Immunosuppressive and anti-angiogenic sphingosine 1-phosphate receptor-1 agonists induce ubiquitinylation and proteasomal degradation of the receptor

Sphingosine 1-phosphate (S1P), a multifunctional lipid mediator, regulates lymphocyte trafficking, vascular permeability, and angiogenesis by activation of the S1P1 receptor. This receptor is activated by FTY720-P, a phosphorylated derivative of the immunosuppressant and vasoactive compound FTY720. However, in contrast to the natural ligand S1P, FTY720-P appears to act as a functional antagonist, even though the mechanisms involved are poorly understood. In this study, we investigated the fate of endogenously expressed S1P1 receptor in agonist-activated human umbilical vein endothelial cells and human embryonic kidney 293 cells expressing green fluorescent protein-tagged S1P1. We show that FTY720-P is more potent than S1P at inducing receptor degradation. Pretreatment with an antagonist of S1P1, VPC 44116, prevented receptor internalization and degradation. FTY720-P did not induce degradation of internalization-deficient S1P1 receptor mutants. Further, small interfering RNA-mediated down-regulation of G protein-coupled receptor kinase-2 and beta-arrestins abolished FTY720-P-induced S1P1 receptor degradation. These data suggest that agonist-induced phosphorylation of S1P1 and subsequent endocytosis are required for FTY720-P-induced degradation of the receptor. S1P1 degradation is blocked by MG132, a proteasomal inhibitor. Indeed, FTY720-P strongly induced polyubiquitinylation of S1P1 receptor, whereas S1P at concentrations that induced complete internalization was not as efficient, suggesting that receptor internalization is required but not sufficient for ubiquitinylation and degradation. We propose that the ability of FTY720-P to target the S1P1 receptor to the ubiquitinylation and proteasomal degradation pathway may at least in part underlie its immunosuppressive and anti-angiogenic properties.     

The role of AMPK and mTOR in nutrient sensing in pancreatic beta-cells

The AMP-activated protein kinase (AMPK) is a central regulator of the energy status of the cell, based on its unique ability to respond directly to fluctuations in the ratio of AMP:ATP. Because glucose and amino acids stimulate insulin release from pancreatic beta-cells by the regulation of metabolic intermediates, AMPK represents an attractive candidate for control of beta-cell function. Here, we show that inhibition of AMPK in beta-cells by high glucose inversely correlates with activation of the mammalian Target of Rapamycin (mTOR) pathway, another cellular sensor for nutritional conditions. Forced activation of AMPK by AICAR, phenformin, or oligomycin significantly blocked phosphorylation of p70S6K, a downstream target of mTOR, in response to the combination of glucose and amino acids. Amino acids also suppressed the activity of AMPK, and this at a minimum required the presence of leucine and glutamine. It is unlikely that the ability of AMPK to sense both glucose and amino acids plays a role in regulation of insulin secretion, as inhibition of AMPK by amino acids did not influence insulin secretion. Moreover, activation of AMPK by AICAR or phenformin did not antagonize glucose-stimulated insulin secretion, and insulin secretion was also unaffected in response to suppression of AMPK activity by expression of a dominant negative AMPK construct (K45R). Taken together, these results suggest that the inhibition of AMPK activity by glucose and amino acids might be an important component of the mechanism for nutrient-stimulated mTOR activity but not insulin secretion in the beta-cell.     

