and electrocardiography (ECG). In contrast to PdxK, PNPO is not affected by thiamine or its derivatives, either in vitro or in vivo. Dephosphorylation of the PdxK Ser285, potentially affecting mobility of the ATP-binding loop, inversely correlates with the enzyme activity. Dephosphorylation of the PdxK Ser213, which is far away from the active site, does not correlate with the activity. The correlations analysis suggests the PdxK Ser213 to be a target of kinase MAP2K1 and phosphatase Ppp1ca. Diurnal effects of thiamine administration on the metabolically linked ThDP- and PLP-dependent enzymes may support the brain homeostatic mechanisms and physiological fitness.


Assuntos
Piridoxal Quinase , Tiamina , Animais , Encéfalo/metabolismo , Mamíferos/metabolismo , Fosfatos , Piridoxal Quinase/química , Piridoxal Quinase/metabolismo , Fosfato de Piridoxal/metabolismo , Fosfato de Piridoxal/farmacologia , Ratos , Tiamina/farmacologia
7.
Eur J Immunol ; 51(2): 342-353, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33169379

RESUMO

The immunological synapse is a transient junction that occurs when the plasma membrane of a T cell comes in close contact with an APC after recognizing a peptide from the antigen-MHC. The interaction starts when CRAC channels embedded in the T cell membrane open, flowing calcium ions into the cell. To counterbalance the ion influx and subsequent depolarization, Kv 1.3 and KCa3.1 channels are recruited to the immunological synapse, increasing the extracellular K+ concentration. These processes are crucial as they initiate gene expression that drives T cell activation and proliferation. The T cell-specific function of the K2P channel family member TASK2 channels and their role in autoimmune processes remains unclear. Using mass spectrometry analysis together with epifluorescence and super-resolution single-molecule localization microscopy, we identified TASK2 channels as novel players recruited to the immunological synapse upon stimulation. TASK2 localizes at the immunological synapse, upon stimulation with CD3 antibodies, likely interacting with these molecules. Our findings suggest that, together with Kv 1.3 and KCa3.1 channels, TASK2 channels contribute to the proper functioning of the immunological synapse, and represent an interesting treatment target for T cell-mediated autoimmune disorders.


Assuntos
Sinapses Imunológicas/imunologia , Canais de Potássio de Domínios Poros em Tandem/imunologia , Animais , Doenças Autoimunes/imunologia , Complexo CD3/imunologia , Cálcio/imunologia , Linhagem Celular Tumoral , Membrana Celular/imunologia , Células Cultivadas , Feminino , Expressão Gênica/imunologia , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/imunologia , Células Jurkat , Canal de Potássio Kv1.3/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T/imunologia
8.
Cell Mol Life Sci ; 77(20): 4133-4142, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31932854

RESUMO

The transcription factors of the nuclear factor κB (NF-κB) family play a pivotal role in the cellular response to DNA damage. Genotoxic stress-induced activation of NF-κB differs from the classical canonical pathway by shuttling of the NF-κB Essential Modifier (IKKγ/NEMO) subunit through the nucleus. Here, we show that DNA-dependent protein kinase (DNA-PK), an enzyme involved in DNA double-strand break (DSB) repair, triggers the phosphorylation of NEMO by genotoxic stress, thereby enabling shuttling of NEMO through the nucleus with subsequent NF-κB activation. We identified serine 43 of NEMO as a DNA-PK phosphorylation site and point mutation of this serine to alanine led to a complete block of NF-κB activation by ionizing radiation (IR). Blockade of DNA-PK by a specific shRNA or by DNA-PKcs-deficient cells abrogated NEMO entry into the nucleus, as well. Accordingly, SUMOylation of NEMO, a prerequisite of nuclear NEMO, was abolished. Based on these observations, we propose a model in which NEMO phosphorylation by DNA-PK provides the first step in the nucleocytoplasmic trafficking of NEMO.


