β subunit affects Na and K affinities of Na/K-ATPase: Na and K affinities of a hybrid Na/K-ATPase composed of insect α and mammalian β subunits.

The affinity for K of silkworm Na/K-ATPase, which is composed of α and β 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 β subunit, a glycosylated protein, had less than 30% identity to the mammalian β subunit, it was suggested that the β subunit was involved in the affinities for Na and K of Na/K-ATPase. To confirm this hypothesis, we examined whether replacing the silkworm β subunit with the mammalian β subunit affected the affinities for Na and K of Na/K-ATPase.

Golgi stress induces upregulation of the ER-Golgi SNARE Syntaxin-5, altered βAPP processing, and Caspase-3-dependent apoptosis in NG108-15 cells.

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.

Inhibition of the human secretory pathway Ca, Mn-ATPase1a by 1,3-thiazole derivatives.

The human Golgi/secretory pathway Ca,Mn-ATPase 1 (hSPCA1) transports Ca and Mn 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.

Cytoplasmic control of Rab family small GTPases through BAG6.

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.

Time lapse imaging analysis of the effect of ER stress modulators on apoptotic cell assessed by caspase3/7 activation in NG108-15 cells.

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.

Data supporting ER stress response in NG108-15 cells involves upregulation of syntaxin 5 expression and reduced amyloid β peptide secretion.

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.

Data for the effects of ER and Golgi stresses on the ER-Golgi SNARE Syntaxin5 expression and on the βAPP processing in cultured hippocampal neurons.

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 β-amyloid precursor protein (βAPP) under ER stress were analyzed by immunological assays.

ER and Golgi stresses increase ER-Golgi SNARE Syntaxin5: Implications for organelle stress and βAPP processing.

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 β-amyloid precursor protein (βAPP), 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.

ER stress response in NG108-15 cells involves upregulation of syntaxin 5 expression and reduced amyloid β peptide secretion.

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.

FRET-based evaluation of Bid cleavage in a single primary cultured neuron.

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.

The Syntaxin 5 isoforms Syx5 and Syx5L have distinct effects on the processing of {beta}-amyloid precursor protein.

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.

Calcium loading capacity and morphological changes in mitochondria in an ischemic preconditioned model.

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).

Ca(2+) buffering capacity of mitochondria after oxygen-glucose deprivation in hippocampal neurons.

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.

Syntaxin 8 has two functionally distinct di-leucine-based motifs.

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.

RNA interference-mediated silencing of the syntaxin 5 gene induces Golgi fragmentation but capable of transporting vesicles.

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.

Syntaxin 5 interacts specifically with presenilin holoproteins and affects processing of betaAPP in neuronal cells.

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.

Syntaxin 5 interacts with presenilin holoproteins, but not with their N- or C-terminal fragments, and affects beta-amyloid peptide production.

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.

Identification of the carboxyl-terminal membrane-anchoring region of HPC-1/syntaxin 1A with the substituted-cysteine-accessibility method and monoclonal antibodies.

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.