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1.
Biochem Biophys Rep ; 32: 101347, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36131851

RESUMO

The affinity for K+ of silkworm Na+/K+-ATPase, which is composed of α and ß subunits, is remarkably lower than that of mammalian Na+/K+-ATPase, with a slightly higher affinity for Na+. Because the α subunit had more than 70% identity to the mammalian α subunit in the amino acid sequence, whereas the ß subunit, a glycosylated protein, had less than 30% identity to the mammalian ß subunit, it was suggested that the ß subunit was involved in the affinities for Na+ and K+ of Na+/K+-ATPase. To confirm this hypothesis, we examined whether replacing the silkworm ß subunit with the mammalian ß subunit affected the affinities for Na+ and K+ of Na+/K+-ATPase. Cloned silkworm α and cloned rat ß1 were co-expressed in BM-N cells, a cultured silkworm ovary-derived cell lacking endogenous Na+/K+-ATPase, to construct a hybrid Na+/K+-ATPase, in which the silkworm ß subunit was replaced with the rat ß1 subunit. The hybrid Na+/K+-ATPase increased the affinity for K+ by 4.1-fold and for Na+ by 0.65-fold compared to the wild-type one. Deglycosylation of the silkworm ß subunit did not affect the K+ affinity. These results support the involvement of the ß subunit in the Na+ and K+ affinities of Na+/K+-ATPase.

2.
Mol Cell Neurosci ; 121: 103754, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35842170

RESUMO

The involvement of secretory pathways and Golgi dysfunction in neuronal cells during Alzheimer's disease progression is poorly understood. Our previous overexpression and knockdown studies revealed that the intracellular protein level of Syntaxin-5, an endoplasmic reticulum-Golgi soluble N-ethylmaleimide-sensitive factor-attachment protein receptor (SNARE), modulates beta-amyloid precursor protein processing in neuronal cells. We recently showed that changes in endogenous Syntaxin-5 protein expression occur under stress induction. Syntaxin-5 was upregulated by endoplasmic reticulum stress but was degraded by Caspase-3 during apoptosis in neuronal cells. In addition, we showed that sustained endoplasmic reticulum stress promotes Caspase-3-dependent apoptosis during the later phase of the endoplasmic reticulum stress response in NG108-15 cells. In this study, to elucidate the consequences of secretory pathway dysfunction in beta-amyloid precursor protein processing that lead to neuronal cell death, we examined the effect of various stresses on endoplasmic reticulum-Golgi SNARE expression and beta-amyloid precursor protein processing. By using compounds to disrupt Golgi function, we show that Golgi stress promotes upregulation of the endoplasmic reticulum-Golgi SNARE Syntaxin-5, and prolonged stress causes Caspase-3-dependent apoptosis. Golgi stress induced intracellular beta-amyloid precursor protein accumulation and a concomitant decrease in total amyloid-beta production. We also examined the protective effect of the chemical chaperone 4-phenylbutylate on changes in amyloid-beta production and the activation of Caspase-3 induced by endoplasmic reticulum and Golgi stress. The compound alleviated the increase in the amyloid-beta 1-42/amyloid-beta 1-40 ratio induced by endoplasmic reticulum and Golgi stress. Furthermore, 4-phenylbutylate could rescue Caspase-3-dependent apoptosis induced by prolonged organelle stress. These results suggest that organelle stress originating from the endoplasmic reticulum and Golgi has a substantial impact on the amyloidogenic processing of beta-amyloid precursor protein and Caspase-3-dependent apoptosis, leading to neuronal cell death.


Assuntos
Precursor de Proteína beta-Amiloide , Proteínas SNARE , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Apoptose , Caspase 3/metabolismo , Complexo de Golgi/metabolismo , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , Proteínas Qa-SNARE/farmacologia , Proteínas SNARE/metabolismo , Proteínas SNARE/farmacologia , Regulação para Cima
3.
Biochem Biophys Res Commun ; 614: 56-62, 2022 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-35567944

