Absence Of Lipids Research Articles

Lipid-free antigen B subunits from echinococcus granulosus: oligomerization, ligand binding, and membrane interaction properties.

BackgroundThe hydatid disease parasite Echinococcus granulosus has a restricted lipid metabolism, and needs to harvest essential lipids from the host. Antigen B (EgAgB), an abundant lipoprotein of the larval stage (hydatid cyst), is thought to be important in lipid storage and transport. It contains a wide variety of lipid classes, from highly hydrophobic compounds to phospholipids. Its protein component belongs to the cestode-specific Hydrophobic Ligand Binding Protein family, which includes five 8-kDa isoforms encoded by a multigene family (EgAgB1-EgAgB5). How lipid and protein components are assembled into EgAgB particles remains unknown. EgAgB apolipoproteins self-associate into large oligomers, but the functional contribution of lipids to oligomerization is uncertain. Furthermore, binding of fatty acids to some EgAgB subunits has been reported, but their ability to bind other lipids and transfer them to acceptor membranes has not been studied.Methodology/Principal FindingsLipid-free EgAgB subunits obtained by reverse-phase HPLC were used to analyse their oligomerization, ligand binding and membrane interaction properties. Size exclusion chromatography and cross-linking experiments showed that EgAgB8/2 and EgAgB8/3 can self-associate, suggesting that lipids are not required for oligomerization. Furthermore, using fluorescent probes, both subunits were found to bind fatty acids, but not cholesterol analogues. Analysis of fatty acid transfer to phospholipid vesicles demonstrated that EgAgB8/2 and EgAgB8/3 are potentially capable of transferring fatty acids to membranes, and that the efficiency of transfer is dependent on the surface charge of the vesicles.Conclusions/SignificanceWe show that EgAgB apolipoproteins can oligomerize in the absence of lipids, and can bind and transfer fatty acids to phospholipid membranes. Since imported fatty acids are essential for Echinococcus granulosus, these findings provide a mechanism whereby EgAgB could engage in lipid acquisition and/or transport between parasite tissues. These results may therefore indicate vulnerabilities open to targeting by new types of drugs for hydatidosis therapy.

Role of phospholipids of subunit III in the regulation of structural rearrangements in cytochrome c oxidase of Rhodobacter sphaeroides.

Subunit III of cytochrome c oxidase possesses structural domains that contain conserved phospholipid binding sites. Mutations within these domains induce a loss of phospholipid binding, coinciding with decreased electron transfer activity. Functional and structural roles for phospholipids in the enzyme from Rhodobacter sphaeroides have been investigated. Upon the removal of intrinsic lipids using phospholipase A2, electron transfer activity was decreased 30-50%. Moreover, the delipidated enzyme exhibited turnover-induced, suicide inactivation, which was reversed by the addition of exogenous lipids, most specifically by cardiolipin. Cardiolipin exhibited two sites of interaction with the delipidated enzyme, a high-affinity site (Km = 0.14 μM) and a low-affinity site (Km = 26 μM). Subunit I of the delipidated enzyme exhibited a faster digestion rate when it was treated with α-chymotrypsin compared to that of the wild-type enzyme, suggesting that lipid removal induces a conformational change to expose the digestion sites further. Upon reaction of subunit III of the enzyme with a fluorophore (AEDANS), fluorescence anisotropy showed an increased rotational rate of the fluorophore in the absence of lipids, indicating increased flexibility of subunit III within the enzyme's tertiary structure. Additionally, Förster resonance energy transfer between AEDANS and a fluorescently labeled cardiolipin revealed that cardiolipin binds in the v-shaped cleft of subunit III in the delipidated enzyme and that it moves closer to the active site in subunit I upon a change in the redox state of the enzyme. In conclusion, these results show that the phospholipids regulate events occurring during electron transfer activity by maintaining the structural integrity of the enzyme at the active site.

The Role of Lipid in Misfolding and Amyloid Fibril Formation by Apolipoprotein C-II.

