RESUMO
A disintegrin and metalloproteinase 10 (ADAM10) is the major α-secretase that catalyzes the amyloid precursor protein (APP) ectodomain shedding in the brain and prevents amyloid formation. Its activity depends on correct intracellular trafficking and on synaptic membrane insertion. Here, we describe that in hippocampal neurons the synapse-associated protein-97 (SAP97), an excitatory synapse scaffolding element, governs ADAM10 trafficking from dendritic Golgi outposts to synaptic membranes. This process is mediated by a previously uncharacterized protein kinase C phosphosite in SAP97 SRC homology 3 domain that modulates SAP97 association with ADAM10. Such mechanism is essential for ADAM10 trafficking from the Golgi outposts to the synapse, but does not affect ADAM10 transport from the endoplasmic reticulum. Notably, this process is altered in Alzheimer's disease brains. These results help in understanding the mechanism responsible for the modulation of ADAM10 intracellular path, and can constitute an innovative therapeutic strategy to finely tune ADAM10 shedding activity towards APP.
Assuntos
Proteínas ADAM/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Membrana/metabolismo , Proteína Quinase C/metabolismo , Proteínas ADAM/química , Proteína ADAM10 , Proteínas Adaptadoras de Transdução de Sinal/química , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Sequência de Aminoácidos , Secretases da Proteína Precursora do Amiloide/química , Animais , Células COS , Chlorocebus aethiops , Proteína 1 Homóloga a Discs-Large , Ativação Enzimática , Células HEK293 , Humanos , Proteínas de Membrana/química , Modelos Moleculares , Dados de Sequência Molecular , Fosforilação , Fosfotreonina/metabolismo , Densidade Pós-Sináptica/metabolismo , Ligação Proteica , Ratos , Sinapses/metabolismoRESUMO
This study was designed to monitor the biochemical profiles of serum and follicular fluid (FF) of postpartum dairy cows during the summer (n=30) and winter (n=30). Blood and FF (follicles ≥ 9 mm) were obtained from Girolando cows at 30, 45, 60, 75 and 90 days postpartum. The samples were collected and analysed to determine glucose, total cholesterol (TC), triglyceride (TG), urea, sodium (Na), potassium (K) and calcium (Ca) levels. Throughout the study, the following clinical variables were measured: rectal temperature (RT), respiratory rate (RR) and body condition score (BCS). In addition, the temperature humidity index (THI) was calculated for each season. During the summer season, THI was higher, BCS decreased, there was an increase in RT, and glucose, urea, Na and K serum levels were decreased (P<0.05). The levels of TC, TG, urea, K and Ca in follicular fluid increased (P<0.05). Positive correlations (P<0.05) were observed between the serum and FF levels for glucose (r=0.29), TC (r=0.24) and Ca (r=0.30). Therefore, the biochemical profile of serum and FF of dairy cows under summer heat-stress conditions demonstrates marked changes that may impair fertility during lactation.
Assuntos
Bovinos , Líquido Folicular/metabolismo , Metaboloma , Período Pós-Parto/metabolismo , Estações do Ano , Soro/metabolismo , Animais , Glicemia/metabolismo , Bovinos/metabolismo , Indústria de Laticínios , Feminino , Líquido Folicular/química , Resposta ao Choque Térmico , Lipídeos/sangue , Período Pós-Parto/sangue , Soro/químicaRESUMO
Membrane associated guanylate kinase proteins (MAGUKs) play a key role in the regulation of the intracellular trafficking and synaptic localization of ionotropic glutamate receptors. In particular, the postsynaptic density-95-like subfamily of MAGUKs (PSD-MAGUKs) organizes ionotropic glutamate receptors and their associated signaling proteins in the postsynaptic density of the excitatory synapse regulating the strength of synaptic activity. Several recent observations clearly put forward the idea that alterations of PSD-MAGUK protein function such as alterations of PSD-MAGUK protein interaction with N-methyl-D-aspartate (NMDA) receptors regulatory subunits are common events in several CNS disorders. With this view, a better knowledge and understanding of PSD-MAGUK function as well as of the molecular events regulating PSD-MAGUK-mediated interactions in the glutamatergic synapse could lead to the identification of new pharmaceutical targets for the therapy of CNS disorders.