RESUMO

The human Golgi/secretory pathway Ca2+,Mn2+-ATPase 1 (hSPCA1) transports Ca2+ and Mn2+ into the Golgi lumen. Studies of the biological functions of hSPCA1 are limited by a lack of selective pharmacological tools for SPCA1 inhibition. The aim of this study was therefore to identify compounds that specifically inhibit hSPCA1 activity. We found that five 1,3-thiazole derivatives exhibited inhibitory action towards the ATP hydrolysis activity of hSPCA1a in a concentration-dependent manner. Among the derivatives tested, compound 1 was the most potent, completely inhibiting hSPCA1a activity with a half-maximal inhibition (IC50) value of 0.8 µM. Compound 1 also partially inhibited the activity of another Ca2+,Mn2+-ATPase (hSPCA2) and Ca2+-ATPase (rSERCA1a), but had no effect on Na+,K+-ATPase or H+,K+-ATPase. Treatment of HeLa cells with compound 1 led to fragmentation of the Golgi ribbon into smaller stacks. In addition, compound 1 mobilized intracellular Ca2+ in HeLa cells that had been pre-treated with thapsigargin. Therefore, based on its selectivity and potency, compound 1 may be a valuable tool with which to further explore the role of SPCA1 in cellular processes.


Assuntos
ATPases Transportadoras de Cálcio , Via Secretória , Adenosina Trifosfatases/metabolismo , Cálcio/metabolismo , Células HeLa , Humanos , Tiazóis/farmacologia
4.
EMBO Rep ; 20(4)2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30804014

RESUMO

Rab family small GTPases are master regulators of distinct steps of intracellular vesicle trafficking in eukaryotic cells. GDP-bound cytoplasmic forms of Rab proteins are prone to aggregation due to the exposure of hydrophobic groups but the machinery that determines the fate of Rab species in the cytosol has not been elucidated in detail. In this study, we find that BAG6 (BAT3/Scythe) predominantly recognizes a cryptic portion of GDP-associated Rab8a, while its major GTP-bound active form is not recognized. The hydrophobic residues of the Switch I region of Rab8a are essential for its interaction with BAG6 and the degradation of GDP-Rab8a via the ubiquitin-proteasome system. BAG6 prevents the excess accumulation of inactive Rab8a, whose accumulation impairs intracellular membrane trafficking. BAG6 binds not only Rab8a but also a functionally distinct set of Rab family proteins, and is also required for the correct distribution of Golgi and endosomal markers. From these observations, we suggest that Rab proteins represent a novel set of substrates for BAG6, and the BAG6-mediated pathway is associated with the regulation of membrane vesicle trafficking events in mammalian cells.


Assuntos
Chaperonas Moleculares/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Membrana Celular/metabolismo , Citoplasma/metabolismo , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Deleção de Genes , Complexo de Golgi/metabolismo , Humanos , Modelos Biológicos , Chaperonas Moleculares/genética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Proteólise , RNA Interferente Pequeno/genética , Ubiquitina/metabolismo , Proteínas rab de Ligação ao GTP/química , Proteínas rab de Ligação ao GTP/genética
5.
Data Brief ; 6: 20-7, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26759824

RESUMO

This paper reports the data from the long term time lapse imaging of neuronal cell line NG108-15 that were treated with apoptosis inducer or various ER stress inducers. Use of the fluorescent reporter for activated caspase3/7 in combination with the conventional light microscope allowed us to investigate the time course of apoptosis induction at the single cell level. Quantitative as well as qualitative data are presented here to show the effect of two different ER stress modulating chemical compounds on caspase3/7-dependent apoptosis in neuronal cell line NG108-15 cells. Additional results and interpretation of our data concerning ER stress and apoptosis in NG108-15 cells can be found in Suga et al. (2015) [1] and in Suga et al. (2015) [2].

6.
Data Brief ; 5: 782-8, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26693512

RESUMO

This data contains insights into the upregulation of the ER-Golgi-soluble N-ethylmaleimide-sensitive factor-attachment protein receptors (ER-Golgi SNAREs) syntaxin 5 (Syx5) by ER stress and the downregulation of Syx5 by apoptosis induction. Use of the protein synthesis inhibitor verified the de novo synthesis of Syx5 under ER stress in NG108-15 cells. We also provide validation data for the increase of Syx5 expression caused by ER stress using different chemical compound and overexpression analysis. Interpretation of our data and further extensive insights into the role of Syx5 in ßAPP processing under ER stress can be found in Suga et al. (2015)[1].