Apolipoproteins are a key component of lipid transport in the circulatory system and share a number of structural features that facilitate this role. When bound to lipoprotein particles, these proteins are relatively stable. However, in the absence of lipids they display conformational instability and a propensity to aggregate into amyloid fibrils. Apolipoprotein C-II (apoC-II) is a member of the apolipoprotein family that has been well characterised in terms of its misfolding and aggregation. In the absence of lipid, and at physiological ionic strength and pH, apoC-II readily forms amyloid fibrils with a twisted ribbon-like morphology that are amenable to a range of biophysical and structural analyses. Consistent with its lipid binding function, the misfolding and aggregation of apoC-II are substantially affected by the presence of lipid. Short-chain phospholipids at submicellar concentrations significantly accelerate amyloid formation by inducing a tetrameric form of apoC-II that can nucleate fibril aggregation. Conversely, phospholipid micelles and bilayers inhibit the formation of apoC-II ribbon-type fibrils, but induce slow formation of amyloid with a distinct straight fibril morphology. Our studies of the effects of lipid at each stage of amyloid formation, detailed in this chapter, have revealed complex behaviour dependent on the chemical nature of the lipid molecule, its association state, and the protein:lipid ratio.

The Role of Annular Lipids in the Mechanism of Membrane Protein Solubilization and Formation of Functionally Competent Proteomicelles

The “solubilization” procedure in which membrane proteins are extracted from their native lipid membranes and transferred into detergent micelles (proteomicelles) is a critical step in the purification of the protein for subsequent structure/function studies. In some notable examples it has become clear, however, that the properties of the proteins in proteomicelles depend on the detergent type and its concentration relative to the residual membrane lipids originating from the preparation. To reveal the molecular mechanism of the solubilization process and the effect of various lipid-to-detergent (L-D) ratios on the properties of the system, we performed extensive atomistic molecular dynamics simulations. We previously explored the properties of micelles formed by dodecyl-β-maltoside (DDM) detergent around the leucine transporter (LeuT), a bacterial homolog of the neurotransmitter:sodium symporter family, and showed how the DDM concentration determines the extent of detergent penetration into the functionally relevant secondary substrate (S2) binding site in LeuT. Here we present computational studies of LeuT embedded in proteomicelles at different L-D ratios exploring configurations of LeuT proteomicelles at different stages of the protein solubilization process. Our results reveal that at a sparse lipid annulus around the transporter, consisting of no more than ∼10 lipids, DDM partitions into the LeuT, engaging with the residues in the S2 site. Higher lipid concentrations protect the S2 site from DDM penetration. In contrast to DDM, MNG-3 (lauryl maltose-neopentyl glycol) detergent, an amphiphile known to better stabilize membrane proteins, does not penetrate the transporter even in the absence of lipids. Parallel experimental measurements of ligand binding to LeuT in the presence of increasing MNG-3 concentrations revealed that the functionality of the S2 site was fully preserved even at the highest (0.5%) MNG-3 concentration probed.

Can Lipids be used as Mobility Standards in Artificial Bilayers?

The literature data and results presented in this article show that in lipidic bilayers lipids diffuse with various diffusion coefficients ranging from D = 3.7 ± 0.4 to 13.9 ± 0.6 μm2s-1, Interestingly, a transmembrane peptide, having nearly the same radius as lipids and whose hydrophobic thickness matches that of the bilayer, exhibits a D value of 9.6 ± 0.4 μm2s-1. Since the lipids and the transmembrane peptide possess a similar diameter and lipids do not span the whole membrane, the diffusion coefficient of peptide is expected to be smaller than that of lipids, which is not the case in our experiments. Our systematic study and reinterpretation of literature data suggests that the slower diffusion of lipids, as compared to that of the transmembrane peptide, is caused by the formation of dynamic lipid nanopatches that diffuse like a single object with an increased radius. These nanopatches form more spontaneously when lipids are saturated. Molecular dynamics simulations confirm our experimental results and show that the nanopatches formation also occurs in bilayers made of single lipid in absence of dye.Consequently, one should be cautious when comparing the mobility of transmembrane proteins and lipids. Transmembrane peptides should be used as a reference instead of lipids.

Improved insulin loading in poly(lactic-co-glycolic) acid (PLGA) nanoparticles upon self-assembly with lipids

