Biomedical subjects

M Joniau

Publications and source records attributed to M Joniau.

At least 19 recordsLinked to original sources

PubMed2001

Structural basis for the appearance of a molten globule state in chimeric molecules derived from lysozyme and alpha-lactalbumin.

The problem as to why alpha-lactalbumin, in the absence of Ca(2+), forms a molten globule intermediate, in contrast to its structural homologue lysozyme, has been addressed by the construction of chimeras of human lysozyme in which either the Ca(2+)-binding loop or a part of helix C of bovine alpha-lactalbumin were transplanted. Previously, we have shown that the introduction of both structural elements together in the lysozyme matrix causes the apo form of the resulting chimera to display molten globule behavior during the course of thermal denaturation. In this article, we demonstrate that this molten globule character is not correlated with the Ca(2+)-binding loop. Also, the Del 101 mutant in which Arg101 was deleted to simulate the alpha-lactalbumin conformation of the connecting loop between helix C and helix D, does not show a stable equilibrium intermediate. Rather, the molten globule character of the chimeras has to be related with a specific part of helix C. More particularly, attention is drawn to the four hydrophobic side-chains I93, V96, I99, and L100, the lysozyme counterparts of which are constituted of less bulky valines and alanine. Our observations are discussed in terms of decreased stability of the native form and increased stability of the intermediate molten globule.

Amino Acid Sequence↗

PubMed2001

The fast folding pathway in human lysozyme and its blockage by appropriate mutagenesis: a sequential stopped-flow fluorescence study.

In this work we were able to show that human lysozyme refolds along two parallel pathways: a fast path followed by 13% of the molecules that leads directly from a collapsed state to the native protein and a slow one for the remaining molecules that involves a partially unfolded intermediate state. However, in the refolding process of LYLA1, a chimera of human lysozyme which possesses the Ca2+-binding loop and helix C of bovine alpha-lactalbumin, the direct pathway is no longer accessible. This indicates that these structural elements, which are located in the interface region between the alpha- and beta-domain of the protein, and their interaction with the environment play an important role in the fast folding of the molecules. These results also shed some light on the conservation of folding patterns amongst structurally homologous proteins. In recent years it was often stated that structurally homologous proteins with high sequence identity follow the same folding pattern. Human lysozyme and LYLA1 have a sequence identity of 87%. However, we have shown that their folding patterns are different. Therefore, a high degree of sequence identity for two proteins belonging to the same family is not a guarantee for an identical folding pattern.

Apoenzymes↗

PubMed2000

Amyloid fibril formation and seeding by wild-type human lysozyme and its disease-related mutational variants.

Wild-type human lysozyme and its two stable amyloidogenic variants have been found to form partially folded states at low pH. These states are characterized by extensive disruption of tertiary interactions and partial loss of secondary structure. Incubation of the proteins at pH 2.0 and 37 degrees C (Ile56Thr and Asp67His variants) or 57 degrees C (wild-type) results in the formation of large numbers of fibrils over several days of incubation. Smaller numbers of fibrils could be observed under other conditions, including neutral pH. These fibrils were analyzed by electron microscopy, Congo red birefringence, thioflavine-T binding, and X-ray fiber diffraction, which unequivocally show their amyloid character. These data demonstrate that amyloidogenicity is an intrinsic property of human lysozyme and does not require the presence of specific mutations in its primary structure. The amyloid fibril formation is greatly facilitated, however, by the introduction of "seeds" of preformed fibrils to the solutions of the variant proteins, suggesting that seeding effects could be important in the development of systemic amyloidosis. Fibril formation by wild-type human lysozyme is greatly accelerated by fibrils of the variant proteins and vice versa, showing that seeding is not specific to a given protein. The fact that wild-type lysozyme has not been found in ex vivo deposits from patients suffering from this disease is likely to be related to the much lower population of incompletely folded states for the wild-type protein compared to its amyloidogenic variants under physiological conditions. These results support the concept that the ability to form amyloid is a generic property of proteins, but one that is mitigated against in a normally functioning organism.

Amyloid↗

PubMed1999

Kinetic and equilibrium intermediate states are different in LYLA1, a chimera of lysozyme and alpha-lactalbumin.

