Palmitoylation of MPP1 (membrane-palmitoylated protein 1)/p55 is crucial for lateral membrane organization in erythroid cells.

S-Acylation of proteins is a ubiquitous post-translational modification and a common signal for membrane association. The major palmitoylated protein in erythrocytes is MPP1, a member of the MAGUK family and an important component of the ternary complex that attaches the spectrin-based skeleton to the plasma membrane. Here we show that DHHC17 is the only acyltransferase present in red blood cells (RBC). Moreover, we give evidence that protein palmitoylation is essential for membrane organization and is crucial for proper RBC morphology, and that the effect is specific for MPP1. Our observations are based on the clinical cases of two related patients whose RBC had no palmitoylation activity, caused by a lack of DHHC17 in the membrane, which resulted in a strong decrease of the amount of detergent-resistant membrane (DRM) material. We confirmed that this loss of detergent-resistant membrane was due to the lack of palmitoylation by treatment of healthy RBC with 2-bromopalmitic acid (2-BrP, common palmitoylation inhibitor). Concomitantly, fluorescence lifetime imaging microscopy (FLIM) analyses of an order-sensing dye revealed a reduction of membrane order after chemical inhibition of palmitoylation in erythrocytes. These data point to a pathophysiological relationship between the loss of MPP1-directed palmitoylation activity and perturbed lateral membrane organization.

Local Shape Preserving Deformations for Augmented Reality Assisted Laparoscopic Surgery.

Image registration is a commonly required task in computer assisted surgical procedures. Existing registration methods in laparoscopic navigation systems suffer from several constraints, such as lack of deformation compensation. The proposed algorithm aims to provide the surgeons with updated navigational information about the deep-seated anatomy, which considers the continuous deformations in the operating environment. We extended an initial rigid registration to a shape-preserving deformable registration pathway by incorporating user interaction and an iterative mesh editing scheme which preserves local details. The proposed deformable registration workflow was tested with phantom and animal trial datasets. A qualitative evaluation based on expert feedback demonstrated satisfactory outcome, and an commensurate execution efficiency was achieved. The improvements offered by the method, couples with its relatively easy implementation, makes it an attractive method for adoption in future pre-clinical and clinical applications of augmented reality assisted surgeries.

Ultrasound evaluation of the radial artery in young adults - A pilot study.

The radial artery is a vessel frequently used in various medical procedures which carry a risk of complications. One such method used to minimize the number of complications is a profound knowledge of the artery's diameter, which allows for appropriate selection of an instrument to use for puncture. The purpose of this study is to determine the diameter of the radial artery and its depth from the skin surface in young adults. MATERIAL AND METHODS: 96 volunteers participated in the study. The average age of the volunteers was 20.43 years. All participants underwent a B-mode ultrasonography to measure the distal radial artery's diameter. Certified anthropometric instruments were used to determine the relation between the artery diameter and the selected basic anthropometric parameters. RESULTS: The mean right arterial diameter for the entire examined population at the level of the styloid process was mean 1.42 (SD 0.26) mm and its distance to the skin was mean 4.00 (SD1.20) mm. Statistical analysis revealed the presence of moderate positive relationship between the diameter of the radial artery and body weight and between the diameter of the radial artery and BMI. A weak correlation between the artery diameter and the width of the wrist was also present. CONCLUSION: In the present work, we aimed to determine the dimensions of the distal section of the radial artery in young people free from symptoms of cardiovascular diseases. The obtained initial study results advise selecting instruments for vascular puncture individually, with special attention paid to slender people with slim wrists.

The effect of the lipid-binding site of the ankyrin-binding domain of erythroid beta-spectrin on the properties of natural membranes and skeletal structures.

It was previously shown that the beta-spectrin ankyrin-binding domain binds lipid domains rich in PE in an ankyrin-dependent manner, and that its N-terminal sequence is crucial in interactions with phospholipids. In this study, the effect of the full-length ankyrin-binding domain of beta-spectrin on natural erythrocyte and HeLa cell membranes was tested. It was found that, when encapsulated in resealed erythrocyte ghosts, the protein representing the full-length ankyrin-binding domain strongly affected the shape and barrier properties of the erythrocyte membrane, and induced partial spectrin release from the membrane, while truncated mutants had no effect. As found previously (Bok et al. Cell Biol. Int. 31 (2007) 1482-94), overexpression of the full-length GFP-tagged ankyrin-binding domain aggregated and induced aggregation of endogenous spectrin, but this was not the case with overexpression of proteins truncated at their N-terminus. Here, we show that the aggregation of spectrin was accompanied by the aggregation of integral membrane proteins that are known to be connected to spectrin via ankyrin, i.e. Na(+)K(+)ATP-ase, IP3 receptor protein and L1 CAM. By contrast, the morphology of the actin cytoskeleton remained unchanged and aggregation of cadherin E or N did not occur upon the overexpression of either full-length or truncated ankyrin-binding domain proteins. The obtained results indicate a substantial role of the lipid-binding part of the beta-spectrin ankyrin-binding domain in the determination of the membrane and spectrin-based skeleton functional properties.

