Importance of phospholipid bilayer integrity in the analysis of protein–lipid interactions

The integrity of supported phospholipid bilayer membranes is of crucial importance for the investigation of lipid–protein interactions. Therefore we recorded the formation of supported membranes on SiO₂ and mica by quartz crystal microbalance and controlled the integrity by atomic force microscopy. This study aims to analyze how membrane defects affect protein–lipid interactions. The experiments focused on a lipid mixture of POPC/DOPC/Chol/POPS/PI(4,5)P₂ (37:20:20:20:3) and the binding of the peripheral membrane associated protein annexin A2. We found that formation of a continuous undisturbed bilayer is an indispensable precondition for a reliable determination and quantification of lipid–protein-interactions. If membrane defects were present, protein adsorption causes membrane disruption and lipid detachment on a support thus leading to false determination of binding constants. Our results obtained for PI(4,5)P₂ and cholesterol containing supported membranes yield new knowledge to construct functional surfaces that may cover nanoporous substrates, form free standing membranes or may be used for lab-on-a-chip applications.     

Influence of HSA and IgG on LDL oxidation studied by size-exclusion chromatography and phospholipid profiling using MALDI tandem-mass spectrometry

In the present study a direct detection approach combining size-exclusion chromatography (SEC) and matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight tandem-mass spectrometry (MALDI-QIT-TOF-MS/MS) was applied to investigate the influence of HSA and IgG on LDL oxidation in vitro. SEC analysis showed an increase of protein aggregation during LDL-oxidation that could be essentially suppressed in the presence of HSA. In parallel, lipid peroxidation measured by TBARS assay over 24 h was inhibited by 95–100% in the presence of HSA but only 0–34% by IgG, respectively. MALDI phospholipid profiles showed considerable decrease of signals from PCs containing sn-2 PUFAs (18:2 or 20:4) accompanied by increase of sn-2 LPCs indicating for specific breakdown of PUFA-containing PLs during LDL-oxidation. These effects were nearly 100% inhibited in the presence of HSA but not by IgG, respectively. Among known pro-atherogenic PL species present in human plasma sphingomyelin (SM16:0) was bound in significant amounts to HSA but not IgG after incubation with oxLDL. Moreover, our investigation showed that LPCs containing SAFAs (16:0 or 18:0) were specifically bound to HSA, while those containing PUFAs (18:2 and 18:3) were preferentially associated with IgG. In summary, the presented methodology provides a promising platform for studying lipid–protein interactions in vivo.     

Biophysical and functional assays for viral membrane fusion peptides

Membrane fusion is a protein catalyzed biophysical reaction that involves the simultaneous intermixing of two phospholipid bilayers and of the aqueous compartments bound by their respective bilayers. In the case of enveloped virus fusogens, short hydrophobic or amphipathic fusion peptides that are components of the larger fusion complex are essential for the membrane merger event. The process of cell–cell membrane fusion and syncytium formation induced by the nonenveloped fusogenic orthoreoviruses is driven by the Fusion-Associated Small Transmembrane (FAST) proteins, which are similarly dependent on the action of fusion peptides. In this article, we describe some simple methods for the biophysical characterization of viral membrane fusion peptides. Liposomes serve as an ideal model system for characterizing peptide–membrane interactions because their size, shape and composition can be readily manipulated. We present details of fluorescence assays used to elucidate the kinetics of membrane fusion as well as complimentary assays used to characterize peptide-induced liposome binding and aggregation.     

High mannose-specific lectin (KAA-2) from the red alga Kappaphycus alvarezii potently inhibits influenza virus infection in a strain-independent manner

The carbohydrate binding profile of the red algal lectin KAA-2 from Kappaphycus alvarezii was evaluated by a centrifugal ultrafiltration–HPLC method using pyridylaminated oligosaccharides. KAA-2 bound exclusively to high mannose type N-glycans, but not to other glycans such as complex type, hybrid type, or the pentasaccharide core of N-glycans. This lectin exhibited a preference for an exposed α1–3 Man on a D2 arm in a similar manner to Eucheuma serra agglutinin (ESA-2), which shows various biological activities, such as anti-HIV and anti-carcinogenic activity. We tested the anti-influenza virus activity of KAA-2 against various strains including the recent pandemic H1N1-2009 influenza virus. KAA-2 inhibited infection of various influenza strains with EC₅₀s of low nanomolar levels. Immunofluorescence microscopy using an anti-influenza antibody demonstrated that the antiviral activity of KAA-2 was exerted by interference with virus entry into host cells. This mechanism was further confirmed by the evidence of direct binding of KAA-2 to a viral envelope protein, hemagglutinin (HA), using an ELISA assay. These results indicate that this lectin would be useful as a novel antiviral reagent for the prevention of infection.     