Polymeric nanoparticles are widely investigated as drug delivery systems for oral administration. However, the hydrophobic nature of many polymers hampers effective loading of the particles with hydrophilic macromolecules such as insulin. Thus, the aim of this work was to improve the loading of insulin into poly(lactic-co-glycolic) acid (PLGA) nanoparticles by pre-assembly with amphiphilic lipids. Insulin was complexed with soybean phosphatidylcholine or sodium caprate by self-assembly and subsequently loaded into PLGA nanoparticles by using the double emulsion-solvent evaporation technique. The nanoparticles were characterized in terms of size, zeta potential, insulin encapsulation efficiency and loading capacity. Upon pre-assembly with lipids, there was an increased distribution of insulin into the organic phase of the emulsion, eventually resulting in significantly enhanced encapsulation efficiencies (90% as compared to 24% in the absence of lipids). Importantly, the insulin loading capacity was increased up to 20% by using the lipid–insulin complexes. The results further showed that a main fraction of the lipid was incorporated into the nanoparticles and remained associated to the polymer during release studies in buffers, whereas insulin was released in a non-complexed form as a burst of approximately 80% of the loaded insulin. In conclusion, the protein load in PLGA nanoparticles can be significantly increased by employing self-assembled protein-lipid complexes.

Abstract 36: Uncovering Lipid-Independent Prothrombinase Function Using Human-Snake Chimeras of Blood Coagulation Factor V

The prothrombinase complex, which converts prothrombin to the key regulatory enzyme thrombin, consists of the serine protease Xa (FXa) and cofactor Va (FVa; A1-A2-A3-C1-C2 domains). The latter dramatically enhances the rate of prothrombin catalysis and exclusively associates with FXa on negatively charged phospholipid surfaces through its lipid-binding C-domains. Interestingly, we have previously shown that the FVa and FXa homologues found in the venom of the Australian snake Pseudonaja textilis bypass the requirement for a membrane surface to achieve complexation. Here we investigated whether the C-domains of venom-derived P. textilis factor V (ptFV) drive its lipid-independent cofactor function and, if so, can sustain this unique property in the setting of human FV. Therefore, we swapped the C-domains of constitutively active B-domainless human FV (hFV) and ptFV and expressed and purified the chimeric variants hFV-ptC and ptFV-hC. Using a purified prothrombinase assay with the lipid-independent substrate prethrombin-1, FV cofactor activity was examined with the corresponding human or venom-derived P. textilis FXa species in the presence or absence of lipids. We hypothesized that the C-domains constrain ptFV in a conformation optimal for lipid-independent cofactor function. Surprisingly, ptFV-hC displayed full cofactor activity, irrespective of the availability of anionic membranes. Similarly, hFV-ptC functioned equivalent to hFV as no gain-of-function in the absence of lipids was observed. Subsequent lipid-titration experiments indicated that whereas both C-domain species promoted maximum rates of substrate conversion at identical phospholipid concentrations, the lipid-affinity of the ptFV C-domains was at least 10-fold reduced as compared to that of hFV. These results demonstrate that the P. textilis C-domains do not play a role in the unique capacity of ptFV to function in the absence of lipids, suggesting that they may be of little relevance to ptFV cofactor function. As such, our findings imply an alternative mode of macromolecular complex assembly for P. textilis venom FVa and FXa and their natural substrate prothrombin, thereby potentially challenging the current paradigm of human prothrombinase assembly and function.

Golgi-Associated plant Pathogenesis Related protein 1 (GAPR-1) forms amyloid-like fibrils by interaction with acidic phospholipids and inhibits Aβ aggregation

Golgi-Associated plant Pathogenesis Related protein 1 (GAPR-1) is a mammalian protein that is a member of the Cysteine-rich secretory proteins, Antigen 5 and Pathogenesis related proteins group 1 (CAP) superfamily of proteins. A role for the common CAP domain in the function of the diverse superfamily members has not been described so far. Here, we show by a combination of independent techniques including electron microscopy, Thioflavin T fluorescence, and circular dichroism that GAPR-1 has the capability to form amyloid-like fibrils in the presence of liposomes containing negatively charged lipids. Surprisingly, GAPR-1 was also shown to bind the amyloid-oligomer specific antibody A11 in the absence of lipids, indicating that GAPR-1 has an intrinsic tendency to form oligomers. This behavior is characteristic for proteins that interfere with Aβ aggregation and indeed we found that GAPR-1 effectively inhibited aggregation of Aβ(1-40) peptide. Immuno-dot blot analysis revealed that GAPR-1 binds to prefibrillar oligomeric Aβ structures during the early stages of fibril formation. Another CAP domain-containing protein, CRISP2, was also capable of forming fibrils, indicating that oligomerization and fibril formation is a shared characteristic between CAP family members. We suggest that the CAP domain may regulate protein oligomerization in a large variety of proteins that define the CAP superfamily.

