Ubiquitin pathway proteins influence the mechanism of action of the novel immunosuppressive drug FTY720 in Saccharomyces cerevisiae

FTY720 is an immunosuppressive drug in clinical development for transplant graft protection in humans. This agent is of particular interest because, unlike currently available regimes, it acts to sequester lymphocytes without causing cytotoxicity or blocking differentiation and growth potential. In an effort to elucidate the mechanism of action of FTY720, and identify its downstream effectors, we have screened genomic libraries and spontaneous mutants of the model system Saccharomyces cerevisiae for resistance to FTY720. We identified several proteins and pathways as being involved in the mechanism of action of FTY720. We show specifically that the two amino acid transporters TAT1 and TAT2, the two ubiquitin proteases UBP5 and UBP11, and the heat shock protein CAJ1 confer growth resistance to FTY720 when overexpressed. Another amino acid transporter, GNP1, and the ubiquitin structural gene UBI4 as well as the ubiquitin ligase RSP5, and its binding protein BUL1 confer growth resistance in a mutated form. Supporting the importance of amino acid transport in the growth resistance phenotype of S. cerevisiae to the immunosuppressive agent FTY720, a prototrophic strain was more resistant to FTY720 than the isogenic auxotroph. To further explore these results, the effects on amino acid uptake and protein degradation were measured in the presence of FTY720. Due to the high conservation of these proteins and pathways between yeast and humans, these results may provide valuable insights into the mechanism of action of FTY720 in lymphocyte sequestration in humans.     

Flaviviral helicase: insights into the mechanism of action of a motor protein

Motor proteins are involved in crucial cell activities, such as cargo transport or nucleic acid remodeling, by converting the free energy of ATP hydrolysis into motion or mechanical work. Flavivirus helicase is a motor protein involved in dsRNA separation during viral replication, thus essential for virus infection. Since a clear vision of the protein activity, in particular of the relationship between ATP cycling and dynamics, is missing, we carried over a molecular dynamics study on Dengue virus helicase in its ATP bound and unbound states. Our simulations show different opening levels of the ssRNA access site to the helicase core. Specifically, we show that ATP induces a closed state into the ssRNA access site, likely involved in the helicase unwinding activity.     

Novel insights into the mechanism of chaperone-assisted protein disaggregation

Cell survival under severe thermal stress requires the activity of a bi-chaperone system, consisting of the ring-forming AAA+ chaperone ClpB (Hsp104) and the DnaK (Hsp70) chaperone system, which acts to solubilize and reactivate aggregated proteins. Recent studies have provided novel insight into the mechanism of protein disaggregation, demonstrating that ClpB/Hsp104 extracts unfolded polypeptides from an aggregate by threading them through its central pore. This translocation activity is necessary but not sufficient for aggregate solubilization. In addition, the middle (M) domain of ClpB and the DnaK system have essential roles, possibly by providing an unfolding force, which facilitates the extraction of misfolded proteins from aggregates.     

BRing it on: new insights into the mechanism of brassinosteroid action

Several recent breakthroughs have filled in key details of the brassinosteroid (BR) response. Identification of BAK1, a BRI1 interacting protein, the negative regulator BIN2, as well as direct targets of BIN2, BZR1 and BES1, provide a link between BR perception at the cell surface and regulation of gene expression in the nucleus. Global expression studies further defined the downstream events in this pathway, confirming the role of several factors acting in negative feedback regulation on BR levels. New links to the plant hormone, auxin, were also uncovered.     

Delayed allograft rejection in mice transgenic for a soluble form of the IL-4 receptor.

Accumulating evidence suggests that in serum and other biological fluids, cytokine binding is a property associated with soluble proteins, including a high-affinity soluble version of the IL-4 receptor (sIL-4R). While it is tempting to speculate that sIL-4R might act as a serum carrier protein or serve to inhibit or modulate IL-4 action, specific biological roles for sIL-4R remain to be established. To further assess the immunoregulatory and therapeutic potential of sIL-4R and other soluble receptors, we have created transgenic mice which constitutively express elevated levels of biologically active sIL-4R. Phenotypic characterization of lymphoid organs in sIL-4R transgenic mice revealed normal numbers of B and T cells and normal surface marker expression. Splenic lymphocytes displayed normal in vitro activities as measured by the PFC response and generation of cytotoxic T cells. In addition, antigen-specific IgE and IgG1 in vivo responses were similar in control and transgenic mice. Despite the apparent developmental normality of the sIL-4R transgenic mice, these animals were markedly deficient in the ability to reject cardiac allografts, suggesting that IL-4 is critical for the generation of alloreactivity. The results further suggest that the ability of sIL-4R to regulate IL-4 activities may be under the control of complex interactions that remain to be elucidated.     

