Is calpain activity regulated by membranes and autolysis or by calcium and calpastatin?

Although the Ca(2+)-dependent proteinase (calpain) system has been found in every vertebrate cell that has been examined for its presence and has been detected in Drosophila and parasites, the physiological function(s) of this system remains unclear. Calpain activity has been associated with cleavages that alter regulation of various enzyme activities, with remodeling or disassembly of the cell cytoskeleton, and with cleavages of hormone receptors. The mechanism regulating activity of the calpain system in vivo also is unknown. It has been proposed that binding of the calpains to phospholipid in a cell membrane lowers the Ca2+ concentration, [Ca2+], required for the calpains to autolyze, and that autolysis converts an inactive proenzyme into an active protease. Recent studies, however, show that the calpains bind to specific proteins and not to phospholipids, and that binding to cell membranes does not affect the [Ca2+] required for autolysis. It seems likely that calpain activity is regulated by binding of Ca2+ to specific sites on the calpain molecule, with binding to each site eliciting a response (proteolytic activity, calpastatin binding, etc.) specific for that site. Regulation must also involve an, as yet, undiscovered mechanism that increases the affinity of the Ca(2+)-binding sites for Ca2+.     

Comparison of protein and lipid composition of the human platelet α-granule membranes and glycerol lysis membranes

Platelet glycerol lysis membranes and α-granule membranes were compared with respect to protein and lipid composition. Crossed immunoelectrophoresis using antibodies against whole platelets, and sodium dodecyl sulphate polyacrylamide gel electrophoresis, revealed the presence of the glycoproteins IIb and IIIa, myosin and an antigen termed G4 in both membrane fractions. The glycoproteins Ia, Ib and IIIb, in addition to β₂-microglobulin and actin, appeared specific for the glycerol lysis membranes, whereas two antigens, termed G8 and G18, were observed only in the α-granule membranes. The localization of glycoprotein IIa was inconclusive. Comparison with the surface-located proteins revealed that the glycerol lysis membranes represented a reasonable approximation to a plasma membrane preparation. Radioactively labelled immunoprecipitates obtained after crossed immunoelectrophoresis of ¹²⁵I-labelled platelets were cut out and applied to sodium dodecyl sulphate electrophoresis on polyacrylamide slab gels. Autoradiography of the dried gels revealed that antigen G4 represented a protein with an average molecular weight of 146 000 in its unreduced state and 132 000 in its reduced state. Antigen G18 represented a protein of molecular weight 130 000–135 000 in the reduced as well as unreduced state. Quantitation of protein and lipids showed that the α-granule membranes contained about one-third as much cholesterol and 2-times as much protein in relation to phospholipids as compared to the glycerol lysis membranes. No significant difference between the two membrane preparations was found as regards the composition of their phospholipids.     

Molecular organization in bacterial cell membranes III. Components of a “soluble” fraction obtained by n-butanol extraction of Streptomyces albus membranes

We have isolated a “soluble” fraction of Streptomyces albus G membranes or membranes previously solubilised by sodium dodecylsulphate, using n-butanol extraction. Polyacrylamide gel electrophoresis in sodium dodecylsulphate of the whole membrane showed a complex protein pattern (about 20–25 bands) with two predominant groups. The “soluble” fraction represented about 25% of the membrane protein and contained part of the major polypeptides. The yield of protein in “soluble” form decreased when membranes were suspended in water and di not significantly change if membranes were reduced with sodium dithionite and then treated with iodoacetamide. A change in relative mobility of some of these polypeptides seemed to occur with membrane delipidation. The proteins of the fraction appear to be glycoproteins as indicated by their simultaneous staining for protein and carbohydrate and the parallel sensitivity to trypsin of both stains. The apparent molecular weights by sodium dodecylsulphate gel electrophoresis of the proteins (glycoproteins) were: 63 000, 40 000 and 17 000. Similar protein patterns were obtained by extraction of the membranes with EDTA and non-ionic detergents. Lipid and nucleotide material were also found in the “soluble” fraction.The “soluble” fraction showed by gel filtration on Sephadex G-200 the existence of different states of aggregation. These states of aggregation revealed the same electrophoretic pattern of proteins, which seemingly corresponded to that of the original fraction (i.e. three protein groups with relative mobilities 0.65, 0.80 and 1.0). Treatment of the samples under different conditions with 1% dodecylsulphate (supplemented or not with 0.5% β-mercaptoethanol) failed to completely dissociate the fraction as shown by Sephadex filtration.     