Effect Of Lipids On The Sequence-Specific Interactions Of Kindlin-3 and Talin-1 With The Cytosolic Tail Of The Integrin β3 Subunit

Activating the platelet integrin αIIbβ3 is an essential step for primary hemostasis. Physiologic αIIbβ3 activation occurs when platelet agonist-generated inside-out signals induce binding of the FERM domains of the cytosolic proteins talin-1 and kindlin-3 to the cytosolic tail (CT) of the β3 subunit of αIIbβ3. While talin-1 binding is thought to activate αIIbβ3 by physically causing separation of the αIIb and β3 cytosolic and transmembrane domains, αIIbβ3 activation in platelets does not occur in the absence of kindlin-3 binding to the β3 CT. Nonetheless, it is unclear whether it is necessary for talin-1 and kindlin-3 to be concurrently bound to the β3 CT in order to activate αIIbβ3, and if that is the case, whether there is a preferred order of binding and whether binding is cooperative. It is noteworthy in this regard that the sequences of the core binding motifs on the β3 CT for the talin-1 and kindlin-3 FERM domains, N744PLY747 and N756ITY759, respectively, are quite similar. To begin to address these questions, we have expressed and purified recombinant forms of the integrin-binding talin-1 head domain (THD) and full-length kindlin-3 and measured their interaction with a peptide corresponding to the β3 CT by surface plasmon resonance (SPR). For these experiments, the β3 CT was anchored to the dextran matrix of a CM5 SPR sensor chip and the equilibrium kinetics of THD and kindlin-3 binding was measured. Analysis of the THD binding data was compatible with two classes of binding sites, a high affinity site with a Kd of 155 nM and a low affinity site with a Kd of 3.5 µM. Similar analysis of kindlin-3 binding was also consistent with two classes of binding sites, a high affinity site with a Kd of 5 nM and a lower affinity site with a Kd of 2.2 µM. Next, we tested the effect of mutating the core binding motifs for the THD and kindlin-3 on the β3 CT. We found that replacing Y759 in the core kindlin-3 binding motif with Ala eliminated high affinity kindlin-3 binding, whereas replacing Y747 in the core THD binding motif with Ala eliminated low affinity kindlin-3 binding. Conversely, the Y747A replacement eliminated high affinity THD binding, while the Y759A replacement eliminated low affinity THD binding. Thus, these experiments demonstrate that the talin-1 and kindlin-3 FERM domains each recognize the general NXXY motif, but their high affinity interactions with this motif are highly sequence-specific. Previously, we found that appending the β3 CT to acidic phospholipids increased its affinity for the THD by three orders of magnitude, likely through interactions involving an extended positively-charged surface on the THD F2 and F3 subdomains. Further, kindlins contain a pleckstrin homology domain with a conserved lipid-binding loop that has been found to be essential for αIIbβ3 activation. Accordingly, we investigated the effect on THD and kindlin-3 binding of tethering the β3 CT to DOPC-coated L1 SPR chips. Unexpectedly, we found that when the β3 CT was tethered to lipid, the Kd for THD binding increased to 430 nM, comparable to the Kd we previously found using isothermal titration calorimetry for THD binding to the β3 CT appended to liposomes. We also found that kindlin-3 binding to the β3 CT tethered to lipids was unexpectedly weaker than binding in the absence of lipid, but it remained approximately 3-fold stronger than THD binding under the same conditions. Previous NMR and hydrogen-deuterium exchange studies of the β3 CT appended to liposomes have revealed that the regions of the β3 CT containing the THD and kindlin-3 binding sites consist of two dynamic amphiphilic helices that are stabilized by interacting with lipid bilayers. Thus, the results presented here suggest that the folding of the β3 CT and the interaction of the folded structure with lipids are important determinants of the strength of the interaction of the THD and kindlin-3 with the β3 CT and consequently are important factors in the regulation of αIIbβ3 activation. Disclosures:No relevant conflicts of interest to declare.

PP58 - Preventive effect of Ellagic acid on blood pressure, oxidative stress and cardiac remodelling in L-NAME induced hypertensive rats

oxidised by H2O2 to (Fe 4þ ) to form perferrylHb and ferrylHb, with the later also bearing a radical on its protein. The relationship between the structural properties of silybin and its derivatives and their ability to prevent oxidation of Hb was investigated in model system in the presence or the absence of lipids. The antioxidant activities of silybin, dehydrosilybin, 23-O-butanoyl and 23-O-palmitoyl silybin derivatives were correlated with their interaction with Hb species. Results are discussed in relation to the potential of dehydrosilybin, silybin and C4 and C16 derivates to prevent activation of Hb to hypevalent heme protein species.