7.
Data Brief ; 5: 114-23, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26504892

RESUMO

This paper reports the data for the effects of organelle stresses on the ER-Golgi-soluble N-ethylmaleimide-sensitive factor-attachment protein receptors (ER-Golgi SNAREs) syntaxin 5 (Syx5) in neuronal cells. Quantitative as well as qualitative data are presented here to verify the upregulation of Syntaxin 5 (Syx5) under ER and Golgi stresses in hippocampal neurons. Changes in the processing of ß-amyloid precursor protein (ßAPP) under ER stress were analyzed by immunological assays. In addition, our data shows the specific increase of Syx5 expression under ER and Golgi stresses. Interpretation of our data and further extensive insights into the role of Syx5 in ßAPP processing under organelle stress can be found in "ER and Golgi stresses increase ER-Golgi SNARE Syntaxin5: Implications for organelle stress and ßAPP processing" [1].

8.
Neurosci Lett ; 604: 30-5, 2015 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-26219982

RESUMO

Unresolved endoplasmic reticulum (ER) stress causes neuronal death and has been implicated in neurodegenerative conditions such as Alzheimer's disease (AD). However, the mechanisms by which stress signals propagate from the ER through the Golgi apparatus and their effects on the transport and processing of AD-related proteins, such as ß-amyloid precursor protein (ßAPP), are unknown. We recently found that in the NG108-15 cell line, ER stress upregulates ER-Golgi-soluble N-ethylmaleimide-sensitive factor-attachment protein receptors (ER-Golgi SNAREs) Syx5 and Bet1. In the present study, we examined the effects of apoptosis and ER stress inducers on the expression of ER-Golgi SNARE proteins and cell viability in a primary culture of rat hippocampal neurons. An apoptosis inducer significantly downregulated the expression of ER-Golgi SNARE Syx5. ER-stress inducers upregulated the expression of Syx5 isoforms and Bet1 proteins via de novo synthesis of their mRNA transcripts. Knockdown of Syx5 during apoptosis or ER stress induction enhanced vulnerability of neurons. Additionally, we examined the effects of Golgi stress on Syx5 expression and ßAPP processing. Golgi stress also induced upregulation of ER-Golgi SNARE Syx5, and concomitantly, suppressed amyloid-ß peptide secretion. These findings suggest that Syx5 is a potential stress responsive factor that participates in ßAPP processing and the survival pathways of neuronal cells.


Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Estresse do Retículo Endoplasmático , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Neurônios/metabolismo , Organelas/metabolismo , Proteínas Qa-SNARE/metabolismo , Animais , Apoptose , Sobrevivência Celular , Hipocampo/citologia , Neurônios/citologia , Cultura Primária de Células , Isoformas de Proteínas/metabolismo , Proteínas Qa-SNARE/genética , Proteínas Qc-SNARE/metabolismo , Ratos Endogâmicos WKY
9.
Exp Cell Res ; 332(1): 11-23, 2015 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-25596448

RESUMO

Endoplasmic reticulum (ER) stress plays a role in the pathogenesis of neurodegenerative diseases such as Alzheimer׳s disease (AD). We previously showed that manipulation of the ER-Golgi-soluble N-ethylmaleimide-sensitive factor-attachment protein receptors (ER-Golgi SNARE) syntaxin 5 (Syx5) causes changes in Golgi morphology and the processing of AD-related proteins. To understand the pathophysiologic significance of these phenomena, we examined whether the expression of Syx5 is altered by ER stress. De novo synthesis of ER-Golgi SNARE Syx5 and Bet1 was induced by various ER stressors. Elevated expression of Syx5 and Bet1 was associated with increased levels of these proteins in vesicular components, including ER-Golgi-intermediate-compartment/vesicular tubular clusters. In addition, ER stress diminished amyloid ß (Aß) peptide secretion. Knockdown of Syx5 expression enhanced the secretion of Aß peptides under condition without ER stress. Moreover, diminished Aß peptide secretion resulting from ER stress was significantly reversed by Syx5 knockdown. These findings suggest that Syx5 plays important roles in ß-amyloid precursor protein processing and in the ER stress response that precedes apoptotic cell death and may be involved in the crosstalk between these two pathways.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Estresse do Retículo Endoplasmático , Fragmentos de Peptídeos/metabolismo , Proteínas Qa-SNARE/metabolismo , Animais , Apoptose , Caspase 3/metabolismo , Linhagem Celular Tumoral , Expressão Gênica , Camundongos , Proteólise , Proteínas Qa-SNARE/genética , Ratos , Regulação para Cima
10.
Neurosci Lett ; 536: 24-8, 2013 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-23262091