Inhibition of Bufo arenarum oocyte maturation induced by cholesterol depletion by methyl-β-cyclodextrin. Role of low-density caveolae-like membranes

The invaginated structure of caveolae seems to provide an optimal environment for hormone binding leading to oocyte meiotic maturation. We conducted a quantitative analysis of lipids and proteins of detergent-free low-density membranes isolated from Bufo arenarum oocytes and we modulated cellular cholesterol to further understand how these domains perform their regulatory functions in the amphibian system. Light membranes derive from the plasma membrane as suggested by the enrichment in the activity of 5′nucleotidase. Lipid analysis by chromatography techniques revealed that this fraction is enriched in phosphatidylserine and cholesterol and that it evidences an important level of sphingomyelin. The finding of a single 21 kDa caveolin in light membranes indicates the presence of caveolae-like structures in B. arenarum oocytes. In support of this finding, c-Src is significantly associated to this fraction. Cholesterol content of oocytes treated with methyl-β-cyclodextrin (MβCD) decreased when compared to control oocytes. Drug treatment inhibited meiotic maturation in a dose-dependent manner and affected the localization of caveolin and c-Src among membrane fractions. Repletion of cholesterol showed a recovery of the ability of MβCD-treated oocytes to mature, particularly at the 25 mM concentration in which reversibility was close to the control level. Results highlight the importance of caveolae-like microdomains for maturation signaling in Bufo oocytes.     

Expansion and apparent fluidity decrease of nuclear membranes induced by low Ca/Mg. Modulation of nuclear membrane lipid fluidity by the membrane-associated nuclear matrix proteins?

Macronuclei isolated from Tetrahymena are contracted in form (average diameter: 10.2 micron) at a final Ca/Mg (3:2)concentration of 5 mM. Lowering the ion concentration to 1 mM induces an expansion of the average nuclear diameter to 12.2 micron. Both contracted and expanded nuclei are surrounded by a largely intact nuclear envelope as revealed by thin-sectioning electron microscopy. Nuclear swelling is accompanied by an expansion of the nuclear envelope as indicated by the decrease in the frequency of nuclear pore complexes from 52.6 to 42.1 pores/micron2 determined by freeze-etch electron microscopy. Contracted nuclear membranes reveal particle-devoid areas (average size: 0.21 micron2) on 59% of their fracture faces at the optimal growth temperature of 28 degrees C. About three-fifths of the number of these smooth areas disappear upon nuclear membrane expansion. Electron spin resonance using 5-doxylstearic acid as a spin label indicates a higher lipid fluidity in contracted than in expa,ded nuclear membranes. Moreover, a thermotropic lipid clustering occurs at approximately 17 degrees C only in expanded nuclear membranes. In contrast to the nuclear membrane- bound lipids, free lipids extracted from the nuclei rigidify with increasing Ca/Mg concentrations. Our findings are compatible with the view that the peripheral layer of the fundamental nuclear protein- framework, the so-called nuclear matrix, can modulate, inter alia, the lipid distribution and fluidity, respectively, in nuclear membranes. We suggest that a contraction of the nuclear matrix's peripheral layer induces a contraction of the nuclear membranes which, in turn, leads to an isothermic lateral lipid segregation within nuclear membranes.     

Tissue-culture fractionation. Zonal centrifugation of Lettrée cells homogenized by glycerol-induced lysis: subfractionation of membranes in sucrose gradients.

1. Lettrée cells were grown intraperitoneally in MF-1 mice. 2. Cells that were loaded with glycerol were swollen in 0.1 M-sucrose and disrupted by Dounce homogenization. 3. Early-passage Lettrée cells were more easily disrupted than late-passage cells by this method, and the former produced larger fragments of plasma membrane. 4. The membranes were fractionated initially in sucrose gradients (on the basis of sedimentation rate) in a BXIV zonal rotor. 5. Fractions from this gradient were further resolved in isopycnic sucrose gradients. 6. Plasma-membrane and endoplasmic-reticulum fractions were recovered in good yield and high purity.     