Assuntos
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteína Quinase Ativada por DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Quinase I-kappa B/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , NF-kappa B/metabolismo , Alanina/metabolismo , Animais , Dano ao DNA/fisiologia , Células HEK293 , Humanos , Camundongos , Células NIH 3T3 , Fosforilação/fisiologia , Serina/metabolismo , Transdução de Sinais/fisiologia
9.
Traffic ; 18(1): 29-43, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27743426

RESUMO

K2P 5.1 channels (also called TASK-2 or Kcnk5) have already been shown to be relevant in the pathophysiology of autoimmune disease because they are known to be upregulated on peripheral and central T lymphocytes of multiple sclerosis (MS) patients. Moreover, overexpression of K2P 5.1 channels in vitro provokes enhanced T-cell effector functions. However, the molecular mechanisms regulating intracellular K2P 5.1 channel trafficking are unknown so far. Thus, the aim of the study is to elucidate the trafficking of K2P 5.1 channels on T lymphocytes. Using mass spectrometry analysis, we have identified 14-3-3 proteins as novel binding partners of K2P 5.1 channels. We show that a non-classical 14-3-3 consensus motif (R-X-X-pT/S-x) at the channel's C-terminus allows the binding between K2P 5.1 and 14-3-3. The mutant K2P 5.1/S266A diminishes the protein-protein interaction and reduces the amplitude of membrane currents. Application of a non-peptidic 14-3-3 inhibitor (BV02) significantly reduces the number of wild-type channels in the plasma membrane, whereas the drug has no effect on the trafficking of the mutated channel. Furthermore, blocker application reduces T-cell effector functions. Taken together, we demonstrate that 14-3-3 interacts with K2P 5.1 and plays an important role in channel trafficking.


Assuntos
Proteínas 14-3-3/metabolismo , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Linfócitos T/metabolismo , Animais , Linhagem Celular , Membrana Celular/metabolismo , Feminino , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Transporte Proteico/fisiologia , Regulação para Cima/fisiologia
10.
Cell Tissue Res ; 375(2): 507-529, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30259139

RESUMO

The subcommissural organ (SCO) is an ancient and conserved brain gland secreting into cerebrospinal fluid (CSF) glycoproteins that form the Reissner fiber (RF). The present investigation was designed to further investigate the dynamic of the biosynthetic process of RF glycoproteins prior and after their release into the CSF, to identify the RF proteome and N-glycome and to clarify the mechanism of assembly of RF glycoproteins. Various methodological approaches were used: biosynthetic labelling injecting 35S-cysteine and 3H-galactose into the CSF, injection of antibodies against galectin-1 into the cerebrospinal fluid, light and electron microscopical methods; isolated bovine RF was used for proteome analyses by mass spectrometry and glycome analysis by xCGE-LIF. The biosynthetic labelling study further supported that a small pool of SCO-spondin molecules rapidly enter the secretory pathways after its synthesis, while most of the SCO-spondin molecules are stored in the rough endoplasmic reticulum for hours or days before entering the secretory pathway and being released to assemble into RF. The proteomic analysis of RF revealed clusterin and galectin-1 as partners of SCO-spondin; the in vivo use of anti-galectin-1 showed that this lectin is essential for the assembly of RF. Galectin-1 is not secreted by the SCO but evidence was obtained that it would be secreted by multiciliated ependymal cells lying close to the SCO. Further, a surprising variety and complexity of glycan structures were identified in the RF N-glycome that further expands the potential functions of RF to a level not previously envisaged. A model of the macromolecular organization of Reissner fiber is proposed.