Rat liver carnitine palmitoyltransferase 1 forms an oligomeric complex within the outer mitochondrial membrane

Carnitine palmitoyltransferase (CPT) 1A catalyzes the rate-limiting step in the transport of long chain acyl-CoAs from cytoplasm to the mitochondrial matrix by converting them to acylcarnitines. Located within the outer mitochondrial membrane, CPT1A activity is inhibited by malonyl-CoA, its allosteric inhibitor. In this study, we investigate for the first time the quaternary structure of rat CPT1A. Chemical cross-linking studies using intact mitochondria isolated from fed rat liver or from Saccharomyces cerevisiae expressing CPT1A show that CPT1A self-assembles into an oligomeric complex. Size exclusion chromatography experiments using solubilized mitochondrial extracts suggest that the fundamental unit of its quaternary structure is a trimer. When studied in blue native-PAGE, the CPT1A hexamer could be observed, however, suggesting that under these native conditions CPT1A trimers might be arranged as dimers. Moreover, the oligomeric state of CPT1A was found unchanged by starvation and by streptozotocin-induced diabetes, conditions characterized by changes in malonyl-CoA sensitivity of CPT1A. Finally, gel filtration analysis of several yeast-expressed chimeric CPTs demonstrates that the first 147 N-terminal residues of CPT1A, encompassing its two transmembrane segments, trigger trimerization independently of its catalytic C-terminal domain. Deletion of residues 1-82, including transmembrane 1, did not abrogate oligomerization, but the latter is limited to a trimer by the presence of the large catalytic C-terminal domain on the cytosolic face of mitochondria. Based on these findings, we proposed that the oligomeric structure of CPT1A would allow the newly formed acylcarnitines to gain direct access into the intermembrane space, hence facilitating substrate channeling.     

Participation of the chaperone Hsc70 in the trafficking and functional expression of ASIC2 in glioma cells

High-grade glioma cells express subunits of the ENaC/Deg superfamily, including members of ASIC subfamily. Our previous work has shown that glioma cells exhibit a basally active cation current, which is not present in low-grade tumor cells or normal astrocytes, and that can be blocked by amiloride. When ASIC2 is present within the channel complex in the plasma membrane, the channel is rendered non-functional because of inherent negative effectors that require ASIC2. We have previously shown that high-grade glioma cells functionally express this current because of the lack of ASIC2 in the plasma membrane. We now hypothesize that ASIC2 trafficking in glioma cells is regulated by a specific chaperone protein, namely Hsc70. Our results demonstrated that Hsc70 co-immunoprecipitates with ASIC2 and that it is overexpressed in glioma cells as compared with normal astrocytes. In contrast, there was no difference in the expression of calnexin, which also co-immunoprecipitates with ASIC2. In addition, glycerol and sodium 4-phenylbutyrate reduced the amount of Hsc70 expressed in glioma cells to levels found in normal astrocytes. Transfection of Hsc70 siRNA inhibited the constitutively activated amiloride-sensitive current, decreased migration, and increased ASIC2 surface expression in glioma cells. These results support an association between Hsc70 and ASIC2 that may underlie the increased retention of ASIC2 in the endoplasmic reticulum of glioma cells. The data also suggest that decreasing Hsc70 expression promotes reversion of a high-grade glioma cell to a more normal astrocytic phenotype.     

Expression, localization and functions in acrosome reaction and sperm motility of Ca(V)3.1 and Ca(V)3.2 channels in sperm cells: an evaluation from Ca(V)3.1 and Ca(V)3.2 deficient mice

In spermatozoa, voltage-dependent calcium channels (VDCC) have been involved in different cellular functions like acrosome reaction (AR) and sperm motility. Multiple types of VDCC are present and their relative contribution is still a matter of debate. Based mostly on pharmacological studies, low-voltage-activated calcium channels (LVA-CC), responsible of the inward current in spermatocytes, were described as essential for AR in sperm. The development of Ca(V)3.1 or Ca(V)3.2 null mice provided the opportunity to evaluate the involvement of such LVA-CC in AR and sperm motility, independently of pharmacological tools. The inward current was fully abolished in spermatogenic cells from Ca(V)3.2 deficient mice. This current is thus only due to Ca(V)3.2 channels. We showed that Ca(V)3.2 channels were maintained in sperm by Western-blot and immunohistochemistry experiments. Calcium imaging experiments revealed that calcium influx in response to KCl was reduced in Ca(V)3.2 null sperm in comparison to control cells, demonstrating that Ca(V)3.2 channels were functional. On the other hand, no difference was noticed in calcium signaling induced by zona pellucida. Moreover, neither biochemical nor functional experiments, suggested the presence of Ca(V)3.1 channels in sperm. Despite the Ca(V)3.2 channels contribution in KCl-induced calcium influx, the reproduction parameters remained intact in Ca(V)3.2 deficient mice. These data demonstrate that in sperm, besides Ca(V)3.2 channels, other types of VDCC are activated during the voltage-dependent calcium influx of AR, these channels likely belonging to high-voltage activated Ca(2+) channels family. The conclusion is that voltage-dependent calcium influx during AR is due to the opening of redundant families of calcium channels.     

