The role of brain-derived neurotrophic factor in mouse oocyte maturation in vitro involves activation of protein kinase B

Brain-derived neurotrophic factor (BDNF) can promote developmental competence in mammalian oocytes during in vitro maturation, but the signal transduction pathways are not clear. In this study, we investigated (using western blots) the effects of BDNF on the phosphorylation of protein kinase B (PKB) and mitogen-activated protein kinase (MAPK) in mouse oocytes and cumulus cells cultured in vitro. Treatment with BDNF enhanced phosphorylation of PKB in oocytes at 2 h (P = 0.0006) and 3 h (P < 0.0001) of in vitro maturation, compared with control oocytes. However, the pan-specific tyrosine kinase (Trk) inhibitor K252a together with BDNF completely inhibited phosphorylation of PKB in the oocytes. Furthermore, BDNF increased phosphorylation of MAPK in oocytes at 16 h of in vitro maturation (P = 0.0041), but K252a together with BDNF did not reduce phosphorylation of MAPK in the oocytes. For cumulus cells, BDNF significantly prolonged the phosphorylation of PKB and MAPK and increased the total amounts of PKB and MAPK proteins after 16 h of in vitro maturation. However, BDNF did not affect apoptosis of the cumulus cells during oocyte maturation in vitro. In conclusion, the PKB pathway is likely to be one signaling cascade activated by BDNF in combination with the TrkB receptor, whereas the MAPK pathway is not involved. These findings may have relevance for BDNF-induced promotion of developmental capacity of in vitro-matured oocytes.     

Immunization method for multi-pass membrane proteins using highly metastatic cell lines

A novel method using metastatic breast cancer cell lines was established for producing monoclonal antibodies (mAbs) against multi-span membrane proteins. Grafting of metastatic cells (MCF7-14) into the mammary gland of BALB/cJ/nu/nu mice induced splenic hypertrophy (1.6–3.0 × 10⁸ cells/spleen [n = 6]). More than half of the mAbs against MCF7-14 cells reacted with the cell membrane. Inducing production of antibodies against the extracellular domain of multi-pass membrane proteins is difficult. Because the protein structure becomes more complex as the number of transmembrane domains increases, preparing antigens for immunization in which the original structure is maintained is challenging. Using highly metastatic MDA-MB231 cells as the host cell line, we produced mAbs against a 12 transmembrane protein, solute carrier family 6 member 6 (SLC6A6), as a model antigen. When SLC6A6-overexpressing MDA-MB231 cells were grafted into nude mice, the number of splenocytes increased to 2.7–11.4 × 10⁸ cells/spleen (n = 10). Seven mAb-producing clones that not only recognized the extracellular domain of SLC6A6 but also were of the IgG subclass were obtained. Immunocytochemistry and flow cytometry analyses revealed that these mAbs recognized the native form of the extracellular domain of SLC6A6 on the cell surface. Our novel immunization method involving highly metastatic cells could be used to develop therapeutic mAbs against other multi-pass membrane proteins.     

A sandwich enzyme-linked immunosorbent assay for the quantification of insoluble membrane and scaffold proteins

Enzyme-linked immunosorbent assays (ELISAs) are applied for the quantification of a vast diversity of small molecules. However, ELISAs require that the antigen is present in a soluble form in the sample. Accordingly, the few ELISAs described so far targeting insoluble proteins such as integral membrane and scaffold proteins have been restricted by limited extraction efficiencies and the need to establish an individual solubilization protocol for each protein. Here we describe a sandwich ELISA that allows the quantification of a diverse array of synaptic membrane and scaffold proteins such as munc13-1, gephyrin, NMDA R1 (N-methyl-d-aspartate receptor subunit 1), synaptic vesicle membrane proteins, and SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors). The assay is based on initial solubilization by the denaturing detergent sodium dodecyl sulfate (SDS), followed by partial SDS removal using the detergent Triton X-100, which restores antigenicity while keeping the proteins in solution. Using recombinant standard proteins, we determined assay sensitivities of 78 ng/ml to 77 pg/ml (or 74-0.1 fmol). Calibration of the assay using both immunoblotting and mass spectroscopy revealed that in some cases correction factors need to be included for absolute quantification. The assay is versatile, allows parallel processing and automation, and should be applicable to a wide range of hitherto inaccessible proteins.     

