Molecular basis for bre5 cofactor recognition by the ubp3 deubiquitylating enzyme

Yeast Ubp3 and its co-factor Bre5 form a deubiquitylation complex to regulate protein transport between the endoplasmic reticulum and Golgi compartments of the cell. A novel N-terminal domain of the Ubp3 catalytic subunit forms a complex with the NTF2-like domain of the Bre5 regulatory subunit. Here, we report the X-ray crystal structure of an Ubp3-Bre5 complex and show that it forms a symmetric hetero-tetrameric complex in which the Bre5 NTF2-like domain dimer interacts with two L-shaped beta-strand-turn-alpha-helix motifs of Ubp3. The Ubp3 N-terminal domain binds within a hydrophobic cavity on the surface of the Bre5 NTF2-like domain subunit with conserved residues within both proteins interacting predominantly through antiparallel beta-sheet hydrogen bonds and van der Waals contacts. Structure-based mutagenesis and functional studies confirm the significance of the observed interactions for Ubp3-Bre5 association in vitro and Ubp3 function in vivo. Comparison of the structure to other protein complexes with NTF2-like domains shows that the Ubp3-Bre5 interface is novel. Together, these studies provide new insights into Ubp3 recognition by Bre5 and into protein recognition by NTF2-like domains.     

Herpes simplex virus type 1 cytoplasmic envelopment requires functional Vps4

The assembly and egress of herpesviruses are complex processes that require the budding of viral nucleocapsids into the lumen of cytoplasmic compartments to form mature infectious virus. This envelopment stage shares many characteristics with the formation of luminal vesicles in multivesicular endosomes. Through expression of dominant-negative Vps4, an enzyme that is essential for the formation of luminal vesicles in multivesicular endosomes, we now show that Vps4 function is required for the cytoplasmic envelopment of herpes simplex virus type 1. This is the first example of a large enveloped DNA virus engaging the multivesicular endosome sorting machinery to enable infectious virus production.     

Pathogenesis of avian influenza (H7) virus infection in mice and ferrets: enhanced virulence of Eurasian H7N7 viruses isolated from humans

Before 2003, only occasional case reports of human H7 influenza virus infections occurred as a result of direct animal-to-human transmission or laboratory accidents; most of these infections resulted in conjunctivitis. An increase in isolation of avian influenza A H7 viruses from poultry outbreaks and humans has raised concerns that additional zoonotic transmissions of influenza viruses from poultry to humans may occur. To better understand the pathogenesis of H7 viruses, we have investigated their ability to cause disease in mouse and ferret models. Mice were infected intranasally with H7 viruses of high and low pathogenicity isolated from The Netherlands in 2003 (Netherlands/03), the northeastern United States in 2002-2003, and Canada in 2004 and were monitored for morbidity, mortality, viral replication, and proinflammatory cytokine production in respiratory organs. All H7 viruses replicated efficiently in the respiratory tracts of mice, but only Netherlands/03 isolates replicated in systemic organs, including the brain. Only A/NL/219/03 (NL/219), an H7N7 virus isolated from a single fatal human case, was highly lethal for mice and caused severe disease in ferrets. Supporting the apparent ocular tropism observed in humans following infection with viruses of the H7 subtype, both Eurasian and North American lineage H7 viruses were detected in the mouse eye following ocular inoculation, whereas an H7N2 virus isolated from the human respiratory tract was not. Therefore, in general, the relative virulence and cell tropism of the H7 viruses in these animal models correlated with the observed virulence in humans.     

Structures of Mycobacterium tuberculosis 1-deoxy-D-xylulose-5-phosphate reductoisomerase provide new insights into catalysis

Isopentenyl diphosphate is the precursor of various isoprenoids that are essential to all living organisms. It is produced by the mevalonate pathway in humans but by an alternate route in plants, protozoa, and many bacteria. 1-deoxy-D-xylulose-5-phosphate reductoisomerase catalyzes the second step of this non-mevalonate pathway, which involves an NADPH-dependent rearrangement and reduction of 1-deoxy-D-xylulose 5-phosphate to form 2-C-methyl-D-erythritol 4-phosphate. The use of different pathways, combined with the reported essentiality of the enzyme makes the reductoisomerase a highly promising target for drug design. Here we present several high resolution structures of the Mycobacterium tuberculosis 1-deoxy-D-xylulose-5-phosphate reductoisomerase, representing both wild type and mutant enzyme in various complexes with Mn(2+), NADPH, and the known inhibitor fosmidomycin. The asymmetric unit corresponds to the biological homodimer. Although crystal contacts stabilize an open active site in the B molecule, the A molecule displays a closed conformation, with some differences depending on the ligands bound. An inhibition study with fosmidomycin resulted in an estimated IC(50) value of 80 nm. The double mutant enzyme (D151N/E222Q) has lost its ability to bind the metal and, thereby, also its activity. Our structural information complemented with molecular dynamics simulations and free energy calculations provides the framework for the design of new inhibitors and gives new insights into the reaction mechanism. The conformation of fosmidomycin bound to the metal ion is different from that reported in a previously published structure and indicates that a rearrangement of the intermediate is not required during catalysis.     

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.