Assuntos
Encéfalo/metabolismo , Proteínas de Membrana/metabolismo , Sinapses/metabolismo , Membranas Sinápticas/metabolismo , Animais , Encéfalo/fisiopatologia , Proteína 4 Homóloga a Disks-Large , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/fisiopatologia , Receptores de Glutamato/metabolismo , Sinapses/ultraestrutura , Membranas Sinápticas/ultraestrutura , Transmissão Sináptica/fisiologiaRESUMO
The integral membrane protein Thlaspi goesingense metal tolerance protein 1 (TgMTP1) has been suggested to play an important role in Zn hyperaccumulation in T. goesingense. Here, we show that the TgMTP1 protein is accumulated to high levels at the vacuolar membrane in shoot tissue of T. goesingense. TgMTP1 is likely to act in the transport of Zn into the vacuole, enhancing both Zn accumulation and tolerance. By specifically expressing TgMTP1 in Arabidopsis thaliana shoots, we show that TgMTP1, localized at the vacuolar membrane, can drive the enhanced shoot accumulation of Zn by initiating a systemic Zn deficiency response. The systematic response includes increased expression of Zn transporters (ZIP3, ZIP4, ZIP5 and ZIP9) in both shoot and root tissue. Furthermore, shoot-specific accumulation of TgMTP1 at the vacuolar membrane also leads to increased resistance to Zn in A. thaliana, probably through enhanced Zn compartmentalization in the vacuole. Such evidence leads to the conclusion that the high levels of TgMTP1 at the vacuolar membrane in shoot tissue of the Zn hyperaccumulator T. goesingense play a role in both Zn tolerance and enhanced Zn uptake and accumulation, via the activation of a systemic Zn deficiency response.
Assuntos
Arabidopsis/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Proteínas de Plantas/metabolismo , Thlaspi/genética , Vacúolos/metabolismo , Zinco/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Proteínas de Transporte de Cátions/genética , Regulação da Expressão Gênica de Plantas , Dados de Sequência Molecular , Proteínas de Plantas/genética , Brotos de Planta/genética , Brotos de Planta/metabolismo , Alinhamento de Sequência , Thlaspi/metabolismo , Vacúolos/genéticaRESUMO
Synapse Associated Protein 97 (SAP97), a member of membrane-associated guanylate kinase (MAGUK) protein family, has been involved in the correct targeting and clustering of ionotropic glutamate receptors (iGluRs) at postsynaptic sites. Calcium/calmodulin kinase II (CaMKII) phosphorylates SAP97 on two major sites in vivo; one located in the N-terminal domain (Ser39) and the other in the first postsynaptic density disc large ZO1 (PDZ) domain (Ser232). CaMKII-mediated phosphorylation of SAP97-Ser39 is necessary and sufficient to drive SAP97 to the postsynaptic compartment in cultured hippocampal neurons. CaMKII-dependent phosphorylation of Ser232 disrupts SAP97 interaction with NR2A subunit, thereby regulating synaptic targeting of this NMDA receptor subunit. Here we show by means of phospho-specific antibodies that SAP97-Ser39 phosphorylation represents the driving force to release SAP97/NR2A complex from the endoplasmic reticulum. Ser39 phosphorylation does not interfere with SAP97 capability to bind NR2A. On the contrary, SAP97-Ser232 phosphorylation occurs within the postsynaptic compartment and is responsible for both the disruption of NR2A/SAP97 complex and, consequently, for NR2A insertion in the postsynaptic membrane. Thus, CaMKII-dependent phosphorylation of SAP97 in different time frames and locations within the neurons controls both NR2A trafficking and insertion.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/farmacologia , Proteínas de Membrana/fisiologia , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Cafeína/farmacologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Células Cultivadas , Cricetinae , Cricetulus , Interações Medicamentosas , Embrião de Mamíferos , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Agonistas de Aminoácidos Excitatórios/farmacologia , Hipocampo/citologia , Imunoprecipitação/métodos , Técnicas In Vitro , N-Metilaspartato/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Neurônios/ultraestrutura , Inibidores de Fosfodiesterase/farmacologia , Fosforilação/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Ratos , Serina/metabolismo , Transfecção/métodosRESUMO
Amyloid precursor protein (APP), ADAM 10, and beta-site-APP cleaving enzyme (BACE) alterations were evaluated in platelets of 31 patients with Alzheimer disease (AD) and 15 age-matched controls. A significant modification of these proteins and enzymes involved in the amyloid cascade was detected from the earliest clinically detectable disease stage. This observation suggests that AD is associated with an early metabolic derangement toward amyloidogenic pathways and supports the potential value of APP and secretase measurements for early diagnosis of AD.