Assuntos
Glicoproteínas/metabolismo , Órgão Subcomissural/fisiologia , Animais , Bovinos , Cisteína/metabolismo , Citoplasma/metabolismo , Epêndima/citologia , Epêndima/metabolismo , Galactose/metabolismo , Galectina 1/metabolismo , Glicoproteínas/ultraestrutura , Glicosilação , Masculino , Polissacarídeos/química , Polissacarídeos/metabolismo , Ratos Sprague-Dawley , Via Secretória , Coloração e Rotulagem , Órgão Subcomissural/ultraestrutura , Radioisótopos de Enxofre/metabolismo , Trítio/metabolismo
11.
J Exp Biol ; 222(Pt 19)2019 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-31488622

RESUMO

The Sap47 gene of Drosophila melanogaster encodes a highly abundant 47 kDa synaptic vesicle-associated protein. Sap47 null mutants show defects in synaptic plasticity and larval olfactory associative learning but the molecular function of Sap47 at the synapse is unknown. We demonstrate that Sap47 modulates the phosphorylation of another highly abundant conserved presynaptic protein, synapsin. Site-specific phosphorylation of Drosophila synapsin has repeatedly been shown to be important for behavioural plasticity but it was not known where these phospho-synapsin isoforms are localized in the brain. Here, we report the distribution of serine-6-phosphorylated synapsin in the adult brain and show that it is highly enriched in rings of synapses in the ellipsoid body and in large synapses near the lateral triangle. The effects of knockout of Sap47 or synapsin on olfactory associative learning/memory support the hypothesis that both proteins operate in the same molecular pathway. We therefore asked if this might also be true for other aspects of their function. We show that knockout of Sap47 but not synapsin reduces lifespan, whereas knockout of Sap47 and synapsin, either individually or together, affects climbing proficiency, as well as plasticity in circadian rhythms and sleep. Furthermore, electrophysiological assessment of synaptic properties at the larval neuromuscular junction (NMJ) reveals increased spontaneous synaptic vesicle fusion and reduced paired pulse facilitation in Sap47 and synapsin single and double mutants. Our results imply that Sap47 and synapsin cooperate non-uniformly in the control of synaptic properties in different behaviourally relevant neuronal networks of the fruitfly.


Assuntos
Comportamento Animal/fisiologia , Proteínas de Drosophila/genética , Drosophila melanogaster/fisiologia , Locomoção/genética , Longevidade/genética , Mutação/genética , Proteínas do Tecido Nervoso/genética , Plasticidade Neuronal/genética , Sinapsinas/metabolismo , Animais , Encéfalo/metabolismo , Ritmo Circadiano/fisiologia , Drosophila melanogaster/genética , Larva/metabolismo , Junção Neuromuscular/metabolismo , Fosforilação , Fosfosserina/metabolismo , Isoformas de Proteínas/metabolismo , Sinapsinas/genética
12.
Biochim Biophys Acta Mol Cell Res ; 1864(8): 1405-1412, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28499918

RESUMO

Cullin 3 (Cul3) belongs to the family of cullins (Cul1-7) providing the scaffold for cullin-RING ubiquitin (Ub) ligases (CRLs), which are activated by neddylation and represent essential E3 ligases of the Ub proteasome system. During adipogenic differentiation neddylated Cul3 accumulates in LiSa-2 preadipocytes. Downregulation of Cul3 and inhibition of neddylation by MLN4924 blocks the formation of lipid droplets (LDs), the lipid storage organelles and markers of adipogenesis. Neddylation of Cul3 coincides with an increase of Rab18, a GTPase associated with LDs. Immunoprecipitation and confocal fluorescence microscopy revealed physical association of Cul3 and Rab18 at the membrane of LDs. RhoA, a suppressor of adipogenesis decreased during differentiation. Our results in LiSa-2 cells, but also mouse embryonic fibroblasts revealed a connection between Cul3, Rab18 and RhoA. Downregulation of Cul3 led to a marked increase in RhoA protein expression after 6days of LiSa-2 cell differentiation, suggesting that Cul3 is involved in the regulation of RhoA stability.