Validity and applicability of membrane model systems for studying interactions of peripheral membrane proteins with lipids

The cell membrane serves, at the same time, both as a barrier that segregates as well as a functional layer that facilitates selective communication. It is characterized as much by the complexity of its components as by the myriad of signaling process that it supports. And, herein lays the problems in its study and understanding of its behavior — it has a complex and dynamic nature that is further entangled by the fact that many events are both temporal and transient in their nature. Model membrane systems that bypass cellular complexity and compositional diversity have tremendously accelerated our understanding of the mechanisms and biological consequences of lipid–lipid and protein–lipid interactions. Concurrently, in some cases, the validity and applicability of model membrane systems are tarnished by inherent methodical limitations as well as undefined quality criteria. In this review we introduce membrane model systems widely used to study protein–lipid interactions in the context of key parameters of the membrane that govern lipid availability for peripheral membrane proteins. This article is part of a Special Issue entitled Tools to study lipid functions.     

A novel small molecule deubiquitinase inhibitor blocks Jak2 signaling through Jak2 ubiquitination

AG490 is a tyrosine kinase inhibitor with activity against Jak2 and apoptotic activity in specific leukemias. Due to its weak kinase inhibitory activity and poor pharmacology, we conducted a cell-based screen for derivatives with improved Jak2 inhibition and activity in animals. Two hits emerged from an initial small chemical library screen, and more detailed structure–activity relationship studies led to the development of WP1130 with 50-fold greater activity in suppressing Jak2-dependent cytokine signaling than AG490. However, WP1130 did not directly suppress Jak2 kinase activity, but mediated Jak2 ubiquitination resulting in its trafficking through HDAC6 to perinuclear aggresomes without cytokine stimulation or SOCS-1 induction. Jak2 primarily contained K63-linked ubiquitin polymers, and mutation of this lysine blocked Jak2 ubiquitination and mobilization in WP1130-treated cells. Further analysis demonstrated that WP1130, but not AG490, acts as a deubiquitinating enzyme (DUB) inhibitor, possibly through a Michael addition reaction. We conclude that chemical modification of AG490 resulted in development of a DUB inhibitor with activity against a DUB capable of modulating Jak2 ubiquitination, trafficking and signal transduction.     

Structure of the E-1-hydroxy-2-methyl-but-2-enyl-4-diphosphate synthase (GcpE) from Thermus thermophilus

Isoprenoids are biosynthesized via the mevalonate or the 2-C-methyl-d-erythritol-4-phosphate (MEP) pathways the latter being used by most pathogenic bacteria, some parasitic protozoa, plant plastids, but not by animals. We determined the X-ray structure of the homodimeric [4Fe–4S] cluster carrying E-1-hydroxy-2-methyl-but-2-enyl-4-diphosphate synthase (GcpE) of Thermus thermophilus which catalyzes the penultimate reaction of the MEP pathway and is therefore an attractive target for drug development. The [4Fe–4S] cluster ligated to three cysteines and one glutamate is encapsulated at the intersubunit interface. The substrate binding site lies in front of an (αβ)₈ barrel. The great [4Fe–4S] cluster-substrate distance implicates large-scale domain rearrangements during the reaction cycle.

Structured summary

gcpEbinds to gcpE by x-ray crystallography (View interaction)     

Tacrine(2)-ferulic acid, a novel multifunctional dimer, attenuates 6-hydroxydopamine-induced apoptosis in PC12 cells by activating Akt pathway