Developmental changes in proteins of purified membranes of chicken lenses and evidence for contamination by cytoplasmic δ-crystallin

Urea-washed membranes from embryonic chick lenses (15 days old) and from the cortical and nuclear regions of adult chicken lenses (1 year) have been prepared by repeated centrifugation through discontinuous density gradients. The protein components of the isolated membranes have been examined by electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate and urea. Proteins with molecular weights of 75 000, 56 000, 54 000, 48 000, 34 000, 32 000, 25 000, and 22 000 were present in all the membrane preparations, although their proportions changed during development. One additional protein, molecular weight 70 000, was seen only in the embryonic lens membranes. The greatest developmental change was the increase in 25 000 molecular weight protein from 12% in the embryonic lens to about 45% in the adult lens. Since it has been suggested that this protein is associated with gap junctions, its increase during development may reflect a corresponding increase in the number of gap junctions in the lens.The 50 000 molecular weight protein of embryonic lens membranes and membranes of adult nuclear lens fibers consisted at least partly of δ-crystallin, since δ-crystallin peptides could be identified in tryptic pepetide maps of the isolated protein after in vitro radioiodination. Peptide maps of the 50 000 molecular weight protein of cortical lens fiber membranes contained no identifiable δ-crystallin peptides, although it is possible that modified δ-crystallin peptides may be present. The level of cytoplasmic contamination of the membrane fraction was estimated by preparing lens membranes in the presence of added δ-[³⁵S]crystallin. The results indicated that cytoplasmic contamination contributes significantly to the presence of δ-crystallin in lens membrane preparations.     

Lipid modifications of proteins – slipping in and out of membranes

Protein lipid modification, once thought to act as a stable membrane anchor for soluble proteins, is now attracting more widespread attention for its emerging role in diverse signaling pathways and regulatory mechanisms. Most multicellular organisms have recruited specific types of lipids and a suite of unique enzymes to catalyze the modification of a select number of proteins, many of which are evolutionarily conserved in plants, animals and fungi. Each of the three known types of lipid modification – palmitoylation, myristylation and prenylation – allows cells to target proteins to the plasma membrane, as well as to other subcellular compartments. Among the lipid modifications, protein prenylation might also function as a relay between cytoplasmic isoprene biosynthesis and regulatory pathways that control cell cycle and growth. Molecular and genetic studies of an Arabidopsis mutant that lacks farnesyl transferase suggest that the enzyme has a role in abscisic acid signaling during seed germination and in the stomata. It is becoming clear that lipid modifications are not just fat for the protein, but part of a highly conserved intricate network that plays a role in coordinating complex cellular functions.     

Production of interferon-β by fibroblast cells on membranes prepared by extracellular matrix proteins

The fibroblast cells from normal human skin were cultured on Langmuir-Blodgett (LB) and cast membranes prepared using extracellular matrix proteins (e.g., collagen, fibronectin, laminin and vitronectin). The cell density of the fibroblast cells cultured on the cast membranes was found to be higher than that on the cast membranes made of fibronectin, vitronectin and collagen-blended membranes. This indicates that not only the primary structure of proteins but the preparation methods of the membranes, i.e., casting and LB methods, are a strong factor affecting cell growth. The concentration and production of interferon-β per unit cell were found to be higher on the LB membranes than on the cast membranes made of the same proteins except in the case of collagen. However, the cell density on the cast membranes was higher than that on the LB membranes. These results appear to result from the suppressed growth of NB1-RGB cells on the LB membranes leading to the enhanced production of interferon-β on the LB membranes. The highest production of interferon-β per unit cell was observed for the NB1-RGB cells on the collagen-blended membranes with fibronectin and vitronectin. The collagen-blended membranes appear to offer a more natural and appropriate environment for NB1-RGB cells to produce interferon-β. This revised version was published online in September 2006 with corrections to the Cover Date.     