Effect of amino acids on the formation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in creatinine/phenylalanine and creatinine/phenylalanine/4-oxo-2-nonenal reaction mixtures

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) formation in mixtures of creatinine, phenylalanine, amino acids and 4-oxo-2-nonenal was studied, to analyse the role of amino acids on the generation of this heterocyclic aromatic amine. When oxidised lipid was absent, cysteine, serine, aspartic acid, threonine, asparagine, tryptophan, tyrosine, proline, and methionine increased significantly (p < 0.05) the amount of PhIP formed in comparison to the control. When lipid was present, only the addition of methionine, glycine, and serine increased significantly (p < 0.05) the amount of PhIP produced, while histidine, cysteine, lysine, tryptophan, tyrosine, and alanine reduced significantly (p < 0.05) PhIP. These results may be a consequence of the different competitive reactions that occur. Thus, in the absence of lipids, thermal decomposition of the amino acids produced reactive carbonyls that converted phenylalanine into phenylacetaldehyde as a key step in the formation of PhIP. When oxidised lipid was present, amino acids competed with phenylalanine for the lipid, and amino acid degradation products were formed, among which alpha-keto acids seemed to play a role in these reactions. These results suggest that PhIP can be produced by several alternative reaction pathways from all major food components, including amino acids and lipids, in addition to carbohydrates.

Effect of oxidation in the presence or absence of lipids on hazelnut and soybean protein detectability by commercial ELISA

Soybean and hazelnut represent major food allergens. Their inadvertent presence in foods might pose a serious threat to allergic consumers. Therefore, reliable detection methods are needed. Commercial enzyme-linked immunosorbent assays (ELISA) are the preferred methods especially by the food industry and enforcement agencies for the detection of contamination levels of food allergens. However, they are susceptible to erroneous results due to the modification of the allergens (proteins) during food processing. In this paper, the impact of protein oxidation on the detectability of soybean and hazelnut was investigated. Lipid-induced oxidation did not have a great impact on the detectability while the more severe hypochlorous acid-induced oxidation led to dramatic decrease in detectability for both hazelnut and soybeans. The observed impact was highly dependent on the type of ELISA kit used.

Novicidin's membrane permeabilizing activity is driven by membrane partitioning but not by helicity: A biophysical study of the impact of lipid charge and cholesterol

We have investigated the interactions between the antimicrobial peptide Novicidin (Nc) and vesicles containing the phospholipid DOPC, with various amounts of DOPG and cholesterol using circular dichroism spectroscopy, calcein release, equilibrium dialysis and isothermal titration calorimetry. Nc adopts a random coil structure in the absence of lipids and in the presence of vesicles containing 100% DOPC. Lipids with 25–40% DOPG induce the highest level of helicity in Nc; higher DOPG levels lead to lower helicity levels and an altered tertiary arrangement of the peptide. However, the ability of Nc to permeabilize vesicles correlates not with helicity but rather with its overall membrane affinity, which is enthalpically favorable but opposed by entropy. Permeabilization declines with increasing mole percentage PG. Changes in helicity correlate with changes in enthalpy, reflecting the enthalpy of helix formation, but not with affinity. There is also a large favorable enthalpic interaction between Nc and lipids in the absence of negative charge and structural changes. Cholesterol slightly reduces membrane permeabilization but has little effect on Nc affinity and secondary structure, and probably protects the membrane by inducing the liquid ordered state. We conclude that helicity is not a prerequisite for activity, and charge–charge interactions are not the only major driving force for AMP interactions with membranes. Our data are compatible with a model in which a superficial binding mode with a large membrane surface binding area per peptide is more efficient than a more intimate embedding within the membrane environment.