For several proteins, a striking resemblance has been observed between the equilibrium partially folded state and the kinetic burst-phase intermediate, observed just after the dead-time in refolding experiments. This has led to the general statement that the conformation of both types of intermediates is similar. We show, at least for one of the proteins investigated here, that, although both states have some common characteristics, they are not identical. LYLA1 is a chimeric protein resulting from the transplantation of the Ca(2+)-binding loop and the adjacent helix C of bovine alpha-lactalbumin into the homologous position (residues 76-102) in human lysozyme. The apo-form of LYLA1 unfolds through a partially folded state, in analogy with the folding behaviour of the structurally homologous alpha-lactalbumin. The folding kinetics of LYLA1 and of its wild-type homologue, human lysozyme, are investigated by means of stopped-flow fluorescence and CD spectroscopy. In the case of human lysozyme, refolding involves parallel pathways as indicated by experiments in the presence of a fluorescent inhibitor. For apo-LYLA1, the burst-phase intermediate is compared with the equilibrium intermediate. At neutral pH, both states correspond, in that an important amount of secondary structure has been established, but the burst-phase intermediate is shown to be significantly less stable than the equilibrium intermediate. At pH 1.85, in the presence of 1.5 M guanidinium hydrochloride (GdnHCl) and at 25 degrees C, the equilibrium partially folded state of LYLA1 is 100% populated. When LYLA1 is rapidly diluted from 6 M GdnHCl to 1.5 M under these conditions, a time-dependent evolution of the fluorescence signal is observed, reflecting the transition from a burst-phase to a different equilibrium intermediate. These results provide strong evidence for the non-identity of both states in this protein.

Animals↗

PubMed1999

Expression, purification, and characterization of the recombinant calcium-binding equine lysozyme secreted by the filamentous fungus Aspergillus niger: comparisons with the production of hen and human lysozymes.

Equine lysozyme (EqL) has been expressed from a synthetic gene and secreted from a heterologous host, the filamentous fungus Aspergillus niger. By including 100 mM Ca2+ in the growth medium, secreted yields of more than 50 mg/liter could be achieved using polyvinylpyrrolidone (PVP) complete medium. In a soya medium yields of up to 150 mg/liter were achieved. The production of recombinant human lysozyme (HuL) from A. niger with yields of over 40 mg/liter was also achieved using PVP medium. Addition of Ca2+ to the growth medium reduced the yield of both HuL and hen egg white lysozyme (HEWL). Sequence differences between the three lysozymes, EqL, HuL, and HEWL, resulted in different susceptibilities to cleavage by A. niger proteases. An improved procedure for the purification of EqL and HuL from A. niger allowed separation of the proteins from pigments produced by the fungus. Detailed spectroscopic analysis, including 2D 1H NMR, for recombinant EqL and recombinant HuL confirm that both proteins possess their native structure and are purified to homogeneity.

Amino Acid Sequence↗

PubMed1998

The perturbations of the native state of goat alpha-lactalbumin induced by 1,1'-bis(4-anilino-5-naphthalenesulfonate) are Ca2+-dependent.

In this work we have studied the interaction of the hydrophobic fluorescent probe 1,1'-bis(4-anilino-5-naphthalenesulfonate) (bis-ANS), with the native state of apo- and Ca2+-bound goat alpha-lactalbumin (GLA). In 10 mM Tris-HCl, pH 7.5, at 4 degrees C in 2 mM EGTA as well as at 37 degrees C in 2 mM Ca2+, the native protein is close to its thermal transition. Therefore, it can be expected that in both conditions the protein is equally susceptible to interaction with bis-ANS. Nevertheless, we have observed a number of interesting differences in the interaction of the dye with the apo and Ca2+ form. Native apo-GLA binds two bis-ANS molecules and in the complex with bis-ANS, the far-UV circular dichroism (CD) spectrum of apo-GLA becomes similar to that of the protein in the molten globule state. In contrast, native Ca2+-GLA binds five bis-ANS molecules and the far-UV CD spectrum of native Ca2+-GLA is conserved for the complex. In both cases, the high activation energies observed in kinetic experiments indicate that upon binding, large parts of the protein structure have to be reorganized. The reduced perturbation of the protein structure in the presence of Ca2+ can be attributed to local stabilization effects.