The influence of different membrane components on the electrical stability of bilayer lipid membranes

A good understanding of cell membrane properties is crucial for better controlled and reproducible experiments, particularly for cell electroporation where the mechanism of pore formation is not fully elucidated. In this article we study the influence on that process of several constituents found in natural membranes using bilayer lipid membranes. This is achieved by measuring the electroporation threshold (V_th) defined as the potential at which pores appear in the membrane. We start from highly stable 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) membranes (V_th ∼ 200 mV), and subsequently add therein other phospholipids, cholesterol and a channel protein. While the phospholipid composition has a slight effect (100 mV ≤ V_th ≤ 290 mV), cholesterol gives a concentration-dependent effect: a slight stabilization until 5% weight (V_th ∼ 250 mV) followed by a noticeable destabilization (V_th ∼ 100 mV at 20%). Interestingly, the presence of a model protein, α-hemolysin, dramatically disfavours membrane poration and V_th shows a 4-fold increase (∼ 800 mV) from a protein density in the membrane of 24 × 10^− 3 proteins/μm². In general, we find that pore formation is affected by the molecular organization (packing and ordering) in the membrane and by its thickness. We correlate the resulting changes in molecular interactions to theories on pore formation.     

Comparative binding of Cry1Ab and Cry1F Bacillus thuringiensis toxins to brush border membrane proteins from Ostrinia nubilalis, Ostrinia furnacalis and Diatraea saccharalis (Lepidoptera: Crambidae) midgut tissue

The European (Ostrinia nubilalis Hübner) and Asian corn borers (Ostrinia furnacalis Guenée) are closely related and display similar sensitivity to Cry1 toxins. In this study, we compared the binding patterns of Cry1Ab and Cry1F toxins between both Ostrinia spp., as well as the expression of putative cadherin- and aminopeptidase-N (APN)-like protein receptors. Additionally, cDNA sequences of these putative toxin receptors from both Ostrinia species were compared. Ligand blots for both species indicated a similar binding pattern for Cry1Ab with the strongest immunoreactive band at 260 kDa in both species. In addition, similar expression of the putative cadherin- and APN-like protein receptors were observed at 260 and 135 kDa, respectively. A high degree of similarity (98% amino acid sequence identity) of cDNA sequences for both putative receptor sequences was observed. The Cry1F ligand blot revealed that O. furnacalis and O. nubilalis BBMV exhibited slightly different binding patterns, with strong binding to putative proteins at 150 and 140 kDa, respectively. Both proteins appeared to also bind Cry1Ab, although the signal intensity was much reduced with Cry1Ab. O. furnacalis showed an additional but weaker band at 210 kDa relative to the 150 kDa band. Diatraea saccharalis (Fabricius), which was used as an outgroup species, exhibited different binding patterns than either Ostrinia species, with both Cry1Ab and Cry1F toxins binding to a 210 kDa protein. These results support the previous experiments indicating that O. nubilalis and O. furnacalis share similar patterns of susceptibility to Cry toxins.     

Interaction of the exported malaria protein Pf332 with the red blood cell membrane skeleton

Intra-erythrocytic Plasmodium falciparum malaria parasites synthesize and export numerous proteins into the red blood cell (RBC) cytosol, where some bind to the RBC membrane skeleton. These interactions are responsible for the altered antigenic, morphological and functional properties of parasite-infected red blood cells (IRBCs). Plasmodium falciparum protein 332 (Pf332) is a large parasite protein that associates with the membrane skeleton and who's function has recently been elucidated. Using recombinant fragments of Pf332 in in vitro interaction assays, we have localised the specific domain within Pf332 that binds to the RBC membrane skeleton to an 86 residue sequence proximal to the C-terminus of Pf332. We have shown that this region partakes in a specific and saturable interaction with actin (K_d = 0.60 µM) but has no detectable affinity for spectrin. The only exported malaria protein previously known to bind to actin is PfEMP3 but here we demonstrate that there is no competition for actin-binding between PfEMP3 and Pf332, suggesting that they bind to different target sequences in actin.     