RESUMO

Apoptosis is a cell death modality that is initiated by the activation of caspases. Theoretically, fluorescence resonance energy transfer (FRET) analysis should be a convenient tool for visualizing the activation of caspase. Since the FRET probe cannot be transfected in primary neuronal cultures effectively, the FRET signal is not sufficiently strong for evaluations. We developed a method of extracting the significant signals from the fluorescent FRET images that enables the initiation of apoptosis to be analyzed. We used primary hippocampal cultures transfected with a vector encoding Bid fused with YFP and CFP. Apoptosis was induced using staurosporine (STS; 1µM). The CFP and YFP signals were observed using an inverted fluorescence microscope and were processed using imaging software for analysis. After the background signal was subtracted, the area of caspase activation and the significant signals were extracted from the localized intense signals originating from mitochondria. The CFP and YFP intensities of a selected area in a single neuron were integrated, and the CFP/YFP ratio was obtained. To confirm caspase activation in a similar experimental setting, a luminescence analysis was also performed. The FRET signals from the cultured neuron were confined to foci, since the Bid linker was specifically localized in the mitochondria. The extracted CFP and YFP signals from the foci were strong enough to be evaluated. The average CFP/YFP ratio in the neuron increased significantly after an STS challenge, from 0.673±0.024 (control) to 1.008±0.134 (STS) (mean±SD) (P<0.05). Our study demonstrated, for the first time, the quantification of Bid cleavage as expressed by FRET in a primary neuron. Since Bid is localized in the mitochondria, the region of interest was restricted to a specific area, enabling the signal to be analyzed. This methodology may be useful for the application of FRET analyses in primary cultured cells.


Assuntos
Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Neurônios/metabolismo , Animais , Apoptose , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/genética , Proteínas de Bactérias/genética , Caspase 8/metabolismo , Células Cultivadas , Ativação Enzimática , Fluorescência , Transferência Ressonante de Energia de Fluorescência , Proteínas de Fluorescência Verde/genética , Hipocampo/citologia , Proteínas Luminescentes/genética , Neurônios/citologia , Ratos , Ratos Wistar , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Análise de Célula Única
11.
J Biochem ; 146(6): 905-15, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19720618

RESUMO

In this study, we examined the interaction of Syntaxin 5L (Syx5L), a Syx5 isoform that has an N-terminal extension containing a di-arginine ER-retrieval motif, with presenilin (PS) and its effects on the processing of beta-amyloid precursor protein (betaAPP). Similar to Syx5, Syx5L bound to PS1 holoprotein but not to its N- or C-terminal fragments. Unlike Syx5, Syx5L overexpression did not cause marked accumulation of intracellular betaAPP holoprotein, and did not inhibit amyloid beta peptide (Abeta) secretion. Analyses using deletion mutants of Syx5L revealed that, in addition to the difference in the intracellular localization between the isoforms, the presence of the N-terminal extension in Syx5L was critical for suppressing its inhibition of betaAPP processing. Treatment of cells that overexpressed Syx5L with brefeldin A, an inhibitor of transport from the ER to the Golgi compartments, resulted in substantial accumulation of intracellular betaAPP holoprotein and reduction in the secretion of Abeta. Although Syx5 and Syx5L share lengthy regions of amino acid identity, they appear to play distinct roles in modulating the metabolism and trafficking of betaAPP in the early secretory compartment.


Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Presenilinas/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Qa-SNARE/metabolismo , Animais , Células Cultivadas , Humanos , Imuno-Histoquímica , Camundongos , Isoformas de Proteínas , Proteínas Qa-SNARE/química , Proteínas Qa-SNARE/genética , Ratos
12.
Neurochem Res ; 34(2): 221-6, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18528755

RESUMO

In an earlier study, we showed that mitochondria hyperpolarized after short periods of oxygen-glucose deprivation (OGD), and this response appeared to be associated with subsequent apoptosis or survival. Here, we demonstrated that hyperpolarization following short periods of OGD (30 min; 30OGD group) increased the cytosolic Ca(2+) ([Ca(2+)](c)) buffering capacity in mitochondria. After graded OGD (0 min (control), 30 min, 120 min), rat cultured hippocampal neurons were exposed to glutamate, evoking Ca(2+)influx. The [Ca(2+)](c) level increased sharply, followed by a rapid increase in mitochondrial Ca(2+) [Ca(2+)](m). The increase in the [Ca(2+)](m) level accompanied a reduction in the [Ca(2+)](c) level. After reaching a peak, the [Ca(2+)](c) level decreased more rapidly in the 30OGD group than in the control group. This buffering reaction was pronounced in the 30OGD group, but not in the 120OGD group. The enhanced buffering capacity of the mitochondria may be linked to preconditioning after short-term ischemic episodes.