Anilino Naphthalenesulfonates↗

PubMed1997

Conformational stability of LYLA1, a synthetic chimera of human lysozyme and bovine alpha-lactalbumin.

LYLA1 is a chimeric protein mainly consisting of residues originating from human lysozyme but in which the central part (Ca(2+)-binding site and helix C) of bovine alpha-lactalbumin has been inserted. The equilibrium unfolding of this hybrid protein has been examined by circular dichroism and tryptophan fluorescence techniques. The reversible denaturation process induced by temperature or by addition of chemical denaturant is three-state in the case of apo-LYLA1 and two-state in the presence of Ca2+. The Ca(2+)-bound form of the chimera exhibits higher stability than both wild-type lysozyme and alpha-lactalbumin. The stability of the apo-form, however, is intermediate between that of the parent molecules. Unfolding of apo-LYLA1 involves an intermediate state that becomes populated to a different extent under various experimental conditions. Combination of circular dichroism with bis-ANS fluorescence experiments has permitted us to characterize the acid state of LYLA1 as a molten globule. Furthermore our results strongly suggest the presence of multiple denatured states depending on external conditions.

Anilino Naphthalenesulfonates↗

PubMed1997

Computation of the binding of fully flexible peptides to proteins with flexible side chains.

Docking algorithms play an important role in the process of rational drug design and in understanding the mechanism of molecular recognition. An important determinant for successful docking is the extent to which the configurational space (including conformational changes) of the ligand/receptor system is searched. Here we describe a new, combinatorial method for flexible docking of peptides to proteins that allows full rotation around all single bonds of the peptide ligand and around those of a large set of receptor side chains. We have simulated the binding of several viral peptides to murine major histocompatibility complex class I H-2Kb. In addition, we have explored the limits of our method by simulating a complex between calmodulin and an 18-residue long helical peptide from calmodulin-dependent protein kinase IIalpha. The calculated peptide conformations generally matched well with the X-ray structures. Essential information about local flexibility and about residues that are responsible for strong binding was obtained. We have frequently observed considerable side-chain flexibility during the simulations, showing the need for a flexible treatment of the receptor. Our method may also be useful whenever the receptor side-chain conformation is not available or uncertain, as illustrated by the docking of an H-2Kb binding nonapeptide to the receptor structure taken from an octapeptide/H-2Kb complex.

Alanine↗

PubMed1996

Lipopolysaccharide-enhanced expression of interleukin-6 in dibutyryl cyclic AMP-differentiated rat C6 glioma.

Rat C6 glioma synthesizes a low basal level of interleukin-6 (IL-6). Stimulation with 10 micrograms/ml of lipopolysaccharide (LPS) and induction of differentiation with 1 mM N6,O2'-dibutyryl cyclic AMP (dbcAMP) for 48 h increased the secreted activity to 400 and 800 U/ml, respectively. An LPS stimulation of dbcAMP-differentiated cells strongly enhanced the secreted activity. Depending on the dbcAMP concentration, the cell number, and the stimulation time, the secreted IL-6 level increased up to 120,000 U/ml. After 48 h of costimulation with 10 micrograms/ml of LPS and 1 mM dbcAMP, northern blotting and immunoassay demonstrated an eightfold increase in IL-6 mRNA concentration and IL-6 immunoreactivity, whereas titration of the biological activity indicated a 100-fold increase in the secreted IL-6 activity. The enhanced secretion of IL-6 is correlated with the induction of differentiation. Chromatography on heparin-Sepharose and on DEAE-5PW separated the secreted activity into several fractions, indicating that they differ in heparin affinity and charge either by posttranslational modifications or by binding to a carrier protein. Each of the partially purified IL-6-like activities could be neutralized by an anti-murine IL-6 antibody. Our observations demonstrate that in vivo inflammatory signals can trigger astrocytes and their precursors to secrete substantially different levels of immunoregulatory cytokines depending on their degree of differentiation.

Animals↗

PubMed1995

A Ca(2+)-binding chimera of human lysozyme and bovine alpha-lactalbumin that can form a molten globule.