The third helix of the murine Hoxc8 homeodomain facilitates protein transduction in mammalian cells

Previously, we have demonstrated that purified Hoxc8 homeoprotein has the ability to penetrate the cellular membrane and can be transduced efficiently into COS-7 cells. Moreover, the Hoxc8 protein is able to form a complex with DNA molecules in vitro and helps the DNA be delivered intracellularly, serving as a gene delivery vehicle. Here, we further analyzed the membrane transduction activity of Hoxc8 protein and provide the evidence that the 16 amino acid (a.a.191-206, 2.23 kDa) third helix of murine Hoxc8 protein is an efficient protein transduction domain (PTD). When the 16 amino acid peptide was fused at the carboxyl terminal of enhanced green fluorescence protein (EGFP), the fusion proteins were transduced efficiently into the primary pig fetal fibroblast cells. The transduction efficiency increased in a concentration-dependent manner up to 1 μM, and appeared to plateau above a concentration of 1 μM. When tandem multimers of PTD, EGFP-PTD(2), EGFP-PTD(3), EGFP-PTD(4), and EGFP-PTD(5), were analyzed at 500 nM of concentration, the penetrating efficiency increased in a dose-dependent manner. As the number of PTDs increased, the EGFP signal also increased, although the signal maintained plateau after EGFP-PTD(3). These results indicate that the 16 amino acid third helix is the key element responsible for the membrane transduction activity of Hoxc8 proteins, and further suggest that the small peptide could serve as a therapeutic delivery vehicle for large cargo proteins.     

Hydrogen-bonding and packing features of membrane proteins: functional implications

The recent structural elucidation of about one dozen channels (in which we include transporters) has provided further evidence that these membrane proteins typically undergo large movements during their function. However, it is still not well understood how these proteins achieve the necessary trade-off between stability and mobility. To identify specific structural properties of channels, we compared the helix-packing and hydrogen-bonding patterns of channels with those of membrane coils; the latter is a class of membrane proteins whose structures are expected to be more rigid. We describe in detail how in channels, helix pairs are usually arranged in packing motifs with large crossing angles (|τ| ≈ 40°), where the (small) side chains point away from the packing core and the backbones of the two helices are in close contact. We found that this contributes to a significant enrichment of Cα-H…O bonds and to a packing geometry where right-handed parallel (τ = −40° ± 10°) and antiparallel (τ = +140° ± 25°) arrangements are equally preferred. By sharp contrast, the interdigitation and hydrogen bonding of side chains in helix pairs of membrane coils results in narrowly distributed left-handed antiparallel arrangements with crossing angles τ = −160° ± 10° (|τ| ≈ 20°). In addition, we show that these different helix-packing modes of the two types of membrane proteins correspond to specific hydrogen-bonding patterns. In particular, in channels, three times as many of the hydrogen-bonded helix pairs are found in parallel right-handed motifs than are non-hydrogen-bonded helix pairs. Finally, we discuss how the presence of weak hydrogen bonds, water-containing cavities, and right-handed crossing angles may facilitate the required conformational flexibility between helix pairs of channels while maintaining sufficient structural stability.     

