Sphingolipids are required for the stable membrane association of glycosylphosphatidylinositol-anchored proteins in yeast

Ongoing sphingolipid synthesis is specifically required in vivo for the endoplasmic reticulum (ER) to Golgi transport of glycosylphosphatidylinositol (GPI)-anchored proteins. However, the sphingolipid intermediates that are required for transport nor their role(s) have been identified. Using stereoisomers of dihydrosphingosine, together with specific inhibitors and a mutant defective for sphingolipid synthesis, we now show that ceramides and/or inositol sphingolipids are indispensable for GPI-anchored protein transport. Furthermore, in the absence of sphingolipid synthesis, a significant fraction of GPI-anchored proteins is no longer associated tightly with the ER membrane. The loose membrane association is neither because of the lack of a GPI-anchor nor because of prolonged ER retention of GPI-anchored proteins. These results indicate that ceramides and/or inositol sphingolipids are required to stabilize the association of GPI-anchored proteins with membranes. They could act either by direct involvement as membrane components or as substrates for the remodeling of GPI lipid moieties.     

Dynamic simulation pericardial bioprosthetic heart valve function

While providing nearly trouble-free function for 10-12 years, current bioprosthetic heart valves (BHV) continue to suffer from limited long-term durability. This is usually a result of leaflet calcification and/or structural degeneration, which may be related to regions of stress concentration associated with complex leaflet deformations. In the current work, a dynamic three-dimensional finite element analysis of a pericardial BHV was performed with a recently developed FE implementation of the generalized nonlinear anisotropic Fung-type elastic constitutive model for pericardial BHV tissues (W. Sun and M.S. Sacks, 2005, [Biomech. Model. Mechanobiol., 4(2-3), pp. 190-199]). The pericardial BHV was subjected to time-varying physiological pressure loading to compute the deformation and stress distribution during the opening phase of the valve function. A dynamic sequence of the displacements revealed that the free edge of the leaflet reached the fully open position earlier and the belly region followed. Asymmetry was observed in the resulting displacement and stress distribution due to the fiber direction and the anisotropic characteristics of the Fung-type elastic constitutive material model. The computed stress distribution indicated relatively high magnitudes near the free edge of the leaflet with local bending deformation and subsequently at the leaflet attachment boundary. The maximum computed von Mises stress during the opening phase was 33.8 kPa. The dynamic analysis indicated that the free edge regions of the leaflets were subjected to significant flexural deformation that may potentially lead to structural degeneration after millions of cycles of valve function. The regions subjected to time varying flexural deformation and high stresses of the present study also correspond to regions of tissue valve calcification and structural failure reported from explanted valves. In addition, the present simulation also demonstrated the importance of including the bending component together with the in-plane material behavior of the leaflets towards physiologically realistic deformation of the leaflets. Dynamic simulations with experimentally determined leaflet material specification can be potentially used to modify the valve towards an optimal design to minimize regions of stress concentration and structural failure.     

Mutations in the rod outer segment membrane guanylate cyclase in a cone-rod dystrophy cause defects in calcium signaling.

Rod outer segment guanylate cyclase 1 (ROS-GC1) is a member of the subfamily of Ca(2+)-regulated membrane guanylate cyclases; and it is pivotal for vertebrate phototransduction. Two opposing regulatory modes control the activity of ROS-GC1. At nanomolar concentrations of Ca(2+), ROS-GC1 is activated by Ca(2+)-binding proteins named guanylate cyclase activating proteins (GCAPs). However, at micromolar concentrations of Ca(2+), ROS-GC1 is stimulated by S100beta [also named calcium-dependent (CD) GCAP]. This mode is not linked with phototransduction; instead, it is predicted to be involved in retinal synaptic activity. Two point mutations, E786D and R787C, in ROS-GC1 have been connected with cone-rod dystrophy (CORD6), with only one type of point mutation occurring in each family. The present study shows that the E786D mutation has no effect on the basal catalytic activity of ROS-GC1 and on its activation by GCAP1 and S100beta; however, the mutated cyclase becomes more activated by GCAP2. The R787C mutation has three consequences: (1) it causes major damage to the basal cyclase activity, (2) it makes the cyclase 5-fold more sensitive to activation by GCAP1; and 3) converts the cyclase into a form that is less sensitive to activation by GCAP2 and S100beta. Thus, the two CORD6-linked mutations in ROS-GC1, which occur at adjacent positions, result in vastly different biochemical phenotypes, and they are connected with very specific molecular defects in the Ca(2+) switching components of the cyclase. These defects, in turn, are proposed to have a profound effect on both the machinery of phototransduction and the retinal synapse. The study for the first time defines the biochemistry of CORD6 pathology in precise molecular terms.     

Mammalian brain plasma membranes : Isolation, enzymatic, and chemical characterization

Two variants of a simplified procedure for the isolation of plasma membrane fractions from monkey and rat brains, are described. The preparations show marked enrichments in the marker enzymes, (Na⁺-K⁺) adenosine triphosphatase, acetylcholinesterase, 5′-nucleotidase and adenylate cyclase. Lipid analysis and a protein electrophoretic pattern are presented. An enzymatic check has been made to assess for contamination by other cellular organelles. The amino acid composition of brain membrane proteins show a resemblance to the reported composition of erythrocyte ghost proteins but differ from myelin proteins.     