In contrast to lysozymes, which undergo two-state thermal denaturation, the Ca(2+)-free form of the homologous alpha-lactalbumins forms an intermediate "molten globule" state. To understand this difference, we have produced a chimera of human lysozyme and bovine alpha-lactalbumin. In the synthetic gene of the former the sequence coding for amino acid residues 76-102 was replaced by that for bovine alpha-lactalbumin 72-97, which represents the Ca(2+)-binding loop and the central helix C. The chimeric protein, LYLA1, expressed in Saccharomyces cerevisiae was homogeneous on electrophoresis and mass spectrometry. Its Ca2+ binding constant was 2.50 (+/- 0.04) x 10(8) M-1, and its muramidase activity 10% of that of human lysozyme. One-dimensional NMR spectroscopy indicated the presence of a compact, well structured protein. From two-dimensional NMR spectra, main chain resonances for 118 of a total of 129 residues could be readily assigned. Nuclear Overhauser effect analysis and hydrogen-deuterium exchange measurements indicated the presence and persistence of all expected secondary structure elements. Thermal denaturation, measured by circular dichroism, showed a single transition temperature for the Ca2+ form at 90 degrees C, whereas unfolding of the apo form occurred at 73 degrees C in the near-UV and 81 degrees C in the far-UV range. These observations illustrate that by transplanting the central part of bovine alpha-lactalbumin, we have introduced into human lysozyme two important properties of alpha-lactalbumins, i.e. stabilization through Ca2+ binding and molten globule behavior.

Amino Acid Sequence↗

PubMed1995

An equilibrium partially folded state of human lysozyme at low pH.

Temperature-induced unfolding of human lysozyme has been monitored by circular dichroism and by nuclear magnetic resonance experiments at a variety of low pH values. The results indicate that, although at pH values above 3 unfolding appears to be consistent with a two-state model, at lower pH values this is not the case. At pH 1.2, for example, unfolding of the tertiary structure occurs at a temperature approximately 10 deg. C lower than that of the secondary structure. At 60 degrees C there is no detectable native tertiary structure remaining for human lysozyme at pH 1.2, although far-UV CD results show preservation of some 40% of the signal attributable to alpha-helical elements in the protein. This indicates the existence of a partially folded state of human lysozyme at low pH that has at least some characteristics of the well-defined molten globule state of the homologous alpha-lactalbumins and of the kinetic intermediates observed in the folding of alpha-lactalbumins and of c-type lysozymes. These results suggest that the absolute distinction between these two groups of proteins in terms of their different unfolding behaviour is not valid, and provide insights into possible features stabilizing such states.

Animals↗

PubMed1995

Polar agents with differentiation inducing capacity potentiate tumor necrosis factor-mediated cytotoxicity in human myeloid cell lines.

Cotreatment or pretreatment of several human myeloid cell lines (KG1, HL60, U937, THP1) with the differentiation inducer DMSO was found to potentiate the antiproliferative and cytotoxic effects of TNF. In addition, TNF-resistant monocytic cell lines could be sensitized to TNF cytotoxicity by DMSO treatment. Other highly polar molecules, known to be potent differentiation inducers, showed similar effects to those of DMSO. The potentiating effect of DMSO was related neither to an up-regulation of TNF receptor expression nor to an alteration in the rate of TNF internalization and degradation. We present evidence that the TNF activities are p55 TNF receptor-mediated and are not due to insertion of TNF into lipid bilayers, an effect that could be susceptible to DMSO, as this component has been described to modify cell membrane characteristics. DMSO-induced potentiation of TNF cytostasis/cytotoxicity was restricted to myeloid leukemia cell lines. In non-myeloid cells such as fibrosarcomas, myosarcomas, thymomas, or carcinomas, DMSO was found either not to alter or to inhibit TNF-induced cell death. The latter results are in good agreement with data reported by others who suggested that DMSO could act as a scavenger of TNF-induced toxic radical formation. The potential correlation in myeloid cells between DMSO-induced changes in the cells' differentiation status and DMSO-enhanced TNF-susceptibility is discussed.