Cryo-EM analysis reveals new insights into the mechanism of action of pyruvate carboxylase

Pyruvate carboxylase (PC) is a conserved multifunctional enzyme linked to important metabolic diseases. PC homotetramer is arranged in two layers with two opposing monomers per layer. Cryo-EM explores the conformational variability of PC in the presence of different substrates. The results demonstrate that the biotin-carboxyl carrier protein (BCCP) domain localizes near the biotin carboxylase (BC) domain of its own monomer and travels to the carboxyltransferase (CT) domain of the opposite monomer. All density maps show noticeable conformational differences between layers, mainly for the BCCP and BC domains. This asymmetry may be indicative of a coordination mechanism where monomers from different layers catalyze the BC and CT reactions consecutively. A conformational change of the PC tetramerization (PT) domain suggests a new functional role in communication. A long-range communication pathway between subunits in different layers, via interacting PT-PT and BC-BC domains, may be responsible for the cooperativity of PC from Staphylococcus aureus.     

Novel insights into the calcium action in cherry fruit development revealed by high-throughput mapping

Plant Molecular Biology - This work provides the first system-wide datasets concerning metabolic changes in calcium-treated fruits, which reveal that exogenously applied calcium may specifically...     

Farnesyltransferase--new insights into the zinc-coordination sphere paradigm: evidence for a carboxylate-shift mechanism

Despite the enormous interest that has been devoted to the study of farnesyltransferase, many questions concerning its catalytic mechanism remain unanswered. In particular, several doubts exist on the structure of the active-site zinc coordination sphere, more precisely on the nature of the fourth ligand, which is displaced during the catalytic reaction by a peptide thiolate. From available crystallographic structures, and mainly from x-ray absorption fine structure data, two possible alternatives emerge: a tightly zinc-bound water molecule or an almost symmetrical bidentate aspartate residue (Asp-297beta). In this study, high-level theoretical calculations, with different-sized active site models, were used to elucidate this aspect. Our results demonstrate that both coordination alternatives lie in a notably close energetic proximity, even though the bidentate hypothesis has a somewhat lower energy. The Gibbs reaction and activation energies for the mono-bidentate conversion, as well as the structure for the corresponding transition state, were also determined. Globally, these results indicate that at room temperature the mono-bidentate conversion is reversible and very fast, and that probably both states exist in equilibrium, which suggests that a carboxylate-shift mechanism may have a key role in the farnesylation process by assisting the coordination/displacement of ligands to the zinc ion, thereby controlling the enzyme activity. Based on this equilibrium hypothesis, an explanation for the existing contradictions between the crystallographic and x-ray absorption fine structure results is proposed.     

Interaction of the antimicrobial peptide pheromone Plantaricin A with model membranes: implications for a novel mechanism of action

Plantaricin A (plA) is a 26-residue bacteria-produced peptide pheromone with membrane-permeabilizing antimicrobial activity. In this study the interaction of plA with membranes is shown to be highly dependent on the membrane lipid composition. PlA bound readily to zwitterionic 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) monolayers and liposomes, yet without significantly penetrating into these membranes. The presence of cholesterol attenuated the intercalation of plA into SOPC monolayers. The association of plA to phosphatidylcholine was, however, sufficient to induce membrane permeabilization, with nanomolar concentrations of the peptide triggering dye leakage from SOPC liposomes. The addition of the negatively charged phospholipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-glycerol POPG (SOPC/POPG; molar ratio 8:2) enhanced the membrane penetration of the peptide, as revealed by (i) peptide-induced increment in the surface pressure of lipid monolayers, (ii) increase in diphenylhexatriene (DPH) emission anisotropy measured for bilayers, and (iii) fluorescence characteristics of the two Trps of plA in the presence of liposomes, measured as such as well as in the presence of different quenchers. Despite deeper intercalation of plA into the SOPC/POPG lipid bilayer, much less peptide-induced dye leakage was observed for these liposomes than for the SOPC liposomes. Further changes in the mode of interaction of plA with lipids were evident when also the zwitterionic phospholipid, 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphoethanolaminne (POPE) was present (SOPC/POPG/POPE, molar ratio 3:2:5), thus suggesting increase in membrane spontaneous negative curvature to affect the mode of association of this peptide with lipid bilayer. PlA induced more efficient aggregation of the SOPC/POPG and SOPC/POPG/POPE liposomes than of the SOPC liposomes, which could explain the attenuated peptide-induced dye leakage from the former liposomes. At micromolar concentrations, plA killed human leukemic T-cells by both necrosis and apoptosis. Interestingly, plA formed supramolecular protein-lipid amyloid-like fibers upon binding to negatively charged phospholipid-containing membranes, suggesting a possible mechanistic connection between fibril formation and the cytotoxicity of plA.     