Aggregation of PrP106–126 on surfaces of neutral and negatively charged membranes studied by molecular dynamics simulations

Prion diseases are neurodegenerative disorders characterized by the aggregation of an abnormal form of prion protein. The interaction of prion protein and cellular membrane is crucial to elucidate the occurrence and development of prion diseases. Its fragment, residues 106–126, has been proven to maintain the pathological properties of misfolded prion and was used as a model peptide. In this study, explicit solvent molecular dynamics (MD) simulations were carried out to investigate the adsorption, folding and aggregation of PrP106–126 with different sizes (2-peptides, 4-peptides and 6-peptides) on the surface of both pure neutral POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and negatively charged POPC/POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol) (3:1) lipids. MD simulation results show that PrP106–126 display strong affinity with POPC/POPG but does not interact with pure POPC. The positively charged and polar residues participating hydrogen bonding with membrane promote the adsorption of PrP106–126. The presence of POPC and POPC/POPG exert limited influence on the secondary structures of PrP106–126 and random coil structures are predominant in all simulation systems. Upon the adsorption on the POPC/POPG surface, the aggregation states of PrP106–126 have been changed and more small oligomers were observed. This work provides insights into the interactions of PrP106–126 and membranes with different compositions in atomic level, which expand our understanding the role membrane plays in the development of prion diseases. This article is part of a Special Issue entitled: Protein Aggregation and Misfolding at the Cell Membrane Interface edited by Ayyalusamy Ramamoorthy.     

Study of the interaction of an α-helical transmembrane peptide with phosphatidylcholine bilayer membranes by means of densimetry and ultrasound velocimetry

We applied precise densimetry and ultrasound velocimetry methods to study the interaction of a synthetic α-helical transmembrane peptide, acetyl-K₂-L₂₄-K₂-amide (L₂₄), with model bilayer lipid membranes. The large unilamellar vesicles (LUVs) utilized were composed of a homologous series of n-saturated diacylphosphatidylcholines (PCs). PCs whose hydrocarbon chains contained from 13 to 16 carbon atoms, thus producing phospholipid bilayers of different thicknesses and gel to liquid-crystalline phase transition temperatures. This allowed us to analyze how the difference between the hydrophobic length of the peptide and the hydrophobic thickness of the lipid bilayer influences the thermodynamical and mechanical properties of the membranes. We showed that the incorporation of L₂₄ decreases the temperature and cooperativity of the main phase transition of all LUVs studied. The presence of L₂₄ in the bilayer also caused an increase of the specific volume and of the volume compressibility in the gel state bilayers. In the liquid crystalline state, the peptide decreases the specific volume at relatively higher peptide concentration (mole ratio L₂₄:PC = 1:50). The overall volume compressibility of the peptide-containing lipid bilayers in the liquid-crystalline state was in general higher in comparison with pure membranes. There was, however, a tendency for the volume compressibility of these lipid bilayers to decrease with higher peptide content in comparison with bilayers of lower peptide concentration. For one lipid composition, we also compared the thermodynamical and mechanical properties of LUVs and large multilamellar vesicles (MLVs) with and without L₂₄. As expected, a higher cooperativity of the changes of the thermodynamical and mechanical parameters took place for MLVs in comparison with LUVs. These results are in agreement with previously reported DSC and ²H NMR spectroscopy study of the interaction of the L₂₄ and structurally related peptides with phosphatidylcholine bilayers. An apparent discrepancy between ²H NMR spectroscopy and compressibility data in the liquid crystalline state may be connected with the complex and anisotropic nature of macroscopic mechanical properties of the membranes. The observed changes in membrane mechanical properties induced by the presence of L₂₄ suggest that around each peptide a distorted region exists that involves at least 2 layers of lipid molecules.     