Assuntos
Adipócitos/metabolismo , Adipogenia/genética , Proteínas Culina/genética , Processamento de Proteína Pós-Traducional , Proteínas rab de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/genética , Adipócitos/citologia , Animais , Diferenciação Celular , Linhagem Celular Tumoral , Proteínas Culina/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Gotículas Lipídicas , Camundongos , Proteína NEDD8 , Cultura Primária de Células , Complexo de Endopeptidases do Proteassoma , Proteólise , Transdução de Sinais , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Ubiquitinas/genética , Ubiquitinas/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo
13.
Learn Mem ; 23(1): 9-20, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26670182

RESUMO

Synapsin is an evolutionarily conserved presynaptic phosphoprotein. It is encoded by only one gene in the Drosophila genome and is expressed throughout the nervous system. It regulates the balance between reserve and releasable vesicles, is required to maintain transmission upon heavy demand, and is essential for proper memory function at the behavioral level. Task-relevant sensorimotor functions, however, remain intact in the absence of Synapsin. Using an odor-sugar reward associative learning paradigm in larval Drosophila, we show that memory scores in mutants lacking Synapsin (syn(97)) are lower than in wild-type animals only when more salient, higher concentrations of odor or of the sugar reward are used. Furthermore, we show that Synapsin is selectively required for larval short-term memory. Thus, without Synapsin Drosophila larvae can learn and remember, but Synapsin is required to form memories that match in strength to event salience-in particular to a high saliency of odors, of rewards, or the salient recency of an event. We further show that the residual memory scores upon a lack of Synapsin are not further decreased by an additional lack of the Sap47 protein. In combination with mass spectrometry data showing an up-regulated phosphorylation of Synapsin in the larval nervous system upon a lack of Sap47, this is suggestive of a functional interdependence of Synapsin and Sap47.


Assuntos
Transtornos da Memória/metabolismo , Memória/fisiologia , Mutação/genética , Sinapsinas/metabolismo , Animais , Animais Geneticamente Modificados , Aprendizagem por Associação , Cromatografia Líquida , Modelos Animais de Doenças , Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Larva , Espectrometria de Massas , Transtornos da Memória/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Odorantes , Fosforilação/genética , RNA Mensageiro/metabolismo , Sinapsinas/genética
14.
J Neurosci ; 35(19): 7487-502, 2015 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-25972175

RESUMO

Adverse life events can induce two kinds of memory with opposite valence, dependent on timing: "negative" memories for stimuli preceding them and "positive" memories for stimuli experienced at the moment of "relief." Such punishment memory and relief memory are found in insects, rats, and man. For example, fruit flies (Drosophila melanogaster) avoid an odor after odor-shock training ("forward conditioning" of the odor), whereas after shock-odor training ("backward conditioning" of the odor) they approach it. Do these timing-dependent associative processes share molecular determinants? We focus on the role of Synapsin, a conserved presynaptic phosphoprotein regulating the balance between the reserve pool and the readily releasable pool of synaptic vesicles. We find that a lack of Synapsin leaves task-relevant sensory and motor faculties unaffected. In contrast, both punishment memory and relief memory scores are reduced. These defects reflect a true lessening of associative memory strength, as distortions in nonassociative processing (e.g., susceptibility to handling, adaptation, habituation, sensitization), discrimination ability, and changes in the time course of coincidence detection can be ruled out as alternative explanations. Reductions in punishment- and relief-memory strength are also observed upon an RNAi-mediated knock-down of Synapsin, and are rescued both by acutely restoring Synapsin and by locally restoring it in the mushroom bodies of mutant flies. Thus, both punishment memory and relief memory require the Synapsin protein and in this sense share genetic and molecular determinants. We note that corresponding molecular commonalities between punishment memory and relief memory in humans would constrain pharmacological attempts to selectively interfere with excessive associative punishment memories, e.g., after traumatic experiences.