Normal hemopoiesis and lymphopoiesis in the combined absence of numb and numblike

The mammalian ortholog of the conserved Drosophila adaptor protein Numb (Nb) and its homolog Numblike (Nbl) modulate neuronal cell fate determination at least in part by antagonizing Notch signaling. Because the Notch pathway has been implicated in regulating hemopoietic stem cell self-renewal and T cell fate specification in mammals, we investigated the role of Nb and Nbl in hemopoiesis using conditional gene targeting. Surprisingly simultaneous deletion of both Nb and Nbl in murine bone marrow precursors did not affect the ability of stem cells to self-renew or to give rise to differentiated myeloid or lymphoid progeny, even under competitive conditions in mixed chimeras. Furthermore, T cell fate specification and intrathymic T cell development were unaffected in the combined absence of Nb and Nbl. Collectively our data indicate that the Nb family of adaptor proteins is dispensable for hemopoiesis and lymphopoiesis in mice, despite their proposed role in neuronal stem cell development.     

A mitochondrial stratigraphy for island southeast Asia

Island Southeast Asia (ISEA) was first colonized by modern humans at least 45,000 years ago, but the extent to which the modern inhabitants trace their ancestry to the first settlers is a matter of debate. It is widely held, in both archaeology and linguistics, that they are largely descended from a second wave of dispersal, proto-Austronesian-speaking agriculturalists who originated in China and spread to Taiwan approximately 5,500 years ago. From there, they are thought to have dispersed into ISEA approximately 4,000 years ago, assimilating the indigenous populations. Here, we demonstrate that mitochondrial DNA diversity in the region is extremely high and includes a large number of indigenous clades. Only a fraction of these date back to the time of first settlement, and the majority appear to mark dispersals in the late-Pleistocene or early-Holocene epoch most likely triggered by postglacial flooding. There are much closer genetic links to Taiwan than to the mainland, but most of these probably predated the mid-Holocene "Out of Taiwan" event as traditionally envisioned. Only approximately 20% at most of modern mitochondrial DNAs in ISEA could be linked to such an event, suggesting that, if an agriculturalist migration did take place, it was demographically minor, at least with regard to the involvement of women.     

Free-range layer chickens as a source of <Emphasis Type="Italic">Campylobacter</Emphasis> bacteriophage

Bacteriophage specific for Campylobacter were isolated from chicken excreta collected from established free-range layer breed stock. Bacteriophage were either propagated on a Campylobacter jejuni host with broad susceptibility to bacteriophage (NCTC 12662) or on Campylobacter isolates from the same samples. Campylobacters were confirmed as being C. jejuni and or C. coli, using a combination of standard biochemical tests and PCR analysis with genus and species specific primers. The bacteriophage displayed differential patterns of susceptibility against reference NCTC strains and contemporary C. jejuni /C. coli isolates from chicken excreta. Electron microscopy demonstrated that the phage possessed icosahedral heads and rigid contractile tails. Pulsed-field gel electrophoresis revealed the bacteriophage genomes to be double stranded DNA in the range of 140 kb in size and the restriction enzyme patterns of the DNAs indicate they are genetically related members of the Myoviridae family. This study showed that Campylobacter bacteriophage could easily be isolated from free-range chickens and form part of their normal microbiological biota of environmentally exposed birds.