Oxidative stress is closely related to the pathogenesis of neurodegenerative disorders such as Parkinson’s disease (PD). In this study, we investigated the neuroprotective effect of tacrine–ferulic acid dimers linked by an alkylenediamine side chain (TnFA, n = 2−7), a series of novel acetylcholinesterase inhibitors, against 6-hydroxydopamine (6-OHDA)-induced apoptosis in PC12 cells. Among these dimers, pre-treatment of tacrine(2)–ferulic acid (T2FA, 3−30 μM) attenuated 6-OHDA-induced apoptosis in a concentration-dependent manner. The activations of glycogen synthase kinase 3β (GSK3β) and extracellular signal-regulated kinase (ERK) were observed after the treatment of 6-OHDA. Both SB415286 (an inhibitor of GSK3β) and PD98059 (an inhibitor of ERK kinase) reduced the neurotoxicity induced by 6-OHDA, indicating that GSK3β and ERK are involved in 6-OHDA-induced apoptosis. T2FA was able to inhibit the activation of GSK3β, but not ERK, in an Akt-dependent manner. Furthermore, LY294002, a phosphoinositide 3-kinase inhibitor, abolished the neuroprotective effect of T2FA. Collectively, these results suggest that T2FA prevents 6-OHDA-induced apoptosis possibly by activating the Akt pathway in PC12 cells.     

Ubiquitin chains in the Dsk2 UBL domain mediate Dsk2 stability and protein degradation in yeast

Ubiquitin-like (UBL)–ubiquitin-associated (UBA) proteins, including Dsk2 and Rad23, act as delivery factors that target polyubiquitinated substrates to the proteasome. We report here that the Dsk2 UBL domain is ubiquitinated in yeast cells and that Dsk2 ubiquitination of the UBL domain is involved in Dsk2 stability, depending on the Dsk2 UBA domain. Also, Dsk2 lacking ubiquitin chains impaired ubiquitin-dependent protein degradation and decreased the interaction of Dsk2 with polyubiquitinated proteins in cells. Moreover, Dsk2 ubiquitination affected ability to restore the temperature-sensitive growth defect of dsk2Δ. These results indicate that ubiquitination in the UBL domain of Dsk2 has in vivo functions in the ubiquitin–proteasome pathway in yeast.     

Brominated aliphatic hydrocarbons and sterols from the sponge Xestospongia testudinaria with their bioactivities

Four brominated aliphatic hydrocarbons (1–4), including a novel brominated ene-tetrahydrofuran named as mutafuran H (1), and five sterols (5–9) were isolated from the South China Sea sponge Xestospongia testudinaria. The structure of 1 was determined on the basis of NMR (¹H, ¹³C NMR, HSQC, HMBC, ¹H–¹H COSY, and NOESY), MS, and optical rotation analysis. Known compounds were identified by comparison of their NMR data with those reported in the literature. Compounds 1–4, and 6–9 were evaluated for their toxicity against Artemia salina larvae, and anti-acetylcholinesterase activity.     

A novel 2,6-diisopropylphenyl-docosahexaenoamide conjugate induces apoptosis in T cell acute lymphoblastic leukemia cell lines

We have previously characterized the effects of 2,6-diisopropylphenyl–docosahexaenoamide (DIP–DHA) conjugates and their analogs on the proliferation and progression of breast cancer cell lines. For this study, we investigated the effects of the DIP–DHA conjugate on 2 representative T cell acute lymphoblastic leukemia (T-ALL) cell lines: CEM and Jurkat. Treatment of both cell lines with DIP–DHA resulted in significantly greater inhibition of proliferation and induction of apoptosis than that of parent compounds, 2,6-diisopropylphenol (DIP) or docosahexaenoate (DHA). Treatment of the cells with DIP–DHA resulted in increased activation of caspase-3, and caspase-7. Furthermore, induction of apoptosis in both cell lines was reversed in the presence of a caspase family inhibitor. Treatment with DIP–DHA reduced mitochondrial membrane potential. These observations suggest that the effects are driven by intrinsic apoptotic pathways. DIP–DHA treatment also downregulated surface CXCR4 expression, an important chemokine receptor involved in cancer metastasis that is highly expressed in both CEM and Jurkat cells. In conclusion, our data suggest that the DIP–DHA conjugate exhibits significantly more potent effects on CEM and Jurkat cells than that of DIP or DHA alone. These conjugates have potential use for treatment of patients with T cell acute lymphoblastic leukemia.     