α-Synuclein interacts differently with membranes mimicking the inner and outer leaflets of neuronal membranes

The toxicity of α-synuclein (α-syn), the amyloidogenic protein responsible for Parkinson's disease, is likely related to its interaction with the asymmetric neuronal membrane. α-Syn exists as cytoplasmatic and as extracellular protein as well. To shed light on the different interactions occurring at the different α-syn localizations, we have here modelled the external and internal membrane leaflets of the neuronal membrane with two complex lipid mixtures, characterized by phase coexistence and with negative charge confined to either the ordered or the disordered phase, respectively. To this purpose, we selected a five-component (DOPC/SM/DOPE/DOPS/chol) and a four-component (DOPC/SM/GM1/chol) lipid mixtures, which contained the main membrane lipid constituents and exhibited a phase separation with formation of ordered domains. We have compared the action of α-syn in monomeric form and at different concentrations (1 nM, 40 nM, and 200 nM) with respect to lipid systems with different composition and shape by AFM, QCM-D, and vesicle leakage experiments. The experiments coherently showed a higher stability of the membranes composed by the internal leaflet mixture to the interaction with α-syn. Damage to membranes made of the external leaflet mixture was detected in a concentration-dependent manner. Interestingly, the membrane damage was related to the fluidity of the lipid domains and not to the presence of negatively charged lipids.     

Investigating the membrane orientation and transversal distribution of 17β-estradiol in lipid membranes by solid-state NMR

17β-Estradiol (E₂) is a potent estrogen, which modulates many important cellular functions by binding to specific estrogen receptors located in the cell nucleus and also on the plasma membrane. We have studied the membrane interaction of E₂ using a combination of solid-state NMR methods. ²H NMR results indicate that E₂ does not cause a condensation effect of the surrounding phospholipids, which is contrary to the effects of cholesterol, and only very modest E₂ induced alterations of the membrane structure were detected. ¹H magic-angle spinning NMR showed well resolved signals from E₂ as well as of POPC in the membrane-lipid layer. Two-dimensional NOESY spectra revealed intense cross-peaks between E₂ and the membrane lipids indicating that E₂ is stably inserted into the membrane. The determination of intermolecular cross-relaxation rates revealed that E₂ is broadly distributed in the membrane with a maximum of the E₂ distribution function in the upper chain region of the membrane. We conclude that E₂ is highly dynamic in lipid membranes and may undergo rotations as it exhibits two polar hydroxyl groups on either side of the molecule.     

Inhibition of peroxidation in linoleic acid membranes by nitroxide radicals,butylated hydroxytoluene,and α-tocopherol

The thermal oxidation of the membranes of linoleic acid vesicles was preceded by a lag period, as long as the membranes contained low levels of preformed peroxides. Incorporation of 0.034 to 0.170 mol% of nitroxide spin label increased the length of this lag between 4.8 and 10.1 times. At the same time, the intensity of the ESR signal fell. The inclusion of as little as 0.04 mol% of butylated hydroxytoluene in the membranes also lengthened the lag period by a factor of 2.5. However, a similar molar proportion of α-tocopherol was without effect. When the linoleic acid from which vesicle membranes were formed contained between 0.45 and 1.43 mol% of peroxide, α-tocopherol produced a significant increase in the lag period, during which the antioxidant was gradually oxidized.     

Biochemical studies of the chromaffin granule. III. Redistribution of lipid phosphate,dopamine-β-hydroxylase and chromogranin a after freezing and thawing of the isolated granule membranes

Freezing and thawing a dialysed suspension of lysed chromaffin granules and sedimented membrane preparations resulted in redistribution of lipid phosphate and protein. By this treatment the high ratios of lipid phosphate/protein in the membrane fragments, isolated on sucrose density gradient from the dialysed suspensions and the sedimented membrane preparation, were reduced from 1.56 to 1.03 μmoles/mg and from 1.97 to 0.83 μmoles/mg, respectively.Multilamellar, liposomal structures could be isolated from the frozen and thawed membrane preparations and were found to sediment in the 0.4 M sucrose layer by density gradient centrigugation. This fraction was without morphological resemblance to the intact chromaffin granules or their membranes and was found to account for 53% of the lipid phosphate, 35% of chromogranin A, 21% of dopamine-β-hydroxylase activity and 12% of the protein of the total preparation. The specific activities of chromogranin A and dopamine-β-hydroxylase in the artificially formed liposomal structures closely resembled that of the solubilized protein and was significantly higher than in the lipid phosphate-depleted membrane fragments recovered in the 1.1 M sucrose layers.It is concluded that freezing and thawing as a means of purifying the isolated granule membranes lead not only to the solubilization of chromogranin A, but also to removal of dopamine-β-hydroxylase activity and lipid phosphate from the labile membrane fragments.