Ice-induced partial unfolding and aggregation of an integral membrane protein

Although the deleterious effects of ice on water-soluble proteins are well established, little is known about the freeze stability of membrane proteins. Here we explore this issue through a combined kinetic and spectroscopic approach using micellar-purified plasma membrane calcium pump as a model. The ATPase activity of this protein significantly diminished after freezing using a slow-cooling procedure, with the decrease in the activity being an exponential function of the storage time at 253 K, with t_½ = 3.9 ± 0.6 h. On the contrary, no significant changes on enzyme activity were detected when a fast cooling procedure was performed. Regardless of the cooling rate, successive freeze-thaw cycles produced an exponential decrease in the Ca²⁺-ATPase activity, with the number of cycles at which the activity was reduced to half being 9.2 ± 0.3 (fast cooling) and 3.7 ± 0.2 (slow cooling). PAGE analysis showed that neither degradation nor formation of SDS-stable aggregates of the protein takes place during protein inactivation. Instead, the inactivation process was found to be associated with the irreversible partial unfolding of the polypeptide chain, as assessed by Trp fluorescence, far UV circular dichroism, and 1-anilino-naphtalene-8-sulfonate binding. This inactive protein undergoes, in a later stage, a further irreversible transformation leading to large aggregates.     

Interactions of Plasmodium falciparum erythrocyte membrane protein 3 with the red blood cell membrane skeleton

Plasmodium falciparum parasites express and traffick numerous proteins into the red blood cell (RBC), where some associate specifically with the membrane skeleton. Importantly, these interactions underlie the major alterations to the modified structural and functional properties of the parasite-infected RBC. P. falciparum Erythrocyte Membrane Protein 3 (PfEMP3) is one such parasite protein that is found in association with the membrane skeleton. Using recombinant PfEMP3 proteins in vitro, we have identified the region of PfEMP3 that binds to the RBC membrane skeleton, specifically to spectrin and actin. Kinetic studies revealed that residues 38-97 of PfEMP3 bound to purified spectrin with moderately high affinity (K_D(kin) = 8.5 × 10^− 8 M). Subsequent deletion mapping analysis further defined the binding domain to a 14-residue sequence (IFEIRLKRSLAQVL; K_D(kin) = 3.8 × 10^− 7 M). Interestingly, this same domain also bound to F-actin in a specific and saturable manner. These interactions are of physiological relevance as evidenced by the binding of this region to the membrane skeleton of inside-out RBCs and when introduced into resealed RBCs. Identification of a 14-residue region of PfEMP3 that binds to both spectrin and actin provides insight into the potential function of PfEMP3 in P. falciparum-infected RBCs.     

Up-regulation of annexin A2 in cholangiocarcinoma caused by Opisthorchis viverrini and its implication as a prognostic marker

Cholangiocarcinoma (CCA), or cancer of the bile ducts, is primarily associated with infection with the liver fluke Opisthorchis viverrini in northeast Thailand. The disease is associated with late presentation, poses challenges for diagnosis and has a high mortality rate - features that highlight the need for tumor markers. At present, there are no specific tumor markers that can indicate the early stages and status of CCA. Proteomic analysis of the proteins expressed on the surface of tumor cells is particularly difficult since proteome-wide analysis of surface membrane proteins has thus far been hampered by the lack of effective strategies to profile hydrophobic membrane proteins. In this study, a sequential protein extraction was utilized to overcome this problem. Membrane protein was extracted from four CCA cell lines with different tumor forming capabilities. The non-tumor H69 biliary cell line was used as a control. Two-dimensional-PAGE followed by MALDI-TOF-MS was used to identify differentially expressed proteins. Among 20 up-regulated membrane proteins identified in the CCA cell lines was ANXA2, a participant in tumor invasion and metastasis in other cancers. Accordingly, ANXA2 was verified in human subjects by probing, using a commercial anti-mouse monoclonal antibody and a tissue microarray of CCA (301 diagnosed cases), where it was found to associate with one of several tumor progression stages as reflected by lymphatic invasion (P = 0.014) and metastasis (P = 0.026). Patients with high expression of ANXA2 had a significantly shorter survival time (P = 0.011). ANXA2 expression in tumors may be useful for predicting the poor outcome of CCA patients.     