Comprehensive trapping of polyubiquitinated proteins using the UIM peptide of ASB2a

An alternative splicing variant of E3 ubiquitin ligase ASB2, termed ASB2a, has a distinct N-terminal sequence containing a ubiquitin-interacting motif (UIM) consensus sequence. Examination of the minimal essential region for binding to polyubiquitinated proteins indicated that the UIM consensus sequence (residues 26–41) alone is not enough, and that amino acids 12–41 from the N-terminus of ASB2a is essential for binding. ASB2a(12–41) peptide was chemically synthesized and coupled to Sepharose 4B via disulfide bonds. This ASB2a(12–41) peptide-coupled affinity resin bound both K48- and K63-linked polyubiquitinated proteins in cell lysates and comprehensively captured polyubiquitinated proteins, including polyubiquitinated β-catenin, I-κB, and EGF receptor, which were eluted with 2-mercaptoethanol under non-denaturing conditions. These results indicate that this UIM affinity purification (designated as ubiquitin-trapping) is a useful method to discover polyubiquitinated proteins and their associated proteins.     

α-Linolenate reduces the dietary requirement for linoleate in the growing rat

Background: We hypothesized that due to the absence of a dietary source of omega-3 fatty acids, the essential fatty acid (EFA) deficiency model leads to an overestimate of linoleic acid (LA) requirements. Methods: over 7 wk, young rats consumed an EFA diet containing either 0 en% linoleate (0LA) and 0 en% α-linolenate (0LNA) or a diet containing 0.5 en% LNA plus one of seven levels of added LA (0.12–4.0 en%; n=6/group).Results: Rats consuming the 0LA–0LNA diet had the lowest final body weight, 34–68% lower LA and arachidonate in plasma and liver, 87% lower LA in epididymal fat, and an 8–20 fold higher eicosatrienoate in plasma, liver and muscle lipids. 0.5LNA completely prevented the lower growth and partly prevented the rise in eicosatrienoate seen in the 0LA–0LNA group.Conclusion: Providing dietary LNA at 0.5 en% reduces the rat's physiological requirement for LA by an estimated factor of at least four (0.5 en% instead of 2 en%). Since LA requirements in humans are also based on the same flawed model of EFA deficiency, it is plausible that they too have been overestimated and should therefore be reinvestigated.     

Crystal structure of the middle domain of human poly(A)-binding protein-interacting protein 1

This communication describes SAXS data based global structures of tetravalent antibody CD4–IgG2 and its dimeric to pentameric complexes with gp120s. Comparison of models brought forth that while the two CD4s grafted on each arm remain tightly packed in the unliganded antibody, they enable binding of first two gp120s preferentially to the same Fab arm in an asymmetric manner. Retention of residues in the CD4–Fab linker earlier reasoned to enable bi-fold collapse of gp120-bound soluble CD4, and observed asymmetry of the (CD4–IgG2)/(gp120)₂ complex suggest that encoded flexibility in CD4–Fab linker is a critical structure–function factor for this broad spectrum neutralizing antibody.     

Ibuprofen and warfarin modulate allosterically ferrous human serum heme-albumin nitrosylation

Ferrous human serum heme–albumin (HSA–heme–Fe(II)) displays globin-like properties. Here, the effect of ibuprofen and warfarin on kinetics of HSA–heme–Fe(II) nitrosylation is reported. Values of the second-order rate constant for HSA–heme–Fe(II) nitrosylation (k_on) decrease from 6.3 × 10⁶ M⁻¹ s⁻¹ in the absence of drugs, to 4.1 × 10⁵ M⁻¹ s⁻¹ and 4.8 × 10⁵ M⁻¹ s⁻¹, in the presence of saturating amounts of ibuprofen and warfarin, respectively, at pH 7.0 and 20.0 °C. From the dependence of k_on on the drug concentration, values of the dissociation equilibrium constant for ibuprofen and warfarin binding to HSA–heme–Fe(II) (i.e., K = 3.2 × 10⁻³ M and 2.6 × 10⁻⁴ M, respectively) were determined. The observed allosteric effects could indeed reflect ibuprofen and warfarin binding to the regulatory fatty acid binding site FA2, which brings about an alteration of heme coordination, slowing down HSA–heme–Fe(II) nitrosylation. Present data highlight the allosteric modulation of HSA–heme–Fe(II) reactivity by heterotropic effectors.     