Assuntos
Aprendizagem por Associação/fisiologia , Aprendizagem da Esquiva/fisiologia , Encéfalo/metabolismo , Memória/fisiologia , Punição , Sinapsinas/fisiologia , Fatores Etários , Animais , Animais Geneticamente Modificados , Encéfalo/citologia , Encéfalo/fisiologia , Discriminação Psicológica , Proteínas de Drosophila/genética , Drosophila melanogaster , Eletrochoque/efeitos adversos , Feminino , Masculino , Mutação/genética , Odorantes , Fosforilação , Interferência de RNA/fisiologia , Sinapsinas/genética
15.
J Neurochem ; 138(1): 124-38, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27062398

RESUMO

Learning and memory processes are accompanied by rearrangements of synaptic protein networks. While various studies have demonstrated the regulation of individual synaptic proteins during these processes, much less is known about the complex regulation of synaptic proteomes. Recently, we reported that auditory discrimination learning in mice is associated with a relative down-regulation of proteins involved in the structural organization of synapses in various brain regions. Aiming at the identification of biological processes and signaling pathways involved in auditory memory formation, here, a label-free quantification approach was utilized to identify regulated synaptic junctional proteins and phosphoproteins in the auditory cortex, frontal cortex, hippocampus, and striatum of mice 24 h after the learning experiment. Twenty proteins, including postsynaptic scaffolds, actin-remodeling proteins, and RNA-binding proteins, were regulated in at least three brain regions pointing to common, cross-regional mechanisms. Most of the detected synaptic proteome changes were, however, restricted to individual brain regions. For example, several members of the Septin family of cytoskeletal proteins were up-regulated only in the hippocampus, while Septin-9 was down-regulated in the hippocampus, the frontal cortex, and the striatum. Meta analyses utilizing several databases were employed to identify underlying cellular functions and biological pathways. Data are available via ProteomeExchange with identifier PXD003089. How does the protein composition of synapses change in different brain areas upon auditory learning? We unravel discrete proteome changes in mouse auditory cortex, frontal cortex, hippocampus, and striatum functionally implicated in the learning process. We identify not only common but also area-specific biological pathways and cellular processes modulated 24 h after training, indicating individual contributions of the regions to memory processing.


Assuntos
Estimulação Acústica , Encéfalo/metabolismo , Aprendizagem por Discriminação/fisiologia , Regulação da Expressão Gênica/fisiologia , Proteoma/metabolismo , Sinapses/metabolismo , Animais , Vias Auditivas/metabolismo , Proteínas do Citoesqueleto/metabolismo , Masculino , Redes e Vias Metabólicas , Camundongos , Camundongos Endogâmicos C57BL , Fosfoproteínas/metabolismo , Transdução de Sinais
16.
Proteome Sci ; 13: 13, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25852303

RESUMO

BACKGROUND: Using auditory discrimination learning in gerbils, we have previously shown that activation of auditory-cortical D1/D5 dopamine receptors facilitates mTOR-mediated, protein synthesis-dependent mechanisms of memory consolidation and anterograde memory formation. To understand molecular mechanisms of this facilitatory effect, we tested the impact of local pharmacological activation of different D1/D5 dopamine receptor signalling modes in the auditory cortex. To this end, protein patterns in soluble and synaptic protein-enriched fractions from cortical, hippocampal and striatal brain regions of ligand- and vehicle-treated gerbils were analysed by 2D gel electrophoresis and mass spectrometry 24 h after intervention. RESULTS: After auditory-cortical injection of SKF38393 - a D1/D5 dopamine receptor-selective agonist reported to activate the downstream effectors adenylyl cyclase and phospholipase C - prominent proteomic alterations compared to vehicle-treated controls appeared in the auditory cortex, striatum, and hippocampus, whereas only minor changes were detectable in the frontal cortex. In contrast, auditory-cortical injection of SKF83959 - a D1/D5 agonist reported to preferentially stimulate phospholipase C - induced pronounced changes in the frontal cortex. At the molecular level, we detected altered regulation of cytoskeletal and scaffolding proteins, changes in proteins with functions in energy metabolism, local protein synthesis, and synaptic signalling. Interestingly, abundance and/or subcellular localisation of the predominantly presynaptic protein α-synuclein displayed dopaminergic regulation. To assess the role of α-synuclein for dopaminergic mechanisms of memory modulation, we tested the impact of post-conditioning systemic pharmacological activation of different D1/D5 dopamine receptor signalling modes on auditory discrimination learning in α-synuclein-mutant mice. In C57BL/6JOlaHsd mice, bearing a spontaneous deletion of the α-synuclein-encoding gene, but not in the related substrains C57BL/6JCrl and C57BL/6JRccHsd, adenylyl cyclase-mediated signalling affected acquisition rates over future learning episodes, whereas phospholipase C-mediated signalling affected final memory performance. CONCLUSIONS: Dopamine signalling modes via D1/D5 receptors in the auditory cortex differentially impact protein profiles related to rearrangement of cytomatrices, energy metabolism, and synaptic neurotransmission in cortical, hippocampal, and basal brain structures. Altered dopamine neurotransmission in α-synuclein-deficient mice revealed that distinct D1/D5 receptor signalling modes may control different aspects of memory consolidation.