The potential of apolipoprotein E4 to act as a substrate for primary cultures of hippocampal neurons

The E4 isoform of apolipoprotein (apoE4) is known to be a major risk factor for Alzheimer's Disease (AD). Previous in vitro studies have shown apoE4 to have a negative effect on neuronal outgrowth when incubated with lipids. The effect of apoE4 itself on the development of neurons from the central nervous system (CNS), however, has not been well characterized. Consequently, apoE4 alone has not been pursued as a substrate for neuronal cultures. In this study, the effect of surface-bound apoE4 on developmental features of rat hippocampal neurons was examined. We show that apoE4 substrates elicit significantly enhanced values in all developmental features at day 2 of culture when compared to laminin (LN) substrates, which is the current substrate-of-choice for neuronal cultures. Interestingly, the adhesion of hippocampal neurons was found to be significantly lower on LN substrates than on glass substrates, but the axon lengths on both substrates were similar. In addition, this study demonstrates that the adhesion- and growth-enhancing effects of apoE4 substrates are not mediated by heparan sulfate proteoglycans (HSPGs), proteins that have been indicated to function as receptors or co-receptors for apoE4. In the absence of lipids, apoE4 appears to use an unknown pathway for up-regulating neuronal adhesion and neurite outgrowth. Our results indicate that apoE4 is better than LN as a substrate for primary cultures of CNS neurons and should be considered in the design of tissue engineered CNS.

Alpha-Synuclein, an Intrinsically Unstructured Protein. How Interesting Can It Be?

N-terminal acetylation of alpha-synuclein (aS), a 140-residue protein implicated in the etiology of Parkinson's disease, is common in mammals. The impact of this modification on the protein's structure and dynamics in free solution and on its membrane binding properties has been evaluated by both NMR and CD spectroscopy. While in contrast to literature reports, no tetrameric form of acetylated aS could be isolated, N-terminal acetylation resulted in ca 15% transient population of alpha-helical structure for its first six residues. The 1H, 15N, and 13C chemical shifts for residues 13-140 remain unaffected by acetylation. Nevertheless, a substantial increase in affinity of aS for negatively charged lipid membranes is observed, likely to be of strong functional significance. A new method for residue-specific NMR probing of lipid binding is demonstrated for aS and assigns a new putative function to this enigmatic protein. Although free aS in the absence of lipids has backbone chemical shifts that are exceptionally close to random coil values, considerable positional variation for the distribution of its backbone torsion angles and the time scale of local reorientation can be deduced from its 3JHH and NOE data.

Replacement of Arg-386 with Gly in Dynamin 1 Middle Domain Reduced GTPase Activity and Oligomer Stability in the Absence of Lipids

Dynamin plays an important role in membrane fission during endocytosis, and its middle domain is involved in the formation of functional oligomers. In this study, we found that replacement of Arg-386 with Gly in the middle domain region of dynamin 1 did not affect the intermolecular interactions of dynamin 1 in the presence of phosphatidylserine-liposomes. But, unexpectedly, this variant showed lower guanosine 5'-triphosphatase activity in the absence of phosphatidylserine-liposomes and enhanced monomer formation from oligomers. Our results indicate that GTPase activity in the absence of lipids is important in the dissociation of oligomer complexes, i.e., reduced basal dynamin 1 GTPase activity is associated with instability of dynamin oligomers.

Magnetic Resonance Spectroscopy for Differentiating Pseudo-Progression from True Progression in GBM on Concurrent Chemoradiotherapy

Neoadjuvant chemo-radiation therapy including temozolomide is commonly used for the treatment of gliomas. However, increased lesion size and contrast enhancement are frequently observed following this therapy and this appearance is termed as 'pseudo-progression'. Since conventional imaging is unable to differentiate pseudo-progression from tumour recurrence, we evaluated the utility of MR spectroscopy (MRS) to differentiate these two pathological entities. Longitudinal MRI and MRS studies prior to and within four months post chemo-radiation therapy including diffusion-weighted imaging and single voxel spectroscopy (short and intermediate echo) were performed in 62 glioblastoma (GBM) patients undergoing chemo-radiation therapy. Clinical follow-up demonstrated four cases of pseudo-progression. In this study, results from these four cases and a known case of tumour recurrence are reported. Metabolite ratios and presence or absence of lipids at 1.3 ppm were used to differentiate between pseudo-progression and tumour recurrence. All four cases of pseudo-progression demonstrated elevated lipid signals on MRS. Additionally, an absence of choline or a low choline/NAA ratio was also observed. In comparison, the patient with tumour recurrence showed lower lipid signals and a high choline/NAA ratio. The presence of elevated lipid signals along with low choline/NAA ratios can aid in differentiation of pseudo-progression from tumour recurrence.

