Cellular functions of NSF: not just SNAPs and SNAREs

N-ethylmaleimide sensitive factor (NSF) is an ATPases associated with various cellular activities protein (AAA), broadly required for intracellular membrane fusion. NSF functions as a SNAP receptor (SNARE) chaperone which binds, through soluble NSF attachment proteins (SNAPs), to SNARE complexes and utilizes the energy of ATP hydrolysis to disassemble them thus facilitating SNARE recycling. While this is a major function of NSF, it does seem to interact with other proteins, such as the AMPA receptor subunit, GluR2, and beta2-AR and is thought to affect their trafficking patterns. New data suggest that NSF may be regulated by transient post-translational modifications such as phosphorylation and nitrosylation. These new aspects of NSF function as well as its role in SNARE complex dynamics will be discussed.     

Identification of HSP90 as a new GABARAPL1 (GEC1)-interacting protein

GABARAPL1 belongs to the small family of GABARAP proteins (including GABARAP, GABARAPL1 and GABARAPL2/GATE-16), one of the two subfamilies of the yeast Atg8 orthologue. GABARAPL1 is involved in the intracellular transport of receptors, via an interaction with tubulin and GABA(A) or kappa opioid receptors, and also participates in autophagy and cell proliferation. In the present study, we identify the HSP90 protein as a novel interaction partner for GABARAPL1 using GST pull-down, mass spectrometry and coimmunoprecipitation experiments. GABARAPL1 and HSP90 partially colocalize in MCF-7 breast cancer cells overexpressed Dsred-GABARAPL1 and in rat brain. Moreover, treatment of MCF-7 cells overexpressed FLAG-GABARAPL1-6HIS with the HSP90 inhibitor 17-AAG promotes the GABARAPL1 degradation, a process that is blocked by proteasome inhibitors such as MG132, bortezomib and lactacystin. Accordingly, we demonstrate that HSP90 interacts and protects GABARAPL1 from its degradation by the proteasome.     

Effects of C-terminal modifications of GEC1 protein and gamma-aminobutyric acid type A (GABA(A)) receptor-associated protein (GABARAP), two microtubule-associated proteins, on kappa opioid receptor expression

We demonstrated previously that GEC1, a member of the microtubule-associated protein (MAP) family, bound to the human κ opioid receptor (hKOPR) and promoted hKOPR cell surface expression by facilitating its trafficking along the secretory pathway. GABA(A) receptor-associated protein (GABARAP), a GEC1 analog, also enhanced KOPR expression, but to a lesser extent. The MAP family proteins undergo cleavage of their C-terminal residue(s), and the exposed conserved glycine forms conjugates with phosphatidylethanolamine, which associate with membranes. Here, we examined whether such modifications were required for GEC1 and GABARAP to enhance hKOPR expression. When transiently transfected into CHO or Neuro2A cells, GEC1 and GABARAP were cleaved at the C termini. G116A mutation alone or combined with deletion of Lys(117) in GEC1 (GEC1-A) or Leu(117) in GABARAP (GABARAP-A) blocked their C-terminal cleavage, indicating that the conserved Gly(116) is necessary for C-terminal modification. The two GEC1 mutants enhanced hKOPR expression to similar extents as the wild-type GEC1; however, the two GABARAP mutants did not. Immunofluorescence studies showed that HA-GEC1, HA-GEC1-A, and HA-GABARAP were distributed in a punctate manner and co-localized with KOPR-EGFP in the Golgi apparatus, whereas HA-GABARAP-A did not. Pulldown assay of GST-KOPR-C-tail with HA-GEC1 or HA-GABARAP revealed that GEC1 had stronger association with KOPR-C-tail than GABARAP. These results suggest that because of its stronger binding for hKOPR, GEC1 is able to be recruited by hKOPR sufficiently without membrane association via its C-terminal modification; however, due to its weaker affinity for the hKOPR, GABARAP appears to require C-terminal modifications to enhance KOPR expression.     