IFN-γ+874 A/T polymorphism and cancer risk: an updated analysis based on 32 case-control studies

IFN-γ is a T-helper 1 cytokine and plays important roles in modulating almost all the immune responses, such as hematopoiesis, T-cell differentiation, antiproliferative, antiviral, and antitumor activities. A single nucleotide polymorphism (+874A/T) which is located in the first intron of the human IFN-γ gene can putatively influence the secretion of IFN-γ. Results from previous studies on the association of +874A/T polymorphism with different cancer types remained contradictory. In order to derive a more precise estimation of the relationship, a meta-analysis was performed by searching PubMed, Embase, CNKI, and Chinese Biomedicine Database. Thirty two studies including 4524 cases and 5684 controls were collected for IFN-γ + 874 A/T polymorphism. Summary odds ratios (OR) and corresponding 95% confidence intervals (CIs) for IFN-γ polymorphism and cancer were estimated using fixed- and random-effects models when appropriate. Overall, no evidence indicated that individuals carrying TT or AT genotypes had significantly increased cancer risk when compared with AA genotype carriers. However, stratified analysis by cancer types indicated a significantly increased risk of breast cancer (TT vs AA: OR = 1.58, 95% CI = 1.10–2.27, P_{heterogeneity} = 0.26; TT vs AT/AA: OR = 1.53, 95%CI = 1.14–2.06, P_{heterogeneity} = 0.19). Moreover, significantly elevated risks were observed in African and European populations when population is concerned. Interestingly, when stratified separately by population-based studies and hospital-based studies, significantly elevated risk was found among population-based studies. This meta-analysis suggests that the IFN-γ + 874 T allele is a low-penetrant risk factor for cancer development.     

Disclosing the distinct interfacial behaviors of structurally and configurationally diverse triazologlycolipids

1- or 6-Triazologluco- and galactolipid derivatives bearing a lipid chain length of 16 carbons were efficiently constructed via click chemistry. The differentiation in their surface pressure-molecular area (π–A) isotherms first implies that these structurally and configurationally diverse amphiphiles adopt different distribution manner at air–water interfaces. The Langmuir–Blodgett (LB) films of the synthesized glycoconjugates on mica surface were subsequently prepared and visualized via atomic force microscopy (AFM), which exhibited diverse topographies and possess different contact angles with water. These data further suggest that the structural variation as well as epimeric identity of triazologlycolipids may result in their distinct interfacial behaviors at the air–solid interface. Furthermore, the addition of increasing amounts of 1-triazologalactolipid 2 to poly-diacetylene (PDA) was determined to impact the π–A isotherm of the latter, prompting us to further fabricate new colorimetrically detectable mixed-type vesicles containing triazologlycolipids for biochemical studies.     

The trend of BMI values of US adults by deciles, birth cohorts 1882–1986 stratified by gender and ethnicity

We estimate trends in BMI values by deciles of the US adult population by birth cohorts 1882–1986 stratified by ethnicity and gender. The highest decile increased by some 18–22 BMI units in the course of the century while the lowest ones increased by merely 1–3 BMI units. For example, a typical African American woman in the 10th percentile and 64 in. (162.6 cm) tall increased in weight by just 12 pounds (5 kg) whereas in the 90th percentile her weight would have increased by 128 pounds (58 kg). Hence, the BMI distribution became increasingly right skewed as the distance between the deciles increased considerably. The rate of change of the BMI decile curves varied greatly over time and across gender and ethnicity. The BMI deciles of white men and women experienced upswings after the two world wars and downswings during the Great Depression and also decelerated after 1970. However, among African Americans the pattern is different during the first half of the century with men's rate of increase in BMI values decreasing substantially and that of females remaining constant at a relatively high level until the Second World War. After the war, though, the rate of change of BMI values of blacks came to resemble that of whites with an accelerating phase followed by a slowdown around the 1970s.     

The chromophore structure of the long-lived intermediate of the C128T channelrhodopsin-2 variant

The photocycle of the light-activated channel, channelrhodopsin-2 C128T, has been studied by resonance Raman (RR) spectroscopy focussing on the intermediates P380 and P353 that constitute a side pathway in the recovery of the parent state. The P353 species displays a UV–vis absorption spectrum with a fine-structure reminiscent of the reduced-retro form of bacteriorhodopsin, whereas the respective RR spectra differ substantially. Instead, the RR spectra of the P380/P353 intermediate couple are closely related to that of a free retinal in the all-trans configuration. These findings imply that the parent state recovery via P380/P353 involves the transient hydrolysis and re-formation of the retinal–protein linkage.