Novel insights into mechanisms of glucocorticoid action and the development of new glucocorticoid receptor ligands

Glucocorticoids (GCs) are potent anti-inflammatory and immunosuppressant agents. Unfortunately, they also produce serious side effects that limit their usage. This discrepancy is the driving force for the intensive search for novel GC receptor ligands with a better benefit-risk ratio as compared to conventional GCs. A better understanding of the molecular mode of GC action might result in the identification of novel drug targets. Genomic GC effects are mediated by transrepression or transactivation, the latter being largely responsible for GC side effects. We here discuss novel GC receptor ligands, such as selective glucocorticoid receptor agonists (SEGRAs), which might optimize genomic GC effects as they preferentially induce transrepression with little or no transactivating activity. In addition to genomic GC effects, GCs also produce rapid genomic-independent activities, termed nongenomic, and we here review the possible implications of a recently reported mechanism underlying nongenomic GC-induced immunosuppression in T cells. It was shown that the synthetic GC dexamethasone targets membrane-bound GC receptors leading to impaired T cell receptor signaling. As a consequence, membrane-linked GC receptors might be a potential candidate target for GC therapy. The ultimate goal is to convert these molecular insights into new GC receptor modulators with an improved therapeutic index.     

Structural insights into the membrane-anchoring mechanism of a cholesterol-dependent cytolysin

Perfringolysin O (PFO), a cytolytic toxin secreted by pathogenic Clostridium perfringens, forms large pores in cholesterol-containing membranes. Domain 4 (D4) of the protein interacts first with the membrane and is responsible for cholesterol recognition. By using several independent fluorescence techniques, we have determined the topography of D4 in the membrane-inserted oligomeric form of the toxin. Only the short hydrophobic loops at the tip of the D4 beta-sandwich are exposed to the bilayer interior, whereas the remainder of D4 projects from the membrane surface and is surrounded by water, making little or no contact with adjacent protein monomers in the oligomer. Thus, a limited interaction of D4 with the bilayer core seems to be sufficient to accomplish cholesterol recognition and initial binding of PFO to the membrane. Furthermore, D4 serves as the fulcrum around which extensive structural changes occur during the formation and insertion of the large transmembrane beta-barrel into the bilayer.     

Kin I kinesins: insights into the mechanism of depolymerization

Kin I kinesins are members of the diverse kinesin superfamily of molecular motors. Whereas most kinesins use ATP to move along microtubules, Kin I kinesins depolymerize microtubules rather than walk along them. Functionally, this distinct subfamily of kinesins is important in regulating cellular microtubule dynamics and plays a crucial role in spindle assembly and chromosome segregation. The molecular mechanism of Kin I-induced microtubule destabilization is as yet unclear. It is generally believed that Kin Is induce a structural change on the microtubule that leads to microtubule destabilization. Recently, much progress has been made towards understanding how Kin Is may cause this structural change, and how ATPase activity is employed in the catalytic cycle.     

Non-coding RNA: insights into the mechanism of methamphetamine neurotoxicity

Molecular and Cellular Biochemistry - Chronic exposure of the methamphetamine has been shown to lead to neurotoxicity in rodents and humans. The manifestations of methamphetamine neurotoxicity...     

New insights into the mechanism for clearance of apoptotic cells

Apoptosis is a physiological mechanism for the removal of unwanted or damaged cells. Apoptotic cells are rarely seen in living tissues, however, because of their rapid and efficient removal by phagocytosis. Phagocytotic cells such as macrophages or dendritic cells recognize apoptotic cells by specific changes of cell surface markers, which usually are not present on normal cells. One such event is the exposure of phosphatidylserine, which moves from the plasma membrane inner leaflet to the outer leaflet in preapoptotic cells. An unresolved problem, however, was the nature of the phosphatidylserine receptor on the phagocytotic cells. In a recent issue of Nature, Fadok et al. have reported the cloning of a phosphatidylserine receptor using an antibody raised against activated macrophages. Antibody treatment of these macrophages blocks this capacity to engulf.