Human DHHC proteins: a spotlight on the hidden player of palmitoylation.

Palmitoylation is one of the most common posttranslational lipid modifications of proteins and we now know quite a lot about it. However, the state of knowledge about the enzymes that catalyze this process is clearly insufficient. This review is focused on 23 human DHHC genes and their products - protein palmitoyltransferases. Here we describe mainly the structure and function of these proteins, but also, to a lesser degree, what the substrates of the enzymes are and whether they are related to various diseases. The main aim of this review was to catalogue existing information concerning the human DHHC family of genes/proteins, making them and their functions easier to understand.

Expression, purification and functional characterization of recombinant human acyl-CoA-binding protein (ACBP) from erythroid cells.

Fatty acyl-CoA esters are extremely important in cellular homeostasis. They are intermediates in both lipid metabolism and post-translational protein modifications. Among these modification events, protein palmitoylation seems to be unique by its reversibility which allows dynamic regulation of the protein hydrophobicity. The recent discovery of an enzyme family that catalyze protein palmitoylation has increased the understanding of the enzymology of the covalent attachment of fatty acids to proteins. Despite that, the molecular mechanism of supplying acyl-CoA esters to this reaction is yet to be established. Acyl-coenzyme A-binding proteins are known to bind long-chain acyl-CoA esters with very high affinity. Therefore, they play a significant role in intracellular acyl-CoA transport and pool formation. The purpose of this work is to explore the potential of one of the acyl-CoA-binding proteins to participate in the protein palmitoylation. In this study, a recombinant form of ACBP derived from human erythroid cells was expressed in E. coli, purified, and functionally characterized. We demonstrate that recombinant hACBP effectively binds palmitoyl-CoA in vitro, undergoing a shift from a monomeric to a dimeric state, and that this ligand-binding ability is involved in erythrocytic membrane phosphatidylcholine (PC) remodeling but not in protein acylation.

A raft-associated species of phosphatidylethanolamine interacts with cholesterol comparably to sphingomyelin. A Langmuir-Blodgett monolayer study.

BACKGROUND: Specific interactions between sphingomyelin (SM) and cholesterol (Ch) are commonly believed to play a key role in the formation of rafts in the biological membranes. A weakness of this model is the implication that these microdomains are confined to the outer bilayer leaflet. The cytoplasmic leaflet, which contains the bulk of phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylinositol (PI), is thought also to harbour half of the membrane cholesterol. Moreover, SLPE (1-stearoyl-2-linoleoyl-sn-glycero-3-phosphatidyl-ethanolamine) has recently been shown to be enriched in isolated detergent-resistant membranes (DRM), and this enrichment was independent of the method of isolation of DRM. METHODOLOGY/PRINCIPAL FINDINGS: Here we present quantitative evidence coming from Langmuir-Blodgett monolayer experiments that SLPE forms complex with Ch similar to that between SM and Ch. The energies of these interactions as calculated form the monolayer studies are highly negative. FRAP analysis showed that NBD-Ch recovery was similar in liposomes composed of DOPC/Ch SM or SLPE but not DPPE, providing further evidence that SLPE may form an l(o) phase in the presence of high Ch concentration. Experiments on the solubility of DOPC liposomes containing DPPE/Ch (1ratio1), SM/Ch (1ratio1) or SLPE/Ch (1ratio1) showed the presence of Triton X-100 insoluble floating fraction (TIFF) in the case of SM/Ch or SLPE/Ch but not in DPPE/Ch containing liposomes. Quantitative determination of particular lipid species in the TIFF fraction confirms the conclusion that SLPE (or similar PE species) could be an important constituent of the inner leaflet raft. CONCLUSION: Such interactions suggest a possible existence of inner-leaflet nanoscale assemblies composed of cholesterol complexes with SLPE or similar unsaturated PE species.