The role of GABARAPL1/GEC1 in autophagic flux and mitochondrial quality control in MDA-MB-436 breast cancer cells

GABARAPL1/GEC1 is an early estrogen-induced gene which encodes a protein highly conserved from C. elegans to humans. Overexpressed GABARAPL1 interacts with GABA_A or kappa opioid receptors, associates with autophagic vesicles, and inhibits breast cancer cell proliferation. However, the function of endogenous GABARAPL1 has not been extensively studied. We hypothesized that GABARAPL1 is required for maintaining normal autophagic flux, and plays an important role in regulating cellular bioenergetics and metabolism. To test this hypothesis, we knocked down GABARAPL1 expression in the breast cancer MDA-MB-436 cell line by shRNA. Decreased expression of GABARAPL1 activated procancer responses of the MDA-MB-436 cells including increased proliferation, colony formation, and invasion. In addition, cells with decreased expression of GABARAPL1 exhibited attenuated autophagic flux and a decreased number of lysosomes. Moreover, decreased GABARAPL1 expression led to cellular bioenergetic changes including increased basal oxygen consumption rate, increased intracellular ATP, increased total glutathione, and an accumulation of damaged mitochondria. Taken together, our results demonstrate that GABARAPL1 plays an important role in cell proliferation, invasion, and autophagic flux, as well as in mitochondrial homeostasis and cellular metabolic programs.     

A bidirectional crosstalk between glioblastoma and brain endothelial cells potentiates the angiogenic and proliferative signaling of sphingosine-1-phosphate in the glioblastoma microenvironment

Glioblastoma is one of the most malignant, angiogenic, and incurable tumors in humans. The aberrant communication between glioblastoma cells and tumor microenvironment represents one of the major factors regulating glioblastoma malignancy and angiogenic properties. Emerging evidence implicates sphingosine-1-phosphate signaling in the pathobiology of glioblastoma and angiogenesis, but its role in glioblastoma-endothelial crosstalk remains largely unknown. In this study, we sought to determine whether the crosstalk between glioblastoma cells and brain endothelial cells regulates sphingosine-1-phosphate signaling in the tumor microenvironment. Using human glioblastoma and brain endothelial cell lines, as well as primary brain endothelial cells derived from human glioblastoma, we report that glioblastoma-co-culture promotes the expression, activity, and plasma membrane enrichment of sphingosine kinase 2 in brain endothelial cells, leading to increased cellular level of sphingosine-1-phosphate, and significant potentiation of its secretion. In turn, extracellular sphingosine-1-phosphate stimulates glioblastoma cell proliferation, and brain endothelial cells migration and angiogenesis. We also show that, after co-culture, glioblastoma cells exhibit enhanced expression of S1P₁ and S1P₃, the sphingosine-1-phosphate receptors that are of paramount importance for cell growth and invasivity. Collectively, our results envision glioblastoma-endothelial crosstalk as a multi-compartmental strategy to enforce pro-tumoral sphingosine-1-phosphate signaling in the glioblastoma microenvironment.     

Depressive symptoms in adolescents with early and continuously treated phenylketonuria: associations with phenylalanine and tyrosine levels

Background

Previous research has suggested an increased risk for individuals with phenylketonuria (PKU) of developing depression and other mood disorders. As PKU can disrupt neurotransmitter synthesis via biochemical mechanisms, depressive symptoms are hypothesised to result from neurotransmitter dysregulation. Whilst adherence (or return) to the phenylalanine-restricted diet may resolve or improve symptoms of depression, data to demonstrate a direct relationship between biochemistry and mood in this population are lacking.

Methods

Thirteen adolescents with early and continuously treated PKU and eight sibling controls were compared in their total reported depressive symptoms. A general executive function assessment was also undertaken in the PKU group. Correlations between depressive symptoms and biochemical markers were examined within the PKU group only.

Results

Correlational analyses within the PKU group demonstrated strong and significant associations between depressive symptoms and long term exposure to either a high phenylalanine:tyrosine ratio, or low tyrosine. Increasing symptoms of depression were also found to be associated with poorer executive function in the PKU sample. However, both groups of adolescents scored within the normal range in symptoms of depression (p > 0.05).

Conclusions

Significant associations were observed between biochemical markers indicating poorer dietary control and increasing depressive symptoms in a sample of adolescents with early and continuously treated PKU, although symptoms of depression remained within the normal range. An association between depressive symptoms and poorer EF was also demonstrated. Further research is needed to establish whether the depressive symptoms observed in this young population represent an emerging (subclinical) risk for major depressive disorder as they age.     