17.
Mediators Inflamm ; 2015: 737310, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26663990

RESUMO

An underlying endothelial dysfunction plays a fundamental role in the pathogenesis of cardiovascular events and is the central feature of atherosclerosis. The protein-based communication between leukocytes and inflamed endothelial cells leading to diapedesis has been largely investigated and several key players such as IL6, TNFα, or the damage associated molecular pattern molecule (DAMP) calprotectin are now well identified. However, regarding cytokine IL27, the controversial current knowledge about its inflammatory role and the involved regulatory elements requires clarification. Therefore, we examined the inflammatory impact of IL27 on primary endothelial cells and the potentially modulatory effect of calprotectin on both transcriptome and proteome levels. A qPCR-based screening demonstrated high IL27-mediated gene expression of IL7, IL15, CXCL10, and CXCL11. Calprotectin time-dependent downregulatory effects were observed on IL27-induced IL15 and CXCL10 gene expression. A mass spectrometry-based approach of IL27 ± calprotectin cell stimulation enlightened a calprotectin modulatory role in the expression of 28 proteins, mostly involved in the mechanism of leukocyte transmigration. Furthermore, we showed evidence for STAT1 involvement in this process. Our findings provide new evidence about the IL27-dependent proinflammatory signaling which may be under the control of calprotectin and highlight the need for further investigations on molecules which might have antiatherosclerotic functions.


Assuntos
Aterosclerose/etiologia , Células Endoteliais/efeitos dos fármacos , Interleucina-27/farmacologia , Complexo Antígeno L1 Leucocitário/farmacologia , Aterosclerose/prevenção & controle , Células Cultivadas , Quimiocina CXCL10/genética , Células Endoteliais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-15/genética , Interleucina-7/genética , Fator de Transcrição STAT1/fisiologia , Fator de Transcrição STAT3/fisiologia
18.
Nat Genet ; 38(6): 668-73, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16699518

RESUMO

Chronic pancreatitis is a common inflammatory disease of the pancreas. Mutations in the genes encoding cationic trypsinogen (PRSS1) and the pancreatic secretory trypsin inhibitor (SPINK1) are associated with chronic pancreatitis. Because increased proteolytic activity owing to mutated PRSS1 enhances the risk for chronic pancreatitis, mutations in the gene encoding anionic trypsinogen (PRSS2) may also predispose to disease. Here we analyzed PRSS2 in individuals with chronic pancreatitis and controls and found, to our surprise, that a variant of codon 191 (G191R) is overrepresented in control subjects: G191R was present in 220/6,459 (3.4%) controls but in only 32/2,466 (1.3%) affected individuals (odds ratio 0.37; P = 1.1 x 10(-8)). Upon activation by enterokinase or trypsin, purified recombinant G191R protein showed a complete loss of trypsin activity owing to the introduction of a new tryptic cleavage site that renders the enzyme hypersensitive to autocatalytic proteolysis. In conclusion, the G191R variant of PRSS2 mitigates intrapancreatic trypsin activity and thereby protects against chronic pancreatitis.