Animals↗

PubMed1995

Potentiation of tumor necrosis factor-mediated cytotoxicity on human myeloid cell lines: effects of interferons versus dimethylsulphoxide.

After prolonged incubation times of 72 h IFN alpha 2a and IFN beta 1 significantly reduced cell growth in the myelomonocytic U937 and THP1 cell lines. IFN gamma showed only slight growth inhibitory activities. IFN activities were potentiated by the highly polar differentiation inducer dimethylsulphoxide, which is similar to our previous study on tumor necrosis factor (TNF). However, in contrast with TNF, none of the interferon types induced cell cytotoxicity or DNA fragmentation. Like DMSO, all interferons potentiated TNF-induced cytotoxicity, IFN beta 1 and IFN gamma being the most potent in this respect. When applied together, DMSO and IFN gamma enhanced TNF-mediated cell lysis in either an additive (in the case of U937, THP1, HL60 cells) or a synergistic (in the case of KG1) manner, suggesting that the mechanisms of the potentiating activity of DMSO and IFN gamma are different. The potential role of the simultaneous use of DMSO-related molecules, and TNF and/or IFN in leukemic cancer chemotherapy is discussed.

Antineoplastic Agents↗

PubMed1994

Polar agents with differentiation-inducing capacity prime myelomonocytic cell lines to lipopolysaccharide-induced cytolysis: the role of endogenous tumor necrosis factor.

Treatment of the human myelomonocytic U937 and THP1 cell lines for 24 h with 180 mM of the differentiation inducer DMSO, resulted in priming these cells to subsequent LPS-induced cytolysis. The observed cytotoxicity was LPS dose-dependent and characterized by a prolonged lag phase with detectable effects only appearing after 8 h. LPS-induced apoptotic cell death in DMSO-pretreated U937 cells as indicated by the appearance of 200 basepair DNA fragments upon agarose gel electrophoresis of total cellular DNA. Furthermore, DMSO pretreatment potentiated the cells' capacity to produce cytokines, especially TNF, upon LPS stimulation. This endogenously present TNF was metabolized by the cells. These observations suggested that the LPS-induced cytostasis/cytotoxicity was mediated through TNF. Indeed, medium conditioned by LPS-stimulated U937-DMSO cells was found to exert a cytotoxic effect on U937-DMSO cells that was completely neutralized by anti-human TNF antiserum. Such TNF-like activities were not only present in the supernatant but also at the level of the cell membrane of LPS-stimulated U937-DMSO cells. Apart from TNF, other exogenously applied recombinant cytokines (IL1, IL6, IFN gamma, GM-CSF) were not cytotoxic to U937-DMSO cells. Thus, DMSO-pretreated myelomonocytic cells become sensitive to LPS-induced cytotoxicity, which is, at least in part, mediated through endogenous TNF.

Cell Death↗

PubMed1993

Complexes of the polyamines spermine, spermidine and putrescine with alpha-lactalbumins.

The effects of polyamines on the spectral properties and thermal stability of different alpha-lactalbumins were measured. Addition of millimolar concentrations of spermine to the Ca(2+)-free (apo) form of bovine or goat alpha-lactalbumin resulted in spectral shifts, in both the far- and near-ultraviolet ranges, similar to those induced by Ca2+ binding. Fluorescence emission spectra of tryptophan residues underwent a pronounced blue shift, concomitant with a decrease in quantum yield. Also, in the presence of spermine, thermal stability was increased. In contrast, in the case of human and to some extent also equine alpha-lactalbumin, the shifts of the CD and fluorescence spectra and the thermal denaturation curves were in the opposite direction. Direct competition of spermine with Mn2+ could be observed for binding to the Ca2+ site of bovine alpha-lactalbumin. As the main binding mode of spermine showed a 1:1 stoichiometry [K1 = (2 +/- 0.5) x 10(4) M-1], we have assumed that binding occurs primarily at this site with one of the distal ammonium groups. In order to accommodate the remaining positively charged ammonium groups of spermine, a binding model was constructed by computer modeling, based on the atomic coordinates of both interacting species. This model makes use of the cluster of negatively charged glutamate residues present in the N-terminal sequence of bovine alpha-lactalbumin, sterically close to the Ca2+ site. The spermine molecule could be nicely fitted in a space delineated by the Ca2+ site, on the one hand, and Glu1, Glu7 and Glu11, on the other. Our model, which has been extended to the shorter polyamines spermidine and putrescine, is able to fully account for the observed stabilization of bovine (and goat) alpha-lactalbumins against thermal denaturation, and for the observed CD and fluorescence spectral shifts. It also explains why human (and equine) alpha-lactalbumin behave differently.