The role of type V collagen fibril as an ECM that induces the motility of glomerular endothelial cells

Although type V collagen (Col V) is present in developing and mature connective tissues of glomeruli, its primary function has not been elucidated yet. The purpose of this study was to elucidate the role of Col V fibrils in glomerular cells. We isolated primary cells from porcine kidney and cultured them on Col V fibrils reconstructed from purified Col V molecules extracted from porcine cornea. Time-lapse observation showed that Col V fibrils induce dynamic movement of glomerular endothelial cells (GEC) by stimulating them to extend long filopodial protrusions and wide lamellipodia. Col V signaling mediated through β1 integrin activated phosphorylation of paxillin at tyrosine 118 (paxillin-pY118) and of focal adhesion kinase at tyrosine 861 (FAKpY861) at the cell periphery; a second Col V signal was mediated through neuroglycan 2 and activated FAKpY397. FAKpY861 was present in loose attachment points between Col V fibrils and GEC, allowing the cells to migrate easily. Activation of FAKpY397 induced incomplete focal adhesion at the centers of cells and caused cell movement. Therefore both signaling pathways facilitated cell motility, which was inhibited by the addition of antibodies to β1 integrin, NG2, and Col V. We suggest that Col V fibrils activate ‘outside-in’ signaling in GEC and induce their dynamic motility.     

The role of GABARAPL1/GEC1 in autophagic flux and mitochondrial quality control in MDA-MB-436 breast cancer cells

GABARAPL1/GEC1 is an early estrogen-induced gene which encodes a protein highly conserved from C. elegans to humans. Overexpressed GABARAPL1 interacts with GABA_A or kappa opioid receptors, associates with autophagic vesicles, and inhibits breast cancer cell proliferation. However, the function of endogenous GABARAPL1 has not been extensively studied. We hypothesized that GABARAPL1 is required for maintaining normal autophagic flux, and plays an important role in regulating cellular bioenergetics and metabolism. To test this hypothesis, we knocked down GABARAPL1 expression in the breast cancer MDA-MB-436 cell line by shRNA. Decreased expression of GABARAPL1 activated procancer responses of the MDA-MB-436 cells including increased proliferation, colony formation, and invasion. In addition, cells with decreased expression of GABARAPL1 exhibited attenuated autophagic flux and a decreased number of lysosomes. Moreover, decreased GABARAPL1 expression led to cellular bioenergetic changes including increased basal oxygen consumption rate, increased intracellular ATP, increased total glutathione, and an accumulation of damaged mitochondria. Taken together, our results demonstrate that GABARAPL1 plays an important role in cell proliferation, invasion, and autophagic flux, as well as in mitochondrial homeostasis and cellular metabolic programs.     

The effect of lead ions on the energy metabolism of human erythrocytes in vitro

The aim of this work was to evaluate the influence of chronic exposure to lead ions on the parameters of energetic status of human erythrocytes in vitro. Umbilical cord erythrocytes were incubated with lead acetate at final lead ion concentrations ranging from 10 to 200 microg/dl. ATP, ADP, AMP, adenosine, GTP, GDP, GMP, guanosine, IMP, inosine, hypoxanthine, NAD and NADP concentrations in erythrocytes were determined using HPLC. Scanning electron micrographs of erythrocytes were taken. The mean concentrations of ATP, GTP, NAD and NADP, and mean values of adenylate energy charge (AEC) and GEC in cells incubated at the presence of lead ions were significantly lower after 20 h of incubation. Concentrations of purine degradation products (Ado, Guo, Ino) and Hyp were significantly higher. It is suggested that lead ions affect the energy metabolism of erythrocytes. Morphological changes in erythrocytes correspond to the increase of lead ions in the incubation mixture and to the decrease of ATP concentration in erythrocytes. A decrease in NAD and ATP concentration in erythrocytes could be a sensitive indicator of energy process disturbance, useful in monitoring in case of chronic lead exposure.     

GEC1, a protein related to GABARAP, interacts with tubulin and GABA(A) receptor

We have previously identified in uterine cells a novel estrogen-regulated gene called gec1. GEC1 presents 87% identity with GABARAP which, so far, was the only protein found to associate with tubulin and GABA(A) receptor. We demonstrated then that GEC1 interacts in vitro with tubulin and GABA(A) receptor, and promotes tubulin assembly and microtubule bundling. Since all polyclonal antibodies failed in discrimination of both proteins GEC1 and GABARAP, a GEC1-GFP fusion protein was used to specifically localize GEC1. GEC1-GFP was distributed over the cytoplasm in perinuclear vesicles with a scattered pattern. Overall, our data show that GEC1 could be a new member of the GABARAP family involved in the transport of GABA(A) receptor.