Amphipol-assisted folding of bacteriorhodopsin in the presence or absence of lipids: functional consequences

Amphipols are short amphipathic polymers designed to stabilize membrane proteins in aqueous solutions in the absence of detergent. Bacteriorhodopsin (BR), a light-driven proton pump, has been denatured, either by direct solubilization of the purple membrane in sodium dodecylsulfate (SDS) solution or by a procedure that involves delipidation with organic solvent followed by transfer to SDS, and renatured in amphipol A8-35. The effect of different renaturation procedures and of the presence or absence of lipids and the cofactor retinal have been investigated. The resulting samples have been characterized by absorbance spectroscopy, size-exclusion chromatography, thermostability measurements, and determination of photocycle kinetics. Transfer to A8-35 can be achieved by SDS precipitation, dilution, or dialysis, the first route resulting in the highest yield of refolding. Functional BR can be refolded whether in the presence or absence of lipids, higher yields being achieved in their presence. Retinal is not required for the protein to refold, but it stabilizes the refolded form and, thereby, improves folding yields. Lipids are not required for BR to perform its complete photocycle, but their presence speeds up the return to the ground state. Taken together, these data indicate that a membrane or membrane-mimetic environment is not required for correct decoding of the chemical information contained in the sequence of BR; functional folding is possible even in the highly foreign environment of lipid-free amphipols. BR interactions with lipids, however, contribute to an effective photocycle.

Haptoglobin Binding Stabilizes Hemoglobin Ferryl Iron and the Globin Radical on Tyrosine β145

Hemoglobin (Hb) becomes toxic when released from the erythrocyte. The acute phase protein haptoglobin (Hp) binds avidly to Hb and decreases oxidative damage to Hb itself and to the surrounding proteins and lipids. However, the molecular mechanism underpinning Hp protection is to date unclear. The aim of this study was to use electron paramagnetic resonance (EPR) spectroscopy, stopped flow optical spectrophotometry, and site-directed mutagenesis to explore the mechanism and specifically the role of specific tyrosine residues in this protection. Following peroxide challenge Hb produces reactive oxidative intermediates in the form of ferryl heme and globin free radicals. Hp binding increases the steady state level of ferryl formation during Hb-catalyzed lipid peroxidation, while at the same time dramatically inhibiting the overall reaction rate. This enhanced ferryl stability is also seen in the absence of lipids and in the presence of external reductants. Hp binding is not accompanied by a decrease in the pK of ferryl protonation; the protonated ferryl species still forms, but is intrinsically less reactive. Ferryl stabilization is accompanied by a significant increase in the concentration of the peroxide-induced tyrosine free radical. EPR spectral parameters and mutagenesis studies suggest that this radical is located on tyrosine 145, the penultimate C-terminal amino acid on the beta Hb subunit. Hp binding decreases both the ferryl iron and free radical reactivity of Hb. Hp protects against Hb-induced damage in the vasculature, not by preventing the primary reactivity of heme oxidants, but by rendering the resultant protein products less damaging.

Immunoreactive Hephaestin and ferroxidase activity are present in the cytosolic fraction of rat enterocytes

Discovered over a decade ago, hephaestin (Heph) has been implicated as a ferroxidase (FOX) vital for intestinal iron absorption. Stringent structural or kinetic data derived from purified, native protein is however lacking, leading to the hypothesis that an alternate, undiscovered form of Heph could exist in mammalian enterocytes. This possibility was tested using laboratory rodent and cell culture models. Cytosolic and membrane fractions were obtained from rat enterocytes and purity of the fractions was assessed. Western blot analyses revealed Heph in cytosol obtained by three different methods, ruling out the possibility of a method-induced artifact being the major contributor to this observation. Absence of two different membrane-proteins, ferroportin 1 and Menke's copper ATPase in cytosol, and the absence of lipids in representative cytosolic samples tested by thin layer chromatography, eliminated significant membrane contamination of cytosol. Further, immunohisto- and immunocyto-chemical analyses identified Heph in rat enterocytes and in two intestinal epithelial cell lines, IEC-6 and Caco-2, intracellularly. Additionally, cytosolic Heph increased upon iron-deprivation but more important, decreased significantly upon copper-deprivation, mimicking the response of membrane-bound Heph. Moreover, FOX activity was present in rat cytosol, and was partly inhibited by anti-Heph antibody. Finally, lack of immunodetectable ceruloplasmin (Cp) by western blot precluded Cp as an underlying cause of this activity. These data demonstrate that rat enterocytes contain a soluble/cytosolic form of Heph possibly contributing to the observed FOX activity.