Amino Acid Sequence↗

PubMed1993

Stability effects associated with the introduction of a partial and a complete Ca(2+)-binding site into human lysozyme.

Two mutants of human lysozyme were synthesized. Mutant A92D, in which Ala92 was substituted by Asp, contains a partial Ca(2+)-binding site and mutant M4, in which Ala83, Gln86, Asn88 and Ala92 were replaced by Lys, Asp, Asp and Asp respectively, contains the complete Ca(2+)-binding site of bovine alpha-lactalbumin. The Ca(2+)-binding constants of wild type human lysozyme and of mutants A92D and M4, measured at 25 degrees C and pH 7.5, were 2(+/- 1) x 10(2) M-1, 8(+/- 2) x 10(3) M-1 and 9(+/- 0.5) x 10(6) M-1 respectively. Information gathered from microcalorimetric and CD spectroscopic measurements indicates that the conformational changes of the M4 mutant lysozyme, induced by Ca2+ binding, are smaller than those observed for bovine alpha-lactalbumin and for the Ca(2+)-binding equine lysozyme. At pH 4.5, the thermostability of both the apo and Ca2+ forms of the A92D human was decreased in comparison with that of native human lysozyme. In particular, within the apo form of this mutant an alpha-helix-containing sequence was destabilized. In contrast, at the same pH the thermostability of the apo and Ca2+ forms of the M4 mutant lysozyme was increased. The epsilon-ammonium group of the Lys83 side chain is assumed to be responsible for the stabilization of the apo form of this mutant.

Amino Acid Sequence↗

PubMed1992

"In vitro" effect of interleukin-1 beta on human glioma cell lines: regulation of cell proliferation and IL-6 production.

The human glioma cell lines U251 and HP591 were chosen as "in vitro" models for functional astrocytes. When cultured in the presence of IL-1 beta these cell lines demonstrated a marked increase in interleukin-6 production and in [3H]-thymidine uptake. The addition of dbcAMP could mimic the first effect of IL-1 beta but at the same time suppressed cell proliferation. These results suggest that IL-1 beta possibly exerts one of its biological effects (IL-6 synthesis) by means of the cyclic AMP pathway.

Bucladesine↗

PubMed1992

Binding characteristics and thermal behaviour of cytochrome-C oxidase, inserted into phospholipid-coated, magnetic nanoparticles.

A strategy for the immobilization of cytochrome-c oxidase, used as a representative membrane-bound enzyme, into so-called magnetoliposomes has been developed. The latter structures consist of a phospholipid bilayer which covers nanometer-sized Fe3O4 colloids. Incorporation of the enzyme into the phospholipid envelope is facilitated by a short sonication step. Upon adsorption, the reaction characteristics of the lipid-depleted enzyme are drastically changed. With double-layered phosphatidylcholine (PC) magnetoliposomes the activity increases by a factor of approximately 5. After a first magnetic fractionation step, approximately 67% of the activity remains with the magnetoliposome retentate. Subsequent magnetophoresis cycles show that the adsorbed enzyme is firmly fixed into the phospholipid coat. Upon immobilization, the thermal behavior is also profoundly affected. The heating inactivation curves show two sigmoidal transition zones. Irrespective of the PC type used, a first inflection point is located near 39 degrees C, whereas a second one, which is located at higher temperatures, clearly depends on the acyl chain length (56 degrees C with dimyristoyl-PC and 60 degrees C for dioleoyl-PC and Ovothin-200). An identical behavior is observed with classical proteoliposomes with an equal phospholipid composition. By contrast, monolayer-coated dimyristoyl-PC magnetic structures are inferior with respect to both their reactivation potency and their ability to strongly affix cytochrome-c oxidase and to improve the thermal stability of the enzyme.

Colloids↗