Assuntos
Cálcio/metabolismo , Glucose/metabolismo , Hipocampo/metabolismo , Mitocôndrias/metabolismo , Neurônios/metabolismo , Oxigênio/metabolismo , Animais , Hipocampo/citologia , Potenciais da Membrana , Ratos , Ratos Wistar
13.
Neurosci Lett ; 448(3): 268-72, 2008 Dec 31.
Artigo em Inglês | MEDLINE | ID: mdl-18955111

RESUMO

The concept of the mitochondrial permeability transition (mPT) has been used to explain cell death induced by calcium deregulation, which is in turn induced by a disruption in the mitochondrial loading capacity of cytosolic calcium (CLC). Whether mitochondria have specific morphologies representing the CLC and the mPT remains controversial. We examined ultrastructural changes in the mitochondria of cultured hippocampal neurons preconditioned with oxygen-glucose deprivation (OGD) for 30 min (30OGD) or 120 min (120OGD). The CLC was then evaluated using simultaneous imaging of the mitochondrial and plasma Ca++ concentrations after the induction of Ca++ influx by the application of glutamate. In the 30OGD group, the CLC increased as the mitochondria rapidly reacted to the increase in plasma Ca++, which was soon cleared. In the 120OGD group, however, the CLC was disturbed because the mitochondrial uptake of Ca was blunted, and the plasma Ca++ was not cleared after glutamate application. We classified the specific morphological changes in the mitochondria according to a previously reported classification. Rounded mitochondria with scarce interior content were observed in the 120OGD group, a model of prolonged lethal OGD, and disruptions in the mitochondrial outer membrane were frequently confirmed, suggesting mPT. The 30OGD group, a model of enhanced CLC in preconditioned neurons, was characterized by round mitochondria with condensed matrices. After glutamate application, the mitochondria became even more rounded with expanded matrices, and outer membrane disruptions were occasionally seen. Our observations suggest that subpopulations of mitochondria with specific morphologies are linked to the CLC and mPT.


Assuntos
Cálcio/farmacologia , Precondicionamento Isquêmico , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Animais , Células Cultivadas , Citosol/efeitos dos fármacos , Citosol/metabolismo , Corantes Fluorescentes , Glutamatos/farmacologia , Hipóxia Encefálica/patologia , Hipóxia Encefálica/psicologia , Mitocôndrias/patologia , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , Membranas Mitocondriais/ultraestrutura , Modelos Neurológicos , Permeabilidade , Ratos , Ratos Wistar
14.
Cell Mol Biol Lett ; 13(1): 144-54, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-17965969

RESUMO

Syntaxin 8 has been shown to form the SNARE complex with syntaxin 7, vti1b and endobrevin. These have been shown to function as the machinery for the homotypic fusion of late endosomes. Recently, we showed that syntaxins 7 and 8 cycle through the plasma membrane, and that the di-leucine-based motifs in the cytoplasmic domain of syntaxins 7 and 8 respectively function in their endocytic and exocytic processes. However, we could not elucidate the mechanism by which syntaxin 8 cycles through the plasma membrane. In this study, we constructed several different syntaxin 8 molecules by mutating putative di-leucine-based motifs, and analyzed their intracellular localization and trafficking. We found a di-leucine-based motif in the cytoplasmic domain of syntaxin 8. It is similar to that of syntaxin 7, and functions in its endocytosis. These results suggest that in the cytoplasmic domain, syntaxin 8 has two functionally distinct di-leucine-based motifs that act independently in its endocytic and exocytic processes. This is the first report on two di-leucine-based motifs in the same molecule acting independently in distinct transport pathways.