A novel prokaryotic expression system for biosynthesis of recombinant human membrane-bound catechol-O-methyltransferase

Membrane proteins constitute 20-30% of all proteins encoded by the genome of various organisms. While large amounts of purified proteins are required for pharmaceutical and crystallization attempts, there is an unmet need for the development of novel heterologous membrane protein overexpression systems. Specifically, we tested the application of Brevibacillus choshinensis cells for the biosynthesis of human membrane bound catechol-O-methyltransferase (hMBCOMT). In terms of the upstream stage moderate to high expression was obtained for complex media formulation with a value near 45 nmol/h/mg for hMBCOMT specific activity achieved at 20 h culture with 37 °C and 250 rpm. Subsequently, the efficiency for reconstitution of hMBCOMT is markedly null in the presence of ionic detergents, such as sodium dodecyl sulphate (SDS). In general, for non-ionic and zwiterionic detergents, until a detergent critic micellar concentration (CMC) of 1.0 mM, hMBCOMT shows more biological activity at lower detergent concentrations while for detergent CMC higher than 1 mM, higher detergent concentrations seem to be ideal for hMBCOMT solubilization. Indeed, from the detergents tested, the non-ionic digitonin at 0.5% (w/v) appears to be the most suitable for hMBCOMT solubilization.     

Mutual structural effect of bilirubin and model membranes by vibrational circular dichroism

In this study, vibrational circular dichroism (VCD) spectroscopy was employed for the first time to study the bilirubin (BR) interaction with model membranes and models for membrane proteins. An enantioselective interaction of BR with zwitterionic 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and sphingomyelin (SPM) liposomes was observed by VCD and electronic circular dichroism (ECD) complemented by absorption and fluorescence spectroscopy. The M-form of BR was preferentially recognized in the BR/DMPC system at concentration above 1 × 10^− 4 M, for lower concentrations the P-form of BR was recognized by the DMPC liposomes. The VCD spectra also showed that the SPM liposomes, which represent the main component of nerve cell membrane, were significantly more disturbed by the presence of BR than the DMPC liposomes—a stable association with a strong VCD signal was observed providing the explanations for the supposed BR neurotoxicity. The effect of time and pH on the BR/DMPC or SPM liposome systems was shown to be essential while the effect of temperature in the range of 15–70 °C was negligible demonstrating the surprisingly high temperature stability of BR when interacting with the studied membranes. The influence of a membrane protein was tested on a model consisting of poly-l-arginine (PLAG) bound in the α-helical form to the surface of 1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol) liposomes and sodium dodecyl sulfate micelles. VCD and also ECD spectra showed that a variety of BR diastereoisomers interacted with PLAG in such systems. In a system of PLAG with micelles composed of sodium dodecyl sulfate, the M-form of bound BR was observed.     

Long-range nonanomalous diffusion of quantum dot-labeled aquaporin-1 water channels in the cell plasma membrane

Aquaporin-1 (AQP1) is an integral membrane protein that facilitates osmotic water transport across cell plasma membranes in epithelia and endothelia. AQP1 has no known specific interactions with cytoplasmic or membrane proteins, but its recovery in a detergent-insoluble membrane fraction has suggested possible raft association. We tracked the membrane diffusion of AQP1 molecules labeled with quantum dots at an engineered external epitope at frame rates up to 91 Hz and over times up to 6 min. In transfected COS-7 cells, >75% of AQP1 molecules diffused freely over ∼7 μm in 5 min, with diffusion coefficient, D_1-3 ∼ 9 × 10⁻¹⁰ cm²/s. In MDCK cells, ∼60% of AQP1 diffused freely, with D_1-3 ∼ 3 × 10⁻¹⁰ cm²/s. The determinants of AQP1 diffusion were investigated by measurements of AQP1 diffusion following skeletal disruption (latrunculin B), lipid/raft perturbations (cyclodextrin and sphingomyelinase), and bleb formation. We found that cytoskeletal disruption had no effect on AQP1 diffusion in the plasma membrane, but that diffusion was increased greater than fourfold in protein de-enriched blebs. Cholesterol depletion in MDCK cells greatly restricted AQP1 diffusion, consistent with the formation of a network of solid-like barriers in the membrane. These results establish the nature and determinants of AQP1 diffusion in cell plasma membranes and demonstrate long-range nonanomalous diffusion of AQP1, challenging the prevailing view of universally anomalous diffusion of integral membrane proteins, and providing evidence against the accumulation of AQP1 in lipid rafts.     