Crystal structure of Mycobacterium tuberculosis YefM antitoxin reveals that it is not an intrinsically unstructured protein

Toxin-antitoxin modules are present on chromosomes of almost all free-living prokaryotes. Some are implicated to act as stress-responsive elements, among their many functional roles. The YefM-YoeB toxin-antitoxin system is present in many bacterial species, where YefM belongs to the Phd family antidote of phage P1, whereas YoeB is a homolog of the RelE toxin of the RelBE system, rather than the Doc system of phage P1. YoeB, a ribonuclease, is believed to be conformationally stable, whereas YefM has been proposed to be a member of intrinsically disordered proteins. The ribonucleolytic activity of YoeB is neutralized by YefM upon formation of the YefM-YoeB complex. We report here the crystal structure of Mycobacterium tuberculosis YefM from two crystal isoforms. Our crystallographic and biophysical studies reveal that YefM is not an intrinsically unfolded protein and instead forms a well-defined structure with significant secondary and tertiary structure conformations. The residues involved in core formation of the folded structure are evolutionarily conserved among many bacterial species, supporting our observation. The C-terminal end of its polypeptide is highly pliable, which adopts different conformations in different monomers. Since at the physiological level YefM controls the activity of YoeB through intricate protein-protein interactions, the conformational heterogeneity in YefM revealed by our structure suggests that these might act a master switch in controlling YoeB activity.     

Plant lectin can target receptors containing sialic Acid, exemplified by podoplanin, to inhibit transformed cell growth and migration

Cancer is a leading cause of death of men and women worldwide. Tumor cell motility contributes to metastatic invasion that causes the vast majority of cancer deaths. Extracellular receptors modified by α2,3-sialic acids that promote this motility can serve as ideal chemotherapeutic targets. For example, the extracellular domain of the mucin receptor podoplanin (PDPN) is highly O-glycosylated with α2,3-sialic acid linked to galactose. PDPN is activated by endogenous ligands to induce tumor cell motility and metastasis. Dietary lectins that target proteins containing α2,3-sialic acid inhibit tumor cell growth. However, anti-cancer lectins that have been examined thus far target receptors that have not been identified. We report here that a lectin from the seeds of Maackia amurensis (MASL) with affinity for O-linked carbohydrate chains containing sialic acid targets PDPN to inhibit transformed cell growth and motility at nanomolar concentrations. Interestingly, the biological activity of this lectin survives gastrointestinal proteolysis and enters the cardiovascular system to inhibit melanoma cell growth, migration, and tumorigenesis. These studies demonstrate how lectins may be used to help develop dietary agents that target specific receptors to combat malignant cell growth.     

Design,parallel synthesis,and crystal structures of biphenyl antithrombotics as selective inhibitors of tissue factor FVIIa complex. Part 1: Exploration of S2 pocket pharmacophores

Factor VIIa (FVIIa), a serine protease enzyme, coupled with tissue factor (TF) plays an important role in a number of thrombosis-related disorders. Inhibition of TF·FVIIa occurs early in the coagulation cascade and might provide some safety advantages over other related enzymes. We report here a novel series of substituted biphenyl derivatives that are highly potent and selective TF·FVIIa inhibitors. Parallel synthesis coupled with structure-based drug design allowed us to explore the S2 pocket of the enzyme active site. A number of compounds with IC₅₀ value of <10 nM were synthesized. The X-ray crystal structures of some of these compounds complexed with TF·FVIIa were determined and results were applied to design the next round of inhibitors. All the potent inhibitors were tested for inhibition against a panel of related enzymes and selectivity of 17,600 over thrombin, 450 over trypsin, 685 over FXa, and 76 over plasmin was achieved. Two groups, vinyl 36b and 2-furan 36ab, were identified as the optimum binding substituents on the phenyl ring in the S2 pocket. Compounds with these two substituents are the most potent compounds in this series with good selectivity over related serine proteases. These compounds will be further explored for structure–activity relationship.     

A Computational,Tissue-Realistic Model of Pressure Ulcer Formation in Individuals with Spinal Cord Injury

People with spinal cord injury (SCI) are predisposed to pressure ulcers (PU). PU remain a significant burden in cost of care and quality of life despite improved mechanistic understanding and advanced interventions. An agent-based model (ABM) of ischemia/reperfusion-induced inflammation and PU (the PUABM) was created, calibrated to serial images of post-SCI PU, and used to investigate potential treatments in silico. Tissue-level features of the PUABM recapitulated visual patterns of ulcer formation in individuals with SCI. These morphological features, along with simulated cell counts and mediator concentrations, suggested that the influence of inflammatory dynamics caused simulations to be committed to “better” vs. “worse” outcomes by 4 days of simulated time and prior to ulcer formation. Sensitivity analysis of model parameters suggested that increasing oxygen availability would reduce PU incidence. Using the PUABM, in silico trials of anti-inflammatory treatments such as corticosteroids and a neutralizing antibody targeted at Damage-Associated Molecular Pattern molecules (DAMPs) suggested that, at best, early application at a sufficiently high dose could attenuate local inflammation and reduce pressure-associated tissue damage, but could not reduce PU incidence. The PUABM thus shows promise as an adjunct for mechanistic understanding, diagnosis, and design of therapies in the setting of PU.