Assuntos
Leucina/química , Leucina/fisiologia , Proteínas Qa-SNARE/química , Proteínas Qa-SNARE/fisiologia , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Animais , Linhagem Celular , Membrana Celular/genética , Membrana Celular/metabolismo , Endocitose/genética , Exocitose/genética , Humanos , Leucina/genética , Leucina/metabolismo , Dados de Sequência Molecular , Estrutura Terciária de Proteína/genética , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , Ratos
15.
FEBS Lett ; 579(20): 4226-34, 2005 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-16081076

RESUMO

It has been suggested that syntaxin 5 (Syx5) participates in vesicular transport. We examined the effects of Syx5 down-regulation on the morphology of the Golgi apparatus and the transport of vesicles in mammalian cells. Knockdown of the Syx5 gene resulted in Golgi fragmentation without changing the level of endoplasmic reticulum (ER)-resident proteins, other Golgi-SNAREs (soluble N-ethylmaleimide-sensitive factor-attachment protein receptors), and coatmer proteins. Strikingly, a major decrease in Syx5 expression barely affected the anterograde transport of vesicular stomatitis virus G (VSVG) protein to the plasma membrane. These results suggest that Syx5 is required for the maintenance of the Golgi structures but may not play a major role in the transport of vesicles carrying VSVG between the ER and the Golgi compartment.


Assuntos
Complexo de Golgi/metabolismo , Proteínas de Membrana/fisiologia , Vesículas Transportadoras/metabolismo , Sequência de Aminoácidos , Retículo Endoplasmático/metabolismo , Complexo de Golgi/ultraestrutura , Humanos , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Dados de Sequência Molecular , Transporte Proteico , Proteínas Qa-SNARE , Interferência de RNA , Vírus da Estomatite Vesicular Indiana/metabolismo , Proteínas Virais/metabolismo
16.
J Neurochem ; 94(2): 425-39, 2005 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15998293

RESUMO

The specific roles of syntaxin 5 (Syx 5) in the interaction with presenilin (PS) and the accumulation of beta-amyloid precursor protein (betaAPP), as well as the secretion of beta-amyloid peptide (Abeta peptide) were examined in NG108-15 cells. Syx 5, which localizes from the endoplasmic reticulum (ER) to the Golgi, bound to PS holoproteins, while the other Syxs studied did not. Among familial Alzheimer's disease (FAD)-linked PS mutants, PS1deltaE9, which lacks the endoproteolytic cleavage site, showed markedly decreased binding to Syx 5. The interaction domains in Syx 5 were mapped to the transmembrane region and to the cytoplasmic region containing the alpha-helical domains, which are distinct from the H3 (SNARE motif). Among all of the Syxs examined, only overexpression of Syx 5 resulted in the accumulation of betaAPP in the ER to cis-Golgi compartment, an attenuation of the amount of the C-terminal fragment (APP-CTF) of betaAPP, and a reduction in the secretion of Abeta peptides. Furthermore, co-expression of Syx 5 with C99 resulted in an increase in APP-CTF and suppressed Abeta secretion. Taken together, these results indicate that Syx 5 may play a specific role in the modulation of processing and/or trafficking of FAD-related proteins in neuronal cells by interaction with PS holoproteins in the early secretory compartment of neuronal cells.


Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Proteínas de Membrana/metabolismo , Neurônios/metabolismo , Secretases da Proteína Precursora do Amiloide , Animais , Ácido Aspártico Endopeptidases/farmacologia , Western Blotting/métodos , Células COS , Compartimento Celular , Chlorocebus aethiops , Cricetinae , Interações Medicamentosas , Endopeptidases , Ensaio de Imunoadsorção Enzimática/métodos , Imuno-Histoquímica/métodos , Imunoprecipitação/métodos , Proteínas de Membrana/classificação , Proteínas de Membrana/farmacologia , Camundongos , Mutagênese/fisiologia , Mutação/fisiologia , Neuroblastoma , Neurônios/efeitos dos fármacos , Biossíntese Peptídica/fisiologia , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Plasmídeos/fisiologia , Presenilina-1 , Estrutura Terciária de Proteína/fisiologia , Proteínas Qa-SNARE , Proteínas Recombinantes de Fusão/metabolismo , Transfecção/métodos , Triglicerídeos/farmacologia , Ácido gama-Aminobutírico/análogos & derivados , Ácido gama-Aminobutírico/farmacologia
17.
Biochem J ; 381(Pt 3): 619-28, 2004 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-15109302