Assuntos
Tripsina/genética , Tripsinogênio/genética , Sequência de Bases , Doença Crônica , Primers do DNA , Haplótipos , Humanos , Hidrólise , Modelos Moleculares , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Tripsina/química , Tripsina/metabolismo , Tripsinogênio/química , Tripsinogênio/metabolismo
19.
Oncogene ; 43(25): 1955-1971, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38730267

RESUMO

Procaspase-8 is a key mediator of death receptor (DR)-mediated pathways. Recently, the role of post-translational modifications (PTMs) of procaspase-8 in controlling cell death has received increasing attention. Here, using mass spectrometry screening, pharmacological inhibition and biochemical assays, we show that procaspase-8 can be targeted by the PRMT5/RIOK1/WD45 methylosome complex. Furthermore, two potential methylation sites of PRMT5 on procaspase-8, R233 and R435, were identified in silico. R233 and R435 are highly conserved in mammals and their point mutations are among the most common mutations of caspase-8 in cancer. The introduction of mutations at these positions resulted in inhibitory effects on CD95L-induced caspase-8 activity, effector caspase activation and apoptosis. In addition, we show that procaspase-8 can undergo symmetric di-methylation. Finally, the pharmacological inhibition of PRMT5 resulted in the inhibitory effects on caspase activity and apoptotic cell death. Taken together, we have unraveled the additional control checkpoint in procaspase-8 activation and the arginine methylation network in the extrinsic apoptosis pathway.


Assuntos
Apoptose , Arginina , Caspase 8 , Proteína-Arginina N-Metiltransferases , Caspase 8/metabolismo , Caspase 8/genética , Arginina/metabolismo , Humanos , Metilação , Proteína-Arginina N-Metiltransferases/metabolismo , Proteína-Arginina N-Metiltransferases/genética , Processamento de Proteína Pós-Traducional
20.
Brain Res ; 1823: 148679, 2024 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-37972846

RESUMO

Emerging evidence highlights the relevance of the protein post-translational modification by SUMO (Small Ubiquitin-like Modifier) in the central nervous system for modulating cognition and plasticity in health and disease. In these processes, astrocyte-to-neuron crosstalk mediated by extracellular vesicles (EVs) plays a yet poorly understood role. Small EVs (sEVs), including microvesicles and exosomes, contain a molecular cargo of lipids, proteins, and nucleic acids that define their biological effect on target cells. Here, we investigated whether SUMOylation globally impacts the sEV protein cargo. For this, sEVs were isolated from primary cultures of astrocytes by ultracentrifugation or using a commercial sEV isolation kit. SUMO levels were regulated: 1) via plasmids that over-express SUMO, or 2) via experimental conditions that increase SUMOylation, i.e., by using the stress hormone corticosterone, or 3) via the SUMOylation inhibitor 2-D08 (2',3',4'-trihydroxy-flavone, 2-(2,3,4-Trihydroxyphenyl)-4H-1-Benzopyran-4-one). Corticosterone and 2-D08 had opposing effects on the number of sEVs and on their protein cargo. Proteomic analysis showed that increased SUMOylation in corticosterone-treated or plasmid-transfected astrocytes increased the presence of proteins related to cell division, transcription, and protein translation in the derived sEVs. When sEVs derived from corticosterone-treated astrocytes were transferred to neurons to assess their impact on protein synthesis using the fluorescence non-canonical amino acid tagging assay (FUNCAT), we detected an increase in protein synthesis, while sEVs from 2-D08-treated astrocytes had no effect. Our results show that SUMO conjugation plays an important role in the modulation of the proteome of astrocyte-derived sEVs with a potential functional impact on neurons.


Assuntos
Vesículas Extracelulares , Proteoma , Proteoma/metabolismo , Astrócitos/metabolismo , Sumoilação , Proteômica , Corticosterona/farmacologia , Vesículas Extracelulares/metabolismo , Neurônios/metabolismo , Dendritos/metabolismo
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