Analysis of Cry8Ka5-binding proteins from Anthonomus grandis (Coleoptera: Curculionidae) midgut

Biotech crops expressing Bacillus thuringiensis Cry toxins present a valuable approach for insect control. Cry8Ka5, which is highly toxic to the cotton boll weevil (Anthonomus grandis), was used as a model to study toxin-ligand interactions. Three Cry-binding proteins were detected after toxin overlay assays. Following de novo sequencing, a heat-shock cognate protein and a V-ATPase were identified, whilst a ∼120 kDa protein remained unknown. Additional Cry8Ka5-binding proteins were visualized by two-dimensional gel electrophoresis ligand blots.     

Electronic Western blot of matrix-assisted laser desorption/ionization mass spectrometric-identified polypeptides from parallel processed gel-separated proteins

Identification of proteins previously separated by one-dimensional (1-D) or two-dimensional gel electrophoresis requires significant manipulations to digest the proteins into their respective peptides and to extract them from the gel prior to mass analysis. This article describes the simultaneous transfer and digestion of proteins directly from 1-D gels onto a membrane ready for matrix-assisted laser desorption/ionization (MALDI) mass spectrometric (MS) analysis. Protein transfer and digestion efficiencies are estimated to be more than 95%. The effectiveness of this procedure is demonstrated by identifying 110 unique proteins derived from a lysate of Escherichia coli and 149 proteins derived from a mouse liver homogenate separated by 1-D sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Using crude mouse liver homogenates, four distinct glutathione S-transferase classes, ranging from 23 to 27 kDa, are identified from a separating gel, indicating the discriminating potential for this method. A Visual Basic program allowed visualization of the identified proteins according to their respective positions on the 1-D gels. In many cases, two or more proteins could be identified within a single band of the SDS gel. The “digital” images generated resemble Western blots without the use of antibodies or signal amplification techniques.     

Insight into hypoxic preconditioning and ischemic injury through determination of nPKCε-interacting proteins in mouse brain

Cerebral hypoxic preconditioning (HPC) provides neuroprotection by intracellular signaling pathways. We previously demonstrated that novel protein kinase Cε (nPKCε) activation participated in cerebral HPC development. In this study, we explore the role of nPKCε in HPC-induced neuroprotection against middle cerebral artery occlusion (MCAO)-induced ischemic injury and identify its possible signaling molecules. A total of 131 adult male BALB/c mice were divided into eight groups: normoxic control (n = 9), HPC (n = 9), HPC + εV1–2 (n = 13), Sham (n = 19), HPC + sham (n = 6), Ischemia (I, 6 h MCAO, n = 31), HPC + I (n = 25) and HPC + εV1–2 + I (n = 19). nPKCε specific inhibitor εV1–2 was administered via intracerebroventricular injection. Western blot, 2,3,5-triphenyltetrazolium chloride staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling were applied to determine nPKCε membrane translocation, infarction volume and programmed cell death (PCD), respectively. Two-dimensional gel electrophoresis (2-De) and matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) were used to identify nPKCε-interacting proteins, followed by bioinformatics analysis of genee ontology (GO) to predict nPKCε-specific signaling pathways. Our results showed that HPC attenuates MCAO-induced brain injuries and stabilized nPKCεmembrane translocation in peri-infarct region, which was abolished by nPKCε-speecific inhibitor εV1–2. Proteomics analysis revealed 8 up- and 3 down-regulated nPKCε-interacting proteins both in cytosolic and particulate fractions of HPC mouse brain. GO analysis predicted 25 significant nPKCε-specific signaling pathways among the 16 identified nPKCε-interacting proteins in brain of HPC mice. This study is the first to report multiple nPKCε-interacting proteins and their signaling pathways in HPC mouse brain, suggesting that nPKCε signaling molecules is responsible for HPC-induced neuroprotection against cerebral ischemic injuries of mice.