RESUMO

Mutations in presenilins 1 and 2 (PS1 and PS2) account for the majority of cases of early-onset familial Alzheimer's disease. However, the trafficking and interaction of PSs with other proteins in the early secretory pathways are poorly understood. Using co-immunoprecipitation, we found that PS bound to Syx5 (syntaxin 5), which is a target-soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor involved in endoplasmic reticulum (ER)-Golgi vesicular transport in vivo. Syx5 interacted only with the full-length PS holoproteins and not with the naturally occurring N- or C-terminal fragments. The PS holoproteins co-immunoprecipitated with the mutant Syx5, which localized to the ER and Golgi compartments, despite the substitution of the transmembrane region with that of syntaxin 1A. In contrast, the transmembrane deletion mutant that localized to the cytosol, but not to the ER or Golgi compartments, did not co-immunoprecipitate the PS holoproteins. The PS1 variant linked to familial Alzheimer's disease (PS1DeltaE9), lacking the region that contains the endoproteolytic cleavage site in the cytoplasmic loop, showed markedly decreased binding to Syx5. Immunofluorescence and sucrose-density-gradient fractionation analyses showed that the full-length PS holoproteins co-localized with Syx5 to the ER and cis-Golgi compartments. Furthermore, Syx5 overexpression resulted in the accumulation of PS holoproteins and the beta-amyloid precursor protein, and reduced the secretion of the Abeta (amyloid beta) peptide in COS-7 cells. In summary, these results indicate that Syx5 binds to full-length PSs and affects the processing and trafficking of beta-amyloid precursor protein in the early secretory compartments.


Assuntos
Peptídeos beta-Amiloides/biossíntese , Proteínas de Membrana/metabolismo , Biossíntese Peptídica/fisiologia , Peptídeos/metabolismo , Animais , Células COS/química , Células COS/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Células HeLa/química , Células HeLa/metabolismo , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Mutação/genética , Presenilina-1 , Presenilina-2 , Mapeamento de Interação de Proteínas/métodos , Estrutura Terciária de Proteína , Proteínas Qa-SNARE , Proteína Amiloide A Sérica/biossíntese , Sintaxina 1 , Transfecção
18.
J Biochem ; 133(3): 325-34, 2003 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-12761168

RESUMO

HPC-1/syntaxin 1A is a member of the syntaxin family, and functions at the plasma membrane during membrane fusion as the target-soluble N-ethylmaleimide-sensitive factor-attachment protein receptor (t-SNARE). We identified the membrane-anchoring region of HPC-1/syntaxin 1A, and examined its role in anchoring of a protein to the plasma membrane. A series of mutants was created from a cysteine-less mutant of HPC-1/syntaxin 1A by substitution of each residue at the C-terminus with cysteine. The accessibility of the thiol-groups in each mutant was analyzed in vivo. The cysteine (C145) within the N-terminal cytosolic segment was labeled, but not that at C271 or C272, or any of those introduced at the C-terminus. The addition of additional residues to the C-terminal tail of HPC-1/syntaxin 1A allowed labeling by thiol-specific reagents. A monoclonal antibody directed against the C-terminal tail peptide did not react with the protein located at the plasma membrane. In addition, subcellular fractionation and immunocytochemical analyses with various transmembrane mutants showed that the C-terminal tail comprising eight amino acids is essential for anchoring of HPC-1/syntaxin 1A to the plasma membrane. These results indicate that the C-terminal membrane-anchoring region, which comprises 23 amino acids, does not traverse the lipid-bilayer and that the C-terminal tail is essential for anchoring of HPC-1/syntaxin 1A to the plasma membrane.


Assuntos
Substituição de Aminoácidos , Anticorpos Monoclonais/metabolismo , Antígenos de Superfície/metabolismo , Cisteína/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Substituição de Aminoácidos/genética , Animais , Anticorpos Monoclonais/genética , Antígenos de Superfície/genética , Células COS , Linhagem Celular Tumoral , Membrana Celular/genética , Membrana Celular/metabolismo , Chlorocebus aethiops , Cisteína/genética , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Mutagênese Sítio-Dirigida , Mutação , Proteínas do Tecido Nervoso/genética , Células PC12 , Estrutura Terciária de Proteína/genética , Ratos , Sintaxina 1
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